Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems

Several atypical antipsychotics exert mood-stabilising effects via the modulation of various monoamine receptors and intracellular signallings. Recent pharmacodynamic studies suggested that tripartite synaptic transmission can contribute to the pathophysiology of schizophrenia and mood disorders, their associated cognitive impairment, and several adverse reactions to atypical antipsychotics. Therefore, to explore the mechanisms underlying the antidepressive mood-stabilising and antipsychotic effects of brexpiprazole (Brex), we determined the effects of subchronic administration of therapeutically relevant concentrations/doses of Brex on the protein expression of 5-HT receptors, connexin43, cAMP levels, and intracellular signalling in cultured astrocytes and rat hypothalamus using ultra-high-pressure liquid chromatography with mass spectrometry and capillary immunoblotting systems. Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) downregulated both 5-HT1A (5-HT1AR) and 5-HT7 (5-HT7R) receptors, in addition to phosphorylated Erk (pErk), without affecting phosphorylated Akt in the astroglial plasma membrane. Subchronic administration of 300 nM Brex decreased and increased phosphorylated AMPK and connexin43, respectively, in the astroglial cytosol fraction. A therapeutically relevant concentration of Brex acutely decreased the astroglial cAMP level, whereas, under the inhibition of 5-HT1AR, Brex did not affect astroglial cAMP levels. However, the 5-HT7R-agonist-induced increased astroglial cAMP level was inhibited by Brex. In contrast to the in vitro study, systemic subchronic administration of effective doses of Brex (3 and 10 mg/kg/day for 14 days) increased the cAMP level but did not affect phosphorylated AMPK in the rat hypothalamus. These results suggest several complicated pharmacological features of Brex. Partial 5-HT1AR agonistic action predominates in the low range of therapeutically relevant concentrations of Brex, whereas in the high range, 5-HT7R inverse agonist-like action is overlapped on the 5-HT1A agonistic action. These unique suppressive effects of Brex on 5-HT7R play important roles in the clinical features of Brex regarding its antidepressive mood-stabilising actions.

Introduction
A novel mood-stabilising atypical antipsychotic agent, brexpiprazole (Brex), 7-[4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydro-2(1H)-quinolinone (Figure 1), has been approved for the treatment of schizophrenia and major depression in several countries, including the USA, EU, Canada, Australia, and Japan [1]. Several meta-analyses and systematic reviews confirmed that the antipsychotic effects of Brex for the treatment of patients with schizophrenia in the acute phase and for relapse prevention are comparable to those of other atypical antipsychotics [2,3]. A recent meta-analysis study provided findings for Brex as an efficacious augmentation agent in antidepressant-resistant major depression, comparable to other mood-stabilising atypical antipsychotics [4]. Indeed, Brex was approved as an adjunctive treatment to oral antidepressants in major depression based on three clinical trials that showed relatively rapid improvement (within six weeks) of The major mechanisms of Brex are considered to be similar to those of aripiprazole: partial agonism with high affinity at the dopamine D2 receptor (D2R) (Ki = 0.30 nM) and 5-HT1A receptor (5-HT1AR; Ki = 0.12 nM) (Table 1) [9,10]. Furthermore, the intrinsic activities at D2R and 5-HT1AR are lower than those for aripiprazole [11]. However, the clinical features of Brex cannot be fully explained by its partial agonisms to D2R and 5-HT1AR alone [12]. A pharmacogenomic study revealed that 5-HT7 receptor (5-HT7R) antagonism contributes to the pathophysiology of mood disorders and schizophrenia [13]. Additionally, functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of several psychiatric disorders [13][14][15]. Indeed, the expression of several monoamine receptors, such as 5-HT1AR, D2R, 5-HT7R, and 5-HT2A receptor , in astrocytes has been identified [12,[16][17][18]. These preclinical findings suggest that the involvement of astroglial monoaminergic transmission provides an opportunity to expand a pathophysiological hypothesis of neuropsychiatric disorders, such as novel monoaminergic tripartite synaptic transmission, including astroglial transmission [13,19].   [27] According to the monoaminergic tripartite synaptic transmission hypothesis, it has been suggested that the pharmacodynamic profile associated with astroglial transmission associated with hemichannels correlates with efficacy in mood disorders [15,19]. Con-nexin43-containing astroglial hemichannels in the plasma membrane fraction of cultured astrocytes were found to be upregulated by several mood-stabilising atypical antipsychotics-clozapine, quetiapine, and zotepine [12,28,29]-but downregulated by the antidepressant vortioxetine [18]. Interestingly, a selective 5-HT-transporter-inhibiting antidepressant, escitalopram, did not affect connexin43 protein expression in the astroglial plasma membrane under the 5-HT-free condition [18]. Contrary to the results of in vitro study, systemic administration of fluoxetine and duloxetine increased the expression of The major mechanisms of Brex are considered to be similar to those of aripiprazole: partial agonism with high affinity at the dopamine D2 receptor (D2R) (Ki = 0.30 nM) and 5-HT1A receptor (5-HT1AR; Ki = 0.12 nM) (Table 1) [9,10]. Furthermore, the intrinsic activities at D2R and 5-HT1AR are lower than those for aripiprazole [11]. However, the clinical features of Brex cannot be fully explained by its partial agonisms to D2R and 5-HT1AR alone [12]. A pharmacogenomic study revealed that 5-HT7 receptor (5-HT7R) antagonism contributes to the pathophysiology of mood disorders and schizophrenia [13]. Additionally, functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of several psychiatric disorders [13][14][15]. Indeed, the expression of several monoamine receptors, such as 5-HT1AR, D2R, 5-HT7R, and 5-HT2A receptor (5-HT2AR), in astrocytes has been identified [12,[16][17][18]. These preclinical findings suggest that the involvement of astroglial monoaminergic transmission provides an opportunity to expand a pathophysiological hypothesis of neuropsychiatric disorders, such as novel monoaminergic tripartite synaptic transmission, including astroglial transmission [13,19]. Table 1. Receptor binding profiles of mood-stabilising atypical antipsychotics. Brexpiprazole (Brex), aripiprazole (APZ), clozapine (CLZ), lurasidone (LUR), quetiapine (QTP), ziprasidone (ZIP), zotepine (ZTP) against serotonin (5-HT) type 1A (5-HT1AR), type 2A (5-HT2AR), type 7 (5-HT7R) receptor, histamine type 1 (H1R) receptor and dopamine receptors type 1 (D1R) and 2 (D2R). Data are equilibrium constant (Ki) values (nM). According to the monoaminergic tripartite synaptic transmission hypothesis, it has been suggested that the pharmacodynamic profile associated with astroglial transmission associated with hemichannels correlates with efficacy in mood disorders [15,19]. Connexin43-containing astroglial hemichannels in the plasma membrane fraction of cultured astrocytes were found to be upregulated by several mood-stabilising atypical antipsychotics-clozapine, quetiapine, and zotepine [12,28,29]-but downregulated by the antidepressant vortioxetine [18]. Interestingly, a selective 5-HT-transporter-inhibiting antidepressant, escitalopram, did not affect connexin43 protein expression in the astroglial plasma membrane under the 5-HT-free condition [18]. Contrary to the results of in vitro study, systemic administration of fluoxetine and duloxetine increased the expression of connexin43 mRNA or protein in the total lysate [30,31], whereas lipopolysaccharide-and corticosterone-induced upregulation of connexin43 was suppressed by fluoxetine [32,33]. These previous findings suggest that the activation and suppression of tripartite synaptic transmission associated with astroglial hemichannels are involved in mood-stabilising and antidepressive actions, respectively [15]. Indeed, a mood-stabilising antipsychotic, lurasidone, exhibits exceptional clinical and preclinical characteristics among the moodstabilising antipsychotics since lurasidone is more effective in its antidepressive action than its antimanic action, possibly due to its inhibitory effects on astroglial L-glutamate release through hemichannels via its 5-HT7R inverse agonistic action [34]. Additionally, the 5-HT7R inverse agonistic action of lurasidone plays an important role in the mechanisms of a lower risk of developing metabolic complications via the suppression of adenosine monophosphate-activated protein kinase (AMPK) signalling [34].

Receptor
Considering the similar clinical features of lurasidone and Brex, an antidepressivedominant action, and a lower risk of developing metabolic complications, the binding affinity of Brex to 5-HT7R (Ki = 3.7 nM) can provide us with information regarding the scientific effectiveness in a pharmacodynamic examination of the effects of Brex on astroglial transmission associated with 5-HT7R [9,10]. Although 5-HT7R-binding atypical antipsychotics have been reported, and many of these agents have shown 5-HT7R inhibition, it has not been clarified whether they are antagonists or inverse agonists [13]. Our recent study demonstrated that Brex weakly enhanced astroglial L-glutamate release through activated astroglial hemichannels, whereas the mechanisms of Brex were not identical to those of clozapine or quetiapine [12]. Clozapine, quetiapine, and zotepine enhanced the trafficking of connexin43 to the plasma membrane via the activation of protein kinase B (Akt) signalling, but the action of Brex was not dependent on Akt signalling [12], similar to that of lurasidone (high-affinity 5-HT7R inverse agonist) [34]. These previous findings suggest that the unclarified effects of Brex on 5-HT7R possibly contribute to the action of Brex on tripartite synaptic transmission associated with astroglial hemichannels. Therefore, the detailed mechanisms of Brex on 5-HT7R and connexin43 remain to be clarified. Based on these previous findings, we aimed to explore the effects of Brex on tripartite synaptic transmission associated with 5-HT7R, determined in the present study via subchronic administration of Brex on astroglial transmission using a capillary immunoblotting system. Additionally, when Brex inhibits AMPK signalling or 5-HT7R, the subsequent effects of subchronic systemic administration of Brex on AMPK signalling in the hypothalamus possibly contribute to the mechanisms of a lower risk of metabolic complications [34].

Effects of Brex on Astroglial Signalling
To clarify the mechanisms underlying the time-dependent effects of Brex on several astroglial proteins associated with 5-HT signalling, the effects of subchronic administration of a therapeutically relevant concentration of Brex on the expression of proteins such as 5-HT1AR, 5-HT7R, phosphorylated extracellular signal-regulated kinase (pErk), phosphorylated protein kinase B (pAkt), and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) in the plasma membrane or cytosol fractions of astrocytes were determined using a capillary immunoblotting system. The therapeutically relevant serum concentrations of Brex are reported to range from 90 nM to 300 nM [35,36]. In accordance with a previous clinical report, in the present study, the cortical primary cultured astrocytes were subchronically (for 7 and 14 days) administered 300 nM Brex [12]. The major procedures of preparation of the cultured astrocytes and the study design are described in the following Section 4.2. according to our previous studies [12,18,34,[37][38][39][40][41][42][43] Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 14 days decreased 5-HT1AR expression, whereas administration for 7 days did not affect its expression (F(2,15) = 21.8(p < 0.01)); however, subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 7 or 14 days decreased 5-HT7R expression in the plasma membrane fractions (F(2,15) = 20.4(p < 0.01)) ( Figure 2). in the plasma membrane fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of 5-HT1AR and 5-HT7R per GAPDH. * p < 0.05, ** p < 0.01: relative to control (Brex-free) by one-way analysis of variance (ANOVA) with Tukey's post-hoc test, and @ p < 0.05: relative to Brex for 14 days by Student's T-test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.
To clarify the mechanisms of time-dependent downregulation of 5-HT1AR and 5-HT7R induced by Brex, the cultured astrocytes were also subchronically administered 300 nM Brex along with the 5-HT1AR antagonist WAY100635 (10 μM) for 14 days. In accordance with our expectations, WAY100635 prevented the downregulation of 5-HT1A and 5-HT7R induced by subchronic administration of Brex ( Figure 2).
The therapeutically relevant concentration of Brex also downregulated both 5-HT1AR and 5-HT7R, but the downregulation of 5-HT7R induced by Brex had a faster onset than did that of 5-HT1AR. The subchronic administration of 5-HT1AR agonist and 5-HT7R inverse agonist downregulated both 5-HT1A and 5-HT7R; however, the downregulation of 5-HT1A and 5-HT7R induced by 5-HT7R inverse agonist is faster in its onset that that induced by 5-HT1AR agonist [18,34]. Taken together with the previous findings, these results suggest that Brex is a candidate 5-HT7R inverse agonist.

Effects of Brex on Intracellular Signal Transduction Protein in the Plasma Membrane Fraction of Astrocytes
Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 7 or 14 days did not affect the protein expression of pAkt (F(2,15) = 1.1(p > 0.1)) ( Figure 3). Contrary to the results for Akt, Brex (300 nM) administration for 14 days decreased pErk levels, whereas that for 7 days did not affect them (F(2,15) = 4.4(p < 0.05)) ( Figure 3). in the plasma membrane fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of 5-HT1AR and 5-HT7R per GAPDH. * p < 0.05, ** p < 0.01: relative to control (Brex-free) by one-way analysis of variance (ANOVA) with Tukey's post-hoc test, and @ p < 0.05: relative to Brex for 14 days by Student's T-test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.
To clarify the mechanisms of time-dependent downregulation of 5-HT1AR and 5-HT7R induced by Brex, the cultured astrocytes were also subchronically administered 300 nM Brex along with the 5-HT1AR antagonist WAY100635 (10 µM) for 14 days. In accordance with our expectations, WAY100635 prevented the downregulation of 5-HT1A and 5-HT7R induced by subchronic administration of Brex ( Figure 2).
The therapeutically relevant concentration of Brex also downregulated both 5-HT1AR and 5-HT7R, but the downregulation of 5-HT7R induced by Brex had a faster onset than did that of 5-HT1AR. The subchronic administration of 5-HT1AR agonist and 5-HT7R inverse agonist downregulated both 5-HT1A and 5-HT7R; however, the downregulation of 5-HT1A and 5-HT7R induced by 5-HT7R inverse agonist is faster in its onset that that induced by 5-HT1AR agonist [18,34]. Taken together with the previous findings, these results suggest that Brex is a candidate 5-HT7R inverse agonist.

Effects of Brex on Intracellular Signal Transduction Protein in the Plasma Membrane Fraction of Astrocytes
Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 7 or 14 days did not affect the protein expression of pAkt (F(2,15) = 1.1(p > 0.1)) ( Figure 3). Contrary to the results for Akt, Brex (300 nM) administration for 14 days decreased pErk levels, whereas that for 7 days did not affect them (F(2,15) = 4.4(p < 0.05)) ( Figure 3).

Effects of Brex on pAMPK in the Astroglial Cytosol Fraction
Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 14 days decreased pAMPK levels in the astroglial cytosol fraction, whereas administration for 7 days did not affect them (F(2,15) = 6.4 (p < 0.01)) ( Figure 4). and phosphorylated extracellular signal-regulated kinase pErk (panel B) in the plasma membrane fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of pAkt and pErk per GAPDH. * p < 0.05: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Effects of Brex on pAMPK in the Astroglial Cytosol Fraction
Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 14 days decreased pAMPK levels in the astroglial cytosol fraction, whereas administration for 7 days did not affect them (F(2,15) = 6.4(p < 0.01)) ( Figure 4). . Effects of subchronic administration (7 and 14 days) of therapeutic relevant concentration of Brex (Brex: 300 nM) on protein expression of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) in the cytosol fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of pAMPK per GAPDH. ** p < 0.01: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Effects of Subchronic Administration of a Therapeutically Relevant Concentration of Brex on Connexin43 Protein Expression in Astrocytes
Subchronic administration of a therapeutically relevant concentration of Brex increased the protein expression of connexin43 in the astroglial plasma membrane [12]. The  and phosphorylated extracellular signal-regulated kinase pErk (panel B) in the plasma membrane fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of pAkt and pErk per GAPDH. * p < 0.05: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Effects of Brex on pAMPK in the Astroglial Cytosol Fraction
Subchronic administration of a therapeutically relevant concentration of Brex (300 nM) for 14 days decreased pAMPK levels in the astroglial cytosol fraction, whereas administration for 7 days did not affect them (F(2,15) = 6.4(p < 0.01)) ( Figure 4). In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of pAMPK per GAPDH. ** p < 0.01: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting. . Effects of subchronic administration (7 and 14 days) of therapeutic relevant concentration of Brex (Brex: 300 nM) on protein expression of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) in the cytosol fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of pAMPK per GAPDH. ** p < 0.01: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Effects of Subchronic Administration of a Therapeutically Relevant Concentration of Brex on Connexin43 Protein Expression in Astrocytes
Subchronic administration of a therapeutically relevant concentration of Brex increased the protein expression of connexin43 in the astroglial plasma membrane [12]. The trafficking process to the plasma membrane of connexin43 is regulated by various protein phosphorylation systems, such as Akt and Erk [14,15,44] signalling [14,15,44]. However, in the present study, subchronic administration of Brex did not affect Akt signalling, while it inhibited Erk signalling. These results suggest that the increased protein expression of connexin43 is probably not modulated by these signalling pathways. Therefore, to clarify the mechanisms behind the increased protein expression of connexin43 in the plasma membrane, the effects of subchronic administration of Brex (300 nM for 14 days) on the protein expression of connexin43 in the astroglial cytosol fraction were examined since the transcription of connexin43 is regulated by AMPK signalling via its activity [34,45].
trafficking process to the plasma membrane of connexin43 is regulated by various protein phosphorylation systems, such as Akt and Erk [14,15,44] signalling [14,15,44]. However, in the present study, subchronic administration of Brex did not affect Akt signalling, while it inhibited Erk signalling. These results suggest that the increased protein expression of connexin43 is probably not modulated by these signalling pathways. Therefore, to clarify the mechanisms behind the increased protein expression of connexin43 in the plasma membrane, the effects of subchronic administration of Brex (300 nM for 14 days) on the protein expression of connexin43 in the astroglial cytosol fraction were examined since the transcription of connexin43 is regulated by AMPK signalling via its activity [34,45].

Figure 5.
Effects of subchronic administration (7 and 14 days) of therapeutic relevant concentration of Brex (300 nM) on protein expression of connexin43 (Cx43) in the cytosol fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD (n = 6) of the relative protein level of Cx43 per GAPDH. * p < 0.05: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Effects of Subchronic Systemic Administration of Brex on pAMPK in the Hypothalamus
The suppressive effects of Brex on AMPK signalling could explain the pathophysiology of the low risk of weight gain with Brex [8]. Therefore, to clarify the possible mecha-  Ordinates indicate mean ± SD (n = 6) of intracellular cAMP level in cultured astrocytes (fmol/mg). ** p < 0.01: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test.

Effects of Subchronic Systemic Administration of Brex on pAMPK in the Hypothalamus
The suppressive effects of Brex on AMPK signalling could explain the pathophysiology of the low risk of weight gain with Brex [8]. Therefore, to clarify the possible mecha-  The suppressive effects of Brex on AMPK signalling could explain the pathophysiology of the low risk of weight gain with Brex [8]. Therefore, to clarify the possible mechanisms of Brex regarding the low risk of weight gain, the effects of subchronic administration of effective doses of Brex on hypothalamic AMPK signalling in vivo were examined. Unexpectedly, subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days did not affect the pAMPK level in the rat hypothalamus (Figure 8).
nisms of Brex regarding the low risk of weight gain, the effects of subchronic administration of effective doses of Brex on hypothalamic AMPK signalling in vivo were examined. Unexpectedly, subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days did not affect the pAMPK level in the rat hypothalamus ( Figure 8).

Effects of Subchronic Systemic Administration of Brex on cAMP Levels in the Hypothalamus
To clarify the mechanisms of the discrepant effects of Brex on AMPK signalling, the effects of subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days on cAMP synthesis in the rat hypothalamus were examined. Subchronic administration of a therapeutically relevant concentration of Brex (10 mg/kg/day) for 14 days increased the cAMP level in the rat hypothalamus, whereas 3 mg/kg/day of Brex did not affect the cAMP level ( Figure 9).

Effects of Subchronic Systemic Administration of Brex on cAMP Levels in the Hypothalamus
To clarify the mechanisms of the discrepant effects of Brex on AMPK signalling, the effects of subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days on cAMP synthesis in the rat hypothalamus were examined. Subchronic administration of a therapeutically relevant concentration of Brex (10 mg/kg/day) for 14 days increased the cAMP level in the rat hypothalamus, whereas 3 mg/kg/day of Brex did not affect the cAMP level ( Figure 9). nisms of Brex regarding the low risk of weight gain, the effects of subchronic administration of effective doses of Brex on hypothalamic AMPK signalling in vivo were examined. Unexpectedly, subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days did not affect the pAMPK level in the rat hypothalamus ( Figure 8).

Effects of Subchronic Systemic Administration of Brex on cAMP Levels in the Hypothalamus
To clarify the mechanisms of the discrepant effects of Brex on AMPK signalling, the effects of subchronic administration of effective doses of Brex (3 and 10 mg/kg/days) for 14 days on cAMP synthesis in the rat hypothalamus were examined. Subchronic administration of a therapeutically relevant concentration of Brex (10 mg/kg/day) for 14 days increased the cAMP level in the rat hypothalamus, whereas 3 mg/kg/day of Brex did not affect the cAMP level ( Figure 9). Ordinates indicate mean ± SD (n = 6) of cAMP level in the rat hypothalamus (fmol/mg). * p < 0.05: relative to control (Brex-free) by one-way ANOVA with Tukey's post-hoc test.

Discussion
Our proposed hypothesis regarding the mechanisms of Brex on astroglial transmission demonstrated by the present and previous studies [12,18,34,41,43] is summarised in Figure 10. demonstrations by in vitro experiments using cultured astrocytes have indicated the possibility that the inhibitory effects of Brex on 5-HT7R play important roles in the clinical actions of Brex via the modulation of astroglial signalling. Figure 9. Effects of subchronically systemic administration (14 days) of effective doses of Brex (3 and 10 mg/kg/day) on cAMP levels in the rat hypothalamus. Ordinates indicate mean ± SD (n = 6) of cAMP level in the rat hypothalamus (fmol/mg). * p < 0.05: relative to control (Brex-free) by oneway ANOVA with Tukey's post-hoc test.

Discussion
Our proposed hypothesis regarding the mechanisms of Brex on astroglial transmission demonstrated by the present and previous studies [12,18,34,41,43] is summarised in Figure 10. demonstrations by in vitro experiments using cultured astrocytes have indicated the possibility that the inhibitory effects of Brex on 5-HT7R play important roles in the clinical actions of Brex via the modulation of astroglial signalling. Chronic administration of a therapeutically relevant concentration of Brex downregulates both 5-HT1AR and 5-HT7R via its 5-HT1AR partial agonistic and possible 5-HT7R inverse agonistic actions, respectively. The downregulation of 5-HT1AR and 5-HT7R by Brex attenuates AMPK, Erk, and Akt signalling in astrocytes.

Effects of Subchronic Administration of Brex on the Expression of 5-HT1AR and 5-HT7R in Astrocytes
This study demonstrated that the subchronic administration of a therapeutically relevant concentration of Brex affected astroglial transmission through the modulation of 5-HT1AR and 5-HT7R functions. Subchronic administration of a therapeutically relevant concentration of Brex downregulated both astroglial 5-HT1AR and 5-HT7R. These pharmacological features of the subchronic administration of Brex on 5-HT1AR and 5-HT7R seem to be similar to those of lurasidone and vortioxetine [34,43]. It has been revealed that Brex is a 5-HT1AR partial agonist with 60% intrinsic activity [10]. In the present study, acute administration of a therapeutically relevant concentration of Brex decreased the astroglial cAMP level. Activation of 5-HT1AR suppresses cAMP synthesis via the inhibition of adenylate cyclase [12,13,18,34,43,46]. Therefore, these results suggest that the 5-HT1AR Figure 10. Proposed hypothesis of the mechanisms of Brex on intracellular signalling in astrocytes. Chronic administration of a therapeutically relevant concentration of Brex downregulates both 5-HT1AR and 5-HT7R via its 5-HT1AR partial agonistic and possible 5-HT7R inverse agonistic actions, respectively. The downregulation of 5-HT1AR and 5-HT7R by Brex attenuates AMPK, Erk, and Akt signalling in astrocytes.

Effects of Subchronic Administration of Brex on the Expression of 5-HT1AR and 5-HT7R in Astrocytes
This study demonstrated that the subchronic administration of a therapeutically relevant concentration of Brex affected astroglial transmission through the modulation of 5-HT1AR and 5-HT7R functions. Subchronic administration of a therapeutically relevant concentration of Brex downregulated both astroglial 5-HT1AR and 5-HT7R. These pharmacological features of the subchronic administration of Brex on 5-HT1AR and 5-HT7R seem to be similar to those of lurasidone and vortioxetine [34,43]. It has been revealed that Brex is a 5-HT1AR partial agonist with 60% intrinsic activity [10]. In the present study, acute administration of a therapeutically relevant concentration of Brex decreased the astroglial cAMP level. Activation of 5-HT1AR suppresses cAMP synthesis via the inhibition of adenylate cyclase [12,13,18,34,43,46]. Therefore, these results suggest that the 5-HT1AR partial agonistic action of Brex is at least partly involved in the downregulation of 5-HT1AR. However, in spite of the downregulation of 5-HT1AR, the suppression of cAMP synthesis was continuously observed after the subchronic Brex administration. These results cannot be interpreted as indicating only 5-HT1AR partial agonistic action of Brex.
The mechanisms of downregulation of 5-HT7R induced by subchronic Brex administration require detailed discussion since the binding affinity of Brex to 5-HT7R has been reported, but the function of Brex on 5-HT7R remains to be clarified [10]. Notably, several mood-stabilising atypical antipsychotics, such as olanzapine, clozapine, and lurasidone, are considered to be 5-HT7R inverse agonists since these three mood-stabilising atypical antipsychotics inhibit 5-HT7R but downregulate 5-HT7R expression [34,47]. In the present study, Brex itself acutely decreased the astroglial cAMP level, whereas, under the 5-HT1AR inhibition by WAY100635 (5-HT1AR antagonist), Brex could also decrease the cAMP level. These results suggest that the inhibitory effects of Brex on cAMP synthesis in astrocytes are generated by not only 5-HT1AR partial agonistic action but also some other mechanism. The application of a 5-HT7R agonist, AS19, and an inverse agonist, SB269970, increased and did not affect astroglial cAMP levels, respectively [12,13,18,34,43,46]. Indeed, Brex pretreatment antagonised the AS19-induced increased cAMP level. Therefore, these results suggest that Brex is a 5-HT7R inverse agonist, similar to clozapine, olanzapine, and lurasidone, since Brex inhibits but downregulates 5-HT7R [18,34,47]. Assuming that Brex is a 5-HT7R inverse agonist, the contradictory effect in which subchronic Brex administration continued to suppress cAMP synthesis despite the downregulation of inhibitory 5-HT1AR can be explained by the inhibition and downregulation of excitatory 5-HT7R induced by its inverse agonistic action.
Based on these previous findings and the binding profile of Brex, a high-affinity partial agonist to D2R and 5-HT1AR, we speculated that Brex increases both Erk and Akt signalling. However, contrary to our expectations, subchronic administration of a therapeutically relevant concentration of Brex did not enhance Akt or Erk signalling. These contradictions between the previous findings and the present results in interpreting the pathophysiology of Brex suggest that a 5-HT7R-inverse agonist-like action of Brex, with inhibition of 5-HT7R function and downregulation of 5-HT7R expression, probably provides the novel pathophysiological strategies [13]. The basis of our hypothesis regarding 5-HT7R inverse agonists has already been supported by lurasidone and vortioxetine [18,34,43,[57][58][59]. Subchronic administration of a therapeutically relevant concentration of lurasidone and vortioxetine suppressed Erk signalling without affecting Akt signalling [18,34,43]. Similar to these two agents, a selective 5-HT7R inverse agonist, SB269970, also inhibited Akt and Erk [18,60] signalling [18,60]. In particular, SB269970 rapidly downregulated 5-HT1AR, 5-HT7R, and Erk signalling [18,43] and generated rapid-acting anti-immobility-like and antidepressant-like effects [61]. Taken together with these previous findings, the present results suggest that the activation of Akt and Erk signalling is not necessarily a mechanism of the clinical action of mood-stabilising atypical antipsychotics. Rather, recent preclinical studies suggest that hyperactivation of Akt signalling plays an important role in the pathophysiology of several serious adverse reactions induced by clozapine, such as cardiotoxicity, non-infectious pneumonia, convulsions, and metabolic syndrome [12,19,28,44].

Impacts of the Suppression of AMPK Signalling on Tripartite Synaptic Transmission
In our previous study, several mood-stabilising atypical antipsychotics, such as clozapine, quetiapine, and zotepine, enhanced the trafficking of connexin43 to the plasma membrane via the activation of Akt signalling [12,28,40]; exceptionally, Brex weakly increased protein expression of connexin43 in the astroglial plasma membrane, but this trafficking process was independent of Akt signalling [12].
On the contrary, vortioxetine and lurasidone enhanced connexin43 expression in astroglial cytosol via the inhibition of AMPK signalling [18,34]. It has been established that AMPK signalling regulates the transcription of various ion channels via the activation of histone deacetylase [45]. The transcription of connexin43 is also regulated by AMPK signalling [34,62]. To clarify the mechanisms by which Brex induced increased connexin43 expression in the astroglial plasma membrane, the present study examined the effects of subchronic administration of Brex on astroglial AMPK signalling and protein expression of connexin43 in the cytosol fraction. In accordance with our expectations, Brex increased the connexin43 level in the cytosol fraction and suppressed AMPK signalling. These actions of Brex on connexin43 are similar to those of the histone deacetylase inhibitor valproate [12,28].

Clinical Implication and Low Risk of Metabolic Syndrome
A recent meta-analysis study reported that Brex is among the better antipsychotics associated with a low risk of metabolic outcomes [8]. AMPK signalling activator in peripheral organs is considered to be a promising candidate anti-obesity agent due to its activation stimulating muscle and hepatic mitochondrial biogenesis and fatty acid oxidation [63]; however, contrary to peripheral organs, in the central nervous system, activation of AMPK signalling in the hypothalamus enhances the secretion of glucagon, corticosterone, and epinephrine, resulting in the activation of hepatic gluconeogenesis and glycogenolysis [64]. AMPK activity in the hypothalamus is regulated by nutrients, anorexigenic and orexigenic signalling, cAMP/PKA, and histamine H1 receptor (H1R) [65,66]. The hypothalamus, which is the main sensor of nutrient concentrations, receives afferents (dopamine, norepinephrine, 5-HT, and histamine) from various basal brain regions [65].
Initially, the weight gain risks of Brex were speculated [67] since the affinity of Brex to H1R was Ki = 19 nM [10] ( Table 1). The inhibition of H1R is considered to be involved in the high risk of weight gain of several atypical antipsychotics via activation of AMPK signalling [65]. Indeed, both clozapine and olanzapine (high-affinity H1R antagonists [65,66]) induced weight gain was compensated by H1R agonist via suppression of AMPK signalling [68][69][70], whereas lurasidone (low binding affinity to H1R) suppresses AMPK signalling in the hypothalamus [34]. Although we could not identify the expression of histamine H1R in the astroglial plasma membrane, therefore, the suppressive effects of Brex on astroglial AMPK signalling cannot deny being over-evaluated due to the lack of expression of H1R in astrocytes. Therefore, to clarify the actual mechanisms of low risk of weight gain of Brex, the subchronic administration of effective dose of Brex (10 mg/kg/day) for 14 days on cAMP levels and AMPK signalling in rat hypothalamus were determined [10,71]. The lack of activation of AMPK signalling in astrocytes and hypothalamus can explain, at least partially, the mechanisms of the low risk of weight gain property of Brex; however, the discrepant effects of Brex on AMPK signalling between astrocyte and hypothalamus need some discussion.
The D2R (Ki = 0.3 nM) and 5-HT1AR (Ki = 0.12 nM) partial agonistic actions of Brex contribute to increasing cAMP level, whereas 5-HT7R inverse agonist-like action (Ki = 3.7 nM) decreases cAMP level (Table 1). Considering these receptor binding profiles of Brex, it is probably a reasonable demonstration that Brex decreased cAMP levels in the astrocytes. Although the results demonstrated by in vitro astroglial experiments strongly suggested that the 5-HT7R inverse agonist-like action of Brex was a candidate mechanism, similar to lurasidone, the results of in vivo hypothalamus experiments deny our expectations since subchronically systemic administration of Brex increased cAMP levels in the hypothalamus. The direct interaction between 5-HT7R and H1R on intracellular signalling has remained to be clarified since the major second messenger system of H1R is inositol phosphate [72,73]. However, activation of H1R weakly increases cAMP synthesis via G βγ subunits from G proteins [73]. Therefore, the binding profile of Brex to these four receptors (D2R, 5-HT1AR, 5-HT7R, and H1R) alone cannot fully explain the effects of Brex on AMPK signalling or cAMP synthesis in the hypothalamus. Additionally, both Akt and Erk signallings also contribute poorly to the effects of Brex on AMPK signalling since these signallings form a negative feedback loop with AMPK signalling [74][75][76].
The present study demonstrated that the effects of Brex on tripartite synaptic transmission are probably y generated by complicated mechanisms compared to those of clozapine, quetiapine, zotepine, and lurasidone [12,28,41]. The therapeutic relevant concentration of Brex weakly enhanced astroglial L-glutamate release through connexin43-containing hemichannel, similar to clozapine, quetiapine, and zotepine [12,28,41]; however, the effect of Brex on trafficking and transcription processes of connexin43 was rather similar to lurasidone and vortioxetine, which suppress connexin43 function via their 5-HT7R inverse agonistic action [18,34]. These results suggest that the 5-HT7R inverse agonist-like action of Brex cannot be ignored as a mechanism of antidepressive-dominant mood-stabilising effects of Brex. In contrast to mechanisms of mood-stabilising action, the lack of stimulatory effects on AMPK signalling (decreased and did not affect AMPK signalling in respective astrocyte and hypothalamus) possibly contributes to a part of the mechanism of low risk of weight gain of Brex; however, rather, the results in the present study strongly indicate the existence of another major mechanism. Therefore, exploring the effects of Brex on the AMPK signalling cascade can identify the novel mechanisms of mood-stabilising action and low risk of weight gain of Brex. We shall explore the effects of acute and chronic effects of Brex on AMPK signalling cascade using in vivo study in the future.

Chemical Agents and Drug Administration
Brex was obtained from Funakoshi (Tokyo, Japan). 5-HT1AR antagonist, WAY100635, 5-HT7R agonist, and AS19 [77] were obtained from CosmoBio (Tokyo, Japan). These three agents were prepared on the day of the experiment and were initially dissolved in dimethyl sulfoxide at 25 mM. The final dimethyl sulfoxide concentration was lower than 0.1% (vol/vol).
The therapeutic relevant serum concentration of Brex was reported to range from 90 nM to 300 nM [35,36]. Based on the clinical findings, in the present study, cultured astrocytes were administrated by 300 nM Brex for 7 or 14 days [12]. According to previous studies, the cultured astrocytes were administrated by 10 µM WAY100635 and 5 µM AS19. Previous studies have reported that the effective dose of Brex was 3 or 10 mg/kg/day [10,71]. Based on the previous reports, in the present study, to explore the dose-dependent effects of systemic subchronic administration of Brex on cAMP levels and AMPK signalling in the rat hypothalamus, rats were subcutaneously administered Brex (3 or 10 mg/kg/day) for 7 and 14 days using an osmotic pump (2ML_1 and 2ML_2, Alzet, Cupertino, CA, USA).

Preparation of Primary Astrocyte Culture
All animal care and experimental procedures described in this report were performed according to the ethical guidelines established by the Institutional Animal Care and Use Committee at Mie University, Japan (no. 2019-3, 24 May 2019) and are reported in accordance with the Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines [78]. Mainly, the preparation of astrocytes followed the protocol of the previous study [12,18,34,43]. Pregnant Sprague-Dawley rats (SLC, Shizuoka, Japan) were housed individually in cages, which were kept in air-conditioned rooms (temperature, 22 ± 2 • C) with a 12 h light/dark cycle and free access to food and water. Cultured astrocytes were prepared from cortical astrocyte cultures of neonatal Sprague-Dawley rats (n = 36), which were sacrificed by decapitation at 0-48 hr of age. The cerebral hemispheres were removed using a dissecting microscope. The cerebral tissue was chopped into fine pieces using scissors and then triturated briefly with a micropipette. The suspension was filtered using 70 µm nylon mesh (BD, Franklin Lakes, NJ, USA) and centrifuged. Then, the pellets were resuspended in 10 mL Dulbecco's modified Eagle's medium (D6546; Sigma-Aldrich, St. Louis, MO, USA) containing 10% foetal calf serum (fDMEM); this procedure was repeated three times. The day after culturing for 14 days (DIV14), contaminated cells were removed by shaking in a standard incubator for 16 h at 200 rpm. Astrocytes were removed from flasks by trypsinisation and seeded directly onto a translucent polyethylene terephthalate (PET) membrane (1.0 µm) with 24 well plates (BD) at a density of 100 cells cm 2 for experiments from DIV14 to DIV28, the culture medium (fDMEM) was changed twice a week, and Brex (300 nM) was administered subchronically (for 7 days, DIV21-28; or for 14 days, DIV14-28) [79]. Previously, our methods detected that the remaining adherent cells included more than 90% glial fibrillary acidic protein-positive and A2B5-negative cells, as detected using immunohistochemical staining [39].

Extraction of Cultured Astrocytes and Rat Hypothalamus
On DIV28, the cultured astrocytes were washed out using ACSF (comprised NaCl 150.0 mM, KCl 3.0 mM, CaCl 2 1.4 mM, MgCl 2 0.8 mM, and glucose 5.5 mM and buffered to pH 7.3 with 20 mM HEPES buffer), and this procedure was repeated three times [79]. After the subchronic administration of effective doses of Brex [43,58,59], the rat hypothalamus was dissected according to the method described by Glowinski and Iversen [80]. To apply the capillary immunoblotting system, after the washout, the cytosol and plasma membrane fractions of cultured astrocytes and dissected rat hypothalamus were extracted using a Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA). To determine the intracellular cAMP level, after the washout, the cultured astrocytes and dissected rat hypothalamus were placed into respective 0.5 mL and 1.5 mL microtubes and homogenised with an ultrasonic cell disrupter (VP-050N, Taitec, Koshigaya, Japan) in chilled 0.1 N HCl. The mixture was centrifuged at 10,000× g for 20 min at 4 • C. Filtered aliquots (5 µL) were injected into the ultra-high-performance liquid chromatography (UHPLC) with a mass spectrometry system (LCMS).

Determination of Intracellular cAMP Levels in Cultured Astrocytes and Rat Hypothalamus
The cAMP levels were determined by UHPLC (Acquity UPLC H-Class system; Waters, Milford, MA, USA) with mass spectrometry (Acquity SQ detector; Waters, Milford, MA, USA). Five microlitres of filtered samples was injected using an autosampler (Acquity UPLC Sample Manager FTN; Waters, Milford, MA, USA). cAMP was separated by UHPLC equipped with a graphite carbon column (particle 3 µm, 150 × 2.1 mm; Hypercarb, Thermo, Waltham, MA, USA) at 40 • C, and the mobile phase was set at 450 µL/min [40]. A linear gradient elution programme was used for over 10 min with mobile phases A (1 mM ammonium acetate buffer, pH 11) and B (acetonitrile). The nitrogen flows of the desolvation and cone were set at 750 and 5 L/h, respectively, and the desolvation temperature was set at 450 • C. The cone voltage for the determination of cAMP (m/z = 330.3) was 42 V.

Data Analysis
All experiments in this study were designed with equally sized animal groups (n = 6) without conducting a formal power analysis, in accordance with previous studies [18,43,58,59]. All values are expressed as the mean ± standard deviation (SD), and a p-value < 0.05 (two-tailed) was considered statistically significant for all tests. Drug levels for acute, subchronic, and chronic administrations were selected on the basis of values reported in previous studies. Where possible, we aimed to randomise and blind the data. Particularly, for the determination of cAMP levels and protein expression, the sample order on the autosampler and Wes was determined using a random number table.
Time-dependent effects of subchronic and chronic administrations of Brex on protein levels (connexin43, 5-HT1AR, 5-HT7R, phosphorylated ERK, and phosphorylated AKT) in the cytosol and plasma membrane fractions were analysed by one-way analysis of variance (ANOVA) with Tukey's multiple comparison, using Bell Curve for Excel ver3.2 (Social Survey Research Information Co., Ltd., Tokyo, Japan). The interaction between Brex and WAY100635 on protein expression was analysed by Student's T-test using Bell Curve for Excel. The concentration-dependent and dose-dependent effects of Brex on cAMP levels and pAMPK levels were also analysed using one-way ANOVA with Tukey's multiple comparison. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology [81].

Nomenclature of Targets and Ligands
Key protein targets and ligands in this report are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, (accessed on 1 June 2020) common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 202/22 [10,34,65,72,74,75,77].

Conclusions
The present study determined the effects of subchronic administration of therapeutic relevant concentration of Brex on astroglial signalling-associated 5-HT receptors to explore mechanisms underlying mood-stabilising antipsychotic effects and low risk of weight gain by Brex. Subchronic administrations of therapeutic relevant concentrations of Brex downregulated both 5-HT1A and 5-HT7R. The response of downregulation of 5-HT7R was faster than that of 5-HT1AR since the downregulations of 5-HT1A and 5-HT7R were observed for 14 days and 7 days, respectively. In the low range of therapeutic relevant concentration of Brex (100 nM), acutely, the 5-HT1AR partial agonistic action was predominant, but in the high range, the 5-HT7R inverse agonistic action was added to the 5-HT1A partial agonistic action. These concentration-dependent complicated actions of Brex contribute to various intracellular signallings in astrocytes. The intracellular signalling of Erk and AMPK were also downregulated by subchronic administration of therapeutic relevant concentration of Brex for 14 days (synchronised with the downregulation of 5-HT1AR), whereas Akt signalling was not affected by Brex. Contrary to in vitro studies, systemically subchronic administration of effective doses of Brex did not affect AMPK signalling but increased cAMP levels in the hypothalamus. These discrepant results between in vitro (astrocyte) and in vivo (thalamus) suggest that the D2R partial agonistic with low intrinsic activity and H1R antagonistic actions of Brex, which lead to weight gain via activation of thalamic AMPK signallings, is probably suppressed by other mechanisms, such as 5-HT7R inverse agonist-like action.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of ethical restrictions.

Conflicts of Interest:
The authors state no conflict of interest.


s2fieldsofstudy: Biology Medicine

text:
Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

Introduction
Cellular communication network factor 2, CCN2, is a cysteine-rich secreted protein and often a down-stream mediator of transforming growth factor-β (TGF-β). Previously known as connective tissue growth factor (CTGF), the biological roles of CCN2 include the regulation of cell proliferation, differentiation, survival, adhesion, extracellular matrix (ECM) accumulation, and angiogenesis. CCN2 has been regarded as a candidate responsible for fibrotic change (Shi-wen et al. 2006). Upregulation of hepatic CCN2 occurs in fibrotic liver in humans (Paradis et al. 1999) and CCN2 expression in liver parenchyma correlated positively with progressive NASH in a monkey model (Chen et al. 2018). Moreover, inhibition of CCN2 attenuated liver fibrogenesis in a carbon tetrachloride-induced liver fibrosis mouse model (Uchio et al. 2004). CCN2 is implicated in a number of end-organ complications in diabetes, especially in tissues 1 3 where accumulation of ECM components occurs including the kidney in nephropathy, and the heart in cardiomyopathy (Frazier et al. 1996).
In previous studies published by our laboratory, CCN2 was reported to be upregulated in a clinically relevant murine high fat fed and diabetes NASH experimental model (Lo et al. 2011). Considering its known biological behaviour, CCN2 may not only be a marker, but also a mediator, in fibrosis progression in NASH. In order to investigate whether CCN2 may be involved in the worsening of NASH pathology, systemic administration of anti-CCN2 neutralizing antibody as an intervention was studied in the HFD alone, diabetes alone and the HFD plus diabetes mouse model (Lo et al. 2011), both in terms of effects on NASH with fibrosis and in exploring potential cellular mediators of CCN2 effects.
CCN2 is one of the six CCN family members, as matricellular associated proteins involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types (Brigstock 2003). CCNs are expressed early in development and are recruited by cells to facilitate different tissue functions, especially in wound healing and several diseases (Yeger and Perbal 2007). Regarding the role of CCN members other than CCN2 in liver disease, CCN1 in a fat fed model has been shown to prevent NAFLD, especially steatosis, to induce apoptosis of hepatic pro-fibrotic activated stellate cells and to reduce the expression of fibrosis markers (Borkham-Kamphorst et al. 2014). CCN3 has been reported to potentially be anti-fibrotic, partly through acting to inhibit pro-fibrotic gene induction as well as through inhibiting CCN2 gene expression (Leask 2009). In contrast, the involvement of CCN4, CCN5 and CCN6 in liver fibrosis is largely unknown. In this study, in addition to the main aim of targeting CCN2 to determine if that approach may prevent NASH fibrosis, we also investigated potential mechanisms of CCN2 effects in NASH liver, including on other CCN members.

Animal and experimental design
Male C57BL/6 J mice aged 5 weeks, a total of n = 45, were purchased from the Animal Resource Centre (Perth, Western Australia), housed with a 12 h light /dark cycle, and provided food and water ad libitum. After 1 week adaptation, the mice were randomly allocated into 2 groups fed either with standard chow (CHOW; 12% kcal fat content) or a high fat diet (HFD; 45% kcal fat content) each for 26 weeks. The HFD was prepared in-house with a formula based on rodent diet no. D12451 from Research Diets (New Brunswick, NJ), as published by ourselves (Lo et al. 2011), and others (Rossmeisl et al. 2003). To induce type 2 diabetes some mice from each group were randomly selected and injected at 15 weeks with low dose streptozotocin (STZ: 3 × 65 mg/ kg i.p.). For the final 10 weeks, from week 16 to week 26, mice randomly selected from the diabetes alone, HFD alone, and HFD plus diabetes (HFD + DM) groups, were injected intraperitoneally with either neutralizing CCN2Ab (150 µg/ per mouse, twice per week, diluted in Phosphate Buffered Saline), or normal control rabbit IgG (150 µg/per mouse, twice per week, diluted in Phosphate Buffered Saline). Animals were weighed and blood glucose levels were checked weekly throughout the study (Lo et al. 2011). Insulin tolerance tests (ITT) were performed 1 week before termination, as previously described (Lo et al. 2011). At termination all mice were anesthetized and mice were euthanased by exanguination and several indices were examined in the collected liver tissue. Both HFD and diabetes induction are required in the model especially to induce robust liver fibrosis in the model (Lo et al. 2011). The mouse groups generated and studied, with respective mouse numbers per group, are shown in Schematic Fig. 1. All animal procedures were approved by the animal ethics committee of Sydney South-West Area Health Services, and Sydney Local Health District, NSW, Australia.

Generation of anti-CCN2 neutralizing antibody
The neutralizing anti-CCN2 polyclonal antibody was commercially generated by IMVS (Adelaide, South Australia). A New Zealand White Rabbit was injected s.c. with 160 µg of full length recombinant human (rh) CCN2 (Wang et al. 2010) in Freund's adjuvant, followed by four further sequential s.c. injections of the 160 µg full length rhCCN2 at 3 week intervals, also in Freund's adjuvant. The antiserum was collected as a terminal bleed and was found to detect full-length rhCCN2 by Western blot (not shown). The IgG total amount purified from rabbit serum was 72 mg. The CCN2Ab as 10 µg/mL of the IgG, when applied to murine H9C2 cardiomyocytes, was found to prevent induction of Collagen I, Collagen III, and Fibronectin mRNA levels at 24 h (not shown), otherwise caused by addition of 500 ng/ mL of rhCCN2 to unconditioned media (Wang et al. 2010).

Quantitative real-time polymerase chain reaction
Total RNA was isolated from liver tissue by PureLink RNA mini Kit (Life technologies, USA) according to the manufacturer's instructions. Total RNA (2000 ng) from each sample was reverse-transcribed to cDNA using Random Hexamer Primer (Invitrogen, CA, USA) and Superscript III reverse transcriptase (Invitrogen). The mRNA levels of MCP-1, TNF-ɑ, IL-1ß, IL-6, Collagen-I, -III, -IV, -VI, FN, CCN2, TGF-ß1, TIMP-1, CCN1 and CCN3, were determined by quantitative real-time PCR using SYBR green fluorophore (Invitrogen). The primer pair sequences used for respective mRNA species are shown in Table 1. In each case, the mRNA of interest expression level was normalized by 18 s ribosomal RNA (Lo et al. 2011). The relative amount of target mRNA in each sample was determined by applying the threshold cycle to the standard curve.

Tissue preparation and histological studies
One portion of liver tissue (left lobe) was fixed in 10% buffered formalin, processed, and then embedded in paraffin. Paraffin sections of 4 µm were stained with Picro Sirius Red (PSR) to evaluate total crosslinked collagen accumulation and stained by immunohistochemistry to detect collagen-I protein. Liver tissue (medial lobe) frozen at collection in OCT was sectioned at 6 µm thickness and stained by immunohistochemistry to detect F4/80 protein. The primary antibodies used were anti-collagen-I (1:400 dilution, AB765P, Millipore, Billerica, USA), and anti-F4/80 (1:500 dilution, ab6640, Abcam, Cambridge, UK). Appropriate biotin conjugated secondary antibodies were followed by Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Collagen by Sirius Red Stain and Collagen-I, and F4/80 staining were scored for overall staining intensity. In brief, slides were examined under light microscopy visually as described. For the same scored measure of interest, all the sections were stained in the same batch and then scored in the same  session. All sections were photographed using an Olympus Provis AX70 microscope (Olympus Optical Co., Japan). For each slide, 10 fields (which collectively cover the majority of the section), were randomly chosen for each sample scoring, at 200×magnification. The sections were viewed by two independent observers blinded to the group source of the sample section. Staining was scored as a value between 0 and 4, where 0 was no staining, and 4 was very intense staining, with reporting as an average of the two observers scores, as previously published (Lo et al. 2011).

Oxidative stress measurement
Malondialdehyde (MDA) content was measured using the thiobarbituric acid reactive substances (TBARS) assay (Sigma-Aldrich, St. Louis, MO) by measuring absorbance value at a 532 nm wavelength. Superoxide-dismutase (SOD) activity was measured using the xanthine oxidase method to measure the absorbance value at 550 nm with the SOD kit (Sigma-Aldrich), according to the manufacturer's instructions.

Statistical analysis
Results are shown as mean ± standard deviation (SD) in Table 2 and as mean ± standard error of the mean (SEM) in Figs. 2, 3, 4, 5, 6, 7, 8 and 9 inclusive. All data were compared using one-way ANOVA followed by post hoc analysis using Bonferroni's multiple comparisons test or an un-paired T-test. Statistical significance was accepted at p < 0.05.

Effects of CCN2Ab on metabolic measures
Body weight of HFD+IgG and HFDDM+IgG mice at week 25, as expected, was greater than Chow mice. In the Chow-diabetes group and the HFD plus diabetes group, as expected, blood glucose levels were elevated in each and they were also insulin resistant as shown with a greater glucose area under the curve (AUC) value than Chow fed mice when subjected to an ITT (Table 2). Notably, none of these metabolic measures were affected by the CCN2Ab compared with the control IgG in each of the respective mouse groups ( Table 2).

Effects of CCN2Ab on hepatic inflammatory markers
While it is mainly thought to be pro-fibrotic, CCN2 has also been reported to induce an inflammatory response (Abraham 2008;Sanchez-Lopez et al. 2009). To assess if inflammatory changes are affected by the anti-CCN2 neutralizing antibody, expression of key hepatic pro-inflammatory cytokines were examined at the mRNA level in liver tissue including TNF-ɑ, MCP-1, IL-1β and IL-6. High-fat feeding induced TNF-ɑ mRNA ~ 3.5 fold compared with Chow + IgG and the induction was inhibited by CCN2Ab in HFD mice. TNF-ɑ mRNA was also significantly elevated ~ 5.1 fold in the HFD + DM + IgG group compared with Chow + IgG and this induction was normalized by CCN2Ab ( Fig. 2A). Intrahepatic MCP-1 mRNA expression was significantly increased in the HFD plus diabetes group by 4.7fold compared with Chow + IgG, and the CCN2Ab treatment inhibited MCP-1 mRNA induction in this group (Fig. 2B). For IL-1β (Fig. 2C) and also IL-6 ( Fig. 2D), there was no changes observed in any group. Mouse macrophage marker F4/80 was assessed by immunohistochemistry (Fig. 2E, G). F4/80 immunopositivity was elevated in HFD+DM+IgG compared with Chow+IgG (p < 0.05; Fig. 2H) and appeared to be partially reversed by the CCN2Ab, but not to statistical significance. In contrast to effects of CCN2 Ab treatment observed on some inflammatory marker mRNA levels in liver, by H&E staining, no effect of the CCN2 Ab treatment on hepatic steatosis compared with control IgG treatment was observed in any mouse group examined (neither HFD, nor in HFD+DM, data not shown).

Effects of CCN2Ab on intrahepatic fibrosis in the high fat feeding with diabetes mouse model
CCN2 as an upstream and downstream mediator of TGF-β bioactivity, can stimulate the expression of many ECM proteins, and also decrease matrix degradation by matrix metalloproteinase through the upregulation of tissue inhibitor of metalloproteinase (TIMP-1) in hepatic stellate cells and myofibroblasts (MFB) (McLennan et al. 2004). Dysregulation of CCN2 is involved in several diseases which involve fibrosis (Chen et al. 2009;Panek 2009;Rachfal and Brigstock 2003). In this study, in order to investigate the effects of targeting CCN2, the impact on ECM was determined by RT-qPCR analysis of mRNA expression of collagen-I, -III, -IV, -VI, CCN2 itself, TGF-β, and TIMP-1. Expression of ECM components is shown in Fig. 3. Collagen-I mRNA expression was significantly increased in the HFD+DM+IgG group ~ 8.4 fold, compared with Chow+IgG and was significantly prevented by CCN2Ab (Fig. 3A). Similarly for collagen-III mRNA levels, HFD+IgG animals showed a non-significant fivefold induction compared with Chow-fed+IgG animals, while this induction was significant in the HFD+DM+IgG group, at 6.7-fold greater than Chow+IgG. The induction of collagen-III mRNA in the HFD+DM+IgG group was significantly prevented by the CCN2Ab (Fig. 3B). High-fat feeding and high-fat feeding with diabetes increased the mRNA levels of both collagen-IV and collagen-VI only as trends, without showing statistical significance. The CCN2Ab showed a trend towards lowering the abundance of these collagens in the HFD + DM group (Fig. 3C, D).
Expression of a series of growth factors and effects of CCN2Ab treatment are shown in Fig. 3. The CCN2 mRNA level was significantly induced in the HFD + DM + IgG group by ~ 2.1-fold compared to Chow + IgG, which was partially inhibited by the CCN2Ab treatment (Fig. 4A). TGF-β mRNA levels showed a non-significant trend towards elevation in the HFD + IgG and HFD + DM + IgG groups, and the CCN2Ab treatment significantly inhibited Data are mean ± SEM as fold change of Chow + IgG. *p < 0.05, significantly different from Chow + IgG; # p < 0.05, significantly different from HFD + DM + IgG the mRNA expression induced in the HFD + DM + IgG group (Fig. 4B). TIMP-1 mRNA levels were elevated in both HFD + IgG and HFD + DM + IgG groups, to 8.7-fold and 10.6-fold respectively, compared with Chow + IgG, but, notably, these changes were not affected by the CCN2Ab treatment (Fig. 4C).
In order to investigate possible effects of neutralizing CCN2Ab on the protein levels of fibrotic markers in the NASH model, PSR staining was used to assess total crosslinked collagen accumulation and Collagen-I protein levels was examined by immunostain. Figure 5 shows representative images of PSR staining in the liver sections from each group of mice. As expected, and as previously observed by our group in this model (Lo et al. 2011), the diabetes alone group did not show differences compared with Chow-fed animals (Fig. 5B). The PSR staining score of the HFD + IgG group was ~ twofold induced compared with Chow + IgG, and it was not affected by the CCN2Ab antibody (Fig. 5C, F and H). In contrast, the combination of HFD + DM + IgG showed a statistically significant induction scores (H). *p < 0.05, significantly different from Chow + IgG; # p < 0.05, significantly different from HFD + DM + IgG in the collagen PSR stain ~ 2.5-fold within the liver lobule sited around the central veins and portal tract as well as across the liver parenchyma, and which was significantly lowered by CCN2Ab therapy (Fig. 5D, G and H). Type-I Collagen is the main collagen induced in NASH and was found to be the most induced in the HFD + DM + IgG group and that induction was statistically significantly inhibited by CCN2Ab only in that group (Fig. 6D, G and H).

Potential mechanisms of NASH prevention by CCN2Ab
CCN2 activates the MAPK cascade in several cell types (Crean et al. 2002;Wahab et al. 2005;Yosimichi et al. 2006), and in other studies some of the CCN2 bioactivity is reported to be mediated by activation of the MAPK signaling pathway (Nagai et al. 2009). To investigate signaling pathways through which CCN2 may contribute to NASH progression, the intrahepatic levels of ERK1/2, p38 and JNK MAPK proteins in HFD + DM mice, with or without CCN2Ab treatment in vivo, were measured by immunoblotting. HFD + DM was the focus of these analyses because the combined metabolic insult causes the greatest induction of fibrosis in the NASH model.
The phosphorylation of ERK in liver lysate was found to be significantly induced in the HFD + DM mouse model compared with normal chow fed mice, and the phosphorylation of ERK was lower following in vivo CCN2Ab treatment ( Fig. 7A and B). In contrast to the findings for ERK phosphorylation, no significant difference in phosphorylation of p38 was found in HFD + DM mice, nor in the antibody treatment group, compared to normal chow fed mice ( Fig. 7C and  D). Total JNK and phospho-JNK were each not significantly changed in the HFD + DM model compared to Chow + IgG. While total JNK was significantly induced in the CCN2Ab treatment group (quantitation not shown), the phospho-JNK to total JNK ratio was not significantly affected by CCN2Ab treatment (Fig. 7E and F).

Effects of CCN2Ab treatment on intrahepatic oxidative stress in the high fat feeding with diabetes mice
Oxidative stress is implicated in the pathogenesis of NAFLD (Sumida et al. 2013). Reactive oxygen species and lipid peroxidation products have been demonstrated to precede necro-inflammatory changes, hepatic stellate cell activation and collagen deposition (Sanchez-Valle et al. 2012). Antioxidant treatment, which decreases oxidative stress, also attenuates the severity of fibrosis in experimental steatohepatitis (Al-Busafi 2012; Vizzutti et al. 2010). As a potentially important pro-fibrotic molecule in hepatic fibrosis, CCN2 is upregulated by oxidative stress in various diseases (Aragno et al. 2008;Elmarakby and Sullivan 2012). However, whether blocking CCN2 activity may ameliorate oxidative stress in NASH has not been clarified.   Fig. 7 Effects of CCN2Ab on MAPK signaling pathway in high fat fed with diabetes mice. A detection of phospho-ERK, total ERK by immunoblot; GAPDH is a protein loading control; C detection of phospho-p38, total p38 by immunoblot; ɑ-tubulin is a protein loading control; E. detection of phospho-JNK, total JNK by immunoblot; GAPDH is a protein loading control. Band intensity in the immunoblot was semi-quantitated using ImageLab (B, D and F). Data is mean ± SEM as fold change of Chow + IgG. *p < 0.05, significantly different from Chow + IgG, # p < 0.05, significantly different from HFD + DM + IgG In our study, we detected oxidative stress by measuring the lipid peroxidation level and by determining antioxidant enzyme activity. MDA concentration, which reflects oxidative stress, compared with the Chow group, was not changed in the HFD + DM group, and was slightly decreased by the CCN2Ab treatment (Fig. 8A). Antioxidant total SOD activity (mitochondrial and cytosolic SOD combined) was significantly decreased in the HFD + DM + IgG group compared with Chow, and trended towards an increase in the CCN2Ab treatment group (Fig. 8B). Cytosolic SOD trended towards decrease in the HFD + DM group and was not affected by the CCN2Ab treatment (Fig. 8C).

Regulation of CCN family member by CCN2Ab treatment in the high fat feeding with type 2 diabetes model
Across CCN family members, CCN protein expression or activities are dysregulated in various pathological conditions, including inflammation and fibrosis (Leask and Abraham 2006). The CCNs have important roles as matricellular proteins, involved in the connection between cell surface and extracellular matrix (ECM) structure and signaling (Leask and Abraham 2006). CCN1 (Kim et al. 2013), CCN2 (Rachfal andBrigstock 2003), and CCN3 (Borkham-Kamphorst et al. 2012) have each been studied to some degree in liver fibrosis. However, the changing profiles in response to blockade of CCN2 bioactivity and possible roles of other CCN family members in NASH development have not been clarified to date. In this study, the respective profile of CCN family member expression in experimental NASH was examined.
As a regulator expressed in myofibroblasts preventing fibrosis through induction of cellular senescence (Borkham-Kamphorst et al. 2014), CCN1 mRNA was unchanged in the CHOW fed diabetes alone group, whereas it was induced in the HFD alone model. CCN1 was also significantly induced in the HFD with added type 2 diabetes model, by ~ 1.7-fold compared with Chow. The CCN2Ab did not have effects in the diabetes nor in HFD alone groups, and it showed only a trend towards lowering CCN1 in the HFD + DM model (Fig. 9A). In addition, CCN1 protein level was induced in the HFD + DM model compared with Chow to ~ 1.3-fold, and was unaffected by CCN2Ab ( Fig. 9C and D). As an antifibrotic molecule in the CCN family, the gene expression of CCN3 showed a trend to being lower in the diabetes alone, HFD alone, and the HFD with diabetes group, each compared with the Chow group. Blocking CCN2 by CCN2Ab showed a trend only to normalisation of the CCN3 gene expression in diabetes alone and HFD alone groups, whereas CCN3 was clearly statistically significantly induced in the HFD + DM group (Fig. 9B). The CCN3 protein level was significantly decreased in the HFD + DM model compared with Chow and the CCN2Ab partially normalized the CCN3 protein level observed in this model ( Fig. 9C and E). The expression levels of CCN4, CCN5 and CCN6 were not significantly changed across DM, HFD or HFD + DM groups, and were not affected by CCN2Ab in any mouse group (data not shown).

Discussion
Intervention with anti-CCN2 neutralizing antibody in two NAFLD models was studied in this work. Data demonstrated that targeting CCN2 bioactivity by the CCN2Ab for 10 weeks provided some protective effects against NASH induced by high fat feeding with diabetes, particularly in inflammation markers and in fibrosis end-points. The main aim in this study was to investigate the role of CCN2 in NASH development and to examine whether its inhibition may be a potential target as a preventive therapy for NASH. Fig. 8 Effects of CCN2Ab on MDA content, and SOD activity in high fat fed with diabetes mice. MDA content (A), total SOD activity (B) and cytosolic SOD activity (C) were measured in high fat feeding with diabetes mice. Data is shown as MDA concentration in Fig. A and as mean ± SEM as fold change of Chow in Fig. B and C. *p < 0.05, significantly different from Chow, by un-paired T-test Some insights into mechanism of action of the CCN2Ab in this NASH model were realised. If there is a central mechanism it is possibly through affecting known proinflammatory and/or pro-fibrotic effects of CCN2. Studies have suggested a potential role for CCN2 in chronic inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, inflammatory kidney disease and neuroinflammatory pathologies (Kular et al. 2011). Leukocyte, especially macrophage, recruitment is an important event involved in inflammation (Muller 2013) and CCN2 can contribute to this phenomenon (Sanchez-Lopez et al. 2009). CCN2 was shown to induce adhesion of peripheral blood activated monocytes and macrophages in vitro, and to promote infiltration of T lymphocytes and monocytes in the renal interstitium in vivo (Sanchez-Lopez et al. 2009;Kular et al. 2011). CCN2 also functions by regulating cytokine and chemokine expression in various cell systems. In cardiomyocytes, CCN2 increases the expression of the pro-inflammatory cytokines TNFα, IL-6, MCP-1 and IL-8 (Wang et al. 2010). To date, the role of CCN2 in the inflammation of NASH is unclear. The current work showed that blocking the bioactivity of CCN2 decreased MCP-1 gene expression and it improved TNF-α levels in the HFD + DM NASH model, suggesting that CCN2 is pro-inflammatory in this model. Related signalling pathways including NF-ĸB need to be investigated in future studies.
In addition to its pro-inflammatory role, CCN2 is regarded as an important mediator involved in fibrotic processes; it stimulates fibroblast growth and ECM protein upregulation in vitro and in vivo. Over-production of CCN2 has been proposed to be important in pathways leading to fibrosis in many tissues including skin, kidney, heart and lung (Leask and Abraham 2004). However, whether CCN2 contributes to fibrosis progression in NASH combined with type 2 diabetes has been unclear. The data in this research shows that blocking CCN2 bioactivity with neutralizing antibody attenuated fibrotic changes, including inhibition of collagen and pro-fibrotic marker gene expression induction and also key ECM and profibrotic protein levels, in the liver. Our laboratory has previously reported that CCN2 may contribute to matrix accumulation not only through ECM formation but also through the inhibition of matrix degradation Detection of CCN1 and CCN3 by immunoblot; GAPDH was a protein loading control (C). Band intensity in the immuno-blot was quantitated using ImageLab (D and E). Data is shown as Mean ± SEM as fold change of Chow + IgG. *p < 0.05, significantly different from Chow + IgG, # p < 0.05, significantly different from HFD + DM + IgG via TIMP-1 induction by CCN2 (McLennan et al. 2004). In this study, in contrast to robust effects of CCN2 bioactivity in human renal mesangial cells (McLennan et al. 2004), TIMP-1 gene expression was only minimally normalized by CCN2Ab treatment in the HFD + DM NASH model. Nonetheless, the results in this study indicate that CCN2 plays an important pro-fibrotic role during NASH fibrosis progression, especially in the HFD + DM model. The MAPK family of serine-threonine protein kinases can be activated by various and multiple extracellular stimuli (Wetzker and Bohmer 2003). Three major MAPK subfamilies, including ERK, p38 and JNK, mediate a number of downstream events such as cellular proliferation, differentiation, and apoptosis by activating transcription factors, in a specific cell type and situation dependent manner (Wetzker and Bohmer 2003;Kyriakis and Avruch 2001). CCN2 has been shown to activate MAPK cascades in several cell types (Crean et al. 2002;Wahab et al. 2005;Yosimichi et al. 2006). We examined whether any of these pathways were involved in matrix regulation in the NASH mouse model by inhibiting CCN2 with the CCN2Ab. The result showed that blocking CCN2 caused lowered phosphorylation levels of ERK. This suggests that CCN2Ab may attenuate liver fibrosis through inhibiting the ERK signalling pathway. The increased level of total JNK in the CCN2Ab treatment group may be due to inhibition of one pathway causing the induction of another pathway, including total levels of protein.
Oxidative stress has been implicated in the pathogenesis of NAFLD (Sumida et al. 2013). The role of oxidative stress in steatosis and its progression to steatohepatitis has been widely discussed. Increased levels of reactive oxygen species (ROS) and lipid peroxidation products (such as MDA) and decreased level of antioxidant enzymes (such as SOD and catalase) and compounds (such as glutathione) have been reported in patients with NASH compared to healthy controls (Videla et al. 2004). In addition, oxidative stress can regulate CCN2 expression in different cell types (Elmarakby and Sullivan 2012;Videla et al. 2004). The data in this study does not clearly address whether CCN2 inhibition downregulates oxidative stress, as overall, detectable changes in oxidative stress measures were small, and the trends towards normalization for the MDA level and SOD activity in the anti-CCN2 neutralizing antibody treatment group were not statistically significant.
In addition to CCN2, emerging studies have reported that the CCN protein family may be a relatively new appreciated class of mediators in inflammation and fibrosis processes (Kular et al. 2011). CCN1 is an important regulator of inflammation and wound repair, commonly being expressed in myofibroblasts of granulation tissue and it regulates fibrosis through cellular senescence (Lau 2011). CCN1 induces the expression of cytokines (TNF-α, IL-1β, IL-6 and IL-12b) and chemokines (MCP3) in murine macrophages, thus helping macrophages to participate as potent inducers and effectors in the Th1 response (Bai et al. 2010). CCN1 also induces the release of multiple growth factors and chemokines in cardiovascular inflammatory process (Grote et al. 2007). CCN3 counteracts CCN2 effects in several cell systems (Kawaki et al. 2008;Riser et al. 2009). It has been demonstrated that CCN3 alone, or in concert with other signals, can regulate neuro-inflammatory responses by exerting both pro-and anti-inflammatory actions (Dreau et al. 2010). Moreover, in a previous study, the overexpression of the CCN3 gene in renal fibroblasts caused a reduction in CCN2 production and the related fibrotic response (Riser et al. 2009). The anti-fibrotic action of CCN3 may be partly through direct inhibition of collagen induction, as well as inhibition of CCN2 mRNA induction, plus by CCN3 directly binding CCN2 protein and preventing CCN2 bioactivity (Abd El Kader, et al. 2013). However, the roles of other CCN family members involved in NASH progression remain unclear. In our study, CCN1 was upregulated in the HFD + DM NASH model suggesting that CCN1 may be a mediator promoting NASH progression, or as a counter-regulatory anti-fibrotic factor. Anti-fibrotic CCN3 was found to be downregulated at the gene and protein level in the HFD + DM model but was induced by CCN2Ab treatment. This indicates that CCN3 levels are inhibited in this pathological state, and inhibiting CCN2 effects can normalise its levels and perhaps the overall CCN3 bioactivity. In contrast, dysregulation of CCN4, CCN5 or CCN6 was not observed in the NASH model, suggesting that they do not play a major role in this model or in NASH fibrosis generally. Thus, in addition to CCN2, CCN1 and CCN3 may be novel therapeutic targets for NASH combined with type 2 diabetes with fibrosis. The precise mechanisms of action of CCN members in NASH progression, alone and in concert, remain to be investigated.
It is notable in this model that the use of the anti-CCN2 antibody prevented about half of the fibrosis induced in the model compared with chow fed mice. It could thus be concluded that CCN2 mediates only part of the fibrosis in the HFD + DM model. The antibody was added at the time diabetes was induced, which is at a time after 15 weeks of the initial high fat feeding and from that perspective it could be considered a form of delayed treatment rather than entirely a prevention regimen. The aim of the current work was to attempt to prevent the induction of NASH with fibrosis caused by adding diabetes to high fat feeding. In general, this did occur-the fibrotic response at the levels of both mRNA expression and collagen stains showed that the antibody prevented any fibrosis induction over and above that seen by HFD alone. Whether a regimen of commencing the CCN2Ab therapy at week 1 of the protocol before high fat feeding, or whether a higher dose or more intense regimen of CCN2Ab may have produced even greater prevention of fibrosis change in NASH is unknown, and was not able to be addressed in this work owing to limiting amounts of polyclonal antibody reagent for in vivo study, which even in the current regimen required 170 mg in total across all mice.
As the CCN2Ab therapy involved a systemic administration of an agent to inhibit CCN2 bioactivity, this suggests that systemic or other organ effects of CCN2 might have been responsible for protective effects observed in the liver. We have previously reported that CCN2 inhibits murine cell adipocyte differentiation in vitro, inducing a cellular profile found in insulin resistance (Tan et al. 2008). Others have found in humans (Yoshino et al. 2019), as we have in mice, that adipose tissue expression of CCN2 correlates with systemic insulin resistance. Whilst it is possible that systemic CCN2 may have mediated the NASH fibrosis in the current work, it is notable that the body weight, blood glucose levels and insulin sensitivity of the mice within each group (HFD, DM, and HFD + DM), were not affected by the CCN2Ab compared with the control IgG (Table 2). This whole body data thus suggests that the anti-CCN2 antibody was not having secondary effects on the liver through systemic glucoregulation and related metabolism, but was directly inhibiting liver pathology, especially in its hepatic anti-fibrotic effect, including in the profile of CCN2Ab preventing ERK-phosphorylation in the HFD + DM mice.
We examined only male mice in the current study as our published NASH fibrosis model induced by was generated in male mice (Lo et al. 2011) and others have preferentially utilised male mice for nutritional models (Rossmeisl et al. 2003) and in CCN liver fibrosis dysregulation research (Kim et al. 2013). Future studies could explore development of a NASH fibrosis model in female mice, including potential effects of estrus, and liver CCN targeting. In addition, blood liver enzymes were not examined as we (Lo et al. 2011) and others (Kim et al. 2013), have not previously found strong associations of these murine measures with histological liver inflammation or fibrosis. Despite these inherent limitations of the CCN2 bioactivity regimen used in a single time course and concentration protocol, the collective data herein indicate that inhibition of CCN2 bioactivity prevents induction of fibrosis in the NASH mouse model that was studied, implicating CCN2 as a mediator in human NASH induced by type 2 diabetes. Future preclinical studies targeting CCN2 expression specifically in the liver and its cell types could complement the studies described in this current work.