s2fieldsofstudy: Biology Medicine

text:
Effects of growth hormone/estrogen/androgen on COVID-19-type proinflammatory responses in normal human lung epithelial BEAS-2B cells

Background COVID-19 is a disease caused by SARS-CoV-2, which can cause mild to serious infections in humans. We aimed to explore the effect of growth hormone (GH)/estrogen/androgen in normal human lung epithelial BEAS-2B cells on COVID-19-type proinflammatory responses. Methods A BEAS-2B COVID-19-like proinflammatory cell model was constructed. After that, the cells were treated with GH, 17β-estradiol (E2), and testosterone (Tes) for 24 h. CCK-8 assays were utilized to evaluate cell viability. The mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 were measured by qRT‒PCR and Western blotting, respectively. ELISAs were performed to determine IL-6, MCP-1, MDA and SOD expression. Flow cytometry was used to measure ROS levels. Finally, MAPK/NF-κB pathway-related factor expression was evaluated. Results The COVID-19-type proinflammatory model was successfully constructed, and 1000 ng/mL RBD treatment for 24 h was selected as the condition for the model group for subsequent experiments. After RBD treatment, cell viability decreased, the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 increased, IL-6, MCP-1, MDA and ROS levels increased, and MDA levels decreased. The mRNA levels of MAPK14 and RELA increased, but the protein levels did not change significantly. In addition, phospho-MAPK14 and phospho-RELA protein levels were also increased. Among the tested molecules, E2 had the most pronounced effect, followed by GH, while Tes showed the opposite effect. Conclusion GH/E2 alleviated inflammation in a COVID-19-type proinflammatory model, but Tes showed the opposite effect.

Introduction
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a serious public health threat globally [1]. SARS-CoV-2 usually spreads through respiratory droplets. The average incubation period is 6.4 days; most patients have mild symptoms, and a few patients develop severe hypoxia, requiring hospitalization and mechanical ventilation [2]. Age, sex, obesity and smoking, and alterations in disease state-related receptor expression may correlate with the incidence and severity of COVID-19 [3]. Clinical data suggest that SARS-CoV-2 infection is generally more severe in adults than in children [4]. It has been speculated that the lower clinical severity in children may be influenced by differential expression of SARS-CoV-2's major host functional receptor, angiotensin-converting enzyme 2 (ACE2), but the data remain conflicting [4]. SARS-CoV-2 enters host cells via ACE2, which is highly expressed in the heart, kidney and lung and shed into plasma [5]. Compared with women, men have a higher incidence, severity, and mortality from COVID-19 [6], but the underlying factors contributing to this sex difference are still being studied.
Differences in the levels of growth hormone (GH) and sex hormones are known to be one of the most significant differences between children and adults. Estrogen and androgen may be closely related to the expression of ACE2 [7]. Emerging evidence suggests that the virus also targets male and female reproductive organs that express ACE2, its main receptor [8]. Reductions in sex steroids (especially in women) and GH (both of which play key roles in immune regulation) are key contributors to the severity of SARS-CoV-2 in older adults [9]. Insulin-like growth factor-1 (IGF1) affects the immune system by controlling the endocrine system [10]. There may be an association between low IGF1 (and possibly GH) and poor prognosis in COVID-19 patients [11]. There is clear evidence that dysregulation of the immune system is a key factor in determining SARS-CoV-2 severity and its association with adult-onset GH deficiency [12]. Galganska H et al. reported that the receptor binding domain of the SARS-CoV-2 spike protein stimulates a COVID-19-like proinflammatory response [13]. In addition, testosterone (Tes), GH, and estrogen are all associated with the TLR4 inflammatory pathway [14][15][16]. However, the effect of GH/estrogen/androgen on COVID-19-type proinflammatory responses is unknown.
Therefore, based on the above background, we wanted to study the effects of GH/estrogen/androgen in BEAS-2B cells on COVID-19-type proinflammatory responses. Our research may provide novel ideas and references for preventing and treating COVID-19.

Cell treatment
Normal human lung epithelial BEAS-2B cells were provided by BLUEFBIO (BFN608009328, China) and cultured in DMEM high glucose medium with 10% FBS and 1% double antibiotics. A COVID-19-type proinflammatory cell model was constructed [13,17,18], and the cells were grouped into the control group (normal cultured cells + solvent control for 12 and 24 h), RBD-500 group (BEAS-2B cells treated with 500 ng/mL recombinant SARS-CoV-2 spike RBD protein with His tag (RBD) for 12 and 24 h), and RBD-1000 group (BEAS-2B cells treated with 1000 ng/mL RBD for 12 and 24 h). To study the effects of GH/estrogen/androgen on COVID-19-type proinflammatory responses in BEAS-2B cells, the cells were further divided into the control group (normal cultured cells + solvent control treatment for 24 h), RBD group (cells treated with 1000 ng/mL RBD for 24 h), RBD + GH group (cells treated with 1000 ng/ mL RBD and 200 ng/mL GH simultaneously for 24 h), RBD + E2 group (cells treated with 1000 ng/mL RBD and 1 nM 17β-estradiol (E2) simultaneously 24 h [6]), and RBD + Tes group (cells treated with 1000 ng/mL RBD and 10 nM Tes simultaneously treated for 24 h [6]).

Cell counting Kit-8 (CCK-8) assay
Different groups of BEAS-2B cells were digested, counted and seeded in a 96-well plate at a density of 1 × 10 4 cells/ well, with 100 µL per well. The cell count assay was performed in replicates. Each group had 3 replicate wells. After culturing adherent cells, 10 µL/well of CCK-8 (#NU679, DOJINDO, Japan) was added, and CCK-8 solution was prepared with complete medium. After incubation at 37 °C and 5% CO 2 , the absorbance value at 450 nm was analyzed on a Bio-Tek microplate reader.

Western blotting
Total protein was extracted from 5.3 × 10 6 BEAS-2B cells using a RIPA kit (P0013B, Beyotime, China). After the protein concentration was determined, proteins were separated by 10% SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes by electrotransfer. For primary antibodies, we used anti-ACE2 The membranes were then incubated with HRP-conjugated secondary antibodies. β-actin was used as the internal reference. The membrane was immersed in ECL chemiluminescent solution (K-12,045-D50, Advantesta, USA) for luminescence visualization. Protein bands were detected using a chemiluminescence imaging system (ChemiScope 6100, Clinx, China). The experiment was repeated three times.

Flow cytometry (FCM)
The level of ROS was assessed according to the ROS assay kit (S0033S, Beyotime, China). First, DCFH-DA (stock concentration 10 mM) was diluted 1:1000 in serum-free medium to a final concentration of 10 µM. Then, the cells treated as indicated above were removed from the cell culture medium, and DCFH-DA was added to cover the cells, followed by incubation in a 37 °C cell incubator for 20 min. The samples were collected by trypsinization. Finally, FCM (A00-1-1102, Beckman, USA) detection was performed using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The level of ROS was analyzed with CyExpert software. The experiment was repeated three times.

Statistical analysis
GraphPad Prism 8.0 software was applied for statistical analysis. The experiments were repeated three times. Measurement data are expressed as the mean ± standard deviation. The experiments were repeated three times. The significance of three or more groups was compared by one-way ANOVA. Tukey's test was adopted for postmortem testing. P < 0.05 indicated a statistically significant difference.

Construction of a COVID-19-type proinflammatory model
First, a COVID-19-type proinflammatory model was established, and cell viability was evaluated by CCK-8 assay. The cell viability of the RBD-500 and RBD-1000 groups decreased at 12 and 24 h compared with that of the control group (Fig. 1 A). Figure 1B J showed that the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 both increased at 12 and 24 h in the RBD-500 group and the RBD-1000 group. Among these, the effect of 1000 ng/mL RBD treatment for 24 h was more significant. Finally, the levels of MCP-1 and IL-6 were assessed by ELISA. After RBD treatment, both MCP-1 and IL-6 levels were increased, and the increase was more evident after 1000 ng/mL RBD treatment for 24 h (Fig. 1 K-1 L). Therefore, 1000 ng/mL RBD treatment for 24 h was selected as the condition for the model group for follow-up experiments.

The role of GH/E2/Tes in a COVID-19-type proinflammatory model
To study the effect of GH/estrogen/androgen in BEAS-2B cells on COVID-19-type proinflammatory responses, we incubated the cells with GH, E2, and Tes. The CCK-8 assay showed that the cell viability decreased after RBD treatment, and the cell viability recovered after adding GH and E2. Among these, E2 had the best effect, followed by GH. While Tes had the opposite effect (Fig. 2 A).  Figure 2B -2J showed that the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 were all increased after RBD treatment. After adding GH and E2, the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 both decreased. Among these, E2 demonstrated the best effect, followed by GH. But Tes had the opposite effect.

GH/E2 alleviated inflammation in a COVID-19-type proinflammatory model
Next, we examined markers related to inflammation and oxidative stress. As shown in Fig. 3 A-3E, after RBD treatment, IL-6, MCP-1, MDA and ROS levels were all increased, and the level of SOD was decreased. After adding GH and E2, IL-6, MCP-1, MDA and ROS levels all decreased, and the level of SOD increased. Among these, E2 had the most obvious therapeutic effect, followed by GH. However, Tes had the opposite effect.

GH/E2 could regulate the MAPK/NF-κB pathway
Finally, we examined levels of MAPK/NF-κB pathwayrelated factors. After RBD treatment, the mRNA expression of MAPK14 and RELA increased, while the protein expression did not change significantly. Simultaneously, the protein expression of phospho-MAPK14 and phospho-RELA was also increased. After the addition of GH and E2, the mRNA expression of MAPK14 and RELA decreased, while the protein expression did not change significantly. In addition, the protein expression of phospho-MAPK14 and phospho-RELA was also decreased (Fig. 4 A-4E). Among these, the role of E2 was the most obvious, followed by GH. But Tes had the opposite effect.

Discussion
COVID-19 is a novel infectious disease characterized by atypical pneumonia caused by SARS-CoV-2, which can cause mild to severe infection in humans [19,20]. COVID-19 has now spread rapidly around the world, posing severe challenges to health care facilities and medical infrastructure. In this research, we studied the effects of GH/estrogen/androgen in normal human lung  epithelial BEAS-2B cells on COVID-19-type proinflammatory responses. We found that after E2/GH treatment, cell viability was increased, the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 were decreased, and inflammation was alleviated. In addition, E2/GH could regulate the MAPK/NF-κB pathway. ACE2 is the receptor of the SARS-CoV-2 spike protein [9]. SARS-CoV-2 binds to ACE2 on host cells via its surface spike protein [21], triggering complex molecular events that lead to excessive inflammation [22]. COVID-19 also targets male and female reproductive organs that express its primary receptor, ACE2, although it is unclear whether this has any effect on human fertility [8]. AGTR1 is a receptor required to support the angiotensin signaling cascade, and TMPRSS2 is a protease capable of promoting viral fusion [23]. During COVID-19, TMPRSS2 is widely expressed in respiratory and gastrointestinal tissues [24]. Moreover, ISG15-dependent activation of the sensor MDA5 was shown to be antagonized by SARS-CoV-2 papain-like protease to evade host innate host immunity [25]. In this study, we found that after E2/GH treated, the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 decreased. This suggested that E2/GH may have a therapeutic effect on COVID-19.
GH deficiency is a common factor in all vulnerable COVID-19 patient populations [26]. Dhindsa S et al. found that lower Tes concentrations during hospitalization were related to elevated disease severity and inflammation in men in a single-center cohort study of COVID-19 patients [27]. Sex hormones reduce the inflammatory response and regulate the expression of ACE2 [28]. The androgen receptor is the transcriptional promoter of TMPRSS2, which can promote SARS-CoV-2 entry into the host [29]. Tes may make men more susceptible to widespread COVID-19 infection than estrogen. Low serum testosterone levels, which should be considered a feature of the hormonal milieu in severe patients, may predispose men, especially older men, to a poor prognosis or death [30]. In contrast, E2 treatment results in serum concentrations equivalent to ovulation or pregnancy, with beneficial immunomodulatory and anti-inflammatory effects in mice and humans [30,31]. In the present study, after treatment of BEAS-2B cells with E2/GH, the IL-6, MCP-1, MDA and ROS levels were all decreased, and SOD was increased, suggesting that E2/ GH treatment alleviated COVID-19-type proinflammatory responses. IL-6 is a proinflammatory factor released in severely ill COVID-19 patients. This cytokine activates the MAPK/ NF-κB-IL-6 pathway [31]. Ma Q et al. reported that Liu Shen capsules can inhibit SARS-CoV-2 infection by downregulating the expression of inflammatory cytokines induced by the virus in vitro, regulating the activity of the MAPK/NF-κB signaling pathway, and inhibiting SARS-CoV-2 infection and are promising new coronavirus control drug candidates [32]. In addition, Sharma VK et al. reported that nanocurcumin effectively inhibited SARS-CoV-2 spike protein-induced cytokine storm by inhibiting the MAPK/NF-κB pathway in epithelial cells [33]. In this study, we found that E2/GH regulated the MAPK/NF-κB pathway. Among these, the role of E2 was the most obvious, followed by GH. But Tes had the opposite effect.
However, this study had some limitations. We only performed in vitro cell experiments to verify the role of GH/ estrogen/androgen in a COVID-19-like proinflammatory model. In vivo animal and human experiments are still lacking. Due to the limitations of time and funding, we cannot solve this problem at present. In the future, we will further explore the role of GH/estrogen/androgen in the COVID-19-like proinflammatory model in animals and humans.
In conclusion, our results showed that E2/GH treatment of BEAS-2B cells increased cell viability, decreased the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15, and alleviated inflammation. In addition, E2/GH could regulate the MAPK/NF-κB pathway. Our research provides a reference and basis for the diagnosis of COVID-19 and a new strategy for treating COVID-19.


Transcriptome Analysis Reveals the Multiple Functions of pBD2 in IPEC-J2 Cells against E. coli

Defensins play an important role in fighting bacteria, and are a good candidate for bactericidal agents. However, the function and mechanism of defensins in regulating host responses against bacteria is unclear. In this study, transcriptome analysis was used to study the comprehensive functions of pBD2 in IPEC-J2 cells against E. coli. In total, 230 differentially expressed genes (DEGs) were identified in IPEC-J2 cells between the control and E. coli groups, and were found by KEGG analysis to be involved in many signaling pathways related to immunity. Furthermore, 812 DEGs were observed between E. coli and E. coli +pBD2 groups, involved in the ribosome, oxidative phosphorylation, and certain disease pathways. Among these, 94 overlapping DEGs were in the two DEG groups, and 85 DEGs were reverse expression, which is involved in microRNA in cancer, while PTEN and CDC6 were key genes according to PPI net analysis. The results of qRT-PCR verified those of RNA-seq. The results indicated that pBD2 plays an important role against E. coli by acting on the genes related to immune response, cell cycle, ribosomes, oxidative phosphorylation, etc. The results provide new insights into the potential function and mechanism of pBD2 against E. coli. Meanwhile, this study provides a certain theoretical basis for research and the development of novel peptide drugs.

Introduction
In the past, the long-term use and abuse of antibiotics in animal husbandry has resulted in serious antibiotic residues and increase of drug-resistant strains, which is currently considered a great threat to public health [1]. Although the use of antibiotics in livestock has been limited as much as possible in recent years, this has brought some other challenges, such as the increase of intestinal diseases and the weakening of growth performance, especially at the weaning stage in the pig industry. It is urgent to develop new antimicrobial agents; host defensins may be one of the methods to solve this problem [2].
Defensins, members of the antimicrobial peptide family, exist widely in nature. They have shown bactericidal activity against gram-positive bacteria, gram-negative bacteria, and even multidrug-resistant bacteria; moreover, they have unique antibacterial mechanisms which make it difficult to induce bacterial resistance [3]. Defensins are part of the innate defense system and play an important role in immune response [2,4,5]. In addition, defensins could regulate the proliferation and migration of epithelial cells, which is very important for overcoming injury, infection and inflammation [4]. These advantages of defensins make them ideal antibiotic substitutes.
Porcine beta defensin 2 (pBD2) is an antibacterial peptide secreted by pigs. pBD2 has shown good antibacterial activity and immunomodulatory function in vitro [6][7][8][9]. pBD2 could inhibit E. coli and S. aureus, as well as some clinically isolated multidrug-resistant bacteria [9]. pBD2 has the strongest antimicrobial activity among the porcine beta defensins (pBDs) detected, including pBD1, pBD2, pBD114, and pBD129 [9][10][11][12]. In addition, pBD2 could alleviate the inflammatory response induced by exogenous stimulation in mice [8]. As a feed additive, pBD2 showed a certain effect on disease resistance and growth promotion, related to the reduction of numbers of harmful bacteria [13]. pBD2 may be a suitable antibiotic substitutes in the pig industry. However, the function and mechanism of how pBD2 protects the host from bacteria remains unclear.
In order to explain the functions and mechanism of pBD2 in the host cells against bacteria, Escherichia coli was selected as an example and intestinal porcine enterocyte cells (IPEC-J2) were selected as the infected model. E. coli is a gram-negative bacteria that exists widely in the intestinal tracts of animals. It is one of the most important pathogenic bacteria causing diarrhea in weaned piglets, which leads to considerable economic losses [14]. The IPEC-J2 cell line was originally isolated from the jejunum of a neonatal unsuckled piglet, and is a non-transformed, permanent intestinal cell line. IPEC-J2 cells are ideal for studying the antibacterial effect of pBD2 on cells [15]. In this experiment, IPEC-J2 cells were challenged with E. coli, and the transcriptome method was used to detect the effect of pBD2 on the gene expression of cells infected by E. coli. Our study helps to understand the functions and mechanisms of defensin in host cells against E. coli.

Analysis of Bactericidal Activity
The recombinant pBD2 was induced and purified following our previous study [9]. The purified pBD2 showed high purity, and the molecular weight was approximately 12 kDα as expected (Figure 1). The recombinant pBD2 showed high bactericidal activity. E. coli were cracked and dead after incubation with 20 µg/mL pBD2 for 1 h and 4 h, the debris was observed, and the bactericidal effect was more obvious at 4 h than those at 1 h ( Figure 2).
Porcine beta defensin 2 (pBD2) is an antibacterial peptide secreted by pigs. pBD2 has shown good antibacterial activity and immunomodulatory function in vitro [6][7][8][9]. pBD2 could inhibit E. coli and S. aureus, as well as some clinically isolated multidrug-resistant bacteria [9]. pBD2 has the strongest antimicrobial activity among the porcine beta defensins (pBDs) detected, including pBD1, pBD2, pBD114, and pBD129 [9][10][11][12]. In addition, pBD2 could alleviate the inflammatory response induced by exogenous stimulation in mice [8]. As a feed additive, pBD2 showed a certain effect on disease resistance and growth promotion, related to the reduction of numbers of harmful bacteria [13]. pBD2 may be a suitable antibiotic substitutes in the pig industry. However, the function and mechanism of how pBD2 protects the host from bacteria remains unclear.
In order to explain the functions and mechanism of pBD2 in the host cells against bacteria, Escherichia coli was selected as an example and intestinal porcine enterocyte cells (IPEC-J2) were selected as the infected model. E. coli is a gram-negative bacteria that exists widely in the intestinal tracts of animals. It is one of the most important pathogenic bacteria causing diarrhea in weaned piglets, which leads to considerable economic losses [14]. The IPEC-J2 cell line was originally isolated from the jejunum of a neonatal unsuckled piglet, and is a non-transformed, permanent intestinal cell line. IPEC-J2 cells are ideal for studying the antibacterial effect of pBD2 on cells [15]. In this experiment, IPEC-J2 cells were challenged with E. coli, and the transcriptome method was used to detect the effect of pBD2 on the gene expression of cells infected by E. coli. Our study helps to understand the functions and mechanisms of defensin in host cells against E. coli.

Analysis of Bactericidal Activity
The recombinant pBD2 was induced and purified following our previous study [9]. The purified pBD2 showed high purity, and the molecular weight was approximately 12 kDα as expected (Figure 1). The recombinant pBD2 showed high bactericidal activity. E. coli were cracked and dead after incubation with 20 μg/mL pBD2 for 1 h and 4 h, the debris was observed, and the bactericidal effect was more obvious at 4 h than those at 1 h ( Figure  2).

Transcriptome Profiling
cDNA libraries were sequenced on the Illumina high-throughput platform, generating significant amounts of high-quality raw data. After removal of adaptor sequences, lowquality, and contaminated reads, only the resulting clean reads were assembled to build transcripts. In these results, 20,721,673-35,369,943 clean data were obtained for samples, and the minimum Q30 was 93.23% (Table 1). The percentage of reads mapped to the reference genome was 95.84-96.55% (Table 2).

Transcriptome Profiling
cDNA libraries were sequenced on the Illumina high-throughput platform, generating significant amounts of high-quality raw data. After removal of adaptor sequences, low-quality, and contaminated reads, only the resulting clean reads were assembled to build transcripts. In these results, 20,721,673-35,369,943 clean data were obtained for samples, and the minimum Q30 was 93.23% (Table 1). The percentage of reads mapped to the reference genome was 95.84-96.55% (Table 2).

Characterization of Differentially Expressed Genes by RNA Sequencing
There were 230 differential expressed genes (DEGs) between the control and E. coli treatments, including 145 significantly upregulated genes and 85 significantly downregulated genes. There were 812 DEGs were between the E. coli and E. coli +pBD2 groups, including 431 significantly upregulated genes and 381 significantly downregulated genes. There were 94 overlapping genes observed by Venn diagram analysis between the two DEG groups.

KEGG Analysis Revealed That Immune Responses of Cells Were Trigger by E. coli
To determine the functions of the DEGs involved, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and corresponding enrichment analysis were performed. KEGG pathway analysis of the DEGs between the E. coli and control groups revealed that the immune pathways were significantly enriched, including NF-kappa B signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, Chemokine signaling pathway, MAPK signaling pathway, etc., suggesting that E. coli caused immune responses of IPEC-J2 cells to eliminate the invading microbes ( Figure 3). These results clearly indicate that the transcriptome of IPEC-J2 cells was changed after infection. Genes involved in the immune signaling pathways are listed in Table 3. The genes related to the immune responses include CXCL2 (chemokine (C-X-C motif) ligand 2), PTGS2 (prostaglandin G/H synthase 2), TNFAIP2 (tumor necrosis factor alpha-induced protein 2), NFKB2 (nuclear factor NF-kappa-B p100 subunit), NFKBIZ (NF-kappa-B inhibitor zeta), IL11 (interleukin-11), CCL20 (C-C motif chemokine ligand 20), etc. According to the PPI (protein-protein interaction) net analysis of the 230 DEGs, NFKB1(Nuclear factor NF-kappa-B p105 subunit), TP53 (tumor protein p53), PTEN (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase) and CDC6 (cell division control protein 6) may be the key genes in IPEC-J2 cells in response to E. coli ( Figure 4).

KEGG Pathway Analysis Revealed That pBD2 Has Multiple Functions in IEPC-J2 Cells against E. coli
There were 812 DEGs between the E. coli and E. coli + pBD2 groups. KEGG pathway analysis revealed DEGs involved in ribosomes, oxidative phosphorylation, and some diseases including Parkinson's disease, Huntington's disease, Alzheimer's disease, Non-alcoholic fatty liver disease (NAFLD), and others ( Figure 5). These results clearly indicate that the transcriptome of IPEC-J2 cells was regulated by pBD2. The 431 upregulated DEGs and 381 downregulated DEGs were analyzed by PPI net, respectively (Figures 6 and 7).

Comparison of pBD2 Effect with E. coli-Induced Transcriptome Changes
There were 94 overlapping DEGs by Venn diagram analysis, which enabled us to identify transcripts specifically regulated by pBD2. Among these 94 DEGs, 85 DEGs were reverse expressed including 35 DEGs upregulated by E. coli and downregulated by pBD2 under E. coli stimulation (E. coli + pBD2 group). The other 50 DEGs were reverse expressed conversely to the above genes in both DEG groups ( Figure 8).
The KEGG pathway analysis of 85 DEGs with reverse expression showed that mi-croRNA in cancer was the only significantly different signaling pathway, including PTEN, CDC25A (cell division cycle 25A), SPRY2 (protein sprouty homolog 2), E2F2 (E2F2 transcription factor), PTGS2, and PLAU (urokinase-type plasminogen activator) ( Figure 9). In addition, PTEN and CDC6 were the hub genes in the PPI network for pBD2 to regulate IPEC-J2 cell responses to E. coli ( Figure 10). In addition, immune-response-related genes among the 85 reverse expressed genes were considered. The immune-response-related genes IL11, PTGS2 and PLAU were all upregulated in the E. coli group compared with the control group, and pBD2 significantly downregulated the expression of these genes compared with the E. coli group.  The KEGG pathway analysis of 85 DEGs with reverse expression showed that mi-croRNA in cancer was the only significantly different signaling pathway, including PTEN, CDC25A (cell division cycle 25A), SPRY2 (protein sprouty homolog 2), E2F2 (E2F2 transcription factor), PTGS2, and PLAU (urokinase-type plasminogen activator) (Figure 9). In addition, PTEN and CDC6 were the hub genes in the PPI network for pBD2 to regulate IPEC-J2 cell responses to E. coli (Figure 10). In addition, immune-response-related genes among the 85 reverse expressed genes were considered. The immune-response-related genes IL11, PTGS2 and PLAU were all upregulated in the E. coli group compared with the control group, and pBD2 significantly downregulated the expression of these genes compared with the E. coli group.

Validation of RNA-seq Data by qRT-PCR
Ten genes were selected for validation of RNA-seq, and the results of qRT-PCR were consistent with those of transcriptomics ( Figure 11).

Validation of RNA-seq Data by qRT-PCR
Ten genes were selected for validation of RNA-seq, and the results of qRT-PCR were consistent with those of transcriptomics ( Figure 11). Figure 10. PPI analysis of the DEGs overlapping between the two DEG groups.

Validation of RNA-seq Data by qRT-PCR
Ten genes were selected for validation of RNA-seq, and the results of qRT-PCR were consistent with those of transcriptomics ( Figure 11).

Discussion
Defensins, a kind of antimicrobial peptide, play an active role in resisting the invasion of pathogenic microorganisms [16]. pBD2 is one of the porcine beta defensins and shows strong antimicrobial activity against E. coli, S. aureus and isolated multidrug-resistant bacteria, with the advantages of high salt-tolerance, thermal stability, and low hemolytic activity, as demonstrated in our previous studies [9]. In addition, pBD2 has an immunomodulatory function [8]. However, the function and mechanisms of defensins in protecting the host from bacteria are still unclear.
In this study, we explored by transcriptomic analyses the effects of defensins on IPEC-J2 cells against E. coli. A total of 230 DEGs were obtained between the control and E. coli groups. The significant signaling pathways were mainly involved in immune responses, including NF-κB signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, chemokine signaling pathway, etc., as demonstrated by KEGG enrichment analysis. The DEGs in these signaling pathways were upregulated, including NFKB1, NFKB2, NFKBIA (NF-kappa-B inhibitor alpha), NFKBIB (NF-kappa-B inhibitor beta), cytokines CXCL2, CCL20, IL1α (Interleukin 1 alpha), CXCL8 (C-X-C motif chemokine ligand 8), etc. Furthermore, NFKB1, TP53, PTEN, and CDC6 were key genes against E. coli, identified in the cells by PPI net analysis. NFKB1 is a key gene in the NF-κB signaling pathway, and PTEN is an important gene in the PI3K phosphatidyl inositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway, which is involved in cell proliferation, differentiation, apoptosis, and immunity [17]. TP53 and PTEN are essential for initiating apoptosis and inflammatory response [18,19]. NFKB1, TP53, and PTEN were all upregulated, and were closely related to inflammatory response. The transcriptomic analyses indicated that immune responses of IPEC-J2 cells were triggered by E. coli, by which cells could eliminate invading pathogens for survival. It has been reported that multiple signaling pathways related to inflammation were activated by E. coli or LPS, resulting in the release of inflammatory cytokines [20][21][22][23][24][25]; those results were consistent with ours. CDC6 was a key gene for cell proliferation by PPI net analysis (Figure 4). In addition to CDC6, CDK18 (cyclin-dependent kinase 18), E2F8 (E2F8 transcription factor), CDCA7 (cell division cycleassociated protein 7), MCM10 (minichromosome maintenance 10 replication initiation factor) and TIMELESS (protein timeless homolog) are all related to cell proliferation according to their functions (Figure 4). It has been reported that CDC6 and CDK18 promoted proliferation of epithelial cells and inhibited apoptosis [26]. These genes were all decreased in the E. coli group, which perhaps suggest that cell proliferation was inhibited by E. coli.
There were 812 DEGs between the E. coli and E. coli +pBD2 groups, including 431 upregulated and 381 downregulated genes. PPI net analysis confirmed that the genes in the upregulated DEGs related to immune responses and cell proliferation, and STAT1 and CREBBP were the key genes ( Figure 6). These genes are involved in many signaling pathways, including JAK-STAT and PI3K-AKT, which are related to immune responses. Our results were consistent with previous findings that defensin regulates immune responses in complex ways [27]. The genes related to cell proliferation in the upregulated DEGs included CDC6, CDK18, CDK14, E2F8, CDCA7, MCM10, TIMELESS, CDC25A, CCNE2 (G1/S-specific cyclin-E2), etc., according to their functions and the PPI net analysis. These genes were all increased by pBD2, which indicates that pBD2 protected cells from E. coli infection by regulating cell proliferation and inhibiting apoptosis, which might be critical for the resolution of injury, infection, and inflammation [2]. pBD2 upregulated DEGs related to immune responses and cell proliferation, which perhaps indicates that pBD2 could promote cell proliferation and inhibit the immune responses caused by E. coli.
The downregulated DEGs were obviously clustered into three groups (ribosome, oxidative phosphorylation, and proteasome) (Figure 7), which indicates that pBD2 had multiple functions in the cells. The ribosome is a complex molecular machine composed of numerous distinct proteins and nucleic acids, and is responsible for the translation of information contained in mRNAs into functional proteins, which play an important role in the execution of gene-expression programs, regulating basic biological processes such as cell growth, cell division, and differentiation. The hyperactivation of ribosome biogenesis has a critical role in cancer initiation and progression [28], and inhibition of ribosome biogenesis represents a potential therapeutic avenue for cancer treatment [29]. pBD2 downregulated the DEGs in the ribosome, perhaps implying that pBD2 could play a role in inhibiting the occurrence of cancer. The DEGs in oxidative phosphorylation (OXPHOS) were related to many diseases, and were also downregulated. OXPHOS was another source of ATP in cells in addition to glycolysis. Although it was reported that OXPHOS is related to cancer, many highly proliferative cell types including many cancer cells can preferentially utilize glycolysis [30][31][32][33]. In addition, OXPHOS inhibitors have heralded novel uses either for treating cancers in which OXPHOS is upregulated or alleviating tumor hypoxia to improve treatment outcomes [34]. In this study, pBD2 could downregulate the DEGs related to OXPHOS, which perhaps further implies that pBD2 could treat cancer. It was reported that peptide antibiotic leucinostatins showed inhibitory action on OXPHOS [35], which is consistent with our results. It was reported that the DEGs in OXPHOS were significantly enriched and upregulated in the large yellow croaker (Larimichthys crocea) when infected by Pseudomonas plecoglossicida, and were also upregulated in the murine model when infected by R. conorii [36,37]. These findings suggest that OXPHOS was related to immune responses and contributed to anti-infection strategies in the hosts. The mechanism underlying this strengthened energy metabolism is unknown, and the DEGs in OXPHOS were not enriched between the control and E. coli infection groups in this study. pBD2 could downregulate the DEGs, perhaps implying that pBD2 could protect the host from excessive inflammatory responses.
The proteasome is a large protein complex, responsible for the degradation of intracellular proteins, which regulates cellular proteostasis through selective degradation of ubiquitylated proteins [38,39]. Thereby, it performs a crucial role in cellular regulation and homeostasis, and has an important function in a variety of basic cellular processes including regulation of cell cycle progression, signal transduction, modulation of immune and inflammatory responses, etc. Malfunction of the ubiquitin-proteasome system (UPS) contributes to various diseases including cancer, inflammation, and neurodegeneration [40]. In this study, pBD2 inhibited the DEGs in the proteasome, and even lowered some ubiquitin expression including UBB (polyubiquitin-B), NEDD8, UBE2J2 (ubiquitin-conjugating enzyme E2 J2), APC2 and APC11 (anaphase-promoting complex subunit 2 and 11), proteasomal ubiquitin receptor UCHL3 (ubiquitin carboxyl-terminal hydrolase isozyme L3), USP19 (ubiquitin carboxyl-terminal hydrolase 19) genes, etc., all perhaps further indicating that pBD2 inhibited inflammations and the occurrence of cancer, as discussed above. It was reported that PR39, one of the porcine antimicrobial peptides, was a non-competitive and reversible inhibitor of 20S proteasome, and inhibited inflammation [41,42]. Our results are consistent with that report. pBD2 downregulated DEGs related to ribosome, oxidative phosphorylation, and proteasome, indicating that pBD2 could inhibit the occurrence of cancer, which needs further research. Some reported defensins have been regarded as a potential therapeutic target for cancer treatment, and attractive novel therapeutic candidates for antimicrobial and anticancer purposes [43,44], which is also consistent with our results.
pBD2 changed the transcriptional response caused by E. coli. There were 85 genes with reverse expression in the two DEGs group; KEGG analysis showed that these overlapping genes included enrichment of the microRNA in cancer (Figure 9). In addition, PTEN and CDC6 were key genes for pBD2 according to PPI net analysis ( Figure 10). It has been reported that defensin regulated the expression of microRNA, and that the expression of miR-34a-5p increased in ethanol-induced liver injury in transgenic (TG) mice with overexpressing human neutrophil peptide 1, compared with WT (wild type) mice [45]. Those results perhaps imply that pBD2 had the ability to regulate gene expression by regulating microRNA, which needs further study. The genes associated with microRNA in cancer included PTEN, CDC25A, SPY2, PLAU, E2F2, and PTGS2. It was reported that PTEN could inhibit immune response and tumors [46]. SPRY2 was upregulated in glioblastoma, and overexpression of SPRY2 is associated with human oral squamous-cell carcinogenesis [47,48]. Dysregulation of PLAU is often accompanied by various cancers, and inhibition of PLAU expression could suppress tumor growth [49]. CDC25A, E2F2, and PTGS2 are all related to the cell cycle, and their dysregulation is related to tumors [50][51][52]. In this study, pBD2 showed anti-tumor potential by upregulating the expression of PTEN and downregulating the expression of SPRY2 and PLAU, and pBD2 was also shown to promote cell proliferation by upregulating the expression of CDC25A, E2F2, and PTGS2, which perhaps might lead to tumorigenesis. Some scholars have reported that defensin had a strong inhibitory effect on cancer [53][54][55], but others had opposite view suggesting that defensin might be related to the occurrence of cancer because the expression of defensins increased abnormally in cancer [56,57]. In addition to CDC25A, E2F2, and PTGS2 relating to the cell cycle, these genes are all related to cell proliferation including CDC6, CDK18, E2F8, CLSPN, CDCA7, MCM10, TIMELESS, KIF18B, etc., as mentioned above ( Figure 10). These genes were all decreased in the E. coli group, and all increased by pBD2, indicating that pBD2 regulated cell proliferation and inhibited apoptosis, playing its role in the resolution of injury, infection, and inflammation [2]. pBD3 enhanced ovarian granulosa cell proliferation and migration [58]. Human β-defensins also stimulated various cellular activities, including keratinocyte proliferation, migration, and wound healing [59]. Those results are consistent with ours, which perhaps indicate that pBD2 could inhibit the occurrence of cancer, and promote cell proliferation to protect cells from infection. The analysis of the overlapping genes with reverse expression was consistent with the above analysis of DEGs in the E. coli and E. coli +pBD2 groups. Our results provide new insights into the potential function and mechanism of pBD2 against E. coli, and the results should be confirmed by further study on mice or human cell lines.

Strains and Cells
E. coli ATCC 25922 was purchased from the Beijing Ordinary Microbiology Strain Store Center (Beijing, China). The IPEC-J2 cells were a gift from Zhanyong Wei at the College of Veterinary Medicine, Henan Agricultural University.

Preparation for pBD2
pBD2 was expressed and purified in our laboratory by affinity chromatography based on the constructed strains BL21(DE3) pLysS-pET30a-pBD2 [9]. The expression products were analyzed by SDS-PAGE, and the protein concentration was determined by bicinchoninic acid (BCA) assay (CW Biotech, Beijing, China).

Bactericidal Activity
The bactericidal activity was analyzed by scanning electron microscopy, as described previously [6]. E. coli ATCC 25922 was cultured in Luria Bertani (LB) medium until logarithmic growth stage at 220 rpm, then E. coli was incubated with 20 µg/mL pBD2 for 1 h or 4 h, and harvested by centrifugation. After being washed for 3 × 10 min in 10 mM PBS buffer, E. coli was fixed with 2.5% glutaraldehyde for 4 h, then dehydrated with gradient concentrations of ethanol, and the samples were observed by scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR, USA).

Cell Culture and Treatment
The IPEC-J2 cells were grown in 1640 medium supplemented with 10% fetal bovine serum (TianHang Biotechnology, Zhejiang, China) and 1% penicillin/streptomycin at 37 • C in an atmosphere of 5% CO 2 . The cells were seeded into a six-well plate and cultured until reaching 80% confluence, then at the logarithmic period (MOI = 50:1) the IPEC-J2 cells were challenged with E. coli or E. coli and 20 µg/mL pBD2 for 2 h in 1640 medium without fetal bovine serum and 1% penicillin/streptomycin. After washing with PBS three times, the cells were cultured for an additional 2 h in the medium without fetal bovine serum and 1% penicillin/streptomycin. After washing three times with PBS, 1 mL of TRIzon was added to the six-well plate, and the cells were lysed by pipetting, then the solute was transferred to RNase-free Eppendorf tubes for RNA extraction.

Library Preparation and Quality Control
RNAs were extracted using TRIzol reagents (CW Biotech, Beijing, China). The extracted RNAs were quantified using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, Waltham, MA, USA). A total of 3 µg RNA was sent to Biomarker Co., Ltd. (Beijing, China) to construct the cDNA libraries. Sequencing was performed on an Illumina HiSeqTM 2500 platform. The original raw data were saved in FASTQ file format. Each sequenced sample included two FASTQ files, containing reads from either end of the cDNA fragments. Quality control was performed to remove adaptor sequences, low-quality, and contaminated reads. Then the clean reads were mapped to the reference genome by alignment software HISAT2.

Analysis of DEGs
The transcript and gene expression levels were measured by FPKM (fragments per kilobase of transcript per million fragments mapped). The DESeq2 R package was used to analyze differential gene expression. The false discovery rate (FDR) was corrected using the Benjamini-Hochberg procedure. The threshold of FDR < 0.05 was used to filter out differential expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs were performed using BMKCloud (www.biocloud.net, accessed on 5 September 2019), based on the KEGG database (http://www.genome.jp/ kegg/, accessed on 5 September 2019), and protein-protein interaction (PPI) networks of the DEGs were constructed on https://www.string-db.org/, accessed on 17 August 2021.

qRT-PCR
The extracted RNAs were converted to complimentary (c) DNA by a reverse transcriptase synthesis kit (DRR047A; TaKaRa Biotechnology, Shiga, Japan). The primers were designed using Primer Premier™ 5.0 (Sigma-Aldrich, Saint Louis, MO, USA) and are detailed in Table 4. Assays were performed with SYBR green dye (QIAGEN, Dusseldorf, Germany) using a real-time PCR cycler (LightCycler 96, Roche, Basel, Switzerland). The program was 95 • C for 30 s, 95 • C for 15 s, 60 • C for 30 s, 72 • C for 15 s, for 40 cycles. The results were analyzed using the 2 −∆∆CT method with TUBA1B (tubulin alpha 1b) as a reference gene [60].

Statistical Analysis
All experiments were conducted with three biological replicates. Data were assessed by analysis of variance using SPSS Statistics 24 (IBM, Armonk, NY, USA).

Conclusions
In this study, IPEC-J2 cells were challenged with E. coli, and the effect of pBD2 on the gene expression of cells infected by E. coli was detected by transcriptome analyses. KEGG enrichment analysis revealed that DEGs between the control and E. coli groups were involved in signaling pathways related to immune responses. The DEGs between the E. coli and E. coli +pBD2 groups were involved in the ribosome, oxidative phosphorylation, and some disease pathways. These results indicate that pBD2 can play an active role in many biological processes including cancer, cell proliferation, and immunity. Furthermore, analysis of the overlapping genes with reverse expression in two DEG groups further revealed that pBD2 was involved in multiple biological processes to protect cells from E. coli. Our results provide new insights into the potential function and mechanism of pBD2 against E. coli. Meanwhile, this study provides a certain theoretical basis for the research and development of novel peptide drugs.
Author Contributions: C.L. designed the experiments and supervised the whole study. S.L. and X.L. performed the cell experiment; S.L. and X.S. performed the RNA extract and qRT-PCR. X.S., Y.L. and F.Z. analyzed the data of RNA-seq. C.L. and X.L. analyzed the data and wrote the paper. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no competing interest.


s2fieldsofstudy: Biology Chemistry

text:
Fisetin Attenuates Arsenic-Induced Hepatic Damage by Improving Biochemical, Inflammatory, Apoptotic, and Histological Profile: In Vivo and In Silico Approach

Arsenic (As) is a toxic metalloid and human carcinogen that may cause hepatotoxicity. Fisetin (3, 3′, 4′, 7-tetrahydroxyflavone) is a phytoflavonoid, which shows diverse therapeutic activities. This study aimed to examine the remedial potential of fisetin against As-instigated hepatotoxicity in adult male rats. To accomplish this aim, albino rats (N = 48) were evenly classified into 4 groups: control group, As (10 mg/kg) group, fisetin (2.5 mg/kg) + As (10 mg/kg) group, and fisetin (2.5 mg/kg) group. After one month of treatment, biochemical assay, total protein content (TPC), hepatic serum enzymes, inflammatory as well as pro- or anti-apoptotic markers, and histopathological profile of hepatic tissues were estimated. As administration disordered the biochemical profile by decreasing activities of antioxidant enzymes i.e., catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), and glutathione (GSH) content while escalating the levels of reactive oxygen species (ROS), and thiobarbituric acid reactive substances (TBARS). TPC was also considerably reduced after exposure to As. Furthermore, As markedly raised the levels of liver serum enzymes such as aspartate transaminase (AST), alkaline phosphatase (ALP), and alanine transaminase (ALT) as well as the levels of inflammatory markers, i.e., nuclear factor- κB (NF-κB), tumor necrosis- α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclo-oxygenase-2 (COX-2) activity. Besides, it lowered the level of antiapoptotic markers (Bcl-2) and upregulated the levels of proapoptotic markers (Bax, Caspase-3, and Caspase-9). Additionally, As exposure led to histopathological damage in hepatic tissues. However, fisetin administration remarkably alleviated all the depicted hepatic damages. For further verification, the screening of several dock complexes was performed by using the GOLD 5.3.0 version. Based on docking fitness and GOLD score, the ranking order of receptor proteins with fisetin compound is superoxide dismutase, interleukin, aspartate aminotransferase, alkaline phosphatase, TNF-alpha, alanine transaminase, cyclo-oxygenase 2, antiapoptotic, and glutathione reductase. Out of these three receptor proteins superoxide dismutase, interleukin, and aspartate aminotransferase showed the best interaction with the fisetin compound. In vivo and in silico outcomes of the current study demonstrated that fisetin could potentially ameliorate As-instigated hepatotoxicity.

Introduction
Arsenic (As) is a noxious metalloid, which is ranked 1 st by the United States (US) Agency for Disease Registry and Toxic Substances as well as US Environmental Protection Agency [1] that affected nearly 200 million people globally Humans get exposed to arsenic via inhalation, skin contact with As-contaminated products, and polluted drinking water (H 2 O) [4]. Moreover, As toxicity depends on the chemical nature of arsenicals (arsenic-comprising compounds), which exist in both organic and inorganic forms with differently charged cations (e.g., As3þ and As5þ) [5]. Overall, the inorganic form of As is more toxic than the organic form of this metal [6].
After absorption from the lungs, As is delivered by the gut into the bloodstream where it (99% of arsenic) binds with red blood cells in circulating fluid, which eventually transports it to other parts of the body [7] and accumulates in different organs, i.e., lungs, liver, kidney, and heart [8].
e liver is a vital organ that tends to retain higher concentrations of As [9]. One of the most generally putative mechanisms to describe As-instigated toxicities is oxidative stress (OS) [10]. OS can cause mitochondrial dysfunction via fibrosis (TGF-β/Smad pathway), inflammation (NF-ĸB, TNF-α, IL-1, and IL-6), apoptosis (AKT-PKB, PI3/AKT, AKT/ERK, MAPK, PKCδ-JNK, and p53 pathways), and necrosis [11]. Besides, As intoxication was shown to weaken the antioxidant defense and damage several macromolecules (deoxyribonucleic acid (DNA), proteins, and lipids), which led to the foundation of the membrane, cell, and tissue dysfunction [12]. Moreover, As exposure may lead to inflammation which also results in liver damage. us, after scrutinizing the numerous sources of As exposure and their damaging impacts on human health, especially on the liver, a study on therapies against As-induced toxicities is needed. e advantage of using in silico methods for drug design is that it takes less time and money to find novel targets. Several biological issues have been resolved using in silico techniques that can characterize interacting molecules and forecast three-dimensional (3D) structures. To ascertain how various target proteins interact with the discovered chemical, in silico investigation was carried out. In this instance, fisetin (3,3′,4′,7-tetrahydroxyflavone) is a phytoflavonoid, which profoundly exists in multiple dietary sources such as apple, persimmon, grape, strawberry, cucumber, onion, and its quantities range from 2 to 160 mg/g with an average everyday consumption estimation of 0.4 mg [13]. It shows a broad range of therapeutic activities that include antioxidant [14], anticarcinogenic [13], antiinflammatory [15], neuroprotective [15], and cardioprotective effects [14]. Up till now, the ameliorative potential of fisetin against arsenic-provoked hepatotoxicity is not available. So, the present investigation proposed to explore the remedial potency of fisetin against As-instigated hepatotoxicity in rats.

Materials and Methods
2.1. Chemicals. As and fisetin were purchased from Germany (Sigma-Aldrich).

Animals.
Sexually mature male albino rats (n � 48) weighing 150 ± 30 g were kept in 12 rats per cage (made of steel) in the animal breeding as well as rearing house of the University of Agriculture, Faisalabad. All the rats were provided with tap water ad libitum as well as standard chow and photoperiod of 12 h light/dark cycle at temperature ranges between 23 and 26°C. Rats were kept in subordination with the European Union protocol (CEE Council 86/609) of animal care and experimentation.

Experimental Protocol
Albino rats (N � 48) were allocated into 4 groups (N � 12) and administered orally the following: control group (Treated with normal saline), As group (10 mg/kg. b. wt. Of As), cotreated group (10 mg/kg b.wt. Of As and 2.5 mg/kg. b.wt. Of fisetin), and only fisetin administered group (2.5 mg/kg.b.wt. Of fisetin). e entire experimental trial was conducted for thirty days. After one month of treatment, hepatic tissues were excised, weighed, and kept till additional analysis.

Biochemical Assay and TPC.
In the hepatic tissues, the activity of CAT was ascertained according to the methodology described by Chance and Maehly [16]. SOD activity was measured by following the process of Kakkar et al. [17]. GSR activity was determined according to the protocol of Carlberg and Mannervik [18]. GSH content was measured via the technique designed by Jollow et al. [19]. Hayashi et al. [20] protocol was used to estimate the level of ROS. e level of TBARS was assessed by following the technique of Iqbal et al. [21]. e TPC of hepatic tissues was quantified according to the Lowry method as modified by Peterson [22].

Liver Serum Enzymes.
e levels of ALT, AST, and ALP were determined in accordance with the commercial kits purchased from Wiesbaden, Germany.

Apoptosis.
e levels of Bcl-2, Bax, Caspase-3, and Caspase-9 were estimated with the help of ELISA kits bought from Cusabio Technology Llc, Houston, TX, USA.

3.5.
Histopathology. For histopathological analysis, initially, hepatic tissues were cleaned in 0.9% chilled saline and placed in 10% formalin solution, subsequently desiccated in mounting concentrations of alcohol, and embedded in paraffin wax. After that, paraffin-encased 5-µm slices were pruned via microtome, and staining was done with the help of hematoxylin-eosin (H&E) stain and observed below the Leica LB microscope at 400X [23].

Statistical Analysis.
e results mean ± standard error (SE) was presented in the tables after applying ANOVA accompanied by Tukey's test to interpret the entire data with the help of Minitab software. Results were declared meaningful at p < 0.05.

In Silico Analysis
e two-dimensional (2D) configuration of fisetin phytocompound retrieved from Pub-Chem (https://pubchem.ncbi.nlm.nih.gov) and treated in the ChemDraw ultra 12.0 and Chem 3D Pro for ionization, minimization, and optimization of ligands. Force field via the module for minimization and optimization of ligands having the lowest energy conformer of the ligand.

Receptor Preparation.
In order to assess the molecular docking, optimum resolution X-ray structures of proteins were obtained from the Protein Databank (RCSB PDB) (https://www.rcsb.org) and underwent the Protein preparation wizard of Maestro (Gold v 5.3.0). is module processed the protein by the addition of hydrogen atoms to the protein structure, removing solvent molecules (H2O), creating disulfide bonds, assigning bond orders, filling missing side chains as well as loops, and generating a protonation state at the cellular level pH (7.4 ± 0.5) using the Epik tool of protein structures for ligands. Following the processing of protein structures, the PDB ID of this 5YTO [24], 1ILR [25], 6WNG (10.2210/pdb6WNG/PDB), 1ANJ [26], 5YOY [27], IBDO [28], 51F9 [29], 6FSO [30], and 6TJL (10.2210/ pdb6TJL/PDB) structures were optimized using GOLD at pH 7.0, and the OPLS3e force field was used to perform restrained minimization for energy minimization and protein structural geometry optimization.

Molecular Docking.
e docking studies were carried out with the use of molecular docking software parameters (https://www.ccdc.cam.ac.uk). Docking simulations were carried out by the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search approach. e initial position, orientation, and torsion of the ligand molecules were determined at random. Every docking experiment was filtered from ten distinct runs that were set to stop followed by a maximum of 1.5Å assessments.
Molecular docking experiments were used to investigate the potential binding/interaction between proteins and ligands. Table S1 illustrates the binding affinity (kcal/mol) of the fisetin (5281614) phytocompound with different receptor proteins.
reedimensional structures of receptor proteins, alkaline phosphate (PDB ID 1ANJ), alanine transaminase (PDB ID, IBDO), cyclooxygenase-2 (PDB ID 51F9), interleukin (PDB ID, 1ILR), TNF-a (PDB ID, 5YOY), superoxide dismutase (PDB ID, 5YTO), antiapoptotic (PDB ID, 6FSO), glutathione reductase (PDB ID, 6TJL), and aspartate aminotransferase (PDB ID, 6WNG) were acquired from the PDB database (Protein Data Bank). Docking calculations were carried out with GOLD version 5.3.0 and BIOVIA discovery studio (http://www. 3dsbiovia.com) for modeling and visualization. e initial position, orientation, and torsion of the ligand molecules were determined at random. Every docking experiment was extracted from a total of ten distinct runs that were set to end after a maximum of 1.5Å evaluations.

Effect of Fisetin on Biochemical Assay and TPC.
e activity of CAT, SOD, GSR, as well as GSH level and TPC, was substantially (p < 0.05) reduced after As intoxication, while the concentration of ROS and level of TBARS were raised as matched with the untreated group. Conversely, fisetin supplementation with As remarkably (p < 0.05) elevated the activity of CAT, SOD, GSR, and GSH content as well as TPC, while considerably (p < 0.05) lowered the levels of ROS and TBARS in the cotreated group as contrasted with the As-induced group. Nonetheless, nonsignificant variation was witnessed among rats of the fisetin-only administrated and the untreated rats (Table 1). Table 2 shows outcomes of the study exposed that hepatic serum levels of AST, ALP, as well as ALT, were substantially (p < 0.05) raised in the As-induced group as matched to the control group. Nevertheless, fisetin treatment substantially (p < 0.05) caused the decline of hepatic enzymes in the cotreated group as matched to the As-intoxicated group. Moreover, a nonsignificant variation was seen between the fisetin-only treated and the control groups. Table 3 demonstrates the outcomes of the investigation that displayed As exposure substantially (p < 0.05) raised the levels of IL-1β, TNF-α, NF-κB, IL-6, and COX-2 activity in the Asinduced group as matched to the control group. Nonetheless, fisetin supplementation markedly (p < 0.05) diminished inflammatory indices in the cotreated (As + fisetin) group as matched to the As group. ere was insignificant (p < 0.05) variation between the fisetin-only treated and the control groups.

Effect of Fisetin on Antiapoptotic and Proapoptotic
Markers. To ascertain the probable antiapoptotic activity of fisetin, a property that presents its protecting impact against As-instigated hepatic tissues apoptosis, we estimated the alterations in the levels of the antiapoptotic marker Bcl-2 and proapoptotic markers, particularly, Bax, Caspase-3, and Caspase-9 ( Table 4). Results of the study exposed that Asinduction considerably (p < 0.05) decreased the antiapoptotic indices, whereas increased the proapoptotic inducers in the As-intoxicated rats as matched with the control rats. Nevertheless, fisetin cotreatment substantially (p < 0.05) restored the level of the above-stated antiapoptotic marker while reducing the levels of proapoptotic markers in the cotreated group as contrasted with the arsenic-induced Evidence-Based Complementary and Alternative Medicine group. However, a nonsignificant alteration was noticed among the mean values of the fisetin-only treated and the untreated rats. Figure 1 shows the comparative changes in the histopathological profile. Outcomes of the investigation presented that As exposure caused necrosis, sinusoid dilation, and apoptosis of hepatocytes along with central venule disruption in the Asinduced rats as matched to the control rats (Figures 1(b)  and 1(a)). Nonetheless, fisetin supplementation substantially (p < 0.05) mitigated the predominance and intensity of histopathological impairments such as diminished dilation of sinusoids with no necrotic cell, central venule disruption, and retrieved the classic architecture of liver cells in the coadministrated (As + fisetin) group as matched to the As group (Figures 1(b) and 1(c)). However, in the fisetin-only treated rats, histological architecture was similar to the control group (Figures 1(d) and 1(a)).

Discussion
Arsenic as a poison is a worldwide health predicament. Chronic As intoxication has been profoundly linked with several disorders and health problems in humans [31]. e overgeneration of intracellular ROS after As exposure mediates multiple alterations in cell functioning by changing signaling molecular antigenic alteration or provokes direct oxidative impairment to molecules [32]. us, antioxidants with remarkable free radical scavenging properties can alleviate As-instigated toxicities [33]. erefore, the current investigation was formulated to estimate the antioxidant potency of fisetin, which is a potential flavonoid with diverse pharmacological properties against As-intoxicated hepatotoxicity in rats.
Outcomes of the current research revealed that As intoxication considerably reduced activities of SOD, CAT, GSR, or GSH content, and TPC while escalating the levels of ROS and TBARS. As evident, the body's defense system is made up of antioxidants that may be enzymatic or    Evidence-Based Complementary and Alternative Medicine nonenzymatic, which act swiftly and neutralize free radicals [34]. SOD, GPX, and CAT are enzymatic antioxidants, while GSH is a nonenzymatic antioxidant [35]. CAT and GPx transformed the hydrogen peroxide (H 2 O 2) into the water [36], whereas the conversion of superoxide anion (O 2 -) into H 2 O 2 is carried out by SOD [37] whereas reduced GSH functions as an anion donator in these redox reactions [38]. GSH is retained by GSR, which renovates reduced GSH from oxidized GSSG for the perpetual functioning of GPx [39]. However, an unnecessary escalation of ROS resulting from deficient antioxidant defense or collapse of the cells' buffering system to retain the redox balance that leads to OS, which consequently commences numerous modifications in biomolecules and ultimately leads to disease conditions [40]. e oxidative damage to lipids is known as LPO [41]. LPO, in turn, may lead to damages that affect membrane integrity as well as fluidity and permeability [42]. However, fisetin provision remarkably alleviated the above-stated biochemical alteration via enhancing the activities of antioxidant defense or total protein content and lowering the levels of ROS and TBARS. is curative effect of fisetin may be due to the presence of one hydroxyl group on its A-ring that sets the lipid-H 2 O interface of the membrane and exhibits equivalent free radical scavenging activity similar to other flavonoids such as quercetin.
us, it inhibited LPO by preventing the additional diffusion of reactive oxygen species into the lipid hydrophobic core [8].
In the current investigation, As exposure caused a remarkable increment in ALP, ALT, and AST levels indicating damage to hepatic tissues. As documented earlier, these enzymes exist in hepatocytes, but their levels are ordinarily low. However, when liver cells are damaged, their membranes become more penetrable as a result their enzymes are liberated into the blood [43]. Our outcomes are in harmony with the results of Un et al. [44], who conveyed similar results, followed by As treatment. However, in the current research, fisetin oral gavage remarkably reduced the levels of hepatic serum enzymes, which may be due to its antioxidant potential.
Inflammation is the reflective response of the body's defense system, which is provoked by internal, i.e., stressed, impaired, or defective functioning of tissues, as well as external sources, i.e., reactive chemicals, allergens, microbes, and ROS [45]. is inflammatory process leads to elevated cell membrane permeability and vasodilatation that causes the nuclear translocation of different leukocytes and inflammatory markers [46]. NF-κB is among the fundamental inflammatory mediators which get triggered instantly in response to the internal or external cellular stimulant, which ultimately increases the levels of TNF-α [46], IL-1β [47], IL-6 [46] and activity of COX-2 [47]. Outcomes of the present investigation showed that As-induction substantially boosted the levels of IL-1β, TNF-α, NF-κB, IL-6, and COX-2 activity. However, fisetin coadministration with As remarkably lowered the elevated levels of inflammatory markers, which showed its anti-inflammatory property.
Apoptosis, a cell death mechanism, which helps to eradicate undesired cells, is accomplished by intrinsic (mitochondrial) and extrinsic (death receptor) pathways [48]. In the current investigation, we assessed apoptosis by Evidence-Based Complementary and Alternative Medicine estimating the level of Bax, Caspase-3, Caspase-9, and Bcl-2. Outcomes showed that As exposure lowered the level of Bcl-2 while boosting the levels of Bax, Caspase-3, and Caspase-9. Bax and Bcl-2 are proteins that are related to the Bcl-2 family. Bcl-2 promotes cellular longevity by stabilizing the opening of the MPT (mitochondrial-permeability-transition) pore complex and defends against Cytochrome c liberation, whereas Bax activates MPT pore and regulates the discharge of Cytochrome c into the cytosol [49], which activate Caspase-9 that cleaves Caspase-3 [50], that eventually leads to apoptosis [51]. As evident, Caspases are cysteine proteases that cut 100 distinct target proteins and provoke apoptosis [52]. us, the anti-or pro-apoptotic Bcl-2/Bax ratio regulates apoptosis [53]. Nevertheless, fisetin mitigated these hepatocytes' apoptosis via down-andupregulating the levels of pro-or anti-apoptotic markers,   Evidence-Based Complementary and Alternative Medicine respectively, in the rat liver. Our outcomes verify the antiapoptotic potential of fisetin. e outcomes of the present research demonstrated that As administration induced intense histopathological impairments in the hepatic tissues. e reason behind these toxic histological alterations is LP, which eventually leads to inflammation and apoptosis in hepatocytes. As-induced hepatic injuries include central venule disruption, apoptosis of hepatocytes, necrosis, and sinusoid dilation. Our results are compatible with Al-Forkan et al. [54], who studied the retention mechanism of As in organs and its effect on liver enzymes, hematology, and histology. However, fisetin treatment remarkably ameliorated the histopathological damages caused by As. Fisetin restored the impairments of its histopathological profile may be due to its free radical quenching, anti-inflammatory, and antiapoptotic, attributes.

Docking Analysis.
ChemDraw Ultra 12.0 and Chem 3D Pro were utilized in GOLD docking for the energy minimization of ligands in accordance with the method adopted by Andleeb et al. (2020). In order to comprehend the efficacy of these receptors, the molecular docking analysis looked at the molecular interactions of the fisetin molecule with different receptor proteins. e protein data repository was considered to obtain the coordinate crystal structure of the receptor proteins (PDB). e GOLD suite version 5.3.0 with a high resolution of 2.70 was then used to load each of these structures one at a time for docking. Using the GOLD 5.3.0 edition, screenings of various dock complexes were done based on docking fitness and GOLD score. e most effective chemical that interacts with the receptor was discovered thanks to the GOLD program. Based on docking score and fitness, the results were evaluated for binding compatibility. e ligand molecule with the highest binding affinity to the receptor molecule was selected as the best medication. e three receptor proteins 5YTO, 1ILR, and 6WNG demonstrated the best interaction with the fisetin compound, with GOLD fitness values of 77.99, 68.50, and 60. 35 and GOLD docking scores of -9.29, -8.96, and -8.90, respectively. e receptor proteins with fisetin compound are ranked in the following order: 5YTO > 1ILR > 6WNG > 1ANJ > 5YOY > IBDO > 51F9 > 6FSO > 6TJL. e best poses created by the discovery studio are depicted in Figure 2 in a 2D depiction of the interactions between proteins and ligands.

Conclusion
Adult male albino rats received the arsenic injection, which resulted in elevated serum enzyme levels, inflammatory and apoptotic indicators, and a worsened histopathological profile. Additionally, an unbalanced state that resulted in hepatic dysfunction was presented by the activity of enzymatic antioxidants, TPC or the levels of ROS, and TBARS. Nevertheless, due to its underlying antioxidant, antiapoptotic, as well as anti-inflammatory potentials, fisetin therapy significantly reduced arsenic-induced deficits in all of the aforementioned measures. [55] Data Availability e data supporting the current study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest. Table S1 illustrates the binding affinity (kcal/mol) of the fisetin (5281614) phytocompound with different receptor proteins. (Supplementary Materials)