Glial cell line-derived neurotrophic factor improves impaired colonic motility in experimental colitis mice through connexin 43
Ulcerative colitis (UC) is a chronic, frequently relapsing-remitting, and non-specific inflammatory disease affecting the intestinal system, typically originating in the rectum. Recent studies have reported an increasing incidence of UC not only in traditionally high-prevalence regions, such as North America and Europe, but also in newly industrialized countries in Asia, possibly attributed to rapid dietary and lifestyle transitions. The clinical symptoms of UC, including weight loss, bloody diarrhea, rectal pain, and abdominal cramps, significantly impair patients’ quality of life across physical, psychological, and social dimensions[ - ]. This aligns with the modern concept of disease clearance, which prioritizes comprehensive recovery beyond symptom control. For instance, the BE-FIT-IBD study highlighted reduced physical activity levels among UC patients, illustrating the broad impact of the disease. Colonic dysfunction has long been recognized as a common complication among individuals with UC. In UC patients, assessment of gastrointestinal motility has revealed reduced postprandial colonic contraction amplitude and altered transit. The entire gastrointestinal tract has been reported to be covered by the enteric nervous system (ENS), which regulates bowel motility. Damage to the ENS in colonic tissue has been identified in UC patients[ - ]. Individuals with UC frequently experience diarrhea due to impaired colonic motility and dysfunction of the intestinal epithelial barrier. These issues are likely interconnected. Changes in the ENS and subsequent colonic motility result from cumulative histological disturbances and structural damage to the gut wall. This leads to clinical symptoms, such as loose stools or diarrhea, abdominal pain, or discomfort, making treatment more challenging[ - ]. However, the exact mechanisms of abnormal colonic motility in UC patients still remain unclear and perplexing. Gap junctions are cylindrical channels between animal cells that allow ions and small molecules to pass from the inside of one cell to the inside of the next. Additionally, they are capable of regulating cell migration, proliferation, and differentiation. Gap junctions have channel clusters formed by the connexin protein family, which generate hemichannels and allow the transfer of ions and signaling chemicals between cells to facilitate intercellular communication. By facilitating the transfer of electrical or second messenger impulses from one cell to another, gap junctions serve crucial roles in coordinating cellular function in numerous organs, including the heart, kidney, liver, and gastrointestinal system. Humans possess at least 21 distinct connexin subtypes, each of which can generate heteromeric hemichannels with distinct connexin subtypes or homomeric channels with the same subtype. Connexin proteins are the fundamental units of hemichannels. Connexin 43 (Cx43), named for its molecular weight of 43 kDa, is the most prevalent and well-studied connexin. Cx43 has been reported to be essential in regulating cell growth and apoptosis. Changes in Cx43 expression in the gastrointestinal tract have been associated with impaired motility, gastrointestinal infections, and inflammatory bowel disease. Furthermore, the Cx43 expression is remarkably declined in the colons of children with Hirschsprung’s disease, leading to disturbances in normal colonic motility. This outcome indicates that Cx43 is involved in gastrointestinal motility regulation. The ENS in the intestinal wall plays a role in organizing gastrointestinal functions through its complex structure. Enteric glial cells (EGCs) and neurons are the two primary cell populations, with the former far outnumbering the latter. Abnormal EGCs have been demonstrated to be an important factor contributing to gastrointestinal motility disorders[ - ]. In the gastrointestinal tract, the Cx43 protein plays a crucial role in facilitating the coordinated contraction of smooth muscle cells and interstitial cells of Cajal by forming gap junctions. Initially, EGCs are identified as the primary source of glial cell line-derived neurotrophic factor (GDNF) secretion, and decreased GDNF levels were observed in intestinal tissue samples from UC patients. As discovered by Chen et al , the GDNF levels were also found to be reduced in the blood of individuals with Parkinson’s disease with constipation, suggesting that low GDNF levels may be a risk factor for constipation in these patients. GDNF can trigger ENS regeneration in mouse models of Hirschsprung’s disease. Furthermore, a previous animal experiment confirms that aging-induced delayed stomach emptying may be linked to EGCs inactivation. GDNF is essential in the viability and maturation of the nervous system. Gap junction channels are controlled by a chemical gating mechanism, responding to cytosolic calcium (Ca 2+ ) concentrations. Active EGCs possess the capacity to produce adenosine triphosphate (ATP), facilitating and propagating the Ca 2+ waves. These waves are transmitted through gap junctions within the EGCs networks, spreading from EGCs to adjacent cells. The purpose of this study was to elucidate the changes in GDNF and Cx43 in colon tissues of UC mice. Additionally, Gap19, a selective inhibitor toward Cx43 hemichannels, was utilized to further investigate whether GDNF exerts its effects on impaired colonic motility through Cx43. These findings may contribute to the development of possible novel drugs or therapies for people with UC with impaired colonic motility. 

Animals A total of 32 male C57BL/6-specific pathogen-free mice, each weighing roughly 25 g and aged 6-8 weeks, were used in this study. These mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Before any surgical or experimental procedures, all mice underwent a 5-day acclimatization period in a specific pathogen-free environment. Subsequently, they were placed in an identical setting with humidity levels of 40%-50%, a temperature range of 22-23 °C, and a 12-hour light and dark cycle. The cages were thoroughly cleaned, and the mice were monitored daily. Prior to this study, approval for all experimental procedures was obtained from the Institutional Animal Care and Use Committee (approval No. 2022175). Efforts were made throughout the study to minimize the distress and discomfort of the mice as much as possible. Induction of UC-like experimental colitis The mice were assigned to four groups at random ( n = 8): The normal control group (CN), the UC model group (UC), the UC + GDNF treatment group (UC + GDNF), and the UC + Gap19 + GDNF treatment group (UC + Gap19 + GDNF). The UC model was constructed according to the description provided in the literature. A 3.5% dextran sodium sulfate (DSS) solution was prepared by dissolving it in distilled water. To induce UC, 3.5% DSS was provided ad libitum in the drinking water of the UC group for 10 days, whereas the CN group received only drinking water under the same conditions. In the UC + GDNF group on the 5 th day, after administering 3.5% DSS, mice were intraperitoneally injected with GDNF at 5 μL/kg/day body weight in 100 μL of 0.9% sodium chloride for 6 consecutive days. Mice in the UC + Gap19 + GDNF group received intraperitoneal injections of Gap19 at 25 mg/kg/day body weight on the 5 th day after the administration of GDNF. Throughout the experimental period, rectal bleeding, diarrhea, and body weight were recorded daily, and the disease activity index (DAI) was calculated using these parameters. The formula for DAI calculation is displayed in Table , with higher DAI values representing more severe UC. Sample acquisition Following overnight fasting, all mice were euthanized via intraperitoneal injection of pentobarbital sodium (75 mg/kg) on day 11. As an indicator of colonic inflammation and damage, colon length was immediately determined. Following careful excision, the entire colon was washed with cold saline and divided into sections for further analyses. For histopathological examination, one tissue portion was preserved in 10% neutral buffered formalin overnight, while other sections were immediately frozen in liquid nitrogen and stored at -80 °C for subsequent biochemical and molecular analyses. Colonic transit time analysis Colonic transit time was quantified using a bead expulsion assay, as previously reported. In this study, this approach was slightly altered. After overnight fasting, a single 2 mm-diameter glass bead was gently inserted into the distal colon (approximately 3 cm from the anus) of each mouse using a plastic rod lubricated with jelly. Following bead insertion, the mice were individually housed in separate cages. Colonic transit time was evaluated by measuring the time between bead insertion and bead expulsion. Histopathological examinations Colon samples preserved in 10% neutral buffered formalin were subjected to routine paraffin embedding. Colon samples were sliced into 4 μm-thick sections and stained with picrosirius red staining, hematoxylin and eosin staining, and periodic acid-Schiff staining. Under a microscope (Eclipse E100, Nikon, Japan), the stained sections were analyzed by two independent investigators blinded to the treatment groups. Representative images were captured to evaluate pathological changes. Histological scoring of the colonic mucosa was measured following the criteria detailed in Table . Transmission electron microscopy Colon tissues from each group were collected and preserved in 2.0% glutaraldehyde. Then, these tissues were rinsed with 0.1 M phosphate buffer, followed by fixation with 1% osmic acid. Transmission electron microscopy was conducted after dehydration, embedding, sectioning, polymerization, and staining with uranyl acetate, as outlined in earlier research. The ultrastructure of the colon tissues was evaluated with a transmission electron microscope (Hitachi HT7700, Japan). Each sample was independently examined by three specialists. Immunohistochemistry staining Following dissection and separation, colonic tissues were rinsed with cold phosphate-buffered saline (PBS). Samples were then fixed with 4% paraformaldehyde solution for stabilization. To reduce background cross-reactivity, the sections underwent 30-minute incubation with 5% bovine serum albumin blocking buffer at ambient temperature. The GDNF and Cx43 levels were analyzed in 4 μm-thick paraffin-embedded colon tissue sections. As instructed by the manufacturer, overnight incubation was conducted at 4 °C with anti-Cx43 (GB12234, 1:200, Servicebio, Wuhan, Hubei Province, China) and anti-GDNF Rabbit primary antibodies (GB11403, 1:200, Servicebio, Wuhan, Hubei Province, China). Thereafter, the sections were immersed in PBS, followed by exposure to a Goat anti-Rabbit IgG secondary antibody for 50 minutes at ambient temperature. Subsequently, three washes with PBS, each lasting 5 minutes, were performed on the sections. Lastly, sections were visualized with 3,3’-Diaminobenzidine, counterstained with hematoxylin, and examined using a microscope (Eclipse E100, Nikon, Japan). Image-Pro Plus 6.0 was utilized for quantifying the immunohistochemistry staining results. Immunofluorescent analysis of Cx43 and GDNF Cx43 hemichannels are predominantly expressed by astrocytes in the central nervous system (CNS), while in the gastrointestinal tract, the morphology of mature EGCs is similar to that of astrocytes in the CNS. Immunofluorescence staining was conducted to examine the exact location and distribution of Cx43 and GDNF in colon tissues. After dewaxing, antigen retrieval was performed using Tris-ethylenediaminetetraacetic acid buffer (potential of hydrogen = 8.0), followed by treatment with 3% body surface area for 30 minutes to block non-specific binding. Then, the colon tissue sections were exposed to a mixture of primary antibodies, including anti-Cx43 (GB12234, Servicebio, Wuhan, Hubei Province, China) and anti-GDNF rabbit primary antibodies (GB11403, Servicebio, Wuhan, Hubei Province, China). Subsequently, the sections underwent 50-minute incubation at ambient temperature with corresponding secondary antibodies: Alexa Fluor ® 488-conjugated goat anti-mouse IgG (H + L) (GB25301, 1:200, Servicebio, Wuhan, Hubei Province, China) and Cy3-conjugated goat anti-rabbit IgG (H + L) (GB21303, 1:200, Servicebio, Wuhan, Hubei Province, China). Following staining with 4’,6-diamidino-2-phenylindole, the sections were sealed using an anti-fluorescence quenching sealing tablet. A fluorescent microscope (Nikon Eclipse C1, Japan) was adopted for analyzing the sections. Relative quantitative real-time polymerase chain reaction Real-time polymerase chain reaction (PCR) was utilized for measuring the relative expression levels of GDNF and Cx43 mRNA in colon samples. RNA extraction was performed on colon samples using Trizol reagent (Invitrogen, Carlsbad, CA, United States) as instructed by the manufacturer. Primer sets for mouse GDNF, Cx43, and glyceraldehyde-3-phosphate dehydrogenase were prepared as detailed in Table . All reactions were conducted through the StepOne™ real-time PCR system (Applied Biosystems, United States). The PCR cycling conditions were as follows: Incubation at 25 °C for 5 minutes, 42 °C for 60 minutes, and 70 °C for 5 minutes. Primer-specific product amplification was confirmed through dissociation curve analysis. The 2 -ΔΔCt method was employed for calculating the relative change in gene expression. Enzyme-linked immunosorbent assay Serum was acquired from whole blood samples by centrifugation at 3000 rpm for 15 minutes at 4 °C. The collected serum was stored at -80 °C. Enzyme-linked immunosorbent assay (ELISA) kits specific to each sample were utilized for determining the C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in the blood, adhering to the manufacturer’s guidelines. The optical density was quantified at 450 nm using a microplate reader (BioTeK, Winooski, United States), and the concentration was determined based on a standard curve. Statistical analysis IBM statistical product and service solutions software (version 25.0) and GraphPad Prism (version 9.5) were employed for statistical analyses. The Shapiro-Wilk test was utilized for evaluating the normality of the data. One-way analysis of variance was applied for multiple comparisons when the data followed a normal distribution. Non-parametric tests, including the Kruskal-Wallis test, were employed when the data did not satisfy normality assumptions. Data were presented as mean ± SD or median (interquartile range). P < 0.05 represented the statistical significance threshold. 

A total of 32 male C57BL/6-specific pathogen-free mice, each weighing roughly 25 g and aged 6-8 weeks, were used in this study. These mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Before any surgical or experimental procedures, all mice underwent a 5-day acclimatization period in a specific pathogen-free environment. Subsequently, they were placed in an identical setting with humidity levels of 40%-50%, a temperature range of 22-23 °C, and a 12-hour light and dark cycle. The cages were thoroughly cleaned, and the mice were monitored daily. Prior to this study, approval for all experimental procedures was obtained from the Institutional Animal Care and Use Committee (approval No. 2022175). Efforts were made throughout the study to minimize the distress and discomfort of the mice as much as possible. 

The mice were assigned to four groups at random ( n = 8): The normal control group (CN), the UC model group (UC), the UC + GDNF treatment group (UC + GDNF), and the UC + Gap19 + GDNF treatment group (UC + Gap19 + GDNF). The UC model was constructed according to the description provided in the literature. A 3.5% dextran sodium sulfate (DSS) solution was prepared by dissolving it in distilled water. To induce UC, 3.5% DSS was provided ad libitum in the drinking water of the UC group for 10 days, whereas the CN group received only drinking water under the same conditions. In the UC + GDNF group on the 5 th day, after administering 3.5% DSS, mice were intraperitoneally injected with GDNF at 5 μL/kg/day body weight in 100 μL of 0.9% sodium chloride for 6 consecutive days. Mice in the UC + Gap19 + GDNF group received intraperitoneal injections of Gap19 at 25 mg/kg/day body weight on the 5 th day after the administration of GDNF. Throughout the experimental period, rectal bleeding, diarrhea, and body weight were recorded daily, and the disease activity index (DAI) was calculated using these parameters. The formula for DAI calculation is displayed in Table , with higher DAI values representing more severe UC. 

Following overnight fasting, all mice were euthanized via intraperitoneal injection of pentobarbital sodium (75 mg/kg) on day 11. As an indicator of colonic inflammation and damage, colon length was immediately determined. Following careful excision, the entire colon was washed with cold saline and divided into sections for further analyses. For histopathological examination, one tissue portion was preserved in 10% neutral buffered formalin overnight, while other sections were immediately frozen in liquid nitrogen and stored at -80 °C for subsequent biochemical and molecular analyses. 

Colonic transit time was quantified using a bead expulsion assay, as previously reported. In this study, this approach was slightly altered. After overnight fasting, a single 2 mm-diameter glass bead was gently inserted into the distal colon (approximately 3 cm from the anus) of each mouse using a plastic rod lubricated with jelly. Following bead insertion, the mice were individually housed in separate cages. Colonic transit time was evaluated by measuring the time between bead insertion and bead expulsion. 

Colon samples preserved in 10% neutral buffered formalin were subjected to routine paraffin embedding. Colon samples were sliced into 4 μm-thick sections and stained with picrosirius red staining, hematoxylin and eosin staining, and periodic acid-Schiff staining. Under a microscope (Eclipse E100, Nikon, Japan), the stained sections were analyzed by two independent investigators blinded to the treatment groups. Representative images were captured to evaluate pathological changes. Histological scoring of the colonic mucosa was measured following the criteria detailed in Table . 

Colon tissues from each group were collected and preserved in 2.0% glutaraldehyde. Then, these tissues were rinsed with 0.1 M phosphate buffer, followed by fixation with 1% osmic acid. Transmission electron microscopy was conducted after dehydration, embedding, sectioning, polymerization, and staining with uranyl acetate, as outlined in earlier research. The ultrastructure of the colon tissues was evaluated with a transmission electron microscope (Hitachi HT7700, Japan). Each sample was independently examined by three specialists. 

Following dissection and separation, colonic tissues were rinsed with cold phosphate-buffered saline (PBS). Samples were then fixed with 4% paraformaldehyde solution for stabilization. To reduce background cross-reactivity, the sections underwent 30-minute incubation with 5% bovine serum albumin blocking buffer at ambient temperature. The GDNF and Cx43 levels were analyzed in 4 μm-thick paraffin-embedded colon tissue sections. As instructed by the manufacturer, overnight incubation was conducted at 4 °C with anti-Cx43 (GB12234, 1:200, Servicebio, Wuhan, Hubei Province, China) and anti-GDNF Rabbit primary antibodies (GB11403, 1:200, Servicebio, Wuhan, Hubei Province, China). Thereafter, the sections were immersed in PBS, followed by exposure to a Goat anti-Rabbit IgG secondary antibody for 50 minutes at ambient temperature. Subsequently, three washes with PBS, each lasting 5 minutes, were performed on the sections. Lastly, sections were visualized with 3,3’-Diaminobenzidine, counterstained with hematoxylin, and examined using a microscope (Eclipse E100, Nikon, Japan). Image-Pro Plus 6.0 was utilized for quantifying the immunohistochemistry staining results. 

Cx43 hemichannels are predominantly expressed by astrocytes in the central nervous system (CNS), while in the gastrointestinal tract, the morphology of mature EGCs is similar to that of astrocytes in the CNS. Immunofluorescence staining was conducted to examine the exact location and distribution of Cx43 and GDNF in colon tissues. After dewaxing, antigen retrieval was performed using Tris-ethylenediaminetetraacetic acid buffer (potential of hydrogen = 8.0), followed by treatment with 3% body surface area for 30 minutes to block non-specific binding. Then, the colon tissue sections were exposed to a mixture of primary antibodies, including anti-Cx43 (GB12234, Servicebio, Wuhan, Hubei Province, China) and anti-GDNF rabbit primary antibodies (GB11403, Servicebio, Wuhan, Hubei Province, China). Subsequently, the sections underwent 50-minute incubation at ambient temperature with corresponding secondary antibodies: Alexa Fluor ® 488-conjugated goat anti-mouse IgG (H + L) (GB25301, 1:200, Servicebio, Wuhan, Hubei Province, China) and Cy3-conjugated goat anti-rabbit IgG (H + L) (GB21303, 1:200, Servicebio, Wuhan, Hubei Province, China). Following staining with 4’,6-diamidino-2-phenylindole, the sections were sealed using an anti-fluorescence quenching sealing tablet. A fluorescent microscope (Nikon Eclipse C1, Japan) was adopted for analyzing the sections. 

Real-time polymerase chain reaction (PCR) was utilized for measuring the relative expression levels of GDNF and Cx43 mRNA in colon samples. RNA extraction was performed on colon samples using Trizol reagent (Invitrogen, Carlsbad, CA, United States) as instructed by the manufacturer. Primer sets for mouse GDNF, Cx43, and glyceraldehyde-3-phosphate dehydrogenase were prepared as detailed in Table . All reactions were conducted through the StepOne™ real-time PCR system (Applied Biosystems, United States). The PCR cycling conditions were as follows: Incubation at 25 °C for 5 minutes, 42 °C for 60 minutes, and 70 °C for 5 minutes. Primer-specific product amplification was confirmed through dissociation curve analysis. The 2 -ΔΔCt method was employed for calculating the relative change in gene expression. 

Serum was acquired from whole blood samples by centrifugation at 3000 rpm for 15 minutes at 4 °C. The collected serum was stored at -80 °C. Enzyme-linked immunosorbent assay (ELISA) kits specific to each sample were utilized for determining the C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in the blood, adhering to the manufacturer’s guidelines. The optical density was quantified at 450 nm using a microplate reader (BioTeK, Winooski, United States), and the concentration was determined based on a standard curve. 

IBM statistical product and service solutions software (version 25.0) and GraphPad Prism (version 9.5) were employed for statistical analyses. The Shapiro-Wilk test was utilized for evaluating the normality of the data. One-way analysis of variance was applied for multiple comparisons when the data followed a normal distribution. Non-parametric tests, including the Kruskal-Wallis test, were employed when the data did not satisfy normality assumptions. Data were presented as mean ± SD or median (interquartile range). P < 0.05 represented the statistical significance threshold. 

GDNF alleviates colitis symptoms in DSS-induced UC mice Colitis symptoms, such as diarrhea, hematochezia, and weight loss, were assessed to evaluate the therapeutic effects of GDNF in DSS-induced colitis. Compared to the CN group, mice in the UC group exhibited reduced physical activity, dull hair, and weight loss (16.28 ± 1.15 vs 26.00 ± 1.06, P < 0.001), along with significantly higher DAI scores and shorter colon lengths (4.85 ± 0.26 vs 7.63 ± 0.25, P = 0.001) (Figure ). In contrast, GDNF treatment resulted in remarkable improvement of clinical symptoms. Mice in the GDNF + UC group showed increased physical activity, reduced hair loss and reversed body weight loss (16.28 ± 1.15 vs 20.01 ± 0.33, P < 0.001). Additionally, GDNF attenuated the elevated DAI scores, reversed colon shortening, and improved overall health. However, these therapeutic effects were not observed in the UC + Gap19 + GDNF group, with no significant improvements in weight loss, DAI scores, or colon shortening. The findings suggested that GDNF could mitigate colitis symptoms in DSS-induced UC mice through Cx43. GDNF protects against colonic histopathological damage and reduces inflammation in DSS-induced UC mice Histopathological changes and inflammatory markers were evaluated to assess the impact of GDNF on colonic tissue structure and inflammation. Histopathological analysis revealed severe tissue damage in the UC group, including multiple superficial ulcers, epithelial cell degeneration, goblet cell loss, and inflammatory cell infiltration (Figure ). In contrast, the UC + GDNF group showed reduced damage with preserved epithelial architecture, with reduced inflammatory cell infiltration and histological score, suggesting that GDNF protected against UC-induced histopathological changes (Figure ). However, in the UC + Gap19 + GDNF group, the pathology was similar to that of the UC group, indicating that Gap19 prevented the therapeutic effects of GDNF on histopathological damage by inhibiting Cx43. Additionally, inflammatory markers were significantly elevated in the UC group in contrast to the CN group, including CRP (89.98 ± 6.17 vs 37.67 ± 4.56, P < 0.001), TNF-α (74.13 ± 4.18 vs 27.07 ± 3.65, P < 0.001), IL-1β (543.73 ± 22.47 vs 221.68 ± 10.77, P < 0.001), and IL-6 (441.46 ± 9.76 vs 112.81 ± 5.37, P < 0.001) (Figure - ). These findings indicated a heightened inflammatory response in the colonic tissues. GDNF treatment led to a decline in these markers, correlating with improved tissue integrity. However, in the UC + Gap19 + GDNF group, the levels of these markers remained elevated, similar to those in the UC group. These findings further suggested that GDNF protected colonic tissue integrity and reduced inflammation in UC mice via Cx43. GDNF improves colonic transit time in DSS-induced UC mice Colonic transit time was measured using the bead expulsion assay to assess the GDNF’s therapeutic effect on colonic motility. Compared to the CN group, colonic transit time (13.13 ± 2.09 minutes) was markedly delayed in the UC group (5.77 ± 0.58 minutes vs 13.13 ± 2.09 minutes, P < 0.001). Treatment with exogenous GDNF considerably improved colonic transit time (7.84 ± 0.98 minutes, P < 0.001), demonstrating its therapeutic efficacy. However, there was no notable improvement in colonic transit time (12.32 ± 1.35 minutes, P = 0.797) in the UC + Gap19 + GDNF group, indicating that Gap19 blocked the therapeutic effects of GDNF on colonic motility by inhibiting Cx43 (Figure ). This outcome suggested that Cx43 was essential for the effect of GDNF in enhancing colonic motility. Reduced GDNF and Cx43 expression in DSS-induced UC mice The GDNF and Cx43 expression in colon tissue was analyzed using real-time PCR. Relative to the CN group, the UC group displayed a remarkable drop in the GDNF (0.60 ± 0.04 vs 0.90 ± 0.10, P < 0.001) and Cx43 (0.61 ± 0.07 vs 0.92 ± 0.06, P < 0.001) mRNA expression. Interestingly, both GDNF and Cx43 expression were markedly raised in the GDNF + UC group (Figure and ). As revealed by the results of immunohistochemistry and immunofluorescent staining, both Cx43 and GDNF were localized not only in the smooth muscle portion of the intestine but also predominantly in colonic epithelial cells (Figure and Figure ). Cx43-positive plaques (green) were colocalized with GDNF (red), suggesting a functional interaction between these two proteins. In the UC group, the Cx43 puncta was markedly sparse and disarrayed in comparison with the CN group; however, this effect was partially attenuated by exogenous GDNF treatment. The UC + Gap19 + GDNF group exhibited a notable decline in Cx43 expression, along with the inhibition of GDNF’s therapeutic effects, confirming that Cx43 is essential in the therapeutic effects of GDNF. GDNF mitigates ultrastructure damage in colonic tissues Transmission electron microscopy was performed to evaluate ultrastructural changes in colon tissues. In the CN group, colon tissues exhibited normal morphology, with intact epithelial cells, clearly identified microvilli, numerous microvilli, plentiful mitochondria, and regular oval euchromatic nucleus. In contrast, the UC group exhibited severe ultrastructural disruptions, including ruptured cell membranes, irregular microvilli, and compromised epithelial integrity (Figure ). GDNF treatment partially mitigated these ultrastructural changes, resulting in fewer damaged cells and more organized microvilli. However, ultrastructural damage in the UC + Gap19 + GDNF group was similar to that observed in the UC group, indicating that Gap19 inhibited the therapeutic effects of GDNF on cell morphology. These findings highlighted the role of GDNF in mitigating colonic ultrastructural damage in UC mice through Cx43. 

Colitis symptoms, such as diarrhea, hematochezia, and weight loss, were assessed to evaluate the therapeutic effects of GDNF in DSS-induced colitis. Compared to the CN group, mice in the UC group exhibited reduced physical activity, dull hair, and weight loss (16.28 ± 1.15 vs 26.00 ± 1.06, P < 0.001), along with significantly higher DAI scores and shorter colon lengths (4.85 ± 0.26 vs 7.63 ± 0.25, P = 0.001) (Figure ). In contrast, GDNF treatment resulted in remarkable improvement of clinical symptoms. Mice in the GDNF + UC group showed increased physical activity, reduced hair loss and reversed body weight loss (16.28 ± 1.15 vs 20.01 ± 0.33, P < 0.001). Additionally, GDNF attenuated the elevated DAI scores, reversed colon shortening, and improved overall health. However, these therapeutic effects were not observed in the UC + Gap19 + GDNF group, with no significant improvements in weight loss, DAI scores, or colon shortening. The findings suggested that GDNF could mitigate colitis symptoms in DSS-induced UC mice through Cx43. 

Histopathological changes and inflammatory markers were evaluated to assess the impact of GDNF on colonic tissue structure and inflammation. Histopathological analysis revealed severe tissue damage in the UC group, including multiple superficial ulcers, epithelial cell degeneration, goblet cell loss, and inflammatory cell infiltration (Figure ). In contrast, the UC + GDNF group showed reduced damage with preserved epithelial architecture, with reduced inflammatory cell infiltration and histological score, suggesting that GDNF protected against UC-induced histopathological changes (Figure ). However, in the UC + Gap19 + GDNF group, the pathology was similar to that of the UC group, indicating that Gap19 prevented the therapeutic effects of GDNF on histopathological damage by inhibiting Cx43. Additionally, inflammatory markers were significantly elevated in the UC group in contrast to the CN group, including CRP (89.98 ± 6.17 vs 37.67 ± 4.56, P < 0.001), TNF-α (74.13 ± 4.18 vs 27.07 ± 3.65, P < 0.001), IL-1β (543.73 ± 22.47 vs 221.68 ± 10.77, P < 0.001), and IL-6 (441.46 ± 9.76 vs 112.81 ± 5.37, P < 0.001) (Figure - ). These findings indicated a heightened inflammatory response in the colonic tissues. GDNF treatment led to a decline in these markers, correlating with improved tissue integrity. However, in the UC + Gap19 + GDNF group, the levels of these markers remained elevated, similar to those in the UC group. These findings further suggested that GDNF protected colonic tissue integrity and reduced inflammation in UC mice via Cx43. 

Colonic transit time was measured using the bead expulsion assay to assess the GDNF’s therapeutic effect on colonic motility. Compared to the CN group, colonic transit time (13.13 ± 2.09 minutes) was markedly delayed in the UC group (5.77 ± 0.58 minutes vs 13.13 ± 2.09 minutes, P < 0.001). Treatment with exogenous GDNF considerably improved colonic transit time (7.84 ± 0.98 minutes, P < 0.001), demonstrating its therapeutic efficacy. However, there was no notable improvement in colonic transit time (12.32 ± 1.35 minutes, P = 0.797) in the UC + Gap19 + GDNF group, indicating that Gap19 blocked the therapeutic effects of GDNF on colonic motility by inhibiting Cx43 (Figure ). This outcome suggested that Cx43 was essential for the effect of GDNF in enhancing colonic motility. 

The GDNF and Cx43 expression in colon tissue was analyzed using real-time PCR. Relative to the CN group, the UC group displayed a remarkable drop in the GDNF (0.60 ± 0.04 vs 0.90 ± 0.10, P < 0.001) and Cx43 (0.61 ± 0.07 vs 0.92 ± 0.06, P < 0.001) mRNA expression. Interestingly, both GDNF and Cx43 expression were markedly raised in the GDNF + UC group (Figure and ). As revealed by the results of immunohistochemistry and immunofluorescent staining, both Cx43 and GDNF were localized not only in the smooth muscle portion of the intestine but also predominantly in colonic epithelial cells (Figure and Figure ). Cx43-positive plaques (green) were colocalized with GDNF (red), suggesting a functional interaction between these two proteins. In the UC group, the Cx43 puncta was markedly sparse and disarrayed in comparison with the CN group; however, this effect was partially attenuated by exogenous GDNF treatment. The UC + Gap19 + GDNF group exhibited a notable decline in Cx43 expression, along with the inhibition of GDNF’s therapeutic effects, confirming that Cx43 is essential in the therapeutic effects of GDNF. 

Transmission electron microscopy was performed to evaluate ultrastructural changes in colon tissues. In the CN group, colon tissues exhibited normal morphology, with intact epithelial cells, clearly identified microvilli, numerous microvilli, plentiful mitochondria, and regular oval euchromatic nucleus. In contrast, the UC group exhibited severe ultrastructural disruptions, including ruptured cell membranes, irregular microvilli, and compromised epithelial integrity (Figure ). GDNF treatment partially mitigated these ultrastructural changes, resulting in fewer damaged cells and more organized microvilli. However, ultrastructural damage in the UC + Gap19 + GDNF group was similar to that observed in the UC group, indicating that Gap19 inhibited the therapeutic effects of GDNF on cell morphology. These findings highlighted the role of GDNF in mitigating colonic ultrastructural damage in UC mice through Cx43. 

In this study, the UC mouse model was successfully established by administering a DSS aqueous solution. The model is widely recognized for its ability to accurately replicate human clinical symptoms of UC, such as bloody diarrhea and weight loss. Experimental findings revealed that colonic transit time was notably delayed in the UC group relative to the CN group. Additionally, pathological damage was observed in the colorectal tissues of UC mice. These indicators confirmed the successful establishment of the UC mouse model. Furthermore, this study demonstrated the beneficial impacts of GDNF on experimental UC in mice. GDNF treatment alleviated colonic morphological changes, improved DAI scores, ameliorated delayed colonic transit time, and reduced colitis-associated weight loss. Therefore, these outcomes indicated that GDNF could be a promising target for treating the aberrant colonic motility observed in colitis mice. However, the therapeutic impacts of GDNF in UC mice were inhibited by Gap19, a specific inhibitor of Cx43 hemichannels. In the UC mice model, IL-6, IL-1β, TNF-α, and CRP are considered key pro-inflammatory cytokines in UC development. The DAI score is commonly used as an indicator of inflammatory activity in animal experiments. The IL-6, IL-1β, TNF-α, and CRP levels in the blood were quantified using the ELISA. ELISA findings, as expected, revealed markedly elevated levels of CRP, IL-6, IL-1β, and TNF-α in UC mice ( P < 0.05). Additionally, the DAI score was notably raised in the UC group. Saber et al  discovered increased P2X receptor 7 expression levels in the colonic tissues of UC rats. Cx43 has been demonstrated to be involved in extracellular ATP release. This ATP, in turn, plays a role in excitatory neurotransmission within the ENS by activating P2X and P2Y receptors. ATP-activated receptors play a well-established role in the inflammatory response, as they have been demonstrated to facilitate the release of pro-inflammatory cytokines from immune cells. Furthermore, this study revealed that GDNF effectively inhibited the excessive release of these pro-inflammatory cytokines in UC mice, indicating that GDNF reduced aberrant inflammatory responses. Increasing evidence indicates that despite the increased frequency of bowel movements, colonic transit time was considerably delayed and smooth muscle contractility was evidently decreased in individuals with UC. The diarrhea symptoms in UC patients may not result from accelerated colonic motility but rather from hypersensitivity and inflammation in the distal colon and rectum that is not compliant, leading to frequent and urgent defecation. Such findings were similar to the results in this study. Nevertheless, the exact signaling mechanism remains elusive[ , , ]. Impaired intestinal motility can exacerbate diarrhea in UC patients, making it difficult to distinguish symptoms directly associated with the inflammatory process, and adversely affecting quality of life. Connexin proteins are the fundamental unit of hemichannels, with Cx43 being the primary gap junction protein in astrocytes. Interestingly, fully developed EGCs exhibit notable morphological and functional similarities to astrocytes in the CNS. EGC, a major component of the ENS, has been shown to possess a highly intricate and sophisticated nature in regulating gastrointestinal motility and maintaining intestinal balance. Neural crest cells, originating from the ectoderm, are widely recognized as multipotent cells capable of migrating to several organs, including the gastrointestinal tract, where they contribute to the formation of the ENS. Bannerman et al  discovered that gap junctions are essential for the survival of neural crest cells. They also found that migrating rat neural crest cells expressed the gap junction component Cx43. Using messenger molecules carried by Cx43, the interstitial cells of Cajal found in the deep muscular plexus area of the rat small intestine promote communication with smooth muscle cells. Recent studies have demonstrated that the Cx43 expression is modified in the colons of children diagnosed with Hirschsprung’s disease. This abnormality leads to improper communication between cells and a disruption in colonic motility. Based on these studies, we hypothesized that Cx43 was involved in regulating gastrointestinal motility. Cx43 is the primary gap junction protein associated with the development of the gastrointestinal motility transmission system. Phosphorylation is the main form in which Cx43 exerts its biological effects in electrical coupling in the gastrointestinal tract. However, the exact mechanism of phosphorylation remains poorly understood. In this study, the Cx43 expression was declined in the colon tissues of UC mice, suggesting that Cx43 might play a protective role in UC development. This study confirmed that GDNF reduced the IL-6, TNF-α, IL-1β, and CRP levels and relieved the morphological changes in UC mice. GDNF, a neurotrophic factor, is mainly produced by EGCs in the gastrointestinal tract. In this study, GDNF up-regulated the Cx43 expression in colonic tissues, including EGCs. GDNF can protect EGCs from apoptosis, and in GDNF-deficient mice, the ENS entirely fails to mature. In DSS-induced UC in rats, Li et al  covered that GDNF significantly alleviated colonic inflammation, partially reversed the loss of enteric neurons, and enhanced delayed intestinal transit. In this study, the enhancement of the intestinal epithelial barrier function might contribute to the anti-inflammatory impact of GDNF. However, the underlying mechanisms remain unclear. Cx43 is crucial in the transmission of regular action potentials. Reduced Cx43 expression can diminish the intercellular communication between myocytes. This study further investigated the relationship between GDNF and Cx43 and the effect of Gap19, a specific inhibitor of Cx43 hemichannels, on experimental UC mice, which was an important mechanism underlying impaired colonic motility in UC. Gap19 effectively and selectively inhibits the opening of Cx43 hemichannels in astrocytes. This study verified that the Cx43 expression was notably reduced. We hypothesized that decreased Cx43 expression would impair the ability of EGCs to communicate and transmit signals, resulting in aberrant colonic motility. Furthermore, administration of Gap19 suppressed the therapeutic effects of GDNF in UC mice. The presence of Cx43 hemichannels is essential for regulating colonic motility in the ENS. As the primary gap junction protein, Cx43 facilitates signal connectivity and transmission across gastrointestinal neurons, smooth muscle networks, and interstitial cells of Cajal in the ENS. Sun et al  confirmed that aging is associated with reduced Cx43 expression in the gastrointestinal tract of mice, and this decline occurred prior to the loss of interstitial cells of Cajal and neurons. Their findings suggested that Cx43 might be an upstream regulation target for managing gastrointestinal motility disorder associated with aging. Additionally, the interstitial cells of Cajal in the deep muscular plexus region of the rat small intestine establish communication among themselves and with smooth muscle cells via messenger molecules passing through Cx43. This study indicated that the Cx43 expression was decreased in UC mice. McClain et al  demonstrated that Cx43 was distributed throughout the EGCs of the colonic myenteric nerve plexus. However, in this study, Cx43 was not only distributed in the EGCs in mouse colonic myenteric nerve plexus but also enriched in epithelial cells, circular muscle layer, and muscularis mucosa. Akbarali et al  clearly confirmed that disturbance of Ca 2+ channel function is a contributing factor to decreased colonic motility. Furthermore, Zhang et al  found that Ca 2+ responses in EGCs were facilitated by Cx43 hemichannels. The absence of Cx43, particularly its channel activity, reduces the propagation of intercellular Ca 2+ waves. Additionally, Cx43 has been shown to be involved in extracellular ATP release and the activation of EGCs is accompanied by ATP release via Cx43[ , , ]. In mice, administration of Cx43 inhibitors restricts the capacity of Ca 2+ to undergo reactive diffusion in EGCs, indicating that Cx43 has a direct role in mediating Ca 2+ responses and an indirect role in facilitating gastrointestinal transit. Future research will further investigate the upstream regulatory mechanism of GDNF to provide deeper insights into the pathological process of UC and identify promising candidates for future medication development. Despite the promising findings of this study, several limitations must be acknowledged. First, the bead expulsion assay was used as the sole indicator of colonic transit capacity. While such an assay is widely employed for its simplicity, it may not fully capture the complexity of colonic motility. Complementary methods, such as radiographic tracking or segmental transit analysis, should be considered in future studies. Second, this study focused on systemic inflammation by measuring serum cytokines but did not assess inflammatory markers within colonic tissues. Local analyses, including cytokine quantification and immunohistochemical studies, could provide more direct insights into the colonic microenvironment and the anti-inflammatory effects of GDNF. Lastly, the upstream regulatory pathways of GDNF and Cx43 were not explored. Further studies targeting these pathways may deepen the understanding of impaired colonic motility in UC and identify new therapeutic targets. Addressing these limitations in future studies will enhance the translational relevance of our findings and contribute to a more comprehensive understanding of colonic motility and inflammatory processes in UC. 

In summary, this study demonstrates a novel role of GDNF in controlling the colonic motility in UC mice. Mechanistically, GDNF is proposed to partially improve impaired colonic motility in UC mice via Cx43. These findings highlight the potential of GDNF to improve abnormal colonic motility in UC patients, identifying Cx43 as a new potential target for UC therapy. Understanding the cellular and molecular drivers of these processes is critical for developing new therapeutic approaches for UC patients. Significant gaps persist in the understanding of UC pathogenesis, providing numerous opportunities for future research to achieve a more comprehensive understanding of cells and molecules. 

