NDRG2 Alleviates Visceral Hypersensitivity In Water-avoidance Stress IBS Mice By Regulating Intestinal Microbiota

【Background】Irritable Bowel Syndrome(IBS)is a prevalent and complex functional gastrointestinal disorder that significantly impacts patients’ quality of life and healthcare resources.The Rome IV criteria reaffirm that the core feature of IBS is visceral hypersensitivity(VHS),which means patients experience discomfort from normal physiological stimuli or have an exaggerated response to noxious stimuli.This sensitivity includes abnormal visceral pain perception and hyperalgesia.Studies have shown that IBS patients generally exhibit lower pain thresholds compared to healthy individuals,and some research suggests that mast cell activation in the gut may be a key mechanism triggering VHS,causing normally harmless gut stimuli to elicit abdominal pain in IBS patients.Additionally,dysbiosis of the gut microbiota and its interactions with the host immune system are increasingly recognized as important factors in the development of VHS.NDRG2(N-Myc downstream regulated gene 2)is a novel tumor suppressor gene that plays a crucial role in regulating the growth,differentiation,and metastasis of various malignancies.In intestinal diseases,NDRG2 may be involved in maintaining the integrity of the intestinal epithelial barrier.In inflammatory bowel disease and colonic tumors,knocking out NDRG2 can increase intestinal mucosal permeability,exacerbating mucosal inflammation.Interestingly,in a model of radiation-induced intestinal injury,NDRG2 knockout reduced the expression of inflammatory factors(such as IL-1β,IL-6,and TNF-α)and improved survival rates in mice.Given the complex role of NDRG2 in intestinal diseases,which is not yet fully understood,we utilize a water-avoidance stress-induced IBS mouse model to explore the specific role of NDRG2 in the pathogenesis of IBS,providing a reference for further studies on the function of NDRG2 in intestinal diseases.【Objectives】1.Establish a water-avoidance stress-induced IBS mouse model and assess the impact on NDRG2 expression within the intestinal epithelium.2.Determine the localization of NDRG2 in the intestinal tract and identify optimal viral delivery methods for modulating NDRG2 expression in intestinal tissues.3.Investigate changes in the fecal microbiota and short-chain fatty acids in water-avoidance stress-induced IBS mice using metagenomic and targeted metabolomic techniques.4.After up-regulating the expression of NDRG2 in the colon,we explored its effect on VHS in IBS mice with water avoidance stress,and utilized multi-omics approaches to investigate its impact on gut microbiota,changes in intestinal tissue gene expression,and fecal short-chain fatty acids.5.Explore whether the role of NDRG2 in reducing visceral hypersensitivity and intestinal inflammation depends on gut microbiota.【Methods】1.Establish an IBS mouse model using the water-avoidance stress method.Evaluate the success of modeling by measuring body weight,fecal water content,and abdominal withdrawal reflex scores.Collect intestinal mucosal tissues from the duodenum,jejunum,ileum,proximal colon,and distal colon of the mice.Assess NDRG2 expression using qPCR and immunohistochemistry.Measure TNF-α,IL-6,and IL-10 levels in the intestinal mucosa using ELISA.Evaluate the histological inflammation scores of each intestinal segment using HE staining.Assess the number of mast cells in the intestinal tissues using immunohistochemical staining for mast cells.Finally,use the GEO database to analyze NDRG2 expression in rat and human intestines.2.To gain a deeper understanding of the physiological function and mechanism of NDRG2 in the intestine,we employed double immunofluorescence staining to clarify the distribution and expression of NDRG2 in different cell types of the intestinal mucosa.In order to up-regulate the expression of NDRG2 in the intestinal mucosa,we utilized three delivery methods: intraperitoneal injection,tail vein injection,and enema,to administer the Adeno-Associated Virus serotype 2/2(AAV2/2)virus.Forty-eight C57BL/6 male mice were randomly selected,and intestinal samples were collected at 15 and 30 days after virus injection.qPCR and immunohistochemistry were used to detect the expression of NDRG2 and Flag protein.Immunohistochemistry results were analyzed using Image J software,and statistical methods were applied to evaluate whether differences between groups were statistically significant.3.To explore changes in the fecal microbiota and short-chain fatty acids(SCFAs)in water-avoidance stress-induced IBS mice,collect fecal samples from both IBS and control mice.Use metagenomic and targeted metabolomic techniques to analyze the impact of water-avoidance stress on the fecal microbiota and SCFAs.Metagenomic analysis will focus on the diversity and species composition of fecal microbes at both the phylum and genus levels,comparing differences in bacteria,archaea,eukaryotes,and viruses between the two groups.Targeted metabolomics will detect significant differences in SCFAs such as acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,isovaleric acid,hexanoic acid,and isohexanoic acid between the groups.4.After upregulating NDRG2 expression in the intestinal mucosa using tail vein injection of AAV2/2,measure body weight,fecal water content,and abdominal withdrawal reflex scores to evaluate the general condition and reduction of visceral hypersensitivity(VHS)in IBS mice.Detect TNF-α,IL-6,and IL-10 levels in proximal colon mucosal tissue using ELISA.Evaluate the histological inflammation scores of the proximal colon using HE staining.Assess the number of mast cells in the proximal colon using immunohistochemical staining.Use metagenomic sequencing to analyze the diversity and species composition of fecal microbes,including bacteria,archaea,eukaryotes,and viruses,at the phylum and genus levels after NDRG2 upregulation.Detect significant differences in SCFAs(acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,isovaleric acid,hexanoic acid,and isohexanoic acid)in fecal samples using targeted metabolomics.Finally,conduct reference transcriptome sequencing on proximal colon tissue samples to preliminarily explore the impact of NDRG2 upregulation on gene expression and related pathways.5.To elucidate the relationship between NDRG2 upregulation and the alleviation of VHS in water-avoidance stress-induced IBS mice through modulation of the gut microbiota,further validation will be performed using pseudo-germ-free animals and fecal microbiota transplantation.This part includes two experiments:Experiment 1: Construct pseudo-germ-free animals using intraperitoneal injection of composite antibiotics.Observe whether NDRG2 upregulation can alleviate visceral hypersensitivity induced by water-avoidance stress.Evaluate the impact on TNF-α,IL-6,IL-10 levels,and mast cell counts using ELISA and immunohistochemical staining for mast cells.Experiment 2: Perform fecal microbiota transplantation by transferring feces from NDRG2-upregulated,water-avoidance stress-exposed mice to pseudo-germ-free animals.Then,induce water-avoidance stress modeling in the recipients and observe whether this fecal transfer can reduce visceral hypersensitivity,mast cell counts,and the expression of TNF-α,IL-6,and IL-10.【Results】1.The water-avoidance stress-induced IBS mice exhibited a decreased growth rate(P< 0.05),increased fecal water content(P < 0.05),and heightened visceral sensitivity(P <0.01).TNF-α and IL-6 were significantly upregulated,and IL-10 was significantly downregulated in the mucosal epithelium of various intestinal segments(P < 0.05).Additionally,there was a significant increase in mast cell numbers(P < 0.05)and a significant decrease in NDRG2 expression(P < 0.05)across all intestinal segments.Analysis of the GEO database revealed a slight but non-significant reduction of NDRG2 expression in the intestinal mucosa of IBS-C and IBS-D patients compared to healthy volunteers in the GSE36701 dataset(P > 0.05).In the GSE38942 dataset,NDRG2 expression in maternal separation-induced IBS rats decreased from 15.46 ± 0.30 to 14.66± 0.60,but the difference was not significant(P = 0.0536).2.NDRG2(red fluorescence)was mainly localized in the cytoplasm,with no co-localization with goblet cells(MUC2)but co-localizing with intestinal stem cells(LGR5),absorptive colonic epithelial cells(CA1),and enteroendocrine cells(CHGA).For gene transduction,we observed two time points(15 and 30 days post-viral delivery).Results indicated that none of the three delivery methods(intraperitoneal injection,tail vein injection,and enema)successfully upregulated NDRG2 expression in the small intestine(duodenum,jejunum,and ileum)at either 15 or 30 days.However,at 15 days post-injection,only the tail vein injection group showed a significant increase in NDRG2 expression in the proximal colon compared to the NC group(P < 0.001).Similarly,at 30 days post-injection,only the tail vein injection group showed a significant increase in NDRG2 expression in the proximal colon(P < 0.001).Notably,the intraperitoneal injection group showed an increasing trend in NDRG2 expression in the proximal colon,but one out of four mice failed to upregulate NDRG2,with upregulation factors of 5.35,6.72,and 7.56 in the three successfully upregulated mice.3.The alpha diversity of the fecal microbiota in water-avoidance stress-induced IBS mice did not show significant changes,while the beta diversity showed significant alterations at the genus and species levels.LEf Se analysis indicated that at the genus level,bacteria in water-avoidance stress-induced IBS mice were dominated by Muribaculum and Lactobacillus,archaea by Methanocorpusculaceae and Thermoplasmatales,and viruses by Kehishuvirus,Burzaovirus,Buchavirus,and Pepyhexavirus.There were no significant changes in the short-chain fatty acids in the feces of water-avoidance stress-induced IBS mice(P > 0.05).4.After upregulating the expression of NDRG2 in the colonic tissue followed by water avoidance stress(WAS),the WAS＿NDRG2 group exhibited a significantly higher body weight gain compared to the WAS＿NC group(P < 0.05),and a significantly reduced fecal water content(P < 0.05).When the volume of water instilled was 0.3 ml or 0.4 ml,visceral sensitivity was significantly decreased(P < 0.05,P < 0.01).Elisa results indicated that upregulation of NDRG2 expression significantly decreased the levels of TNF-α and IL-6 while increasing IL-10 expression in the colonic mucosa of WAS-induced IBS mice(P < 0.05).Immunohistochemical analysis of mast cells showed that NDRG2 upregulation significantly reduced the number of mast cells in the colonic tissue of WAS-induced IBS mice(P < 0.01).Metagenomic analysis revealed that upregulation of NDRG2 had no significant effect on α-diversity but caused significant changes in β-diversity.LEf Se analysis indicated that in the WAS＿NDRG2 group,the predominant bacterial genera included Candidatus Scatovivens,Candidatus Arthromitus,Subdoligranulum,Enterobacterales,Morganellaceae,and Proteus.There was no significant effect on the short-chain fatty acids in the feces(P > 0.05).Finally,transcriptomic sequencing results showed significant differences in gene expression between the NDRG2 overexpression group(WAS＿NDRG2)and the control group(WAS＿NC).These differentially expressed genes were enriched in several key biological pathways and functions,particularly those related to immune responses and extracellular matrix interactions,such as cancer-related pathways,ECM-receptor interaction,and the PI3K-Akt signaling pathway.5.Results included two parts.Experiment 1: After "clearing" the gut microbiota using composite antibiotics(abx),NDRG2 upregulation did not alleviate visceral hypersensitivity or increase fecal water content induced by water-avoidance stress(P > 0.05)and did not reverse the upregulation of TNF-α and IL-6 induced by water-avoidance stress(P > 0.05).Mast cell numbers did not decrease(P > 0.05).Experiment 2: Fecal microbiota transplantation from NDRG2-upregulated,water-avoidance stress-exposed mice to pseudo-germ-free animals reversed the increase in visceral hypersensitivity and fecal water content in water-avoidance stress-induced IBS mice(P < 0.01),reduced the high expression of TNF-α and IL-6,and significantly decreased mast cell numbers(P < 0.001).【Conclusion】1.Water-avoidance stress-induced IBS mice exhibited reduced growth rates,increased fecal water content,enhanced visceral sensitivity,upregulated TNF-α and IL-6,downregulated IL-10,increased mast cell numbers in the intestinal mucosa,and decreased NDRG2 expression.2.NDRG2 is primarily localized in the cytoplasm and co-localizes with intestinal stem cells,absorptive colonic epithelial cells,and enteroendocrine cells.Tail vein injection of AAV2/2 successfully upregulated NDRG2 expression in the proximal colon but not in the small intestine.3.Water-avoidance stress altered the beta diversity but not the alpha diversity of the fecal microbiota in mice,affecting the composition of the microbiota but not fecal short-chain fatty acids.4.Upregulating NDRG2 expression significantly improved growth rates,reduced fecal water content,and decreased visceral sensitivity in water-avoidance stress-induced IBS mice,reduced TNF-α and IL-6 expression,increased IL-10 expression,and decreased mast cell numbers.Transcriptome sequencing revealed significant differences in gene expression involving immune response and extracellular matrix-related pathways.5.The alleviation of visceral hypersensitivity and intestinal inflammation in water-avoidance stress-induced IBS mice by upregulating NDRG2 requires the involvement of the gut microbiota.Antibiotic clearance of the gut microbiota prevented NDRG2 from reducing visceral hypersensitivity and intestinal inflammation in water-avoidance stress-induced mice.