| ObjectivesDiarrhea-predominant irritable bowel syndrome(IBS-D)with recurrent abdominal pain,abdominal distension and diarrhea as the cardinal symptom,it belongs to the category of spleen deficiency and diarrhea in traditional Chinese medicine.The predisposing factors include social environment,psychology,intestinal infection,inheritance,etc.Intestinal barrier function disorder and visceral hypersensitivity are important pathophysiological basis.The abnormal intestinal barrier function causes increased intestinal permeability,which is primarily manifested by visceral hypersensitivity as pathological feature.However,the specific regulatory mechanism is not current entirely clear,which needs to be further investigated.Recent research found miRNA-29a plays a key role in the regulation of intestinal permeability,and the confirmed target genes include Nuclear Factor Kappa B Repressing Factor(NKRF)and Tight Junction Protein 1(CLDN1).However,the regulating approach of related structural proteins,affecting intestinal machinery and immune barrier is not clear.And whether this regulatory relationship can change the visceral hypersensitivity of IBS-D has not been reported.Our previous research showed that the main prescription for tonifying spleen and anti-diarrhea Changji'an formula has obvious curative effect of IBS-D patients.Changji'an formula can modulate the function of 5-hydroxytryptamine(5-HT)and mast cell degranulation,down regulate the expression of Nuclear Factor Kappa B(NF-?B)and CLDN1,but the mechanism of its role in regulating intestinal sensitivity and reducing permeability is not fully understood.This study intends to investigate the potential mechanism of miRNA-29a and target genes in IBS-D through regulating intestinal permeability and visceral sensitivity in clinical study,in vivo and in vitro experiments.We intend to preliminary clarify the mechanism of Changji'an formula in regulating intestinal barrier function disorder and visceral hypersensitivity.It also has important clinical implication to explore miRNA-29a as a potential biological diagnostic marker of IBS-D.Methods1.Investigate the relationship between colon mucosa ultra-morphological structure of IBS-D patients and miRNA-29a with target genes.According to the integration and elimination criteria the mucosa biopsy specimens of IBS-D patients and healthy volunteer were collected.The changes of colon mucosa ultra-morphological structure of IBS-D patients were observed by transmission electron microscope(TEM).Expression of miRNA-29a,CLND1,NKRF and(zonula occluden1)ZO-1 were measured with reverse transcription?quantitative polymerase chain reaction(RT-qPCR).Western blot was performed to detect the protein levels of CLDN1and NKRF.2.Establish IBS-D model in miRNA-29 knockout mice,investigate the role of miRNA-29a and its target genes in intestinal permeability and intestinal sensitivity in mice.Water avoidance stress(WAS)-induced visceral hypersensitivity IBS-D mouse model was established.Mice were randomly divided into four groups(control,WAS,miRNA-29a–/–,WAS+miRNA 29–/–),with six mice in each group.Mice were sacrificed after modeling.Abdominal withdrawal reflex(AWR)was measured to evaluate the visceral sensitivity.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the activity of diamine oxidase(DAO)and D-lactate(D-LA)in serum.Immunofluorescence was used to measure CLDN1 and NKRF expression levels in colon tissues.RT?qPCR was performed to assess the gene expression level of miRNA-29a,CLDN1,NKRF and ZO-1.Western blot was used to measure the protein levels of Transient receptor potential vanilloid 1(TRPV1)and Protease activated receptor 1(PAR2).3.Verify the regulatory relationship of miRNA-29a and its target genes in vivo.Human intestinal epithelial cell line NCM460 was used.Cells were transfected with miRNA-29a-3p mimic and miRNA-29a-3p inhibitor respectively.Cell transfection efficiency was measured.The levels of miRNA-29a,CLDN1and NKRF were measured by RT-qPCR,and the protein expression level of CLDN1 and NKRF were measured by western blot.4.Investigate the change of colon mucosa ultra-morphological structure,increased intestinal permeability and visceral hypersensitivity effected by Changji'an formula based on miRNA-29a and target genes in IBS-D rats.4.1 IBS-D rat model was established with neonatal SD rats,by using the three factors method including maternal separation,acetic acid stimulation and immobilization stress.All modeling rats were randomly divided into four group,including the model control group,the pinaverium bromide group(positive comparison group,0.018g/kg),the high dose of Changji'an formula group(33.48g/kg),and the low dose of Changji'an formula group(16.74g/kg),with eight mice per group.All medication lasted for 14successive days by gavage.Sterile water was given to the model control group and another eight normal littermates.After medication,rats body weight,sucrose preference test and AWR scores of rectal distention test were performed to evaluate the visceral sensitivity.The changes of colon mucosa ultra-morphological structure were observed by TEM.4.2 Neonatal SD rats were performed the three factors method to established IBS-D model.Rats grouping and medication were the same as above.Rats were sacrificed after modeling and medication.ELISA was used to analyze the activity of DAO and D-LA in serum.Immunohistochemistry was used to examine the expression of CLDN1 and ZO-1 in colon tissues.The m RNA level of miRNA-29a,CLDN1,MKRF and ZO-1 in colon tissues were assessed using RT?qPCR.Western blot was used to measure the protein levels of CLDN1 and NKRF.Results1.Ultra-morphological structure of IBS-D patients'colon mucosa showed intestinal barrier function damaged.Gene expression level of miRNA-29a was upregulated and CLDN1,NKRF,ZO-1 was downregulated in colon tissues.TEM showed that microvilli on the surface of colon mucosa of healthy volunteers were distributed evenly,arranged orderliness and the density was uniform.IBS-D patients colon mucosa microvilli showed depletion in numbers and of various lengths,with the disperse arrangement.Visualized the staining of the tight junctions(TJs)among colonic enterocytes,it was identified that the staining was strong and continuous in the healthy volunteer group,while the TJs were faint and discontinuous and gap junctions were broadening in the IBS-D patients'group.It was observed that the relative content of miRNA-29a in the IBS-D patients'colon mucosa was significantly upregulated compared with colon tissues of healthy volunteer.In addition,the CLDN1,ZO-1 and NKRF m RNA expression levels were significantly downregulated compared with the IBS-D group.The protein expression level of CLDN1 and ZO-1 were also downregulated compared with the IBS-D group.2.Mi RNA-29a and its target genes regulated the intestinal permeability and visceral sensitivity in miRNA-29a knockout mice.ELISA results showed the serum content of DAO and D-LA in WAS group increased.Serum content of DAO and D-LA decreased in WAS+miRNA29-/-group compared with WAS group.Serum content of DAO and D-LA are usually used to evaluate the function of intestinal barrier,which indicated miRNA-29a has the function of regulating intestinal permeability.AWR scores in WAS group increased compared with the control group.Compared with WAS group,the AWS scores decreased in WAS+miRNA29-/-group,which indicated miRNA-29a has the effect of regulating visceral hypersensitivity.RT-qPCR showed the m RNA expression levels of CLDN1,NKRF in colon tissues decreased in WAS group,while the level of NF-?B was measured increase.Compared with WAS group,m RNA expression levels of CLDN1 and NKRF were measured increase in WAS+miRNA29-/-group,while the level of NF-?B was measured decrease.Western blot showed the protein expression levels of PAR2 and TRPV1 in colon tissues were observed increase in WAS group.Compared with WAS group,protein levels of PAR2 and TRPV1 were measured decrease in WAS+miRNA29-/-group.Immunofluorescence showed the expression levels of CLDN1 and NKRF in colon tissues were observed decrease in WAS group.Compared with WAS group,expression levels of CLDN1 and NKRF increased in WAS+miRNA29-/-group.The above results suggested that miRNA-29a may effect intestinal permeability and visceral sensitivity through regulating CLDN and NKRF.3.The regulatory effects of miRNA-29a with CLDN1 and NKRF in NCM460 cells.RT-qPCR showed that the m RNA levels of CLDN1 and NKRF were downregulated after transfection of miRNA-29a-3p mimic in NCM460 cells.And CLDN1 and NKRF m RNA expressions were observed increase after transfection of miRNA-29a-3p inhibitor.Trends of protein levels were observed consistent in western blot and Immunofluorescence with gene expressions.4.Effect of Changji'an formula on intestinal permeability and visceral sensitivity in IBS-D model rats.The HE stains of colon tissue and AWR scores indicated that the IBS-D model was established.Compared with control group,the sucrose preference values were measured decrease and AWR scores were increased in IBS-D model rats.Compared with model group,the sucrose preference values were measured increase in both high and low dose of Changji'an formula group,as well as the pinaverium bromide group,while the AWR scores were measured decrease in each group.No obvious pathological changes were observed in colon tissue morphology.Colon mucosa ultra-morphological structure of control group showed intercellular tight junction structure was integrated and the density was uniform.The tight junction of model group was partial teared,gap junction was broadened.Changji'an formula alleviated the impair of ultra-morphological structure of colon mucosa on a certain degree of IBS-D model rats.Compared with control group,the serum content of DAO and D-LA increased,the miRNA-29a expression level increased in model group.The gene expression level of CLDN1,NKRF and ZO-1 were measured decrease,as well as the protein level of CLDN1 and NKRF.Compared with model group,both high and low dose of Changji'an formula reduced the serum content of DAO and D-LA,downregulated the gene expression level of miRNA-29 in colon tissues,as well as the pinaverium bromide group.The gene expression levels of CLDN1,NKRF and ZO-1,as well as the protein level of CLDN1 and NKRF were measured increased in rats'colon tissue after administration of both high and low dose of Changji'an formula and pinaverium bromide.to IBS-D rats.Conclusion1.The abnormal intestinal barrier function of IBS-D patients may be related to the regulation of miRNA-29a and target gene.2.Mi RNA-29a plays an important role in reversing intestinal permeability and visceral sensitivity in WAS-induced IBS-D model mice through regulating NKRF and CLDN1.3.miRNA-29a had the negative regulatory effects of CLDN1 and NKRF in NCM460cells.4.Administration of Changji'an formula could attenuate symptom of diarrhea due to spleen deficiency,as well as reduce the intestinal permeability and visceral sensitivity of IBS-D rats.IBS-D rats manifests increasing intestinal permeability and visceral hypersensitivity,which may regulate by miRNA-29a and its target genes.Changji'an formula could reduce the level of miRNA-29a,enhance the gene and protein expression of CLDN1 and NKRF in IBS-D rats colon tissue,which reveal that Changji'an formula could regulate miRNA-29a and its target genes.This regulation has a certain degree of correlation with intestinal permeability and visceral sensitivity in IBS-D rats.In conclusion,the results of our study indicated that miRNA-29a and target genes have regulatory effects of intestinal permeability and visceral sensitivity in IBS-D.Changji'an formula could regulate miRNA-29a and its target genes to relieve intestinal permeability and visceral hypersensitivity in IBS-D. |
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