| Purpose:From the whole,cellular,bacterial and molecular levels,the treatment mechanism of Shugan Jianpi Decoction on the IBS-D rats was investigated.By observing the general state,symptoms and histological structure,serum inflammatory index,abundance and diversity of intestinal flora,expression of short-chain fatty acids in IBS-D rats,and observing the intestinal mucosal barrier tight junction protein ZO-1,Claudin-1 and LPS/ TLR4/ My D88 /NF-κB pathway,the mechanism of Shugan Jianpi recipe in treating irritable bowel syndrome was discussed.Material and method: 1 Animal grouping and modeling There were 36 SPF grade SD rats,which were divided into blank group（BC）,model group（MC）,high dose group of Shugan Jianpi Decoction（ZYH）,medium dose group of Shugan Jianpi Decoction（ZYM）,and low dose group of Shugan Jianpi Decoction（ZYL）in each group.Except for the BC group,the other five groups were combined with chronic binding + tail clip stimulation + senna by gavage.2 Intervention method After 14 days of modeling,each group began treatment according to corresponding intervention measures.Rats in BC group and MC group were given normal saline by gavage;Rats in ZYH group were given Chinese medicine decoction by gavage,the concentration was 4.10g/ml;ZYM group was given Chinese decoction by gavage,the concentration was 2.05 g/ml;ZYL group rats were gavaged with decoction of traditional Chinese medicine at the concentration of 1.03g/m L;In PC group,rats were given the diluted suspension of Bifidobacterium tetralogy viable tablets,the concentration was 0.47g/m L.The gastric perfusion volume was 10 ml/kg/d.The intervention lasted for 14 days.3 Sampling and index detection All experimental rats were evaluated before sampling and then tested for efficacy.Blood collection from abdominal aorta was used by ELISA to detect IL-1β,IL-6,TNF-α,and LPS in serum;stool samples were frozen at-80℃.The fecal sample was analyzed by 16 s DNA;short chain fatty acids by liquid phase mass spectrometry;colonic tissue was fixed in 4% paraformaldehyde for immunoexpression of ZO-1,Claudin-1,TLR4,My D88,NF-κBp65 and p-NF-κBp65.Detection of ZO-1 and Claudin-1 protein expression by RT-PCR.Western Blot was used to detect the protein expression of TLR4,My D88,NF-κBp65and p-NF-κBp65.Results: Paper 1 Pharmacodynamic study of Shugan Jianpi Decoction in the treatment of IBS-D rats with liver depression and spleen deficiency 1 Changes in general state,food consumption,body weight,diarrhea index and AWR score of rats in each group After modeling,rats in MC group suffered from mental fatigue,dry and dull coat color,decreased food intake,slow weight gain,increased stool frequency,watery stool or shapeless stool.The score of abdominal wall rutting reflex,the horizontal and vertical scores of rats in open field test,the score of sugar-water preference test,and weight gain were significantly decreased,while the score of diarrhea index and the score of rats in tail suspension test were significantly increased（P<0.05）.No obvious pathological changes were observed in colon tissue under microscope,indicating successful modeling.It indicates that the modeling is successful.After the intervention of Shugan Jianpi Decoction,the rats in each group were in good mental state,increased activity,gradually improved and shiny hair color,loud voice,no obvious irritation,increased food intake,and gradually formed feces.The food intake and weight gain were significantly increased（P<0.05）,and the diarrhea index and abdominal wall reflex score were significantly decreased.2 Changes in the content of inflammatory cytokines（IL-1β,IL-6,TNF-α）in serum After the intervention of Shugan Jianpi Decoction,compared with BC group,serum levels of IL-1β,IL-6 and TNF-α in MC group were significantly increased（P<0.05）.Compared with MC group,serum levels of IL-1β,IL-6 and TNF-α in Shugan Jianpi Decoction high,medium and low dose groups were significantly decreased（P<0.05）,and the high dose group had the best effect.Paper 2 Effect of Shugan Jianpi Decoction on the intestinal flora of IBS-D rats liver depression and spleen deficiency 1 Shugan Jianpi Decoction improved the similarity of OTU composition between IBS-D rats and blank rats,increased the F/B value of IBS-D model rats,promoted the growth of Ruminococcus1,which can produce butyrate,and also promoted the growth of beneficial bacteria Lactobacillus,Prevotella9,Turicibacter,RuminococcaceaeUCG-014,and inhibited the growth of harmful bacteria Alloprevotella and Other.2 In this study,we found that LachnospiraceaeNK4A136group was more abundant in the intestinal microflora of PC group,which was positively correlated with the inflammatory indicators of IL-6,IL-IL-1B and TNF-a（P<0.05）,suggesting that it could be used as a marker of the intestinal microbiota of IBS-D rats.However,Turicibacter and Clostridiumsensustricto1 were negatively correlated with IL-1 β and IL-6（P<0.05）.PrevotellaceaeUCG-001 was negatively correlated with AWR in IBS-D model rats（p<0.001）,and Intestinimonas was positively correlated with IBS-D diarrhea（P<0.05）.3 The structure of IBS-D rats changed after intervention with Shugan Jianpi Decoction,and the function of 16 Sr function indicated that the expression of Carbohydrate Metabolism pathway was enhanced in the ZY group compared with the BC group.Paper 3 Effect of Shugan Jianpi Decoction on short chain fatty acids in IBS-D rats with liver depression and spleen deficiency Compared with the BC group,the intestinal levels of SCFAs and the total amount of SCFAs except valeric acid were significantly increased.Compared with the MC group,the levels of acetate,propionate,isobutyrate,butyrate,isovaleric,valeric acid and total levels of SCFAs were significantly reduced in the ZYH group and PC group（P<0.05）.Paper 4 Effect of Shugan Jianpi Decoction on intestinal mucosa of IBS-D rats with liver depression and spleen deficiency 1 Immunohistochemistry and Western Blot detection of TLR4,My D88,NF-κBp65,p-NF-κBp65 and their protein expression: compared with BC group,TLR4,My D88,NF-κBp65 and p-NF-κBp65 and their protein expression all increased significantly in the MC group（P<0.001,P<0.001）;Compared with the MC group,TLR4,My D88,NF-κBp65,p-NF-κBp65 and their protein expression of ZYH、ZYM、ZYL、PC groups decreased to varying degrees（P<0.001,P<0.001）,Among them,it was the most obvious in the ZYH group.2 RT-PCR method was used to detect the m RNA expression of ZO-1 and Claudin-1: Compared with the BC group,the levels of ZO-1 and Claudin-1 m RNA in the MC group was significantly reduced,and the difference was statistically significant（P <0.001,P <0.001）.The results showed that the levels of tight junction proteins ZO-1 and Claudin-1 m RNA decreased after diarrhea in the IBS-D model rats.Compared with MC group,ZO-1 and Claudin-1 in ZYH group and PC group,with significant difference（P <0.01,P <0.001）.The results showed that both Chinese medicine and western medicine could increase the tight junction protein ZO-1 and Claudin-1 m RNA levels in the IBS-D model rats.3 The detection of ZO-1 and Claudin-1 protein expression by Western Blot: compared with the BC group,the expression levels of ZO-1 and Claudin-1 protein in the MC group were significantly reduced,and the difference was statistically significant（P <0.001,P <0.001）.The results showed that the tight junction proteins ZO-1 and Claudin-1 protein levels decreased after diarrhea in IBS-D model rats.Compared with MC group,ZO-1 and Claudin-1 protein expression were significantly increased in ZYH group and PC group,with significant difference（P <0.01,P <0.001）.The results showed that both TCM and western medicine could increase the tight junction protein ZO-1 and Claudin-1 proteins in rat IBS-D model.4 The level of serum LPS was measured by ELISA method: in MC rats compared with BC rats P<0.001);compared with MC rats,LPS decreased in ZYH and PC rats（P<0.01）.Conclusion: 1 "Chronic binding,tail clip stress combined with diarrhea leaf gavage" can successfully replicate the combined model of liver depression and spleen deficiency in IBS-D rats.2 Shugan Jianpi Decoction can significantly improve the food intake and weight gain of IBS-D rats with liver depression and spleen deficiency,increase the intestinal pain threshold of rats,reduce visceral sensitivity,and improve the symptoms of liver depression and spleen deficiency in rats.And it can inhibit and reduce IL-1β、IL-6 and TNF-α Expression.It is suggested that Shugan Jianpi Decoction can effectively improve abdominal pain,diarrhea and other discomfort symptoms of IBS-D rats,and inhibit low-grade inflammation,and the high-dose group has the best treatment effect.3 Based on 16 S r DNA technology,Shugan Jianpi Decoction can adjust the abundance and diversity of intestinal flora structure of IBS-D model rats,and predict the enhanced expression of Carbohydrate Metabolism pathway.4 Shugan Jianpi Decoction can reduce the content of short-chain fatty acids in the intestine of rats in the IBS-D model group,which may be due to the involvement of regulating the structure of intestinal flora,promoting the growth of beneficial bacteria in the intestine,regulating immunity,anti-inflammation,and protecting the intestinal mucosal barrier.5 The liver and spleen can inhibit LPS/TLR4/My D88/NF-κB signaling pathway activation and upregulate the expression of tight junction protein factors ZO-1 and Claudin-1. |
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