Based On Endoplasmic Reticulum Autophagy To Study The Mechanism Of Tongxie Yaofang On IBS-D Liver Stagnation And Spleen Deficiency Syndrome

Background:Irritable bowel syndrome(IBS)is a clinically common functional gastrointestinal disease.Symptoms include abdominal discomfort and related changes in stool characteristics or frequency.It affects 4-10%of the global population.For most people,the symptoms of IBS will recur and alleviate,which brings a huge mental and economic burden to people.The onset of IBS is more common in people with mental illness and young adult women than the general population.Irritable bowel syndrome with predominant diarrhea(IBS-D)is the most common subtype of IBS.The pathophysiological mechanisms of IBS are diverse,and there is currently no specific conclusion.The concept of bio-psycho-society believes that this disease is the product of psychosocial factors and changes in multiple levels of the intestinal physiopathological interaction.Abnormal gastrointestinal motility,brain-enteric nervous system regulation,increased visceral sensitivity,intestinal infection and inflammation,mental and psychological factors,intestinal microecology,destruction of intestinal mucosal barrier function,etc.are closely related to it.The damage of intestinal mucosal barrier and the increase of intestinal permeability are one of the main pathogenesis.A variety of pathogens and toxins enter the body through the intestinal mucosa,causing a series of immune reactions.Intestinal mucosal permeability and visceral hypersensitivity also interact,resulting in a series of abdominal pain and diarrhea symptoms.Endoplasmic reticulum(ER)is an important organelle in eukaryotic mammalian cells and a key part of the biosynthesis and folding of new transmembrane proteins and secreted proteolipids.Various stress factors may cause the accumulation of misfolded proteins in the endoplasmic reticulum,which in turn triggers the endoplasmic reticulum stress(ERS)response.When the ERS continues,unfolded or misfolded proteins are overproduced,and the cell It will initiate the apoptotic pathway,affect the secretory function of intestinal Paneth cells and goblet cells,cause inflammation,apoptosis and intestinal mucosal barrier damage.ER autophagy plays a role in ER homeostasis.ER autophagy may protect the intestinal mucosal barrier by alleviating the intestinal epithelial cell apoptosis caused by ERS.Therefore,using ERS and ER autophagy as an entry point to study the damage mechanism of the intestinal mucosal barrier can provide some new ideas and methods for the treatment of IBS-D.According to the main clinical manifestations of IBS-D,it can be classified into the category of "diarrhea" and "abdominal pain" in traditional Chinese medicine.The syndrome of liver depression and spleen deficiency is the most common syndrome type in clinical practice.TXYF is a classic prescription for the clinical treatment of IBS-D with liver depression and spleen deficiency,which has achieved good curative effect in clinical practice.Then,what is the mechanism of TXYF to alleviate the symptoms of IBS-D?Whether it plays a role by regulating endoplasmic reticulum autophagy and stress,affecting the intestinal mucosal barrier,these issues are worthy of in-depth study.Part one:clinical studyObjectiveTo observe the efficacy and safety of TXYF in the treatment of patients with liver depression and spleen deficiency syndrome of IBS-D;to clarify the expression of serum biomarkers and endoplasmic reticulum stress marker protein GRP78 in patients with IBS-D,and the effect of TXYF on serum biomarkers Intervention.MethodsAdopt the exploratory test method of self-control study.Give TXYF granules 1 bag/time,2 times/day,4 weeks of treatment,and 4 weeks of follow-up.The IBS-SSS scale and the Bristol stool trait scale were used as the main efficacy indicators,and the SF-36,functional gastrointestinal disease PRO scale and TCM symptom score scale were used as secondary efficacy indicators for evaluation;serum before and after taking the medicine was collected The serum of healthy volunteers was used to detect the levels of zonulin,I-FABP,DAO,IL-6,IL-8,IL-10,and TNF-α in the serum using the Elisa method;collected IBS-D patients and healthy volunteers Intestinal mucosal tissue,pathological changes were observed by HE staining;GRP78 expression was observed by immunohistochemistry.ResultsIBS-SSS:there were significant differences in IBS-SSS scores at baseline,week2,week4 and week8(P<0.001).Bristol stool form scale:there were significant differences in the scores of baseline,week2,week4 and week8(P<0.001).SF-36 scale:the scores of PF,RP,GH,VT,RE,MH and HT of SF-36 scale in baseline,week2,week4 and week8 were statistically significant(P<0.05).There was no significant difference in the scores of BP and SF(P>0.05).Functional gastrointestinal disease PRO:the scores of dyspepsia,general condition,defecation,psychological and social function of functional gastrointestinal disease PRO scale in baseline,week2,week4 and week8 were statistically significant(P<0.05).There was no significant difference in reflux dimension(P>0.05).TCM symptom score:baseline,week2,week4 and follow-up period(week8)were statistically significant in the scores of lower abdominal pain,fatigue,tired and lazy,spontaneous perspiration and sleepy(P<0.05,P<0.01).The difference of the scores of other symptoms were not statistically significant(P>0.05).HE:healthy people’s colonic mucosa has a complete structure,with tight connections between epithelial cells,regular arrangement of glands,and no obvious edema,congestion,erosion and ulcers.In patients with IBS-D,the structure of the colonic mucosa is incomplete,with thickened mucosa,hyperplasia of glands,and dilated submucosal blood vessels.Serum Elisa test:After taking TXYF,compared with baseline,the patient’s serum zonulin,I-FABP,DAO,IL-6,IL-8,and TNF-α levels decreased(P<0.001),and IL-10 levels increased(P<0.001);But compared with the healthy control group,the differences in serum zonulin,I-FABP,DAO,IL-6,IL-8,TNF-α,and IL-10 levels were still statistically significant after taking the drug(P<0.001).Immunohistochemical:Compared with the healthy control group,the average optical density of GRP78 in the IBS-D patient group increased,and the difference was statistically significant(P<0.001).Conclusion1.TXYF has a good effect in treating patients with IBS-D with liver depression and spleen deficiency syndrome in terms of abdominal pain,abdominal distension and frequency,stool form,quality of life,and mental health.It can also improve the symptoms of TCM like lower abdominal pain,fatigue,tired and lazy,spontaneous perspiration,sleepy,hypochondriac pain,depression/nervous,insomnia,belching,lack of appetite,chilly sensation and abdominal distension.2.Pathological changes appear in the intestinal tissue of patients with IBS-D,the structure of the intestinal mucosa is incomplete,the mucosa is thickened,the glands are proliferated,and the submucosal blood vessels are dilated.The serum levels of intestinal permeability-related proteins zonulin,I-FABP,and DAO increased,and the intestinal permeability increased.Serum levels of IL-6,IL-8,TNF-α increased,and IL-10 levels decreased,indicating that there is low-grade inflammation in the intestinal tissues and tight junction damage.The above-mentioned situation can be improved after the intervention of TXYF.3.The expression of ERS marker protein GRP78 in intestinal tissue of IBS-D patients is increased,which indicates that ERS occurs continuously in intestinal tissue of IBS-D patients.Part two:In vivo experimentsObjectiveTo clarify the intervention effect of TXYF on IBS-D model rats with liver-stagnation and spleen-deficiency syndrome;and to explore the mechanism of TXYF in improving intestinal mucosal barrier and intestinal permeability from endoplasmic reticulum stress and endoplasmic reticulum autophagy.MethodsThe model of IBS-D rats with liver depression and spleen deficiency syndrome was established by the method of neonatal maternal separation(NMS)+4%acetic acid enema+chronic unpredictable psychological stress.TXYF was used to intervene for 14 days.The general condition,body weight,fecal water content and Abdominal withdrawal reflex(AWR),food intake,grasping,two-bottle choice,D-xylose absorb were measured.The establishment of the model and the efficacy of traditional Chinese medicine were evaluated.Immunohistochemical method was used to observe the expression of GRP78 in rat colon tissue;transmission electron microscope was used to observe the position and quantity of endoplasmic reticulum and autophagosomes in rat colon epithelial cells;immunofluorescence double-labeling method was used to observe autophagosomes and endothelial cells.Plasma reticulum co-localization;Western blot was used to detect the expression of GRP78,Claudin-4,Occludin,LC3,p62,Beclinl,E-Caderhin protein in rat colon tissue.ResultsIn the normal group,the hair color was bright,soft and clean,the mental state was normal,the activity was good,and the feces were jujube like.In the model group,the hair color was dark and dry,the mental state was poor,the activity was reduced,the feces were loose and the perianal stains were obvious.Compared with the normal group,the weight of rats in the model group decreased significantly(P<0.01);the fecal water content increased significantly(P<0.01);the AWR score increased significantly(P<0.05,P<0.01);the food intake decreased significantly(P<0.05,P<0.01);the grasping force was significantly reduced,the difference was statistically significant(P<0.001);the preference of sugar water was significantly reduced,the difference was statistically significant(P<0.001);the urine D-xylose absorb was significantly reduced,the difference was statistically significant(P<0.001).After 14 days of TXYF intervention,compared with the model group,the weight of rats in TXYF-L group increased,and the difference was statistically significant(P<0.05);the fecal water content and AWR score of TXYF-L,TXYF-M,TXYF-H group and rapamycin group were decreased;the intake of food,preference of sugar water and D-xylose absorb in urine of rats were increased;the difference was statistically significant(P<0.05);the grasping of TXYF-L,TXYF-M group and rapamycin group were increased,the difference was statistically significant(P<0.01)。The results of GRP78 immunohistochemistry showed that compared with the model group,the optical density of GRP78 in TXYF-L,TXYF-M group and rapamycin group was lower,and the difference was statistically significant(P<0.01,P<0.001).The number of autophagosomes was higher in TXYF-L group,TXYF-M group and rapamycin group than that in normal group,model group and 3-mA group;immunofluorescence double labeling results showed that compared with model group,TXYF-L,TXYF-M and rapamycin group had a higher number of autophagosomes.The expression of Atg8 increased and the colocalization with endoplasmic reticulum increased.Western blot results showed that compared with the model group,the expression of GRP78 in TXYF-L,TXYF-M,TXYF-H and rapamycin groups decreased,and the difference was not statistically significant(P>0.05).Compared with the model group,the expressions of claudin-4 protein,occludin protein,LC3-II/Iratio,beclin-1 and E-caderhin in TXYF-L,TXYF-M and TXYF-H groups and rapamycin group were up-regulated,but the differences were not statistically significant(P>0.05).The expression of p62 protein in TXYF-L,TXYF-M and TXYF-H groups and rapamycin group all decreased,but the difference was not statistically significant(P>0.05).Conclusion:1.The combination of disease and syndrome rat model established by the NMS+4%acetic acid enema+ chronic unpredictable psychological stress conforms to the characteristics of IBS-D and liver depression and spleen deficiency syndrome.It is an ideal model for the observation of therapeutic effect of Chinese herbal compound intervention and the study of disease syndrome mechanism.2.TXYF can improve the general state,weight,food intake,fecal water content,intestinal visceral sensitivity,intestinal permeability and fecal water content of rats.It also plays a role in improving the pathological manifestations of intestinal mucosa and alleviating mild inflammation.3.TXYF can regulate the level of ER autophagy,promote the production of autophagosomes and ER autophagy,relieve ERS,regulate ER homeostasis and cell homeostasis,inhibit cell apoptosis caused by ERS,enhance intestinal mucosal barrier,and reduce intestinal permeability.