| ObjectiveIrritable Bowel Syndrome(IBS-D)is one of the most common functional gastrointestinal diseases,and its underlying molecular mechanisms are not yet clear.Jianpi Qushi Paste is an empirical formula used in clinical practice to treat diarrhea with liver depression and spleen deficiency,with significant clinical effects.Traditional Chinese medicine believes that liver depression and spleen deficiency are the main cause of IBS-D.Therefore,this experiment first improves the quality control methods of Jianpi Qushi Paste,providing an experimental basis for the establishment of quality standards for Jianpi Qushi Paste.Subsequently,by establishing a model of diarrhea-type irritable bowel syndrome with liver stagnation and spleen deficiency,the therapeutic effect and mechanism of Jianpi Qushi Paste on IBS-D model rats were preliminarily explored,and its safety was preliminarily evaluated,providing an experimental basis for the clinical application of Jianpi Qushi Paste.Methods1.According to the methods of Chinese Pharmacopoeia and based on thin-layer chromatography,Puerariae Radix,Coptis chinensis Franch.,RCorydalis Rhizoma,and Caulis Polygoni Multiflori in Jianpi Qushi Paste were identified.Based on the principle of high performance liquid chromatography,the content of puerarin in the paste was determined with C18 column as the chromatographic column,methanol-water(18:82)as the mobile phase,column temperature of 40 ℃,flow rate of 1.0 ml/min,and detection wavelength of 250 nm.2.The model rats of IBS-D rats of liver depression and spleen-deficiency type were reproduced regarding the three-factor method(maternal separation +acetic acid stimulation+restraint stress).The rats were randomly divided into the blank group,model group,and Jianpi Qushi Paste group,which were administered by gavage for 14 days.The model was evaluated by recording the rats’ body weight,stool Bristol typing score,measuring the stool water content,sugar water preference value,and intestinal pain threshold,and the effect of Jianpi Qushi Paste on the treatment of IBS-D model rats was preliminarily evaluated;The safety of Jianpi Qushi Paste was preliminarily evaluated by measuring organ index and observing HE staining pathological sections.3.The model rats of IBS-D rats of liver depression and spleen-deficiency type were reproduced regarding the three-factor method(maternal separation +acetic acid stimulation+restraint stress).The rats were randomly divided into blank group,model group,Bu Pi Yi Chang Wan group,Pivium Bromide group,and Jianpi Qushi Paste low and high dose groups.After modeling and 14 days of continuous gavage administration,the fecal water content,intestinal pain threshold,and sugar-water preference were measured in each group,and then the rats were sacrificed.Determination of 5-hydroxytryptamine(5-HT),interleukin-1(IL-1β),interferon-β(IFN-β)Protein levels in the colon of rats in each group were determined by ELISA.5-HT1 A receptor,5-HT2 A receptor,and tumor necrosis factorα(TNF-α)mRNA expression in the colon were determined by q-PCR;The expression levels of mRNA and proteins of Zonula Occludens(ZO-1),Claudin-1and the mRNA and proteins related to cyclic GMP-AMP synthase/STING(cGAS /STING)pathway in the colon tissue of rats in each group were determined by q-PCR and Western Blot.Results1.The TLC identification method established by our research can effectively identify Puerariae Radix,Coptis chinensis Franch.,RCorydalis Rhizoma,and Caulis Polygoni Multiflori in Jianpi Qushi Paste which has clear spots,without negative results interference.According to the HPLC method we established,in the range of 22.526 μg/ml to 225.260μg/ml,the injection volume of puerarin reference substance had a good linear relationship with the peak area,the average content of the puerarin in three batches of samples is 2.640mg/g.2.After modeling,the rats’ body weight,sugar preference value,and intestinal pain threshold were lower than those of the blank group(P<0.05).After 14 days of continuous gavage of Jianpi Qushi Paste with a clinical equivalent dose,compared with the model group,the body weight of rats in the Jianpi Qushi Paste group increased(P<0.05 for male rats,P<0.05 for female rats),but there were still differences compared with the blank group(P<0.05 for male rats,P<0.05 for female rats),and the score of stool Bristol typing,the stool moisture content decreased(P<0.05),the threshold of intestinal pain and the preference value of sugar water increased(P<0.05).The safety of the drug was evaluated by organ coefficient and HE pathological section.The main organ structure of rats in each group was normal,and there was no generative pathological change.Compared with the blank group,the liver coefficient,thymus coefficient and spleen coefficient of the model group were significantly increased(P<0.05),but there was no significant difference(P=0.243).Compared with the model group,the liver coefficient of rats in the Jianpi Qushi Paste group decreased(P<0.05),and the thymus coefficient had a downward trend,but there was no significant difference(P>0.05).Compared with the model group,the kidney coefficient of the paste group decreased(P<0.05),but there was no significant difference compared with the blank group(P>0.05).3.Compared with the male rats in the model group,the high and low dose of Jianpi Qushi Paste can increase the body weight of male rats(P<0.05),which is equivalent to the positive drug group(P>0.05);Compared with the body weight of female rats in the model group,the body mass of female rats in each treatment group has an upward trend,but there is no significant difference(P>0.05).Compared with the model group,the water content of stool in the Jianpi Qushi Paste group decreased(P<0.05),the threshold of intestinal pain increased(P<0.05),and the sugar-water preference value in the low-dose group increased(P<0.05).The water content of stool in the high-dose group of the Jianpi Qushi Paste group was significantly different from that in the Bu Pi Yi Chang Wan(P<0.05),and the sugar-water preference value was not different from that in the model group(P>0.05),and was significantly different from that in the blank group(P<0.05).Compared with the blank group,the protein content of rat colon 5-HT and IL-1β in the model group increased(P<0.05).Compared with the model group,the level of 5-HT in the colon of the high and low-dose groups of Jianpi Qushi Paste decreased significantly(P<0.05).The content of colon IL-1β was significantly lower in the high-dose group of the paste than in the model group(P<0.05),but there is no difference when comparing the colon IL-1β in the low-dose group of the paste to the model group(P>0.05)and the blank group(P>0.05).Compared with the blank group rats,the expression of the colon TNF-α,5-HT1 A receptor,and 5-HT2 A receptor mRNA of model rats increased(P<0.05).Compared with the model group rats,the expression of the high and low-dose group TNF-α,5-HT1 A receptor,and 5-HT2 A receptor mRNA decreased(P<0.05).Compared with the model group,the expression of ZO-1 and Claudin-1 mRNA and protein in the Jianpi Qushi Paste group increased(P<0.05).The expression of protein level IFN-β,cGAS,STING mRNA,STING,TBK1,p-TBK1 protein increased in the model group(P<0.05)and decreased in the high and low-dose groups of the Jianpi Qushi Paste(P<0.05).Conclusion1.The TLC and HPLC identification methods we set up are simple,accurate,and specific,and can be used as the preliminary quality control method of Jianpi Qushi Paste.2.The clinical equivalent dose of Jianpi Qushi Paste has a therapeutic effect on IBS-D model rats with high safety.However,considering the changes in the kidney coefficient of rats in the Jianpi Qushi Paste group,it is recommended that clinical use should be combined with the patient’s disease status.3.The therapeutic effect of the clinically equivalent dose of Ji anpi Qushi Paste on IBS-D model rats is better than that of the clinically equivalent dose of Jianpi Qushi Paste and is equivalent to that of Jian Pi Yi Chang Wan,which is a positive Chinese medicine in this study.Its mechanism may be related to the regulation of 5-HT,tight junction protein,and cGAS/STING pathway. |
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