Study On Mechanism Of LiuJunAnWei Fomula In Preventing And Treating Chemotherapy-induced Diarrhea And Its Clinical Differention And Treatment

Objectives:1.To investigate the material basis and action targets of Liujunanwei Formula for the prevention and treatment of chemotherapy-associated diarrhea.2.To observe the protective effect of Liujunanwei Formula on the small intestinal mucosa of CID mice and to explore its efficacy mechanism based on apoptotic cells,inflammatory factors and the mechanical barrier of intestinal mucosa.3.This article reviews the syndrome differentiation rule of Liujun’an Wei fang in treating chemotherapy-related diarrhea.Methods:1.The chemical constituents of Liujun’an Weifang were identified by mass spectrometry,and the data were collected and analyzed by Analyst 1.6 software and Peak View 2.1.2.The potential targets of Liujun’an Weifang in the treatment of chemotherapy-related diarrhea were screened by network pharmacological method.The protein-protein interaction network was constructed and analyzed by GO and Kegg.The Drug-target-pathway model was constructed by using Cytoscape 3.6.0 software.3.60 SPF grade C57BL/6J male mice were randomly stratified by body mass into six groups.Except for the blank control group,the other 5 groups were injected intraperitoneally with 50mg/kg of fluorouracil injection for 7 consecutive days,and mice with decreased body mass and diarrhea were successfully modled.Fluorouracil was injected intraperitoneally 1 week before the start of the positive control drug montelukast, Liujunanwei-high,medium and low doses of gavage,the blank control group and the model group were gavaged with equal amounts of sterile water for14 consecutive days.Mice were observed daily for diarrhea during the intervention.On day 15,FITC-dextran 0.4 g/kg was gavaged,and 12 hours later,blood was removed from the eyes after anaesthesia.200ul of plasma was isolated and the fluorescence intensity of FITC was measured immediately using an enzyme marker.After dissecting the mice,the jejunum tissues were fixed in formaldehyde solution and frozen at-80 degrees Celsius in liquid nitrogen for the following tests:(1)Mucosal morphology observation:the fixed jejunum tissues were stained with HE to observe the mucosal morphological structures such as villi and crypts.(2)Intestinal mucosal permeability:Intestinal permeability was measured by measuring the fluorescence intensity of plasma FITC and calculating the concentration of FITC-D in plasma according to the standard curve.(3)Apoptotic cell assay:Immunohistochemical observation of bax and bcl-2 protein expression in mouse jejunal crypt structures.(4)Inflammatory factor assay:protein immunoblotting assay to detect iNOS and NF-κB protein expression in mouse jejunal tissue.(5)Tight junction protein assay:ZO-1,Occludin,claudin-1 protein expression in mouse jejunum tissues by immunofluorescence and protein immunoblotting.4.Sharing a typical case in the team’s clinical study.Results:1.Based on the high-resolution mass spectrometry technique,197 compounds were initially identified in this study from the extract of Liujunanwei Formula。2.Based on the network pharmacology,82 potential targets of Lioujunan Gastric Formula for the treatment of chemotherapy-associated diarrhea were screened.The target nodes of PIK3C and NOS2 are large and may be important targets of Lioujunan Gastric Formula for the treatment of chemotherapy-associated diarrhea,and PI3K-Akt signaling pathway and NF-kappa B signaling pathway may be important pathways of Lioujunan Gastric Formula for the treatment of chemotherapy-associated diarrhea.3.The results of animal experiments showed that:compared with the blank control group,the structural integrity of intestinal mucosa was significantly impaired in the model group(p<0.01);the plasma FITC-D concentration was significantly increased in the model group(p<0.01),the expression of bax was significantly increased,and the expression of bcl-2 was weakly positive and significantly decreased(p<0.01);the amount of ZO-1,Occludin,and claudin-1 protein amount was significantly decreased(p<0.01);inflammatory factors iNOS,NF-κB expression was significantly increased(p<0.01);compared with the model group,the high dose group of Liujunan stomach had high villi height and less crypt destruction(p<0.05);plasma FITC-D concentration in mice was reduced,the difference was not statistically significant(p>0.05);bax expression was significantly reduced and bcl-2 expression was significantly increased(p<0.05);ZO-1,Occludin,claudin-1 protein amounts were significantly increased;inflammatory factors iNOS,NF-κB expression was significantly reduced(p<0.01).4.The main pathogenesis of chemotherapy-associated diarrhea is:spleen deficiency as the basis and dampness and toxicity as the symptoms,a spleen and kidney yang deficiency is the key point of clinical diagnosis in the evolution of the disease from mild to severe.Liujunanwei formula is the main prescription,which has obvious curative effect.Conclusion:1.The highest proportion of flavonoids and terpenoids in Liugunanwei Formula are important chemical components,which are the specific pharmacological substance basis for its clinical action and are closely related to clinical efficacy.2.Using network pharmacology to explore the mechanism of action of Liugunanwei Formula to prevent chemotherapy-associated diarrhea,it can be found that the active ingredients of Liugunanwei Formula can act on different targets and affect multiple pathways,suggesting that Liugunanwei Formula exerts its effect on chemotherapy-associated diarrhea through the synergistic regulation of multiple ingredients,multiple targets and multiple pathways.3.Animal experiments have shown that Liugunanwei Formula can improve intestinal permeability,reduce apoptosis in mice intestinal tissues,improve the inflammatory response of intestinal tissues and repair the mechanical barrier of intestinal mucosa,which has a significant improvement effect on chemotherapy-related diarrhea.4.Through the sharing of cases,we can sort out the identification characteristics of chemotherapy-related diarrhea and the clinical diagnosis and treatment ideas of the combination of disease and evidence,so as to provide a basis for the better application of Liugunanwei Formula in clinical treatment.