Study Of Biological Basic Of Ulcerative Colitis With Spleen Qi Deficiency Based On Metabolism And Preliminary Study On Mechanism Of Shenling Baizhu San

Background and purposeThe study of TCM syndrome is a bridge between clinical and basic theory,and the study of TCM syndrome is helpful to provide a scientific basis for the objectification of TCM syndrome.Spleen-qi deficiency syndrome is one of the common basic syndromes of ulcerative colitis(UC).This study explored the biological basis of UC with spleen-qi deficiency syndrome based on metabolism and explored the mechanism of Shenling Baizhu San on UC with spleen-qi deficiency.MethodClinical researchThe patients with UC who were admitted to the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine were evaluated based on the "Alternative Diagnosis Items of Basic Symptoms of Ulcerative Colitis" which was obtained from the previous research.The clinical manifestations of spleen-qi deficiency syndrome were explored.31 UC patients with spleen-qi deficiency syndrome,30 patients with damp-heat syndrome,and 27 healthy controls were included and their serum was collected for untargeted metabolomics.Animal experiment30 male C57BL/6 mice,6-8 weeks old,clean grade,were randomly divided into blank group(Ctrl group),UC group(DSS group),spleen-qi deficiency group(SD group),UC with spleen-qi deficiency group(DSS+SD group)and Shenling Baizhu San group(SLS group).The models of UC were prepared by feeding Dextran sulphate sodium(DSS)solution.The models of UC with spleen-qi deficiency were prepared by combining DSS solution with eating disorders and exhausted swimming.The change in body weight and length of the colon were recorded and the disease activity index was evaluated.The colonic histopathological changes were observed using H&E staining;the relative expression of mRNA of inflammatory factors was detected using the qRT-PCR method.In addition,the weight of the spleen,spleen index,the weight of thymus,and thymus index of each mouse were recorded.18 male C57BL/6 mice aged 6-8 weeks were randomly divided into Ctrl group,DSS+SD group,and SLS group.The serum metabolites of mice among three groups were detected using untargeted metabolomics.The Na+-K+-ATPase and Ca+-Mg+-ATPase activity was detected using a spectrophotometer;the copy number of mitochondrial DNA of colon tissue was detected using qRT-PCR method;the relative expression of SIRT1,PGC-1α and TFAM were detected by qRT-PCR and Western blot method;the relative expression of ZO-1 and Mucin-2 were detected using immunohistochemical;the secretion of goblet cells and mucus layer in colon tissue was observed using Alcian blue/Periodic acid Schiff staining.ResultsClinical researchA total of 236 patients with ulcerative colitis were included in this study,including 131 males and 105 females,with a median age of 40.5 years old.The median of body mass index was 21.22 and the median of disease duration was 3 years.According to the Montreal classification,58 patients had ulcerative proctitis,72 patients had left-sided UC and 106 patients had extensive UC.The common symptoms of UC patients with spleen-qi deficiency syndrome included indigestion,incomplete defecation,diarrhea,sticky stools,tenesmus,bloody stools,diarrhea due to abdominal pain,abdominal pain,and fatigue.The results of untargeted metabolomics analysis showed that compared with the healthy control,the average abundance of 1-Linoleoylglycerophosphocholine,Lysophospholipid,Sphinganine,N-palmitoyl-phosphoethanolamine,O-Arachidonoyl Glycidol,2-Linoleoyl Glycerol upregulated,while the average abundance of Docosatrienoic Acid and Palmitic amide downregulated in UC patients with spleen-qi deficiency.Compared with UC with damp-heat syndrome,the average abundance of the above-mentioned metabolites in UC patients with spleen-qi deficiency syndrome significantly reduced except for 2-Linoleoyl Glycerol increased.Animal experimentThe UC mice with spleen-qi deficiency syndrome showed some symptoms such as reduced food intake,weight loss,dry and yellow hair,and unresponsiveness,as well as spleen weight,spleen index,thymus weight,and thymus index were significantly reduced compared with Ctrl group,which was in line with the performance of spleen-qi deficiency.In addition,the length of colon of the UC mice with spleen-qi deficiency was found to be shortened,inflammatory infiltration was observed in the colon tissue,and the relative expression of IL-1β,TNF-α,and IL6 was increased compared with the Ctrl group,which indicated that the model of UC with spleen-qi deficiency syndrome had been successfully prepared.Compared with the DSS+SD group,the SLS group showed elevated spleen weight,spleen index,thymus weight and thymus index,which suggested that Shenling Baizhu San can effectively alleviate spleen-qi deficiency syndrome of UC.Compared with the DSS+SD group,there were longer colons,reduced inflammatory infiltration of the colon tissue and decreased relative expression of IL-1β,TNF-α,IL-6 mRNA in SLS group,which indicated that Shenling Baizhu San can effectively alleviate the intestinal inflammation in UC mice with spleen-qi deficiency syndrome.The results of untargeted metabolomics showed that the average abundances of 1-Linoleoylglycerophosphocholine,Lysophospholipid,Sphinganine,O-Arachidonoyl Glycidol increased in the DSS+SD group compared with the Ctrl group.After Shenling Baizhu San intervention,the average abundance of the above-mentioned metabolites showed downregulation in the SLS group.Compared with the Ctrl group,the activity of Na+-K+-ATPase and Ca+-Mg+-ATPase of the DSS+SD group significantly reduced;compared with the DSS+SD group,both activity of Na+-K+-ATPase and Ca+-Mg+-ATPase in the SLS group increased significantly.The copy number of mtDNA showed the same tendency as ATPase.Compared with the Ctrl group,the relative expressions of PGC-1α,SIRT1,and TFAM mRNA and protein in the DSS+SD group were significantly reduced.After the intervention of Shenling Baizhu San,the relative expressions of PGC-1α,SIRT1,TFAM mRNA and protein increased significantly in the SLS group,which suggested that the mice of DSS+SD group had dysfunction of mitochondrial biogenesis and Shenling Baizhu San could promote mitochondrial biogenesis in UC mice with spleen-qi deficiency syndrome.Compared with the Ctrl group,the positive expression of ZO-1 and Mucin-2 in DSS+SD group showed a significant reduction,as well as the intestinal mucus layer was thinner and the number of goblet cells decreased,which indicated there were intestinal barrier damage in the UC with spleen-qi deficiency syndrome model.Compared with the DSS+SD group,the positive expression of ZO-1 and Mucin-2 increased,the goblet cell count upregulated and the mucus layer became thicker in SLS group.It was suggested that Shenling Baizhu San could alleviate the intestinal barrier damage and promote mucosal healing of UC with spleen-qi deficiency model.Conclusions1.The main clinical manifestations of UC with spleen-qi deficiency included:indigestion,incomplete defecation,diarrhea,sticky stool,dull abdominal pain,and fatigue.2.Compared with the healthy control group,there were lipid metabolism disorders in the UC patients with spleen-qi deficiency and the average abundances of 1-Linoleoylglycerophosphocholine,Ly sophospholipid,Sphinganine,N-palmitoyl-phosphoethanolamine,and O-Arachidonoyl Glycidol showed significantly altered.O-Arachidonoyl Glycidol,2-Linoleoyl Glycerol,and Palmitic amide were the potential biomarkers of UC with spleen-qi deficiency.3.The combination of DSS,eating disorder,and exhausted swimming was an effective method to prepare the UC model with spleen-qi deficiency syndrome.4.Shenling Baizhu San could alleviate intestinal inflammation and lipid metabolism disorders inUC mice with spleen-qi deficiency.5.The mechanism of Shenling Baizhu San on UC with spleen-qi deficiency may be related to regulating the SIRT1/PGC-1α pathway to promote mitochondrial biogenesis and to protect the intestinal barrier.