| Objective: The objective of this study is to investigate the pharmacodynamic substances and mechanism of fritillaria bulb alkaloids in ameliorating ulcerative colitis.Methods: 1.Effect of fritillaria bulb total alkaloids on ameliorating ulcerative colitis:The impact of fritillaria bulb total alkaloids(8 mg/Kg)on the DSS-induced ulcerative colitis model in mice was evaluated through measurements of body weight,disease activity index,colonic length,and intestinal histopathological scores.Additionally,the composition and diversity of microorganisms in the colon were analyzed using 16 S r RNA sequencing technology.2.Research on the efficacy of fritillaria bulb main alkaloids in ameliorating ulcerative colitis: The effects of different dosages of fritillaria bulb main alkaloids(Peimine 2 mg/Kg,6 mg/Kg;Peiminine 0.25 mg/Kg,0.75 mg/Kg;Peimisine 5 mg/Kg,15 mg/Kg;Sipeimine 3mg/Kg,9 mg/Kg)on the DSS-induced ulcerative colitis model in mice were assessed by evaluating body weight,disease activity index,colonic length,and intestinal histopathological scores.Additionally,the impact of each alkaloid on the Jak-Stat signaling pathway was evaluated by Western-blot and Immunohistochemistry.3.Research on the mechanism of Peimisine in ameliorating ulcerative colitis: The effects of Peimisine on the DSS-induced ulcerative colitis model in mice were assessed by evaluating body weight,disease activity index,colonic length,intestinal histopathological scores,and the levels of inflammatory factors(MCP-1,TNF-α,IL-1β,IL-6,IFN-γ and LPS)in serum.The impact of Peimisine on the Jak-Stat signaling pathway was evaluated using Western-blot and Immunohistochemistry.Additionally,the levels of macrophages,M1 macrophages,Th 17 cells and Treg cells in intestinal tissue were assessed through Westernblot and Immunofluorescence.Furthermore,the composition and diversity of microorganisms in the colon were analyzed by using 16 S r RNA sequencing technology.4.Research on the effect and mechanism of Peimisine fecal microbiota transplantation in ameliorating ulcerative colitis: Ulcerative colitis mouse model was induced by DSS after four antibiotics cleared intestinal flora,then treated the model mouse with fecal bacteria supernatant(0.1 m L/20 g)or peiminae(15 mg/Kg).Weight change and disease activity index were monitored,colon length was measured,and pathological damage to intestinal tissue was evaluated by using H&E staining.Microbial composition and diversity in colon contents were analyzed by using 16 S r RNA sequencing technology.Serum metabolite changes were detected by metabolomics,gene transcription changes in colon tissues were detected by transcriptomics,and the levels of IL-17 A,ACT1,TRAF6 and c/EBP-β in mouse colon tissues were detected by Western-blot.Results: 1.Fritillaria bulb total alkaloids significantly improved symptoms in a DSSinduced ulcerative colitis model in mice.This was demonstrated by reducing in weight change rate,disease activity index,colonic length shortening,intestinal histopathological scores,and alleviating of gut microflora dysbiosis,improving of potential beneficial bacteria,reducing of potentially harmful bacteria.2.All four alkaloids demonstrated the ability to ameliorate symptoms in a DSS-induced ulcerative colitis model in mice,as evidenced by reducing weight change rate,disease active index,colonic length shortening,and intestinal histopathological scores.Of note,the high dose of Peimisine showed the most effective results in symptoms and intestinal tissue injury of the DSS-induced ulcerative colitis model mouse,while even the low dose was also effective.Stat1/3 and p-Stat1/3 play crucial roles in the Jak-Stat signaling pathway.They can induce inflammatory cytokines secretion,affect macrophage polarization and T cell differentiation.Immunohistochemistry and Western-blot results revealed that Peimisine significantly inhibited the expressions of Stat1,p-Stat1(Tyr701),Stat3,p-Stat3(Tyr705)and p-Stat3(Ser727)in colonic tissues.These results preliminarily determined that Peimisine maybe the primary pharmacodynamic substance responsible for ameliorating ulcerative colitis in fritillaria bulb alkaloids and achieves this effect through regulating the Jak-Stat signaling pathway.3.Peimisine significantly ameliorated the symptoms of DSS-induced ulcerative colitis in a mouse model.This was evidenced by reducing weight change rate,disease activity index,colonic length shortening,and intestinal histopathological scores.Peimisine ameliorated the symptoms of colitis mice by inhibiting the Jak-Stat signaling pathway,reversing gut microbiota alterations,suppressing Macrophage M1 polarization,maintaining the Th17/Treg cell balance,and reducing sustained inflammatory cytokines-related inflammatory injury.4.Peimisine fecal microbiota transplantation has a significant alleviating effect on ulcerative colitis mouse model,specifically reducing the rate of weight change,decreasing the active disease index score,suppressing the length of the colon shorten and reducing the histopathological score of the intestinal tissue.The dominant colonizing strain of Peimisine fecal microbiota transplantation is Lachnospiraceae＿NK4A136＿group.The mechanism of Peimisine fecal microbiota transplantation alleviating ulcerative colitis may be that Lachnospiraceae＿NK4A136＿group participates in amino acid metabolism,produces shortchain fatty acids,inhibits the levels of IL-17 A,ACT1,TRAF6,and c/EBPβ in the IL-17 signaling pathway,reduces the expression of pro-inflammatory factors,and alleviates inflammation.Conclusion: In this study,we selected the fritillaria bulb,a renowned medicinal plant from Jilin Province,as our research subject.Our findings indicate that the total alkaloids of fritillaria bulb have a significant effect on ameliorating ulcerative colitis.Peimisine was identified as the main pharmacodynamic substance.Peimisine exerts its effects by inhibiting the Jak–Stat signaling pathway,suppressing Macrophage M1 polarization,maintaining the Th17/Treg cell balance,reversing gut microbiota alterations,and reducing sustained inflammatory cytokines-related inflammatory injury.The dominant colonization strain after Peimisine fecal microbiota transplantation was Lachnospiraceae＿NK4A136＿group,which could inhibit IL-17 signaling pathway,reduce the expression of pro-inflammatory factors,and alleviate inflammation by participating in amino acid metabolism to produce shortchain fatty acids. |
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