Protective Effects Of Nuciferine On Anti-inflammatory Of In Vitro And In Vivo And DSS Induced Colitis In Mice

Lotus leaf is mainly used to treat symptoms such as splenasthenic diarrhea,summer humidity alvine flux,heat polydipsia and hematochezia.Nuciferine is an isoquinoline alkaloid extracted from lotus leaf and has a wide range of pharmacological activities,such as anti-cancer,anti-inflammation,anti-oxidation,reducing fat and weight,anti-virus,promoting immunity,maintaining the steady state of intestinal flora,etc.Therefore,the aim of this study was to investigate the effect of nuciferine on alleviating colitis induced by dextran sodium sulfate(DSS)in mice.The results would provide the basis for the research and development of new enteritis drugs and the basic mechanism of Chinese medicine.Firstly,the inflammation model of RAW264.7 cell was induced by lipopolysaccharide.The content of NO,TNF-αand IL-6 were tested for verifying the anti-inflammatory effect of lotus leaf alkaloid in vitro.Secondly,animal test was followed.The male BALB/C mice were randomly divided into 5 groups with 10 mice in each group:Control group(Control),high-dose treatment group(H-Nuc+DSS),low-dose treatment group(L-Nuc+DSS),DSS Group(DSS)and nuciferine control group(Nuc).Mice enteritis model was induced by 5%DSS for 7 days.The mice in the H-Nuc+DSS and L-Nuc+DSS groups were intraperitoneally injected with 20 mg/m L and 10 mg/m L of Neferine for 7 days.The changes of body weight of mice during model construction and treatment were recorded.After the treatment,the mice were killed and the length of colon was measured for evaluating the disease activity index(DAI).The pathological section of colon was made and observed.The proliferation of cell in spleen and mesenteric lymph nodes was measured by MTT method.The number of CD3~+,CD4~+and CD8~+cells,Th1,Th2,Th17 and Treg in the spleen,B220 and Ig A in mesenteric lymph nodes were measured by Flow cytometry.The intestinal microflora were analyzed by 16S r RNA sequencing.The results of the study were as follows:1.The results of cell culture in vitro showed that the content of NO,TNF-αand IL-6 were significantly increased induced by LPS(P<0.05).After intervention by nuciferine for 24 hours,different concentrations of nuciferine could reverse the situation to some extent compared with the control group.The results showed that lotus leaf alkaloid had a good anti-inflammatory effect on lipopolysaccharide-induced macrophage inflammation model,compared with LPS group,2.Treatment with high-dose and low-dose nuciferine could alleviate inflammatory bowel disease induced by DSS,including the overall state of diarrhea mice were significantly improved,the value of DAI was reduced,and the pathological characteristics were ameliorated.It was also found that nuciferine could promote T cell proliferation in spleen and mesenteric lymph nodes.The Flow cytometry results showed that nuciferine enhanced the levels of B220 and Ig A cells and regulated homeostasis of CD4~+/CD8~+,Th1/Th2,Th17/Treg in DSS-induced intestinal inflammation.3.According to the analysis of 16S r RNA sequencing,it was found that the abundance of Firmicutes and Actinobacteria decreased and the abundance of Bacteroidtes increased in DSS group at the phylum level.Compared with the control group,the abundance of Firmicutes and Actinobacteria tended to normal after the treatment with different concentration of nuciferine.At the genus level,the number of lachnospira,Bilophila,Halomonas were increased and the number of Bacteroides,Parabacteroides,Parasutterella were decreased in the DSS group,and the trend was reversed after treatment with high-dose and low-dose nuciferine.In conclusion,nuciferine could significantly ameliorate the intestinal inflammatory reaction induced by DSS in mice.This effect may be related to regulating intestinal flora and promoting the balance of Th17/Treg and Th1/Th2 immune axis.