Alleviation Effect And Mechanism Of Total Flavonoids In Seabuckthorn Leaves In Mice With DSS-induced Ulcerative Colitis

Ulcerative colitis is a complex multifactorial disease with unknown etiology.It is more common in western countries,but the incidence rate in China is gradually increasing,causing huge economic losses,reducing patients' quality of life and increasing the risk of colorectal cancer.At present,the therapeutic drugs of UC mainly play a role by suppressing inflammation and regulating immunity.Although its efficacy is certain,there are still many problems,including many adverse side effects and high price.Therefore,it is urgent to develop novel UC therapeutics.In recent years,traditional Chinese medicine treatment has shown its unique advantages due to its effectiveness,safety and low cost.Objective To investigate the anti-inflammatory effect and mechanisms of total flavone of seabuckthorn leaves on ulcerative colitis induced by Dextran Sulfate Sodium(DSS)in mice.Methods(1)In vivo experiments: 3.5%(w / v)DSS in drinking water was used to induce acute ulcerative colitis.Forty female BABL / C mice were randomly divided into 4 groups(10 mice each): control group(Ctrl);model group(DSS);total flavonoids of sea-buckthorn leaves + DSS group(Fla + DSS);mesalazine + DSS Group(Mes + DSS).The body weight,stool viscosity,and bleeding of each mouse were observed daily during the establishment of the DSS model.After the model was established,the mice were sacrificed,and the colorectal length of each group of mice was photographed.Severity of colitis was assessed using the Disease activity index(DAI)and colonic length.Real-time PCR technology was used to detect the proinflammatory cytokine(IL-1?,IL-6,TNF-a)m RNA levels in mouse colon tissue.The serum levels of proinflammatory cytokines(IL-1?,IL-6,TNF-a)in the serum of mice were measured by ELISA.Western blot was used to detect NLRP3,ASC,Caspase-1and IL-1? protein levels in colon tissues.The HE method was used to detect the degree of pathological damage in the colon of mice.(2)In vitro experients: After RAW264.7macrophages were pretreated with a gradient of total flavones of seabuckthorn leaves,lipopolysaccharide(LPS)and adenosine triphosphate(ATP)were used to establish a RAW264.7 macrophage inflammation model.Real-time PCR was used to detect the m RNA levels of proinflammatory cytokines(IL-1?,IL-6,TNF-a)in macrophages.The content of proinflammatory cytokines(IL-1?,IL-6,TNF-a)in cell was detected by ELISA.Western blot was used to detect the levels of p-IKBa,p-IKK? / ? and NLRP3 signaling pathway-related proteins in macrophages.Results 1.Total flavones(Fla)of seabuckthorn leaves improved the clinical symptoms andpathological damage of DSS-induced ulcerative colitis in mice.The results showed that,compared with the control group,the disease activity index of DSS group increased significantly,and the colon tissue was seriously damaged.However,compared with the DSS group,the disease activity index of total flavonoids +DSS group and mesalazine +DSS group were significantly reduced,which significantly reversed the DSS-induced shortening of colon length.At the same time,compared with the DSS group,the total flavonoids of seabuckthorn leaves and methalazine reduced the pathological damage caused by DSS.2.Total flavonoids of seabuckthorn leaves inhibited proinflammatory cytokines in serum and colon tissues of DSS-induced ulcerative colitis.After the completion of the modeling,the contents of IL-1? in the DSS group were significantly increased,and the contents of IL-1? in the DSS group were significantly decreased after the treatment of total flavonoids of seabuckthorn leaves,while the contents of TNF-a and IL-6 were not affected.In the colon tissues,compared with the DSS group,the total flavonoids of seabuckthorn leaves inhibited the m RNA level of IL-1?.3.Total flavonoids of seabuckthorn leaves inhibited the NLRP3 signaling pathway in the colon tissues.Compared with the control group,the expression of NLRP3 signaling pathway related proteins in the colon tissues of the DSS group was increased,and the NLRP3 level was significantly reduced by the treatment of total flavonoids of seabuckthorn leaves.4.Total flavonoids of seabuckthorn leaves inhibited the production of proinflammatory cytokines by RAW264.7 in macrophages induced by LPS + ATP.The results showed that different concentrations of total flavones of seabuckthorn leaves(6.4?g / m L)(12.8 ?g / m L,25.6 ?g /m L,51.2 ?g / m L)pretreatment can significantly inhibit the secretion levels and m RNA levels of TNF-a,IL-6 and IL-1? in the macrophage RAW264.7 caused by LPS + ATP.5.The total flavonoids of seabuckthorn leaves inhibit the NF-k B / NLRP3 signaling pathway of macrophage RAW264.7.Compared with LPS + ATP treatment alone,the pretreatment of total flavonoids in seabuckthorn leaves can significantly inhibit Ik Ba phosphorylation.Further study results showed that the pretreatment of total flavonoids of seabuckthorn leaves could significantly inhibit the expression of NLRP3 pathway related proteins in macrophages RAW264.7 induced by LPS+ATP.Conclusion The total flavonoids of seabuckthorn leaves may inhibit the clinical symptoms of DSS-induced ulcerative colitis mice by inhibiting the NLRP3 signaling pathway of the colon tissue of mice,inhibiting proinflammatory cytokines,and increasing the integrity of the intestinal mucosa.Further results showed that the inhibitory effect of total flavones of seabuckthorn leaves on NLRP3 was also verified in mouse macrophage RAW264.7 in vitro.