| Objective: To take Geranium strictipes R.Knuth as a research object,clarify its active site.To observe the effects of Geranium strictipes R.Knuth on pathological changes,inflammatory factors,TLR4/NF-κB and Nrf2/HO-1 signaling pathway by establishing a model of acute ulcerative colitis in rats and explore the prevention and therapy mechanism of G.strictipes on ulcerative colitis rats.Methods: 1.A UV-visible spectrophotometric method was established for the determination of tannins,flavonoids,polysaccharides,and alkaloids in the several polar extracts of Geranium strictipes R.Knuth.2.The HPLC spectrums of different parts of Geranium strictipes R.Knuth were established.The anti-inflammatory activities of different parts of Geranium strictipes R.Knuth were determined by xylene-induced mice ear swelling method.The spectral-effect correlation between HPLC characteristic peaks of different parts and their anti-inflammatory effects were analyzed by bivariate correlation analysis and partial least squares regression analysis.3.Dextran sulphate sodium(DSS)was used to induce acuteulcerative colitis(UC)in SD rats through 2 methods of free drinking and intragastric administration.The general condition of rats,disease activity index score(DAI),colon length,colon tissue gross score and pathology score were used to evaluate the results of colon morphology and pathology of UC rats.Biochemical detection was used to detect the activity of MPO in serum of UC rats.MDA content,SOD and GSH-Px activities in colon tissue.4.The rat model with acute UC was induced by free drinking of DSS.48 rats were randomly divided into normal group,model group,EAEG groups(low,medium,high)and sulfasalazine(SASP)group.Each group of 8 rats was used to detect inflammation in rat colon tissues by observing the general conditions of rats,disease activity index score(DAI),colon length,colon tissue gross score,pathological score,peripheral blood cell content,and enzyme-linked immunosorbent assay(ELISA)to detect factor(TNF-α,IL-1β,IL-6,IL-10)contents for evaluating the preventive effect of EAEG on UC rats.5.Establishment of rat acute UC model was through free drinking of DSS.48 rats were randomly divided into normal group,model group,EAEG groups(low,medium,high)and sulfasalazine(SASP)group.Biochemical assay was used to detect MDA content,SOD and GSH-Px activities in rat colon tissues,and RT-qPCR was used to detect related cytokines for TLR4,NF-κB p65,TNF-α,IL-6 and INF-γ mRNA expression levels in the TLR4/NF-κB signaling pathway,Nrf2 and HO-1 mRNA expression levels in the Nrf2/HO-1 signaling pathway.Western Blot method was used to detect TLR4,P-NF-κB p65,NF-κB p65 and IL-6 protein expression levels to investigate the mechanism of Geranium strictipes R.Knuth to prevent and treat UC rats.Results: 1.The total ethanol extracts and different polar parts in Geranium strictipes R.Knuth mainly contains tannins,flavones,polysaccharides and alkaloids.Among which the tannins in ethyl acetate was higher(57.52%),and the flavonoids in ethanol was higher(44.33%).The content of polysaccharides in water part was higher(78.48%),and the content of alkaloid in chloroform part was higher(1.04%).2.Different parts of Geranium strictipes R.Knuth all exhibited anti-inflammatory effects in which the ethyl acetate part worked best.The 27 characteristic chromatographic peaks in the HPLC spectrums were selected,and 13 peaks were significantly correlated with the anti-inflammatory rate,the peaks ranked as No.1(gallic acid),2(methyl gallate),3(catechin),8,11,13,15,17,19(pentagalloyl glucose),20,21,25 and 27.3.Two modeling methods can create acute UC model.Among them,The free-drinking group DAI score was significantly increased(P<0.01),colon length was significantly shorter(P<0.05),larger colonic tissue score and pathology score were significantly increased(P<0.01),the contents of WBC,RBC and LY cells of peripheral blood cells were significantly increased(P<0.05),MPO activity of serum was significantly increased(P<0.01),MDA content of colon tissue was significantly increased(P<0.05),the activities of SOD and GSH-Px were significantly decreased(P<0.05)compared with normal group;And the colonic tissue showed severe inflammation compared with gastric perfusion group.DAI score,colon histopathological score,MPO activity,MDA content and GSH-Px activity were significantly different compared with gastric perfusion group,and these results were more similar to Cooper modeling standards.4.Compared with the normal group,the DAI score in the model group was significantly increased(P<0.01),the colon length was significantly shortened(P<0.01),the gross score of the colon tissue and the pathological score were significantly increased(P<0.01),and the peripheral blood cells were significantly increased.The contents of WBC,LY and GR increased significantly(P<0.05),the activity of MPO in serum increased significantly(P<0.01),and the levels of TNF-α,IL-1β,and IL-6 in colon tissues increased(P<0.05).and IL-10 content decreased(P<0.01).After drug intervention,all indicators of each drug group were improved to some extent.5.Compared with the normal group,the related cytokines TLR4,NFκB p65,TNF-α,IL-6 and INF-γ mRNA expression levels in the TLR4/NF-κB signaling pathway were significantly increased of the model group(P<0.01),IL-10 mRNA expression levels were significantly reduced(P<0.01),TLR4、NFκB p-p65/p65 and IL-6 protein expression levels were significantly increased(P<0.01).And the MDA content in the intestine of the model group increased(P<0.05),the activity of SOD and GSH-Px were significantly decreased(P<0.01).The Nrf2,HO-1 mRNA expression levels in the Nrf2/HO-1 signaling pathway were significantly reduced(P<0.01).After drug intervention,all indicators of each drug group were improved to some extent.Conclusion: 1.The content determination method established in this study is simple and accurate,which lays the foundation for the comprehensive quality evaluation of Geranium strictipes R.Knuth.2.The anti-inflammatory activity of the ethyl acetate site of Geranium strictipes R.Knuth isstrongest,which might bethe result of the coaction of multiple components.3.The rat acute UC induced by free drinking 5% DSS for 7 days is an ideal UC model,which might be served for investigation of the drug treatment and pathogenesis effect of acute UC.4.EAEG can prevent and treat ulcerative colitis in rats caused by DSS,and can alleviatein flammation,ulcers and edema in UC rats,and reduce colon tissue damage.5.The mechanism of EAEG on UC rats may be related to the inhibition of TLR4/NF-κB signaling path way and the activation of Nrf2/HO-1 signaling pathway. |
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