尖叶假龙胆不同组分对酒精性肝损伤模型大鼠的保护作用研究

Study on theprotective effects of different components of gentianella acuta on alcoholic liver disease model rats

目的研究尖叶假龙胆不同组分拮抗酒精性肝损伤(alcoholic liver disease,ALD)效果并分析其机理,确定尖叶假龙胆治疗ALD的最佳疗效组分。方法采用渗漉法对尖叶假龙胆水、30%、60%和90%乙醇提取液的不同极性部位进行分离富集。健康雄性SD大鼠随机分为空白组、模型组、阳性药组、全草组、水洗脱组、30%乙醇洗脱组、60%乙醇洗脱组和90%乙醇洗脱组8组。实验组大鼠给予梯度酒精灌胃,建立慢性ALD模型,空白组给予等量生理盐水8周。从第5周起,以尖叶假龙胆水煎液及渗漉法水洗脱部位及30%、60%、90%乙醇洗脱部位对大鼠进行灌胃,阳性药对照组给予等量复方蛋氨酸胆碱片灌胃,第8周后取材。肝脏称重计算肝指数;电镜观察肝组织病理变化;酶联免疫吸附测定法检测血清中谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)、甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、白介素6(interleukin-6,IL-6)、白介素8(interleukin-8,IL-8)、白介素10(interleukin-10,IL-10)、转化生长因子-β1(transforming growth factor-β,TGF-β1)及肝组织匀浆中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)、乙醇脱氢酶(alcohol dehydrogenase,ADH)、乙醛脱氢酶(acetaldehyde dehydrogenase,ALDH)含量;免疫组化法检测细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白表达。结果(1)相较模型组,全草组ALT、AST、TG、TC、IL-6、IL-8、TGF-β1、MDA含量和ICAM-1、TNF-α蛋白表达降低,IL-10、SOD、GSH、ADH、ALDH含量升高,肝脏指数降低,均具有显著差异(P<0.05)。(2)相较模型组,水洗脱组TG、ALT、IL-6、TGF-β1和MDA含量降低,ADH含量升高,均具有显著差异(P<0.05),其余指标无明显差异(P>0.05)。(3)相较模型组,30%乙醇洗脱组TG、ALT、IL-6、TGF-β1和MDA含量降低,GSH、ADH、ALDH含量升高,均具有显著差异(P<0.05),其余指标无明显差异(P>0.05)。(4)相较模型组,60%乙醇洗脱组ALT、AST、TG、TC、IL-6、IL-8、TGF-β1、MDA含量以及ICAM-1、TNF-α蛋白表达降低,IL-10、SOD、GSH、ADH、ALDH含量升高,肝脏指数降低,均具有显著差异(P<0.05)。(5)相较模型组,90%乙醇洗脱组ALT、AST、TG、TC、IL-6、IL-8、TGF-β1、MDA含量以及ICAM-1、TNF-α蛋白表达降低,IL-10、SOD、GSH、ADH和ALDH含量升高,肝脏指数降低,均具有显著差异(P<0.05)。(6)在降低TG、MDA和升高ADH方面全草组与90%乙醇洗脱组具有显著差异(P<0.05),在降低TG、MDA、IL-6和升高ADH、ALDH方面60%乙醇洗脱组与90%乙醇洗脱组具有显著差异(P<0.05)。结论尖叶假龙胆组分具有拮抗ALD效果,不同组分作用效果有所差异,其中最优组分为90%乙醇洗脱组,其拮抗ALD机制可能是通过调节脂代谢系统,平衡氧化—抗氧化系统,提高酒精代谢率,抑制促炎细胞因子的生成,促进抗炎细胞因子生成以及抑制TNF-α、ICAM-1蛋白表达从而发挥作用。

Objective To study the antagonism effects of different components of Gentianella acuta on chronic alcoholic liver disease(ALD)and analyze its mechanisms,so as to determine the best therapeutic component of Gentianella acuta in the treatment of ALD.Methods Percolation method was used to separate and enrich the different polar parts of the water,30%,60%and 90%ethanol extract.The male SD rats were randomly divided into eight groups:the blank group,the model group,the positive drug group,the whole herb group,the water-eluting group,the 30%ethanol-eluting group,the 60%ethanol-eluting group and the 90%ethanol-eluting group.The rats in the experimental group were given gradient alcohol by gavage to establish the chronic ALD model,and the rats in the blank group were given the same amount of normal saline for 8 weeks.From the 5th week onwards,the rats were given gavage with the water decoction of Gentianella acuta and the water-eluted site by osmosis method,and the eluted site with 30%,60%and 90%ethanol,and the positive drug control group was given the same amount.The compound methionine choline tablets were administered orally,and the materials were taken after the 8th week.The liver was weighed to calculate the liver index;pathological changes of liver tissue were observed by electron microscope;the contents of AST,ALT,TG,TC,IL-6,IL-8,IL-10,TGF-β1 in serum and MDA,SOD,GSH,ADH and ALDH in liver tissue were detected by enzyme-linked immunosorbent assay.The protein expressions of ICAM-1 and TNF-αwere detected by histochemical method.Results(1)Compared with the model group,the contents of ALT,AST,TG,TC,IL-6,TGF-β1,MDA and the protein expression of TNF-αwere decreased in the whole herb group,while the contents of IL-10,GSH,ADH and ALDH increased,liver index were decreased(P<0.01);SOD content was increased,IL-8 content and ICAM-1 protein expressions were decreased(P<0.05).(2)Compared with the model group,the contents of TG and IL-6 in the water-eluting group were decreased(P<0.01);the contents of ALT,TGF-β1 and MDA were decreased,and the content of ADH was increased(P<0.05);other indicators had no significant difference(P>0.05).(3)Compared with the model group,the contents of TG and IL-6 in the 30%ethanol elution group were decreased,while the contents of ADH were increased(P<0.01);The contents of ALT,TGF-β1 and MDA were decreased,while the contents of GSH and ALDH were increased(P<0.05),but no significant differences in other indicators(P>0.05).(4)Compared with the model group,the contents of ALT,AST,TG,TC,IL-6,TGF-β1,MDA and TNF-αprotein expression were decreased in the 60%ethanol elution group,and the contents of IL-10,SOD,GSH,ADH,ALDH were increased,liver index decreased(P<0.01);IL-8 content and ICAM-1 protein expression were decreased(P<0.05).(5)Compared with the model group,the contents of ALT,AST,TG,TC,IL-6,IL-8,TGF-β1,MDA and the protein expressions of ICAM-1 and TNF-αin the 90%ethanol elution group were decreased;The contents of IL-10,SOD,GSH,ADH and ALDH were increased(P<0.01);the liver index was decreased(P<0.05).(6)There were significant differences between the whole herb group and the 90%ethanol elution group in reducing TG,MDA and increasing ADH(P<0.05).In terms of reducing TG,MDA,IL-6 and increasing ADH,ALDH,the 60%ethanol elution group and the 90%ethanol elution group had significant differences(P<0.05).Conclusion The components of Gentianella acuta can antagonize ALD,and the effects of different components were different.The optimal component was the 90%ethanol elution group.The mechanism of antagonizing ALD may be through regulating the lipid metabolism system and balancing the oxidation-Antioxidant system,improving alcohol metabolism rate,inhibiting the production of pro-inflammatory cytokines,promoting the production of anti-inflammatory cytokines and inhibit the expression of TNF-α,ICAM-1 protein to play a role.