清脂护肝方对大鼠非酒精性脂肪性肝炎的作用研究

Role of “Qingzhi Hugan Prescription” in nonalcoholic steatohepatitis of rats model

目的探讨清脂护肝方对大鼠非酒精性脂肪性肝炎的作用及其机制。方法 58只雄性SD大鼠,均喂养高脂饲料,予以12只作对应。将造模成功的大鼠再随机分为模型组、低剂量、中剂量和高剂量清脂护肝方组,分别给予0.9%NaCl溶液灌胃,清脂护肝方组予清脂护肝方药液灌胃6周;观察记录大鼠的行为表现及体质量变化。干预结束后次日处死所有大鼠并留取血清和肝脏,检测清脂护肝方对NASH大鼠肝生化指标的影响。免疫组织化学方法检测NASH大鼠肝组织中SSeCKS、Hck、Lyn、Fgr的表达水平。体外实验:对库普弗细胞株采用不同浓度不同时间的CCL4刺激,用优选方案的清脂护肝方药物血清培养后检测SSeCKS、Hck、Lyn、Fgr的表达。结果与正常对照组相比,模型组大鼠体重明显增加(P<0.05);模型组ALT、AST、TBIl均较正常对照组升高(P<0.05);模型组TG、CH、LDL均明显上升(P<0.01),HDL亦有所降低(P<0.05)。与模型组相比,中大剂量清脂护肝方组体重均有减轻,其中大剂量组的体重减轻最为明显(P<0.01);清脂护肝方各剂量治疗组ALT与模型组比较明显降低(P<0.01),大剂量治疗组AST与模型组比较明显降低(P<0.01);清脂护肝方中、大剂量组的血清TBIL较模型组显著下降(P<0.01);与模型组比较,清脂护肝方组的TG、LDL有所降低(P<0.05),尤其大剂量组降低相对更明显;清脂护肝方各剂量组CH有所降低,但仅有大剂量组有统计学差异(P<0.05)。ICC结果表明:Lyn、Hck在kupffer细胞中有表达,并与炎症程度呈正相关,清脂护肝方药物血清培养后表达减少,Fgr和SSeCKS在kupffer细胞中无明显表达。ICC结果表明:Hck、Lyn在正常肝脏组织中低表达,模型组中高表达,用药组无论是大、中、小剂量组,在抑制Hck、Lyn两种Src相关激酶成员的阳性表达是有显著效果,特别是大剂量组,与模型组比较有显著意义(P<0.01);模型组SSeCKS蛋白的表达显著低于正常组,在治疗组中的表达介于模型组和正常组,同时伴随Lyn、Hck表达的升高呈减少趋势,提示SSeCKS与Src家族相关激酶(Lyn、Hck)存在某种上下游抑制关系;而清脂护肝方对Fgr的表达无明显影响,用药前后无统计学意义(P>0.05)。结论清脂护肝方能减轻肝脏的炎性反应,明显改善肝功能和血脂指标,其机制可能与SSeCKS、Lyn、Hck信号通路有关,且高剂量时疗效更优。

Objective To investigate the role and mechanism of “Qingzhi Hugan Prescription”in nonalcoholic steatohepatitis of rats model.Methods In vivo experiment：Fifty-eight SD male rats except control group,all rats were treated with high-fat for 8 weeks and the models were established.The models were randomized into model group,“Qingzhi Hugan Prescription”groups （large,medium and small dose）.The model ang control group were gavaged with 0.9% NaCl solution.The “Qingzhi Hugan Prescription”groups were gavaged with decotions for 6 weeks.The mouse changes in body weight and behaviors were recorded.The next day after the end of the intervention,all rats were sacrificed and the serum and livers were obtained.The expression of Hck、Lyn 、Fgr and SSeCKS in the rat liver tissue were deceted by Immunohistochemistry.In vitro experiment：model group of Kupffer cells after stimulation with different concentrations at different times of CCL4 were training with optimization of the “Qingzhi Hugan Prescript-ion”medicated serum,the expression of Hck、Lyn 、Fgr and SSeCKS in kupffer cells were deceted by Immunocytochemistry.Results Compared with the control group,the model group weight was obviously increased （P 〈0.05 ）,ALT,AST,TBIL in rats model group were higher than control group （P 〈0.05 ）.Compared with normal control group,the TG,CH and LDL of model group were significantly increased （P 〈0.01 ）,and HDL was reduced （P 〈 0.05）.Compared with the model group,the TG,LDL of “Qingzhi Hugan Prescription”groups were reduced （P 〈0.05 ）,especially high dose group;The CH of each dose group of “Qingzhi Hugan Prescription”was decreased,but only the large dose group was statistically difference （P 〈0.05 ）.The results of ICC showed that：Lyn,Hck were expressed in kupffer cells,and associ-ated with inflammation was positively,after training with the “Qingzhi Hugan Prescription”medicated serum,the expression were reduced.But the expression of Fgr and SSeCKS was not obvious in kupffer cell-s.The results of IHC indicated that：compared with model group,whatever the large、medium and small dose of the Chinese medicine groups, especially the large dose of “Qingzhi Hugan Prescription”group,the positive expression of Hck and Lyn were decreased markedly（P 〈 0.01 ）;Compared with the normal group,the expression of SSeCKS in model group was decreased markedly;In the Chinese medicine groups,the SSeCKS were expressed between the model group and normal group;It showed a trend of decrease along with the rise of the expression of Lyn and Hck in the rat liver tissue with nonalcoholic fatty liver disease;Compared with model group,effect of “Qingzhi Hugan Prescription”on the expression of Fgr was insignificant（P 〉 0.05 ）.Conclusion “Qingzhi Hugan Prescription”especially the large dose group can efficiently improve the inflammatory reaction in the rats model of nonalcoholic steatohepatitis,liver function,blood lipids index wereimproved significantly,and the mechanism may be related with SSeCKS、Lyn and Hck signaling pathways.The higher the dose,the more significant the efficacy.