基于方证相关探讨茵陈蒿汤调控库普弗细胞功能及MAPK通路抗肝纤维化的作用机制

摘要

Objective:Our previous study showed significantly efficacy of series formula of Yin-chen-hao Decoction (YCHD)on fibrosis models in rats induced dimethynitrosamine (DMN).From the perspective of formula-efficacy-syndrome,we proposed a hy-pothesis ‘corresponding permit on formula-syndrome,gene regulation of formula on suffering body follow‘error free repair’princi-ple’and explore the mechanism of action of YCHD.Methods:Liver fibrosis was induced in rats with DMN.YCHD was adminis-trated intragastrically for 2 weeks for rats.At the end of the fourth week,all of the animals were sacrificed and their liver samples were collected for histology and gene chip analyses.Results:Compared with the normal group,the blood serum ALT level of the rats was remarkably increased (P<0.01 )four weeks after modeling;the inflammatory cells of hepatic tissue was remarkably infil-trated according to pathological observance,the deposition of collagen was remarkable (P<0.01 ).There were remarkable up-reg-ulation of the expression of the genes including Interleukin 1 beta (IL-1 b),Cd68,Tumor necrosis factor receptor superfamily, member 1 4 (Tnfrsf1 4),Tumor necrosis factor receptor superfamily,member 9 (Tnfrsf9),Fas,Cd1 4,Connective tissue growth factor (Ctgf),Collagen,type I,alpha 2(Col1 a2),Insulin-like growth factor binding protein,acid labile subunit(Igfals),Insu-lin-like growth factor 1 (Igf1 ),Insulin-like growth factor binding protein 1 (Igfbp1 ),Matrix metallopeptidase 1 2(Mmp1 2),Matrix metallopeptidase 2(Mmp2),Matrix metallopeptidase 23(Mmp23),Chemokine ligand 21 (Ccl21 )and Protein kinase C,beta (Prkcb)in hepatic tissue,and mitogen-activated protein kinase (MAPK)pathway was activated.Yin-Chen-Hao Decoction signif-icantly reduced the level of blood serum ALT inducted by DMN,restrained the infiltration and necrosis of the inflammatory cells of hepatic tissue,inhibited collagen deposit,decreased the expression of the genes including IL-1 b,Cd68,Tnfrsf1 4,Tnfrsf9,Fas, Cd1 4,Ctgf,Col1 a2,Igfals,Igf1 ,Igfbp1 ,Mmp1 2,Mmp2,Mmp23,Ccl21 and Prkcb,and restrained the activation of MAPK pathway.Conclusion:The hypothesis ‘corresponding permit on formula-syndrome,gene regulation of formula on suffering body follow‘error free repair’principle’was verified.The MOA of anti-fibrotic of Yin-Chen-Hao Decoction was regulation of the KCs action and MAPK pathway.%目的：基于前期茵陈蒿汤类方抗肝硬化的方证病理基础结果，围绕“方-证相关”的学术内涵，提出了“方证相关时，方剂对机体基因的调控遵循‘无差错修复’原则”的假说；并探讨茵陈蒿汤对DMN诱导大鼠肝纤维化形成阶段肝组织库普弗细胞（Kupffer Cells，KCs）相关基因表达及对丝裂原活化蛋白激酶（MAPK）通路的影响。方法：采用wistar大鼠，于每周前3天连续腹腔注射0.5％二甲基亚硝胺（Dimethylnitrosamine，DMN）制备大鼠肝纤维化模型，2周末取模型组6只做动态观察。第3周初开始，在持续造模的同时给予茵陈蒿汤干预治疗到4周末，正常组与模型对照组给予等量生理盐水。4周末在3％戊巴比妥钠腹腔注射麻醉情况下，杀鼠取材，检测肝功能、肝组织病理、肝组织羟脯氨酸含量、胶原半定量，并采用基因芯片检测分析茵陈蒿汤对模型大鼠肝组织基因表达谱的影响。结果：与正常组比较，造模4周大鼠血清肝功能水平明显升高（P＜0.01）；病理观察肝组织炎细胞显著浸润，胶原显著沉积（P＜0.01），肝组织白介素1（IL-1 b）、Cd68、肿瘤坏死因子受体超家族成员14（Tnfrsf14）、肿瘤坏死因子受体超家族成员9（Tnfrsf9）、TNF受体超家族成员6（Fas）、Cd14、结缔组织生长因子（Ctgf）、Ⅰ型胶原α2（Col1 a2）、胰岛素样生长因子结合蛋白（Igfals）、胰岛素样生长因子1（Igf1）、胰岛素样生长因子结合蛋白1（Igfbp1）、基质金属蛋白酶12（Mmp12）、基质金属蛋白酶2（Mmp2）、基质金属蛋白酶23（Mmp23）、趋化因子配体21（Ccl21）、蛋白激酶Cβ（Prkcb）基因表达明显上调，MAPK通路被激活。经2周治疗后茵陈蒿汤能显著降低DMN诱导的大鼠血清肝功能水平，抑制组织炎细胞的浸润与坏死，胶原沉积，并下调了肝组织IL-1 b、Cd68、Tnfrsf14、Tn-frsf9、Fas、Cd14、Ctgf、Col1 a2、Igfals、Igf1、Igfbp1、Mmp12、Mmp2、Mmp23、Ccl21、Prkcb 基因的表达，抑制了MAPK通路的活化。通过全基因芯片的分析，在茵陈蒿汤干预治疗后基因表达得到了不同程度的修复。结论：验证了“方证相关时，方剂对机体基因的调控遵循‘无差错修复’原则”的假说；茵陈蒿汤显著抑制DMN 诱导肝纤维化形成，其机制可能是调控了KCs，抑制相关炎症因子的释放，同时可能参与调控MAPK通路，从而达到抗肝纤维化的作用。