益气逐瘀解毒颗粒对肝纤维化模型大鼠GIV及下游信号通路PKA/CREB表达的影响

Study on the effect of Yiqi Zhuyu Jiedu granule by regulating GIV and PKA/CREB signal pathway on hepatic fibrosis in a carbon tetrachloride-induced rat model

目的观察益气逐瘀解毒颗粒对四氯化碳(carbon tetrachloride,CCl 4)诱导的肝纤维化模型大鼠的抗纤维化作用及其对与Gα相互作用的囊泡相关蛋白(Gα-interacting vesicle-associated protein,GIV)、下游信号通路蛋白激酶A(protein kinase A,PKA)/环磷酸腺苷反应元件结合蛋白(cyclic adenosine monophosphate response element binding protein,CREB)表达水平的影响并探讨其作用机制。方法SD雄性大鼠予以CCl 4橄榄油混合物腹部皮下注射7周诱导肝纤维化模型,造模成功后随机分为益气逐瘀解毒颗粒高、中、低剂量组,扶正化瘀组及模型对照组,另设阴性对照组,共6组,每组大鼠8只。益气逐瘀解毒颗粒高、中、低剂量组分别予益气逐瘀解毒颗粒10.8 g/(kg·d)、5.4 g/(kg·d)、2.7 g/(kg·d)灌胃给药,扶正化瘀组予扶正化瘀胶囊0.405 g/(kg·d)灌胃给药,其余组等量蒸馏水灌胃,持续5周后处死大鼠收集标本。检测外周血丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST);肝组织苏木精—伊红(hematoxylin-eosin,HE)、MASSON染色检测肝纤维化程度;免疫组化法检测肝组织α-平滑肌激动蛋白(alpha smooth muscle actin,α-SMA)含量,并采用实时荧光定量PCR法(real time quantitative PCR,RT-qPCR)和蛋白免疫印迹法分别检测大鼠肝组织中Ⅰ型胶原α1链(collagen type I alpha 1 chain,Col1A1)、Ⅰ型胶原α2链(collagen type I alpha 2 chain,Col1A2)、GIV、PKA、CREB mRNA和蛋白表达水平。结果益气逐瘀解毒颗粒可减少肝纤维化模型大鼠胶原纤维增生及肝细胞损伤,且与模型对照组相比,益气逐瘀解毒颗粒各剂量组α-SMA含量显著下降(P<0.05),Col1A1、Col1A2、GIV表达显著下调(P<0.05),益气逐瘀解毒颗粒高、中剂量组PKA表达上调(P<0.05),中剂量组CREB表达显著上调(P<0.05);与扶正化瘀组比较,益气逐瘀解毒颗粒高、中剂量组α-SMA含量显著下降(P<0.05),高剂量组GIV表达下调(P<0.05),中剂量组PKA表达上调(P<0.05)。结论益气逐瘀解毒颗粒对CCl 4诱导的肝纤维化模型大鼠具有抗肝纤维化作用,可能与调节GIV表达及其下游信号通路PKA/CREB的表达有关。

Objective Study the effect and mechanism of Yiqi Zhuyu Jiedu granule by regulating GIV and PKA/CREB signal pathway on hepatic fibrosis in the carbon tetrachloride(CCl 4)-induced rat model.Methods Hepatic fibrosis was induced in male Sprague-Dawley rats by subcutaneous injection of mixture of CCl 4 and Olive oil twice a week for 7 weeks.The animals were randomly divided into six groups(each group,n=8)as follows:high,middle and low dose group each was treated with different dose of Yiqi Zhuyu Jiedu granule by gavage,the Fuzheng Huayu capsule group was treated with Fuzheng Huayu capsule,the model group and the control group was treated with distilled water.After 5 weeks,Alanine aminotransferase(ALT),aspartate aminotransferase(AST)were analyzed by ELISA and automatic biochemical instrument.The effect of Yiqi Zhuyu Jiedu granule on hepatic fibrosis was evaluated by histomorphologically using hematoxylin and eosin(HE)staining and MASSON staining,and the collagen proportionate area was quantified.The content of alpha smooth muscle actin(α-SMA)in liver tissue was detected by immunohistochemistry.In addition,fibrosis-related factors,such as Collage typeⅠalpha 1 chain(Col1A1),Collage typeⅠalpha 2 chain(Col1A2),Gα-interacting vesicle-associated protein(GIV),protein kinase(PKA)and cAMP response element binding protein(CREB)expression level,were evaluated using the method of real-time polymerase chain reaction and Western blot.Results Yiqi Zhuyu Jiedu granules could decrease collagen fiber hyperplasia and hepatocyte injury in liver fibrosis model rats.Compared with the model group,the content ofα-SMA in each group of Yiqi Zhuyu Jiedu granules group was significantly decreased(P<0.05),and the expression of Col1A1,Col1A2 and GIV was significantly down-regulated(P<0.05).The expression of PKA in the high and medium dose groups was up-regulated(P<0.05),and the expression of CREB in the medium dose group was significantly up-regulated(P<0.05).Compared with the Fuzheng Huayu group,the content ofα-SMA in the the high and middle dose groups of Chinese medicine compound was decreased significantly(P<0.05),the expression of GIV in the high dose group was down-regulated(P<0.05),and the expression of PKA in the middle dose group was up-regulated(P<0.05).Conclusion Yiqi Zhuyu Jiedu granule has anti-hepatic fibrosis effect on CCl 4-induced liver fibrosis model rats,which may be related to the regulation of GIV expression and its downstream signaling pathway PKA/CREB expression.