乌骨藤总皂苷抑制大鼠肝纤维化的分子机制研究

Molecular mechanism study of total saponins from marsdenia tenacissima on the inhibition of hepatic fibrosis in rats

目的:研究从中药乌骨藤中提取总皂苷(saponins from marsdenia tenacissima,SMT)对四氯化碳(carbon tetrachloride,CCl4)诱导的肝纤维化模型大鼠的影响及作用机制。方法:60只SD大鼠随机分为正常对照组、模型组、SMT低剂量组(10 mg·kg-1)和SMT高剂量组(40 mg·kg-1),每组15只。通过皮下注射CCl4制备大鼠肝纤维化模型,按0.3 m L·100 g-1,每周2次,共12周。造模4周后给药组开始灌胃(ig)给予相应剂量的SMT,同时正常对照组和模型组ig给予等量生理盐水。12周后处死大鼠,测定血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartic aminotransferase,AST)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、谷胱甘肽-过氧化物酶(glutathione peroxidase,GSH-Px)、透明质酸(hyaluronic acid,HA)、层黏连蛋白(laminin,LN)和四型胶原(collagen IV,CⅣ)含量;HE染色观察实验大鼠肝组织病理,Masson染色观察肝脏胶原沉积变化;Western Blot法检测肝组织α-SMA,p-smad2,p-smad3,p-p38MAPK和p-PI3K的蛋白表达水平。结果:病理结果显示,正常组大鼠肝细胞排列整齐、呈放射状排布,可见完整的肝小叶结构和中央静脉,未见明显的炎性和坏死特征以及纤维组织增生;模型组大鼠肝可见明显的细胞变性、坏死和脂肪变性特征,Masson染色可见明显的胶原沉积和肝细胞疏松;SMT各剂量组的大鼠肝组织病理较模型组明显好转,肝细胞坏死及炎细胞浸润程度明显减轻,汇管区胶原沉积减少,尤其SMT高剂量组。血清学指标检测结果显示,与正常组比较,模型组大鼠的血清ALT,AST,HA,LN和CⅣ水平均明显升高(P<0.01);与模型组比较,SMT低剂量组和SMT高剂量组的ALT,AST,HA,LN和CⅣ水平均显著降低,差异具有统计学意义(P<0.01)。Western Blot实验结果显示,与正常组比较,模型组大鼠肝组织的α-SMA,p-Smad2,p-Smad3,p-p38MAPK,p-PI3K的蛋白表达水平均显著升高(P<0.01);与模型组比较,SMT低剂量组和SMT高剂量组的α-SMA,p-Smad2,p-Smad3,p-p38MAPK,pPI3K蛋白表达水平均显著降低,差异具有统计学意义(P<0.01)。结论:SMT对CCl4诱导的大鼠肝纤维化有明显的抑制作用,可减轻模型大鼠肝脏病理损伤程度、降低实验大鼠的血清ALT,AST,HA,LN和CⅣ水平和肝组织α-SMA蛋白的表达水平,机制可能与其调节肝TGF-β/smads,PI3K/Akt和有丝分裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)信号转导通路有关。本研究可为肝纤维化的预防和治疗提供新思路和一定的理论、实验基础。

Objective:To study the effects and molecular mechanism of total saponins extracted from Marsdenia tenacissima(SMT)on liver fibrosis in rats induced by carbon tetrachloride(CCl4).Methods:Sixty SD rats were randomly divided into normal control group,model group,SMT low dose group(10 mg·kg-1)and SMT high dose group(40 mg·kg-1),with 15 rats in each group.Liver fibrosis model of rat was prepared by subcutaneous injection of CCl4at 0.3 m L·100 g-1,twice a week for 12 weeks.After 4 weeks of modeling,the drug-administered groups were intragastrically administered with the corresponding dose of the drug(SMT),while the normal control group and the model group were given the same amount of physiological saline.After 12 weeks,rats were sacrificed and the levels of alanine aminotransferase(ALT),aspartic aminotransferase(AST),superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px),hyaluronic acid(HA),laminin(LN)and collagen IV(CⅣ)in serum were detected.The pathological changes of liver tissues of rats were observed by HE staining,and the changes of liver collagen deposition were observed by Masson staining.The protein expression levels ofα-SMA,p-smad2,p-smad3,p-p38MAPK and p-PI3K in liver tissues were detected by Western Blot.Results:Pathological results showed that the liver cells of normal group arranged neatly and radially,showing intact hepatic lobular structure and central vein,and no obvious inflammatory and necrotic features and fibrous tissue hyperplasia were observed.Significant cell degeneration,necrosis and steatosis were observed in the livers of model group,and significant collagen deposition and hepatocyte loosening were also observed.The liver pathology of rats in each dose group of SMT was significantly improved compared with the model group.The degree of hepatocyte necrosis and inflammatory cell infiltration was significantly reduced.Moreover,the collagen deposition in the portal area was reduced,especially in the SMT high dose group.Serological indicators showed that the serum levels of ALT,AST,HA,LN and CIV in model group were significantly higher than those in normal group(P<0.01).Compared with model group,the levels of ALT,AST,HA,LN and CIV in SMT low-dose group and SMT highdose group were significantly lower,and the differences were statistically significant(P<0.01).Western Blot showed that the expression levels ofα-SMA,p-Smad2,p-Smad3,p-p38MAPK and p-PI3K in the livers of model group were significantly higher than those in the normal group(P<0.01).Compared with the model group,the expression levels ofα-SMA,p-Smad2,p-Smad3,p-p38MAPK,and p-PI3K protein in SMT low-dose group and SMT high-dose group were significantly lower,and the differences were statistically significant(P<0.01).Conclusion:SMT can significantly inhibit the liver fibrosis induced by CCl4in rats.It can alleviate the pathological damage of liver in model rats,reduce the serum levels of ALT,AST,HA,LN and CIV,and the protein expression level ofα-SMA in liver tissue.The mechanism may be related to its regulation of liver TGF-β/smads,PI3K/Akt and MAPK signal transduction pathways.This study provides new ideas,as well as theoretical and experimental basis for the prevention and treatment of liver fibrosis.