青蒿琥酯在大鼠体内外抗肝纤维化的作用

Effect of artesunate on rat liver fibrogenesis in vitro and in vivo

目的观察青蒿琥酯(artesunate,Art)对大鼠肝纤维化的治疗作用及其对大鼠原代肝星状细胞(hepatic stellate cells,HSCs)增殖的影响,并探讨其作用机制。方法建立牛血清白蛋白(bovine serum albumin,BSA)免疫性肝纤维化大鼠模型,分为正常对照组、模型对照组及青蒿琥酯低、中、高剂量组;给药组分别给予不同剂量的青蒿琥酯灌胃,正常和模型对照组给予同体积蒸馏水灌胃,每日1次,连续2个月;酸水解法检测其肝组织胶原蛋白含量,测定血清白蛋白(albumin,Alb)、丙氨酸氨基转移酶(alanine amino transferase,ALT)及天门冬氨酸氨基转移酶(aspartate amino transferase,AST)水平;苏木精-伊红染色法(hematoxylin-eosin staining,HE染色)和胶原染色法对肝组织切片染色。分离大鼠HSCs,以培养活化HSCs。用MTT法测定HSCs增殖率,消化法测定细胞培养上清液中的羟脯氨酸(hydroxyproline,Hyp)含量,Western blot和RT-PCR法检测HSCs p53表达。结果与正常对照组相比,模型对照组大鼠血清Alb水平降低(P<0.05),ALT、AST水平增高(P<0.05);与模型对照组相比,青蒿琥酯低、中、高剂量组血清AST水平降低(P<0.05),大鼠肝组织胶原蛋白含量降低(P<0.05)。青蒿琥酯对培养活化的HSCs有抑制作用,且呈剂量和时间依赖性(P<0.05);青蒿琥酯作用24 h后,HSCs分泌羟脯氨酸减少(P<0.05),p53 mRNA表达增加(P<0.05)。结论青蒿琥酯在大鼠体内、体外均有抗肝纤维化作用,该作用与其增加HSCs p53的表达有关。

Aim To study the effect of artesunate on immuno-injured hepatic fibrosis induced by bovine ser-um albumin in rat model and the effect of artesunate on hepatic stellate cells ( HSCs ) proliferation, so as to provide experimental evidence for clinical application of artesunate and the treatment of hepatic fibrosis. Methods The model of immuno-injured hepatic fibro-sis induced by bovine serum albumin was established in Wistar rats. Rats were randomly divided into 5 groups:normal group, model group, low dose of arte-sunate, middle dose of artesunate and high dose of ar-tesunate. Drugs were given to the corresponding thera-peutic groups, and then were continued once a day for two months. Distilled water was given to the rats of normal and model groups according to the same meth-od. Liver tissues were used for measuring the content of collagen, the rat serum activities of albumin( Alb) , alanine aminotransferase ( ALT ) and aspartate amin-otransferase(AST). Liver tissue’ s pathological chan-ges were observed by HE and collagen staining. Isola-ted and cultured rat primary HSCs in the flask for 10 days to make cells activated, MTT assay was used to detect rate of cellular proliferation; concentration of hydroxyproline in supernatant was detected by digestive method; the expression of p53 was investigated by&nbsp;Western blot and RT-PCR. Results Serum levels of Alb in model group were significantly lower ( P <0. 05 ) , and levels of ALT and AST in model group were significantly higher ( P <0. 05 ) compared with normal group. Levels of AST in low, middle and high dose groups(3. 2, 9. 6, 28. 8 mg·kg-1 ) were signifi-cantly lower(P <0. 05) compared with model group, and levels of ALT in high dose groups were significant-ly lower(P<0. 01) compared with model group. The contents of collagen in model groups were significantly higher(P<0. 01) compared with normal group, while the contents of collagen in therapy groups significantly decreased ( P < 0. 05 ) compared with model group. Activated HSCs treated with various concentrations of artesunate (150, 175, 200 μmol·L-1 ) were inhibi-ted on dose and time-effect relationships. Production/secretion of hydroxyproline decreased after HSCs was treated by artesunate for 24 h; the expression of p53 was up-regulated showed by Western blot and RT-PCR in artesunate treated cells. Conclusion Artesunate brings about anti-fibrosis in vitro and in vivo by increas-ing the expression of p53 .