含氧化苦参碱大鼠血清通过阻断PI3K/AKT通路抑制砷对LX2人肝星状细胞增殖活化的刺激作用  

Rat serum containing oxymatrine inhibits the proliferation of LX2 human hepatic stellate cells induced by sodium arsenite via blocking PI3K/AKT pathway

作　　者：杨柳 张景允 韩萧萧 李晨驰 刘冉阳 马子华 韩冰 谢汝佳 杨勤 YANG Liu;ZHANG Jingyun;HAN Xiaoxiao;LI Chenchi;LIU Ranyang;MA Zihua;HAN Bing;XIE Ruja;YANG Qin(Department of Pathophysiology,Guizhou Provincial Key Laboratory of Pathogenesis&Drug Research on Common Chronic Diseases,Guizhou Medical University,Guiyang 550000,China)

机构地区：[1]贵州医科大学基础医学院病理生理学教研室,贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳550000

出　　处：《细胞与分子免疫学杂志》2022年第7期598-604,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81460484)。

摘　　要：目的 探讨含氧化苦参碱(OM)大鼠血清对砷致LX2人肝星状细胞增殖活化的影响及其可能机制。方法 SD大鼠分别给予100 mg/kg OM及等容积生理盐水灌胃以制备含OM血清及空白血清。采用100μmol/L亚砷酸钠处理LO2人胚胎肝细胞株,24 h后收集上清液,上清液与正常培养液以1∶4的比例孵育LX2细胞24 h建立间接染砷组细胞模型。设立空白血清组(160 mL/L空白血清)、间接染砷组(160 mL/L空白血清联合染砷刺激)、含OM血清低剂量组(80 mL/L空白血清、 80 mL/L含OM血清联合染砷刺激),含OM血清高剂量组(160 mL/L含药血清联合染砷刺激)。采用噻唑蓝(MTT)法检测细胞增殖能力,流式细胞术检测细胞周期,Western blot法检测LX2细胞α平滑肌肌动蛋白(α-SMA)、 B细胞淋巴瘤因子2(Bcl2)、 Bcl2相关X蛋白(BAX)、细胞周期蛋白D1(cyclin D1)、磷脂酰肌醇3激酶(PI3K)、磷酸化蛋白激酶B(p-AKT)的蛋白表达。结果间接染砷处理后LX2细胞增殖率升高,G1期比例减少,凋亡比例减少,α-SMA、 PI3K、p-AKT、 cyclin D1、 Bcl2表达均明显上调、 BAX表达降低;OM血清处理后,G1期细胞比例增加,细胞凋亡比例增加,BAX蛋白表达明显增加,其他蛋白表达均明显下调,高剂量组更为显著。结论 含OM血清能有效抑制砷对LX2肝星状细胞的增殖刺激作用,促进其凋亡,可能与抑制PI3K/AKT信号通路有关。Objective To investigate the effect of rat serum containing oxymatrine(OM) on the activation of LX2 human hepatic stellate cells induced by sodium arsenite and its mechanism. Methods SD rats were gavaged with 100 mg/kg OM or equal volume of normal saline to prepare OM-containing serum and blank serum. LO2 human embryonic liver cell line was treated with 100 μmol/L sodium arsenite for 24 hours, and then the supernatant was collected. LX2 cells were incubated with the mixture of the supernatant and normal medium at the ratio of 1∶4 for 24 hours to establish the cell model of indirect arsenic exposure. Blank serum group(160 mL/L blank serum), indirect arsenic exposure group(160 mL/L blank serum with arsenic exposure), low-dose OM-containing serum group(80 mL/L blank serum and 80 mL/L OM-containing serum with arsenic exposure), high-dose OM-containing serum group(160 mL/L medicated serum with arsenic exposure) were set up. MTT assay and flow cytometry were used to detect cell proliferation and cell cycle, respectively. Western blot analysis was performed to detect the protein expressions of α-SMA, Bcl2, BAX, cyclin D1, PI3K, and phospho-AKT(p-AKT) in LX2 cells. Results After indirect arsenic treatment, the proliferation rate of LX2 cells increased, the proportion of G1 phasedecreased, the proportion of apoptosis decreased, the expression of α-SMA, PI3K, p-AKT, cyclin D1, Bcl2 were significantly up-regulated, and the expression of BAX decreased. After OM-containing serum treatment, the proportion of cells in G1 phase increased, the proportion of apoptosis increased, the expression of BAX protein increased significantly, and the expression of other proteins were significantly down-regulated, especially in the high-dose group. Conclusion OM-containing serum can effectively inhibit the proliferation of LX2 hepatic stellate cells induced by arsenite and promote their apoptosis, which may be related to the blocking of PI3K/AKT signaling pathway.

关 键 词：氧化苦参碱(OM) 亚砷酸钠 血清 肝纤维化 增殖活性 

分 类 号：R285.5[医药卫生—中药学] R392-33[医药卫生—中医学] R575[生物学—细胞生物学] Q25