二氢石蒜碱对棕榈酸诱导H9c2细胞炎性损伤的保护作用及其机制  

Protective Effects and Mechanism of Dihydrolycorine on Inflammatory Injury of H9c2 Cells Induced by Palmitic Acid

作　　者：徐文芳 金红花 XU Wen-fang;JIN Hong-hua(College of Pharmacy,Yanbian University,Yanji 133002,China;Department of Pharmacy,Affiliated Hospital of Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学药学院,吉林延吉133002 [2]延边大学附属医院药学部,吉林延吉133002

出　　处：《中药材》2022年第10期2460-2465,共6页Journal of Chinese Medicinal Materials

基　　金：国家自然科学基金资助项目(81860077)

摘　　要：目的:研究二氢石蒜碱对棕榈酸诱导H9c2细胞炎性损伤的影响并探讨其机制。方法:建立棕榈酸诱导H9c2细胞炎性损伤细胞模型,采用CCK-8法筛选二氢石蒜碱的最佳给药浓度。将培养的H9c2细胞随机分为正常对照组、模型组、LY294002组、雷帕霉素组及二氢石蒜碱低、中、高浓度组。蛋白免疫印迹法检测H9c2细胞中TLR4、p-PI3K、p-AKT、p-mTOR蛋白表达;免疫荧光染色及流式细胞术检测二氢石蒜碱对棕榈酸诱导H9c2细胞内活性氧(ROS)产生水平的影响。结果:采用棕榈酸100μmol/L建立H9c2细胞炎性损伤模型,CCK-8实验筛选出二氢石蒜碱组在1、10、50μmol/L下细胞存活率都在80%以上,分别确定为低、中、高给药浓度。蛋白免疫印迹实验表明,二氢石蒜碱预处理能显著下调棕榈酸诱导下H9c2细胞的TLR4、p-PI3K、p-mTOR蛋白表达,上调p-AKT蛋白表达(P<0.05或P<0.01),其作用呈浓度依赖性;PI3K/AKT抑制剂LY294002可抑制p-AKT蛋白表达,mTOR抑制剂雷帕霉素可抑制p-mTOR蛋白表达(P<0.01)。免疫荧光染色及流式细胞术结果表明二氢石蒜碱对炎性损伤H9c2细胞内ROS产生有显著的抑制作用(P<0.01)。结论:二氢石蒜碱对棕榈酸诱导的H9c2细胞炎性损伤具有保护作用,其机制可能与抑制TLR4、p-PI3K、p-mTOR蛋白表达,促进AKT蛋白磷酸化,抑制炎性损伤H9c2细胞内ROS的产生相关。Objective: To study the effect of dihydrolycorine on inflammatory injury of H9c2 cells induced by Palmitic acid and explore the mechanism.Methods: The inflammatory injury cell model of H9c2 cells induced by Palmitic acid was established, and the optimal concentration of dihydrolycorine was screened by CCK-8 method.The cultured H9c2 cells were randomly divided into normal control group, model group, LY294002 group, rapamycin group, low, medium and high concentration dihydrolycorine groups.The protein expressions of TLR4,p-PI3K,p-AKT and p-mTOR in H9c2 cells were detected by Western Blot.The effect of dihydrolycorine on the production of reactive oxygen species(ROS) in H9c2 cells induced by palmitic acid was analyzed by immunofluorescence staining and flow cytometry.Results: The inflammatory injury model of H9c2 cells was established by 100 μmol/L Palmitic acid, and the cell survival rate of dihydrolycorine group was above 80% at 1,10 and 50 μmol/L,which were determined as low, medium and high dose, respectively.Western Blot showed that pretreatment with dihydrolycorine significantly down-regulated the expressions of TLR4,p-PI3 K and p-mTOR proteins in H9c2 cells induced by Palmitic acid, and up-regulated the expression of p-AKT protein in a concentration dependent manner(P<0.05 or P<0.01).PI3K/AKT inhibitor LY294002 inhibited the expression of p-AKT protein, and mTOR inhibitor rapamycin inhibited the expression of p-mTOR protein(P<0.01).The results of immunofluorescence staining and flow cytometry showed that dihydrolycorine could significantly inhibit the production of ROS in inflammatory H9c2 cells(P<0.01).Conclusion: Dihydrolycorine has a protective effect on Palmitic acid-induced inflammatory injury of H9c2 cells, the mechanism may be related to the inhibition of TLR4,p-PI3K,p-mTOR proteins expressions, the promotion of AKT protein phosphorylation, and the inhibition of ROS production in inflammatory injury of H9c2 cells.

关 键 词：二氢石蒜碱 棕榈酸 H9c2细胞炎性损伤 TLR4 p-PI3K P-AKT P-MTOR 活性氧 

分 类 号：R285[医药卫生—中药学]