摘要
目的:研究哈蟆油蛋白酶解物(ORPH)对乙醇诱导的L-02细胞损伤相关通路蛋白表达影响的作用机制研究。方法:复合酶解法制备ORPH,以400 mmol·L^(-1)乙醇建立L-02肝细胞损伤模型,细胞增殖与活性检测试剂盒-8(CCK-8)检测细胞活力;流式细胞术检测细胞凋亡、周期变化,JC-1/Hochest染色观察形态学变化;蛋白免疫印迹法(Western blot)检测凋亡相关蛋白、丝裂原活化蛋白激酶(MAPK)信号通路及细胞焦亡相关蛋白在L-02肝细胞中的表达变化,研究ORPH对乙醇所致肝细胞损伤的保护作用及机制。结果:CCK-8法结果表明400 mmol·L^(-1)乙醇可在12 h内明显引起L-02肝细胞损伤;与空白组比较,模型组L-02细胞活力显著下降(P<0.01),细胞处于G0/G1期的细胞占比显著升高(P<0.01),细胞总凋亡率显著升高(P<0.01),线粒体膜电位下降;B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax),细胞色素C(Cyt C),半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)凋亡相关蛋白的表达明显升高(P<0.05,P<0.01),MAPK信号通路相关蛋白c-Jun氨基末端激酶(JNK),p38丝裂原活化蛋白激酶(p38 MAPK)明显上调(P<0.05,P<0.01),且焦亡蛋白Caspase-1,白细胞介素-1β(IL^(-1)β)表达明显增强(P<0.05),Bcl-2显著下降(P<0.01);与模型组比较,ORPH处理组细胞周期阻滞有所改善(P<0.05,P<0.01),细胞总凋亡率显著下降(P<0.01),线粒体膜电位升高,呈剂量相关性;Bax,Cyt C,Caspase-3蛋白表达明显降低(P<0.05,P<0.01),Bcl-2蛋白表达升高(P<0.05,P<0.01),MAPK信号通路相关蛋白及焦亡相关蛋白表达均呈下降趋势(P<0.05,P<0.01)。结论:ORPH可通过抑制乙醇诱导L-02肝细胞引起的氧化应激肝细胞损伤,改善恢复线粒体膜电位,降低线粒体介导的细胞凋亡通路蛋白的表达,并抑制MAPK信号通路相关蛋白、焦亡通路相关蛋白表达,从而降低乙醇诱导L-02肝细胞的线粒体功能障碍及炎症反应并减轻氧化应激,对酒精性肝损伤起到治疗作用。
Objective:To study the protective effect and mechanism of Ranae Oviductus protein hydrolysate (ROPH) on the expression of pathway-related proteins in ethanol-induced L-02 cell injury.Method:The ROPH was prepared by compound enzymatic hydrolysis.L-02 cell injury model was induced with400 mmol·L^(-1)ethanol.Cell viability was detected by cell counting kit-8(CCK-8)assay.Cell cycle and apoptosis were examined by flow cytometry.JC-1/Hochest staining was employed for qualitative investigation.The expression of related proteins in apoptosis,mitogen-activated protein kinase(MAPK)signaling pathway,and pyroptosis in L-02 cells was detected by Western blot.Result:The results of the CCK-8 assay showed that400 mmol·L^(-1)ethanol could induce L-02 cell injury within 12 hours.Compared with the blank group,the modelgroup showed decreased viability of L-02 cells(P<0.01),elevated percentage of the cell cycle in the G0/G1phase(P<0.01),increased total cell apoptosis rate(P<0.01),reduced mitochondrial membrane potential(P<0.01),up-regulated expression of apoptosis-related proteins[B-cell lymphoma-2(Bcl-2)-associated X protein(Bax),Cytochrome C(Cyt C),and cysteine-dependent aspartate specific protease-3(Caspase-3)](P<0.05,P<0.01)and MAPK signaling pathway-related proteins[C-Jun amino-terminal kinase(JNK)and p38 MAPK](P<0.05,P<0.01),and potentiated expression of pyrolysis-related proteins Caspase-1 and interleukin-1β(IL^(-1)β)(P<0.05).Compared with the model group,the ROPH treatment group exhibited improved cell cycle arrest(P<0.05,P<0.01),diminished total cell apoptosis rate(P<0.01),elevated mitochondrial membrane potential in a dose-dependent manner,down-regulated expression of Bax,Cyt C,and Caspase-3 proteins(P<0.05,P<0.01),up-regulated expression of Bcl-2 protein(P<0.05,P<0.01),and a downward trend in expression of proteins related to MAPK signaling pathway and pyrolysis(P<0.05,P<0.01).Conclusion:ROPH could inhibit oxidative stress-triggered liver injury in ethanol-induced cells by improving mitochondrial membrane potential,red
作者
张怡
郑冉
于奇
焦丽丽
李波
刘达
李宜平
ZHANG Yi;ZHENG Ran;YU Qi;JIAO Li-li;LI Bo;LIU Da;LI Yi-ping(Changchun University of Chinese Medicine,Changchun 130117,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第15期43-50,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
吉林省科技发展项目(20170309005YY)。
关键词
哈蟆油蛋白酶解物(ORPH)
酒精性肝细胞损伤
氧化应激
线粒体凋亡
炎症反应
Ranae Oviductus protein hydrolysate(ROPH)
alcoholic liver cell injury
oxidative stress
mitochondrial apoptosis
inflammatory response
