Roles of Drp1 and Mfn2 in palmitate induced rat hepatocyte injury and the effects of curcumin derivative L6H4 on it
ZHENG Jingyu1, WU Huan1, TANG Wen1, LIU Cheng1, DING Tingting1, DAI Shuang1, WU Ling1, ZHU Zaisheng2, LI Jianmin1.
1.Department of Pathology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 2.Department of Gerontology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015
ZHENG Jingyu1,WU Huan1,TANG Wen, et al. Roles of Drp1 and Mfn2 in palmitate induced rat hepatocyte injury and the effects of curcumin derivative L6H4 on it[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2016, 46(7): 469-475,481.
Abstract：Objective: To explore the roles and mechanism of mitochondrial dynamin-related protein 1 (drp1) and mitochondrial fusion protein 2 (Mfn2) in palmitate (PA) induced rat hepatocyte injury in the model of nonalcoholic-fatty-liver-disease (NAFLD) in vitro, and the effects of curcumin derivative L6H4 on it. Methods: Rat BRL-3A hepatocytes were cultured with difference concentrations of PA and oleate (OA) and PA+ curcumin derivative L6H4 mixed medium for 24 hours respectively. Cell vitality was detected by MTT assay and the optimal concentration of PA for establishing non alcoholic fatty liver model in vitro and drug intervention of curcumin derivative L6H4 were screened. Cells were divided into 4 groups: control group (CON group), oleate group (OA group), palmitate group (PA group) and curcumin derivative L6H4 intervention group (L6H4 group), and were cultured with normal medium, containing oleate medium, containing palmitate medium and containing palmitate and curcumin derivative L6H4 mixed medium for 24 hours, respectively. Total SOD activity was detected by hydroxylamine method. The content of MDA was analyzsed by thiobarbituric acid method. The expression of mRNA and protein of Drp1, Mfn2, Bcl-2, Bax, Caspase-3, TNF-α of hepatocyte were detected by real-time PCR and Western blot respectively. Results: Different concentrations of PA had certain inhibitory effect on BRL-3A cells growth (P<0.05), and showed a clear dose dependence manner, 0.1 mmol/L PA was the cut-off point, while 0.05 mmol/L OA had no obvious effect. 0.05 mmol/L PA was selected as the optimal concentration to establish the model of NAFLD in vitro. The cell viability was higher than 75% when the concentration of curcumin derivative L6H4 was lower than 10 µmol/L. The cell viability dropped to below 50% (P<0.05) when the concentration of curcumin derivatives L6H4 was higher than 20 µmol/L, 10 µmol/L L6H4 was the cut-off point. Therefore, 5 µmol/L curcumin derivative L6H4 was chosen for subsequent L6H4 interfere concentration. Compared with the CON group, MDA content of cellular homogenization and the expressions of mRNA and protein of Drp1, Bax, Caspase-3, TNF-α were increased (P<0.05) significantly, T-SOD activity and the expressions of mRNA and protein of Mfn2, Bcl-2 were decreased (P<0.05) significantly in PA group. Compared with the PA group, MDA content of cellular homogenization and the expressions of mRNA and protein of Drp1, Bax, Caspase-3, TNF-α were decreased significantly (P<0.05), T-SOD activity and the expressions of mRNA and protein of Bcl-2 were increased significantly (P<0.05) in L6H4 group, but the expression of Mfn2 had no significant difference (P>0.05). Conclusion: Curcumin derivative L6H4 plays an important role in attenuation hepatocyte injury induced by PA, which is associated with its decrease the lipid peroxidation, inhibition mitochondrial fission and suppression the downstream of mitochondrial apoptotic pathway and signal transduction of inflammatory factor. It may be one of the underlying mechanism for curcumin derivative L6H4 to treat NAFLD.
