护肝清脂片通过调控蛋白激酶 C-δ的激活改善游离脂肪酸诱导的 L02 肝细胞内质网应激
Hugan Qingzhi medication ameliorates free fatty acid-induced L02 hepatocyte endoplasmic reticulum stress by regulating the activation of PKC-δ.
机构信息
Department of Pharmacy, People's Hospital of Longhua, Shenzhen, 518109, Guangdong, China.
Department of Pharmacy, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, 518107, Guangdong, China.
出版信息
BMC Complement Med Ther. 2020 Dec 11;20(1):377. doi: 10.1186/s12906-020-03164-3.
BACKGROUND
Previous studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD). Moreover, the results of proteomic analysis confirmed that various proteins in endoplasmic reticulum stress (ERS) pathway were activated and recovered by HQT. However, its mechanism remains confused. The purpose of this study was to explore the effects of HQT-medicated serum on hepatic ERS and its relevant mechanisms.
METHODS
L02 cells were induced by Free Fatty Acid (FFA) for 24 h to establish a model of hepatic ERS and pretreated with the drug-medicated rat serum for 24 h. Accumulation of intracellular lipid was evaluated using Oil Red O staining and Triglyceride detection kit. The morphological changes of ER were observed by TEM. PKC-δ was silenced by specific siRNA. Western blot and RT-qPCR were applied to detect the expression of markers related to ERS, calcium disorder, steatosis and insulin resistance. The fluorescence of Ca influx was recorded using fluorescence spectrophotometer.
RESULTS
HQT-medicated serum significantly decreased the intracellular TG content. Furthermore, it caused significant reduction in the expression of ERS markers and an improvement in ER structure of L02 cells. PKC-δ was activated into phosphorylated PKC-δ in FFA-induced L02 hepatocytes while these changes can be reversed by HQT-medicated serum. Silencing PKC-δ in L02 cells can restore the expression and activity of SERCA2 in ER and down-regulate the expression of IP3R protein to maintain intracellular calcium homeostasis, so as to relieve FFA-induced ERS and its lipid accumulation and insulin resistance.
CONCLUSIONS
The results concluded that HQT-medicated serum exerts protective effects against hepatic ERS, steatosis and insulin resistance in FFA-induced L02 hepatocyte. And its potential mechanism might be down-regulating the activation of PKC-δ and stabilization of intracellular calcium.
背景
先前的研究发现，护肝清脂片（HQT）对非酒精性脂肪肝（NAFLD）具有显著的降脂和抗氧化作用。此外，蛋白质组学分析的结果证实，HQT 能激活并恢复内质网应激（ERS）途径中的各种蛋白。然而，其机制仍不清楚。本研究旨在探讨 HQTM 对肝 ERS 的作用及其相关机制。
方法
用游离脂肪酸（FFA）诱导 L02 细胞 24 h 建立肝 ERS 模型，用含药大鼠血清预处理 24 h。油红 O 染色和甘油三酯检测试剂盒评估细胞内脂质蓄积。TEM 观察 ER 形态变化。用特异性 siRNA 沉默 PKC-δ。Western blot 和 RT-qPCR 检测与 ERS、钙紊乱、脂肪变性和胰岛素抵抗相关的标志物表达。荧光分光光度计记录 Ca 内流荧光。
结果
HQTM 明显降低了细胞内 TG 含量。此外，它显著降低了 ERS 标志物的表达，改善了 L02 细胞的 ER 结构。PKC-δ 在 FFA 诱导的 L02 肝细胞中被激活为磷酸化 PKC-δ，而这些变化可被 HQTM 逆转。在 L02 细胞中沉默 PKC-δ 可恢复 ER 中 SERCA2 的表达和活性，并下调 IP3R 蛋白的表达，以维持细胞内钙稳态，从而缓解 FFA 诱导的 ERS 及其脂质蓄积和胰岛素抵抗。
结论
HQTM 对 FFA 诱导的 L02 肝细胞中肝 ERS、脂肪变性和胰岛素抵抗具有保护作用。其潜在机制可能是下调 PKC-δ 的激活和稳定细胞内钙。
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