Abstract
BackgroundPrevious 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.MethodsL02 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 Ca2+ influx was recorded using fluorescence spectrophotometer.ResultsHQT-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.ConclusionsThe 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.
Highlights
Previous studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD)
HQT-medicated serum reduces Free Fatty Acid (FFA)-induced L02 hepatocyte steatosis In order to examine the optimum concentration of HQT serum for L02 hepatocyte, we performed a lactate dehydrogenase (LDH) release assay to measure the cytotoxicity of HQT-medicated rat sera
In comparison to normal culture medium containing 10% FBS, 10% vehicle serum have no harm to L02 cells (p > 0.05, Fig. 1a), when the concentration reached over 20% the content of LDH have increased sharply (p < 0.001, Fig. 1a)
Summary
Previous studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD). Sustained and irreversible ERS plays an essential role in NAFLD pathologic changes which activates multiple signaling pathways [5]. It would interfere with the secretion of triglyceride from liver, indirectly slow down the metabolism of triglycerides by inducing insulin resistance, and promote the expression of inflammatory factors, and deteriorate the progression of NAFLD to NASH [6, 7]. In a previous study of methionine- and choline- deficient (MCD) dietary induced NASH [11], crucial features for the pathophysiology of NAFLD progression, such as TG accumulation in the liver, oxidative stress and hepatocytes apoptosis, were repressed in PKC-δ−/− mice. An internal link between PKC-δ and the pathology of NASH or ERS remains to be expounded
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