Hepatotoxic Mechanisms of Ephedrine and Dihydroceramide through Impaired Autophagy

Abstract

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common chronic liver disease worldwide. The pathogenic mechanisms underlying the progression of NAFLD are not fully understood. Therefore, the aims of this study are to assess the relationship between oxidative stress and autophagy in human hepatocytes during NAFLD. Autophagy also is an adaptive response under stressful conditions, and basal level of autophagy ensures the physiological turnover of old and damaged organelles. Recent studies have shown that autophagy plays a role in NAFLD. Thus, autophagic pathway can be a novel therapeutic target for liver disease. <br/> The herb Ephedra sinica (also known as Chinese ephedra or Ma Huang), used in traditional Chinese medicine, contains alkaloids identical to ephedrine and pseudoephedrine as its principal active constituents. Ephedrine is known as an effective diet component. However, recent studies have reported that ephedrine has various side effects in the cardiovascular and nervous systems. In addition, herbal Ephedra, a plant containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. Many studies reported clinical cases, however, the cellular mechanism of liver toxicity by ephedrine remains unknown. This study investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. Ephedrine triggered mitochondrial oxidative stress and depolarization. Mitochondrial swelling and autolysosome were observed in ephedrine treated cells. Ephedrine also inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Parkin siRNA recovered the ephedrine-induced mitochondrial damage. Excessive mitophagy lead to cell death through imbalance of autophagic flux. Moreover, antioxidants and reducing Parkin level could serve as therapeutic targets for ephedrine-induced hepatotoxicity.<br/> Sphingolipids are a family of lipids that play essential roles as critical regulators in metabolic disorders. Some sphingolipids are known key factors in metabolic dysfunction. However, the precise effect of dihydroceramide on NAFLD remains unknown. Here, these results report how dihydroceramide in autophagosome accumulation activates fibrogenesis in human liver Chang cells treated with free fatty acids (FFA). According to LC/MS lipid profiling, FFA increased the levels of sphingolipids and triacylglycerol (TG). To demonstrate the potential role of dihydroceramide metabolism in autophagy, several sphingolipid synthesis inhibitors were used. Increased dihydroceramide led to impairment of autophagic flux, resulting in increased TG storage in lipid droplets (LD) and upregulated expression of fibrosis markers. Hepatic stellate cells (HSCs, LX-2 cells) were co-cultured with Chang cells to assess the potential fibrogenic response to dihydroceramide, Treatment with rapamycin recovered autophagic flux in Chang cells and fibrogenesis in the co-culture system. These results identified a critical function of dihydroceramide metabolism in autophagy. It could play an important role in the progression of NAFLD associated with lipid over-accumulation. Therefore, preventing autophagic flux by regulating dihydroceramide could be a potential strategic approach for providing therapy for NAFLD.<br/> Taken together, these findings demonstrate that oxidative stress induces hepatic toxicity through impairment of autophagy. Also, increased dihydroceramide induces fibrosis response through impaired autophagic flux. Consequently, this study evaluated hepatotoxicity through impaired autophagic flux. Therefore, these results strongly suggest that therapies aimed to restore the autophagic flux might prevent or attenuate the progression of NAFLD.