Gymnemic acid I triggers mechanistic target of rapamycin-mediated ß cells cytoprotection through the promotion of autophagy under high glucose stress.

Gymnemic acid I (GA I) is a bioactive component of Gymnema sylvestre. It is an Indian traditional medicinal herb which has antidiabetic effect. However, the molecular mechanism is remaining to be elucidated. Here, we showed that high glucose promoted the rate of apoptosis, GA I decreased the apoptosis under the high glucose stress. Our further study explored that GA I increased the number of autophagosome and the ratio of light chain 3-I (LC3-I)/LC3-II in MIN-6 cells under the normal or high glucose stress by the methods of western blot analysis and immunofluorescence. It induced autophagy flux and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase ß-1 (p70 S6K/S6K1), which is a substrate of mTOR. GA I decreased the rate of apoptosis and the activity of caspase-3 under the high glucose stress. The inhibition of apoptosis and caspase-3 activity by GA I were increased after treating with autophagy inhibitor in mouse islet ß cells MIN-6. Our data suggested that GA I-induced autophagy protected MIN-6 cells from apoptosis under high glucose stress via inhibition the phosphorylation activity of mTOR.

Organosulfur compounds induce cytoprotective autophagy against apoptosis by inhibiting mTOR phosphorylation activity in macrophages.

Organosulfur compounds (OSCs) are the bioactive components of garlic. Some OSCs have apoptotic or autophagy-inducing effects. Autophagy plays roles in both cytoprotection and apoptosis-related cell death, and the interaction between autophagy and apoptosis is important in the modulation of immune responses. The mechanism of an OSC-mediated effect via the interaction of autophagy and apoptosis is unknown. In this study, the effects of five OSC compounds on autophagy in the macrophage cell line RAW264.7 and primary macrophages were investigated. We found that S-allylcysteine (SAC), diallyl disulde (DADS) and diallyl tetrasulfide (DTS) treatment increased the number of autophagosomes of RAW264.7 cells, inhibited the phosphorylation of ribosomal protein S6 kinase beta-1 (p70S6K/S6K1) which is a substrate of mammalian target of rapamycin (mTOR), and significantly enhanced autophagy flux. The induction of autophagy by SAC, DADS and DTS was inhibited by stably knocking down the expression of autophagy-related gene 5 (ATG5) with short hairpin RNA (shRNA). Further experiments confirmed that SAC, DADS and DTS also induced apoptosis in RAW264.7 cells. The induction of apoptosis and Caspase 3 activity by SAC, DADS and DTS were increased by stably knocking down of ATG5 expression with shRNA in RAW264.7 cells or treating with 5 mM 3-MA in primary macrophages. Our results suggest that SAC, DADS and DTS induce both autophagy and apoptosis. The autophagy induction protects macrophages from apoptosis by inhibiting mTOR phosphorylation activity to maintain the mass of immune cells.

Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy.

Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs). The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN) is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl) is widely used to treat rheumatoid arthritis (RA). However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP) in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA) was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3) puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS)-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.