Bifendate inhibits autophagy at multiple steps and attenuates oleic acid-induced lipid accumulation.

Some traditional Chinese medicines exert roles in the therapy of liver diseases by modulating autophagy. Bifendate (DDB), a synthetic intermediate of Schisandrin C extracted from Schisandrae chinensis, is clinically used to treat hepatitis in China. While DDB is a positive control to research some potential hepatoprotective agents, its related molecular mechanisms are unknown. In this study, we show that DDB inhibited autophagosome-lysosome fusion, lysosome acidification and autophagic lysosome reformation. Moreover, DDB attenuated oleic acid-induced lipid droplet accumulation. These findings reveal the effects of DDB on the autophagy-related processes and lysosomal function, and also provide a possibility to understand the bioactivity mechanism of DDB in the future.

Resveratrol Attenuates High-Fat Diet-Induced Hepatic Lipotoxicity by Upregulating Bmi-1 Expression.

Resveratrol (RES), a natural polyphenol phytoalexin, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD). However, its roles on protection of liver from lipotoxicity and underlying mechanism are not fully understood. In this study, we investigated the impacts of RES on alleviating hepatic lipotoxicity and corresponding molecular mechanism. Impacts of RES on oleic acid (OA)-induced lipotoxicity were assessed in L02 cells and C57BL/6J mice, respectively. In L02 cells, lipotoxicity was assessed by detection of apoptosis, mitochondrial function, oxidative stress and ROS-related signaling. In mice, lipotoxicity was evaluated by detecting hepatic function, serum enzyme activity, and reactive oxygen species (ROS) levels. We found that RES reduced OA-induced apoptosis, mitochondrial dysfunction, ROS generation, and DNA damage in L02 cells. RES also decreased expression of cleaved caspase-3 and p53 and increased expression of B-cell lymphoma 2 (Bcl-2). Importantly, RES protected mice from high-fat diet-induced hepatic lipotoxicity, demonstrated by reduced ROS levels and lipid peroxidation. Mechanically, B lymphoma Mo-MLV insertion region 1 (Bmi-1) expression and antioxidative superoxide dismutase were increased after RES treatment. Further mechanistic analysis indicated that protection effects of RES against OA-induced lipotoxicity were abrogated by Bmi-1 small interference RNA (siRNA) in L02 cells. SIGNIFICANCE STATEMENT: Results from clinical studies about the effect of RES on NAFLD are inconsistent and inconclusive. This study confirms the protective role of RES as an anti-ROS agent and its ability to alleviate DNA damage through a pathway involving p53/p21 signaling. Further mechanistic analysis indicated that protection effects of RES were relative with Bmi-1. This is the first study on the role of Bmi-1 in the pathogenesis of NAFLD and the target of resveratrol against NAFLD.

Recycling of autophagosomal components from autolysosomes by the recycler complex.

Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.

STING controls energy stress-induced autophagy and energy metabolism via STX17.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

Delivery of miR-375 and doxorubicin hydrochloride by lipid-coated hollow mesoporous silica nanoparticles to overcome multiple drug resistance in hepatocellular carcinoma.

Multidrug resistance (MDR) due to overexpression of P-glycoprotein (P-gp) is a major obstacle that hinders the treatment of hepatocellular carcinoma (HCC). It has been shown that miR-375 inhibits P-gp expression via inhibition of astrocyte elevated gene-1 (AEG-1) expression in HCC, and induces apoptosis in HCC cells by targeting AEG-1 and YAP1. In this study, we prepared lipid-coated hollow mesoporous silica nanoparticles (LH) containing doxorubicin hydrochloride (DOX) and miR-375 (LHD/miR-375) to deliver the two agents into MDR HCC cells in vitro and in vivo. We found that LHD/miR-375 overcame drug efflux and delivered miR-375 and DOX into MDR HepG2/ADR cells or HCC tissues. MiR-375 delivered by LHD/miR-375 was taken up through phagocytosis and clathrin- and caveolae-mediated endocytosis. Following release from late endosomes, it repressed the expression of P-gp in HepG2/ADR cells. The synergistic effects of miR-375 and hollow mesoporous silica nanoparticles (HMSN) resulted in a profound increase in the uptake of DOX by the HCC cells and prevented HCC cell growth. Enhanced antitumor effects of LHD/miR-375 were also validated in HCC xenografts and primary tumors; however, no significant toxicity was observed. Mechanistic studies also revealed that miR-375 and DOX exerted a synergistic antitumor effect by promoting apoptosis. Our study illustrates that delivery of miR-375 using HMSN is a feasible approach to circumvent MDR in the management of HCC. It, therefore, merits further development for potential clinical application.

Efficacy of Resveratrol Supplementation against Non-Alcoholic Fatty Liver Disease: A Meta-Analysis of Placebo-Controlled Clinical Trials.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with rising prevalence. Increasing evidence has demonstrated that resveratrol, a dietary phytochemical, is capable of attenuating NAFLD development and progression; however, results from clinical studies are inconsistent and inconclusive. Here, we conducted a meta-analysis to evaluate the efficacy of resveratrol on NAFLD, using several parameters to provide new insights for clinical application. We systematically searched EMBASE, PubMed, Science Citation Index, Elsevier, and Cochrane Library databases for studies published up to date (July 2016), in English, to identify and screen eligible, relevant studies. Either a fixed-effect model or random model was used to estimate mean difference (MD) and 95% confidence intervals (CIs) for the effect of resveratrol on NAFLD. Four randomized, double-blinded, placebo-controlled trials involving 156 patients were included in the meta-analysis. Levels of low-density lipoprotein (MD = 0.47, 95% CI: 0.21, 0.74, P < 0.05) and total cholesterol (MD = 0.49, 95% CI: 0.18, 0.80, P < 0.05) were higher in the resveratrol treatment groups than in placebo control groups, whereas other parameters were not altered. Overall, this study indicates that resveratrol treatment has negligible effects on attenuating NAFLD, given the small improvement in NAFLD features. More high-quality clinical trials of resveratrol for NAFLD are required to confirm these results.

Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets.

Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells (HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis.

Gold nanoparticles delivered miR-375 for treatment of hepatocellular carcinoma.

MiR-375 is a tumor suppressor miRNA that is downregulated in hepatocellular carcinoma (HCC). However, due to the lack of effective delivery strategies, miR-375 replacement as a therapy for HCC has not been investigated. In the present study, we have developed a straightforward strategy to deliver miR-375 into HCC cells by assembling miR-375 mimics on the surface of AuNPs and forming AuNP-miR-375 nanoparticles. AuNP-miR-375 exhibits high cellular uptake and preserves miR-375's activities to suppress cellular proliferation, migration/invasion, and colony formation, and to induce apoptosis in HCC cells. Furthermore, AuNP-delivered miR-375 efficiently downregulated its target genes through RNA interference. In primary and xenograft tumor mouse models, AuNP-miR-375 showed high tumor uptake, therapeutic efficacy, and no apparent toxicity to the host mice. In conclusion, our findings indicate that AuNPs is a reliable strategy to deliver miR-375 into HCC cells and tissue, and that AuNP-miR-375 has the potential in the clinic for treatment of unresectable HCC.

Knockdown of Bmi1 inhibits bladder cancer cell growth both in vitro and in vivo by blocking cell cycle at G1 phase and inducing apoptosis.

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.