Resveratrol Ameliorates Lipid Droplet Accumulation in Liver Through a SIRT1/ ATF6-Dependent Mechanism

Background/Aims: Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, and an increased accumulation of LDs in the liver is closely linked to hepatic steatosis. Our previous studies suggested that resveratrol (RSV) supplement could improve hepatic steatosis, but the underlying mechanism, particularly which related to LD accumulation, has not yet been elucidated. Methods: A high-fat diet (HFD) and palmitic acid were used to induce hepatic steatosis in mouse liver and hepatocytes, respectively. The effects of RSV on LD accumulation were analyzed in vivo and in vitro. The effects of RSV on the expression levels of LD-associated genes (ATF6, Fsp27β/CIDEC, CREBH, and PLIN1) were measured by qRT-PCR and western blot assays, followed by KD or overexpression of SIRT1 and ATF6 with small interfering RNAs or overexpressed plasmids, respectively. The dual luciferase reporter assay, chromatin immunoprecipitation assay, coimmunoprecipitation, and proximity ligation assay were utilized to clarify the mechanism of transcriptional regulation and possible interaction between SIRT1 and ATF6. Results: There was a significant increase in the accumulation of LDs in liver and hepatocytes during the process of HFD-induced steatosis, respectively, which was significantly inhibited by RSV supplementation. RSV notably activated SIRT1 expression and decreased the expression levels of ATF6, Fsp27β/CIDEC, CREBH, and PLIN1, which are associated with LD accumulation. Interestingly, the inhibitory effects of RSV on LD accumulation and the associated expression of genes in hepatocytes were abrogated or strengthened with SIRT1 silencing or overexpression, respectively. On the contrary, the benefits of RSV in hepatocytes were eliminated or aggravated when transfected with the overexpressed ATF6 or ATF6 siRNA, respectively. Furthermore, we found that RSV stimulated SIRT1 expression significantly, which was followed by increased deacetylation and inactivation of ATF6, resulting in a positive feedback loop for SIRT1 transcription associated with ATF6 binding to the SIRT1 promoter region. Conclusion: Taken together, these findings indicate that RSV supplementation improves hepatic steatosis by ameliorating the accumulation of LDs, and this might be partially mediated by a SIRT1/ATF6-dependent mechanism

Resveratrol promotes hepatic lipid decomposition by regulating ATGL/CGI58 and Rab7/LC3β through perilipin 2

Objective To investigate the effect of resveratrol (RSV) on enhancing hepatic lipolysis and lipophagy through perilipin 2 (PLIN2), and elucidate its underlying mechanism. Methods Human liver cell line HHL-5 was used to establish a cellular model of steatosis, and then the cells were divided into control group, palmitic acid (PA, model) group, siPLIN2 transfection group and RSV treatment groups (5, 10, 20, 40 μmol/L). CCK-8 assay, intracellular TG detection and Bodipy staining were adopted to detect the effects of different concentrations of RSV on cell viability and lipid degradation of the model cells. The expression of hepatic lipid decomposition related molecules and the changes of their average fluorescence intensity were determined by real-time qPCR, Western blotting and immunofluorescence staining, respectively. In addition, PLIN2 siRNA transfection technology was performed to verify the pathway of RSV promoting hepatic lipid decomposition. Results As compared with the control group, the intracellular TG content and the average fluorescence intensity of lipid droplets were notably increased in the PA group (P < 0.05), while the expression of hepatic lipolysis and lipophagy related molecules including PLIN2, ATGL, CGI58, Rab7, and LC3β were significantly decreased, with their mean fluorescence intensity and co-localization were declined as well (P < 0.05). After the treatment of 40 μmol/L RSV, the lipid accumulation of steatosis HHL-5 cells was reduced (P < 0.05), whereas the expression of lipolysis and lipophagy related molecules, their average fluorescence intensity and co-localization were elevated (P < 0.05). However, the knockdown of PLIN2 blocked RSV from regulating lipolysis and lipophagy related molecules and improving hepatocyte steatosis (P < 0.05). Conclusions RSV promotes the expression and interaction of ATGL/CGI58 and Rab7/LC3β through upregulating PLIN2, and thus enhances hepatic lipolysis and lipophagy

Effect of fruit juice on glucose control and insulin sensitivity in adults: a meta-analysis of 12 randomized controlled trials.

BACKGROUND: Diabetes mellitus has become a worldwide health problem. Whether fruit juice is beneficial in glycemic control is still inconclusive. This study aimed to synthesize evidence from randomized controlled trials on fruit juice in relationship to glucose control and insulin sensitivity. METHODS: A strategic literature search of PubMed, EMBASE, and the Cochrane Library (updated to March, 2014) was performed to retrieve the randomized controlled trials that evaluated the effects of fruit juice on glucose control and insulin sensitivity. Study quality was assessed using the Jadad scale. Weighted mean differences were calculated for net changes in the levels of fasting glucose, fasting insulin, hemoglobin A1c (HbA1c), and homeostatic model assessment of insulin resistance (HOMA-IR) using fixed- or random-effects model. Prespecified subgroup and sensitivity analyses were performed to explore the potential heterogeneity. RESULTS: Twelve trials comprising a total of 412 subjects were included in the current meta-analysis. The numbers of these studies that reported the data on fasting glucose, fasting insulin, HbA1c and HOMA-IR were 12, 5, 3 and 3, respectively. Fruit juice consumption did not show a significant effect on fasting glucose and insulin concentrations. The net change was 0.79 mg/dL (95% CI: -1.44, 3.02 mg/dL; P = 0.49) for fasting glucose concentrations and -0.74 µIU/ml (95% CI: -2.62, 1.14 µIU/ml; P = 0.44) for fasting insulin concentrations in the fixed-effects model. Subgroup analyses further suggested that the effect of fruit juice on fasting glucose concentrations was not influenced by population region, baseline glucose concentration, duration, type of fruit juice, glycemic index of fruit juice, fruit juice nutrient constitution, total polyphenols dose and Jadad score. CONCLUSION: This meta-analysis showed that fruit juice may have no overall effect on fasting glucose and insulin concentrations. More RCTs are warranted to further clarify the association between fruit juice and glycemic control

Role of PCSK9 in sulforaphane attenuating palmitic acid-induced autophagic flux in hepatic cells

Objective To determine the effect and underlying mechanism of sulforaphane (SFN) on hepatocyte injury and autophagy. Methods HHL5 cells were treated with 200 μmol/L palmitic acid (PA) for 24 h to establish a hepatocyte injury model. Then the model was treated with 5 μmol/L SFN and co-cultured with 200 μmol/L PA for 24 h. So there were 3 groups of cells, that is, control group, PA group, and PA+SFN group. Cell viability, malonaldehyde (MDA) content, and production of reactive oxygen species (ROS) were measured with CCk-8 assay, MDA reagent kit, and CellROXTM Deep Red Reagent, respectively. qPCR was used to detect the transcription levels of IL-1β and TNF-α, and Western blotting was employed to measure the protein expression of SQSTM1 and LC3II. The total RNA of the cell was extracted by TRIzol reagent to sequence the whole gene transcriptome, and the RNA sequence was analyzed to figure out differentially expressed genes. And the results were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, HHL5 cells were pretreated with PCSK9 siRNA for 24 h, then the mRNA level of PCSK9 was measured by qPCR, and the expression of SQSTM1 and LC3 II were measured by Western blotting. Results Compared with the PA group, the cell survival rate was increased, and the levels of MDA and ROS were decreased significantly in the PA+SFN group (P < 0.05). The transcriptional levels of IL-1β and TNF-α were reduced obviously in the PA+SFN group (P < 0.05). The expression of SQSTM1 was decreased and that of LC3II was increased in the PA+SFN group. KEGG enrichment analysis suggested that differential expressed genes were enriched on the autophagy pathway. PCSK9 was selected as our candidate gene. Compared with the control group, the transcriptional level of PCSK9 was increased in the PA group, while was decreased significantly in the PA+SFN group (P < 0.05). The level was elevated in the PA group but decreased after SFN intervention. After PCSK9 knockdown, the ROS level in the PA group was decreased compared with that in the PA group without siPCSK9 treatment, which was consistent with the trend of SFN-induced ROS reduction. After PCSK9 knockdown, the expression level of LC3II was increased, which was consistent with the trend of SFN-induced autophagy flux recovery. Conclusion SFN can attenuate hepatocyte injury induced by PA, which may be related to the inhibition of PCSK9 at transcriptional level, thus regulating autophagic flux

3,6-Dihydroxyflavone regulates microRNA-34a through DNA methylation

Abstract Background Breast cancer is the common cancer in China. In previous study, we determined that 3,6-dihydroxyflavone (3,6-DHF) increases miR-34a significantly in breast carcinogenesis, but the mechanism remains unclear. Methods We used qRT-PCR to analyze miR-34a and ten-eleven translocation (TET)1, TET2, TET3 levels in breast cancer cells. With a cellular breast carcinogenesis model and an experimental model of carcinogenesis in rats, TET1 levels were evaluated by western blot analysis and immunofluorescence. TET1 and 5hmC (5-hydroxymethylcytosine) levels were evaluated by immunofluorescence in nude mouse xenografts of MDA-MB-231 cells. Chromatin immunoprecipitation(ChIP) assayed for TET1 on the TET1 promoter, and dot blot analysis of DNA 5hmC was performed in MDA-MB-231 cells. We evaluated the mechanism of 3,6-DHF on the expression of tumor suppressor miR-34a by transfecting them with DNA methyltransferase (DNMT)1 plasmid and TET1 siRNA in breast cancer cells. Methylation-specific PCR detected methylation of the miR-34a promoter. Results First, we found that 3,6-DHF promotes the expression of TET1 during carcinogen-induced breast carcinogenesis in MCF10A cells and in rats. 3,6-DHF also increased TET1 and 5hmC levels in MDA-MB-231 cells. Further study indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibited the 3,6-DHF reactivation effect on expression of miR-34a in breast cancer cells. Methylation-specific PCR assays indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibit the effect of 3,6-DHF on the demethylation of the miR-34a promoter. Conclusions Our study showed that 3,6-DHF effectively increases TET1 expression by inhibiting DNMT1 and DNA hypermethylation, and consequently up-regulates miR-34a in breast carcinogenesis

Dihydromyricetin ameliorates liver fibrosis via inhibition of hepatic stellate cells by inducing autophagy and natural killer cell-mediated killing effect

Abstract Background This study investigated the mechanisms underlying the preventive effect of dihydromyricetin (DHM) against liver fibrosis involving hepatic stellate cells (HSCs) and hepatic natural killer (NK) cells. Methods A carbon tetrachloride (CCl4)-induced liver fibrosis model was established in C57BL/6 mice to study the antifibrotic effect of DHM based on serum biochemical parameters, histological and immunofluorescence stainings, and the expression of several fibrosis-related markers. Based on the immunoregulatory role of DHM, the effect of DHM on NK cell activation ex vivo was evaluated by flow cytometry. Then, we investigated whether DHM-induced autophagy was involved in HSCs inactivation using enzyme-linked immunosorbent assays, transmission electron microscopy, and western blot analysis. Thereafter, the role of DHM in NK cell-mediated killing was studied by in vitro coculture of NK cells and HSCs, with subsequent analysis by flow cytometry. Finally, the mechanism by which DHM regulates NK cells was studied by western blot analysis. Results DHM ameliorated liver fibrosis in C57BL/6 mice, as characterized by decreased serum alanine transaminase and aspartate transaminase levels, decreased expressions of collagen I alpha 1 (CoL-1α1), collagen I alpha 2 (CoL-1α2), tissue inhibitor of metalloproteinases 1 (TIMP-1), α-smooth muscle actin (α-SMA) and desmin, as well as increased expression of matrix metalloproteinase 1 (MMP1). Interestingly, HSCs activation was significantly inhibited by DHM in vivo and in vitro. As expected, DHM also upregulated autophagy-related indicators in liver from CCl4-treated mice. DHM also prevented TGF-β1-induced activation of HSCs in vitro by initiating autophagic flux. In contrast, the autophagy inhibitor 3-methyladenine markedly abolished the antifibrotic effect of DHM. Surprisingly, the frequency of activated intrahepatic NK cells was significantly elevated by DHM ex vivo. Furthermore, DHM enhanced NK cell-mediated killing of HSCs by increasing IFN-γ expression, which was abolished by an anti-IFN-γ neutralizing antibody. Mechanistically, DHM-induced IFN-γ expression was through AhR-NF-κB/STAT3 pathway in NK cells. Conclusion These results demonstrated that DHM can ameliorate the progression of liver fibrosis and inhibition of HSCs activation by inducing autophagy and enhancing NK cell-mediated killing through the AhR-NF-κB/STAT3-IFN-γ signaling pathway, providing new insights into the preventive role of DHM in liver fibrosis

Equol Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice by Inhibiting Endoplasmic Reticulum Stress via Activation of Nrf2 in Endothelial Cells.

The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway

Flavonoids, flavonoid subclasses and breast cancer risk: a meta-analysis of epidemiologic studies.

Studies have suggested the chemopreventive effects of flavonoids on carcinogenesis. Yet numbers of epidemiologic studies assessing dietary flavonoids and breast cancer risk have yielded inconsistent results. The association between flavonoids, flavonoid subclasses (flavonols, flavan-3-ols, etc.) and the risk of breast cancer lacks systematic analysis.We aimed to examine the association between flavonoids, each flavonoid subclass (except isoflavones) and the risk of breast cancer by conducting a meta-analysis.We searched for all relevant studies with a prospective cohort or case-control study design published before July 1(st), 2012, using Cochrane library, MEDLINE, EMBASE and PUBMED. Summary relative risks (RR) were calculated using fixed- or random-effects models. All analyses were performed using STATA version 10.0.Twelve studies were included, involving 9 513 cases and 181 906 controls, six of which were prospective cohort studies, and six were case-control studies. We calculated the summary RRs of breast cancer risk for the highest vs lowest categories of each flavonoid subclass respectively. The risk of breast cancer significantly decreased in women with high intake of flavonols (RR=0.88, 95% CI 0.80-0.98) and flavones (RR=0.83, 95% CI: 0.76-0.91) compared with that in those with low intake of flavonols and flavones. However, no significant association of flavan-3-ols (RR=0.93, 95% CI: 0.84-1.02), flavanones (summary RR=0.95, 95% CI: 0.88-1.03), anthocyanins (summary RR=0.97, 95% CI: 0.87-1.08) or total flavonoids (summary RR=0.98, 95% CI: 0.86-1.12) intake with breast cancer risk was observed. Furthermore, summary RRs of 3 case-control studies stratified by menopausal status suggested flavonols, flavones or flavan-3-ols intake is associated with a significant reduced risk of breast cancer in post-menopausal while not in pre-menopausal women.The present study suggests the intake of flavonols and flavones, but not other flavonoid subclasses or total flavonoids, is associated with a decreased risk of breast cancer, especially among post-menopausal women

Characteristics of 12 randomized controlled trials included in analysis.

<p>The studies by Reshef (2005), Cerda (2006), Hollis (2010), Dohadwala (2010), Dohadwala (2011), and Basu (2011) used fruit juice with polyphenols as main nutrients, and the studies by Summer (2005), Bannni (2006), Basu (2010a), Basu (2010b), Gonzalez-Ortiz (2011), and Morand (2011) used fruit juice with multi-nutrients (polyphenols, vitamins, and sugar et al.); a usual diet was similar to a conventional diet.</p><p>NR, not reported.</p