Xinyu Liang

Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor α (TNF/TNFα)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 β 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A1 (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.

Ampelopsin Induces Cell Growth Inhibition and Apoptosis in Breast Cancer Cells through ROS Generation and Endoplasmic Reticulum Stress Pathway

Ampelopsin (AMP), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. In this study, we investigated its anti-cancer activity in human breast cancer cell lines, and explored the underlying mechanism of this action. Our results showed that treatment with AMP dose-dependently inhibited cell viability and induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells without cytotoxicity in human normal breast epithelial cells MCF-10A. Meanwhile, AMP dose- dependently triggered reactive oxygen species (ROS) generation in both breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) strongly attenuated AMP-induced ROS production, along with cell growth inhibition and apoptosis. Furthermore, AMP was observed to activate endoplasmic reticulum (ER) stress, as evidenced by the up-regulation of ER stress-related proteins, including GRP78, p-PERK, p-elF2α, cleaved ATF6α and CHOP, while knockdown of ATF6α or PERK markedly down-regulated AMP-induced CHOP expression. Blocking ER stress using 4-phenylbutyric acid not only down-regulated AMP-induced GRP78 and CHOP expression, but also significantly decreased AMP-induced cell growth inhibition and apoptosis, whereas ER stress inducer thapsigargin played opposing effects. Additionally, NAC inhibited AMP-induced ER stress by down-regulating GRP78 and CHOP expression. Conversely, blocking ER stress using CHOP siRNA decreased AMP-induced ROS production and cell apoptosis. Taken together, these results demonstrate that AMP has anti-tumor effects against breast cancer cells through ROS generation and ER stress pathway, which therefore provide experimental evidences for developing AMP as a new therapeutic drug for breast cancer.

Dihydromyricetin improves skeletal muscle insulin resistance by inducing autophagy via the AMPK signaling pathway.

Skeletal muscle insulin resistance (SMIR) plays an important role in the pathogenesis of type 2 diabetes. Dihydromyricetin (DHM), a natural flavonoid, exerts various bioactivities including anti-oxidative and hepatoprotective effects. Herein, we intended to determine the effect of DHM on SMIR and the underlying mechanisms. We found that DHM increased the expression of phosphorylated insulin receptor substrate-1, phosphorylated Akt and glucose uptake capacity in palmitate-treated L6 myotubes under insulin-stimulated conditions. The expression of light chain 3, Beclin 1, autophagy-related gene 5 (Atg5), the degradation of sequestosome 1 and the formation of autophagosomes were also upregulated by DHM. Suppression of autophagy by 3-methyladenine and bafilomycin A1 or Atg5 and Beclin1 siRNA abolished the favorable effects of DHM on SMIR. Furthermore, DHM increased the levels of phosphorylated AMP-activated protein kinase (AMPK) and Ulk1, and decreased phosphorylated mTOR levels. AMPK inhibitor compound C (CC) and AMPK siRNA abrogated DHM-induced autophagy, subsequently suppressed DHM-induced SMIR improvement. Additionally, DHM inhibited the activity of F1F0-ATPase thereby activating AMPK. Finally, the results of in vivo study conducted in high fat diet-fed rats were consistent with the findings of in vitro study. In conclusion, DHM improved SMIR by inducing autophagy via the activation of AMPK signaling pathway.

Ampelopsin-induced autophagy protects breast cancer cells from apoptosis through Akt-mTOR pathway via endoplasmic reticulum stress.

Our previous study has shown that ampelopsin (AMP), a flavonol mainly found in Ampelopsis grossedentata, could induce cell death in human breast cancer cells via reactive oxygen species generation and endoplasmic reticulum (ER) stress pathway. Here, we examined whether autophagy is activated in AMP‐treated breast cancer cells and, if so, sought to find the exact role and underlying molecular profile of autophagy in AMP‐induced cell death. Our results showed that AMP treatment activated autophagy in MDA‐MB‐231 and MCF‐7 breast cancer cells, as evidenced by the accumulation of autophagosomes, an increase of microtubule‐associated protein 1 light chain 3 beta‐2 (LC3B‐II) and the conversion of LC3B‐I to LC3B‐II, the degradation of the selective autophagic target p62/SQSTM1, and the formation of green fluorescent protein (GFP)‐LC3 puncta. Blockage of autophagy augmented AMP‐induced cell death, suggesting that autophagy has cytoprotective effects. Meanwhile, AMP treatment suppressed Akt‐mammalian target of rapamycin (mTOR) pathway as evidenced by dose‐ and time‐dependent decrease of the phosphorylation of Akt, mTOR and ribosomal protein S6 kinase (p70S6K), whereas Akt activator insulin‐like growth factor‐1 (IGF‐1) pretreatment partially restored Akt‐mTOR pathway inhibited by AMP and decreased AMP‐inuduced autophagy, signifying that AMP activated autophagy via inhibition of the Akt‐mTOR pathway. Additionally, blocking ER stress not only reduced autophagy induction, but also alleviated inhibition of the Akt‐mTOR pathway induced by AMP, suggesting that activation of ER stress was involved in induction of autophagy and inhibition of the Akt‐mTOR pathway. Taken together, these findings indicate that AMP induces protective autophagy in human breast cancer cells through Akt‐mTOR pathway via ER stress.

Estrogen Receptor and PI3K/Akt Signaling Pathway Involvement in S-(-)Equol-Induced Activation of Nrf2/ARE in Endothelial Cells

S-(-)equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-)equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-)equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-)equol (10–250 nM) increases nuclear factor-erythroid 2-related factor 2 (Nrf2) as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1) and NAD(P)H (nicotinamide-adenine-dinucleotide-phosphate) quinone oxidoreductase 1 (NQO1). Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-)equol exposure. S-(-)equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and LY294002) increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-)equol. In addition, DPN treatment (an ERβ agonist) induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP) impaired S-(-)equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-)equol significantly improved cell survival in response to H2O2 or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-)equol to protect against H2O2-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-)equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases.

Ampelopsin protects endothelial cells from hyperglycemia‐induced oxidative damage by inducing autophagy via the AMPK signaling pathway

Diabetic angiopathy is a major diabetes-specific complication that often begins with endothelial dysfunction induced by hyperglycemia; however, the pathological mechanisms of this progression remain unclear. Ampelopsin is a natural flavonol that has strong antioxidant activity, but little information is available regarding its antidiabetic effect. This study focused on the effect of ampelopsin on hyperglycemia-induced oxidative damage and the underlying mechanism of this effect in human umbilical vein endothelial cells (HUVECs). We found that hyperglycemia impaired autophagy in HUVECs through the inhibition of AMP-activated protein kinase (AMPK), which directly led to endothelial cell damage. Ampelopsin significantly attenuated the detrimental effect of hyperglycemia-induced cell dysfunction in a concentration-dependent manner in HUVECs. Ampelopsin significantly upregulated LC3-II, Beclin1, and Atg5 protein levels but downregulated p62 protein levels in HUVECs. Transmission electron microscopy and confocal microscopy indicated that ampelopsin notably induced autophagosomes and LC3-II dots, respectively. Additionally, the autophagy-specific inhibitor 3-MA, as well as Atg5 and Beclin1 siRNA pretreatment, markedly attenuated ampelopsin-induced autophagy, which subsequently abolished the protective effect of ampelopsin against hyperglycemia in HUVECs. Moreover, ampelopsin also increased AMPK activity and inhibited mTOR (mammalian target of rapamycin) complex activation. Ampelopsin-induced autophagy was attenuated by the AMPK antagonist compound C but strengthened by the AMPK agonist AICAR (5-minoimidazole-4-carboxamide ribonucleotide). Furthermore, AMPK siRNA transfection eliminated ampelopsins alleviation of cell injury induced by hyperglycemia. The protective effect of ampelopsin against hyperglycemia-induced cell damage, which functions by targeting autophagy via AMPK activation, makes it a promising pharmacological treatment for type-2 diabetes.

Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis.