Mechanism Of Anti-inflammatory Effects Of Morin Hydrate In Atherosclerosis

BackgroundsAtherosclerosis (AS) is the most common pathological process associated with serious cardiovascular diseases, such as stroke, myocardial infarction and peripheral vascular disease, which is seriously harm to heath. Current research reveals that atherosclerosis initiates the inflammatory injury of endothelial cells, and participates in all of progression of atherosclerosis. Therefore, the researches on mechanism of anti-inflammation and drug therapies are serious highlights in atherosclerosis. Morin hydrate (MO), a bioflavonoid, is isolated as a yellowish pigment from in a number of fruits, vegetables, and herbs of the Moraceae family, which has been reported to exert a variety of beneficial pharmacological effects including anti-oxidation, anti-inflammation, antitumor and apoptosis induction. However, t whether MO has anti-AS action based its anti-inflammation and the molecular mechanism of anti-inflammation is unclear.Autophagy, or called self-digestion, is a cellular pathway involved in protein and organelle degradation in eukaryotic cell, with an astonishing number of connections to human disease, including myocardial remodeling, ischemia-reperfusion, cancer, aging and neurodegenerative diseases, such as Parkinson disease (PD), Huntington disease (HD) and Alzheimer’s disease (AD). The presence of MO has recently been shown to increase the level of intracellular cAMP. We speculate that MO may exert anti-inflammation mediated by autophagy induction via activating cAMP/PKA/AMPK/SIRT1 signaling pathway. Therefore, this study mainly focuses on investigatingthe anti-AS effects of MO in vivo and its molecular mechanism of anti-inflammatory effects in vitro.MethodsIn vitro assay:TNF-induced inflammation on primary human umbilical vein endothelial cells to mimic the inflammatory injury of atherosclerosis. CCK-8 assay was performed to determine the non-cytotoxic concentrations of TNF-a and morin hydrate to be used in experiments. ELISA and Western blotting analysis were employed to detect effects of morin hydrate on the expressions of inflammatory cytokines (ICAM-1, MMP-9 and COX-2) in HUVECs exposed to TNF-a. The increase of MAP1LC3B2 expression and the decrease of SQSTM1/P62 were regarded as autophagic biomarkers. Autophagic inhibitors (3-methyladenine, chloroquine, ATG and BECN1 siRNA), and LysoTracker Green fluorescent dye were used to determine effects of morin hydrate on autophagy in normal HUVECs and TNF-stimulated HUVECs. The expressions of PKA, AMPK, p-AMPK, SIRT1 was detected by western blotting analysis. In addition, SIRT1 inhibitors (EX527 and VPP), ADCY activator (KH7), and PDE4 inhibitors (Rolipram), ELISA was used to evaluate the relationship betweenthe anti-inflammation, autophagy induction of morin hydrate and cAMP/PKA/AMPK/SIRT1 signaling pathway in HUVECs.In vivo assay:The effects of morin hydrate in vivo were performed in an ApoE-/-atherosclerosis mouse model fed with high fat diet. The mice were randomly divided into three groups (n=6/group):vehicle (2% tween 80) group,30 mg/kg and 100 mg/kg MO groups. Either MO or vehicle was treated by p.o. for ten weeks. At the end of the experiment, body weight was obtained, blood samples were taken from the retro-orbital sinus of each mouse into a heparin-containing tube. Serum lipid levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were measured by biochemistry automatic analyzer. For en face analysis, the aortas from the ascending arch to the iliac bifurcation were prepared and cleaned of peripheral tissue, opened longitudinally, pinned flat, and stained with Oil-Red O for examination of pathological changes and lipid deposition.ResultsIn vitro studies:TNF-a at concentrations of up to 20 ng/mL and MO at concentrations of up to 20 μM was tolerated by HUVECs without significant change of cell viability. Thus, TNF-α (10 ng/mL) were used to induce inflammation on HUVECs with no effect on cell viability. Preincubation of the cells with MO (1,10, 20 μM) before exposure to TNF-a significantly suppressed the expressions of ICAM-1, MMP-9 and COX-2, as well as soluble ICAM-1 level, in a MO dose-dependent manner in comparison to TNF-treated cells. Subsequently, we found that treatment of the cells with MO increased the expression of MAP1LC3B2 and decreased the expression of SQSTM1/p62 in dose-and time-dependent manners, which indicated that MO could induce autophagy in both normal HUVECs and TNF-stimulated HUVECs. In the presence of CQ, the autophagy induction of MO in TNF-stimulated HUVECs was inhibited significantly. In addition,3-MA, CQ, ATG5 or BECN1 siRNA attenuated the inhibition of MO on the increased expressions of inflammatory factors (ICAM-1, MMP-9 and COX-2) in TNF-stimulated HUVECs. Pretreatment of the cells with MO with or without TNF-a remarkably increased cAMP levels compared with control group. Expression levels of PKA, p-AMPK (phosphorylated AMPK) and SIRT1 in HUVECs untreated or treated with TNF-a were increased in response to MO in a dose-and time-dependent manner. After treatment with SIRT1 inhibitor (EX527 or VPP), both the anti-inflammation and autophagy induction of MO were inhibited. These results indicated that MO exerted anti-inflammatory effects on HUVECs by autophagy induction through activation of cAMP/PKA/AMPK/SIRT1 signaling pathway.In vivo studies:After treatment of MO for 10 weeks, no differences of body weight occurred between MO-treated and-untreated mice. Elevation of TG, TC and LDL levels were observed in mice with high fat diet. MO decreased the serum levels of LDL, TC, and TG significantly in comparison with the vehicle group. The relative en face lesion area of the aorta was reduced in MO-treated group compared with the vehicle group, and 100 mg/kg dose group of MO showed stronger reduction than 30 mg/kg dose group of MO.ConclusionIn conclusion, MO reduces the expressions of inflammatory cytokines (ICAM-1, COX-2 and MMP-9) in TNF-stimulated HUVECs. The anti-inflammatory effect of MO is at least partly dependent on autophagy induction and is involved in cAMP/PKA/AMPK/SIRT1 signal transduction. The anti-inflammatory effect of MO in atherosclerosis is associated with autophagy induction through activation of the cAMP/PKA/AMPK/SIRT1 signaling pathway. Thus, morin hydrate was a promising anti-atherosclerosis agent for AS treatment.