| Insulin resistance is a marker of metabolic disorders and is a risk factor for metabolic syndrome such as obesity,diabetes,and cardiovascular disease.Epidemiological studies have shown that consumption of vegetables and fruits rich in flavonoids is associated with physical health benefits,which has the effect of preventing insulin resistance and inhibiting obesity and other metabolic diseases.The health effects of the diet are related to the interaction of multiple active ingredients in food.Quercetin and epigallocatechin-3-gallate(EGCG)are two flavonoids that are highly abundant in foods and edible plants.Previous studies have found that they have synergistic anti-diabetic effects.However,the underlying mechanisms of interaction between quercetin and EGCG remain unclear.Studies have found that phytochemicals in human diets can activate or inhibit the expression of different micro RNAs and their target genes,thereby altering different biological processes involved in cell function.Therefore,based on our previous study,we established an obese model in C57BL/6J mice induced by high fat and high fructose diet(HFFD),and systematically analyzed the protective effects of quercetin and EGCG on obesity,glycolipid homeostasis and insulin resistance.In order to explore its intrinsic molecular mechanism,palmitic acid(PA)induced Hep G2 hepatocytes insulin resistance model and tumor necrosis factor-α(TNF-α)induced 3T3-L1 mature adipocytes insulin resistance model were established.The targets and signaling pathways of quercetin and EGCG synergistically improve insulin resistance and glucose and lipid metabolism disorders at the post-transcriptional level were analyzed from the perspective of micro RNA,which provided a new theoretical basis for elucidating the interactive mechanism of quercetin and EGCG.The main results are summarized as follows:(1)The protective effect of quercetin and EGCG on obese mice induced by high calorie diet.The results showed that dietary supplementation of quercetin and EGCG for 10 weeks was significantly(P < 0.05)reversed HFFD-induced weight gain,increased fasting glucose level,decreased glucose tolerance,abnormal lipid level and insulin resistance in obese mice,and the effect was stronger(P < 0.05)than quercetin or EGCG dietary supplement alone.The combined effects of quercetin and EGCG on obesity,glucose and lipid homeostasis and insulin resistance were preliminarily confirmed.(2)The ameliorative effect and the molecular mechanism of quercetin and EGCG on hepatic insulin resistance and glucose metabolism in obese mice.The results showed that cotreatment of quercetin and EGCG could reverse the abnormal liver structure,the reduction of glycogen content and the accumulation of lipid droplet in HFFD-induced obese mice.Further analysis showed that the combination of quercetin and EGCG could improve the expression of genes involved in glucose and lipid metabolism.In addition,quercetin combined with EGCG significantly(P < 0.05)increased the activities of antioxidant enzymes and reduced the accumulation of malondialdehyde(MDA)in liver tissue,which was closely related to the combination of quercetin and EGCG regulated the Keap1/Nrf2/ARE signaling pathway to resist HFFD-induced oxidative stress in liver tissue.In addition,dietary supplementation of quercetin and EGCG improved glucose metabolism and hepatic insulin resistance by activating IRS-1/AKT/FOXO1/PEPCK/G6 Pase and IRS-1/AKT/GLUT4 signaling pathways.(3)Micro RNAs mediated the synergistic protection of quercetin and EGCG on PAinduced Hep G2 insulin resistance.The results showed that the combination of quercetin and EGCG(5 μM + 5 μM)significantly(P < 0.05)reversed the increase of glucose content in PA(0.25 m M)induced Hep G2 cells,and there was no significant(P > 0.05)difference in cell survival rate among different treatment groups.Further analysis about the effects of quercetin and EGCG on glucose homeostasis,the results showed that the combined treatment significantly(P < 0.05)promoted glycogen synthesis,hexokinase(HK)activity and inhibited gluconeogenic enzyme activity.The combination of quercetin and EGCG inhibited gluconeogenesis by regulating the expression of FOXO1,PEPCK and G6 Pase.Further analysis showed that when FOXO1 was silenced,the synergistic inhibitory effect of quercetin and EGCG on glucose content was disappeared,and the inhibitory ability of quercetin and EGCG on the expression of PEPCK and G6 pase was significantly(P < 0.05)enhanced.The above results indicated that FOXO1 plays a key role in the synergistic effect of quercetin and EGCG.Next,we used multiple bioinformatics tools to find mi RNAs that could target FOXO1.Through screening,overlap analysis,and RT-q PCR,the results shown that co-treatment quercetin and EGCG significantly(P < 0.05)promoted the expression of mi R-27a-3p and mi R-96-5p,and a direct targeted regulatory relationship between FOXO1 and mi R-27a-3p or mi R-96-5p was confirmed.Mi R-27a-3p and mi R-96-5p could jointly regulate FOXO1 to participate in the synergistic protective effect of quercetin and EGCG.When mi R-27a-3p and mi R-96-5p were inhibited together,the synergistic effect of quercetin and EGCG on reducing glucose content disappeared,and the synergistic ability of quercetin and EGCG to inhibit FOXO1 expression was significantly(P < 0.05)weakened.These results suggested that quercetin and EGCG synergistically inhibited gluconeogenesis and improved hepatic insulin resistance by regulating mi R-27a-3p and mi R-96-5p to target FOXO1.(4)The ameliorative effect and the molecular mechanism of quercetin and EGCG on adipose tissue insulin resistance and lipid metabolism in obese mice.Results showed that cotreatment quercetin and EGCG improved adipocyte expansion and lipid droplet accumulation in obese mice.Dietary supplementation of quercetin and EGCG could improve lipid metabolism by inhibiting the expression of lipogenic genes(ACC,FAS),promoting lipolysis key enzyme genes(HSL)and fatty acid oxidation key enzyme genes(CPT-1).In addition,quercetin and EGCG significantly(P < 0.05)inhibited the expression of pro-inflammatory cytokines IL-6,IL-1β and TNF-α,and suppressed obesity related low-grade systemic inflammation.Further analysis showed that quercetin and EGCG increased insulin sensitivity and alleviated adipose tissue insulin resistance by activating PPARγ/PI3K/AKT/GLUT4 signaling pathway.(5)Micro RNAs mediates the synergistic protection of quercetin and EGCG against TNF-α-induced insulin resistance in 3T3-L1 mature adipocytes.The results showed that the combination of quercetin and EGCG significantly(P < 0.05)promoted glucose uptake capacity and significantly(P < 0.05)reduced the expression of pro-inflammatory factors in adipocytes.The combination of quercetin and EGCG activated the PPARγ/PI3K/AKT/GLUT4 signaling pathway.Further analysis showed that mi R-130-3p had a direct targeting relationship with PPARγ.Overexpression of mi R-130-3p significantly(P < 0.05)inhibited the ability of quercetin and EGCG to promote glucose uptake and the synergistic ability of quercetin and EGCG to promote the expression of PPARγ.These results suggested that quercetin and EGCG activated insulin signaling pathway by targeting mi R-130-3p/PPARγ pathway to alleviate insulin resistance in adipocytes. |
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