| With many nutrients and bioactive substances,Hippophae rhamnoides L.is widely used as food and folk medicine for a long time.And the seed oil of Hippophae rhamnoides L.has been extracted as a product due to its biological activities,including anti-inflammatory,antioxidant,hepatoprotective,anti-hyperlipidaemic activities.After oil extraction,a large amount of seed residues riched in active substances such as flavonoids are abandoned.In order to make full use of Hippophae rhamnoides L.,flavonoids-enriched extract from seed of Hippophae rhamnoides L.(FSH)was further screened out based on its beneficial effect on glucose metabolism.In our previous studies,FSH exhibited hypoglycemic effect in different diabetic animal models,which may be related to the improvement of insulin resistance.However,its effective substance and underlying mechanism still remain unclear.Therefore,in the present study,the effects of FSH on insulin sensitivity as well as its underlying mechanism were investigated using palmitate treated C2C12 myotubes.Meanwhile,screening and functional characterization of main effective substances from FSH were also investigated.First,the in vitro insulin resistance model of C2C12 myotubes was induced by 0.5m M palmitate for 24 hours,and the expression of key proteins in insulin signaling pathway were detected by Western blotting.It was found that the phosphorylation level of AKT(Ser473)mediated by insulin was decreased under the stimulation of palmitate,while the phosphorylation level of IRS1(Ser307)was increased,resulting in inhibition of insulin signaling pathway.FSH intervention exhibited a positive prevention effect on insulin resistance of C2C12 myotubes induced by palmitate.As showed in result,FSH treatment promoted insulin-mediated glucose uptake,upregulated phosphorylation level of AKT(Ser473)and downregulated phosphorylation level of IRS1(Ser307),which manifested amelioration of insulin resistance.At the meantime,FSH activated AMPK,and decreased the expression levels of endoplasmic reticulum(ER)stress-related proteins Bip.Moreover,palmitate induced activation of JNK and IRE1 were inhibited by FSH treatment,indicating this extract improves ER stress.After knocking down AMPK with si-AMPKα1 and si-AMPKα2,the improvement of ER stress and insulin resistance was also weakened.All these have demonstrated that the ER stress and insulin resistance amelioration activity of FSH is AMPK-dependent.After that,UPLC-MS/MS technology was used to detect the plant polyphenol metabolome.Based on the relative content,literature query,network pharmacology analysis and experimental verification,we aim to identify the active substance of FSH which improving insulin resistance and further screen out quality markers.The results showed that gallocatechin,quercetin and caffeic acid could activate AMPK as well as reverse the reduction of insulin-mediated AKT phosphorylation under the stimulation of palmitate,thus improving insulin signaling pathway and enhancing insulin sensitivity.Considering the efficacy,dose dependence and stability of experimental results,gallocatechin was screened to investigate the mechanism of improving insulin resistance.After knocking down AMPK with si-AMPKα1 and si-AMPKα2,the improving effect of gallocatechin on insulin resistance was also weakened.All these have demonstrated that insulin resistance ameliorating activity of gallocatechin is AMPK-dependent.In summary,FSH improves palmitate-induced ER stress and insulin resistance in C2C12 myotubes by targeting to AMPK.Gallocatechin,quercetin and caffeic acid could be used as quality markers to some extent,providing reference for the establishment of quality control standards.This study provided experimental data for the insulin resistance ameliorating activity of FSH,further clarified its underlying mechanism and effective substance.And these results provided scientific evidence for the deep utilization of Hippophae rhamnoides L.. |
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