Preventive Effect And Mechanism Of Coreopsis Tinctoria Tea On Insulin Resistance Of 2 Type Diabetes Mellitus

Coreopsis tinctoriaNutt (Asteraceae) (CT) is a plant native to North America that has spread worldwide. Native Indians have used this plant to treat several disorders, including diarrhea, internal pain, and bleeding, to strengthen blood and as an emetic And an infusion of CT flowering tops is traditionally used in Portugal to control hyperglycemia. In China it has been recorded in the xinhua materia medica outline with the traditional function of heat-clearing, detoxicating and dampness dysentery. The effects of CT are mainly attributed to its flavonoid-like polyphenols; however, the effects of drinking CT tea on insulin sensitivity and hepatic glucose metabolism are still unknown. The aim of the present study is trying to figure out the preventive effect and mechanism of Coreopsis tinctoria tea on insulin resistance of 2 type diabetes.In the present study, the metabolomic profiles of insulin-resistant rats fed a HFD and a CT-supplemented diet (HFD supplemented with CT drinking) for 8 weeks were investigated. Serum samples for clinical biochemistry and liver samples for histopathology and liquid chromatography-mass spectrometry based metabolomic research were collected. Combined metabolomic, Western blot, and quantitative real-time PCR analyses revealed that CT treatment significantly improved the glucose homeostasis by, on the one hand, through inhibiting the expression of gluconeogenic pathway key proteins glucose-6-phosphatase and phosphoenolpyruvate carboxykinase and, on the other hand, via regulating the mRNA or protein levels of the Krebs cycle critical enzymes (citrate synthase, succinate dehydrogenase complex, subunit A, flavoprotein, and dihydrolipoamide S-succinyltransferase). These results provide metabolic evidence of the complex pathogenic mechanism involved in hepatic insulin resistance and that the supplementation with CT improves insulin resistance at a global scale.The chemical constituents of CT were investigated by various kinds of chromatographic methods such as, silica gel column chromatography, reversed phase MCI resin column chromatography and Sephadex LH-20 gel chromatography. The structures of isolated were elucidated on basis of the methods of 1H-NMR and 13C-NMR. 15 compounds were obtained from ethanolic extract of Coreopsis tinctoria and 11compounds were identified as follows:luteolin, okanin,3’,4’,8-trihydroxy flavone-7-O-β-D-glucopyranoside, quercetin, isookanin -7-O-β-D-glucopyranose, marein, 2S-3’,5’,7-trihydroxyflavanone, β-sitosterol, stigmasterol, butein and stigmasterol-3-O-β-D-glucopyranoside.The in-vitro IR activities of all the compounds were tested on high glucose induced HepG2 cell line by MTT assay. The results showed that the falavanomarein and marein have potential preventive activity on high glucose-induced insulin resistance, those two flavonoids could be the main active compounds of CT. The mechanism of marein on prevention IR of high glucose-induced HepG2 cells were investigated mainly by Western blot. The expression of IRS-1、p-IRS-1、p-AS160、p-AMPK、p-Akt、Akt、GLUT1、 p-FoxO1、 FoxO1、G6Pase and PEPCK were determined. Marein could increase glucose consumption of IR-HepG2 cells and the levels of p-AS160, GLUT1, p-AMPK, p-FoxO1 compared with HG group. Marein-stimulated AMPK phosphorylation was not blocked by LKB1 siRNA in HepG2 cells. IRS tyrosine kinase inhibitor, PI3K and AMPK inhibitors could significantly inhibit marein increased HepG2 cells glucose consumption. As a conculsion, marein could activate insulin signaling pathway, promote glycogen synthesis and inhibit gluconeogenesis pathways to improve IR of HepG2 cells.In addition, this paper also studied the effect of marein, a chalcone compound from Coreopsis tinctoria, on MG-induced PC12 cell injury. The data showed that marein attenuated MG-induced changes in the mitochondrial membrane potential (△Ψm), mitochondrial permeability transition pores (mPTPs), intracellular Ca2+ levels and the production of reactive oxygen species (ROS), glutathione (GSH)/glutathione disulfide (GSSG), ATP and the increase in percentage of apoptotic cells. Marein also increased glyoxalase I (Glo1) activity, phosphorylated AMP-activated protein kinase (p-AMPK) and Bcl-2 expression and diminished the activation of Bax, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD). Importantly, pretreatment of the cells with marein diminished the compound C-induced inactivation of p-AMPK. Molecular docking simulation showed that marein interacted with the y subunit of AMPK. In conclusion, we found for the first time that marein protected PC12 cells against MG-induced oxidative stress and apoptosis. The mechanism underlying the protective effect of marein was at least partly attributed to a reduction of damage to the mitochondria function, enhancement MG detoxification system and activation of AMPK signal pathway to inhibit apoptosis.These results indicate that marein may become a potent lead compound for treating diabetic encephalopathy via reducing MG toxicity.