| Purpose: To establish the vascular endothelial cell EA.hy926model of Insulin Resistance(IR)by concentrations Palmitic Acid(PA)and to learn the relationship between total flavonoids oftartary buckwheat(TFTB)and IRS2/eNOS signaling pathway by experimental techniques andmolecular biology methods. Discuss the mechanism of total flavonoids of tartary buckwheatto improve insulin resistance from the genetic level and to provide experimental evidence forclinical medicine with the bitter buckwheat total flavonoids as a treatment for insulinresistance.Materials and methods:EA.hy926cells were cultured in vitro and randomly divided intocontrol group, palmitic acid induced insulin resistance group, total flavonoids of tartarybuckwheat group and metformin group. All groups were grown in complete mediumcontaining10%fetal bovine serum(FBS) and50nmol/L Insulin(INS).The culture mediumwas changed2to3days later. Each group was added palmitic acid whose final concentrationis600μmol/L，except the control group. Total flavonoids of tartary buckwheat group wasadded whose final concentration was respective31.25μg/ml,62.5μg/ml,125μg/ml andMetformin group was added whose final concentration was2mmol/L.Check the proliferationrate of the cells by MTT；Detect the content of NO in supernatant by nitrate reductase；Detectthe IRS2,PI3K,Akt,eNOS protein expression by Immunohistochemistry. TheIRS2,PI3K,Akt,eNOS mRNA and protein expression levels were determined by RT-PCR andWestern blotting, respectively. By studying the relationship between total flavonoids oftartary buckwheat(TFTB)and IRS2/eNOS signaling pathway and exploring the molecularmechanisms of insulin resistance， provide experimental evidence for the treatment of TypeⅡ diabetes and the application of total flavonoids of tartary buckwheat.Results:1Each group cell MTT results showed that: compareding with control group, theproliferation rate of cell was significantly lower in insulin resistance group (P<0.05) and theinhibition rate could reach50%. The difference was significant which makes sense on thestatistics. Comparing the low, medium and high rate total flavonoids of tartary buckwheat groups with the insulin resistance group, the difference was significant (P<0.05). Comparingwith insulin resistance group, the proliferation rate of cell was significantly higher inmetformin group (P<0.05).There was no difference between total flavonoids of tartarybuckwheat group and metformin group (P>0.05).2Each group cell nitrate reductase resultsshowed that: comparing with control group, the NO content in supernatant was significantlylower in insulin resistance group (P<0.05). The difference was significant. Comparing totalflavonoids of tartary buckwheat with the insulin resistance group, the difference wassignificant (P<0.05) except the low dose groups. Comparing with insulin resistance group, theNO content in supernatant was significantly higher in metformin group (P<0.05).Thedifference was not significant between middle total flavonoids of tartary buckwheat group andmetformin group (P>0.05).3The effect of the total flavonoids of tartary buckwheat oninsulin resistance EA.hy926cells IRS2expression：RT-PCR results showed that the mRNAexpression of IRS2were significantly lower in the insulin resistance group (P<0.05). Thedifference was significant. Comparing with the insulin resistance group, the difference of theIRS2mRNA expression was significant (P<0.05) in the high dose total flavonoids of tartarybuckwheat group and the metformin group. There was no difference between the totalflavonoids of tartary buckwheat group and the metformin group (P>0.05).Each group cellprotein results showed that: comparing with the control group, the IRS2protein expressionwas significantly lower in the insulin resistance group (P<0.05). The difference wassignificant. Comparing with the insulin resistance group, the difference of the IRS2proteinexpression was significant (P<0.05) in the high dosage total flavonoids of tartary buckwheatgroup and the metformin group. There was no difference between the total flavonoids oftartary buckwheat group and the metformin group (P>0.05).4The effect of the totalflavonoids of tartary buckwheat on insulin resistance EA.hy926cells PI3K expression：RT-PCR results showed that the mRNA expression of PI3K were significantly lower in theinsulin resistance group (P<0.05). The difference was significant. Comparing with the insulinresistance group, the difference of the PI3K mRNA expression was significant (P<0.05) in thehigh dose total flavonoids of tartary buckwheat group and the metformin group. There was nodifference between the total flavonoids of tartary buckwheat group and the metformin group(P>0.05).Each group cell protein results showed that: comparing with the control group, the PI3K protein expression was significantly lower in the insulin resistance group (P<0.05). Thedifference was significant. Comparing with the insulin resistance group, the difference of thePI3K protein expression was significant (P<0.05) in the high dosage total flavonoids of tartarybuckwheat group and the metformin group. There was no difference between the totalflavonoids of tartary buckwheat group and the metformin group (P>0.05).5The effect of thetotal flavonoids of tartary buckwheat on insulin resistance EA.hy926cells Aktexpression：RT-PCR results showed that the mRNA expression of Akt were significantlylower in the insulin resistance group (P<0.05). The difference was significant. Comparingwith the insulin resistance group, the difference of the Akt mRNA expression was significant(P<0.05) in the high dose total flavonoids of tartary buckwheat group and the metformingroup. There was no difference between the total flavonoids of tartary buckwheat group andthe metformin group (P>0.05).Each group cell protein results showed that: comparing withthe control group, the Akt protein expression was significantly lower in the insulin resistancegroup (P<0.05). The difference was significant. Comparing with the insulin resistance group,the difference of the Akt protein expression was significant (P<0.05) in the high dosage totalflavonoids of tartary buckwheat group and the metformin group. There was no differencebetween the total flavonoids of tartary buckwheat group and the metformin group (P>0.05).6The effect of the total flavonoids of tartary buckwheat on insulin resistance EA.hy926cells eNOS expression：RT-PCR results showed that the mRNA expression of eNOS weresignificantly lower in the insulin resistance group (P<0.05). The difference was significant.Comparing with the insulin resistance group, the difference of the eNOS mRNA expressionwas significant (P<0.05) in the high dose total flavonoids of tartary buckwheat group and themetformin group. There was no difference between the total flavonoids of tartary buckwheatgroup and the metformin group (P>0.05).Each group cell protein results showed that:comparing with the control group, the eNOS protein expression was significantly lower in theinsulin resistance group (P<0.05). The difference was significant. Comparing with the insulinresistance group, the difference of the eNOS protein expression was significant (P<0.05) inthe high dosage total flavonoids of tartary buckwheat group and the metformin group. Therewas no difference between the total flavonoids of tartary buckwheat group and the metformingroup (P>0.05). Conclusions:1Palmitic acid can effectively inhibit proliferation rate of EA.hy926cell. PA also canestablish the vascular endothelial cell EA.hy926model of insulin resistance(IR) andprevent the insulin entering the cells in order to exercise its biological function.2Total flavonoids of tartary buckwheat can improve insulin resistance EA.hy926cellsexpressing mRNA by IRS2/eNOS signaling pathway.3Total flavonoids of tartary buckwheat can improve insulin resistance EA.hy926cellsexpressing protein by IRS2/eNOS signaling pathway.4Total flavonoids of tartary buckwheat can increase cells release of NO through affecting onIRS2/eNOS signaling pathway and improve insulin resistance state. Keeping thehomeostasis of vascular microenvironment. |
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