The Effects Of Ginkgo Biloba Extract On TTG In Diabetic Rat Kidney And Screening Of Effective Components In Vitro

In recent years, some research show Ginkgo biloba extract may ameliate diabetic kidneyfunction and structural changes, but the mechanism is not clear. Tissue transgl-utaminases(tTG) can made protein Cross-linking by catalytic the rection between protein glutamine orpolyurethane and lysine residues, thus resistant to hydrolysis of matrix-degrading enzymes, theresult is that extracellular matrix (ECM) accumulated, However, The basic lesion of diabetic n-ephropathy (DN) is extracellular matrix accu-mulation. tTG’s activity in glomerular of diab-etic patients and animal models is increased significantly, indicates that extracellular matrix ac-cumulation process have the effect of tTG. The purpose of this study is to explore that whetheror not tTG invo-lved with protection effect of Ginkgo biloba extract on kidney, as well as itsmechanism. Furthermore, we screened the different components of the GBE from theperspective of cell proliferation and accumulation of ECM.Experiment1（Chapter2）: Extracts of Ginkgo biloba extract inExfluence on expression oftTG in kidney of diabetic rats.Replicated diabetes mellitus rat model with STZ (50mg/kg), a part of group intr-agastricadministration of Ginkgo biloba extract (GBE)100mg/kg,the model group are rats in normal.The effect of GBE on diabetic rats will be saw after12weeks. Results displayed that urinaryprotein of GBE group reduced (P<0.05), compared with the diabetes group, in PAS staining, e-xtracellularmatrix (ECM)’s expression was reduced significantly, and the expression of tTG w-as reduced (P<0.01). Tip that the protective effect of kidney may be related to tTG reduceing.Experiment2（Chapter3）:The effect ion of Ginkgo biloba extract on over-expression ofrat mesangial cells’ tTG induced by high glucose and the mechanism of TGF-β、CTGF invo-lveed.Culture the rat glomerular mesangial cells HBZY-1in vitro, detect the Content of tTG、TGF-β、CTGF、FN、Col Ⅳ of HBZY-1on the effect of high glucose and different conce-ntration of GBE by ELISA. Result shows low concentration（0.39μg/ml）of GBE can re-duce the expression of tTG、TGF-β、CTGF、FN、Col Ⅳ, roughly in dose-dependent.Andanalysis found the content of FN、Col Ⅳ both related closely with tTG, TGF-β1, CTGF. West-ern Blot also shows that GBE can also inhibit the expression of tTG protein and this effect is indose-dependent. To study the relationship between tTG and TGF-β、CTGF, we use RNA inte-rference silence the expression of TGF-β, CTGF of HBZY-1cells cultured in high glucose. Re- sult shows after TGF-βsiRNA interference significantly reduce tTG protein expression inducedby high glucose (P <0.01), after CTGFsiRNA interference tTG protein expression did not cha-nge significantly. tTG protein of GBE+TGF-βsiRNA interference group did not change sign-ificantly compared to the TGF-βsiRNA interference group, tTG prot-ein of GBE+CTGF siR-NA interference group dicrased significantly compared to the CTGFsiRNA interfe-rence gro-up（P<0.01）. Suggest that high glucose-induced tTG protein expression mainly by TGF-β1mediated,but the intervention effect of Ginkgo biloba extract mainly be mediated by TGF-β1.Experiment3（Chapter4）: Screening of effective ingredients of Ginkgo biloba extractFirst, using the MTT assay different concentration:0、3.125μg/ml,6.25μg/ml12.5μg/ml,25μg/ml,50μg/ml of different Ginkgo biloba extract ingredients’(Total of Ginkgo biloba extr-act, total flavonoids, total lactone, total flavonoids hydrolyzate, quercetin, kaempferol, isorha-mnetin, ginkgolide A, B, C) effect on rat mesangial cell proliferation induced by high glucose(30Mm), result shows that in addition to aglyc-ones quercetin, kaempferol, isorhamnetin, totalflavonoids hydrolyzate, other compo-nents, including a total of Ginkgo biloba extract, totalflavonoids, total fat, lactone A, B, C had no strong cytostatic. Total Ginkgo biloba the extract(ginaton) only in high concentration (50μg/ml) inhibit the proliferation of mesangial cells, totalflavonoids’ proliferate inhibition is stronger than the total extract of Ginkgo biloba, and can in-hibit proliferation in only25μg/ml, Lactone C can inhibite proliferation50μg/ml.Then, For th-ese components which have no strong inhibition in cell proliferation, we detect their effect onaccumulation of mesangial cells’ ECM in high glucose, The experime-ntal concentrations are0、0.05、0.5、5、50μg/ml，Total of Ginkgo biloba extract (ginaton), total flavonoids in a certainconcentration range (0.5~50μg/ml) have an relaxation effect on accumulation of mesangial c-ells’ ECM in high glucose. Total lipid only at50μg/ml inhibit content of Col Ⅳ, Lactone A, Bhave no significant inhibition on ECM accumulation, lactone C only at50μg/ml suppressed thecontent of Col Ⅳand FN.According to the research we get following conclusions：1. Ginkgo biloba extract has a protective effect on kidney of diabetic rats2. Ginkgo biloba extract can alleviate the accumulation of diabetic rats renal ECM, Themechanism may be associated with down expression of tTG.3. Ginkgo biloba extract (ginaton) inhibit rat mesangial cells’ ECM accumulation inducedby high glucose（0.39μg/ml-50μg/ml）, and in dose-dependent. The content of ECMcomponents： FN、Col Ⅳ closely related with tTG, TGF-β, CTGF levels.4. The tTG protein of Mesangial cells induced by high glucose over expression ismediated by TGF-β1, and the intervention effect of Ginkgo biloba extract on tTG is mainly mediated by TGF-β15. Ginkgo biloba extract (Ginaton) only in the high concentration (50μg/ml) inhibitedhigh glucose-induced mesangial cell proliferation and inhibit mesangial cells’ ECMaccumulation in low concentration (0.5μg/ml onwards).6. The total flavonoids of Ginkgo biloba extract plays a major role in the inhibition of cellproliferation and ECM accumulation, total internal fat had no significant effect. The totalflavonoids inhibit kidney mesangial cell proliferation in high concentration; lowconcentrations (from0.5μg/ml) inhibit the content of mesangial cell ECM.7. Aglycones inhibit mesangial cell proliferation is much stronger than the glucoside. Theaglycone the monomer quercetin, kaempferol, isorhamnetin, and total flavonoids hydrolyzatesignificantly inhibited mesangial cell proliferation, and in concentration-dependent.Innovation points:In this study, for the first time from the angle of of tTG induction of ECM accumulationstudy the protective effect of Ginkgo biloba extract on rats with diabetic nephropathy, and useRNA interference technology to explore the mechanism of TGF-β, CTGF and tTG in diabeticnephropathy. Studies have shown ginkgo biloba extract can alleviate the accumulation of ratsdiabetic renal ECM, associated with down tTG expression. But Ginkgo biloba extract tTGintervention mainly mediated by TGF-β.1. In this study, for the first time from the cultured in high glucose mesangial cellproliferation and ECM accumulation angle screening the different components of the Ginkgobiloba extract, the results showed that the total Ginkgo biloba extract mainly inhibit cellproliferation at high concentrations (50μg/ml), inhibit ECM accumulation in low concentration(from0.5μg/ml), the GBE play a major role in the ginkgo flavonoids,and the aglycone role fargreater than glycosides.