Study On The Regulation Of Trigonelline On Wnt/β-catenin Signaling In Glomerular Mesangial Cells Under High Glucose Conditions

Diabetic nephropathy(DN)is one of the common complic ations of diabetes and it is the main cause of end-stage renal disease(ESRD).The main pathological change is excessive proliferation of mesangial cells(MCs),extracellular matrix(ECM)accumulation,and basement membrane thickening.High glucose conditions cause mesangial cells dysfunction,extracellular matrix protein synthesis and degradation unbalanced and the accumulations of ECM in the mesangial area and basement membrane area.Then structure and function of glomerular morphology pathological change.It eventually lead to the occurrence of glomerulosclerosis.Trigonelline is an active ingredient of the leguminous plant fenugreek.It has functions of reducing blood glucose,lowering cholesterol,promoting the regeneration of nerve tissue,anti-cancer,anti-oxidation,sedative and so on.However,the mechanism of its effect on the mesangial cel s under high glucose condition is not clear.Objective: To investigate the effects of Trigonelline on the proliferation,apoptosis and extracellular matrix expres sion of human glomerular mesangial cells induced by high glucose and its possible mechanism.Methods: 1.Immunocytochemistry and ELISA method were used to detect the expression and secretion of FN and Col IV.2.Cell proliferation was detected by MTT assay.Cell cycle was detected by flow cytometry.3.m RNA and protein expression of Wnt5 a,β-catenin,TCF4,Cyclin D1 and CDK4 were detected by RT-PCR and Western blot.4.Cell apoptosis was detected by flow cytometry and TUNEL assay.Results: 1.After 30 m M high glucose incubation for 24 h and 48 h,the proliferation of mesangial cells got significantly promoted,and the percentage of cells in S phase increased,while trigonelline could blocked cells in G0/G1 phase,which significantly inhibits the high-glucose induced excessive proliferation of mesangial cel s.2.30 m M high glucose incubation for 48 h could stimulate the expression and secretion of FN and Col IV in mesangial cells and promote the accumulation of ECM,while trigonelline could significantly downregulate the abnormal expression and secretion of FN and Col IV caused by high glucose conditions,so that reduced ECM accumulation and protected mesangial cel s.3.The m RNA and protein expression of Wnt5 a,β-catenin,TCF4,Cyclin D1 and CDK4 were significantly increased under high glucose conditions.The Wnt/β-catenin pathway-related factors were significantly up-regulated when β-catenin overexpression plasmid was transfected into mesangial cells.Trigonelline had a significant inhibitory effect on Wnt/β-catenin pathway in mesangial cells under high glucose conditions.The inhibition effect to Wnt/β-catenin pathway after silencing β-catenin and interfered with trigonelline was still significant.4.After 30 m M high glucose incubation for 72 h,the cell survival rate in high glucose group decreased compared with that in the normal glucose group.Early apoptosis of mesangial cells increased under high glucose conditions,detected by flow cytometry and TUN EL assay.W hile trigonelline inhibited high glucose-induced apoptosis significantly;overexpression of β-catenin also increased early cell death.Apoptotic rates,but trigonelline could reduce apoptosis caused by β-catenin overexpression.The apoptosis of mesangial cells was significantly reduced compared with that in the high glucose group after interfered with the Wnt/β-catenin pathway inhibitor ICG-001.Conclusions: 1.30 m M high glucose conditions could promote the abnormal proliferation of HMCs and the overexpression and secretion of FN and Col IV,while trigonelline inhibited high glucose-induced hyperproliferatio n and ECM accumulation.2.30 m M high glucose condition could accelerate the cell cycle progression,while trigonelline treatment could down-regulate the expression of Cyclin D1 and CDK4,then blocked the cell cycle from G1 phase to S phase,So trigonelline could inhibit the abnormal proliferation induced by high glucose and over-expression of β-catenin 3.30 m M high glucose condition and β-catenin overexpression could activate Wnt/β-catenin signaling pathway,while trigonelline inhibited Wnt/β-catenin signaling through regulating the expression of β-catenin,TCF4 and other related factors.4.30 m M high glucose conditions and overexpression of β-catenin could induce apoptosis.But trigonelline couldreduce the apoptosis rate.