Role Of Caveolae In High Glucose/TGF-β₁-induced Extracellular Matrix Production In Rat Mesangial Cells

ObjectiveTo Investigate the role of caveolae in high glucose(HG)/Transforing growth factor(TGF-β1)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs),and to investigate the mechanisms underlying.MethodsSynchronized Rat MCs were divided into following groups:(1)Normal glucose group;(2) High glucose group.Each group were cultured0、12、24、48h. MCs were treated with High glucose/TGF-β1of differrent concentration for1.5h and then were treated with High glucose (30mmol/L) or TGF-β1(lOng/mL) for0-3h.To avoid the interference of different osmotic pressure,mannitol were used to adjust osmotic pressure to30mmol/L.β-MCD pretreated MCs were divided into following two groups:(3)HG+Methyl-(3-cyclodextrin (β-MCD) group;(4)HG+P-MCD+Cholesterol (Chol) group.Each group continued to cultivate cells1、12、24、48h.Western blotting was used to detect the protein expression of caveolin-1(Cav-1)、phosphocaveolin-1(p-cav-lyl4)、collagen type1(Col-1).The mRNA expression of Cav-1was deter分�'�ed by real-time PCR. ELISA was used to deter分�'�e the level of FN in the supernatent.Results1. Compared with NG group, Fibronection concentration in HG/TGF-β1group was significantly increased(P<0.01,respectively).Compared with HG Oh group, HG increased Collagen-1generation in a time-dependent manner (P<0.05) Compared with TGF-β10h group, TGF-β1increased Collagen-1generation in a time-dependent manner.(P<0.05)2.Compared with TGF-β10h group, Caveolin-1mRNA and protein levels were decreased after TGF-β1treatment (P>0.05,respectively). There was no significant changes in Caveolin-1mRNA and protein levels after HG treatment.(P<0.01,respectively)3. HG and TGF-β1of different concentration induced phosphorylation of Caveolin-1in a dose-dependent manner(P<0.01); After High glucose (30mmol/L) or TGF-β1(10ng/mL) treatment, phosphorylation of Caveolin-1is evident during1-2h(P<0.01).4. Compared with HG (30mmol/L) group, the phosphorylation level of Caveolin-1of β-MCD (pretreatment,1h)+HG (30mmol/L) group is significantly increased(P<0.01); Phosphorylated caveolin-1of β-MCD+HG+Chol (15μg/mL) group is higher than β-MCD+HG group.(P<0.01)5. compared with TGF-β1(10ng/mL) group, the phosphorylation level of Caveolin-1of β-MCD (pretreatment1h)+TGF-β1(10ng/mL) group is significantly increased(P<0.01); Phosphorylated caveolin-1of β-MCD+TGF-β1+Chol (15μ g/mL) group is higher than β-MCD+TGF-β1group.(P<0.01)ConclusionsHG/TGF-β1induced ECM (Col-1and FN)production in a time dependent mannerTGF-β1treatment decreased Caveolin-1mRNA and protein expression,while HG has no effect on that.Both HG and IGF-β1could induce phosphoralation of Caveolin-1transitorily,which present a dose dependent relationship.β-MCD blocked the phosphoralation of Caveolin-1and the upregulation of FN induced by HG/TGF-β1,but has no effect on Col-1expression. Excessive cholesterol treatment could partly disturb the effects of β-MCD.