| Objective: Chronic kidney disease(CKD)can lead to progressive and irreversible nephron loss,renal tissue damage,and then renal dysfunction,and eventually end-stage renal failure,which has become a global public health problem.Renal fibrosis is the central link and important feature of the occurrence and development of a variety of chronic kidney diseases,and it is one of the common pathways to end-stage renal failure.Because the diagnosis of renal fibrosis is still lack of early and accurate specific markers,and its treatment is also lack of pertinence,it is of great significance to clarify its pathogenesis for the prevention and early intervention of the occurrence and development of the disease.Transforming growth factor-β(TGF-β1),a member of transforming growth factor-β family,is widely expressed in renal cells and plays an important role in the deposition of renal interstitial matrix by inhibiting the degradation of extracellular matrix.As a protein kinase,cyclin dependent protein kinase 5 plays its physiological role by phosphorylating the corresponding substrate.Our previous study found that high glucose,TGF-β1 and other factors can cause p35 to break into P25,and then activate Cdk5,making its kinase activity abnormally increased,which plays an important role in the occurrence and development of podocyte injury and apoptosis in diabetic nephropathy.On this basis,this study focused on the role of TGF-β1 regulating Cdk5 expression in mesangial extracellular matrix deposition in order to further clarify the mechanism of TGF-β1 promoting renal fibrosis and provide theoretical basis for the effective prevention and treatment of renal fibrosis.Methods: Human mesangial cells were cultured in vitro and stimulated by10 ng/ml TGF-β1.Experimental groups:1.To observe the effect of TGF-β1 on the expression of Cdk5 protein and m RNA: HMC was inoculated into 6-well plate and stimulated with 10 ng/ml TGF-β1 for 0,3,6,12 and 24 hours respectively.The expression of Cdk5 and p35/25 protein was detected by Western blot and immunofluorescence,and the expression of Cdk5 m RNA was detected by real time-PCR.2.To observe the effect of Cdk5 on the expression of collagen IV and FN in mesangial cells: Cdk5 wild-type plasmid was transfected and blank control group and plasmid control group were set up.The protein expression of collagen IV and FN was detected by Western blot and immunofluorescence.3.To observe the effect of roscovitine on the expression of collagen IV and FN in HMC stimulated by TGF-β1: HMC was divided into control group,TGF-β1 stimulation group(10 ng/ml TGF-β1 stimulation for 24 h),TGF-β1+roscovitine group(10 ng/ml TGF-β1 and 10 μm Roscovitine mixed medium stimulation for 24 h).The expression of collagen Ⅳ and FN was detected by Western blot and immunofluorescence.The expression of collagen IV and FN m RNA was detected by real time-PCR.4.To observe the effect of Cdk5 knockdown plasmid on the expression of collagen IV and FN in HMC stimulated by TGF-β1: they were divided into control group,TGF-β1+plasmid control group,vector group and TGF-β1+DN-Cdk5 group.TGF-β1+vector group and TGF-β1+DN-Cdk5 group were transfected with corresponding plasmids for 24 hours,and then stimulated with TGF-β1 for 24 hours.The protein expression of collagen IV and FN was detected by Western blot and immunofluorescence,and the m RNA expression of collagen IV and FN was detected by real time-PCR.Results:1.The effect of TGF-β1 on the expression of Cdk5 and p35/p25 in mesangial cells: after 10 ng/ml TGF-β1 treated mesangial cells for 0,3,6,12 and 24 hours,the expression of Cdk5 protein was detected by Western blot in a time-dependent manner.P35/p25 is a specific co-agonist of Cdk5.After treatment with TGF-β1,the expression of p35 protein was consistent with that of Cdk5 and increased in a time-dependent manner;the expression of p25 protein decreased at 12 h and reached the peak at 24 h.Real time-PCR was used to detect the expression of Cdk5 m RNA in mesangial cells treated with TGF-β1 for 0,12 and 24 h.Compared with 0 h,the expression of Cdk5 m RNA increased significantly after 12 h and 24 h of TGF-β1 treatment(p<0.05),and increased by 3.67 times after 24 h of TGF-β1 treatment.The expression of Cdk5 and p35/p25 were significantly increased after 24 hours of TGF-β1 stimulation.Cdk5 was activated after TGF-β1 stimulated mesangial cells.2.The effect of Cdk5 on the expression of collagen IV and FN in mesangial cells: compared with blank control group and blank plasmid transfection group,the expression of collagen IV and FN in Cdk5 wild-type expression plasmid transfected group was significantly increased by Western blot(p<0.05).The results of immunofluorescence also showed that the expression of collagen IV(red fluorescence)and FN(green fluorescence)protein was significantly increased after transfection of Cdk5 plasmid.3.After inhibiting of Cdk5 kinase activity,the effect of TGF-β1 on the expression of collagen IV and FN in mesangial cells: compared with the blank control group,Western blot results showed that the expression of collagen IV and FN in mesangial cells was significantly increased after stimulation with TGF-β1(p<0.05),while the expression of collagen IV and FN in mesangial cells was significantly decreased after treatment with roscovitine(p<0.05).The results of real time-PCR showed that the m RNA expression of collagen IV and FN was increased significantly after TGF-β1 stimulation(p<0.05),while the m RNA expression decreased after roscovitine treatment(p<0.05).The results of immunofluorescence showed that compared with the control group,the expression of collagen IV(red fluorescence)and FN(green fluorescence)was increased significantly after TGF-β1 stimulation.After roscovitine interventing,the expression of collagen IV and FN was decreased.4.After transfecting of Cdk5 inhibitory plasmid,the effect of TGF-β1 on the expression of collagen IV and FN in mesangial cells: compared with the blank control group,the expression of collagen IV and FN in mesangial cells was significantly increased in TGF-β1+blank plasmid transfection group by Western blot(p<0.05),while the expression of collagen IV and FN was significantly decreased after transfection of Cdk5 inhibitory plasmid(p<0.05).The results of real time-PCR showed that the expression of collagen IV and FN m RNA in TGF-β1+blank plasmid transfection group was significantly increased(p<0.05),and the expression of collagen IV and FN m RNA in Cdk5 inhibitory plasmid transfection group was decreased(p<0.05).The results of immunofluorescence showed that compared with the control group,the expression of collagen IV(red fluorescence)and FN(green fluorescence)in TGF-β1+blank plasmid transfection group was significantly increased,while the expression of collagen IV and FN decreased after transfection of Cdk5 inhibitory plasmid.Conclusions:1.The expression of Cdk5 protein and m RNA and the expression of collagen IV and FN in human mesangial cells were increased by TGF-β1.2.Inhibition of Cdk5 kinase activity or transfection of Cdk5 inhibitory plasmid could reduce the expression of collagen IV and FN.3.Cdk5 may play an important role in TGF-β1-induced glomerular mesangial extracellular matrix deposition. |
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