Study On The Expression And Function Of Different Subtypes Of TGF-β In The Heart Of Diabetic Mic

Objective: To investigate the differential expression of three TGF-isoforms in the hearts of diabetic mice.Methods:(1)The protein and m RNA expression of CollagenⅠ and Collagen Ⅲ were detected by Western Blot and RT-qPCR in the heart tissues of diabetic mice and normal mice.(2)Detection of TGF-β1,TGF-β2 and TGF-β3 expression levels in mouse heart tissues by Western Blot,RT-qPCR and immunofluorescence.(3)MCFs were treated with 30 m M HG,and the cell viability of MCFs was detected by CCK-8method;the expression levels of CollagenⅠ,Collagen Ⅲ and TGF-β1,TGF-β2 and TGF-β3 were detected by Western Bolt and RT-qPCR.(4)After treatment of MCFs with different concentrations of TGF-β1,TGF-β2 and TGF-β3 for 24 h,the cell viability of MCFs was detected by CCK-8 assay,and the expression of Collagen Ⅰ and Collagen Ⅲ was detected by Western Blot and RT-qPCR.(5)The phosphorylation levels of AMPK,Smad2,and p38 MAPK and the protein expression of Smad7 and MMP9 in heart tissues of diabetic mice and in HG-treated MCFs were detected by Western Blot.(6)After the transfection of si RNA into MCFs by liposome transfection method for 24 h,the cells were collected after 24 h of HG treatment.The protein expression of TGF-β1,TGF-β3,CollagenⅠ,Collagen Ⅲ,Smad7,and MMP9,and the phosphorylation levels of AMPK,Smad2,and p38 MAPK in MCFs were detected by Western Blot.Results: 1.The expression of Collagen Ⅰ,Collagen Ⅲ,TGF-β1 and TGF-β3 protein and m RNA were significantly upregulated in the heart tissues of diabetic mice,and the expression levels of TGF-β3 protein and m RNA were significantly higher than those of TGF-β1,while the expression of TGF-β2 protein and m RNA did not change.2.The cell viability was strongest when 30 m M HG treated MCFs for 24 h.The expression levels of Collagen Ⅰ,Collagen Ⅲ,TGF-β1 and TGF-β3 were all upregulated,and the expression level of TGF-β3 was higher than that of TGF-β1,while the expression of TGF-β2 was unchanged.3.After treating MCFs cells with different concentrations of TGF-β1,TGF-β2 and TGF-β3 for 24 h,5ng·m L-1of TGF-β1,TGF-β2 and TGF-β3 cells were the most active,and 5ng·m L-1 of TGF-β1and TGF-β3 could significantly increase the protein expression of Collagen Ⅰ and Collagen Ⅲ,while TGF-β2 failed to promote the protein expression of Collagen Ⅰ and Collagen Ⅲ.4.The si RNAs of TGF-β1 and TGF-β3 were transfected separately or simultaneously into MCFs for 24 h and then treated with HG for 24 h.The si RNAs of TGF-β1 and TGF-β3 were able to inhibit the expression of CollagenⅠ and Collagen Ⅲ and the si RNA of TGF-β3 was able to inhibit the protein expression of CollagenⅠ and Collagen Ⅲ more effectively.5.Phosphorylation of Smad2,p38 MAPK and MMP9 protein expression levels were significantly upregulated and phosphorylation of AMPK and Smad7 protein expression levels were significantly downregulated in heart tissues of diabetic mice and in HG-treated MCFs,whereas si RNAs for TGF-β1and TGF-β3 were able to suppress phosphorylation of Smad2,p38 MAPK and protein expression levels of MMP9 and restore phosphorylation of AMPK and protein expression levels of Smad7.However,the si RNA of TGF-β3 was able to inhibit the phosphorylation level of Smad2 and p38 MAPK more effectively,and the si RNA of TGF-β1 was more capable of restoring the phosphorylation level of AMPK than TGF-β3.Conclusions :(1)Elevated expression of CollagenⅠ,Collagen Ⅲ,TGF-β1,and TGF-β3 in cardiac tissues of diabetic mice and in HG-treated MCFs,and TGF-β3 had a stronger expression pattern in diabetic mouse heart tissue and HG-induced MCFs.(2)TGF-β1 and TGF-β3 can promote the expression of CollagenⅠ and Collagen Ⅲ,and the ability of TGF-β3 to promote the expression of CollagenⅠ and Collagen Ⅲ is greater than that of TGF-β1.(3)There are differences in the regulation of collagen synthesis and degradation signaling pathways by TGF-β1 and TGF-β3.TGF-β3 could more effectively promote the phosphorylation level of Smad2 and p38 MAPK,inhibit the phosphorylation level of AMPK and the protein expression of Smad7,and TGF-β1 could more effectively inhibit the phosphorylation level of AMPK.