| Diabetic nephropathy(diabetic nephropathy,DN)is one of the most common and serious chronic complications of diabetes.Glomerular mesangial cell proliferation and extracellular matrix(ECM)accumulation and glomerulosclerosis are important pathological bases for diabetic nephropathy.Meanwhile,TGF-?1 is the most important fibrogenesis factor.Recent studies show that mi R-130 b is significantly decreased in the kidney tissues of DN mice and the peripheral blood of clinical DN patients,it may be altered by TGF-?1.TGF-?1 is a key regulator of DN induction,which aggravates renal fibrosis.It mainly regulates downstream target genes such as ColI,ColIV and FN through TGF-?1/Smads pathway,but its intrinsic regulatory mechanism is not yet clear.Therefore,it is of great significance to understand and prevent diabetic nephropathy by clarifying the interaction between TGF-?1/Smads and mi R-130 b.OBJECTIVE: The aim of this study is to investigate the intrinsic relationship between the fibrosis of human glomerular mesangial cells(HMC)after stimulation of high glucose,TGF-?1and miR-130 b mimics/inhibitor,so as to reveal the role of mi R-130 b in the fibrosis of DN,and to provide new ideas for the prevention,diagnosis and treatment of human DN.METHOD:1.In the experiment,we obtained the consent of the ethical Association of Henan University of Science and Technology and the patients themselves.We performed HE,Masson staining and immunohistochemistry for diabetic glomerulosclerosis and normal kidney tissues in the pathology department,and observed the difference between the lesions and normal tissues.2.HMC cells were cultured in high glucose(25 mmol/l)and low glucose(5.5 mmol/l)in vitro,and the relationship between high concentration or low concentration of glucose and HMC cell fibrosis was observed.3.TGF-?1 was used as intervention factor.We set up TGF-?1(0ng/ml,10ng/ml,30 ng/ml)24h,48 h test group and intervention group without TGF-?1 respectively.The relationship between miR-130 b and fibroblast protein factor and TGF-?1 of HMC cells were observed.4.As an experimental group,miR-130 b mimics and inhibitors were transfected into HMC cells respectively.Mi R-130 b control was used as a control group.The relationship between miR-130 b and fibrosis was observed.5.Under the induction of TGF-?1,miR-130 b mimics,inhibitor and miR-130 b control were transfected respectively,and the relationship between miR-130 b and fibrotic protein factor inducedby TGF-?1 was observed.6.Under the induction of TGF-?1,miR-130 b mimics,inhibitor and miR-130 b control were transfected respectively,and the expression of Smad2/3/4 was observed under the induction of TGF-?1.RESULTS:1.HE staining results showed,comparison of pathological sections between renal tissue in patients with diabetic glomerulosclerosis and normal renal tissue,staining of ColI,ColIV,FN fibrosis protein factor;glomerular sclerosis,basement membrane tickened and stained red,mesangial matrix hyperplasia.Masson staining results showed,comparison of the staining results of ColI,Col IV,FN fibrosis protein factor in patients with diabetic glomerulosclerosis and normal renal tissue,glomerular sclerosis,dyed blue were significantly increased and stout,indicated that fibrosis tissues were increasing.Immunohistochemical results showed,comparison of the staining results of ColI,Col IV,FN fibrosis protein factor in patients with diabetic glomerulosclerosis and normal renal tissue,obvious increase in brown granules around Mesangial tissue.Mesangial thickening compared with normal tissue.Another proof of tissue fibrosis in patient with Diabetic glomerulosclerosis.2.The expression of miR-130 b in HMC cells cultured with high glucose compared with low glucose were as follows: 1.00,1.42±0.03;the expression of Col I mRNA were 1.00,1.43±0.02,ColIV mRNA expression were 1.00,1.49±0.05,the expression of FN mRNA were 1.00,1.89±0.06;Western blot of gray value analysis results showed that compared with low glucose group HMC cells cultured with high glucose group,the expression of Col I,ColIV protein and FN protein were1.00,1.36±0.01;1.00,1.53±0.04;1.00,1.93±0.05;there were significant differences between the groups(P<0.01);immune the fluorescence results show that the high glucose group compared with the low glucose group had no difference in ColI,ColIV,FN blue staining nuclei cytoplasm pale,green fluorescent brighter,enhanced fibrosis.3.The expression of miR-130 b in the mi R-130 b control group and the miR-130 b mimics group were 1.00,5.11±0.70,respectively.The expressions of ColI mRNA were 1.00,1.61 ±0.19,and the expressions of ColIV mRNA were 1.00,3.17±1.00;FN mRNA were 1.00,3.84±0.93;The transfection of miR-130 b control group compared with miR-130 b inhibitor group,the expression of mi R-130 b were 1.00,0.31±0.02.Col I mRNA were 1.00,0.58±0.02,and ColIV mRNA were1.00,0.85±0.03.and FN mRNA were 1.00,0.09±0.04.By analyzing the gray value of Western blot results,we found that the expression of ColIprotein in mi R-130 b control group was 1.00 and miR-130 b mimics group was 1.52±0.01.The expressions of Col IV protein were 1.00,1.22±0.02;and the expressions of FN protein were 1.00,1.98±0.06.The expressions of ColI protein in mi R-130 b control group was 1.00 and miR-130 b inhibitors group was 0.66±0.02.The amount of Col IV protein were 1.00,0.39±0.01,and the amount of FN protein were 1.00,0.60±0.10.Immunofluorescence results showed that mi R-130 b mimics group compared with mi R-130 b control group,the ColI,ColIV,FN fibrosis protein blue stained nuclei with light green fluorescent cytoplasm more bright.MiR-130 b inhibitor group compared with mi R-130 b control group,ColI,ColIV,and FN fibrosis protein cytoplasm of the green fluorescence were very weak.4.When 10 ng/ml and 30 ng/ml TGF-?1 were treated with 24 h and 48 h respectively,the expression of miR-130 b were 1.00,1.48±0.27,1.51±0.04,1.24±0.06,2.33±0.23,2.60±0.21,respectively.The expressions of Col I mRNA were: 1.00,1.27±0.11,1.81±0.11,2.09±0.12,3.32±0.30,2.28±0.23.The expression of ColIV mRNA were: 1.00,1.37±0.01,1.51±0.17,1.38±0.06,1.69±0.32,1.52±0.58.The expression of FN mRNA were 1.00,1.31±0.09,1.68±0.06,2.25±0.18,4.72±0.70,4.21±0.21,respectively.By analyzing the gray value of Western blot results,we found that the expressions of col I were 1.00,1.26±0.07,1.12±0.06,2.54±0.09,3.04±0.15,2.85±0.22,respectively.The expressions of colIV were 1.00,1.67±0.18,3.14±0.08,3.02±0.12,4.66±0.12,4.52±0.10,respectively.The expressions of FN protein were: 1.00,1.31±0.03,1.43±0.02,1.34±0.03,2.08±0.02,1.41±0.03,respectively.Immunofluorescence results showed that Col I,Col IV and FN protein did not show more bright red fluorescence with the increase of TGF-?1 concentration and treatment time,and the strongest fibrosis was observed when 10ng/ml TGF-?1 induced 48 h.5.After TGF-?1 induced,miR-130 b control group compared with miR-130 b mimics group,the expressions of miR-130 b were: 1.00,17.40±2.16.The expressions of Col I m RNA were:1.00,2.36±0.22;the expressions of Col IV mRNA were: 1.00,1.21±0.04;the expressions of FN mRNA were 1.00,2.21±0.31.mi R-130 b control group compared with mi R-130 b inhibitor group,the expressions of miR-130 b were: 1.00,0.40±0.02,the expressions of Col I mRNA were: 1.00,0.54±0.03,the expressions of ColI mRNA were: 1.00,0.70±0.11,the expressions of FN mRNA were: 1.00,0.48±0.11,respectively.After the induction of TGF-?1,we analyzed the gray value of the Western blot band.miR-130 b control group compared with miR-130 b mimics group,the expressions of Col I were:1.00,1.27±0.01.The expressions of Col IV were: 1.00,2.89±0.18;the expressions of FN were: 1.00,1.28±0.03.miR-130 b control group compared with miR-130 b inhibitor group,Col I protein expressions were 1.00,0.90±0.01;the expressions of ColIV were 1.00,0.71±0.05;the expressions of FN protein were 1.00,0.60±0.01,respectively.After the induction of TGF-?1,fibrosis protein expression,such as ColI,Col IV and FN can be seen from our immunofluorescence results.MiR-130 b mimics group compared with miR-130 b control group,the red fluorescence of cytoplasm more bright,fibrosis strengthened;the mi R-130 b inhibitor group compared with the miR-130 b control group,the cytoplasmic red fluorescence of ColI,ColIV,FN fibrotic protein were dim and almost did not appear,and the fibrosis were weakened.6.Without the induction of TGF-?1,the transfected of miR-130 b control group and miR-130 b mimics group,the expressions of Smad2 m RNA were: 1.00,2.69±0.16,the expressions of Smad3 mRNA were: 1.00,2.78±0.12;the expressions of Smad4 mRNA were: 1,2.24±0.09.After the induction of TGF-?1,transfection of mi R-130 b control group than in miR-130 b mimics group,the expressions of Smad2 mRNA were: 1,1.40±0.06,the expressions of Smad3 mRNA were: 1,1.28 ±0.02,the expressions of Smad4 mRNA were: 1,1.37±0.05.No TGF-beta 1 induced by transfection of mi R-130 b control group than the mi R-130 b inhibitor group,the expressions of Smad2 mRNA were: 1,0.24±0.01,the expressions of Smad3 mRNA were: 1,0.26±0.01,the expressions of Smad4 mRNA were: 1,0.24±0.02;after TGF-?1 induction,the miR-130 b control group compared with miR-130 b inhibitor group,the expressions of Smad2 mRNA were: 1,0.74±0.04,the expressions of Smad3 mRNA were: 1,0.82±0.06,the expressions of Smad4 mRNA were: 1,0.73±0.05.The Western blot band gray value analysis,TGF-?1 induced by transfection of mi R-130 b control group than the mi R-130 b mimics group,the expressions of P-Smad2 mRNA were: 1,2.31±0.09,the expressions of t-Smad2 mRNA were: 1,2.06±0.05,P-Smad3 mRNA respectively were: 1,2.10±0.11.The expressions of t-Smad3 mRNA were: 1,1.56±0.01,the expressions of Smad4 mRNA were: 1,3.24±0.19;TGF-?1 induced after transfection of mi R-130 b control group than the mi R-130 b mimics group,P-Smad2 mRNA expressions were: 1,1.40±0.06,the expressions of t-Smad2 mRNA were: 1.1.36±0.05,the expressions of P-Smad3 mRNA were: 1,1.35±0.05,the expressions of t-Smad3 mRNA were: 1,1.19±0.01,the expressions of Smad4 mRNA were: 1,1.17±0.04.No TGF-beta 1 induced by transfection of miR-130 b control group than the mi R-130 b inhibitor group,the Western blot band gray value analysis,the expressions of P-Smad2 mRNAwere: 1,0.81±0.04,the expressions of t-Smad2 mRNA were: 1,0.76±0.05,the expressions of P-Smad3 mRNA respectively were: 1,0.90±0.08,the expressions of t-Smad3 mRNA were: 1,0.54±0.03,the expressions of Smad4 m RNA were: 1,0.47±0.03;TGF-?1 induced after transfection of mi R-130 b control group than the miR-130 b mimics group,P-Smad2 mRNA expressions were: 1,0.80±0.05,the expressions of t-Smad2 mRNA were: 1,0.91±0.02,the expressions of P-Smad3 mRNA were: 1,0.68±0.03,the expressions of t-Smad3 mRNA were: 1,0.71±0.02,the expressions of Smad4 mRNA were: 1,0.90±0.04.Immunofluorescence results showed that TGF-?1 after induction,miR-130 b mimics group compared with miR-130 b control group,Col I,ColIV,FN fibrosis protein nuclei were blue staining and bright,the red fluorescence of the cytoplasm were more also brighter;mi R-130 b inhibitor group compared with miR-130 b control group,Col I,Col IV,FN fibrosis protein in nucleus and cytoplasm were all dim red fluorescence.CONCLUSION:1.There was a positive correlation between fibrosis of miR-130 b and HMC cells;2.TGF-? 1 could induce fibrosis of HMC cells,and in several experimental groups,10 ng /ml TGF-? 1 induced the fibrosis of HMC cells at 48 h;3.Inhibiting miR-130 b could inhibit the fibrosis induced by TGF-? 1,but had no effect on the expression of TGF-? 1 mRNA;4.TGF-? 1 could induce the fibrosis of HMC cells,and TGF-? 1 could induce the fibrosis of HMC cells when the concentration of TGF-? 1 was 10 ng/ml for 48 h;5.TGF-? and miR-130 b may play an important role in promoting the fibrosis of HMC cells,which is mediated by TGF-? / Smads signaling pathway,and Smad3 plays an important role. |
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