Study of Correlation Between the Expression of Smad2 and Collagen Type Ⅳ Secretion of Rat Glommerular Mesangial Cell Stimulated by Transforming Growth Factor-β1 and the Effect of Interventions by Leflunomide

doi:10.3877/cma.j.issn.1673-5250.2013.04.003

Objective

To observe the correlation between the expression levels of Smad2 and collagen type Ⅳ (ColⅣ)secretion of rat glomerular mesangial cell(GMC) stimulated by TGF-β1, whether leflunomide(LEF) influences the expression levels of Smad2 and ColⅣ secretion of rat GMC stimulated.

Methods

After establishing the cultured GMC of rat in vitro, the seventh passages of cells were used in the experiment after identification. The experiment incluced 4 groups: TGF-β1 group(cell cultured medium and 5 μg/mL TGF-β1 were added), LEF-1 group(low dose of LEF treated group, besides normal cell cultured medium and 5 ng/mL TGF-β1, 5 μg/mL LEF were added), LEF-2 group(high dose of LEF treated group, besides normal cell cultured medium and 5 ng/mL TGF-β1, 50 μg/mL LEF were added), cotrol group (nomal cell cultured medium was added). Each group was collected at 15 min, 30 min, 1 h, 2 h and 6 h. ColⅣ secretion was detected by the enzyme-linked immunosorbent-assay (ELISA). The expressions levels of P-Smad2 protein were analyzed by indirect immunofluorescence, real time(RT)-PCR were used to detect the mRNA expression of Smad2.

Results

①At each time, ColⅣ secretion level was singnificantly higher in the protein expression in TGF-β1 group than that in control group(P<0.05), but singnificantly lower than that in LEF-1 group and LEF-2 group (P<0.05). ②In control group, phosphorylation(P)-Smad2 protein expressed a little, while stimulation of cells staining positive in TGF-β1 group was singnificantly higher than those in control group(P<0.05). Compared with the stimulation, the expressions of P-Smad2 in LEF-1 group and LEF-2 group were significantly decreased (P<0.05). ③Expression of Smad2 mRNA in TGF-β1 group was higher than those other three groups (P<0.01), but no significant difference in the expressions were found between those in LEF-1 group and LEF-2 group (P>0.05). ④The content of ColⅣ secretion was positively correlated with the expression of Smad2(r=0.707, P<0.05).

Conclusions

TGF-β1 can increase the expressions of Smad2 and ColⅣsecretion.LEF may inhibit the expression levels of Smad2 and ColⅣ secretion induced by TGF-β1, thereby reducing the ECM deposition and offering experiment evidence for the renal protection.

Key words: glommerular mesangial cell, transforming growth factor-β1, Smad2, collagen Ⅳ, leflunomide

图1 Smad2 mRNA表达的扩增和熔解曲线(A: Smad2 mRNA扩增曲线；B: β-actin mRNA扩增曲线；C: Smad2 mRNA熔解曲线；图1-D: β-actin mRNA熔解曲线)

Figure 1 Amplification and melting curve of Smad2 mRNA by fluorescent quantitation PCR(A: Amplification curve of Smad2 mRNA；B: Amplification curve of β-actin mRNA; C: Melting curve of Smad2 mRNA; D: Melting curve of β-actin mRNA)

图2 肾小球系膜细胞smad2与β-actin扩增产物的凝胶电泳图

Figure 2 Amplified PCR product of glomerular mesangial cell smad2 and β-actin mRNA with agarose gel electrophoresis

表1 4组肾小球系膜细胞上清液Ⅳ型胶原含量变化(±s，ng/mL)

Table 1 Changes of collagen Ⅳ in glomerular mesangial cell the supernatant at different time point among 4 groups(±s，ng/mL)

组别	15 min	30 min	1 h	2 h	6 h
TGF-β1	37.90±2.12^a	53.84±3.56^a	48.01±1.15^a	42.09±5.49^a	36.36±3.20^a
LEF-1组	30.82±2.30^bc	31.98±1.44^bc	25.88±1.57^bc	28.98±1.97^c	26.76±1.67^c
LEF-2组	30.91±2.77^bc	28.78±1.67^bc	24.48±4.66^bc	23.10±2.11^c	25.46±0.19^c
对照组	31.30±3.15	33.82±1.30	36.04±2.61	27.56±2.89	24.77±0.29

表1 4组肾小球系膜细胞上清液Ⅳ型胶原含量变化(±s，ng/mL)

Table 1 Changes of collagen Ⅳ in glomerular mesangial cell the supernatant at different time point among 4 groups(±s，ng/mL)

组别	15 min	30 min	1 h	2 h	6 h
TGF-β1	37.90±2.12^a	53.84±3.56^a	48.01±1.15^a	42.09±5.49^a	36.36±3.20^a
LEF-1组	30.82±2.30^bc	31.98±1.44^bc	25.88±1.57^bc	28.98±1.97^c	26.76±1.67^c
LEF-2组	30.91±2.77^bc	28.78±1.67^bc	24.48±4.66^bc	23.10±2.11^c	25.46±0.19^c
对照组	31.30±3.15	33.82±1.30	36.04±2.61	27.56±2.89	24.77±0.29

图3 各模型组与对照组肾小球系膜细胞不同时间点P-Smad2免疫荧光染色(荧光显微镜，×200)

Figure 3 Immunofluoressence picture of P-Smad2 in glomerular mesangial cell at different time point (immunofluorescence microscope, ×200)

图4 各模型组与对照组肾小球系膜细胞Smad2 mRNA的表达水平比较

Figure 4 Comparison of expression of Smad2 mRNA of glomerular mesangial cell between TGF group, LEF-1 group ，LEF-2 group and control group

图5 TGF-β1组与对照组肾小球系膜细胞Smad2 mRNA的表达水平比较

Figure 5 Comparison of expression of Smad2 mRNA of glomerular mesangial cell between TGF-β1 group and control group

图6 LEF-1组与对照组肾小球系膜细胞Smad2 mRNA的表达水平比较

Figure 6 Comparison of expression of Smad2 mRNA of glomerular mesangial cell between LEF-1 group and control group

图7 LEF-2组与对照组肾小球系膜细胞Smad2 mRNA的表达水平比较

Figure 7 Comparison of expression of Smad2 mRNA of glomerular mesangial cell between LEF-2 and control group

1	Derynck R, Zhang YE.Smad-dependent and Smad-independent pathways in TGF-β family Signalling[J]．Nature，2003，425(6958)：577-584.
2	Hansen KE, Cush J, Singhal Aet al.The safety and efficacy of leflunomide in combination with infliximab in rheumatoid arthritis[J]．Arthritis Rheum，2004，51(2)：228-232.
3	Zhang HD, Wang WM, Jin YM，et al. Effects of TGF-B1 on expression of inflammation-related chemokines in renal tubular epithelial cells[J]. Chin J Nephrol, 2008，24(2)：138-139.
4	Xiao ZJ, Jia YZ, Dong JX, et al. Expression and localization of smad2/3 and P-smad2/3 in human kidney[J]. Chin J Histochem Cytochem, 2008, 17(4): 345-348.
5	Lan HY.Tubular epithelial-myofibroblast transdifferentia-tionmechanisms in proximal tubule cells[J]．Curr Opin Nephro Hypertens，2003，12(1): 25-29.
6	Sun L, Zhang D, Liu F, et al.Low-dose paclitaxel ameliorates fibrosis in the remnant kidney model by down-regulating miR-192[J]. J Pathol，2011，225(3)：364-377.
7	Zao D, Yu L, Weng ZY, et al. Correlation between expression of transforming growth factor β gene and response to steroid therapy in children with nephritic syndrome[J]. J Clin Pediatr, 2006, 24(4): 260-263.
8	Lin J, Wang NN, Tan RY, et al. Myofibroblast transition of human kidneytubular cells regulated by smad mediated signaling pathway[J]. Chin J Nephrol Dial Transplant, 2005, 14(3): 230-234.
9	Lu J, Liang H, Liu SZ，et al，.Action of transforming growth factor-1 in renal fibrosis on human mesengial proliferative glomerulonephritis[J]. J Zhejiang Univ, 2004，3(1)：46-51.
10	Zhang JH, Chen ZQ, Sun YF，et al. Effect of herb of supplement qi and nourish yin eliminating mass and dredge nels and collaterals on the mesangial cells secreting extracellular matrix[J]．Chin J Gerontol，2010，30(3)：620-622.
11	Wand J, Guo JH, Meng W，et al. Expression of the TGF-β1, MMP-2 ，collage IV deposit in rental fibrosis formation in CAN and its cliniconolog significance[J]．Chin J Organ Transplant, 2006，27(8)：478-480.
12	Chui TG, Hou FF, Ni ZH, et al. Treatment of proliferative lupus nephritis with leflunomide and steroid a prospective multi-ceater controlled clinical trial[J]. Chin J Intern Med，2005, 44(9): 672-676.
13	Wang ZL, Wang MB, Liu P, et al.Leflunomide for idiopathic frequent-relapsing nephrotic syndrome in children[J]．J Clin Nephrolog, 2012，12(1)：24-25.
14	Tang Y, Lou TQ, Wang C，et al. Study on protective effects and its mechanism of leflunomide on renal tissue in rat IgA nephropathy model[J]．Chin Critic Care Med, 2008，20(5)：283-286.
15	Xu L, LYU XH, Ne RH, et al. The effect of leflunomide on renal expression of TGF-β1 in rats with 5/6 nephrectomy[J]. Chin J Integrat Tradition West Nephrol, 2009, 10(5): 409-412.

[1]	Zhenhua Zhang, Jun Hou, Jianming Lai. Curative effect of leflunomide combined with methotrexate in treatment of juvenile idiopathic arthritis[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2020, 14(04): 403-407.
[2]	Wen-hui HU, Li YU, Zhi-hong HAO, Yue-qiang WEN, Yao ZHANG, Rong-yan CHEN, You-xiang ZHANG. Influence of Leflunomide on the Expression of Smad2 of Rat Glomerular Mesangial Cell Induced by Transforming Growth Factor β₁[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2010, 06(06): 411-416.
[3]	Donghui Wu, Youyuan Wang, Song Fan, Zhaoyu Lin, Jinsong Li. MicroRNA-639 regulates epithelial-mesenchymal transition of tongue squamous carcinoma[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(01): 21-28.
[4]	Rui Duan, Faguang Jin. TGF-1/Smad2 signal pathway being involved in apoptosis induced by seawater drowning-induced acute lung injury[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(02): 154-158.
[5]	Qiang Wang, Xin Chen, Xiaoxue Weng, Yongze Zhuang, Guoqing Yu. Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(06): 329-334.
[6]	Hui-ling WANG, Ying-liang OU, Zhi-xiang JIAN. Expression and significance of MMP-2 and collagen Ⅳ protein in hepatocellular carcinoma[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2012, 01(01): 46-50.
[7]	Mengjie Jiang, Zhijun Qian, Si Xu, Wei Liang. Experimental study on the treatment of frozen shoulder model Rabbit with low-intensity pulsed ultrasound[J]. Chinese Journal of Shoulder and Elbow(Electronic Edition), 2023, 11(01): 30-34.
[8]	Yi Liu, Yini Wen, Yinghui Wu. Relationship of cytokines of peripheral blood helper T cells and regulatory T cells with renal damage in children with Henoch-Schönlein purpura[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2023, 12(05): 271-275.
[9]	Zhenyu Wu, Yafen Hu, Xiaofen Dong, Yuanfang Ma. Predictive values of serum CTGF, TGF-β1 and MMP2 levels on renal interstitial fibrosis in diabetes nephropathy[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2022, 11(06): 332-337.
[10]	Yu Dong, Shunlai Shang, Qinggang Li, Xiangmei Chen. Therapeutic effects of leflunomide combined with methylprednisolone in patients with IgA nephropathy[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2020, 09(03): 102-106.
[11]	Yuzhen Chen, Jiayun Xu. The efficacy and safety of leflunomide combined with low or moderate doses of glucocorticoids in the treatment of chronic progressive IgA nephropathy[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2019, 08(05): 208-212.
[12]	Xiaojian Feng, Ying Yang, Lihua Wang, Ruixia Yuan. The role and mechanism of Wnt signaling in the regulation of bone marrow-derived macrophages involved in renal fibrosis[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2018, 07(03): 126-130.
[13]	Na Li, Xiaojing Jia, Xing Feng, Yan Han, Lijun Zhao, Jianjun Cui. Effect of bone marrow mesenchymal stromal cells on gout kidney[J]. Chinese Journal of Clinicians(Electronic Edition), 2017, 11(11): 1894-1901.
[14]	Xiaohua Pei, Tao Zhang, Ke Jin, Yun Bai, Fei Gao, Bei Zhu, Weihong Zhao. Diagnostic characteristics of pneumocystis carinii pneumonia with non-human immunodeficiency virus infection and literature review[J]. Chinese Journal of Diagnostics(Electronic Edition), 2022, 10(04): 224-228.

Viewed

Full text

Abstract

Please choose a citation manager

Content to export