新型多肽AMPP2在TGF-β1诱导的系膜细胞增殖中的作用及其机制

doi:10.11958/20231143

摘要/Abstract

摘要：

目的探究新型多肽AMPP2在转化生长因子β1（TGF-β1）诱导的小鼠系膜细胞增殖中的作用及相关机制。方法体外用TGF-β1（10 μg/L）及AMPP2（10 ng/L）干预系膜细胞，分为Control组、AMPP2组、TGF-β1组及TGF-β1＋AMPP2组。CCK-8法检测各组系膜细胞增殖活力；Western blot法检测各组细胞周期蛋白依赖性激酶（CDK）-4、CDK-6、细胞增殖核抗原（PCNA）、α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（COL-Ⅰ）、纤维连接蛋白（FN）及磷酸化的SMAD同源物3/SMAD同源物3（p-SMAD3/SMAD3）的蛋白表达水平；qPCR法检测各组α-SMA、COL-Ⅰ、FN的mRNA表达水平。结果与Control组相比，TGF-β1组系膜细胞增殖活力增加（P<0.05），与TGF-β1组相比，TGF-β1＋AMPP2组系膜细胞增殖活力下降（P<0.05）；与Control组相比，TGF-β1组CDK-4、CDK-6、PCNA蛋白以及α-SMA、COL-Ⅰ、FN蛋白和mRNA表达水平升高（P<0.05），与TGF-β1组相比，TGF-β1＋AMPP2组上述蛋白及mRNA表达水平均下降（P<0.05）；与Control组相比，TGF-β1组p-SMAD3/SMAD3水平显著升高（P<0.05），与TGF-β1组相比，TGF-β1＋AMPP2组p-SMAD3/SMAD3的蛋白表达量显著降低（P<0.05）。结论 AMPP2可能通过调控TGF-β1/SMAD3通路抑制系膜细胞增殖。

关键词: 转化生长因子β1, 肾小球系膜细胞, 细胞增殖, 肾炎, 紫癜, 过敏性, 肽类, AMPP2

Abstract:

Objective To explore the effect and mechanism of anti-mesangial cell-proliferation-peptide 2 (AMPP2) on mesangial cell proliferation induced by transforming growth factor β1 (TGF-β1). Methods Mesangial cells were cultured in vitro and treated with TGF-β1 (10 μg/L) and AMPP2 (10 ng/L). According to different intervention factors, mesangial cells were divided into four groups: the control group, the AMPP2 group, the TGF-β1 group and the TGF-β1＋AMPP2 group. The proliferation activity of mesangial cells was detected by CCK-8. The relative protein expression of cyclin dependent kinase 4 (CDK-4), cyclin dependent kinase 6 (CDK-6), proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), collagen-Ⅰ (COL-Ⅰ) and fibronectin (FN) were examined by Western blot assay. The relative mRNA expression of α-SMA, COL-Ⅰ and FN were detected by qPCR. Results Compared with the control group, proliferation activity of mesangial cells was significantly increased in the TGF-β1 group (P<0.05). The proliferation activity of mesangial cells was markedly decreased in the TGF-β1＋AMPP2 group compared with that of the TGF-β1 group (P<0.05). Compared with the control group, protein levels of CDK-4, CDK-6, PCNA, α-SMA, COL-Ⅰ and FN in cells were significantly increased in the TGF-β1 group (P<0.05), as well as the mRNA levels of α-SMA, COL-Ⅰ and FN (P<0.05). In the TGF-β1＋AMPP2 group, the protein and mRNA levels of α-SMA, COL-Ⅰ and FN and the protein levels of CDK-4, CDK-6 and PCNA were markedly decreased compared with those of the TGF-β1 group (P<0.05). Compared with the control group, levels of p-SMAD3/SMAD3 was remarkably upregulated in the TGF-β1 group (P<0.05), while levels of p-SMAD3/SMAD3 was remarkably downregulated in the TGF-β1＋AMPP2 group compared with those of the TGF-β1 group (P<0.05). Conclusion AMPP2 may inhibit mesangial cell proliferation by regulating TGF-β1/SMAD3 pathway.

Key words: transforming growth factor beta1, mesangial cells, cell proliferation, nephritis, urpura, Schoenlein-Henoch, peptides, AMPP2

中图分类号:

图/表 13

表1 qPCR引物序列

Tab.1 Sequences of qPCR primers

基因	引物序列（5′→3′）	产物大小/bp
α-SMA	上游：CAGCAAACAGGAATACGACGAA	168
α-SMA	下游：AACCACGAGTAACAAATCAAAGC	168
COL-Ⅰ	上游：GTCAGACCTGTGTGTTCCCTACTCA	99
COL-Ⅰ	下游：TCTCTCCAAACCAGACGTGCTTC	99
FN	上游：GCAAGAAGGACAACCGAGGAAA	126
FN	下游：GGACATCAGTGAAGGAGCCAGA	126
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC	171
β-actin	下游：ATGGAGCCACCGATCCACA	171

图1 2组系膜细胞中CDK-4、CDK-6及PCNA蛋白表达

Fig.1 The protein expression levels of CDK-4, CDK-6 and PCNA in mesangial cells of two groups

表2 2组系膜细胞中CDK-4、CDK-6及PCNA蛋白表达水平比较

Tab.2 Comparison of protein expression levels of CDK-4, CDK-6 and PCNA in mesangial cells between two groups （n=3，$\bar{x}±s$）

组别	CDK-4	CDK-6	PCNA
Control组	0.66±0.05	0.69±0.05	0.49±0.03
TGF-β1组	1.14±0.07	1.05±0.05	0.97±0.12
t	32.600^＊＊	8.310^＊	6.733^＊

图2 2组系膜细胞中α-SMA、COL-Ⅰ及FN 蛋白表达

Fig.2 The protein expression levels of α-SMA, COL-Ⅰ and FN in mesangial cells of two groups

表3 2组系膜细胞α-SMA、COL-Ⅰ及FN蛋白及mRNA表达水平比较

Tab.3 Comparison of protein and mRNA expression levels of α-SMA, COL-Ⅰ and FN in mesangial cells between two groups （n=3，$\bar{x}±s$）

组别	蛋白			mRNA
组别	α-SMA	COL-Ⅰ	FN	α-SMA	COL-Ⅰ	FN
Control组	0.56±0.03	0.62±0.02	0.47±0.09	1.01±0.00	1.01±0.01	1.00±0.00
TGF-β1组	1.05±0.09	1.03±0.04	0.99±0.05	4.36±0.44	4.21±0.35	4.64±0.74
t	14.930^＊＊	21.230^＊＊	7.386^＊	13.340^＊＊	15.870^＊＊	8.520^＊＊

表4 4组系膜细胞不同时间点增殖情况比较

Tab.4 Comparison of mesangial cell proliferation at different time points between four groups （n=3，$\bar{x}±s$）

组别	0 h	24 h	48 h	72 h	F
TGF-β1+0 ng/L AMPP2组	1.00±0.00	1.47±0.05^A	1.59±0.03^AB	1.58±0.06^AB	164.300^＊＊
TGF-β1+1 ng/L AMPP2组	1.00±0.00	1.46±0.11^A	1.48±0.04^aA	1.40±0.03^aA	174.300^＊＊
TGF-β1+10 ng/L AMPP2组	1.00±0.00	1.42±0.16^A	1.19±0.01^aAB	1.17±0.04^aAB	157.700^＊＊
TGF-β1+100 ng/L AMPP2组	1.00±0.00	1.32±0.02^aA	1.29±0.04^aA	1.27±0.04^aAB	96.870^＊＊
F		20.810^＊＊	112.400^＊＊	45.840^＊＊

图3 AMPP2对细胞活力的影响 A：Control组；B：AMPP2组；C：TGF-β1组；D：TGF-β1＋AMPP2组；a与Control组比较，b与TGF-β1组比较，P<0.05；图4—6同。

Fig.3 Effects of AMPP2 on mesangial cell proliferative activity

图4 4组系膜细胞中CDK-4、CDK-6及PCNA蛋白表达

Fig.4 The protein expression levels of CDK-4, CDK-6 and PCNA in mesangial cells in four groups

表5 4组系膜细胞CDK-4、CDK-6及PCNA蛋白表达水平比较

Tab.5 Comparison of protein expression levels of CDK-4, CDK-6 and PCNA in mesangial cells between four groups （n=3，$\bar{x}±s$）

组别	CDK-4	CDK-6	PCNA
Control组	0.85±0.05	1.09±0.13	0.79±0.15
AMPP2组	0.81±0.11	1.09±0.22	0.78±0.26
TGF-β1组	1.26±0.10^a	1.78±0.15^a	1.62±0.17^a
TGF-β1＋AMPP2组	0.86±0.14^b	1.30±0.15^b	0.96±0.22^b
F	16.420^＊＊	11.480^＊＊	11.390^＊＊

图5 4组系膜细胞中α-SMA、COL-Ⅰ及FN蛋白表达

Fig.5 The protein expression levels of α-SMA, COL-Ⅰ and FN in mesangial cells of four groups

表6 4组系膜细胞α-SMA、COL-Ⅰ及FN蛋白表达水平比较

Tab.6 Comparison of mRNA expression levels of α-SMA, COL-Ⅰ and FN in mesangial cells between four groups （n=3，$\bar{x}±s$）

组别	α-SMA	COL-Ⅰ	FN
Control组	0.63±0.08	0.85±0.10	0.67±0.05
AMPP2组	0.60±0.05	0.81±0.12	0.60±0.01
TGF-β1组	1.20±0.11^a	1.23±0.08^a	1.24±0.19^a
TGF-β1＋AMPP2组	0.76±0.13^b	0.94±0.16^b	0.89±0.09^b
F	32.660^＊＊	12.520^＊＊	20.940^＊＊

表7 4组系膜细胞α-SMA、COL-Ⅰ及FN mRNA表达水平比较

Tab.7 Comparison of mRNA expression levels of α-SMA, COL-Ⅰ and FN in mesangial cells between four groups （n=3，$\bar{x}±s$）

组别	α-SMA	COL-Ⅰ	FN
Control组	0.99±0.03	1.02±0.01	0.92±0.03
AMPP2组	0.90±0.06	0.88±0.10	0.92±0.11
TGF-β1组	4.03±0.45^a	4.37±0.25^a	4.12±0.60^a
TGF-β1＋AMPP2组	1.58±0.14^b	1.68±0.52^b	1.73±0.34^b
F	98.530^＊＊	110.800^＊＊	54.750^＊＊

图6 4组系膜细胞中p-SMAD3/SMAD3蛋白表达

Fig.6 The protein expression levels of p-SMAD3/SMAD3 in mesangial cells in four groups

参考文献 19

[1]	PILLEBOUT E, SUNDERKOTTER C. IgA vasculitis[J]. Semin Immunopathol, 2021, 43(5):729-738. doi:10.1007/s00281-021-00874-9.
[2]	GARDNER-MEDWIN J M, DOLEZALOVA P, CUMMINS C, et al. Incidence of Henoch-Schonlein purpura,Kawasaki disease,and rare vasculitides in children of different ethnic origins[J]. Lancet, 2002, 360(9341):1197-1202. doi:10.1016/S0140-6736(02)11279-7.
[3]	YANG X, LI Q, HE Y, et al. Individualized medication based on pharmacogenomics and treatment progress in children with IgAV nephritis[J]. Front Pharmacol, 2022, 13:956397. doi:10.3389/fphar.2022.956397.
[4]	ZHANG H, DENG Z, WANG Y. Molecular insight in intrarenal inflammation affecting four main types of cells in nephrons in IgA nephropathy[J]. Front Med(Lausanne), 2023, 10:1128393. doi:10.3389/fmed.2023.1128393.
[5]	SUN G X, DING R, LI M, et al. Ghrelin attenuates renal fibrosis and inflammation of obstructive nephropathy[J]. J Urol, 2015, 193(6):2107-2115. doi:10.1016/j.juro.2014.11.098.
[6]	YUAN Q, REN Q, LI L, et al. A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling[J]. Nat Commun, 2022, 13(1):438. doi:10.1038/s41467-022-28096-z.
[7]	LI Y K, MA D X, WANG Z M, et al. The glucagon-like peptide-1 (GLP-1)analog liraglutide attenuates renal fibrosis[J]. Pharmacol Res, 2018, 131:102-111. doi:10.1016/j.phrs.2018.03.004.
[8]	施会敏, 鱼敏逸, 曲高婷, 等. 紫癜性肾炎患儿血清中多肽的差异表达研究[J]. 徐州医科大学学报, 2019, 39(4):239-244.
	SHI H M, YU M Y, QU G T, et al. Differential expression of peptides in serum of children with purpura nephritis[J]. Acta Academiae Medicinae Xuzhou, 2019, 39(4):239-244. doi:10.3969/j.issn.2096-3882.2019.04.002.
[9]	ZULIANI-ALVAREZ L, MIDWOOD K S. Fibrinogen-related proteins in tissue repair:how a unique domain with a common structure controls diverse aspects of wound healing[J]. Adv Wound Care(New Rochelle), 2015, 4(5):273-285. doi:10.1089/wound.2014.0599.
[10]	SCINDIA Y M, DESHMUKH U S, BAGAVANT H. Mesangial pathology in glomerular disease:targets for therapeutic intervention[J]. Adv Drug Deliv Rev, 2010, 62(14):1337-1343. doi:10.1016/j.addr.2010.08.011.
[11]	ONG C H, THAM C L, HARITH H H, et al. TGF-β-induced fibrosis:A review on the underlying mechanism and potential therapeutic strategies[J]. Eur J Pharmacol, 2021, 911:174510. doi:10.1016/j.ejphar.2021.174510.
[12]	YE X, LI J, LIU Z, et al. Peptide mediated therapy infibrosis:Mechanisms,advances and prospects[J]. Biomed Pharmacother, 2023, 157:113978. doi:10.1016/j.biopha.2022.113978.
[13]	LI D, NIE H, JIANG K, et al. Molecular characterization and expression analysis of fibrinogen related protein(FREP)genes of Manila clam(Ruditapes philippinarum)after lipopolysaccharides challenge[J]. Comp Biochem Physiol C Toxicol Pharmacol, 2020, 228:108672. doi:10.1016/j.cbpc.2019.108672.
[14]	SORIA J, MIRSHAHI S, MIRSHAHI S Q, et al. Fibrinogen alphaC domain:Its importance in physiopathology[J]. Res Pract Thromb Haemost, 2019, 3(2):173-183. doi:10.1002/rth2.12183.
[15]	TIAN Y, HONG M, JING S, et al. Clinical and prognostic effect of plasma fibrinogen in renal cell carcinoma:a meta-analysis[J]. Biomed Res Int, 2017, 2017:9591506. doi:10.1155/2017/9591506.
[16]	GAO J, WANG Y, DONG Z, et al. A novel differential diagnostic model based on multiple biological parameters for immunoglobulin A nephropathy[J]. BMC Med Inform Decis Mak, 2012, 12:58. doi:10.1186/1472-6947-12-58.
[17]	VILAR R, FISH R J, CASINI A, et al. Fibrin(ogen)in human disease:both friend and foe[J]. Haematologica, 2020, 105(2):284-296. doi:10.3324/haematol.2019.236901.
[18]	MIDGLEY A C, ROGERS M, HALLETT M B, et al. Transforming growth factor-beta1 (TGF-beta1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor(EGFR)and CD44 co-localization in lipid rafts[J]. J Biol Chem, 2013, 288(21):14824-14838. doi:10.1074/jbc.M113.451336.
[19]	HU H H, CHEN D Q, WANG Y N, et al. New insights into TGF-β/Smad signaling in tissue fibrosis[J]. Chem Biol Interact, 2018, 292:76-83. doi:10.1016/j.cbi.2018.07.008.