Effects and mechanism of AMPP2 on mesangial cell proliferation induced by TGF-β1

doi:10.11958/20231143

Abstract

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

CLC Number:

Figures/Tables 13

References 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.

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

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

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^＊

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^＊

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^＊＊