The Molecular Mechanisms Of SEA-induced Senescence In Hepatic Stellate Cells

Objective The study was designed to investigate the effects and possible mechanisms of Schistosoma japonicum soluble egg antigens(SEA)on the senescence of human hepatic stellate cell line LX-2.Methods1.Cultured human hepatic stellate cell line LX-2 cells were treated with SEA at different concentrations(5,10,20 ?g/mL)of the experiment,and then the following experiments were carried out.(1)Liver fibrosis-associated proteins were analyzed by Western blot.(2)The impacts of SEA on LX-2 cells were determined by Senescence-associated ?-galactosidase(SA-?-Gal)staining.(3)The effects of SEA on senescence-associated cell cycle arrest were assessed by flow cytometry.(4)Proliferation of LX-2 cells was measured by MTT.2.The LX-2 cells were stimulated by SEA,p53 shRNA and STAT3 siRNA respectively,and the following tests were carried out.(1)After SEA stimulation,the expression of senescence-associated proteins STAT3,P-STAT3,p53,P-p53 and p21 were analyzed by Western blot.(2)Detecting the expression of p53,P-p53,p21 and STAT3 protein in LX-2 cells after the knock down of p53.(3)The expression of STAT3 and p53 protein were observed in LX-2 cells after the knock down of STAT3.(4)The Senescence-associated ?-galactosidase staining was observed upon the knock down of P53 and STAT3 in LX-2 cells.(5)The interaction of p53 and SOCS3 was analyzed by immunoprecipitation.3.The LX-2 cells were stimulated by SEA,p27 siRNA and SKP2 overexpression plasmid respectively.(1)LX-2 cells were treated with SEA for 48 h,and then the expression of SKP2,P-Akt and FoxO3 a protein were detected by Western blot.(2)The localization of FoxO3 a in LX-2 cells was observed by immunofluorescent assay.(3)LX-2 cell senescence was observed by Senescence-associated ?-galactosidase staining after the knock down of p27 by p27 siRNA.(4)SKP2 overexpression plasmid was constructed and transfected into LX-2 cells to observe the cellular senescence and the expression of p27 protein.Results1.SEA inhibited the expression of liver fibrosis-associated proteins,such as ?-SMA and pro-collagen I.Senescence-associated ?-galactosidase staining method showed that the proportion of the SA-?-Gal positive cells was increased in LX-2 cells after the treatment with SEA.Besides,the results of flow cytometry assay showed that the proportion of G1 phase cells increased,while the proportion of S phase cells decreased upon the SEA stimulation.The results of MTT method showed that SEA could inhibit the proliferation of LX-2 cells.2.SEA induced an increased expression of senescence-associated proteins(P-STAT3,P-p53,p21),and immunofluorescence assay showed that SEA induced the nuc lear transfer of STAT3 in the LX-2 cells.Phosphorylated STAT3 was elevated upon SEA stimulation,while loss of STAT3 decreased the level of p53,as well as activities of Senescence-associated ?-galactosidase in HSCs.Apart from these observations,knockdown of p53 inhibited the expression of p21 and the absence of p53 failed to induce senescence of activated-HSCs.Results from immunoprecipitation analys is demonstrated the direct interaction between SOCS3 and p53.3.SEA upregulated the expression of p27 but downregulated the expression of the P-Akt,SKP2 and FoxO3 a in LX-2 cells.Immunofluorescence results showed that SEA treatment led to the nuclear transfer of FoxO3 a in LX-2 cells.The knockdown of p27 by p27 siRNA inhibited the senescence of LX-2 induced by SEA,and the proportion of the SA-?-Gal positive cells decreased significantly.Besides,the high expression of p27 and activities of SA-?-Gal were reversed by overexpression of SKP2.Conclusions1.SEA induces the senescence of LX-2 cells;2.SEA induces the senescence of LX-2 cells through STAT3-p53-p21 pathway;3.SEA induces the senescence of LX-2 cells through Akt-FoxO3a-SKP2-p27 pathway.