The Mechanism Of The HSCs Senescence Induced By The Recombinant Protein Sjp40 From Schistosoma Japonicum Eggs

Objective The study was designed to explore the effect of S.japonicum egg protein p40(Sjp40)on cell senescence and the underlying mechanisms of Sjp40-induced senescence in activated LX-2 cells.Methods(1)Cells were exposed to the different concentration of Sjp40(5,10,20,40?g/mL)for 48 h and Western Blot assay was used to observe the expression of ?-SMA and collagen type I viewed as markers of HSCs activation,and obtain the best concentration of Sjp40;(2)The level of cleaved-Caspase-3 analysis and SA-?-Gal assay were applied to investigate the ability of Sjp40 to induce apoptosis and senescence in LX-2 cells respectively,and the proliferation capacity of LX-2 cells was analyzed by MTT assay(ETO and OVA were served as a positive and negative stimulus to treat cells,respectively);(3)The cell cycle phase distribution of LX-2 with or without Sjp40 was measured by flow cytometry and the expression of cell cycle regulatory proteins about G1-S phase transition was observed by Western Blot;(4)Total proteins extracted from LX-2 cells treated with or without Sjp40 were analyzed byWestern Blot assay with the specific antibodies against p53,P-p53,p21 and p16.The effects of p53 silencing on the Sjp40-induced aging of LX-2 cells were analyzed by SA-?-Gal assay and the protein levels of P-p53 and p21 following p53 knockdown were examined by Western Blot assay(experiment grouping: Control,Sjp40,Sh-Con,Sh-Con+Sjp40,Sh-p53,Sh-p53+Sjp40,OVA);(5)The protein levels of STAT3 and P-STAT3 were investigated by Western Blot assay and the Sjp40-induced translocation of STAT3 was detected by immunofluorescence assay;(6)The expression levels of P-STAT3,p53,P-p53 and p21 following silencing STAT3 were measured by Western Blot and Sjp40-induced aging of LX-2following STAT3 silencing was analyzed by SA-?-Gal assay(experiment grouping: Control,Sjp40,Si-Con,Si-Con+Sjp40,Si-STAT3,Si-STAT3+Sjp40,OVA);(7)The forced expression of TLR-4 via LPS,and the protein level of P-p53,the most critical aging-associated molecular in this study,were examined via Western Blot,and the effect of overexpression of TLR-4 on the number of SA-?-Gal positive cells being exposed to Sjp40 was measured by SA-?-Gal assay(experiment grouping: Control,Sjp40,LPS,LPS+Sjp40,OVA);(8)Total proteins extracted from LX-2 cells treated with or without Sjp40 were analyzed by Western Blot assay with the specific antibodies against P27,P-ERK,SKP2 and P-Rb.The effects of P27 silencing on the Sjp40-induced aging of LX-2cells were analyzed by SA-?-Gal assay and the protein levels of P27 and SKP2 following P27 knockdown were examined by Western Blot assay(experiment grouping: Control,Sjp40,Si-Con,Si-Con+Sjp40,Si-P27,Si-P27+Sjp40,OVA);(9)The Sjp40-induced senescence of LX-2 cells following SKP2 over-expression was analyzed by SA-?-Gal assay and the protein levels of P27 and markers of HSCs activation following SKP2 over-expression were examined by Western Blot assay(experiment grouping: Control,Sjp40,pcDNA3.1,pcDNA3.1+Sjp40,pcDNA3.1-SKP2,pcDNA3.1-SKP2+Sjp40,OVA);(10)The expression of intercellular adhesion molecule and ligand molecule of the NK cell receptor was measured by Western Blot,and the potential interaction between LX-2 and YT cells was observed by immunofluorescence assay.The NK-mediated cytotoxicity effect was assessed via crystal violet staining.Results(1)Sjp40 treatment could down-regulate the expression of ?-SMA and collagen type I measured by Western Blot assay;(2)The cells exposed to Sjp40 dramatically increased the SA-?-Gal activity,which proved that Sjp40 could induce the senescence of LX-2 cells.MTT assay demonstrated that the proliferation capacity of LX-2 cells had been markedly suppressed after being exposed to Sjp40.Flow cytometry showed that the number of G1-arrested LX-2 cells in Sjp40-treated group exhibited an obvious increase compared with the control group.Western Blot assay showed that the expression of phosphorylation of Rb and cyclin A was markedly reduced in Sjp40-treated cells;(3)Sjp40could induce the activation of P-STAT3/P-p53/p21 signaling and up-regulate the protein levels of P-STAT3,P-p53 and p21.In addition,we also observed an obvious translocation of STAT3 from cytoplasm to the cellular nucleus in the presence of Sjp40.Si-RNA knockdown of STAT3 or Sh-RNA knockdown of p53 could reduce the number of SA-?-Gal positive cells being treated with Sjp40 and partially rescued the Sjp40-induced senescence in LX-2 cells;(4)The protein level of TLR-4 was decreased in the Sjp40-treated LX-2cells and over-expression of TLR-4 via LPS could down-regulate the protein level of P-p53 induced by Sjp40,which is regarded as the most critical aging-associated molecular in this study.Forced expression of TLR-4 could reduce the number of SA-?-Gal positive cells being exposed to Sjp40 and partially rescue the Sjp40-inducedsenescence in LX-2 cells;(5)Sjp40 could induce the activation of SKP2/P27 signaling,and up-regulate the level of P27 and down-regulate the level of SKP2 in LX-2 cells.Si-RNA knockdown of P27 or over-expression of SKP2 could reduce the number of SA-?-Gal positive cells being treated with Sjp40 and partially rescued the Sjp40-induced senescence in LX-2 cells;(6)The protein levels of adhesion molecule ICAM-1 and ligand molecule MICA in LX-2 cells were up-regulated by being exposed to Sjp40,which might enhance the elimination effect of NK against HSCs via cell-cell adhesion and receptor-ligand activation after the induction of cell aging.Conclusions(1)Sjp40 could induce the senescence of HSCs;(2)Sjp40 could promote HSCs senescence via STAT3/p53/p21pathway;(3)Sjp40 also could induce the senescence of HSCs by SKP2/P27 pathway;(4)Senescent HSCs could be cleared by NK cells by direct contact.