Research On The Effects Of Soybean Agglutinin On Integrin-Mediated Cell Proliferation And Apoptosis Mechanism In IPEC-J2

Soybean agglutinin(SBA)is an anti-nutritional substance contained in soybean and soybean products.Such anti-nutritional substance can bind to the surface of intestinal epithelial cells in mammalian,alter the structure and metabolism of the cells,and affect the metabolic process of the intestnal epithelial cells.Integrins are transmembrane heterodimeric glycoprotein,and integrin-mediated cell adhesion is one prerequisite factor for the biological function of the cell.To elucidate whether SBA affect the health of the porcine intestinal epithelial cell through the integrins,three experimental researches were performed.In this research,intestinal epithelial cell from piglets(IPEC-J2)was selected as the cell mode in vitro to explore the species of integrin subunits that expressed on IPEC-J2;to investigate the effects of these integrin subunits on cell proliferation,cell cycle,cell apoptosis and SBA-induced cell cycle and cell apoptosis alterations in IPEC-J2;to research the relationship and connection type between SBA and integrin subunits.These researches provided a theoretical basis and data support for further revealing the SBA anti-nutritional mechanism.Experiment one: Identification of the integrin subunits in IPEC-J2In this study,IPEC-J2 membrane proteins were isolated by a native membrane protein extraction kit,and separated using 1D SDS-PAGE.According to the molecular weight of integrins,bands with a molecular weight of 80-220 kDa were collected and analyzed by electro-spray ionization tandem mass spectrometry(ESI-Q-TOF MS/MS).The results were further identified by immunoblotting.The results from mass spectrometry and immunoblotting showed a total of five integrin subunits that distributed in IPEC-J2 cell membranes were successfully identified,they were integrin ?2,?3,?6,?1 and ?4.Experiment two: Effects of integrins on cell proliferation,cell cycle,cell apoptosis and SBA-induced cell cycle and cell apoptosis alterations in IPEC-J2.After treated with 0,0.125,0.25,0.5,1.0 or 2.0 mg/mL SBA for 24 h,the effects of SBA with different levels on IPEC-J2 cell proliferation were determined by CCK-8 kits,and the cell cycle and cell apoptosis results were analyzed using flow cytometry.The changes in the mRNA expression of cell cycle related genes after SBA stimulation were analyzed by q-PCR.After treated with 0,5,10,or 20 ?g/mL integrin(?2,?3,?6,?1 or ?4)functional inhibitors for 24 h,CCK-8 kits were used to explore the effects of integrin inhibitors on IPEC-J2 cell proliferation and screen the optimal concentrations of integrin inhibitors.SBA and integrin inhibitors(?2,?3,?6,?1 or ?4)with the optimal concentrations were used in integrin functional inhibitor test.The cells were divided into four groups: 0 mg/mL SBA(control),SBA treatment,integrin(?2,?3,?6,?1 or ?4)inhibitor treatment,and integrin(?2,?3,?6,?1 or ?4)inhibitor+SBA treatment.After 24 h treatment,the roles of the integrin inhibitors on IPEC-J2 cell proliferation,cell cycle,cell apoptosis and SBA-induced cell cycle and apoptosis alterations in IPEC-J2 were analyzed.The results indicated that SBA declined IPEC-J2 cell proliferation by arresting the cells at G0/G1 phase of cell cycle(P<0.05),and induced cell apoptosis(P<0.05).Moreover,SBA lowered mRNA expression of cell cycle-related gene CDK4,Cyclin E and Cyclin D1(P<0.05).Integrin ?2,?3,?6,?1 and ?4 were crucial for maintaining normal cell proliferation,cell cycle and cell apoptosis in IPEC-J2.Restrain of either these five subunits by their inhibitors significantly induced G0/G1 phase arrest(P<0.05),lowered cell proliferation(P<0.05),and caused cell apoptosis(P<0.05)in IPEC-J2.Furthermore,integrin ?2,?6 and ?1 were involved in the block of G0/G1 phase induced by SBA,and integrin ?2,?3,?6,?1 and ?4 were involved in SBA-induced cell apoptosis in IPEC-J2.Experiment three: Research of the connection type and interaction between SBA and integrins.After treated with 2.0 mg/mL SBA for 24 h,the mRNA expression of integrins(?2,?3,?6,?1 and ?4)were identified by q-PCR.SBA-specific binding proteins on IPEC-J2 cell membranes were separated using Co-Immunoprecipitation method and analyzed with C-MS-MS(Q-E)detection.q-PCR was applied to detect the effects of 2.0 mg/mL SBA on ?-actinin-2(ACTN2)gene expression.After treated with siRNA sequences of the ACTN2 for 24 h,q-PCR were used to determine the relationship between ACTN2 and integrins(?2,?3,?6,?1,and ?4)gene expression.The results showed that SBA significantly lowered mRNA expression of integrin ?2,?3,?6,?1 and ?4(P<0.05).Sixty-seven kinds of SBA specific binding proteins on IPEC-J2 membranes were identified.Integrins were not detected in SBA receptors,indicating there had no direct connection between integrins and SBA.However,integrin-associated protein,?-actinin-2(ACTN2),was detected in SBA receptors and its mRNA expression was lowered when treated with SBA(P<0.05).Additionally,siRNA treatment of ACTN2 caused a reduction in mRNA expression of integrin ?2,?3,?6,?1 and ?4 subunits(P<0.05),suggesting that SBA influenced the relationship between ACTN2 and integrin though the reduction of ACTN2 gene expression,then induced these integrin mRNA expression decrease in IPEC-J2.In conclusion,the integrin subunits that expressed in IPEC-J2 cell membranes were integrin ?2,?3,?6,?1 and ?4.These integrins were important for maintaining normal cellular metabolic process in IPEC-J2.SBA can indirectly induced the alterations of integrin gene expression and functions through the connection with ACTN2,and then affect the biological functions(such as cell proliferation,cell cycle and apoptosis)of IPEC-J2.The current research provided an important basis for exploring the SBA anti-nutritional mechanism.Integrins can be used as a new research ideas to improve SBA-induced cell biological functions alterations.