Genistein Affect The Apoptosis Of LPS-activated Macropahge Through Regulating TIPE2/Akt

Objective:To investigate the effect of genistein（GEN）on the apoptosis of lipopolysaccharides（LPS）-activated macrophages and whether the potential mechanism is related to regulating TIPE2/Akt.Methods:1.Apply 1 000 ng · mL^-1LPS to RAW264.7 cells for 24 h,use RT-qPCR to detect IL-6 and TNF-α mRNA expression levels,Western Blot to detect iNOS and COX-2 protein expression,and construct LPS activated macrophage model.GEN（10 μM）was used to pre-incubate RAW264.7 cells for 1 h,2 h,4 h,and 8 h respectively,and then treated with LPS(1 000 ng · mL^-1)for 24 h.Cell viability was detected by CCK8,RT-qPCR and Western Blot were used to detect the apoptotic proteins Cleaved caspase-3,CHOP,Bax,Bcl-2 and Cleaved caspase-8 mRNA and protein expression.In order to study the effects of GEN on the activated Macrophage Apoptosis,the apoptosis rate was detected by Annexin V-FITC / PI,TUNEL staining to detect the apoptosis index.TIPE2 is an immunonegative regulator,in order to clarify the regulatory role of TIPE2 in GEN regulating macrophage apoptosis,RAW264.7 cells were incubated with GEN for 2 h,and then treated with LPS for another 24 h.In order to observe effects of GEN on TIPE2 expression and Akt phosphorylation of activated macrophages,RT-qPCR was used to detect the mRNA expression of TIPE2,Western Blot was used to detect the protein expression of TIPE2,Akt,p-Akt,and Immunofluorescence was used to detect TIPE2 expression.2.Infect RAW264.7 cells with TIPE2 overexpression or knock down lentiviral particles,screened with purinomycin,and observe the expression of GFP protein under fluorescence microscope.RT-qPCR and Western Blot were used to detect TIPE2 mRNA and protein expression.Treated with LPS for 24 h,CCK8 kit to measure cell viability,Annexin V-FITC / PI to detect apoptosis rate,TUNEL to detect apoptosis index,Western Blot to detect Cleaved caspase-3,CHOP,Bax,Bcl-2,Cleaved caspase-8,Akt,p-Akt protein expression.IGF-1(100 ng · mL^-1)pretreated TIPE2 overexpress cells for 2 h,and then co-treated with LPS for 24 h.Cell viability was measured by CCK8 kit.The protein expression of Cleaved caspase-3,CHOP,Bax,Bcl-2,Cleaved caspase-8 were detected by Western Blot,verified the effect of TIPE2 on Akt phosphorylation in activated macrophages.3.GEN pretreated RAW264.7 cells infected with TIPE2 KD lentivirus for 2 hours and co-incubation with LPS(1 000 ng·mL^-1)for 24 hours.Cell viability was measured by the CCK8 kit.Cleaved caspase-3,CHOP,Bax,Bcl-2,and Cleaved caspase-8 protein expressions were detected by Western Blot.IGF-1 and GEN combined treatment RAW264.7 cells infected with TIPE2 NC,TIPE2 OE,TIPE2 NC and TIPE2 KD lentivirus for 2 hours and then incubation with LPS for 24 h.Cell viability was measured by CCK8 kit.Western Blot was used to detect Cleaved caspase-3,CHOP,Bax,Bcl-2,and Cleaved caspase-8 protein expressions,to investigate whether GEN affects activated macrophage apoptosis through the TIPE2 / Akt pathwayResults:1.LPS(1 000 ng · mL^-1)treated RAW264.7 cells for 24 hours,IL-6 and TNF-α mRNA expression increased,iNOS and COX-2 protein expression increased,indicating that LPS can effectively activate RAW264.7 cells that can be used in subsequent experiments.Compared with the control group,LPS significantly increased the RAW264.7 cell viability,decreased the apoptotic rate and apoptosis index,reduced the expression of apoptotic proteins Cleaved caspase-3,CHOP,Bax,Cleaved caspase-8,and increased Bcl-2 expression.GEN significantly reduced the vitality of activated macrophages in a time-dependent manner,increased the apoptotic rate and apoptosis index,promoted the expression of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8,inhibited Bcl-2 expression.while GEN can effectively promote the apoptosis of activated macrophages after 2 hours（P <0.05）,so we choose 2 h as the treatment time for GEN.Compared with the control group,LPS significantly down-regulated TIPE2 mRNA and protein expression,up-regulated p-Akt levels,and Akt had no significant change;GEN had no significant effect on the expression of TIPE2,Akt,and p-Akt in unactivated cells,while GEN significantly increased the TIPE2 mRNA and protein expression level of activated macrophages,reduced p-Akt expression and did not affect the Akt expression.2.Compared with the TIPE2 empty vector group,the TIPE2 expression of TIPE2 overexpression group was up-regulated about 2 times,the TIPE2 expression of TIPE2 knockdown group was down-regulated about 70%.TIPE2 overexpression significantly inhibits the cell viability of activated macrophages,increases the apoptotic rate and apoptosis index,reduces the expression of p-Akt and Bcl-2,and increases the expression of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8;IGF-1 can Effectively reverse the effects of TIPE2 overexpression on activated macrophage.TIPE2 knockdown can significantly promote the cell viability of activated macrophages,reduced the apoptosis rate and apoptosis index,increase the expression of p-Akt and Bcl-2,and reduce the expression of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8.3.Compared with the TIPE2 empty vector + GEN + LPS group,the cell viability of the TIPE2 knockdown + GEN + LPS group increased the cell viability and the expression of Bcl-2,while the expression of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8 decreased.Compared with the LPS group,the cell viability and Bcl-2 expression of the LPS + IGF-1 group were significantly increased,and the expressions of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8 were decreased.Compared with the LPS + GEN group,LPS + GEN + IGF increased the cell viability and Bcl-2 expression,while the expressions of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8 were decreased.Compared with the RAW264.7 + GEN + LPS group,the cell viability of TIPE2 overexpression + GEN + LPS group was weakened,Bcl-2 expression was reduced,Cleaved caspase-3,CHOP,Bax,Cleaved caspase-8 expression were increased,and TIPE2 overexpression + GEN + LPS + IGF-1 had opposite results to TIPE2 overexpression + GEN + LPS group;compared with the RAW264.7 + GEN + LPS group,the cell viability of the TIPE2 knockdown + GEN + LPS group was up-regulated,Bcl-2 expression increased,and the expressions of Cleaved caspase-3,CHOP,Bax,and Cleaved caspase-8 were decreased,and the results of TIPE2 knockdown + GEN + LPS + IGF-1 group were more obvious than those of TIPE2 knockdown + GEN + LPS group.Conclusions: GEN may promote LPS-activated macrophage apoptosis by increasing the expression of TIPE2 and decreasing the activity of p-Akt.