Protective Effect Of C-Phycocyanin And Apo-Phycocyanin Subunit On Programmed Necrosis Of GC-1 Spg Cells

Objective:To investigate the effects of C-phycocyanin(C-PC)and apo-phycocyanin subunit on programmed necrosis against oxidative damage of mouse spermatogonium(GC-1 spg)induced by H₂O₂.Methods:Through genetic engineering technology,C-phycocyanin subunit genes were cloned into p GEX-4T-1 vector,and transformed into E.coli BL21 strain to construct its prokaryotic expression vector,thus realizing the efficient expression of apo-phycocyanin subunit in spirulina in engineering bacteria.GST-Sefinose column was used to analyze and purify the target protein.Western blot was used to analyze the purity of the target protein.GC-1 spg cells were treated with 600?M H₂O₂ to establish oxidative stress injury model.They were divided into 5 groups according to different treatment groups:control group,H₂O₂ group,C-PC+H₂O₂ group,GST-CPC?+H₂O₂ group,Nec-1+H₂O₂ group.CCK-8 assay was used to detect cell activity.The production of cell reactive oxygen species(ROS)in living cells of each group was detected under fluorescence microscope by 2',7'-dichloro fluorescent yellow diacetate(DCFH-DA).Mitochondrial membrane potential was determined by JC-1.Cell necrosis rate was detected by Annexin V-FITC/PI.The expression levels of programmed necrosis associated proteins RIP-1,RIP-3 and p-MLKL in GC-1 spg cells were detected by Western blot.Results:1.The recombinant plasmid was transformed into E.coli BL21 and successfully induced the expression of the recombinant protein with IPTG,and purified to obtain the apo-phycocyanin?and?subunit.2.Compared with the control group,the survival rate of cells in H₂O₂ group was significantly reduced(P<0.01).While compared with the H₂O₂group,the cell survival rate of C-PC+H₂O₂ group,GST-CPC?+H₂O₂ group and Nec-1+H₂O₂ group were increased(P<0.01),and there was no significant difference in GST+H₂O₂ group and GST-CPC?+H₂O₂group.3.Fluorescence microscope observation of DCFH-DA results showed that compared with the control group,the intensity of intracellular green fluorescence in H₂O₂ group was significantly increased(P<0.01),The green fluorescence of C-PC+H₂O₂ group,GST-CPC?+H₂O₂ group and Nec-1+H₂O₂group was significantly decreased compared with the H₂O₂ group,the intracellular ROS level is reduced(P<0.01).4.Compared with the control group,JC-1 mitochondrial membrane potential observed under fluorescence microscope decreased significantly in H₂O₂group(P<0.05),the mitochondrial membrane potential of C-PC+H₂O₂ group,GST-CPC?+H₂O₂ group and Nec-1+H₂O₂ group increased compared with the H₂O₂group(P<0.05).5.Annexin V-FITC/PI double staining results showed that the cell necrosis rate of GC-1 cells in H₂O₂ group was significantly higher than those in the control group(P<0.01),the cell necrosis rate of C-PC+H₂O₂ group,GST-CPC?+H₂O₂group and Nec-1+H₂O₂ group was significantly lower compared with the H₂O₂ group(P<0.01).6.Western blot analysis showed that the protein expression levels of RIP-1,RIP-3 and p-MLKL in H₂O₂ group were significantly increased compared with the control group(P<0.05),the protein expression levels of RIP-1,RIP-3 and p-MLKL were decreased significantly in C-PC+H₂O₂ group,GST-CPC?+H₂O₂ group and Nec-1+H₂O₂group compared with the H₂O₂ group(P<0.05).Conclusion:C-phycocyanin and apo-phycocyanin?subunit can protect GC-1 spg cells from oxidative stress by inhibiting programmed necrosis.