基于NF-κB信号通路对M1型巨噬细胞极化的影响探讨柴芪益肝颗粒调控对HepG2细胞的作用机制OA

Objective To investigate the mechanism of ChaiQiYiGanKeLi(CQYGKL)modulating NF-κB signaling pathway mediating M1-type macrophage polarization on HepG2 cells.Methods Tissues from 20 patients with hepatocellular carcinoma(including hepatocellular carcinoma tissues and their paracancerous tissues)were collected,and the expression of M1-type macrophage-associated markers(CD16,CD68)in hepatocellular carcinoma tissues and their paracancerous tissues were detected by immunohistochemistry.Preparation of CQYGKL-containing serum was prepared and set aside.The survival rate of HepG2 cells after intervention with different concentrations of CQYGKL-containing serum was detected by CCK8 method.A cell co-culture system was established,and the effect of CQYGKL on the migration of HepG2 cells in the co-culture system was detected by cell scratch assay.The cells in the co-culture system were intervened with different factors and were divided into:control group,blank serum group,CQYGKL group,BAY 11-7082 group,PMA group and PMA+CQYGKL group,and the cells in the lower chamber of Transwell were collected after 48 h of intervention for the subsequent experiments.①RT-qPCR was used to detect the expression of Caspase-3,Bcl-2,N-cadherin,E-cadherin,Vimentin,MMP7,CD68,CD16,iNOS and NF-κBp65 mRNA in the cells of each group.②The expression of Caspase-3,Bcl-2,N-cadherin,E-cadherin,Vimentin,MMP7,CD68,CD16,P-NF-κBp65 and NF-κBp65 proteins were detected by Western blot in each group of cells.③The expression of iNOS in the cell supernatant of each group was detected by ELISA assay.Results The expression of M1-type macrophage markers(CD16 and CD68)was significantly lower in hepatocellular carcinoma tissues compared with that in paraneoplastic tissues(P<0.01).The results of CCK-8 experiment showed that after 24 h of intervention,the survival rate of HepG2 cells was significantly decreased when the concentration of CQYGKL-containing serum was more than 20%compared with the control group(P<0.01);And after 48 h of intervention,the survival rate of HepG2 cells was significantly decreased when the concentration of CQYGKL-containing serum was more than 10%compared with the control group(P<0.01).The results of the scratch experiment showed that compared with the control group,there was no significant decrease in the healing rate of the blank serum group for either 24 h or 48 h of intervention(P>0.05),whereas CQYGKL significantly reduced the healing rate of HepG2 cells for both 24 and 48 h of intervention(P<0.01).Following intervention with different factors in a co-culture system,no statistically significant differences in mRNA and protein expression levels of Caspase-3,Bcl-2,N-cadherin,E-cadherin,Vimentin,MMP7,CD68,CD16,iNOS,and P-NF-κBp65 were observed between the control group and the blank serum group,nor between the CQYGKL group and the BAY 11-7082 group(P>0.05).Compared with the control group,both CQYGKL and BAY 11-7082 significantly downregulated the mRNA and protein expression of Bcl-2,N-cadherin,Vimentin,MMP7,and P-NF-κBp65(P<0.01 or P<0.05),while upregulating the expression of Caspase-3,E-cadherin,CD68,CD16,and iNOS(P<0.01).In contrast,PMA promoted the expression of Bcl-2,N-cadherin,Vimentin,MMP7,and P-NF-κBp65(P<0.01 or P<0.05)and suppressed the expression of Caspase-3,E-cadherin,CD68,CD16,and iNOS(P<0.01 or P<0.05).Notably,compared with the PMA group,co-treatment with CQYGKL and PMA inhibited the expression of Bcl-2,N-cadherin,Vimentin,MMP7,and P-NF-κBp65(P<0.01 or P<0.05),and enhanced the expression of Caspase-3,E-cadherin,CD68,CD16,and iNOS(P<0.01 or P<0.05).Conclusion CQYGKL can promote the apoptosis of HepG2 cells in the co-culture system and inhibit their metastasis.The mechanism of action may be related to its ability to inhibit the activation of the NF-κB signaling pathway in the co-culture system and further promote the polarization of M0 macrophages to M1-type macrophages in the co-culture system.