山柰酚通过激活NLRP3-Caspase-1信号轴介导焦亡增强胃癌细胞对奥沙利铂的化疗敏感性OA

Objective To investigate kaempferol enhances the chemosensitivity of gastric cancer cells to oxaliplatin by promoting pyroptosis through regulation of the NOD-like receptor thermal protein domain associated protein 3(NLRP3)-Caspase-1 signaling axis.Methods The effect of kaempferol and oxaliplatin on the proliferation of gastric cancer cells(MKN-45,HGC-27)was detected by CCK-8 assay.The synergistic effect between kaempferol and oxaliplatin was analyzed using the Synergy Finder online software.LDH release assay was used to measure LDH activity.Interleukin-1β(IL-1β)content was detected by ELISA.Cell morphology and intracellular ultrastructure were observed using scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The protein expression levels of NLRP3,Caspase-1,cleaved-Caspase-1,gasdermin D(GSDMD),GSDMD-NT,gasdermin E(GSDME),IL-18,and IL-1β were detected by Western Blot.Results Compared with the control group(0 μmol·L-1),after treatment with kaempferol(20-140 μmol·L-1)or oxaliplatin(5-35 μmol·L-1)for 24,48,and 72 hours,the relative viability of both cell lines was significantly reduced(P＜0.05,P＜0.01)in a time-and concentration-dependent manner.The ZIP synergy score for the combined treatment of HGC-27 cells with kaempferol and oxaliplatin was 15.694,and for MKN-45 cells was 26.865.Both scores were greater than 10,indicating a synergistic effect.Compared with the control group,the kaempferol-treated groups showed increased release of LDH and IL-1β(P＜0.05,P＜0.01),and the release increased with higher kaempferol concentrations(P＜0.05,P＜0.01).Compared with the kaempferol-only or oxaliplatin-only groups,the combined treatment group(kaempferol+oxaliplatin)showed increased release of LDH and IL-1β(P＜0.05,P＜0.01).The two gastric cancer cell lines treated with kaempferol and oxaliplatin exhibited different morphological features under SEM and TEM.MKN-45 cells tended to show morphological characteristics of pyroptosis,while HGC-27 cells leaned more towards features of ferroptosis.Compared with the control group,treatment of both gastric cancer cell lines with different concentrations of kaempferol significantly increased the protein expression levels of NLRP3,Caspase-1,cleaved-Caspase-1,GSDMD,GSDMD-NT,GSDME,IL-1β,and IL-18(P＜0.05,P＜0.01).The increase in the expression of cleaved-Caspase-1 and GSDMD-NT relative to their full-length counterparts(Caspase-1 and GSDMD)was particularly notable.Compared with the blank control group,the kaempferol group,oxaliplatin group,and the combined treatment group all showed significantly increased protein expression levels of NLRP3,Caspase-1,cleaved-Caspase-1,GSDMD,GSDMD-NT,GSDME,IL-1β,and IL-18(P＜0.05,P＜0.01).Again,the increase in cleaved-Caspase-1 and GSDMD-NT was more pronounced relative to their pro-forms.The combined treatment group(kaempferol+oxaliplatin)showed the most potent effect(P＜0.05,P＜0.01).Conclusion Kaempferol may enhance the sensitivity of gastric cancer cells to oxaliplatin by promoting pyroptosis through activation of the NLRP3-Caspase-1 signaling axis.