摘要
目的:探究芍药苷(paeoniflorin,PF)对脂多糖(lipopolysaccharide,LPS)/尼日利亚菌素(nigericin,Nig)诱导的肾小管上皮细胞(HK-2)焦亡的影响及其作用机制。方法:选择对数生长期且生长状态良好的3~5代HK-2细胞。采用10 mg/L LPS联合10μmol/L Nig刺激HK-2细胞,建立细胞焦亡模型。设置4个实验组进行对比研究:(1)对照(control,Con)组:细胞正常培养,不做处理;(2)模型组(LPS组):10 mg/L LPS+10μmol/L Nig处理细胞;(3)PF组:50μmol/L PF预处理细胞4 h后加入10 mg/L LPS和10μmol/L Nig继续孵育细胞24 h;(4)PI3K激动剂740Y-P组:50μmol/L PF及20μmol/L 740Y-P预处理细胞4 h后加入10 mg/L LPS和10μmol/L Nig继续孵育细胞24 h。造模24 h后收集细胞与上清液,显微镜观察细胞形态变化;ELISA测定上清液中白细胞介素1β(interleukin-1β,IL-1β)和IL-18水平;Western blot检测焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、cleaved caspase-1及消皮素D的N端片段(N-terminal fragment of gasdermin D,GSDMD-N)表达水平,通路蛋白磷脂酰肌醇3-激酶(phosphatidylinositiol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB/AKT)和核因子κB(nuclear factor-κB,NF-κB)p65磷酸化及总蛋白表达;免疫荧光检测细胞核p-p65表达水平。结果:光镜下Con组细胞形态正常,呈上皮样排列;LPS组细胞肿胀,有伪足样突起;PF组较LPS组细胞肿胀减轻;740Y-P组较PF组细胞肿胀加重。电镜下Con组细胞形态规则,胞膜完整;LPS组细胞肿胀明显,膜泡及焦亡孔道形成;PF组较LPS组细胞肿胀减轻,细胞膜完整性改善。ELISA、Western blot及免疫荧光示LPS组较Con组IL-1β和IL-18,焦亡相关蛋白(NLRP3、cleaved caspase-1和GSDMD-N),通路蛋白(p-PI3K、p-AKT和pp65),及细胞核p-p65表达均显著升高(P<0.01);PF组较LPS组上述炎症因子、焦亡及通路蛋白及细胞核p-p65表达均显著降低(P<0.01);740Y-P组较PF组上述指标表达显著升高(P<0.05或P<0.01)。结论:芍药苷通过抑制PI3K/AKT/NF-κB通路减轻LPS/Nig诱导的HK-2细胞焦亡。
AIM:To investigate the effects of paeoniflorin(PF)on pyroptosis in renal tubular epithelial cells(HK-2)induced by lipopolysaccharide(LPS)/nigericin(Nig)and explore its underlying mechanisms.METHODS:HK-2 cells at passages 3~5 in logarithmic growth phase with favorable growth status were selected.HK-2 cells were stimulated with 10 mg/L LPS combined with 10μmol/L Nig to establish a cell pyroptosis model.Four experimental groups were established for comparative research:(1)control(Con)group:cells cultured normally without treatment;(2)model(LPS)group:cells treated with 10 mg/L LPS+10μmol/L Nig;(3)PF group:cells pretreated with 50μmol/L PF for 4 hours,then 10 mg/L LPS and 10μmol/L Nig added to continue incubation for 24 h;(4)PI3K agonist 740Y-P(740Y-P)group:cells were pretreated with 50μmol/L PF and 20μmol/L 740Y-P for 4 h,then 10 mg/L LPS and 10μmol/L Nig added to continue during incubation for 24 h.After 24 h of modeling,cells and supernatants were collected.Cell morphology changes were observed under microscope.ELISA was used to measure interleukin-1β(IL-1β)and IL-18 levels in supernatants.Western blot was performed to detect expression levels of pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),cleaved caspase-1,and N-terminal fragment of gasdermin D(GSDMD-N),as well as phosphorylation and total protein expression of pathway proteins phosphatidylinositiol 3-kinase(PI3K),protein kinase B(PKB/AKT)and nuclear factor-κB(NF-κB)p65.Immunofluorescence was used to detect nuclear p-p65 expression levels.RESULTS:Under light microscopy,the Con group cells displayed normal morphology with an epithelial-like arrangement,the LPS group cells demonstrated swelling accompanied by pseudopod-like protrusions,the PF group showed diminished swelling compared to the LPS group,and the 740Y-P group exhibited increased swelling compared to the PF group.Under electron microscopy,the Con group cells maintained regular morphology with intact cell membranes,the LPS group cells revealed pronounced swelling with membrane vesicle formation and pyroptosis pore development,and the PF group showed attenuated swelling compared to the LPS group with enhanced cell membrane integrity.ELISA,Western blot and immunofluorescence results demonstrated that the LPS group exhibited significantly elevated levels of IL-1βand IL-18,pyroptosis-related proteins(NLRP3,cleaved caspase-1 and GSDMD-N),pathway proteins(p-PI3K,p-AKT and p-p65),and nuclear p-p65 expression compared to the Con group(P<0.01).The PF group showed significantly decreased expression of the aforementioned inflammatory mediators,pyroptosis and pathway proteins,and nuclear p-p65 compared to the LPS group(P<0.01).The 740Y-P group demonstrated significantly increased expression of these parameters compared to the PF group(P<0.05 or P<0.01).CONCLUSION:Paeoniflorin alleviates LPS/Nig-induced pyroptosis in HK-2 cells through inhibition of PI3K/AKT/NF-κB signaling pathway.
作者
李若男
谢菁
孟照宇
王朝阳
马玉清
LI Ruonan;XIE Jing;MENG Zhaoyu;WANG Chaoyang;MA Yuqing(The First Clinical Medical College of Lanzhou University,Lanzhou 730000,China;Department of Anesthesiology,The First Hospital of Lanzhou University,Lanzhou 730000,China)
出处
《中国病理生理杂志》
北大核心
2025年第12期2399-2407,共9页
Chinese Journal of Pathophysiology
基金
甘肃省自然科学基金资助项目(No.24JRRA1075)
兰州大学第一医院院内基金(No.ldyyyn2022-5)。
