Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling path...Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.展开更多
目的 探讨复方蛇龙胶囊抑制Bax/Caspase-3信号通路减轻膜性肾病大鼠肾组织细胞凋亡的影响。方法 抽取10只6周龄SPF级雄性SD大鼠作为正常组,其余40只采用注射阳离子化牛血清白蛋白(Alb)构建膜性肾病大鼠模型,采用随机数字表法将其分为模...目的 探讨复方蛇龙胶囊抑制Bax/Caspase-3信号通路减轻膜性肾病大鼠肾组织细胞凋亡的影响。方法 抽取10只6周龄SPF级雄性SD大鼠作为正常组,其余40只采用注射阳离子化牛血清白蛋白(Alb)构建膜性肾病大鼠模型,采用随机数字表法将其分为模型组,雷公藤多苷片组,复方蛇龙胶囊低、高剂量组,每组10只。除正常组和模型组灌服等量生理盐水外,其余各组灌胃给予相应药物,连续4周。HE染色、Masson染色、透射电镜及扫描电镜观察各组肾组织病理学变化;比较各组大鼠尿微量白蛋白(mAlb)和血肌酐(SCr)、甘油三酯(TG)、总胆固醇(TC)、Alb、总蛋白(TP)含量;比较各组大鼠肾组织细胞凋亡率;比较各组大鼠肾组织Nephrin、Podocin、Bcl-2、Bax和Caspase-3 m RNA表达水平。结果 与正常组比较,模型组大鼠尿m Alb含量升高(P<0.05);血清Alb、TP含量降低,TC、TG和SCr含量升高(P<0.05)。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠尿m Alb含量降低(P<0.05);血清Alb、TP含量升高,TC、TG和SCr含量降低(P<0.05)。与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组尿m Alb降低(P<0.05);血清Alb、TP含量升高,SCr含量降低(P<0.05)。正常组大鼠HE染色可见肾小球结构完整;Masson染色未见组织纤维化。模型组大鼠HE染色可见肾小球体积增大,肾小管可见空泡变性,并伴有蛋白管型;Masson染色可见组织纤维化,蓝色胶原纤维组织出现。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠肾小球体积增大、肾小管空泡变性及组织纤维化有所减轻。正常组基底膜结构完整,足细胞结构正常;模型组肾小球大面积水肿,基底膜增厚,足突融合变宽,足细胞结构严重损伤,细胞骨架蛋白裸露;与模型组比较,复方蛇龙胶囊低、高剂量组大鼠基底膜增厚程度减轻,足细胞形态结构趋于正常。与正常组比较,模型组大鼠肾组织细胞凋亡率升高(P<0.05);与模型组比较,复方蛇龙胶囊低、高剂量组大鼠肾组织细胞凋亡率降低(P<0.05);与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组大鼠肾组织细胞凋亡率降低(P<0.05)。与正常组比较,模型组大鼠Nephrin、Podocin、Bcl-2 m RNA表达水平降低,Caspase-3、Bax m RNA表达水平升高,Bcl-2/Bax比值降低(P<0.05)。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠Nephrin、Podocin、Bcl-2 m RNA表达水平升高,Caspase-3、Bax m RNA表达水平降低,Bcl-2/Bax比值升高(P<0.05)。与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组Nephrin、Podocin m RNA表达水平升高,Bcl-2/Bax比值升高(P<0.05)。结论 复方蛇龙胶囊能有效改善膜性肾病大鼠蛋白尿,显著减轻肾脏病理损伤,延缓膜性肾病进展,其机制可能与抑制足细胞凋亡有关。展开更多
基金supported by grants from startup fund program at Beijing University of Chinese Medicine(90011451310011)key research fund for drug discovery in Chinese medicine at Beijing University of Chinese Medicine(1000061223476)startup fund program at Beijing University of Chinese Medicine(90020361220006).
文摘Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.
文摘目的 探讨复方蛇龙胶囊抑制Bax/Caspase-3信号通路减轻膜性肾病大鼠肾组织细胞凋亡的影响。方法 抽取10只6周龄SPF级雄性SD大鼠作为正常组,其余40只采用注射阳离子化牛血清白蛋白(Alb)构建膜性肾病大鼠模型,采用随机数字表法将其分为模型组,雷公藤多苷片组,复方蛇龙胶囊低、高剂量组,每组10只。除正常组和模型组灌服等量生理盐水外,其余各组灌胃给予相应药物,连续4周。HE染色、Masson染色、透射电镜及扫描电镜观察各组肾组织病理学变化;比较各组大鼠尿微量白蛋白(mAlb)和血肌酐(SCr)、甘油三酯(TG)、总胆固醇(TC)、Alb、总蛋白(TP)含量;比较各组大鼠肾组织细胞凋亡率;比较各组大鼠肾组织Nephrin、Podocin、Bcl-2、Bax和Caspase-3 m RNA表达水平。结果 与正常组比较,模型组大鼠尿m Alb含量升高(P<0.05);血清Alb、TP含量降低,TC、TG和SCr含量升高(P<0.05)。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠尿m Alb含量降低(P<0.05);血清Alb、TP含量升高,TC、TG和SCr含量降低(P<0.05)。与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组尿m Alb降低(P<0.05);血清Alb、TP含量升高,SCr含量降低(P<0.05)。正常组大鼠HE染色可见肾小球结构完整;Masson染色未见组织纤维化。模型组大鼠HE染色可见肾小球体积增大,肾小管可见空泡变性,并伴有蛋白管型;Masson染色可见组织纤维化,蓝色胶原纤维组织出现。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠肾小球体积增大、肾小管空泡变性及组织纤维化有所减轻。正常组基底膜结构完整,足细胞结构正常;模型组肾小球大面积水肿,基底膜增厚,足突融合变宽,足细胞结构严重损伤,细胞骨架蛋白裸露;与模型组比较,复方蛇龙胶囊低、高剂量组大鼠基底膜增厚程度减轻,足细胞形态结构趋于正常。与正常组比较,模型组大鼠肾组织细胞凋亡率升高(P<0.05);与模型组比较,复方蛇龙胶囊低、高剂量组大鼠肾组织细胞凋亡率降低(P<0.05);与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组大鼠肾组织细胞凋亡率降低(P<0.05)。与正常组比较,模型组大鼠Nephrin、Podocin、Bcl-2 m RNA表达水平降低,Caspase-3、Bax m RNA表达水平升高,Bcl-2/Bax比值降低(P<0.05)。与模型组比较,复方蛇龙胶囊低、高剂量组大鼠Nephrin、Podocin、Bcl-2 m RNA表达水平升高,Caspase-3、Bax m RNA表达水平降低,Bcl-2/Bax比值升高(P<0.05)。与复方蛇龙胶囊低剂量组比较,复方蛇龙胶囊高剂量组Nephrin、Podocin m RNA表达水平升高,Bcl-2/Bax比值升高(P<0.05)。结论 复方蛇龙胶囊能有效改善膜性肾病大鼠蛋白尿,显著减轻肾脏病理损伤,延缓膜性肾病进展,其机制可能与抑制足细胞凋亡有关。
