BACKGROUND Acute liver failure(ALF)has a high mortality with widespread hepatocyte death involving ferroptosis and pyroptosis.The silent information regulator sirtuin 1(SIRT1)-mediated deacetylation affects multiple b...BACKGROUND Acute liver failure(ALF)has a high mortality with widespread hepatocyte death involving ferroptosis and pyroptosis.The silent information regulator sirtuin 1(SIRT1)-mediated deacetylation affects multiple biological processes,including cellular senescence,apoptosis,sugar and lipid metabolism,oxidative stress,and inflammation.AIM To investigate the association between ferroptosis and pyroptosis and the upstream regulatory mechanisms.METHODS This study included 30 patients with ALF and 30 healthy individuals who underwent serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)testing.C57BL/6 mice were also intraperitoneally pretreated with SIRT1,p53,or glutathione peroxidase 4(GPX4)inducers and inhibitors and injected with lipopolysaccharide(LPS)/D-galactosamine(D-GalN)to induce ALF.Gasdermin D(GSDMD)^(-/-)mice were used as an experimental group.Histological changes in liver tissue were monitored by hematoxylin and eosin staining.ALT,AST,glutathione,reactive oxygen species,and iron levels were measured using commercial kits.Ferroptosis-and pyroptosis-related protein and mRNA expression was detected by western blot and quantitative real-time polymerase chain reaction.SIRT1,p53,and GSDMD were assessed by immunofluorescence analysis.RESULTS Serum AST and ALT levels were elevated in patients with ALF.SIRT1,solute carrier family 7a member 11(SLC7A11),and GPX4 protein expression was decreased and acetylated p5,p53,GSDMD,and acyl-CoA synthetase long-chain family member 4(ACSL4)protein levels were elevated in human ALF liver tissue.In the p53 and ferroptosis inhibitor-treated and GSDMD^(-/-)groups,serum interleukin(IL)-1β,tumour necrosis factor alpha,IL-6,IL-2 and C-C motif ligand 2 levels were decreased and hepatic impairment was mitigated.In mice with GSDMD knockout,p53 was reduced,GPX4 was increased,and ferroptotic events(depletion of SLC7A11,elevation of ACSL4,and iron accumulation)were detected.In vitro,knockdown of p53 and overexpression of GPX4 reduced AST and ALT levels,the cytostatic rate,and GSDMD expression,restoring SLC7A11 depletion.Moreover,SIRT1 agonist and overexpression of SIRT1 alleviated acute liver injury and decreased iron deposition compared with results in the model group,accompanied by reduced p53,GSDMD,and ACSL4,and increased SLC7A11 and GPX4.Inactivation of SIRT1 exacerbated ferroptotic and pyroptotic cell death and aggravated liver injury in LPS/D-GalNinduced in vitro and in vivo models.CONCLUSION SIRT1 activation attenuates LPS/D-GalN-induced ferroptosis and pyroptosis by inhibiting the p53/GPX4/GSDMD signaling pathway in ALF.展开更多
目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分...目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分院普外科手术切除的60例临床CRC组织及癌旁组织,应用实时荧光定量PCR(q PCR)技术检测CRC组织及癌旁组织中GPX1 m RNA水平。在高表达GPX1 m RNA的HT29细胞株用慢病毒携带sh RNA感染的方法构建敲除GPX1的细胞株,瞬时转染法在低表达GPX1 m RNA的LOVO细胞系构建过表达GPX1的细胞株,并在GPX1 m RNA及蛋白水平进行验证。通过MTS法检测CRC细胞的增殖能力的变化,用划痕实验、Transwell侵袭实验分别检测CRC细胞迁移侵袭能力的变化,同时用Western blotting检测E-钙黏蛋白和波形蛋白表达的变化。结果:CRC组织中GPX1 m RNA表达水平显著低于癌旁组织(0.051±0.024 vs 0.142±0.051,P<0.01)。敲除GPX1后,HT29细胞的增殖能力显著增强(12.901±2.790 vs 6.617±2.462,P<0.01)、侵袭能力显著增强[(384.7±37.9)vs(209.2±31.2)个,P<0.01]、迁移能力显著增强[(0.139±0.025)vs(0.251±0.038)mm,P<0.01]、E-钙黏蛋白表达下调(P<0.01)、波形蛋白表达上调(P<0.05)。过表达GPX1的LOVO细胞的增殖能力显著降低(P<0.01)、迁移和侵袭能力下降(均P<0.05)、E-钙黏蛋白上调(P<0.01)、波形蛋白表达下调(P<0.01)。结论:GPX1 m RNA在人CRC组织中低表达,GPX1负向调控CRC细胞的增殖、迁移和侵袭,其在CRC中可能发挥抑癌作用。展开更多
Previous studies have reported upregulation of heme oxygenase-1 in different central nervous system injury models.Heme oxygenase-1 plays a critical anti-inflammatory role and is essential for regulating cellular redox...Previous studies have reported upregulation of heme oxygenase-1 in different central nervous system injury models.Heme oxygenase-1 plays a critical anti-inflammatory role and is essential for regulating cellular redox homeostasis.Metformin is a classic drug used to treat type 2 diabetes that can inhibit ferroptosis.Previous studies have shown that,when used to treat cardiovascular and digestive system diseases,metformin can also upregulate heme oxygenase-1 expression.Therefore,we hypothesized that heme oxygenase-1 plays a significant role in mediating the beneficial effects of metformin on neuronal ferroptosis after spinal cord injury.To test this,we first performed a bioinformatics analysis based on the GEO database and found that heme oxygenase-1 was upregulated in the lesion of rats with spinal cord injury.Next,we confirmed this finding in a rat model of T9 spinal cord compression injury that exhibited spinal cord nerve cell ferroptosis.Continuous intraperitoneal injection of metformin for 14 days was found to both upregulate heme oxygenase-1 expression and reduce neuronal ferroptosis in rats with spinal cord injury.Subsequently,we used a lentivirus vector to knock down heme oxygenase-1 expression in the spinal cord,and found that this significantly reduced the effect of metformin on ferroptosis after spinal cord injury.Taken together,these findings suggest that metformin inhibits neuronal ferroptosis after spinal cord injury,and that this effect is partially dependent on upregulation of heme oxygenase-1.展开更多
目的评价褪黑素对神经病理性疼痛夜间加重的影响,并通过特异性沉默信息调节因子1(SIRT1)-脑和肌肉ARNT样蛋白1(BMAL1)通路探讨其机制。方法96只SPF级雄性C57/B6小鼠,随机分为3组:假手术(S)组、神经病理性疼痛模型(NP)组和NP模型+褪黑素...目的评价褪黑素对神经病理性疼痛夜间加重的影响,并通过特异性沉默信息调节因子1(SIRT1)-脑和肌肉ARNT样蛋白1(BMAL1)通路探讨其机制。方法96只SPF级雄性C57/B6小鼠,随机分为3组:假手术(S)组、神经病理性疼痛模型(NP)组和NP模型+褪黑素治疗(NP+M)组;术前试验小鼠置于指定光照模式环境中;采用12 h光照与12 h黑暗交替环境,持续至少两周,将自然时间转换为授时时间(ZT),光照起点定为ZT0;S组小鼠仅分离坐骨神经,NP组和NP+M组采用坐骨神经慢性缩窄性损伤制备小鼠NP模型,NP+M组术后注射10 mg/kg褪黑素;Western blot法检测术后14 d各时点脊髓的SIRT1、BMAL1和谷胱甘肽过氧化酶1(Gpx1)表达量的变化;术后通过免疫荧光技术对脊髓背角SIRT1和脊髓神经元标志物神经元特异性核蛋白(NeuN)、小胶质细胞激活标记物离子钙结合适配器分子1(iba-1)、星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)进行共染色,并检测各时点iba-1以确定小胶质细胞激活状态。结果与NP组ZT10时点相比,NP组小鼠术后14 d ZT22时点SIRT1、BMAL1和Gpx1降低(P<0.05);与NP组ZT14时点相比,NP+M组ZT14时点SIRT1与BMAL1升高(P<0.05),而Gpx1于ZT18时点升高(P<0.05)。SIRT1在脊髓背角与小胶质细胞共表达;与ZT10时点相比,NP组小鼠术后14 d ZT22时点小胶质细胞表达降低(P<0.05);与ZT10时点相比,NP+M组小鼠术后14 d ZT22时点小胶质细胞表达差异无统计学意义。结论褪黑素可以减轻神经病理性疼痛夜间加重,其机制可能与激活小胶质细胞SIRT1-BMAL1通路蛋白表达有关。展开更多
基金Supported by National Natural Science Foundation of China,No.82060123Doctoral Start-up Fund of Affiliated Hospital of Guizhou Medical University,No.gysybsky-2021-28+1 种基金Fund Project of Guizhou Provincial Science and Technology Department,No.[2020]1Y299Guizhou Provincial Health Commission,No.gzwjk2019-1-082。
文摘BACKGROUND Acute liver failure(ALF)has a high mortality with widespread hepatocyte death involving ferroptosis and pyroptosis.The silent information regulator sirtuin 1(SIRT1)-mediated deacetylation affects multiple biological processes,including cellular senescence,apoptosis,sugar and lipid metabolism,oxidative stress,and inflammation.AIM To investigate the association between ferroptosis and pyroptosis and the upstream regulatory mechanisms.METHODS This study included 30 patients with ALF and 30 healthy individuals who underwent serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)testing.C57BL/6 mice were also intraperitoneally pretreated with SIRT1,p53,or glutathione peroxidase 4(GPX4)inducers and inhibitors and injected with lipopolysaccharide(LPS)/D-galactosamine(D-GalN)to induce ALF.Gasdermin D(GSDMD)^(-/-)mice were used as an experimental group.Histological changes in liver tissue were monitored by hematoxylin and eosin staining.ALT,AST,glutathione,reactive oxygen species,and iron levels were measured using commercial kits.Ferroptosis-and pyroptosis-related protein and mRNA expression was detected by western blot and quantitative real-time polymerase chain reaction.SIRT1,p53,and GSDMD were assessed by immunofluorescence analysis.RESULTS Serum AST and ALT levels were elevated in patients with ALF.SIRT1,solute carrier family 7a member 11(SLC7A11),and GPX4 protein expression was decreased and acetylated p5,p53,GSDMD,and acyl-CoA synthetase long-chain family member 4(ACSL4)protein levels were elevated in human ALF liver tissue.In the p53 and ferroptosis inhibitor-treated and GSDMD^(-/-)groups,serum interleukin(IL)-1β,tumour necrosis factor alpha,IL-6,IL-2 and C-C motif ligand 2 levels were decreased and hepatic impairment was mitigated.In mice with GSDMD knockout,p53 was reduced,GPX4 was increased,and ferroptotic events(depletion of SLC7A11,elevation of ACSL4,and iron accumulation)were detected.In vitro,knockdown of p53 and overexpression of GPX4 reduced AST and ALT levels,the cytostatic rate,and GSDMD expression,restoring SLC7A11 depletion.Moreover,SIRT1 agonist and overexpression of SIRT1 alleviated acute liver injury and decreased iron deposition compared with results in the model group,accompanied by reduced p53,GSDMD,and ACSL4,and increased SLC7A11 and GPX4.Inactivation of SIRT1 exacerbated ferroptotic and pyroptotic cell death and aggravated liver injury in LPS/D-GalNinduced in vitro and in vivo models.CONCLUSION SIRT1 activation attenuates LPS/D-GalN-induced ferroptosis and pyroptosis by inhibiting the p53/GPX4/GSDMD signaling pathway in ALF.
文摘目的:探讨谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)在结直肠癌(colorectal cancer,CRC)组织中的表达水平及其对CRC细胞株HT29和LOVO细胞增殖、迁移及侵袭能力的影响。方法:收集2015年6月至2016年3月间海口市人民医院滨江分院普外科手术切除的60例临床CRC组织及癌旁组织,应用实时荧光定量PCR(q PCR)技术检测CRC组织及癌旁组织中GPX1 m RNA水平。在高表达GPX1 m RNA的HT29细胞株用慢病毒携带sh RNA感染的方法构建敲除GPX1的细胞株,瞬时转染法在低表达GPX1 m RNA的LOVO细胞系构建过表达GPX1的细胞株,并在GPX1 m RNA及蛋白水平进行验证。通过MTS法检测CRC细胞的增殖能力的变化,用划痕实验、Transwell侵袭实验分别检测CRC细胞迁移侵袭能力的变化,同时用Western blotting检测E-钙黏蛋白和波形蛋白表达的变化。结果:CRC组织中GPX1 m RNA表达水平显著低于癌旁组织(0.051±0.024 vs 0.142±0.051,P<0.01)。敲除GPX1后,HT29细胞的增殖能力显著增强(12.901±2.790 vs 6.617±2.462,P<0.01)、侵袭能力显著增强[(384.7±37.9)vs(209.2±31.2)个,P<0.01]、迁移能力显著增强[(0.139±0.025)vs(0.251±0.038)mm,P<0.01]、E-钙黏蛋白表达下调(P<0.01)、波形蛋白表达上调(P<0.05)。过表达GPX1的LOVO细胞的增殖能力显著降低(P<0.01)、迁移和侵袭能力下降(均P<0.05)、E-钙黏蛋白上调(P<0.01)、波形蛋白表达下调(P<0.01)。结论:GPX1 m RNA在人CRC组织中低表达,GPX1负向调控CRC细胞的增殖、迁移和侵袭,其在CRC中可能发挥抑癌作用。
文摘Previous studies have reported upregulation of heme oxygenase-1 in different central nervous system injury models.Heme oxygenase-1 plays a critical anti-inflammatory role and is essential for regulating cellular redox homeostasis.Metformin is a classic drug used to treat type 2 diabetes that can inhibit ferroptosis.Previous studies have shown that,when used to treat cardiovascular and digestive system diseases,metformin can also upregulate heme oxygenase-1 expression.Therefore,we hypothesized that heme oxygenase-1 plays a significant role in mediating the beneficial effects of metformin on neuronal ferroptosis after spinal cord injury.To test this,we first performed a bioinformatics analysis based on the GEO database and found that heme oxygenase-1 was upregulated in the lesion of rats with spinal cord injury.Next,we confirmed this finding in a rat model of T9 spinal cord compression injury that exhibited spinal cord nerve cell ferroptosis.Continuous intraperitoneal injection of metformin for 14 days was found to both upregulate heme oxygenase-1 expression and reduce neuronal ferroptosis in rats with spinal cord injury.Subsequently,we used a lentivirus vector to knock down heme oxygenase-1 expression in the spinal cord,and found that this significantly reduced the effect of metformin on ferroptosis after spinal cord injury.Taken together,these findings suggest that metformin inhibits neuronal ferroptosis after spinal cord injury,and that this effect is partially dependent on upregulation of heme oxygenase-1.
文摘目的评价褪黑素对神经病理性疼痛夜间加重的影响,并通过特异性沉默信息调节因子1(SIRT1)-脑和肌肉ARNT样蛋白1(BMAL1)通路探讨其机制。方法96只SPF级雄性C57/B6小鼠,随机分为3组:假手术(S)组、神经病理性疼痛模型(NP)组和NP模型+褪黑素治疗(NP+M)组;术前试验小鼠置于指定光照模式环境中;采用12 h光照与12 h黑暗交替环境,持续至少两周,将自然时间转换为授时时间(ZT),光照起点定为ZT0;S组小鼠仅分离坐骨神经,NP组和NP+M组采用坐骨神经慢性缩窄性损伤制备小鼠NP模型,NP+M组术后注射10 mg/kg褪黑素;Western blot法检测术后14 d各时点脊髓的SIRT1、BMAL1和谷胱甘肽过氧化酶1(Gpx1)表达量的变化;术后通过免疫荧光技术对脊髓背角SIRT1和脊髓神经元标志物神经元特异性核蛋白(NeuN)、小胶质细胞激活标记物离子钙结合适配器分子1(iba-1)、星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)进行共染色,并检测各时点iba-1以确定小胶质细胞激活状态。结果与NP组ZT10时点相比,NP组小鼠术后14 d ZT22时点SIRT1、BMAL1和Gpx1降低(P<0.05);与NP组ZT14时点相比,NP+M组ZT14时点SIRT1与BMAL1升高(P<0.05),而Gpx1于ZT18时点升高(P<0.05)。SIRT1在脊髓背角与小胶质细胞共表达;与ZT10时点相比,NP组小鼠术后14 d ZT22时点小胶质细胞表达降低(P<0.05);与ZT10时点相比,NP+M组小鼠术后14 d ZT22时点小胶质细胞表达差异无统计学意义。结论褪黑素可以减轻神经病理性疼痛夜间加重,其机制可能与激活小胶质细胞SIRT1-BMAL1通路蛋白表达有关。
