基于NLRP3/GSDMD信号通路研究人参皂苷Rb1对高糖诱导的胰岛β细胞焦亡的影响

目的: 研究人参皂苷Rb1(GRb1)抑制高糖诱导的胰岛β细胞焦亡及对NLRP3/GSDMD信号通路的调节机制。方法: 高糖(50 mmol·L^-1)诱导大鼠胰岛细胞瘤细胞(rat insulinoma cells, INS-1)焦亡模型,并构建NLRP3过表达质粒转染INS-1细胞,采用倒置显微镜观察GRb1对细胞形态变化的影响;采用ELISA法检测GRb1对细胞上清液中乳酸脱氢酶(LDH)、白细胞介素-1β(IL-1β)、胰岛素水平的影响;采用Western blot法和qRT-PCR法检测GRb1对INS-1细胞中核苷酸结合域样受体蛋白3(nucleotide binding domain like receptor protein 3, NLRP3)和切割蛋白D(gasdermin D, GSDMD)表达的影响。结果: 在高糖环境下,GRb1可减轻INS-1细胞形态学改变,显著增加INS-1细胞胰岛素分泌,降低细胞上清液中IL-1β、LDH水平(P＜0.05),且呈剂量依赖性;GRb1呈剂量依赖性抑制高糖诱导的INS-1细胞中GSDMD及上游调节因子NLRP3的表达(P＜0.05);过表达NLRP3后可显著逆转高糖环境下GRb1对INS-1细胞的部分保护性作用(P＜0.05)。结论: GRb1能够改善高糖诱导的INS-1细胞焦亡,减轻炎症反应,其机制可能与抑制NLRP3/GSDMD信号通路有关,从而防治糖尿病及其并发症的发生。     

灯盏花乙素对J774A.1巨噬细胞中ATP诱导的炎症小体活化和细胞焦亡的影响

目的以LPS致敏的小鼠巨噬细胞J774A.1作为炎症细胞模型,研究灯盏花乙素对ATP诱导的炎症小体活化和细胞焦亡的影响及其机制。方法利用碘化丙锭(PI)染色法检测LPS+ATP诱导的小鼠J774A.1巨噬细胞发生细胞焦亡的情况;免疫印迹法检测细胞裂解液和上清中IL-1β、caspase-1、HMGB1等蛋白的表达水平;基于微珠的免疫测定法(CBA)检测细胞上清中IL-1β的分泌水平。结果 ATP能够明显诱导LPS致敏的J774A.1巨噬细胞中caspase-1活化、成熟IL-1β(17 ku)和HMGB1释放至培养上清中,并诱导细胞焦亡;而灯盏花乙素预处理能够剂量依赖性地抑制ATP诱导的caspase-1活化以及成熟IL-1β、HMGB1的释放,并抑制细胞焦亡;同时,经腺苷酸环化酶抑制剂MDL12330A和蛋白激酶A(PKA)抑制剂H89处理,可以逆转灯盏花乙素对ATP诱导的细胞焦亡的抑制作用。结论灯盏花乙素通过调节PKA活性,抑制NLRP3炎症小体的活化与细胞焦亡,从而发挥抗炎作用。     

羟基红花黄色素A对糖氧剥夺/复糖复氧诱导的BV2细胞炎性反应的机制研究

目的 探讨羟基红花黄色素A(HSYA)对糖氧剥夺/复糖复氧(OGD/R)诱导的小胶质细胞(MG)炎性反应作用机制的研究。方法 构建BV2细胞OGD/R模型模拟脑缺血再灌注损伤，BV2细胞分为正常对照组(常规培养)、模型组(OGD/R模型)、HSYA药物干预组(OGD/R模型+25μmol·L^-1 HSYA)、TREM2激动剂组(5μmol·L^-1 Hsp60+OGD/R模型)、HSYA+TREM2激动剂组(5μmol·L^-1 Hsp60+OGD/R模型+25μmol·L^-1 HSYA)。用蛋白质印迹法(Western blot)检测髓样细胞触发受体2(TREM2)、Toll样受体4(TLR4)、核因子-κB(NF-κB)蛋白表达情况，用双重免疫荧光染色检测一氧化氮合酶(iNOS)和精氨酸酶1(Arg1)荧光染色数量；用酶联免疫吸附(ELISA)法检测各组细胞白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和IL-10的表达。结果 正常对照组、模型组、HSYA药物干预组、TREM2激动剂...     

实时动态法分析七味红花殊胜丸抑制肝癌细胞线粒体代谢

目的：观察七味红花殊胜丸对人肝癌SMMC-7721细胞线粒体代谢酶的影响。方法：运用实时动态法分析七味红花殊胜丸对细胞活性的影响及各给药组不同时间IC₅₀值的变化情况。采用MTT法检测七味红花殊胜丸对SMMC-7721细胞的抑制率;ELISA法检测七味红花殊胜丸对SMMC-7721细胞线粒体代谢和凋亡的影响,Western Blot法检测凋亡蛋白的表达状况。结果：实时动态检测结果提示,七味红花殊胜丸5 mg·mL^-1作用48 h对SMMC-7721细胞的抑制率最高（P<0.01）;ELISA检测结果提示,七味红花殊胜丸能降低SMMC-7721细胞内ATP、ATPase、线粒体呼吸链复合物Ⅰ/Ⅱ/Ⅲ/Ⅳ酶的表达量,影响细胞的能量代谢;升高促凋亡蛋白Bax、Caspase-3和Caspase-9的表达量,降低抗凋亡蛋白Bcl-2的表达量,诱导细胞凋亡。结论：七味红花殊胜丸可通过影响细胞的能量代谢,诱导SMMC-7721细胞凋亡。     

去氢骆驼蓬碱诱发PC12细胞凋亡时对线粒体融合分裂的影响

目的 探讨去氢骆驼蓬碱(HM)对PC12细胞线粒体功能损伤和线粒体融合分裂相关蛋白表达水平的影响。方法 PC12细胞分为细胞对照组、HM组、线粒体分裂抑制剂Mdivi-1组、HM+Mdivi-1组、线粒体裂变激动剂WY14643组、HM+WY14643组,药物浓度均为1、10、25、50、100μmol·L^-1,处理24 h,噻唑蓝(MTT)法检测细胞存活率,显微镜观察细胞形态;MitoTracker Red探针染色观察线粒体形态和纵横轴长度比值,JC-1染色检测线粒体膜电位,试剂盒检测ROS、ATP水平和乳酸脱氢酶(LDH)活性,免疫荧光染色法和Western blotting检测胱天蛋白酶3(caspase-3)、促凋亡蛋白(Bax)、细胞色素C(cyt-c)和线粒体融合蛋白(Mfn2)、线粒体分裂蛋白(Drp-1)的表达水平;电穿孔法转染Drp1的干扰序列,筛选转染效果好的siRNA序列,协同药物干预,通过荧光法、MTT法、免疫印迹法检测相关指标。结果 MTT结果显示,与细胞对照组比较,HM组、Mdivi-1组、HM+Mdivi-1组、WY14643组和HM...     

乌帕替尼对氧糖剥夺/复氧后BV2细胞极化的影响

目的研究乌帕替尼对氧糖剥夺再复氧(OGD/R)后BV2小胶质细胞极化及炎症的影响,并探讨其作用机制。方法实验分对照组、OGD组和乌帕替尼组3组。BV2细胞经OGD/R处理后,噻唑蓝试剂(MTT)检测细胞生存率,划痕实验观察细胞迁移能力,实时荧光定量多聚核苷酸链式反应(qPCR)检测BV2细胞M1型极化标志物(CD11b、CD32、iNOS)和M2型极化标志物(Arg-1、IL-10、CD206)的mRNA水平,酶联免疫吸附测定(ELISA)检测培养基中IL-1β、IL-6、TNF-α含量,蛋白质印迹法(Western Blot)检测JAK1/STAT6通路相关蛋白表达水平。结果乌帕替尼能增加OGD/R后BV2细胞的生存率(P<0.05),能减少BV2细胞向M1型极化(P<0.05)。乌帕替尼可明显降低OGD/R诱导的BV2细胞的迁移能力(P<0.05),减少OGD/R诱导BV2细胞分泌的炎症因子:IL-1β、IL-6、TNF-α(P<0.05)。乌帕替尼提高共培养PC12细胞的生存率(P<0.05)。乌帕替尼可显著抑制由OGD/R诱导激活BV2细胞p-JAK1和p-STAT6蛋白的表达水平(P<0.05)。结论乌帕替尼可减少OGD/R诱导BV2细胞向M1型极化和减少炎症反应,与JAK1/STAT6通路有关。     

荜茇酰胺对H1975非小细胞肺癌细胞增殖的影响

目的 探讨荜茇酰胺对体外培养的H1975非小细胞肺癌细胞增殖的影响及其机制.方法 采用不同浓度荜茇酰胺(0、1.25、2.5、5、10、20、30、40和50 μmol/L)孵育H1975细胞72 h,MTT法检测细胞生存率.采用荜茇酰胺0、2.5、5和10 μmol/L孵育H1975细胞,细胞集落形成实验和划痕实验分别测定细胞增殖和迁移情况,流式细胞术测定细胞凋亡情况和活性氧(ROS)水平,倒置显微镜观察ROS荧光水平、细胞焦亡情况和ROS变化对细胞焦亡的影响,Western blot法分析加斯德明E(GSDME)、GSDME-N端、GSDMD和前体Caspase-3蛋白表达的变化.结果 荜茇酰胺能够浓度依赖性地抑制细胞生长、集落形成和细胞迁移,诱导细胞凋亡和焦亡,提高细胞内ROS水平,下调GSDME、前体Caspase-3和GSDMD蛋白表达,上调GSDME-N端蛋白表达(P＜0.05).降低ROS水平后,抑制细胞焦亡(P＜0.05).结论 荜茇酰胺能促进H1975细胞凋亡,通过提高细胞内ROS水平诱导细胞焦亡;其机制可能与ROS作用于GSDMD/GSDME蛋白有关.     

雷公藤甲素对类风湿关节炎成纤维样滑膜细胞线粒体自噬、NLRP3炎症小体活化和细胞焦亡的影响

目的 探究雷公藤甲素对类风湿关节炎成纤维样滑膜细胞（FLSs）线粒体自噬、NOD样受体蛋白3(NLRP3)炎症小体活化和细胞焦亡的影响。方法 将FLSs细胞和类风湿关节炎FLSs细胞进行传代培养。将类风湿性关节炎FLSs细胞采用0、10、20、40 ng/mL雷公藤甲素进行处理。采用蛋白质印迹法（Western blotting）检测线粒体自噬相关蛋白LC3B、p62、PHB2、NLRP3的蛋白表达。采用酶联免疫吸附试验（ELISA）检测炎症因子白细胞介素（IL）-1β和IL-18的水平。采用流式细胞术检测细胞焦亡情况,并采用Western blotting法检测焦亡相关蛋白GSDMD和GSDMD-N表达。结果 采用10、20、40ng/mL雷公藤甲素处理类风湿关节炎FLSs后,雷公藤甲素各剂量组均能显著减弱类风湿关节炎FLSs细胞的线粒体自噬相关蛋白LC3B的表达,上调p62和PHB2的表达;NLRP3表达水平均显著降低,IL-1β和IL-18水平显著降低;细胞焦亡率显著降低;GSDMD蛋白表达显著上升,GSDMD-N蛋白表达下降（P<0.05、0.01、0.001）,且呈剂量...     

星形胶质细胞焦亡与抑郁症发生的相关性

目的研究星形胶质细胞焦亡在抑郁症发生发展中的作用。方法采用野生型(WT)、胱天蛋白酶1~(-/-)、GSDMD~(-/-)、星形胶质细胞过表达GSDMD-N的GSDMD~(-/-)等多种模式小鼠制备CMS模型,通过行为学、免疫组化、蛋白水平、多重染色等多种方法研究焦亡在抑郁症中的作用及机制。结果 CMS 2周小鼠海马脑区星形胶质细胞活化但未发生焦亡;CMS 4周小鼠海马脑区星形胶质细胞发生焦亡;CMS 6周小鼠海马脑区星形胶质细胞焦亡愈加明显;CMS6周小鼠海马脑区小胶质细胞及神经元不发生焦亡;CMS2,4和6周小鼠海马脑区胱天蛋白酶1、IL-1β表达量随着造模时间的延长也显著增加;FLX可改善CMS小鼠抑郁样行为;CMS小鼠海马区星形胶质细胞焦亡,FLX改善星形胶质细胞凋亡;FLX降低CMS小鼠海马脑区凋亡相关蛋白表达;胱天蛋白酶1敲除显著改善CMS小鼠糖水偏好、强迫游泳不动时间、悬尾不动时间及旷场运动总路线;胱天蛋白酶1敲除减轻CMS小鼠海马脑区星形胶质细胞凋亡;GSDMD敲除显著改善CMS小鼠糖水偏好、强迫游泳不动时间、悬尾不动时间及旷场总路线;GSDMD敲除缓解CMS小鼠海马区星形胶质细胞凋亡;GSDMD敲除小鼠海马星形胶质细胞特异性过表达GSDMD-N,恢复GSDMD敲除CMS小鼠抑郁样行为。结论 CMS小鼠海马区星形胶质细胞发生凋亡;GSDMD介导的星形胶质细胞凋亡参与抑郁的发生发展。     

瓜子金皂苷己对连二亚硫酸钠致氧糖剥夺/复供诱导PC12细胞凋亡的抑制作用

目的观察瓜子金皂苷己(polygalasaponin F,PGSF)对氧糖剥夺/复供(oxygen-glucose deprivation and reperfusion,OGD/R)所诱导的PC12细胞凋亡的作用及其机制。方法以连二亚硫酸钠合并无糖Earle’s液造成氧糖剥夺,继而恢复为完全培养基以建立体外OGD/R模型,以Hoechst33342/PI双染及流式细胞术观察细胞受损和凋亡情况;以JC-1荧光染色法测定细胞线粒体膜电位(mitochondrial membranepotential,MMP);以Western blot法检测凋亡相关蛋白的表达。结果 PGSF可明显改善细胞形态并降低细胞受损和凋亡百分率,抑制MMP水平的降低,增加Bcl-2/Bax表达比值。结论 PGSF对OGD/R诱导的PC12细胞凋亡具有明显抑制作用,机制与其调节Bcl-2和Bax的表达及稳定MMP有关。     

干扰素调节因子1通过调控细胞焦亡参与小鼠肝脏缺血再灌注损伤

摘要：目的　探讨小鼠肝脏缺血再灌注损伤（LIRI）过程中干扰素调节因子-1（IRF-1）对焦亡的影响。方法　（1）24只C57BL/6正常雄性小鼠按照随机数字表法分为假手术（Sham）组与缺血再灌注（IR）2 h、6 h和12 h组,每组6只。Sham组不进行IR处理,其他3组采用血管夹夹闭肝中叶、左叶脉管（门静脉、动脉与胆道）60 min后分别再灌注2、6、12 h建立LIRI模型。全自动生化仪检测各组血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）;酶联免疫吸附测定（ELISA）法检测血清白细胞介素-1β（IL-1β）水平;HE染色观察肝脏病理改变;免疫组织化学染色检测IRF-1表达情况;TUNEL染色检测肝脏细胞凋亡和焦亡情况;Western blot检测IRF-1、半胱天冬氨酸蛋白酶-1（Caspase-1）、Cleaved-Caspase-1及gasdermin D（GSDMD）蛋白水平;透射电镜检测肝组织细胞形态变化。（2）将小鼠正常肝脏AML12细胞分别进行过表达和沉默IRF-1表达后进行模拟IR处理,分为过表达IRF-1+IR组、空载体+IR组、IRF-1 siRNA+IR组及其对照组（NC+IR组）。Western blot检测4组细胞IRF-1、Caspase-1、Cleaved-Caspase-1和GSDMD蛋白水平;透射电镜检测细胞形态变化;流式细胞术检测细胞焦亡情况;酶标仪检测细胞上清乳酸脱氢酶（LDH）释放量。结果　（1）体内实验结果显示,与Sham组比较,IR6 h组和12 h组血清ALT、AST和IL-1β水平均有明显升高（P＜0.05）,IRF-1、Caspase-1和GSDMD蛋白表达升高（P＜0.05）;同时肝细胞出现肿胀、排列紊乱、空泡、片状坏死和红细胞淤积并伴有核膜损伤和线粒体肿胀。（2）体外AML12细胞过表达IRF-1后Cleaved-Caspase-1和GSDMD的表达增加,而敲低IRF-1表达后Caspase-1和GSDMD表达随之减少（P＜0.05）。沉默IRF-1表达后,与NC+IR组相比,IRF-1 siRNA+IR组细胞质膜和核膜的破坏减轻,细胞焦亡率下降,LDH释放量减少约20%（P＜0.05）。结论　IRF-1可通过调节Caspase-1和GSDMD的表达以及IL-1β的释放导致小鼠LIRI。     

Glycyrrhizin attenuates hepatic ischemia-reperfusion injury by suppressing HMGB1-dependent GSDMD-mediated kupffer cells pyroptosis

Gasdermin D (GSDMD), a genetic substrate for inflammatory caspases, plays a central role in pyroptosis of macrophages and release of interleukin‑1β (IL-1β), but was mainly referred to microbial infection. High mobility group box-1 (HMGB1), served as an alarm molecule during various pathological process, has been widely recognized to be involved in liver ischemia-reperfusion (I/R). Glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, was recently referred to have ability to prevent I/R induced liver injury by inhibiting HMGB1 expression and activity. However, the mechanisms responsible for damage amelioration subsequently to HMGB1 inhibition during liver I/R remain enigmatic. This study was designed to explore the functional role and molecular mechanism of glycyrrhizin in the regulation of I/R induced liver injury. We found that liver I/R promotes GSDMD-mediated pyroptotic cell death of Kupffer cells, which was inhibited by glycyrrhizin. Interestingly, endogenous HMGB1, not exogenous one, was involved in hypoxia-reoxygenation (H/R) induced pyroptosis. Moreover, GSDMD knockdown protects kupffer cells against H/R induced pyroptosis in vitro. Here, we report, for the first time, that glycyrrhizin attenuated tissue damage and kupffer cells pyroptosis during liver ischemia-reperfusion injury (LIRI) and identify a previously unrecognized HMGB1- dependent GSDMD- mediated signaling pathway in the mechanism of kupffer cells pyroptosis induced by H/R. Our findings provide the first demonstration of GSDMD-determined pyroptotic cell death responsible for I/R induced release of IL-1β and this would provide a mandate to better understand the unconventional mechanisms of cytokine release in the sterile innate immune system.     

瑞舒伐他汀通过调控UCP2-SIRT3通路减轻脑缺血/再灌注对神经元线粒体的损伤

目的探讨瑞舒伐他汀(rosuvastatin,RS)通过UCP2-SIRT3信号通路在脑缺血/再灌注(CIR)神经元线粒体损伤中的作用及其机制。方法建立SH-SY5Y细胞的脑梗死再灌注模型(OGD/R),给予不同浓度RS(40和2.5μmol·L^-1)分别处理,观察两组中细胞增殖和凋亡的变化及UCP2和SIRT3分子的表达;构建UCP2沉默细胞系,研究不同浓度RS对UCP2沉默前后细胞形态和线粒体膜电位的影响、以及SIRT3分子、线粒体外膜异位酶20(TOMM20)和线粒体合成相关蛋白(Drp1、Opa1和PGC1)的表达变化。结果RS能提高OGD/R细胞的存活率、抑制细胞凋亡、改变细胞形态、稳定细胞线粒体膜电位;增加OGD/R细胞中UCP2、SIRT3分子和TOMM20蛋白表达,并诱导Drp1和Opa1 mRNA的表达,抑制PGC1 mRNA的表达;沉默UCP2后能明显降低OGD/R细胞的存活率及TOMM20蛋白的表达,降低Drp1和Opa1 mRNA的表达,使PGC1 mRNA的表达增加。结论RS通过调控UCP2-SIRT3通路减轻CIR对神经元线粒体的损伤,发挥神经细胞保护作用。     

积雪草酸联合奥沙利铂对结肠癌HCT116细胞凋亡、自噬和焦亡的调控作用研究

目的:探讨积雪草酸(asiatic acid,AA)联合奥沙利铂(oxaliplatin,OXP)对结肠癌HCT116细胞增殖、凋亡、自噬和焦亡的调控作用.方法:采用CCK8法检测AA、OXP、AA与OXP联用对HCT116细胞增殖的影响;采用Hoechst染色法和Annexin V/PI双染法检测细胞凋亡;JC-1染...     

瑞舒伐他汀通过UCP2-SIRT3信号调控脑I/R对神经元的损伤

目的:研究瑞舒伐他汀对脑缺血-再灌注损伤的保护作用及作用机制。方法:(1)建立脑梗死及OGD/R细胞模型,检测不同浓度瑞舒伐他汀对细胞增殖及细胞凋亡的影响;(2)用不同浓度瑞舒伐他汀处理OGD/R细胞模型,观察瑞舒伐他汀对细胞形态和细胞中UCP2/SIRT3表达和定位的影响;(3)构建UCP2沉默的细胞系,观察细胞线粒体形态和细胞中TOMM20及SIRT3分子表达与定位,研究瑞舒伐他汀在OGD/R细胞模型中发挥保护作用的通道和机制;(4)检测线粒体膜电位,PCR检测线粒体生成基因PGC1、Drp1和Opa1表达,研究瑞舒伐他汀对线粒体的保护作用。结果:(1)不同浓度瑞舒伐他汀均可以明显减低OGD/R细胞凋亡,提高细胞存活率;(2)瑞舒伐他汀通过影响细胞UCP2和SIRT3表达,进而发挥细胞保护作用,使细胞免受OGD/R损伤;(3)瑞舒伐他汀通过调控UCP2影响TOMM20表达,增加线粒体跨膜转运和能量代谢,增强线粒体功能,提高细胞存活;(4)瑞舒伐他汀阻止OGD/R细胞膜电位下降,保护线粒体,改善细胞状态,减少细胞凋亡。结论:瑞舒伐他汀通过调控UCP2/SIRT通路来抑制OGD/R细胞线粒体损伤,从而发挥神经元保护作用。     

Melatonin prevents endothelial cell pyroptosis via regulation of long noncoding RNA MEG3/miR-223/NLRP3 axis

OBJECTIVE Atherosclerosis(AS) is an inflammatory disease linked to endothelial dysfunction.Melatonin is reported to possess substantial anti-inflammatory properties, which has proven to be effective in AS. Emerging literature suggests that pyroptosis plays a critical role during AS progression. However, whether pyroptosis contributes to endothelial dysfunction and the underlying molecular mechanisms remained unexploited.This study was designed to investigate the antipyroptotic effects of melatonin in atherosclerotic endothelium and to elucidate the potential mechanisms. METHODS ApoE-/-mice were fed a high-fat diet to establish an atherosclerotic model, then divided into normal diet(ND) group, normal diet+melatonin(ND+melatonin) group, high fat diet(HFD)group and high fat diet+melatonin(HFD+melatonin) group.After 12 weeks, HE and oil Red O staining were used to detect the formation of atherosclerosis; qRT-RCR and Western blotting were used to detect the expression of NLRP3, ASC, IL-1β, IL-18, GSDMD, NF-κB, miR-223 and MEG3 in aortic endothelium; The luciferase assay was used to detect the binding of mi R-223 to MEG3. Human aortic endothelial cells(HAECs) were pretreated with ox-LDL. After melatonin treatment, qRT-RCR was used to detect the expression of mi R-223 and MEG3. Western blotting was used to detect NLRP3, ASC, c-caspase1,p-caspase1, GSDMD expression. In addition, after overexpressing MEG3 and knocking out mi R-223, the pyroptosis of HAECs was also detected. RESULTS We found intragastric administration of melatonin for 12 weeks markedly reduced the atherosclerotic plaque in aorta.Meanwhile, melatonin also attenuated the expression of pyroptosis-related genes, including NLRP3, ASC,cleaved caspase1, NF-κB/GSDMD, GSDMD N-termini,IL-1β, and IL-18 in aortic endothelium of melatonin-treated animals. Consistent antipyroptotic effects were also observed in ox-LDL-treated human aortic endothelial cells(HAECs). We found that lnc RNA MEG3 enhanced pyroptosis in HAECs. Moreover, MEG3 acted as an endogenous sponge by sequence complementarity to suppress the function of mi R-223 and to increase NLRP3 expression and enhance endothelial cel pyroptosis. Furthermore, knockdown of mi R-223 blocked the antipyroptotic actions of melatonin in ox-LDL-treated HAECs. CONCLUSION Our results suggest that melatonin prevents endothelial cell pyroptosis via MEG3/mi R-223/NLRP3 axis in atherosclerosis, and therefore, melatonin replacement might be considered a new strategy for protecting endothelium against pyroptosis, thereby for the treatment of atherosclerosis associated with pyroptosis.     

Scutellarin inhibits caspase-11 activation and pyroptosis in macrophages via regulating PKA signaling

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus, acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1β) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1β and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.KEY WORDS: Caspase-11, Macrophages, Gasdermin D, Pyroptosis, Scutellarin, PKA signaling     

NLRP6 expressed in astrocytes aggravates neurons injury after OGD/R through activating the inflammasome and inducing pyroptosis

NLRP6, the nucleotide oligomerization domain-like receptor family pyrin domain containing 6, has a substantiable effect on inflammation and host defense against microorganisms. In our previous study, NLRP6 promotes inflammation after cerebral I/R injury in a MCAO model. However, the effect of NLRP6 in different nerve cells subjected to OGD/R needs to be further understood. Here, evidence shows that the expression of NLRP6 is increased in different nerve cells subjected to OGD/R, and mainly expressed in astrocytes. NLRP6 may up-regulate inflammation factors (IL-1β, Il-8) via the form of inflammasomes in astrocytes after OGD/R. Then, primary neuron-astrocyte co-culture model under OGD/R in vitro was performed, and we found that NLRP6 decreased the neurons viability and aggravated apoptosis of neurons. Mechanically, NLRP6 could induce pyroptosis to regulate the survival of neurons through activating caspase-1.     

重建线粒体动态平衡对NK细胞NK2型活化影响

目的立于“线粒体功能紊乱-细胞异常活化”视角,探讨NK细胞NK2型活化机制。方法将NK-92MI细胞分为空白组、TSLP组、1、5、10μmol·L^-1 Mdivi-1组。ELISA法测定各组IL-4、IL-5、IFN-γ水平;蛋白印迹法分析各组p-Drp1、MnSOD蛋白表达;DHE染色及流式检测各组ROS水平;激光共聚焦观察各组线粒体形态。结果ELISA显示,与对照组比较,TSLP组IL-4、IL-5水平明显升高,IFN-γ水平下调(P<0.05);与TSLP组比较,5、10μmol·L^-1 Mdivi-1组IL-4、IL-5水平降低,10μmol·L^-1 Mdivi-1组IFN-γ浓度有所回升(P<0.05)。DHE染色及流式显示,TSLP组ROS水平较对照组升高;而5、10μmol·L^-1 Mdivi-1组ROS水平较TSLP组降低(P<0.05)。共聚焦显示,TSLP刺激后,细胞内大量圆球状线粒体生成,5、10μmol·L^-1 Mdivi-1干预后该现象得到改善。蛋白印迹显示,TSLP组p-Drp1水平明显上调,MnSOD表达下降,Mdivi-1干预则有效逆转了上述变化。结论线粒体动态失衡可能是NK细胞异常活化内在机制之一,其或是调控NK细胞NK2型活化介导的变应性炎症反应的重要靶点。