IRF-1对LPS诱导的小鼠巨噬细胞凋亡和自噬的调节作用及其机制研究
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摘要
研究背景脓毒症导致的多器官衰竭,是重症感染、烧伤、严重创伤等患者的主要死亡原因。但是其病理生理改变复杂,具体发病机制并不十分明确,给有效、针对的靶向性治疗带来一定困难。大量研究表明,免疫细胞凋亡是脓毒症发生发展过程中免疫抑制状态的主要原因。免疫抑制状态导致机体免疫系统对原发感染和继发感染控制的失败,从而导致死亡。其中,已证实脓毒症导致的巨噬细胞凋亡,与其细菌清除能力和炎症细胞因子释放能力的下降有密切关系,是脓毒症导致死亡的重要原因之一。除了凋亡之外,另外一种细胞生物学行为,自噬,也在脓毒症中激活,并被认为发挥保护性作用,其保护机制可能与拮抗细胞凋亡的发生有关。对巨噬细胞凋亡和自噬的调节,在一定程度上,决定了脓毒症的发生发展及预后。干扰素调节因子-1(IRF-1)在脂多糖(LPS)诱导的小鼠脓毒症模型中具有损伤性作用。IRF-1基因敲除的小鼠,腹腔内注射LPS后,终末脏器损伤明显减轻,死亡率明显降低,其保护作用被证实与促炎症因子TNF-α和IFN-γ释放的减少有关。除了促进炎症因子释放外,IRF-1还是一种重要的促凋亡因子,可以促进肝细胞、淋巴细胞、乳腺癌细胞、胆囊癌细胞等原代细胞或肿瘤细胞系的凋亡。但在脓毒症模型中,IRF-1对巨噬细胞凋亡和自噬的调节作用,国内外未见相关报道。本研究通过LPS腹腔内注射建立小鼠脓毒症动物模型,以LPS干预的鼠巨噬细胞株RAW264.7细胞为体外模型,探讨IRF-1对LPS诱导的小鼠巨噬细胞的凋亡和自噬的调节作用,并进一步探讨LPS诱导巨噬细胞IRF-1激活的信号传导通路,以及IRF-1调节巨噬细胞凋亡和自噬的机制。
第一章小鼠脓毒症模型中IRF-1对巨噬细胞凋亡和自噬的调节作用
目的腹腔内注射LPS后,观察IRF-1基因敲除(KO)小鼠和匹配的C57BL/6野生型(WT)小鼠的生存率及终末脏器损伤,并观察各组小鼠腹腔巨噬细胞、肺泡巨噬细胞、脾脏巨噬细胞中凋亡和自噬的发生水平。
方法8-10周龄雄性野生型C57BL/6小鼠和IRF-1KO小鼠,每组各12只,腹腔内注射LPS(20mg/kg),建立脓毒症动物模型,观察96h生存率。给予NS或LPS(20mg/kg)腹腔内注射16h后,分别获取各组小鼠肺组织,HE染色观察肺组织病理学改变；同时,分别收集各组小鼠腹腔巨噬细胞、肺泡巨噬细胞、脾脏巨噬细胞,检测凋亡和自噬的发生水平。Western blot检测其cleaved caspase-3的水平、LC3-Ⅱ/Ⅰ的比值；TUNEL法检测腹腔巨噬细胞凋亡指数(AI)的变化,激光共聚焦显微镜(confocal)检测腹腔巨噬细胞LC3B的颗粒聚集情况,电镜(TEM)检测腹腔巨噬细胞中线粒体损伤和自噬的发生情况,
结果WT小鼠腹腔内注射LPS后,96h的生存率为25%。而IRF-1KO小鼠,腹腔内注射LPS后,无死亡现象发生,急性肺损伤明显改善。Western blot结果显示,与WT小鼠相比,给予LPS刺激后,IRF-1KO小鼠中,肺泡巨噬细胞、脾脏巨噬细胞、腹腔巨噬细胞的cleaved caspase-3水平明显降低,而LC3-Ⅱ/Ⅰ的比值明显增高。此外,TUNEL法显示,较WT小鼠,IRF-1KO组小鼠腹腔巨噬细胞凋亡指数明显降低。TEM结果显示,较WT小鼠,IRF-1KO组小鼠腹腔巨噬细胞自噬发生水平明显增高。共聚焦显微镜亦证实,IRF-1KO组小鼠腹腔巨噬细胞中LC3B的荧光强度和聚集程度增高。
结论采用腹腔内注射LPS的方法,成功建立了小鼠脓毒症模型。IRF-1基因敲除显著改善了脓毒症小鼠的预后和终末脏器损伤。在脓毒症模型中,IRF-1参与了小鼠巨噬细胞凋亡和自噬的调节。较WT小鼠相比,IRF-1KO小鼠的巨噬细胞凋亡发生水平明显降低,自噬的发生水平明显提高。IRF-1对巨噬细胞凋亡和自噬的调节,可能是其影响脓毒症预后的重要原因。
第二章自噬对LPS诱导鼠巨噬细胞株RAW264.7细胞损伤的保护性作用研究
目的验证巨噬细胞自噬在脓毒症模型中的作用,观察自噬对LPS诱导的鼠巨噬细胞株RAW264.7细胞损伤的保护性作用。
方法分别使用自噬抑制剂3-MA(5mM)和Beclin-1siRNA抑制鼠巨噬细胞株RAW264.7细胞自噬的发生水平,给予LPS(500ng/ml)刺激16h后,Western blot检测LC3-Ⅱ/Ⅰ的比值和cleaved caspase-3水平,使用MTT法检测各组细胞活力。
结果较对照组,LPS处理后RAW264.7细胞的自噬水平明显增高,LC3-Ⅱ/Ⅰ的比值增加。使用自噬抑制剂3-MA预处理,以及使用siRNA特异性干扰自噬相关蛋白Beclin-1表达后,LPS诱导的自噬发生明显受到抑制,LC3-Ⅱ/Ⅰ的比值明显降低,cleaved caspase-3水平明显增高。MTT结果显示抑制自噬后,LPS诱导的细胞死亡明显加重。
结论使用自噬抑制剂3-MA或是小干扰RNA抑制自噬相关蛋白的表达,均可以明显抑制LPS诱导的巨噬细胞自噬的发生,促进其凋亡的发生。抑制自噬后,LPS诱导的巨噬细胞死亡明显加重,提示自噬在LPS诱导的巨噬细胞损伤中具有保护性作用,其保护作用可能与拮抗凋亡的发生有关。
第三章LPS诱导鼠巨噬细胞株RAW264.7细胞IRF-1的表达及信号传导通路研究
目的观察LPS诱导的鼠巨噬细胞株RAW264.7细胞中IRF-1的表达,并探讨其信号传导通路。
方法第一部分,体外培养RAW264.7细胞,使用LPS(500ng/ml)刺激不同时间(0—8h)后,Western blot检测IRF-1的表达水平。使用不同浓度的LPS(10ng/ml-1000ng/ml)刺激4h后,Western blot检测IRF-1的表达水平。第二部分,将RAW264.7细胞分成四组：control siRNA组,TLR4siRNA组,LPS+control siRNA组,以及LPS+TLR4siRNA组。siRNA转染RAW264.7细胞24h后,使用LPS(500ng/ml)刺激4h,Western blot检测IRF-1和TLR4的表达水平。第三部分,将RAW264.7细胞分成四组：control siRNA组,TRIF siRNA组,LPS+control siRNA组,以及LPS+TRIF siRNA组。siRNA转染及LPS刺激同上,Western blot检测IRF-1和TRIF的表达水平。第四部分,将RAW264.7细胞分成四组：control siRNA组,MyD88siRNA组,LPS+control siRNA组,以及LPS+MyD88siRNA组。siRNA转染及LPS刺激同上,Western blot检测IRF-1和MyD88的表达水平。
结果LPS诱导RAW264.7细胞的IRF-1表达,呈时间、剂量依赖性,其在LPS刺激4h后表达达峰；在LPS浓度为500ng/ml表达达峰,继续增加LPS的量并不能提高IRF-1的表达。小干扰RNA干预TLR4、TRIF、MyD88表达后,Western blot检测显示,较control siRNA转染组,TLR4siRNA、TRIF siRNA、MyD88siRNA转染组,TLR4、TRIF、MyD88的表达明显下降。LPS+TLR4siRNA组和LPS+TRIF siRNA组,较LPS+control siRNA组,Western blot检测显示IRF-1表达明显下降。而LPS+MyD88siRNA组,较LPS+control siRNA组,IRF-1的表达水平无明显变化。
结论LPS诱导RAW264.7细胞IRF-1的表达呈时间依赖性和剂量依赖性。LPS通过TLR4-TRIF依赖性、MyD88非依赖性信号通路诱导IRF-1的表达。
第四章IRF-1/NO调节鼠巨噬细胞株RAW264.7细胞凋亡和自噬及其机制研究
目的观察IRF-1及其下游因子NO对鼠巨噬细胞株RAW264.7细胞凋亡和自噬的调节作用,并探讨其作用机制。
方法第一部分,使用腺病毒AdIRF-1和AdlacZ,按照不同的MOI(100,200)感染RAW264.7细胞48h。Western blot检测IRF-1表达。选择MOI为200感染RAW264.7细胞48h后,Western blot检测pmTOR, mTOR,p-P70S6,P70S6,LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3,8,9的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞中LC3B的颗粒聚集情况。第二部分,使用腺病毒AdIRF-1和AdlacZ按照MOI为200感染RAW264.7细胞48h后,收集细胞及上清液。Western blot检测iNOS表达,Griess Assay检测上清液中NO的含量。第三部分,使用内源性NO供体SNAP,按照不同的浓度(100μM—800μM)处理RAW264.7细胞16h后,Western blot检测pmTOR, mTOR,LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3,8,9的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞中LC3B的颗粒聚集情况。第四部分,使用iNOS抑制剂L-NIL(200μM)预处理RAW264.7细胞1h后,给予LPS(500ng/ml)刺激16h。Western blot检测LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞内LC3B的颗粒聚集情况。
结果与病毒载体AdlacZ相比,腺病毒AdIRF-1感染48h可以诱导RAW264.7细胞特异性高表达IRF-1,而且不产生明显的细胞毒性,因此选择该MOI进行后续实验。与AdlacZ组相比,AdIRF-1感染RAW264.7细胞后,Western blot显示,pmTOR/p-P70S6蛋白表达水平增高,LC3-Ⅱ/Ⅰ的比值降低。Arrayscan显示AdIRF-1感染组,LC3B颗粒聚集被显著抑制。同时,Western blot显示AdIRF-1感染组,cleaved caspase-8,9,3水平显著提高,caspase-3活性显著提高。AdIRF-1感染RAW264.7细胞48h后,Western blot显示iNOS显著激活,Griess Assay显示上清液中NO含量明显增加,证实在RAW264.7细胞中,IRF-1是iNOS/NO的上游调节因子。采用无血清的培养液预处理RAW264.7细胞12h后,使用不同剂量的内源性NO供体SNAP处理RAW264.7细胞16h,Western blot显示pmTOR蛋白表达水平增高,LC3-Ⅱ/Ⅰ的比值明显降低,Arrayscan显示SNAP处理组,LC3B颗粒聚集被显著抑制,该反应呈剂量依赖性。同时,Western blot显示SNAP处理组,cleaved caspase-8,9,3水平显著提高,caspase-3活性显著提高,该反应亦呈剂量依赖性。使用iNOS抑制剂L-NIL预处理RAW264.7细胞1h,随后给予LPS刺激。较LPS处理组,L-NIL预处理后,LC3-Ⅱ/Ⅰ的比值明显增高,cleaved caspase-3激活被抑制。Arrayscan显示L-NIL预处理组,LC3B颗粒聚集显著增多,caspase-3活性显著降低。
结论IRF-1及其下游因子NO,通过激活mTOR/P70S6通路抑制鼠巨噬细胞株RAW264.7细胞自噬的发生。同时,IRF-1/NO通过激活cleaved caspase-9,8,进一步激活cleaved caspase-3,参与了内源性凋亡和外源性凋亡途径,从而促进RAW264.7细胞凋亡的发生。
Background Sepsis is a leading cause of death in intensive care units. Unfortunately, the precise mechanism of the dysregulated host response in sepsis is complex and remains ambiguous.
Apoptosis is one of the best characterized mechanisms that contribute to sepsis induced end organ damage, immune cell death and eventual lethality. Dysregulated apoptotic immune cell death may be a key mechanism of immunosuppression during sepsis. Both peritoneal macrophages and liver macrophages from septic mice show increased apoptosis which is associated with a decreased capacity for cytokine release and bacteria elimination.
Another cellular process, autophagy, is also activated in sepsis and is primarily believed to play a protective role. Autophagy has been well demonstrated to be essential for cellular homeostasis, cell defense during severe infection. The inhibition of autophagy results in an accelerated apoptotic cell death while increased autophagy allows for cell survival by inhibiting apoptosis, suggesting that autophagy plays primarily a protective role by limiting cellular damage and death. The balance between autophagy and apoptosis can influence cellular survival. Interestingly, recent studies indicate that crosstalk exists between apoptosis and autophagy via shared signaling components and regulatory factors; however, whether redundancy exists in the upstream signals that control these two processes in sepsis is undetermined.
IRF-1KO mice protect against lethal dose LPS administration. IRF-1participates in mortality associated with disease models mediated by TNF-α and IFN-γ. In regards to apoptosis, IRF-1functions as a pro-apoptotic signal in vitro in both primary and tumor cell lines. The present study investigates the role that IRF-1plays in regulating autophagy and apoptosis in murine macrophages the setting of LPS stimulation.
Chapter One IRF-1regulates apoptosis and autophagy in murine macrophages in sepsis
Objective To compare the survival rate, end organ damage and macrophages apoptosis and autophagy between IRF-1KO and matched WT mice following lethal dose LPS administration.
Methods8-10weeks male WT and IRF-1KO mice,12mice for each group, were injected intraperitoneally with a lethal dose of LPS (20mg/kg). Survival rates96h after LPS administration were assessed. Following LPS administration for16h, lung tissues were collected and acute lung injury was analyzed by H&E staining. The peritoneal macrophages, alveolar macrophages and splenic macrophages were harvested. Cleaved caspase-3levels, the ratio of LC3-Ⅱ/Ⅰ were analyzed by western blot. Apoptosis index (AI) of peritoneal macrophages was analyzed by TUNEL staining. The autophagic flux of peritoneal macrophages was analyzed by LC3B staining and transmission electron microscope.
Results IRF-1KO mice experienced significantly improved survival outcomes and end organ damage following LPS administration. Peritoneal macrophages, alveolar macrophages and splenic macrophages isolated from IRF-1KO mice exhibited decreased caspase-3cleavage following LPS administration. Peritoneal macrophages isolated from IRF-1KO mice exhibited less TUNEL positive cells. On the contrary, peritoneal macrophages, alveolar macrophages and splenic macrophages isolated from IRF-1KO mice demonstrated an increase in the ratio of LC3-Ⅱ/Ⅰ as compared to WT macrophages. Peritoneal macrophages isolated from IRF-1KO mice experienced increased autophagic flux as demonstrated by increased cytoplasmic LC3B. Also, WT peritoneal macrophages demonstrate significant mitochondrial damage while KO peritoneal macrophages demonstrate mitophagy.
Conclusion IRF-1KO mice are protected from lethal dose LPS challenge, which experienced improved survival rate and end organ damage. IRF-1KO macrophages demonstrate increased autophagic flux and decreased apoptosis following LPS administration. IRF-1regulates the balance between apoptosis and autophagy in macrophage, which may be a protective mechanism in IRF-1KO mice.
Chapter Two Decreased autophagic flux in RAW264.7cells leads to apoptotic cell death in response to LPS stimulation
Objective To explore the function of autophagy in LPS induced apoptotic cell death in RAW264.7cells.
Methods RAW264.7cells were pretreated with the autophagy inhibitor3-methyladenine (3-MA) or Beclin-1siRNA, and then subjected to LPS (500ng/ml) stimulation for16h. LC3-Ⅱ/Ⅰ and caspase-3cleavage was analyzed by western blot and cell viability was analyzed by MTT assay.
Results Compared with the control group, LPS treatment can induce autophagic flux and apoptosis as demonstrated by increased LC3-Ⅱ/Ⅰ ratio and caspase-3cleavage. Autophagy inhibitors,3-MA pretreatment or Beclin-1siRNA transfection, exhibited inhibition of LPS induced autophagic flux as demonstrated by LC3-Ⅱ/Ⅰratio. Furthermore, inhibition of autophagy led to increased apoptosis as demonstrated by caspase-3cleavage and increased cell death as shown by MTT cell viability assay results
Conclusion LPS can induce autophagy and apoptosis in RAW264.7cells. Autophagy inhibitors3-MA or Beclin-1siRNA transfection can inhibit LPS-induced autophagy. Inhibited of LPS induced autophagy can lead to increased apoptosis and cell death in RAW264.7cells, suggesting that autophagy plays a protective role in the LPS treatment and its protective effect is associated with the inhibition of apoptotic cell death.
Chapter Three LPS induced IRF-1expression in RAW264.7cells and the signaling pathway
Objective To investigate the signaling transduction pathway of LPS induced IRF-1expression in RAW264.7cells.
Methods The first part, with LPS (500ng/ml) stimulation at different time (0-8h) after, IRF-1expression was detected by western blot. With different concentrations of LPS (10ng/ml-1000ng/ml) stimulation for4h, IRF-1expression was detected by western blot. The second part, the RAW264.7cells were divided into four groups:control siRNA group, TLR4siRNA group, LPS+control siRNA group, and LPS+TLR4siRNA group. RAW264.7cells were transfected with siRNA for24h, following LPS (500ng/ml) stimulation for4h, IRF-1and TLR4expression by detected by western blot. The third part, the RAW264.7 cells were divided into four groups:control siRNA group, TRIF siRNA group, LPS+control siRNA group, and LPS+TRIF siRNA group. IRF-1and TRIF expression were detected by western blot. The fourth part, the RAW264.7cells were divided into four groups:control siRNA group, MyD88siRNA group, LPS+control siRNA group, and LPS+MyD88siRNA group. IRF-1expression and MyD88were detected by western blot.
Results LPS induced IRF-1expression in RAW264.7cells occurs in a time-dependent and dose-dependent manner. It gets peak at4h and with500ng/ml LPS stimulation. Western blot analysis showed that, compared with control siRNA transfection group, in TLR4siRNA, TRIF siRNA, MyD88siRNA transfected group, TLR4, TRIF, MyD88expression were significantly decreased. Compared with LPS+control siRNA group, LPS+TLR4siRNA group and LPS+TRIF siRNA group showed significantly decreased IRF-1expression. In the LPS+MyD88siRNA group, compared with LPS+control siRNA group, IRF-1expression did not change.
Conclusion LPS induced IRF-1expression in RAW264.7cells occurs in a time-dependent and dose dependent manner. IRF-1activation in response to LPS stimulation is mediated through the TLR4signaling pathway in a TRIF dependent/MyD88independent manner.
Chapter Four IRF-1/NO regulates apoptosis and autophagy in murine macrophages and its mechanism
Objective To investigate the regulatory function of IRF-1and its downstream NO on apoptosis and autophagy in murine macrophages.
Methods The first part, RAW264.7cells were infected with AdIRF-1and AdlacZ with different MOI (100,200) for48h. IRF-1expression was detected by western blot. RAW264.7cells were infected with MOI of200for48h, pmTOR, mTOR, p-P70S6, P70S6, LC3-Ⅱ/Ⅰ ratio and cleaved caspase-3,8,9were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used. The second part, RAW264.7cells were infected by AdIRF-1and AdlacZ with MOI of200for48h, iNOS expression was analyzed by western blot, NO production in the supernatant was analyzed by Griess Assay. The third part, endogenous NO donor, SNAP, with its different concentrations (100μM-800μM) treatment in RAW264.7cells for16h, pmTOR, mTOR, LC3-Ⅱ/Ⅰ ratio and cleaved caspase-3,8,9were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used. The fourth part, iNOS inhibitor L-NIL (200μM) pretreated RAW264.7cells for1h, following LPS (500ng/ml) stimulation for16h, LC3-Ⅱ/Ⅰ ratio and caspase-3cleavage were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used.
Results Compared with AdlacZ, AdIRF-1infection can induce expression of IRF-1in RAW264.7cells. Overexpression of IRF-1can lead to the activation of pmTOR/p-P70S6, decreased LC3-Ⅱ/Ⅰ ratio. AdIRF-1infection inhibits autophagy as demonstrated by LC3B Arrayscan. Meanwhile, overexpression of IRF-1can lead to increased cleavage of caspase-8,9and3by western blot. Caspase-3activity kit confirmed significantly increased caspase-3activity. AdIRF-1infection can also lead to significant activation of iNOS and NO production, which confirmed IRF-1, is the upstream of iNOS/NO. With the different doses of SNAP treatment for16h, western blot showed increased levels of pmTOR and decreased LC3-Ⅱ/Ⅰ ratio. Arrayscan confirmed significantly decreased LC3B with SNAP treatment. The inhibition of autophagy with SNAP treatment was dose-dependent. Meanwhile, western blot showed increased cleavage of caspase-8,9and3with SNAP treatment. Caspase-3activity kit confirmed increased caspase-3activity with SNAP treatment. The effect was dose-dependent. Pretreated RAW264.7cells with iNOS inhibitor L-NIL for1h, and then followed by LPS treatment. Compared with LPS treatment, in the L-NIL pretreatment group, LC3-Ⅱ/Ⅰ ratio was significantly increased and caspase-3cleavage was inhibited. LC3B Arrayscan confirmed increased autophagy and caspase-3kit confirmed decreased caspase-3activity in the L-NIL pretreatment group.
Conclusion IRF-1and its downstream NO inhibited autophagy by activating mTOR/p70S6activity in RAW264.7cells. IRF-1/NO through the activation of cleaved caspase-9,8, and further activation of cleaved caspase-3, involved in both intrinsic and extrinsic apoptotic pathways.
第一章小鼠脓毒症模型中IRF-1对巨噬细胞凋亡和自噬的调节作用
目的腹腔内注射LPS后,观察IRF-1基因敲除(KO)小鼠和匹配的C57BL/6野生型(WT)小鼠的生存率及终末脏器损伤,并观察各组小鼠腹腔巨噬细胞、肺泡巨噬细胞、脾脏巨噬细胞中凋亡和自噬的发生水平。
方法8-10周龄雄性野生型C57BL/6小鼠和IRF-1KO小鼠,每组各12只,腹腔内注射LPS(20mg/kg),建立脓毒症动物模型,观察96h生存率。给予NS或LPS(20mg/kg)腹腔内注射16h后,分别获取各组小鼠肺组织,HE染色观察肺组织病理学改变；同时,分别收集各组小鼠腹腔巨噬细胞、肺泡巨噬细胞、脾脏巨噬细胞,检测凋亡和自噬的发生水平。Western blot检测其cleaved caspase-3的水平、LC3-Ⅱ/Ⅰ的比值；TUNEL法检测腹腔巨噬细胞凋亡指数(AI)的变化,激光共聚焦显微镜(confocal)检测腹腔巨噬细胞LC3B的颗粒聚集情况,电镜(TEM)检测腹腔巨噬细胞中线粒体损伤和自噬的发生情况,
结果WT小鼠腹腔内注射LPS后,96h的生存率为25%。而IRF-1KO小鼠,腹腔内注射LPS后,无死亡现象发生,急性肺损伤明显改善。Western blot结果显示,与WT小鼠相比,给予LPS刺激后,IRF-1KO小鼠中,肺泡巨噬细胞、脾脏巨噬细胞、腹腔巨噬细胞的cleaved caspase-3水平明显降低,而LC3-Ⅱ/Ⅰ的比值明显增高。此外,TUNEL法显示,较WT小鼠,IRF-1KO组小鼠腹腔巨噬细胞凋亡指数明显降低。TEM结果显示,较WT小鼠,IRF-1KO组小鼠腹腔巨噬细胞自噬发生水平明显增高。共聚焦显微镜亦证实,IRF-1KO组小鼠腹腔巨噬细胞中LC3B的荧光强度和聚集程度增高。
结论采用腹腔内注射LPS的方法,成功建立了小鼠脓毒症模型。IRF-1基因敲除显著改善了脓毒症小鼠的预后和终末脏器损伤。在脓毒症模型中,IRF-1参与了小鼠巨噬细胞凋亡和自噬的调节。较WT小鼠相比,IRF-1KO小鼠的巨噬细胞凋亡发生水平明显降低,自噬的发生水平明显提高。IRF-1对巨噬细胞凋亡和自噬的调节,可能是其影响脓毒症预后的重要原因。
第二章自噬对LPS诱导鼠巨噬细胞株RAW264.7细胞损伤的保护性作用研究
目的验证巨噬细胞自噬在脓毒症模型中的作用,观察自噬对LPS诱导的鼠巨噬细胞株RAW264.7细胞损伤的保护性作用。
方法分别使用自噬抑制剂3-MA(5mM)和Beclin-1siRNA抑制鼠巨噬细胞株RAW264.7细胞自噬的发生水平,给予LPS(500ng/ml)刺激16h后,Western blot检测LC3-Ⅱ/Ⅰ的比值和cleaved caspase-3水平,使用MTT法检测各组细胞活力。
结果较对照组,LPS处理后RAW264.7细胞的自噬水平明显增高,LC3-Ⅱ/Ⅰ的比值增加。使用自噬抑制剂3-MA预处理,以及使用siRNA特异性干扰自噬相关蛋白Beclin-1表达后,LPS诱导的自噬发生明显受到抑制,LC3-Ⅱ/Ⅰ的比值明显降低,cleaved caspase-3水平明显增高。MTT结果显示抑制自噬后,LPS诱导的细胞死亡明显加重。
结论使用自噬抑制剂3-MA或是小干扰RNA抑制自噬相关蛋白的表达,均可以明显抑制LPS诱导的巨噬细胞自噬的发生,促进其凋亡的发生。抑制自噬后,LPS诱导的巨噬细胞死亡明显加重,提示自噬在LPS诱导的巨噬细胞损伤中具有保护性作用,其保护作用可能与拮抗凋亡的发生有关。
第三章LPS诱导鼠巨噬细胞株RAW264.7细胞IRF-1的表达及信号传导通路研究
目的观察LPS诱导的鼠巨噬细胞株RAW264.7细胞中IRF-1的表达,并探讨其信号传导通路。
方法第一部分,体外培养RAW264.7细胞,使用LPS(500ng/ml)刺激不同时间(0—8h)后,Western blot检测IRF-1的表达水平。使用不同浓度的LPS(10ng/ml-1000ng/ml)刺激4h后,Western blot检测IRF-1的表达水平。第二部分,将RAW264.7细胞分成四组：control siRNA组,TLR4siRNA组,LPS+control siRNA组,以及LPS+TLR4siRNA组。siRNA转染RAW264.7细胞24h后,使用LPS(500ng/ml)刺激4h,Western blot检测IRF-1和TLR4的表达水平。第三部分,将RAW264.7细胞分成四组：control siRNA组,TRIF siRNA组,LPS+control siRNA组,以及LPS+TRIF siRNA组。siRNA转染及LPS刺激同上,Western blot检测IRF-1和TRIF的表达水平。第四部分,将RAW264.7细胞分成四组：control siRNA组,MyD88siRNA组,LPS+control siRNA组,以及LPS+MyD88siRNA组。siRNA转染及LPS刺激同上,Western blot检测IRF-1和MyD88的表达水平。
结果LPS诱导RAW264.7细胞的IRF-1表达,呈时间、剂量依赖性,其在LPS刺激4h后表达达峰；在LPS浓度为500ng/ml表达达峰,继续增加LPS的量并不能提高IRF-1的表达。小干扰RNA干预TLR4、TRIF、MyD88表达后,Western blot检测显示,较control siRNA转染组,TLR4siRNA、TRIF siRNA、MyD88siRNA转染组,TLR4、TRIF、MyD88的表达明显下降。LPS+TLR4siRNA组和LPS+TRIF siRNA组,较LPS+control siRNA组,Western blot检测显示IRF-1表达明显下降。而LPS+MyD88siRNA组,较LPS+control siRNA组,IRF-1的表达水平无明显变化。
结论LPS诱导RAW264.7细胞IRF-1的表达呈时间依赖性和剂量依赖性。LPS通过TLR4-TRIF依赖性、MyD88非依赖性信号通路诱导IRF-1的表达。
第四章IRF-1/NO调节鼠巨噬细胞株RAW264.7细胞凋亡和自噬及其机制研究
目的观察IRF-1及其下游因子NO对鼠巨噬细胞株RAW264.7细胞凋亡和自噬的调节作用,并探讨其作用机制。
方法第一部分,使用腺病毒AdIRF-1和AdlacZ,按照不同的MOI(100,200)感染RAW264.7细胞48h。Western blot检测IRF-1表达。选择MOI为200感染RAW264.7细胞48h后,Western blot检测pmTOR, mTOR,p-P70S6,P70S6,LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3,8,9的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞中LC3B的颗粒聚集情况。第二部分,使用腺病毒AdIRF-1和AdlacZ按照MOI为200感染RAW264.7细胞48h后,收集细胞及上清液。Western blot检测iNOS表达,Griess Assay检测上清液中NO的含量。第三部分,使用内源性NO供体SNAP,按照不同的浓度(100μM—800μM)处理RAW264.7细胞16h后,Western blot检测pmTOR, mTOR,LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3,8,9的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞中LC3B的颗粒聚集情况。第四部分,使用iNOS抑制剂L-NIL(200μM)预处理RAW264.7细胞1h后,给予LPS(500ng/ml)刺激16h。Western blot检测LC3-Ⅱ/Ⅰ的比值以及cleaved caspase-3的表达水平。使用试剂盒检测caspase-3活性,使用Arrayscan检测细胞内LC3B的颗粒聚集情况。
结果与病毒载体AdlacZ相比,腺病毒AdIRF-1感染48h可以诱导RAW264.7细胞特异性高表达IRF-1,而且不产生明显的细胞毒性,因此选择该MOI进行后续实验。与AdlacZ组相比,AdIRF-1感染RAW264.7细胞后,Western blot显示,pmTOR/p-P70S6蛋白表达水平增高,LC3-Ⅱ/Ⅰ的比值降低。Arrayscan显示AdIRF-1感染组,LC3B颗粒聚集被显著抑制。同时,Western blot显示AdIRF-1感染组,cleaved caspase-8,9,3水平显著提高,caspase-3活性显著提高。AdIRF-1感染RAW264.7细胞48h后,Western blot显示iNOS显著激活,Griess Assay显示上清液中NO含量明显增加,证实在RAW264.7细胞中,IRF-1是iNOS/NO的上游调节因子。采用无血清的培养液预处理RAW264.7细胞12h后,使用不同剂量的内源性NO供体SNAP处理RAW264.7细胞16h,Western blot显示pmTOR蛋白表达水平增高,LC3-Ⅱ/Ⅰ的比值明显降低,Arrayscan显示SNAP处理组,LC3B颗粒聚集被显著抑制,该反应呈剂量依赖性。同时,Western blot显示SNAP处理组,cleaved caspase-8,9,3水平显著提高,caspase-3活性显著提高,该反应亦呈剂量依赖性。使用iNOS抑制剂L-NIL预处理RAW264.7细胞1h,随后给予LPS刺激。较LPS处理组,L-NIL预处理后,LC3-Ⅱ/Ⅰ的比值明显增高,cleaved caspase-3激活被抑制。Arrayscan显示L-NIL预处理组,LC3B颗粒聚集显著增多,caspase-3活性显著降低。
结论IRF-1及其下游因子NO,通过激活mTOR/P70S6通路抑制鼠巨噬细胞株RAW264.7细胞自噬的发生。同时,IRF-1/NO通过激活cleaved caspase-9,8,进一步激活cleaved caspase-3,参与了内源性凋亡和外源性凋亡途径,从而促进RAW264.7细胞凋亡的发生。
Background Sepsis is a leading cause of death in intensive care units. Unfortunately, the precise mechanism of the dysregulated host response in sepsis is complex and remains ambiguous.
Apoptosis is one of the best characterized mechanisms that contribute to sepsis induced end organ damage, immune cell death and eventual lethality. Dysregulated apoptotic immune cell death may be a key mechanism of immunosuppression during sepsis. Both peritoneal macrophages and liver macrophages from septic mice show increased apoptosis which is associated with a decreased capacity for cytokine release and bacteria elimination.
Another cellular process, autophagy, is also activated in sepsis and is primarily believed to play a protective role. Autophagy has been well demonstrated to be essential for cellular homeostasis, cell defense during severe infection. The inhibition of autophagy results in an accelerated apoptotic cell death while increased autophagy allows for cell survival by inhibiting apoptosis, suggesting that autophagy plays primarily a protective role by limiting cellular damage and death. The balance between autophagy and apoptosis can influence cellular survival. Interestingly, recent studies indicate that crosstalk exists between apoptosis and autophagy via shared signaling components and regulatory factors; however, whether redundancy exists in the upstream signals that control these two processes in sepsis is undetermined.
IRF-1KO mice protect against lethal dose LPS administration. IRF-1participates in mortality associated with disease models mediated by TNF-α and IFN-γ. In regards to apoptosis, IRF-1functions as a pro-apoptotic signal in vitro in both primary and tumor cell lines. The present study investigates the role that IRF-1plays in regulating autophagy and apoptosis in murine macrophages the setting of LPS stimulation.
Chapter One IRF-1regulates apoptosis and autophagy in murine macrophages in sepsis
Objective To compare the survival rate, end organ damage and macrophages apoptosis and autophagy between IRF-1KO and matched WT mice following lethal dose LPS administration.
Methods8-10weeks male WT and IRF-1KO mice,12mice for each group, were injected intraperitoneally with a lethal dose of LPS (20mg/kg). Survival rates96h after LPS administration were assessed. Following LPS administration for16h, lung tissues were collected and acute lung injury was analyzed by H&E staining. The peritoneal macrophages, alveolar macrophages and splenic macrophages were harvested. Cleaved caspase-3levels, the ratio of LC3-Ⅱ/Ⅰ were analyzed by western blot. Apoptosis index (AI) of peritoneal macrophages was analyzed by TUNEL staining. The autophagic flux of peritoneal macrophages was analyzed by LC3B staining and transmission electron microscope.
Results IRF-1KO mice experienced significantly improved survival outcomes and end organ damage following LPS administration. Peritoneal macrophages, alveolar macrophages and splenic macrophages isolated from IRF-1KO mice exhibited decreased caspase-3cleavage following LPS administration. Peritoneal macrophages isolated from IRF-1KO mice exhibited less TUNEL positive cells. On the contrary, peritoneal macrophages, alveolar macrophages and splenic macrophages isolated from IRF-1KO mice demonstrated an increase in the ratio of LC3-Ⅱ/Ⅰ as compared to WT macrophages. Peritoneal macrophages isolated from IRF-1KO mice experienced increased autophagic flux as demonstrated by increased cytoplasmic LC3B. Also, WT peritoneal macrophages demonstrate significant mitochondrial damage while KO peritoneal macrophages demonstrate mitophagy.
Conclusion IRF-1KO mice are protected from lethal dose LPS challenge, which experienced improved survival rate and end organ damage. IRF-1KO macrophages demonstrate increased autophagic flux and decreased apoptosis following LPS administration. IRF-1regulates the balance between apoptosis and autophagy in macrophage, which may be a protective mechanism in IRF-1KO mice.
Chapter Two Decreased autophagic flux in RAW264.7cells leads to apoptotic cell death in response to LPS stimulation
Objective To explore the function of autophagy in LPS induced apoptotic cell death in RAW264.7cells.
Methods RAW264.7cells were pretreated with the autophagy inhibitor3-methyladenine (3-MA) or Beclin-1siRNA, and then subjected to LPS (500ng/ml) stimulation for16h. LC3-Ⅱ/Ⅰ and caspase-3cleavage was analyzed by western blot and cell viability was analyzed by MTT assay.
Results Compared with the control group, LPS treatment can induce autophagic flux and apoptosis as demonstrated by increased LC3-Ⅱ/Ⅰ ratio and caspase-3cleavage. Autophagy inhibitors,3-MA pretreatment or Beclin-1siRNA transfection, exhibited inhibition of LPS induced autophagic flux as demonstrated by LC3-Ⅱ/Ⅰratio. Furthermore, inhibition of autophagy led to increased apoptosis as demonstrated by caspase-3cleavage and increased cell death as shown by MTT cell viability assay results
Conclusion LPS can induce autophagy and apoptosis in RAW264.7cells. Autophagy inhibitors3-MA or Beclin-1siRNA transfection can inhibit LPS-induced autophagy. Inhibited of LPS induced autophagy can lead to increased apoptosis and cell death in RAW264.7cells, suggesting that autophagy plays a protective role in the LPS treatment and its protective effect is associated with the inhibition of apoptotic cell death.
Chapter Three LPS induced IRF-1expression in RAW264.7cells and the signaling pathway
Objective To investigate the signaling transduction pathway of LPS induced IRF-1expression in RAW264.7cells.
Methods The first part, with LPS (500ng/ml) stimulation at different time (0-8h) after, IRF-1expression was detected by western blot. With different concentrations of LPS (10ng/ml-1000ng/ml) stimulation for4h, IRF-1expression was detected by western blot. The second part, the RAW264.7cells were divided into four groups:control siRNA group, TLR4siRNA group, LPS+control siRNA group, and LPS+TLR4siRNA group. RAW264.7cells were transfected with siRNA for24h, following LPS (500ng/ml) stimulation for4h, IRF-1and TLR4expression by detected by western blot. The third part, the RAW264.7 cells were divided into four groups:control siRNA group, TRIF siRNA group, LPS+control siRNA group, and LPS+TRIF siRNA group. IRF-1and TRIF expression were detected by western blot. The fourth part, the RAW264.7cells were divided into four groups:control siRNA group, MyD88siRNA group, LPS+control siRNA group, and LPS+MyD88siRNA group. IRF-1expression and MyD88were detected by western blot.
Results LPS induced IRF-1expression in RAW264.7cells occurs in a time-dependent and dose-dependent manner. It gets peak at4h and with500ng/ml LPS stimulation. Western blot analysis showed that, compared with control siRNA transfection group, in TLR4siRNA, TRIF siRNA, MyD88siRNA transfected group, TLR4, TRIF, MyD88expression were significantly decreased. Compared with LPS+control siRNA group, LPS+TLR4siRNA group and LPS+TRIF siRNA group showed significantly decreased IRF-1expression. In the LPS+MyD88siRNA group, compared with LPS+control siRNA group, IRF-1expression did not change.
Conclusion LPS induced IRF-1expression in RAW264.7cells occurs in a time-dependent and dose dependent manner. IRF-1activation in response to LPS stimulation is mediated through the TLR4signaling pathway in a TRIF dependent/MyD88independent manner.
Chapter Four IRF-1/NO regulates apoptosis and autophagy in murine macrophages and its mechanism
Objective To investigate the regulatory function of IRF-1and its downstream NO on apoptosis and autophagy in murine macrophages.
Methods The first part, RAW264.7cells were infected with AdIRF-1and AdlacZ with different MOI (100,200) for48h. IRF-1expression was detected by western blot. RAW264.7cells were infected with MOI of200for48h, pmTOR, mTOR, p-P70S6, P70S6, LC3-Ⅱ/Ⅰ ratio and cleaved caspase-3,8,9were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used. The second part, RAW264.7cells were infected by AdIRF-1and AdlacZ with MOI of200for48h, iNOS expression was analyzed by western blot, NO production in the supernatant was analyzed by Griess Assay. The third part, endogenous NO donor, SNAP, with its different concentrations (100μM-800μM) treatment in RAW264.7cells for16h, pmTOR, mTOR, LC3-Ⅱ/Ⅰ ratio and cleaved caspase-3,8,9were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used. The fourth part, iNOS inhibitor L-NIL (200μM) pretreated RAW264.7cells for1h, following LPS (500ng/ml) stimulation for16h, LC3-Ⅱ/Ⅰ ratio and caspase-3cleavage were detected by western blot. Caspase-3activity detection and LC3B Arrayscan were also used.
Results Compared with AdlacZ, AdIRF-1infection can induce expression of IRF-1in RAW264.7cells. Overexpression of IRF-1can lead to the activation of pmTOR/p-P70S6, decreased LC3-Ⅱ/Ⅰ ratio. AdIRF-1infection inhibits autophagy as demonstrated by LC3B Arrayscan. Meanwhile, overexpression of IRF-1can lead to increased cleavage of caspase-8,9and3by western blot. Caspase-3activity kit confirmed significantly increased caspase-3activity. AdIRF-1infection can also lead to significant activation of iNOS and NO production, which confirmed IRF-1, is the upstream of iNOS/NO. With the different doses of SNAP treatment for16h, western blot showed increased levels of pmTOR and decreased LC3-Ⅱ/Ⅰ ratio. Arrayscan confirmed significantly decreased LC3B with SNAP treatment. The inhibition of autophagy with SNAP treatment was dose-dependent. Meanwhile, western blot showed increased cleavage of caspase-8,9and3with SNAP treatment. Caspase-3activity kit confirmed increased caspase-3activity with SNAP treatment. The effect was dose-dependent. Pretreated RAW264.7cells with iNOS inhibitor L-NIL for1h, and then followed by LPS treatment. Compared with LPS treatment, in the L-NIL pretreatment group, LC3-Ⅱ/Ⅰ ratio was significantly increased and caspase-3cleavage was inhibited. LC3B Arrayscan confirmed increased autophagy and caspase-3kit confirmed decreased caspase-3activity in the L-NIL pretreatment group.
Conclusion IRF-1and its downstream NO inhibited autophagy by activating mTOR/p70S6activity in RAW264.7cells. IRF-1/NO through the activation of cleaved caspase-9,8, and further activation of cleaved caspase-3, involved in both intrinsic and extrinsic apoptotic pathways.
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