NALP3炎症小体参与小鼠肝脏缺血再灌注损伤的作用及其机制

发布时间：2018-01-16 16:03

本文关键词：NALP3炎症小体参与小鼠肝脏缺血再灌注损伤的作用及其机制　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：肝脏缺血再灌注损伤(LIRI)是一个级联炎症反应过程,发病机制复杂,涉及多种因素的参与及相互作用,包括：肝脏微血管紊舌L Kupffer细胞激活、活性氧簇(ROS)增多、炎性细胞因子[如肿瘤坏死因子(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)、白细胞介素6(IL-6)]及高迁移率组蛋白B1(HMGB1)等。炎症小体(inflammasome)是由多种蛋白组成的复合体,能激活炎性caspase复合物,caspase-1能被NALP3及NALP1炎症小体激活。活化的caspase-1能将proIL-1β和proIL-18分别剪切为IL-1β和IL-18,也有文献报道其能将proIL-33剪切为IL-33。NALP3炎症小体能识别许多内源性和外源性危险信号,包括细菌RNA、ATP、尿酸(urid acid, UA)、铝、石棉、硅石等。另外,胞浆内低浓度钾离子(钾离子外流)和ROS升高能激活NALP3炎症小体。近期研究表明,多种以发热、贫血及急性反应蛋白增高为特征的系统性炎症与IL-1β大量释放相关。缺血再灌注过程可释放多种激活炎症小体的启动因子,如钙聚集、钾离子外流、ROS及多种危险信号(如HMGB1、DNA及RNA)等,故推测炎症小体可能参与缺血再灌注损伤。文献已报道：炎症小体激活所释放IL-1β和IL-18在小鼠LIRI中是重要的启动因子,某些针对IL-1β和IL-18的治疗策略显示较好疗效；IL-1β在缺血再灌注损伤中发挥关键作用,IL-1β基因敲除小鼠能明显减缓缺血再灌注损伤；IL-1受体拮抗基因敲除小鼠缺血再灌注损伤明显加重。迄今,有关NALP3炎症小体是否参与肝脏缺血再灌注损伤,以及干预NALP3对小鼠肝脏缺血再灌注损伤的影响,均尚未见报道。本课题建立小鼠LIRI动物模型,并构建针对小鼠NALP3的干扰质粒,观察NALP3炎症小体参与LIRI的作用,并初步探讨其机制。结果发现：NALP3炎症小体在小鼠肝脏缺血再灌注所致无菌性炎症及组织损伤中起重要作用,其机制为：NALP3炎症小体参与IL-1β和IL-18等细胞因子成熟、释放；NALP3炎症小体参与肝脏细胞凋亡及炎性细胞浸润。本课题所获研究成果为临床上探索防治LIRI的策略提供了新的实验依据。一.NALP3炎症小体参与小鼠LIRI 建立雄性C57/BL小鼠LIRI模型,同时设立假手术对照组。 1.小鼠LIR后血清ALT/AST变化及肝脏形态学改变小鼠肝脏缺血1h再灌注6h后收集血清检测谷丙转氨酶(ALT)及谷草转氨酶(AST)变化,同时制作肝脏石蜡切片,HE染色观察形态学改变。结果显示：模型组ALT/AST明显高于假手术组,形态学观察发现模型组出现大量肝细胞变性、凋亡、坏死及炎性细胞浸润。提示小鼠肝脏缺血再灌注损伤模型制作成功。 2.小鼠LIR后不同时间点IL-1β水平分别采集小鼠肝脏缺血1h再灌注1h、3h、6h及24h后血样,ELISA检测IL-1β水平。结果显示：与假手术对照组相比,血清IL-1β在1h后开始升高,6h达高峰,24h后回落至1h水平(p0.05)。 3.小鼠LIR后不同时间点NALP3蛋白表达小鼠肝脏缺血1h再灌注6h、12h及24h后分别取小鼠肝脏组织,免疫印记法检测NALP3蛋白水平。结果发现：与假手术组对照,小鼠缺血1h再灌注后NALP3蛋白开始上调,持续到12h,以6h最为明显(p0.05)。 4.小鼠肝脏缺血再灌注损伤后ROS变化小鼠肝脏缺血1h再灌注6h后分离非实质细胞(NPC), DCF染色,流式细胞术检测ROS含量。结果显示,与假手术对照组相比,缺血lh再灌注6h后ROS明显增高。以上结果提示,NALP3炎症小体参与小鼠LIRI过程。二、NALP3基因干扰载体的构建及鉴定 1.针对小鼠NALP3基因干扰序列的筛选利用siRNA设计软件筛选针对小鼠NALP3基因的3条干扰序列及1条无关序列,分别合成并命名为siRNA1(S1), siRNA2(S2), siRNA3(S3)及scramble siRNA(SS),利用脂质体2000分别转染CHO细胞,Western-blot检测目的蛋白NALP3沉默效果,发现siRNA3沉默效果最佳,scramble siRNA无明显影响。故选择siRNA3序列为NALP3有效干扰序列。 2.特异性pNALP3shRNA干扰载体的构建及鉴定分别将siRNA3特异性干扰序列及scramble siRNA无关序列构建入干扰载体pNALP3shRNA及pshRNANC中,经酶切鉴定及测序验证其正确性。利用小鼠巨噬细胞核转试剂盒分别转染pNALP3shRNA禾(?)pshRNANC至巨噬细胞,设立：nock组对照。荧光实时定量PCR检测NALP3 mRNA水平,Western blot检测其蛋白水平。结果发现,pNALP3shRNA干扰载体能特异性沉默NALP3基因(p0.05),而对其他相关基因(NALP1b、NLRC4等)无影响。提示pNALP3shRNA干扰载体具有沉默特异性。 3. pNALP3shRNA干扰载体对巨噬细胞分泌IL-1β的影响利用小鼠巨噬细胞核转试剂盒转染pNALP3shRNA和pshRNANC至巨噬细胞,设mock组对照。48h后继续给予LPS及ATP刺激,检测上清IL-11β含量。结果发现,转染pNALP3shRNA组IL-1β含量明显降低(p0.05)。三、沉默NALP3基因对小鼠LIRI的影响及其机制 1.体内干扰NALP3基因对小鼠LIRI的保护作用 (1) pNALP3shRNA对小鼠肝脏的沉默效果尾静脉高压注射pNALP3shRNA质粒后48h,制作小鼠缺血1h再灌注6h动脉模型,实时定量PCR、Western blot、免疫组化及流式细胞术检测肝组织NALP3表达。结果表明：与生理盐水及pshRNANC组相比,pNALP3shRNA组NALP3表达明显下降；免疫组化显示NALP3主要表达于肝脏Kupffer细胞和肝窦内皮细胞。流式细胞术检测发现,Kupffer细胞和肝窦状内皮细胞NALP3表达均被抑制。 (2) pNALP3shRNA预处理对小鼠缺血再灌注损伤后肝功能的影响小鼠肝脏缺血1h再灌注6h、12h及24h后收集血清及肝组织制作切片。检测ALT/AST水平及HE染色。结果表明：与生理盐水及pshRNANC对照组相比,pNALP3shRNA组血清ALT/AST.水(?)平明显降低(p0.01),HE染色显示肝细胞变性坏死及炎性细胞浸润明显减少。提示pNALP3shRNA预处理小鼠能明显减轻肝脏缺血再灌注损伤。 2.沉默NALP3基因对小鼠LIRI保护作用的可能机制 (1) pNALP3shRNA预处理对小鼠LIRI后血清IL-1β、IL-18水平的影响小鼠缺血1h再灌注6h后,血清IL-1β和IL-18水平均升高；pNALP3shRNA预处理能明显降低血清IL-1β和IL-18水平(p0.05),、Vestern blot检测发现pNALP3shRNA预处理能降低活化的Caspase-1水平。体外实验证实,pNALP3-shRNA能抑制巨噬细胞分泌IL-1β(p0.05)。 (2) pNALP3shRNA对小鼠血清细胞因子及HMGB1水平的影响与生理盐水及pshRNANC对照组相比,pNALP3shRNA预处理组小鼠缺血1h再灌注6h后血清TNF-α和IL-6水平明显减低,同时肝组织HMGB1表达明显降低(p0.05)。再灌注后1h提取肝脏核蛋白,凝胶迁移率(EMSA)实验检测NF-κB的DNA结合活性,结果显示,pNALP3shRNA组NF-κB活性明显减低(p0.05)。 (3) pNALP3shRNA对小鼠肝细胞凋亡的影响小鼠缺血1h再灌注6h后,切取肝组织,4%多聚甲醛固定,制作石蜡切片,TUNEL法检测肝细胞凋亡。结果发现,与生理盐水组及pshRNANC组对比,pNALP3shRNA预处理能明显减轻小鼠缺血1h再灌注6h后肝细胞凋亡(p0.05)。 (4) pNALP3shRNA对小鼠肝脏巨噬细胞及中性粒细胞浸润的影响小鼠缺血1h再灌注6h后,切取肝组织,4%多聚甲醛固定,制作石蜡切片,免疫组化检测巨噬细胞(F4/80)和中性粒细胞(Gr-1)浸涧。结果发现, pNALP3sh-RNA预处理能明显降低巨噬细胞和中性粒细胞浸润(p0.05)。四、结论 1. NALP3炎症小体参与小鼠LIRI发生、发展。 2. pNALP3shRNA预处理能保护小鼠肝脏缺血再灌注损伤。 3.上述保护作用的可能机制为：抑制炎性因子IL-1β、IL-18、TNF-α,、IL-6及HMGB1释放；抑制NF-κB活性；减少肝细胞凋亡；减少炎性细胞浸润。
[Abstract]:Hepatic ischemia - reperfusion injury ( LIRI ) is a cascade of inflammatory reaction processes , pathogenesis is complex , involves the participation and interaction of various factors , including : the activation of the liver microvessels , the increase of reactive oxygen species ( ROS ) , the inflammatory cytokines , such as tumor necrosis factor ( TNF - 伪 ) , interleukin - 1尾 ( IL - 1尾 ) , interleukin - 18 ( IL - 18 ) , interleukin - 6 ( IL - 6 ) and high - mobility group protein , B1 , and so on . Inflammatory caspase - 1 can be activated by NALP3 and NALP1 . The activated caspase - 1 can cut proIL - 1尾and proIL - 18 into IL - 1尾 and IL - 18 respectively . It is also reported that it is capable of cutting proIL - 1尾and proIL - 18 into IL - 1尾 and IL - 18 . It is also reported that it can identify many endogenous and exogenous risk signals including bacterial RNA , ATP , uric acid ( UA ) , aluminum , asbestos , silica , etc . In addition , low concentration of potassium ( potassium ion efflux ) and ROS increase in cytoplasm can activate NALP3 inflammatory small body . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . IL - 1尾 and IL - 18 play a key role in ischemia - reperfusion injury . In this study , we established an animal model of mouse LIRI , and constructed an interfering plasmid against NALP3 in mice , and observed the role of NALP3 inflammatory small body in LIRI . It was found that NALP3 inflammation was involved in the maturation and release of cytokines such as IL - 1尾 and IL - 18 . The results showed that NALP3 - inflammatory small body was involved in the apoptosis of liver cells and inflammatory cell infiltration . A male C57 / BL mouse LIRI model was established and sham operation control group was established . 1 . Serum ALT / AST changes and hepatic morphological changes after LIR in mice The results showed that ALT / AST in model group was significantly higher than that in sham operation group . The results showed that ALT / AST in model group was significantly higher than that in sham operation group . The results showed that ALT / AST in model group was significantly higher than that in sham operation group . 2 . IL - 1尾 levels at different time points after LIR in mice The levels of IL - 1尾 and IL - 1尾 in liver of mice were measured at 1h , 3h , 6h and 24h after reperfusion . The results showed that the serum IL - 1尾 increased after 1 h , peaked at 6 h , and returned to 1 hour after 24 h ( p < 0.05 ) . 3 . NALP3 protein expression at different time points after LIR in mice The results showed that the NALP3 protein was up - regulated after 1 h of ischemia in mice and 12 h after reperfusion . The results showed that the NALP3 protein was up - regulated after 1 h of ischemia - reperfusion in mice and was the most obvious in 6 h ( p < 0.05 ) . 4 . ROS changes after liver ischemia / reperfusion injury in mice The content of ROS was detected after reperfusion for 6 hours after reperfusion in the liver of the mice , and the ROS content was detected by flow cytometry . The results showed that ROS increased significantly after 6 hours of ischemia - reperfusion compared with the sham - operated control group . The results suggested that NALP3 was involved in the process of LIRI in mice . The construction and identification of NALP3 gene interference vector 1 . Screening of Mouse NALP3 Gene Interference Sequence Three interfering sequences and 1 unrelated sequences of NALP3 gene of mouse were screened by siRNA design software . siRNA1 ( S1 ) , siRNA2 ( S2 ) , siRNA3 ( S3 ) and siRNA ( SS ) were synthesized and named respectively . Construction and identification of specific pNALP3shRNA interference vector pNALP3shRNA and pshRNANC were transfected into pNALP3shRNA and pshRNANC respectively . The results showed that pNALP3shRNA interfering vector could specifically silence NALP3 mRNA level and Western blot was used to detect NALP3 mRNA level . 3 . Effect of pNALP3shRNA interfering vector on IL - 1尾 secretion in macrophages The results showed that the content of IL - 1尾 in pNALP3shRNA group was significantly lower than that of pNALP3shRNA and pshRNANC to macrophages ( p < 0.05 ) . The effect of silencing NALP3 gene on LIRI in mice and its mechanism were found . 1 . Protective effect of NALP3 gene on LIRI in mice ( 1 ) Effect of pNALP3shRNA on Mouse Liver The expression of NALP3 in liver tissue was detected by RT - PCR , Western blot , immunohistochemistry and flow cytometry . The results showed that the expression of NALP3 in the pNALP3shRNA group was significantly decreased compared with normal saline and pshRNANC group . The expression of NALP3 was inhibited by flow cytometry . The expression of NALP3 was inhibited by flow cytometry . ( 2 ) Effect of pretreatment of pNALP3shRNA on liver function after ischemia / reperfusion injury in mice The levels of ALT / AST and water ( ? ) were significantly decreased in the pNALP3shRNA group ( P0.01 ) . The results showed that the serum ALT / AST and water ( ? ) level of pNALP3shRNA group were significantly decreased ( P0.01 ) compared with normal saline and pshRNANC control group . 2 . Possible mechanism of silencing NALP3 gene on the protection of LIRI in mice ( 1 ) Effect of pretreatment of pNALP3shRNA on serum levels of IL - 1尾 and IL - 18 after LIRI in mice The levels of IL - 1尾 and IL - 18 in serum were increased after 1 hour reperfusion in mice . The pretreatment of pNALP3shRNA could significantly reduce the levels of IL - 1尾 and IL - 18 in serum ( P < 0.05 ) . The results showed that pNALP3 shRNA could inhibit the secretion of IL - 1尾 ( p . 05 ) . ( 2 ) Effect of pNALP3shRNA on serum cytokines and serum levels in mice Compared with normal saline and pshRNANC control group , the levels of serum TNF - 伪 and IL - 6 were significantly decreased after reperfusion for 6 h after reperfusion in the pretreatment group of pNALP3shRNA . The DNA binding activity of NF - 魏B was detected at 1h after reperfusion . The results showed that the activity of NF - 魏B in pNALP3shRNA group was significantly lower ( p < 0.05 ) . ( 3 ) Effect of pNALP3shRNA on Apoptosis of Mouse Hepatocytes The results showed that the pretreatment of pNALP3shRNA could significantly reduce the apoptosis of hepatocytes after 1 hour reperfusion ( p . 05 ) . ( 4 ) Effect of pNALP3shRNA on the infiltration of macrophages and neutrophils in mouse liver The results showed that pNALP3sh - RNA pretreatment significantly decreased the infiltration of macrophages and neutrophils ( p . 05 ) . Conclusion : The results showed that pNALP3sh - RNA pretreatment could significantly reduce the infiltration of macrophages and neutrophils ( p . 05 ) . 1 . NALP3 inflammation was involved in the development and development of LIRI in mice . 2 . The pretreatment of pNALP3shRNA can protect the liver ischemia - reperfusion injury in mice . 3 . The possible mechanism of the above protection is to inhibit the release of inflammatory cytokines such as IL - 1尾 , IL - 18 , TNF - 伪 , IL - 6 , and IL - 6 , inhibit NF - 魏B activity , reduce hepatocellular apoptosis , and reduce inflammatory cell infiltration .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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