摘要
1.背景与目的
急性肺损伤(acute lung injury, ALI)/急性呼吸窘迫综合症(acute respiratory distress syndrome, ARDS)是由炎症失控而引起的临床危急重症,目前尚缺乏有效的特异性治疗。Toll样受体(Toll like receptors, TLRs)是固有免疫系统中的主要病原模式受体之一,TLR信号通路的过度激活是导致炎症反应爆发失控的重要机制。单免疫球蛋白白介素-1受体相关蛋白(single immunoglobin IL-1 receptor related protein, SIGIRR)是新近发现的Toll样受体/白介素-1受体(Toll like receptor/Interleukin-1 receptor, TIR)超家族成员,又称为TLR-IL-1R8 (TIR8),是TLR信号通路中的负性调控分子。但目前有关SIGIRR在急性肺损伤中的确切作用尚未见报道。SIGIRR的负性调控机制可能与其“俘获”TLR-IL-1R (TIR)信号通路中的关键组分而抑制TIR信号的转导有关,但具体的分子机制仍有待进一步阐明。SIGIRR的胞内段TIR域是其发挥功能的重要结构,目前已证实,SIGIRR能通过胞内段TIR域与TIR信号通路中IL-1R或TLR4受体复合物发生相互作用。酵母双杂交系统是研究蛋白质间相互作用的高效的新技术,通过以已知功能的蛋白质基因筛选双杂交cDNA文库,可发现与已知蛋白分子有相互作用的新分子并推测新分子可能的功能。鉴于此,本课题拟通过构建腺病毒表达载体上调SIGIRR在肺内的表达,在内毒素急性肺损伤小鼠模型中确定SIGIRR的作用;通过酵母双杂交系统,在人肺cDNA文库中筛选与SIGIRR有相互作用的蛋白并对有意义的阳性克隆进行初步研究;以期阐明SIGIRR在ALI中的确切作用及SIGIRR负性调控TIR信号的机制,并为ALI/ARDS的防治提供新的作用靶点。
2.方法
(1)重组SIGIRR基因腺病毒表达载体的构建与鉴定
以pCMV-SPORT6-mSIGIRR为模板保真扩增小鼠源性murine SIGIRR (mSIGIRR)基因,经过酶切和连接反应构建重组质粒pDC316-mSIGIRR,并行酶切和测序鉴定;采用AdMaxTM包装系统,将穿梭质粒pDC316-mSIGIRR和骨架质粒pBHGlox_E1,3Cre共转入包装293细胞中,构建携带mSIGIRR基因的重组腺病毒载体Ad.mSIGIRR;通过PCR扩增、病毒颗粒吸光度值测定及TCID50的方法,对重组腺病毒Ad.mSIGIRR的质量和滴度进行鉴定;建立Ad.mSIGIRR体内外的感染模型,利用Western blot、免疫组化和肺组织切片HE染色的方法,对Ad.mSIGIRR在真核细胞与肺内的表达效力及对宿主肺部病理改变的影响进行检测。
(2) SIGIRR过表达对小鼠内毒素性急性肺损伤的保护作用
用经鼻滴注的方法,以4×107 PFU/只的剂量建立小鼠重组腺病毒Ad.mSIGIRR感染模型,同时用空病毒载体Ad.V作为对照。感染48 h后,以LPS (15 mg/kg)腹腔注射复制内毒素性急性肺损伤模型。在LPS刺激后的各个时间节点,用RT-PCR、Western blot和免疫组化的方法比较Ad.mSIGIRR组和Ad.V组之间SIGIRR的表达情况;同时对比两组间的肺组织病理改变、肺损伤评分情况以及模型小鼠的7天存活率的变化;用ELISA法和硝酸还原酶法分别检测两组肺内和血清中TNF-α及肺内NO含量变化;用邻连茴香胺-比色法检测两组肺内MPO活性变化;用EMSA和ELISA两种方法比较两组间肺内核转录因子NFκB的活性。
(3) SIGIRR相互作用蛋白的筛选与初步研究
以pReceiver-LV19-SIGIRR质粒为模板保真扩增获得人SIGIRR胞内区(480-1230 bp)基因片段,经过酶切和连接反应构建重组诱饵质粒pSos-SIGIRR,并行酶切和测序鉴定;按照stratagene公司CytoTrap yeast two-hybrid体系的说明行温度敏感型酵母菌的表型与回复突变鉴定以及诱饵质粒自激活检测后,将诱饵质粒pSos-SIGIRR与人肺cDNA文库质粒,共转化感受态酵母细胞筛选鉴定与SIGIRR胞内区相互作用的蛋白,免疫共沉淀的方法进一步验证筛选结果;应用生物信息学的方法,分析比对阳性克隆序列,同时查阅相关文献选取有意义的阳性克隆为对象进行后继研究;用RT-PCR的方法检测paralemmin-3在人肺泡上皮细胞系(A549细胞)中有无表达;用Realtime-PCR的方法比较A549细胞中,LPS刺激后SIGIRR和paralemmin-3的表达变化;用RNA干扰的方法下调A549细胞中paralemmin-3的表达,观察paralemmin-3对LPS诱导的A549细胞炎症因子释放的影响。
3.结果
(1)成功构建了携带小鼠源性SIGIRR基因的重组腺病毒表达载体Ad.mSIGIRR,病毒颗粒数和感染滴度分别为:4.65×1011 VP/ mL和4×109 PFU/mL,符合实验要求;Western blot和免疫组化的方法分别检测到mSIGIRR在体内外的高效表达,且经鼻滴注感染宿主后,不会引起宿主肺组织发生病理变化。
(2) SIGIRR组成性表达于正常小鼠肺组织,主要分布于肺上皮细胞。LPS腹腔注射后,小鼠肺内SIGIRR的表达下调,但与正常小鼠及Ad.V对照组相比,Ad.mSIGIRR组小鼠肺内SIGIRR表达显著增加(P < 0.05);与Ad.V对照组相比,Ad.mSIGIRR组小鼠肺组织的病变减轻、炎症介质TNF-α和NO含量减少、肺内MPO和NFκB活性降低、肺损伤小鼠7天存活率显著提高(P < 0.05)。
(3)通过酵母双杂交实验,共筛选到4个与SIGIRR胞内区之间存在可能相互作用的阳性克隆,分别是:Homo sapiens mitochondrion, complete genome;Homo sapiens cyclin H (CCNH), mRNA;Homo sapiens paralemmin-3 (PALM3), mRNA;Homo sapiens v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS), mRNA;其中Homo sapiens mitochondrion, complete genome为非编码序列,KRAS为Cytotrap酵母双杂交系统中的背景性假阳性蛋白,CCNH为细胞周期素H定位于细胞核,与SIGIRR缺少共同物理空间定位。而PALM3为paralemmin蛋白家族成员,定位于细胞膜,与SIGIRR有共同物理空间定位,因此选择PALM3作为后继研究的对象;免疫共沉淀结果进一步验证了PALM3与SIGIRR间的相互作用;RT-PCR检测证实PALM3在A549细胞中有表达,LPS刺激后,SIGIRR的表达下调而PALM3的表达上调;沉默PALM3的表达,减少LPS诱导的A549细胞炎性细胞因子TNF-α、IL-6、IL-1β的释放(P < 0.05)。
4.结论
(1) LPS刺激后,小鼠肺内SIGIRR的表达下调,增强小鼠肺内SIGIRR的表达能通过负性调控LPS-TLR4信号的转导而对内毒素性急性肺损伤起保护作用;
(2)通过酵母双杂交及免疫共沉淀实验,我们证实PALM3是SIGIRR新的相互作用分子,LPS刺激上调A549细胞中PALM3的表达,SIGIRR与PALM3之间的相互作用可能是SIGIRR负性调控TLRs信号通路的机制之一。
1. Background and objective
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical critical diseases. At the present, there are not specific therapies for ALI/ARDS. Toll-like receptors (TLRs) are one of important pathogen pattern recognition receptors in innate immune system. The excessive activation of TLR-signaling plays a key role in uncontrolled inflammatory response. Single immunoglobin IL-1 receptor related protein (SIGIRR). SIGIRR, also named Toll-like receptor/Interleukin-1 receptor 8 (TIR8), is one of recently discovered TIR superfamily members and is a negative regulator for TLR signaling. However, the precise and potential effect of SIGIRR on lung injury remains unclear. Trapping the crucial components of Toll-like receptor/Interleukin-1 receptor (TIR) signaling by SIGIRR may be one of possible mechanisms of SIGIRR negative regulation, but the detailed molecular mechanisms of SIGIRR negative regulation require further investigations. It has been proved that the intracellular TIR domain of SIGIRR can interact with IL-1R/TLR4 complexes in the TIR signaling. Yeast two hybrid system is an effective method for studying the interaction between two proteins. Using yeast two-hybrid screening, we can find novel proteins that interacted with the functionally known protein and speculate the possible roles of these novel proteins. With these in mind, the present study was performed to investigate the effect of enhanced SIGIRR expression on LPS-induced ALI in mice by constructing recombinant adenoviral vector that carries the mSIGIRR, to further identify intracellular protein that interacts with SIGIRR in human lung cDNA library by using a yeast two-hybrid screen and then to study the novel binding proteins of SIGIRR superficially. Our expected results will provide direct and precise evidences about the role of SIGIRR in LPS-induced ALI/ARDS, a novel insight into the molecular mechanisms of
SIGIRR negative regulation, and new targets for the prevention and treatment of ALI/ARDS.
2. Methods
(1) Construction and identification of recombinant adenoviral vector (Ad.mSIGIRR) An adenovirus was generated that contained a single open reading frame encoding a murine SIGIRR (mSIGIRR). First, cDNA coding for mSIGIRR was excised from pCMV-SPORT6-mSIGIRR and ligated into the adenoviral shuttle plasmid pDC316 to generate the plasmid pDC316-mSIGIRR. And the pDC316-mSIGIRR was identified by DNA sequencing and dual-site endonuclease digestion. Recombinant viruses were then generated using HEK293 packaging cells co-transfected with pDC316-mSIGIRR and a plasmid containing cDNA for adenoviral proteins (pBHGlox) via the AdMax system. The quality and titer of Ad.mSIGIRR were identified by polymerase chain reaction (PCR), determination of the viral particle absorbance, and means of 50% tissue culture infectious dose, respectively. Ad.mSIGIRR was used to infect A549 cells and to establish respiratory adenovirus infected mice models. Then the expression activity of Ad.mSIGIRR in vitro and in vivo was detected by Western blot and immunohistochemistry. Meanwhile, the pulmonary histological changes of adenovirus infected mice models were determined by routine pathological examination.
(2) Enhanced expression of SIGIRR ameliorates LPS-induced ALI in mice
BALB/c mice were lightly anesthetized by ether inhalation, and inoculated intranasally with 4×10~7 PFU (in 50μL) of Ad.mSIGIRR (group Ad.mSIGIRR) or Ad.V (group Ad.V). Forty-eight hours after intranasal administration of viruses, LPS at 15 mg/kg was injected into the peritoneal cavity of conscious mice to establish ALI mouse model. The mice were sacrificed at 0, 2, 6, 12, 24, and 48 h after LPS challenge. Blood and lung samples were harvested aseptically for subsequent experiments. The expression of SIGIRR was determined by RT-PCR, Western blot and immunohistochemistry analysis. Meanwhile, the differences of pulmonary histological changes and 7-day survival rate between two groups were compared. The levels of TNF-αin lung and serum, the concentration of NO in lung, and the activity of MPO and NFκB were also examined by using corresponding methods.
(3) The screening and preliminary study of SIGIRR interacting proteins
Human SIGIRR (480–1230bp) cDNA encoding the intracellular domain of SIGIRR was amplified from pReceiver-LV19-SIGIRR by PCR and ligated into pSos vector to generate the bait plasmid pSos-SIGIRR. And the pSos-SIGIRR was identified by DNA sequencing and dual-site endonuclease digestion. After the mutational revertant and phenotypic verification of cdc25H yeast cells and the transcriptional activation of bait plasmid were performed, the bait and the cDNA library were co-transformed into yeast cells. Yeast cultures and two-hybrid procedures were carried out according to the procedure of the protocol of CytoTrap yeast two-hybrid system (Stratagene). The screening results were analyzed by bioinformatics method and further confirmed by co-immunoprecipitation. In consideration of the subcellular localization, one clone encoding the partial cDNA fragment of paralemmin-3 (PALM3) was chosen as the object of subsequent study. The PALM3 expression in a human alveolar epithelial cell line (A549 cells) was detected by RT-PCR. The expression patterns of SIGIRR and PALM3 were compared by means of Real-time RT-PCR. To test whether PALM3 has a role in the LPS-mediated inflammatory cytokine gene expression, the expression of PALM3 expression in A549 cells was knocked down by siRNA. Then the levels of cytokines (TNF-α, IL-6 and IL-1β) in cultured supernatants were examined by ELISA.
3. Results
(1) The recombinant adenovirus vector carries that murine SIGIRR, Ad.mSIGIRR, was constructed successfully. The titer and the number of viral particle of Ad.mSIGIRR were 4×10~~9 PFU/m and 4.65×10~(11) VP/ mL, respectively, which met the requirement of subsequent experiments. The adenovirus delivery system had no effect on pulmonary inflammation and histological changes.
(2) SIGIRR was constitutively expressed in normal murine lung and was mainly detected in the pulmonary epithelial cells. After LPS stimulation, SIGIRR protein and mRNA levels were downregulated. Compared with those in group Ad.V, SIGIRR mRNA and protein expression were enhanced in group Ad.mSIGIRR (P < 0.05). Enhanced expression of SIGIRR significantly decreased the levels of TNF-αand NO, inhibited the activation of NF-κB, attenuated pulmonary pathological changes and neutrophil infiltration, and improved the outcome of ALI (P < 0.05).
(3) Using yeast two-hybrid screening, four clones were identified as the potential interaction protein of SIGIRR. They were Homo sapiens mitochondrion, complete genome; Homo sapiens cyclin H (CCNH), mRNA; Homo sapiens paralemmin-3 (PALM3), mRNA; and Homo sapiens v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), mRNA, respectively. Among these positive clones, Homo sapiens mitochondrion, complete genome was noncoding sequence. KRAS was a false positive clone in the background of Cytotrap Y2H system. The protein expression of CCNH located in cellular nucleus, while that of PALM3 located in cellular membrane. PALM3 was selected for the subsequent study. The interaction between PALM3 and SIGIRR was confirmed by co-immunoprecipitation. PALM3 was expressed in A549 cells. The expression of SIGIRR was downregulated but that of PALM3 was upregulated by LPS-stimulation in A549 cells. Silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation (P < 0.05).
4. Conclusion
(1) The expression of SIGIRR is downregulated post LPS-challenge. SIGIRR overexpression has a protective effect on LPS-induced ALI in mice by regulating the LPS-TLR4 signaling.
(2) PALM3 should be a novel interacting partner of SIGIRR. The PALM3 mRNA expression is upregulated by LPS-stimulation in a human alveolar epithelial cell line (A549 cells). The interaction between SIGIRR and PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR.
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