【作者】 邢微微；

【导师】 于晓妉； 徐东刚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 细胞生物学， 2011， 博士

【摘要】 白介素-22 (Interleukin-22, IL-22)是IL-10细胞因子家族成员之一,两者具有22%的同源性。不同于传统意义上的“白介素”,IL-22是一种新型的免疫介质,它由激活的Th细胞(Th1,Th17,Th22细胞)以及天然免疫细胞(NK细胞,ILC细胞)分泌产生,但却并不作用于这些细胞。IL-22主要作用于少数组织器官(皮肤,肝脏,胰腺,小肠,肺脏等)的实质细胞,发挥促炎,天然免疫调节,组织保护及修复等功能。在表皮炎症如银屑病中,IL-22作为一种重要的炎性介质,在其病理生理中扮演关键角色。而在Crohn’s病,溃疡性结肠炎以及自身免疫性心肌炎等疾病中IL-22则发挥保护作用。目前认为IL-22促炎或组织修复保护的双重功能可能与具体的组织类型及局部的细胞因子环境密切相关。在肝脏,IL-22似乎是肝细胞的存活因子,多种毒性物质诱导的肝损伤模型以及肝部分切除模型均证实IL-22可以促进肝细胞的存活与再生。然而目前对于IL-22的肝保护作用机制了解甚少。我们对IL-22的结构和功能及其在常见肝病中的作用和机制进行了深入研究。在IL-22的结构和功能研究方面,在已知IL-22晶体结构基础上,利用Homology程序(Insight II 2000软件包, Octane2图形工作站)从理论上模拟IL-22的胞外区三维结构,通过表观静电分析,可及性表面积分析并结合细胞因子结构特征对IL-22可能的受体结合区进行分析,将潜在的受体结合区定位于LoopAB N末端的67~72位氨基酸。我们通过点突变构建了一系列IL-22突变体,通过分析其促细胞增殖活性及其对STAT3转录活性的影响,发现该区域氨基酸位点变化可影响其生物学功能。综合计算机模建与生物学功能分析的结果,我们确定该区域为IL-22潜在的受体结合位点。多种毒性物质诱导的实验性肝损伤模型均证实IL-22的肝细胞保护作用,然而不同肝脏疾病具有不同的病理生理特点,IL-22在其它肝病中作用如何?其可能的机制是什么?对这些问题的深入研究有助于拓展对IL-22功能及作用机制的认识,为筛选新的肝病治疗药物提供思路。酒精滥用是一个世界性的问题,全球每年约有3.2%死亡病例与酒精相关,酒精性肝病是酒精滥用最主要的临床表现,在我国其发病率仅次于病毒性肝炎,造成极大的医疗负担。尽管针对酒精性肝病的研究已进行数十年,但至今尚无有效的治疗药物。为评价IL-22在酒精性肝病中的作用,我们分别建立了大鼠慢性酒精性肝炎模型及小鼠急性酒精性肝损伤模型。在前期研究的基础上,我们采用酒精,高脂及碳酰铁联合诱导建立大鼠酒精性肝炎模型,结果表明随着造模时间的延长,大鼠血清转氨酶水平进行性升高,组织病理损伤不断加重,32周时出现肝细胞气球样变,大量中性粒细胞浸润以及Mallory小体形成等典型的酒精性肝炎病理学指征,提示模型建立成功。在此基础上我们利用IL-22对其进行治疗评价,结果表明IL-22可以降低血清转氨酶,总胆固醇及甘油三酯的升高,明显改善肝组织病理学变化。在整个治疗期间,IL-22处理并未引起大鼠一般状态及体重发生明显变化。在利用慢性酒精性肝炎模型对IL-22作用进行评价后,我们又建立小鼠急性酒精性肝损伤模型,进一步分析IL-22在酒精诱导的肝损伤中的作用。结果显示,急性酒精灌注可以诱导血清转氨酶(ALT,AST,GGT)升高,囊泡状肝脂变的出现,肝细胞凋亡增加,肝组织氧化应激及TNF-α表达升高,而IL-22治疗可明显抑制上述不利变化。暴发性肝炎是由于病毒性肝炎,酒精或其他肝毒性物质诱发的一种临床危重症,由于缺乏有效的预防及治疗手段,预后极差。GalN/LPS诱导的小鼠急性致死性肝损伤模型是临床暴发性肝炎的经典动物模型,我们利用这一模型对IL-22在其中的作用及可能的机制做进一步研究。结果表明,IL-22可明显降低GalN/LPS诱导的暴发性肝炎的死亡率,明显改善肝组织病理损伤,降低血清转氨酶的升高,减轻肝组织的氧化应激,抑制肝细胞的凋亡,降低血清炎性因子TNF-α的升高。机制研究表明IL-22可以激活肝组织STAT3的磷酸化,诱导抗凋亡蛋白Bcl-xL,抗氧化因子HO-1及Ref-1的表达上调。上述结果提示IL-22可能通过抑制炎性因子的升高,诱导抗凋亡及抗氧化蛋白的表达来发挥肝保护作用。为深入研究IL-22作用机制,我们又利用GalN/LPS诱导的暴发性肝炎的体外模型,即TNF/ActD诱导的HL7702细胞凋亡模型对IL-22的肝保护作用机制进一步分析,结果表明IL-22可以抑制TNF/ActD诱导的HL7702细胞的凋亡,其可能机制是IL-22通过STAT3信号路径,诱导抗凋亡蛋白Bcl-xL及HO-1的表达而发挥其保护作用。综上所述,我们利用计算机同源模建分析预测了IL-22潜在的受体结合区,对上述区域进行突变,构建了系列IL-22突变体。通过检测IL-22系列突变体的促增殖活性及STAT3转录活性确定了这一区域为IL-22受体结合区。利用慢性酒精性肝炎模型,急性酒精性肝损伤模型以及暴发性肝炎模型对IL-22在上述肝病中的作用进行了研究,结果表明IL-22可以明显减轻上述肝病模型的病理损伤,有效改善肝功,其保护机制可能与抑制炎性因子的表达,抗凋亡及抗氧化作用相关。鉴于IL-22明确的肝保护作用及其靶点的局限性,IL-22具有成为肝病治疗药物的潜力。更多还原

【Abstract】 Interleukin-22 (IL-22) is a member of the Interleukin-10 (IL-10) cytokine family and its sequence has 22% identity with human IL-10. However, unlike traditional“interleukin”, IL-22 seems to be a novel type of immune mediator which is produced by activated Th22, Th1 and Th17 cells as well as natural killer cells and ILC cells. IL-22 although is produced by the above immune cells does not affect these cells. The main target of IL-22 is the parenchymal cells on several tissues including skin, liver pancreas and small intestine, where it favors the antimicrobial defense, regeneration, and protection against damage. In the epidermal inflammation such as psoriasis, IL-22 was an important pro-inflammatory mediator which played a critical role in the development of diseases. In contrast, IL-22 was demonstrated to be a protective factor in the Crohn’s disease, ulcerative colitis and experimental autoimmune myocarditis (EAM). Current studies suggested that the dual nature of IL-22 likely depended on the inflammatory context, which included the kind of organ and of the presence of other cytokines. In the liver, IL-22 seems to be a survival factor for hepatocyte. Several liver disease models including toxins-induced hepatitis and hepatectomy model all demonstrated that IL-22 could increase the survival and regeneration of hepatocyte. However, the hepatoprotetive mechanisms of IL-22 still remain obscure.In this study, relationship between structure and function of IL-22 as well as its roles in the common liver diseases were thoroughly demonstrated. Based on the known crystal structure of IL-22, we used Homology Program (Insight II 2000 Package, Octane2 Graphics workstations) to simulate the extracellular three-dimensional structure of IL-22. Through analyzing the surface electricity and accessible surface area of above-mentioned domain and structural characteristics of cytokines, we anticipated that the potential receptor binding domain of IL-22 located at the N terminal of loopAB (Asn67–Val72). We constructed a series of IL-22 mutants by point mutation towards the potential receptor binding domain. By analyzing the promoting cell proliferation activity and STAT3 transcriptional activity of these IL-22 mutants, we found that the change of the region affected its functions. Combined with computer modeling and biological functions analysis, we speculated that the region might be the potential receptor binding domain of IL-22.Several toxin-induced experimental hepatitis models have demonstrated IL-22 to be a protective factor for hepatocyte. However, since various liver diseases have different pathophysiological features, which role will IL-22 play in other liver diseases and what is its corresponding mechanism? Depth study on these issues will help to expand the knowledge on functions and possible mechanisms of IL-22.Alcohol abuse is a global problem, each year about 3.2% of global deaths is related to alcohol. Alcoholic liver disease (ALD) is one of the major medical complications of alcohol abuse, and its incidence in our country is just below the viral hepatitis. Although the studies on ALD have been conducted for several decades, there are still no effective therapeutic drugs. We utilized alcoholic hepatitis in rats and acute alcohol-induced liver injury in mice to investigate the role of IL-22 in ALD respectively. Alcohol, fat diet and carbonyl iron were jointly applied to induce alcoholic hepatitis in rat. The results showed that multiple factors lead to continuously increased serum transaminase levels and serious histopathological changes. On 32 weeks, typical indications for alcoholic hepatitis including ballooned hepatocytes, neutrophils infiltration and formation of Mallory bodies were observed. Subsequently, this model was used to evaluate the hepatoprotective function of IL-22, and we found that IL-22 could significantly lower serum transaminase activities, total cholesterol and triglyceride levels and improve pathological changes of liver tissue. Moreover, during the course of treatment, we did not observe significant changes of general condition and body weight in rats. In addition, the results from acute alcohol-induced liver injury in mice indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-αproduction. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration.Fulminant hepatic failure (FHF), induced by bacteria, viral hepatitis, alcohol and other hepatotoxic drugs, is a dramatic clinical syndrome, which remains an extremely poor prognosis and high mortality due to lack of effective preventives and therapies. D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced acute lethal liver injury in mice has been widely used as experimental animal model to investigate the underlying mechanisms of clinical fulminant hepatic failure. This study examined the hepatoprotective effects of IL-22 against FHF and explored its potential mechanisms. Results showed that IL-22 pretreatment remarkably decreased the death rate, suppressed the elevation of serum ALT levels and improved histopathologic changes. In addition, IL-22 could also lessen hepatic oxidative stress, inhibit liver apoptosis and decrease the serum TNF-αlevel. And the hepatoprotective mechanisms may be related to the hepatic STAT3 activation and subsequent induction of Bcl-xl, HO-1 and Ref-1.These data indicated the protective effect of IL-22 could be due to attenuation of oxidative stress, anti-inflammation and inhibition of hepatocyte apoptosis. To further investigate the mechanisms behind the hepatoprotective effect of IL-22, we analyzed the effects of IL-22 on the apoptosis induced by TNF/ActD in HL7702 cell. The results showed that IL-22 could inhibit the apoptosis of HL7702 cells induced by TNF/ActD and the mechanism may be related to the phosphorylation of STAT3 and expression of Bcl-xL and HO-1.In summary, we used a computer homology modeling analysis to anticipate the potential receptor binding domain of IL-22. By analyzing the promoting cell proliferation activity and STAT3 transcriptional activity of IL-22 mutants, we found that the region might be the potential receptor binding domain of IL-22. In addition, we used three different models of liver diseases to investigate the hepatoprotective effects of IL-22 and found that IL-22 could protect against the above liver injury. And its hepatoprotective effects may be related to anti-inflammation, attenuation of oxidative stress and inhibition of hepatocyte apoptosis. Due to its hepatoprotective functions and limited targets, IL-22 may be an ideal therapeutic agent in the treatment of liver damage.更多还原