【作者】 李红岩；

【导师】 高润平；

【作者基本信息】 吉林大学 ， 内科学， 2019， 博士

【摘要】 研究背景及目的:酒精性慢性胰腺炎(ACP)的发病率逐年升高。乙醇可激活PSC被认为是ACP形成的独立危险因素,然而5-10%的长期嗜酒者发生胰腺纤维化。内毒素脂多糖(LPS)是ACP发生的重要触发因素之一,体内、外研究证明LPS可引起PSC激活和合成Col1增加。TGF-β1是重要的致纤维化因子,通过PSC自分泌和旁分泌调节自身激活过程,增加α-SMA和Col1表达。近年来的研究发现IL-6在许多疾病纤维化的发生和发展过程中发挥重要作用。IL-6通过其受体(IL-6R)激活JAK2/STAT3信号通路可促进肝脏、肺、肾脏等多器官纤维化,但是IL-6及JAK2/STAT3通路在ACP胰腺纤维化的作用和机制尚不十分明确。本研究旨在通过人和大鼠整体水平与巨噬细胞(Mφ)和胰腺星状细胞(PSC)细胞水平研究IL-6/JAK2/STAT3通路在ACP胰腺纤维化发生学的作用与机制。材料和方法:将33只120±20 g雄性SD大鼠随机分为3组:(1)对照饲料喂养组,(2)Lieber-DeCarli酒精饲料喂养组,(3)对照饲料喂养+LPS反复注射组。LPS组于8,9,10周末每周经尾静脉注射LPS(3mg/kg)1次,其它两组尾静脉注射生理盐水1 ml。10周末接受LPS或生理盐水24 h后3组大鼠被异氟烷麻醉,留取胰腺组织块。其中部分组织块放入10%中性福尔马林液固定,用于制备石蜡切片,部分组织块放入液氮中保存。采用本研究室经RSV启动子/增强子驱动SV T抗原建立的永生化人胰腺星状细胞系(HP-1细胞)进行体外研究。采用淋巴细胞分离液分离人末梢血单核细胞,经粒细胞巨噬细胞集落刺激因子(GM-CSF)诱导培养6天获得巨噬细胞(Mφ)用于ALC和LPS体外造模。采用免疫组织化学检测人胰腺组织IL-6表达,免疫双染用于识别PSC表达IL-6R,免疫细胞化学法分别检测Mφs和HP-1细胞IL-6和IL-6R表达。ELISA法检测大鼠胰腺组织、人血清Mφs和HP-1细胞IL-6、TGF-β1、α-SMA和Col1α1产生。采用RT-qPCR检测HP-1细胞IL-6、TGF-β1、α-SMA和Col1α1 mR NA转录水平。Western blot法检测HP-1细胞p-JAK2、JAK2、p-STAT3、STAT3、p-Smad2、Smad2、p-Smad3、Smad3的含量。结果:在人ACP胰腺免疫组织学研究发现:ACP患者与健康对照者相比胰腺组织IL-6及其受体表达上调,IL-6+Mφs和IL-6+PSCs与IL-6R+Mφs和IL-6R+PSCs数量均明显增加(各组P<0.001)。进一步研究发现:不伴有或伴有内毒素血症ACP患者血清IL-6,TGF-β1和Col1α1水平明显升高(各组P<0.001),升高的IL-6与TGF-β1或Col1α1水平呈正相关(r=0.6609,P<0.05;r=0.6293,P<0.05;r=0.6422,P<0.01;r=0.5006,P<0.05)。在乙醇或LPS制备的大鼠胰腺炎模型研究发现:乙醇和LPS还可使大鼠胰腺组织IL-6,TGF-β1和Col1α1合成增加(各组P<0.001),增加的IL-6与TGF-β1或Col1a1含量呈正相关(r=0.7369,P<0.01;r=0.6445,P<0.05;r=0.7380,P<0.01;r=0.6344,P<0.05)。体外研究发现:乙醇或LPS均可诱导人Mφs和HP-1细胞IL-6R表达上调与IL-6和TGF-β1分泌增加(各组P<0.001)。外源性IL-6可诱导HP-1细胞分泌TGF-β1,而TGF-β1也可诱导HP-1细胞分泌IL-6。IL-6中和抗体可部分抑制乙醇或LPS诱导HP-1细胞α-SMA和Col1α1 mR NA转录和蛋白合成(各组P<0.001),提示IL-6是ALC和LPS诱导HP-1细胞合成α-SMA和Col1α1的一种途径。进一步研究发现IL-6经IL-6R/JAK2/STAT3通路诱导TGF-β1合成。TGF-β1ab可明显抑制IL-6诱导HP-1细胞产生α-SMA和Col1α1,推测TGF-β1是IL-6诱导HP-1细胞合成α-SMA和Col1a1的中间介导者。IL-6能诱导HP-1细胞延迟出现Smad2/3磷酸化,而小干扰RNA si-Smad2能有效抑制IL-6诱导的α-SMA mR NA表达,si-Smad3能有效抑制IL-6诱导的Col1α1 mR NA表达(两者P<0.001)。结论:IL-6是ACP胰腺纤维化形成的重要因素之一。IL-6经IL-6R/JAK2/STAT3通路诱导TGF-β1合成,通过上调TGF-β1/Smads通路促进PSC激活和Col1合成,本研究提示IL-6可能作为防治乙醇相关胰腺纤维化的一个靶点。更多还原

【Abstract】 Background and aims: Morbidity of alcoholic chronic panreatitis(ACP)is getting higher and higher.Although alcohol is considered as an independent risk factor of ACP,only 5–10% of chronic alcoholics suffered from pancreatic fibrosis.Endotoxin lipopolysaccharide(LPS)is one of the most important triggers of ACP.In vivo and in vitro studies have shown that LPS could induce activation of PSC and increase synthesis of Collagen type I(Col1).TGF-β1 as an important pro-fibrotic factor that is produced in an autocrine and paracrine fashion,could increase the expression of α-SMA and Col1 in PSC.Recent studies have found that interleukin-6(IL-6)plays an important role in the fibrogenesis of many diseases.IL-6 activates the JAK2/STAT3 signaling pathway through IL-6 receptor(IL-6R),which promotes the fibrogenesis of liver,lung and kidney.So far the effect of IL-6/JAK2/STAT3 pathway on the fibrogenesis of ACP has not been fully clarified.The purpose of this study was to elucidate the role and mechanism of IL-6 and its signaling pathway on the fibrogenesis in ACP patients and alcohol-fed rat model as well as in vitro PSC and macrophage(Mφ)models.Methods: Thirty-three male SD rats(120 ± 20g)were randomly divided into 3 groups: control diet group,Lieber-DeCarli alcohol liquid diet group andcontrol diet plus LPS group.The rats of control diet plus LPS group were injected with LPS(3 mg/kg)weekly via the tail vein at the end of week 8,9,or10.The rats of other two groups were injected with 1 ml normal saline weekly at the same time point.All rats were sacrificed at 24 h after normal saline or LPS challenge.Pancreatic tissues were fixed in 10% neutral buffer formalin for preparation of paraffin sections or snap-frozen in liquid nitrogen.A stable activated human PSC line(designated as HP-1 cell)was used in this study,which was established by RSV promoter/enhancer-driven SV40 T antigen expression.To prepare monocyte-derived Mφs,human peripheral blood mononuclear cells(PBMCs)were isolated using human lymphocyte separation medium and then cultured in RPMI 1640 culture medium containing 10 ng/ml GM-CSF for 6 day.Thereafter the Mφ culture was stimulated with alcohol or LPS for additional 24 h.Immunohistochemistry was used to detect IL-6R expression in human pancreatic tissues.Double immunostains was used to recognize the expression of IL-6R in PSCs.Immunocytochemistry was used to determine the expression of IL-6 or IL-6R in Mφs or HP-1 cells.ELISA was used to determine the production of IL-6,TGF-β1,α-SMA and Col1a1 in Mφs,HP-1 cells,rat pancreatic tissues or human sera.Real-time quantitative PCR(RT-qPCR)was used to determine the levels of IL-6,TGF-β1,α-SMA and Col1α1 mR NAs in HP-1 cells.Western blot was used to detect the synthesis of p-JAK2,JAK2,p-STAT3,STAT3,p-Smad2,Smad2,p-Smad3 and Smad3proteins in HP-1 cells.Results: Immunohistochemical stain showed enhanced expression of IL-6protein and its receptor in the pancreas of ACP patients as compared with healthy controls(HC),The number of IL-6+Mφs and IL-6+PSCs or IL-6R+Mφs and IL-6R+PSCs of ACP patients were significantly increased as compared with HC(all P<0.001).Our further study found higher levels of IL-6,TGF-β1 and Col1α1 in the sera of ACP patients as compared with HC(all P<0.001)and positive correlations between IL-6 level and either TGF-β1 or Col1α1 content in ACP without or with endotoxemia patients(r=0.6609,P<0.05;r=0.6293,P<0.05;r=0.6422,P<0.01;r=0.5006,P<0.05).In vivo study showed increased contents of IL-6,TGF-β1 and Col1α1 in the pancreases of ALC-fed rats or rats with LPS injections(all P<0.001)and positive correlations between IL-6content and either TGF-β1 or Col1α1 content in the pancreases of ALC or LPS rats(r=0.7369,P<0.01;r=0.6445,P<0.05;r=0.7380,P<0.01;r=0.6344,P<0.05).In vitro study showed enhanced expression of IL-6R and increased secretion of IL-6 and TGF-β1 in both Mφs and HP-1 cells treated by ALC or LPS(all P<0.001).Mutually induced synthesis between exogenous TGF-β1and IL-6 in HP-1 cells was observed.The expression of α-SMA and Col1α1mR NAs and proteins were partially inhibited by IL-6 neutralizing antibody(IL-6ab)in HP-1 cells treated by ALC or LPS(all P<0.001),suggesting IL-6 as a stimulator of α-SMA and Col1α1 synthesis in ALC or LPS-treated HP-1 cells.Further study demonstrated that IL-6 induced TGF-β1 synthesis through IL-6R/JAK2/STAT3 pathway.The production of α-SMA and Col1α1 in IL-6-treated HP-1 cells was significantly antagized by TGF-β1 neutralizing antibody(TGF-β1ab),suggesting TGF-β1 as a mediator of IL-6-inducedα-SMA and Col1α1 synthesis in the HP-1 cells.IL-6 could induce a delayed Smad2/3 phosphorylation in HP-1 cells.The expression of α-SMA and Col1α1mR NA induced by IL-6 was significantly suppressed by small interfere Smad2(si-Smad2)and si-Smad3 respectively(both P<0.001).Conclusion: ACP is a key player in the fibrogenesis of ACP.IL-6 induces TGF-β1 production by IL-6R/JAK2/STAT3 pathway.IL-6 also promotes PSC activation and collagen I production through up-regulation of TGF-β1/Smads pathway.This study demonstrates that IL-6 may be a potential therapeutic target for pancreatic fibrosis.更多还原