[1]刘彦,吴皓,龚建平,等.阻断Kupffer细胞的TIM-4蛋白功能对小鼠肝脏缺血再灌注损伤的影响[J].第三军医大学学报,2019,41(01):48-55.
 LIU Yan,WU Hao,GONG Jianping,et al.inhibition of TIM-4 in Kupffer cells alleviates hepatic ischemia-reperfusion injury in mice[J].J Third Mil Med Univ,2019,41(01):48-55.
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阻断Kupffer细胞的TIM-4蛋白功能对小鼠肝脏缺血再灌注损伤的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第01期
页码:
48-55
栏目:
基础医学
出版日期:
2019-01-15

文章信息/Info

Title:
inhibition of TIM-4 in Kupffer cells alleviates hepatic ischemia-reperfusion injury in mice
作者:
刘彦吴皓龚建平李旭宏
成都市第五人民医院消化内科;重庆医科大学附属第二医院肝胆外科;重庆三峡中心医院百安分院外科
Author(s):
LIU Yan WU Hao GONG Jianping LI Xuhong

Department of Gastroenterology, Fifth People’s Hospital of Chengdu, Chengdu, Sichuan Province, 611130; Department of Hepatobiliary Surgery, Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010; Department of General Surgery, Bai’an Branch of Chongqing Three Gorges Central Hospital, Chongqing, 404000, China

关键词:
T细胞免疫球蛋白黏蛋白4Kupffer细胞肝缺血再灌注损伤核转录因子&kappaB
Keywords:
T-cell immunoglobulin and mucin-domain-containing molecule 4 Kupffer cells hepatic ischemia-reperfusion injury nuclear factor-&kappaB
分类号:
R364.12; R392.32; R657.3
文献标志码:
A
摘要:

目的    探讨阻断Kupffer细胞(Kupffer cells,KCs)的TIM-4蛋白功能对小鼠肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury,HIRI)的影响。方法    建立小鼠肝缺血再灌注损伤模型,术后Western blot检测KCs和树突状细胞(dendritic cells,DCs)TIM-4表达,免疫组化检测肝组织TIM-4表达;IR小鼠模型采用随机数字表法分为Sham组(PBS处理)、Ctr Ig组(TIM-4同型对照抗体处理)以及TIM-4 mAb组(TIM-4抗体处理),术后检测血清肝功、炎症指标、肝组织病理变化、肝细胞凋亡以及NF-κB通路相关蛋白的表达。结果    KCs表达TIM-4 蛋白随再灌注时间逐渐增加,然而DCs表达TIM-4不随时间变化;组化结果提示TIM-4主要表达于肝血窦巨噬细胞;与Ctr Ig组比较,TIM-4 mAb组血清肝功、促炎因子产物水平明显减低,而抗炎因子IL-10以及抗氧化应激产物CAT、SOD逐渐增高,且差异具有统计学意义(P<0.05);肝组织病理学结果示Ctr Ig组部分炎症细胞浸润,肝血窦轻度充血,TIM-4 mAb组无明显变化;TUNEL结果示,TIM-4 mAb组凋亡程度明显低于Ctr Ig组,Ctr Ig组和TIM-4 mAb组细胞早期凋亡程度分别为(25.56±1.26)%、(5.35±0.33)%,且两组差异具有统计学意义(P<0.05);阻断KCs TIM-4功能后,KCs TLR4以及下游p-IKKα、p-IκBα、p-p65蛋白表达水平逐渐减低;另外,TPCA-1(IKK-2阻断剂)进行预处理小鼠,进一步验证了阻断KCs TIM-4可通过抑制NF-κB信号通路减轻IRI程度。结论    阻断KCs TIM-4的功能能够抑制NF-κB炎症通路的激活,改善IR炎症反应以及肝细胞凋亡,从而对HIRI起到一定的保护作用。

Abstract:

Objective    To investigate the effects of functional inhibition of TIM-4 in Kupffer cells(KCs) in protecting against hepatic ischemia-reperfusion injury in mice and explore the mechanism. Methods   We determined the expression of TIM-4 in KCs and dendritic cells(DCs) in a mouse model of hepatic ischemiareperfusion injury using Western blotting and immunohistochemistry. The mouse models were treated with PBS(sham group), a monoclonal antibody of TIM-4(TIM-4 mAb group) or a control antibody(Ctr Ig group), and the liver functions, inflammatory factors and products of oxidative stress were assayed. HE staining was used to observe the pathological changes in the liver tissue, and TUNEL and flow cytometry were used to detect apoptosis of the hepatocytes. Western blotting was used to detect the expression of nuclear factor-κB(NF-κB) pathway-related proteins. Results    In the mouse model of hepatic ischemiareperfusion injury, the expression of TIM-4 protein in KCs was increased progressively as the reperfusion time extended, while TIM-4 expression in the DCs showed no significant variations with time. Immunohistochemical staining identified TIM-4 expression mainly in the hepatic sinusoidal macrophages; The serum levels of hepatic function-related factors, pro-inflammatory cytokines in the hepatic homogenate, and oxidative stress products were significantly reduced, while anti-inflammatory cytokine(IL-10) and anti-oxidative stress products(CAT and SOD) were increased significantly in TIM-4 mAb group as compared with Ctr Ig group(P<0.05). Histopathological examination of the liver tissues revealed inflammatory cell infiltration and mild congestion in the hepatic sinusoids in Ctr Ig group, but no obvious pathological changes in TIM-4 mAb group. The apoptotic index was significantly lower in TIM-4 mAb group than in Ctr Ig group; the early apoptosis rates in the 2 groups were(5.35±0.33)% and(25.56±1.26)%, respectively, showing a significant difference between them(P<0.05). Functional blocking of TIM-4 in the KCs lowered the expression levels of TLR4 and the downstream p-IKKα, p-IκBα, p-p65 proteins. Pre-treatment of the mice with TPCA-1, an IKK-2 blocker, further validated that blocking TIM-4 in the KCs inhibited NF-κB signaling pathway and improved hepatic ischemia-reperfusion injury.  Conclusion     Functional inhibition of TIM-4 in the KCs can inhibit the activation of NF-κB inflammatory pathway and reduce the inflammatory response and hepatocyte apoptosis to protect against hepatic ischemia-reperfusion injury in mice.

参考文献/References:

[1]FENG J, YAO W, ZHANG Y, et al. Intravenous anesthetics enhance the ability of human bone marrowderived mesenchymal stem cells to alleviate hepatic ischemiareperfusion injury in a receptordependent manner[J]. Cell Physiol Biochem, 2018, 47(2): 556-566. DOI: 10.1159/000489989.
[2]DENG W S, XU Q, LIU Y E, et al. Effects of melatonin on liver function and lipid peroxidation in a rat model of hepaticischemia/reperfusion injury[J]. Exp Ther Med, 2016, 11(5): 1955-1960.
[3]SHIMADA S, WAKAYAMA K, FUKAI M, et al. Hydrogen gas ameliorates hepatic reperfusion injury after prolonged cold preservation in isolated perfused rat liver[J]. Artif Organs, 2016, 40(12): 1128-1136. DOI: 10.1111/aor.12710.
[4]LI Z, JU Z, FRIERI M. The Tcell immunoglobulin and mucin domain(Tim) gene family in asthma[J]. Allergy Asthma Proc, 2013, 34(1): e21-e26. DOI: 10.2500/aap.2013.34.3646.
[5]MEYERS J H, SABATOS C A, CHAKRAVARTI S, et al. The TIM gene family regulates innate and adaptive immunity[J]. Trends Mol Med, 2005, 11(8): 362-269.
[6]CHEN G S, QI H Z. Effect of Kupffer cells on immune tolerance in liver transplantation[J]. Asian Pac J Trop Med, 2012, 5(12): 970-972. DOI: 10.1016/S19957645(12)60184-9.
[7]LI P Z, LI J Z, LI M, et al. An efficient method to isolate and culture mouse Kupffer cells[J]. Immunol Lett, 2014, 158(1/2): 52-56. DOI: 10.1016/j.imlet.2013.12.002.
[8]YANG W, CHEN J, MENG Y, et al. Novel targets for treating ischemiareperfusion injury in the liver[J]. Int J Mol Sci, 2018, 19(5): e1302. DOI: 10.3390/ijms19051302.
[9]ZHANG J, XU P, SONG P, et al. CCL2CCR2 signaling promotes hepatic ischemia/reperfusion injury[J]. J Surg Res, 2016, 202(2): 352-362. DOI: 10.1016/j.jss.2016.02.029.
[10]XIE F, LI Z P, WANG H W, et al. Evaluation of liver ischemiareperfusion injury in rabbits using a nanoscale ultrasound contrast agent targeting ICAM1[J]. PLoS ONE, 2016, 11(4): e0153805. DOI: 10.1371/journal.pone.0153805.
[11]ZHANG Y, LIU Y, CHEN H, et al. TIM-1 attenuates the protection of ischemic preconditioning for ischemia reperfusion injury in liver transplantation[J]. Am J Transl Res, 2017, 9(8): 3665-3675.
[12]DEWITZ C, MLLERHACKBARTH K, SCHWEIGERT O, et al. T-cell immunoglobulin and mucin domain 2(TIM-2) is a target of ADAM10mediated ectodomain shedding[J]. FEBS J, 2014, 281(1): 157-174. DOI: 10.1111/febs.12583.
[13]KIKUSHIGE Y, MIYAMOTO T, YUDA J, et al. A TIM3/Gal9 autocrine stimulatory loop drives selfrenewal of human myeloid leukemia stem cells and leukemic progression[J]. Cell Stem Cell, 2015, 17(3): 341-352. DOI: 10.1016/j.stem.2015.07.011.
[14]RONG S, PARK J K, KIRSCH T, et al. The TIM1: TIM-4 pathway enhances renal ischemiareperfusion injury[J]. J Am Soc Nephrol, 2011, 22(3): 484-495. DOI: 10.1681/ASN.2010030321.
[15]FOKS A C, ENGELBERTSEN D, KUPERWASER F, et al. Blockade of Tim-1 and Tim-4 enhances atherosclerosis in low-density lipoprotein receptor-deficient mice[J]. Arterioscler Thromb Vasc Biol, 2016, 36(3): 456-465. DOI: 10.1161/ATVBAHA.115.306860.
[16]TIETJEN G T, GONG Z, CHEN C H, et al. Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4[J]. Proc Natl Acad Sci USA, 2014, 111(15): e1463-e1472. DOI: 10.1073/pnas.1320174111.
[17]SAVILL J, GREGORY C. Apoptotic PS to phagocyte TIM-4: eat me[J]. Immunity, 2007, 27(6): 830-832.
[18]ABE Y, KAMACHI F, KAWAMOTO T, et al. TIM-4 has dual function in the induction and effector phases of murine arthritis[J]. J Immunol, 2013, 191(9): 4562-4572. DOI: 10.4049/jimmunol.1203035.
[19]TANIGUCHI K, KARIN M. NF-κB, inflammation, immunity and cancer: coming of age[J]. Nat Rev Immunol, 2018, 18(5): 309-324. DOI: 10.1038/nri.2017.142.
[20]ROTHSCHILD D E, MCDANIEL D K, RINGELSCAIA V M, et al. Modulating inflammation through the negative regulation of NFκB signaling[J]. J Leukoc Biol, 2018. [Epub ahead of print]. DOI: 10.1002/JLB.3MIR0817-346RRR.

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更新日期/Last Update: 2019-01-14