β-arrestin-2通过抑制自噬减轻小鼠肝脏缺血-再灌注损伤

王励; 汪根树

doi:10.3969/j.issn.1674-7445.2015.03.003

β-arrestin-2通过抑制自噬减轻小鼠肝脏缺血-再灌注损伤

doi: 10.3969/j.issn.1674-7445.2015.03.003

王励,
汪根树^,

510630 广州, 中山大学附属第三医院肝移植中心中山大学器官移植研究所广东省器官移植研究中心

基金项目:

国家自然科学基金面上项目 81170422

广州市科技计划项目 2014J4100128

详细信息

通讯作者:
汪根树, Email:wgsh168@163.com

中图分类号: R617
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- 被引次数: 0
出版历程
- 收稿日期: 2015-04-10
- 网络出版日期: 2021-01-19
- 刊出日期: 2015-05-15

β-arrestin-2 alleviates mouse hepatic ischemia-reperfusion injury by inhibiting autophagy

Wang Li,
Wang Genshu^,

Liver Transplantation Center, the Third Affiliated Hospital of Sun Yat-sen University, Organ Transplantation Institute of Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, Guangzhou 510630, China

More Information

Corresponding author: Wang Genshu, Email:wgsh168@163.com

摘要

摘要: 目的探讨β-arrestin-2对小鼠缺血-再灌注(IR)肝脏自噬的影响及其在肝脏缺血-再灌注损伤(IRI)中的作用。方法采用β-arrestin-2野生型(WT)及基因敲除(KO)小鼠制作肝脏IR模型(70%肝脏缺血90 min后恢复肝脏血流)。实验分为4组:即WT小鼠假手术组(WT+Sham组)、WT小鼠IR组(WT+IR组)、KO小鼠假手术组(KO+Sham组)、KO小鼠IR组(KO+IR组), 每组各18只。再灌注6、12、24 h收集各组小鼠血清和肝组织标本。通过检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平、肝组织苏木素-伊红(HE)染色和病理学分析判断肝脏损伤程度, 用免疫组织化学(免疫组化)染色和蛋白免疫印迹法检测自噬关键蛋白轻链3(LC3)的表达、透射电子显微镜(透射电镜)观察肝组织中的自噬体来分析判断肝脏自噬活动情况。结果与Sham组相比, IR组再灌注后各时间点血清ALT、AST显著升高(均为P < 0.01), KO+IR组较WT+IR组升高更明显(均为P < 0.01)。肝组织HE染色显示, WT+Sham组和KO+Sham组再灌注后各时间点肝细胞形态、肝小叶结构正常; KO+IR组和WT+IR组再灌注后6 h肝细胞轻、中度肿胀, 肝窦稍扩张, 12 h肝细胞重度肿胀, 炎症细胞浸润, 片状损伤区域明显, 24 h肝索排列趋于规则; 与WT+IR组比较, KO+IR组再灌注后各时间点的损伤程度更严重和范围更大。免疫组化染色和蛋白免疫印迹法结果显示再灌注后6、12 h自噬关键蛋白LC3的表达呈上升趋势, 24 h表达略有下降, 其中KO+IR组的表达水平高于WT+IR组。肝组织透射电镜结果显示再灌注后各时间点IR组的自噬体数量均明显高于Sham组(均为P < 0.01), KO+IR组的自噬体数量较WT+IR组明显增多(P < 0.05)。结论 β-arrestin-2可能通过抑制自噬来减轻小鼠肝脏IRI。
- 肝移植 /
- 缺血-再灌注损伤 /
- β-arrestin-2 /
- 自噬
Abstract: Objective To investigate the impact of β-arrestin-2 on hepatic autophagy after ischemia-reperfusion(IR) and its effect on mouse hepatic ischemia-reperfusion injury(IRI). Methods β-arrestin-2 wild type(WT) and knock out(KO) mice were used to build a mouse model of hepatic IR(70% hepatic warm ischemia for 90 min). Mice were divided into four groups: WT mice sham-operated group(WT+Sham group), WT mice IR group(WT+IR group), KO mice sham-operated group(KO+Sham group) and KO mice IR group(KO+IR group), 18 mice in each group. Serum and liver tissues were collected at 6, 12 and 24 h after reperfusion. The alanine aminotransferase(ALT), aspartate aminotransferase(AST) measurement and liver tissues hematoxylin-eosin(HE) staining and pathology analysis were used to estimate hepatic injury. The expression of light chain(LC)3, the key protein of autophagy, were detected by immunohistochemical(IHC) staining and western blot. Autophagosomes in liver tissues were evaluated by transmission electron microscopy(TEM). Results Compared with Sham groups, the levels of serum ALT and AST significantly increased in IR groups at each time point (all in P < 0.01). The levels of KO+IR group were higher than those of WT+IR group at each time point (all in P < 0.01). The result of liver tissue HE staining showed that liver cell morphology and lobular architecture was normal in WT+Sham group and KO+Sham group at each time point after reperfusion. Liver cells were light or moderate swelling with liver sinus expansion in KO+IR group and WT+IR group at 6 h after reperfusion. And liver cells were severe swelling with inflammatory cells infiltration, and flake damage area is obvious at 12 h after reperfusion. Liver rope arranged regularly at 24 h after reperfusion. The degree of hepatic injury in KO+IR group was more serious than WT+IR group. IHC staining and western blot analysis showed that the expression levels of LC3 increased in IR groups at 6, 12 h but slightly decreased at 24 h after reperfusion. And the expression levels of KO+IR group were higher than WT+IR group. TEM result show that autophagosomes in IR groups were obviously more than those in Sham groups(both in P < 0.01). The counts of autophagosomes in KO+IR group were more than those of WT+IR group(P < 0.05). Conclusions β-arrestin-2 may alleviate mouse hepatic IRI by inhibiting autophagy.
- Liver transplantation /
- Ischemia-reperfusion injury /
- β-arrestin-2 /
- Autophagy

HTML全文

图 1 arrestin-2 WT和KO小鼠基因型鉴定结果

注：MW为分子量marker，NC为阴性对照，PC为阳性对照，WT为野生型，KO为敲除型，HET为杂合子

Figure 1. Identification of β-arrestin-2 genotype WT and KO mice

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图 2 各组小鼠缺血-再灌注后血清ALT、AST水平的比较

注：IR组与Sham组比较，^aP < 0.01；KO+IR组与WT+IR组比较，^bP < 0.01

Figure 2. Comparison of the levels of serum ALT and AST among mice in each group after ischemia-reperfusion

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图 3 各组小鼠缺血-再灌注后肝组织病理学的变化(HE, ×200)

Figure 3. Changes of liver tissue pathology of mice in each group after ischemia-reperfusion

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图 4 各组小鼠缺血-再灌注后肝组织LC3的表达(DAB, ×200)

Figure 4. Expression of LC3 in liver tissue of mice in each group after ischemia-reperfusion

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图 5 各组小鼠肝组织免疫组化染色LC3阳性细胞数量的比较

注：IR组与Sham组比较，^aP < 0.01；KO+IR组与WT+ IR组比较，^bP < 0.01

Figure 5. Comparison of the number of LC3 positive cells of liver tissue of mice among each group detected by immunohistochemical staining

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图 6 各组小鼠缺血-再灌注后肝组织LC3的蛋白表达水平

Figure 6. The expression level of LC3 of mice in each group after ischemia-reperfusion

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图 7 透射电镜观察缺血-再灌注后各组小鼠肝组织自噬体表达(TEM, ×8 900)

Figure 7. The expression of autophagosomes of mice in each group after ischemia-reperfusion evaluated by transmission electron microscopy

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图 8 各组小鼠缺血-再灌注后肝组织自噬体数量的比较

注：IR组与Sham组比较，^aP < 0.01；KO+IR组与WT+IR组比较，^bP < 0.01

Figure 8. Comparison of the number of autophagosomes of mice among each group after ischemia-reperfusion

下载: 全尺寸图片幻灯片

参考文献(21)

[1]	饶建华, 王学浩.T淋巴细胞在缺血-再灌注损伤中的重要作用[J].器官移植, 2014, (5):261-265. http://www.organtranspl.com/browse/detail/cat/29/id/187 Rao JH, Wang XH. Important effects of T lymphocyte in ischemia-reperfusion injury[J]. Organ Transplant, 2014, 5(5):261-265. http://www.organtranspl.com/browse/detail/cat/29/id/187
[2]	Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy[J]. Nat Cell Biol, 2013, 15(7):713-720. doi: 10.1038/ncb2788
[3]	Wang JH, Behrns KE, Leeuwenburgh C, et al. Critical role of autophage in ischemia/reperfusion injury to aged livers[J]. Autophagy, 2012, 8(1):140-141. doi: 10.4161/auto.8.1.18391
[4]	Cheng P, Wang F, Chen K, et al. Hydrogen sulfide ameliorates ischemia/reperfusion-induced hepatitis by inhibiting apoptosis and autophagy pathways[J]. Mediators Inflamm, 2014:935251. https://www.hindawi.com/journals/mi/2014/935251/
[5]	Klein SR, Piya S, Lu Z, et al. C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy[J]. Oncogene, 2015, DOI: 10.1038/onc.2014.452.
[6]	Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, et al. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration[J]. Cell Signal, 2014, 26(12):2694-2701. doi: 10.1016/j.cellsig.2014.08.019
[7]	Zhang L, Wang H, Xu J, et al. Inhibition of cathepsin S induces autophagy and apoptosis in human glioblastoma cell lines through ROS-mediated PI3K/AKT/mTOR/p70S6K and JNK signaling pathways[J]. Toxicol Lett, 2014, 228(3):248-259. doi: 10.1016/j.toxlet.2014.05.015
[8]	Kang DS, Tian X, Benovic JL. β-Arrestins and G protein-coupled receptor trafficking[J]. Methods Enzymol, 2013, 521:91-108. doi: 10.1016/B978-0-12-391862-8.00005-3
[9]	Zhan X, Kook S, Gurevich EV, et al. Arrestin-dependent activation of JNK family kinases[J]. Handb Exp Pharmacol, 2014, 219:259-280. doi: 10.1007/978-3-642-41199-1
[10]	Lefkowitz RJ. Arrestins come of age:a personal historical perspective[J]. Prog Mol Biol Transl Sci, 2013, 118:3-18. doi: 10.1016/B978-0-12-394440-5.00001-2
[11]	Luedde T, Kaplowitz N, Schwabe RF. Cell death and cell death responses in liver disease:mechanisms and clinical relevance[J]. Gastroenterology, 2014, 147(4):765-783. doi: 10.1053/j.gastro.2014.07.018
[12]	Yang M, Antoine DJ, Weemhoff JL, et al. Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice[J]. Liver Transpl, 2014, 20(11):1372-1382. doi: 10.1002/lt.23958
[13]	Wang Y, Shen J, Xiong X, et al. Remote ischemic preconditioning protects against liver ischemia-reperfusion injury via heme oxygenase-1-induced autophagy[J]. PLoS One, 2014, 9(6):98834. doi: 10.1371/journal.pone.0098834
[14]	Liu A, Fang H, Wei W, et al. Ischemic preconditioning protects against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy[J]. Crit Care Med, 2014, 42(12):762-771. doi: 10.1097/CCM.0000000000000659
[15]	Sun K, Xie X, Liu Y, et al. Autophagy lessens ischemic liver injury by reducing oxidative damage[J]. Cell Biosci, 2013, 3(1):26. doi: 10.1186/2045-3701-3-26
[16]	Shen M, Lu J, Dai W, et al. Ethyl pyruvate ameliorates hepatic ischemia-reperfusion injury by inhibiting intrinsic pathway of apoptosis and autophagy[J]. Mediators Inflamm, 2013, 2013:461536. http://www.medscape.com/medline/abstract/24453420
[17]	Yang X, Zhou G, Ren T, et al. β-arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways[J]. Apoptosis, 2012, 17(9):1019-1026. doi: 10.1007/s10495-012-0741-2
[18]	Wu JX, Shan FX, Zheng JN, et al. β-arrestin promotes c-Jun N-terminal kinase mediated apoptosis via a GABA(B)R·β-arrestin·JNK signaling module[J]. Asian Pac J Cancer Prev, 2014, 15(2):1041-1046. doi: 10.7314/APJCP.2014.15.2.1041
[19]	Zeng LX, Tao J, Liu HL, et al. β-arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis[J]. Mucosal Immunol, 2014, DOI: 10.1038/mi.2014.104.
[20]	Yonekawa T, Thorburn A. Autophagy and cell death[J]. Essays Biochem, 2013, 55:105-117. doi: 10.1042/bse0550105
[21]	Thorburn A. Autophagy and its effects:making sense of double-edged swords[J]. PLoS Biol, 2014, 12(10):1001967. doi: 10.1371/journal.pbio.1001967