CAJ | 학술논문

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。目的：探討β-arrestin-2對小鼠缺血-再灌註（IR）肝髒自噬的影響及其在肝髒缺血-再灌註損傷（IRI）中的作用。方法採用β-arrestin-2野生型（WT）及基因敲除（KO）小鼠製作肝髒 IR 模型（70％肝髒缺血90 min 後恢複肝髒血流）。實驗分為4組：即 WT 小鼠假手術組（WT ＋Sham 組）、WT 小鼠 IR 組（WT ＋IR 組）、KO 小鼠假手術組（KO ＋Sham 組）、KO 小鼠 IR 組（KO ＋IR 組），每組各18隻。再灌註6、12、24 h 收集各組小鼠血清和肝組織標本。通過檢測血清丙氨痠轉氨酶（ALT）、天鼕氨痠轉氨酶（AST）水平、肝組織囌木素-伊紅（HE）染色和病理學分析判斷肝髒損傷程度，用免疫組織化學（免疫組化）染色和蛋白免疫印跡法檢測自噬關鍵蛋白輕鏈3（LC3）的錶達、透射電子顯微鏡（透射電鏡）觀察肝組織中的自噬體來分析判斷肝髒自噬活動情況。結果與Sham 組相比，IR 組再灌註後各時間點血清 ALT、AST 顯著升高（均為 P ＜0.01），KO ＋IR 組較 WT ＋IR 組升高更明顯（均為 P ＜0.01）。肝組織 HE 染色顯示，WT ＋Sham 組和 KO ＋Sham 組再灌註後各時間點肝細胞形態、肝小葉結構正常；KO ＋IR 組和 WT ＋IR 組再灌註後6 h 肝細胞輕、中度腫脹，肝竇稍擴張，12 h 肝細胞重度腫脹，炎癥細胞浸潤，片狀損傷區域明顯，24 h 肝索排列趨于規則；與WT ＋IR組比較，KO ＋IR 組再灌註後各時間點的損傷程度更嚴重和範圍更大。免疫組化染色和蛋白免疫印跡法結果顯示再灌註後6、12 h 自噬關鍵蛋白 LC3的錶達呈上升趨勢，24 h 錶達略有下降，其中KO ＋IR 組的錶達水平高于 WT ＋IR 組。肝組織透射電鏡結果顯示再灌註後各時間點 IR 組的自噬體數量均明顯高于 Sham 組（均為 P ＜0.01），KO ＋IR 組的自噬體數量較 WT ＋IR 組明顯增多（P ＜0.05）。結論β-arrestin-2可能通過抑製自噬來減輕小鼠肝髒 IRI。
목적：탐토β-arrestin-2대소서결혈-재관주（IR）간장자서적영향급기재간장결혈-재관주손상（IRI）중적작용。방법채용β-arrestin-2야생형（WT）급기인고제（KO）소서제작간장 IR 모형（70％간장결혈90 min 후회복간장혈류）。실험분위4조：즉 WT 소서가수술조（WT ＋Sham 조）、WT 소서 IR 조（WT ＋IR 조）、KO 소서가수술조（KO ＋Sham 조）、KO 소서 IR 조（KO ＋IR 조），매조각18지。재관주6、12、24 h 수집각조소서혈청화간조직표본。통과검측혈청병안산전안매（ALT）、천동안산전안매（AST）수평、간조직소목소-이홍（HE）염색화병이학분석판단간장손상정도，용면역조직화학（면역조화）염색화단백면역인적법검측자서관건단백경련3（LC3）적표체、투사전자현미경（투사전경）관찰간조직중적자서체래분석판단간장자서활동정황。결과여Sham 조상비，IR 조재관주후각시간점혈청 ALT、AST 현저승고（균위 P ＜0.01），KO ＋IR 조교 WT ＋IR 조승고경명현（균위 P ＜0.01）。간조직 HE 염색현시，WT ＋Sham 조화 KO ＋Sham 조재관주후각시간점간세포형태、간소협결구정상；KO ＋IR 조화 WT ＋IR 조재관주후6 h 간세포경、중도종창，간두초확장，12 h 간세포중도종창，염증세포침윤，편상손상구역명현，24 h 간색배렬추우규칙；여WT ＋IR조비교，KO ＋IR 조재관주후각시간점적손상정도경엄중화범위경대。면역조화염색화단백면역인적법결과현시재관주후6、12 h 자서관건단백 LC3적표체정상승추세，24 h 표체략유하강，기중KO ＋IR 조적표체수평고우 WT ＋IR 조。간조직투사전경결과현시재관주후각시간점 IR 조적자서체수량균명현고우 Sham 조（균위 P ＜0.01），KO ＋IR 조적자서체수량교 WT ＋IR 조명현증다（P ＜0.05）。결론β-arrestin-2가능통과억제자서래감경소서간장 IRI。
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.TEMresult 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.