缺血后处理对肝硬化大鼠肝脏缺血-再灌注损伤保护作用的免疫机制

doi:10.3877/cma.j.issn.2095-3232.2014.06.010

目的

探讨缺血后处理（IPO）对肝硬化大鼠肝脏缺血-再灌注损伤（IRI）的保护作用及其免疫机制。

方法

按随机数字表法将30只肝硬化模型SD大鼠随机分为IPO组、IRI组和单纯肝切除组（肝切组），各10只。IPO组先切除40％肝脏，阻断第一肝门20 min，然后反复缺血-再灌注3次，最后持续再灌注；IRI组，切除40％肝脏，阻断第一肝门20 min后持续再灌注；肝切组切除40％肝脏。分别于术后6、24 h抽取大鼠下腔静脉血，检测ALT、AST、分化群（CD）4⁺、CD8⁺、调节性T细胞（Treg）百分率、白细胞介素（IL）-4、IL-10水平。3组比较采用单因素方差分析，两两比较采用LSD-t检验。

结果

恢复灌注6 h后，IPO组的ALT、AST分别为（1 623±378）、（1 993±469）U/L，IRI组相应为（2 690±549）、（3 020±577）U/L，IPO组ALT、AST明显低于IRI组（LSD-t= -4.21，-3.72；P<0.05）。恢复灌注24 h后，IPO组的ALT、AST分别为（307±76）、（555±137）U/L，IRI组相应为（518±105）、（1 050±355） U/L，IPO组ALT、AST亦明显低于IRI组（LSD-t=-4.06，-3.37；P<0.05）。恢复灌注6 h后，IPO组CD4⁺、CD8⁺、CD4⁺/ CD8⁺比值、Treg、IL-4、IL-10分别为（57±5）%、（25±3）%、2.3±0.5、（8.9±0.4）%、（1.27±0.25）mg/L、（0.61±0.03）mg/L，IRI组相应为（52±6）%、（12±3）%、4.5±0.8、（7.3±0.3）%、（0.66±0.11）mg/L、（0.34±0.06）mg/L，IPO组CD8⁺、Treg、IL-4、IL-10较IRI组明显升高，CD4⁺/ CD8⁺比值明显降低（LSD-t= 7.74，6.67，5.52，9.31，-6.69；P<0.05）。

结论

IPO可能通过减轻免疫损伤发挥对肝硬化大鼠肝脏IRI的保护作用。

关键词: 再灌注损伤, 大鼠, 肝硬化，实验性, 缺血后处理, T淋巴细胞亚群, 白细胞介素4, 白细胞介素10

Objective

To investigate the protective effects and its mechanism of ischemia postconditioning (IPO) in hepatic ischemia-reperfusion injury (IRI) in liver cirrhotic rats.

Methods

Thirty liver cirrhotic Sprague-Dawley (SD) rats were randomly divided into three groups according to the random table methods: ischemia postconditioning (IPO) group, IRI group, and pure hepatectomy (PH) group with 10 rats in each group. Rats in IPO group underwent partial hepatectomy (40%), and the first portal was occluded for 20 min, then 3 times of ischemia-reperfusion were performed, and finally continuous reperfusion. Rats in IRI group underwent partial hepatectomy (40%), and the first portal was occluded for 20 min, then continuous reperfusion was performed. Rats in PH group underwent partial hepatectomy (40%) only. Inferior vena cava blood was collected at 6, 24 h after operation. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), proportion of cluster of differentiation (CD) 4⁺, CD8⁺, regulatory T cells (Treg) and levels of interleukin (IL)-4, IL-10 were tested. Comparison of three groups was conducted by one way analysis of variance and pairwise comparison by LSD-t test.

Results

After 6 h reperfusion, levels of ALT and AST in IPO group [(1 623±378) , (1 993±469) U/L] were significantly lower than those in IRI group [(2 690±549) , (3 020±577) U/L] (LSD-t=-4.21, -3.72; P<0.05). After 24 h reperfusion, levels of ALT and AST in IPO group [(307±76) , (555±137) U/L] were still significantly lower than those in IRI group [(518±105) , (1 050±355) U/L] (LSD-t=-4.06, -3.37; P<0.05). After 6 h reperfusion, proportion of CD4⁺, CD8⁺, CD4⁺/CD8⁺ ratio, Treg, and levels of IL-4, IL-10 in IPO group were (57±5) %, (25±3) %, 2.3±0.5, (8.9±0.4) %, (1.27±0.25) mg/L, (0.61±0.03) mg/L, respectively. While in IRI group, they were (52±6)%, (12±3) %, 4.5±0.8, (7.3±0.3) %, (0.66±0.11) mg/L, (0.34±0.06) mg/L, respectively. In IPO group, CD8⁺, Treg, IL-4 and IL-10 increased significantly, while CD4⁺/CD8⁺ ratio decreased significantly, compared with those in IRI group (LSD-t= 7.74, 6.67, 5.52, 9.31, -6.69; P<0.05).

Conclusion

IPO can prevent from hepatic IRI injury in liver cirrhotic rats through decreasing the immune injury.

Key words: Reperfusion injury, Rats, Liver cirrhosis, experimental, Ischemic postconditioning, T-lymphocyte subsets, Interleukin-4, Interleukin-10

表1 IPO组、IRI组和肝切组各时间点血ALT、AST的变化（U/L,±s ）

组别	鼠数	ALT		AST
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	1 623±378	307±76	1 993±469	555±137
IRI组	10	2 690±549	518±105	3 020±577	1 050±355
肝切组	10	545±112	256±60	965±152	725±129

表1 IPO组、IRI组和肝切组各时间点血ALT、AST的变化（U/L,±s ）

组别	鼠数	ALT		AST
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	1 623±378	307±76	1 993±469	555±137
IRI组	10	2 690±549	518±105	3 020±577	1 050±355
肝切组	10	545±112	256±60	965±152	725±129

表2 IPO组、IRI组和肝切组各时间点T淋巴细胞亚群和细胞因子的变化（±s）

组别	鼠数	CD4⁺（%）		CD8⁺（%）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	57±5	39±5	25±3	18±3
IRI组	10	52±6	33±4	12±3	17±2
肝切组	10	40±6	36±5	19±2	18±2
组别	鼠数	CD4⁺/CD8⁺比值		Treg（%）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	2.3±0.5	2.2±0.4	8.9±0.4	7.4±0.5
IRI组	10	4.5±0.8	2.0±0.3	7.3±0.3	9.5±0.7
肝切组	10	2.2±0.2	2.1±0.4	7.1±0.4	7.5±0.5
组别	鼠数	IL-4（mg/L）		IL-10（mg/L）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	1.27±0.25	0.70±0.17	0.61±0.03	0.29±0.07
IRI组	10	0.66±0.11	0.69±0.21	0.34±0.06	0.55±0.08
肝切组	10	0.73±0.14	0.71±0.10	0.31±0.05	0.23±0.06

表2 IPO组、IRI组和肝切组各时间点T淋巴细胞亚群和细胞因子的变化（±s）

组别	鼠数	CD4⁺（%）		CD8⁺（%）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	57±5	39±5	25±3	18±3
IRI组	10	52±6	33±4	12±3	17±2
肝切组	10	40±6	36±5	19±2	18±2
组别	鼠数	CD4⁺/CD8⁺比值		Treg（%）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	2.3±0.5	2.2±0.4	8.9±0.4	7.4±0.5
IRI组	10	4.5±0.8	2.0±0.3	7.3±0.3	9.5±0.7
肝切组	10	2.2±0.2	2.1±0.4	7.1±0.4	7.5±0.5
组别	鼠数	IL-4（mg/L）		IL-10（mg/L）
组别	鼠数	6 h	24 h	6 h	24 h
IPO组	10	1.27±0.25	0.70±0.17	0.61±0.03	0.29±0.07
IRI组	10	0.66±0.11	0.69±0.21	0.34±0.06	0.55±0.08
肝切组	10	0.73±0.14	0.71±0.10	0.31±0.05	0.23±0.06

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Immunity mechanism of ischemia postconditioning in preventing from hepatic ischemia-reperfusion injury in liver cirrhotic rats

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Immunity mechanism of ischemia postconditioning in preventing from hepatic ischemia-reperfusion injury in liver cirrhotic rats