西罗莫司预处理减轻大鼠肝脏缺血再灌注损伤的作用及其机制

Protective mechanism of sirolimus pretreatment against liver ischemia-reperfusion injury in rats

目的探讨西罗莫司（SRL）预处理减轻大鼠肝脏缺血再灌注损伤的作用及其机制。方法将SD大鼠随机分为4组，每组12只。假手术对照组：开腹后仅用生理盐水纱布覆盖切口60min，关腹；假手术SRL组：手术方式同假手术对照组；实验对照组：开腹后夹闭肝门静脉左支、肝动脉左支及左肝管，60min后开放血管；实验SRL组：手术方式同实验对照组。两SRL组大鼠术前2周开始给予SRL2mg·kg^-1·d^-1灌胃，术前6h加用1次，而丽对照组同期仅给予等体积无菌生理盐水灌胃。术后24h分别采集各组大鼠的血液和肝组织，检测血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平，光镜下观察肝组织的病理学变化，采用流式细胞术检测肝组织中CD4^＋CD25^＋T淋巴细胞占单个核细胞的比例，采用实时聚合酶链反应检测肝组织中Foxp3 mRNA的表达，采用酶联免疫吸附试验检测血清中转化生长因子-β（TGF-β）和白细胞介素10（IL-10）的含量。结果假手术对照组和假手术SRL组血清ALT和AST均处于较低水平，而实验SRL组和实验对照组明显升高，且实验SRL组低于实验对照组（P〈0．05）。假手术对照组和假手术SRL组肝组织结构正常，实验对照组可见明显的片状坏死，实验SRL组肝小叶结构基本完整，未见明显细胞坏死。假手术对照组、假手术SRL组、实验对照组和实验SRL组CD4^＋CD25^＋T淋巴细胞占单个核细胞的比例分别为（6．12±1．87）％、（22．36±6．75）％、（4．53±1．02）％和（13．29±3．16）％。假手术SRL组Foxp3 mRNA的相对表达量以及血清中TGF—β和1L-10的含量明显高于假手术对照组（P〈0．05），实验SRL组明显高于实验对照组（P〈0．05）。结论SRL可以减轻大鼠肝脏缺血再灌注损伤，其机制可能与SRL诱导体内CD4^＋CD25^＋Foxp3^＋调节性T淋巴细胞的分化以及增加TGF-β和IL-10的分泌抑制炎症反应有关。

Objective To investigate the protective effect of sirolimus pretreatment against liver isehemia-reperfusion （I/R） injury in rat model and the possible mechanism. Methods Forty-eight male SD rats were randomized into four groups （12/group）： A： sham group with saline,B., sham group with sirolimus,C： saline-operated group,D： sirolimus-operated group. The rats were pretreated with either saline or sirolimus （2 mg·kg^-1·d^-1 ） by oral garage for two weeks. The rat partial liver model of I/R injury was established,and the samples were collected at the 24th h after the I/R. The serum ALT and AST levels were determined, the histologic changes were observed by HE staining under the light microscopy, the frequency of CD4^＋ CD25^＋ T cells among mononuelear cells in liver tissue was analyzed by using flow cytometry, the expression of Foxp3 mRNA was detected in liver tissue by real time PCR,and the serum TGF-β, IL-10 levels were measured by enzyme-linked immunosorbent assay （ELISA）. Results Serum ALT and AST levels were significantly decreased and the histological damage was significantly alleviated in the sirolimus-operated group as compared with saline-operated group （P〈 0. I}5）. The percentage of CD4^＋ CD25^＋ T cells among mononuclear cells in groups A,B,C,and D was （6. 12 ±1.87）%,（22. 36 ± 6. 75）%, （4. 53 ± 1. 02）% and （ 13. 29± 3. 16）% respectively in liver tissue. The expression levels of the Foxp3 mRNA were significantly higher in sirolimus group than in saline group （P〈 0. 05）. The ELISA showed that sirolimus could significantly increase the levels of TGF-β and IL-10 （P〈0. 05）. Conclusion Pretreatment of sirolimus can effectively protect against liver ischemia-reperfusion injury in rats,which may be related to induction of CD4^＋ CD25^＋ Foxp3^＋ T regulator cells by sirolimus, and the increase of TGF-β and IL-10 secretion to inhibit the inflammatory response.