青蒿琥酯对大鼠肾脏缺血再灌注后纤维化改变的保护作用

Protective effect of artesunate on fibrosis induced by renal ischemia and reperfusion injury

目的探讨青蒿琥酯对大鼠肾脏缺血再灌注后纤维化改变的保护作用及其机制。方法将18只大鼠随机分为3组，每组6只。假手术组：戊巴比妥钠麻醉（40ms／kg）后，取腹正中切口进入腹腔，游离双侧肾脏，切除右肾后缝合腹壁；缺血再灌注组：在切除右肾后，用无创伤血管夹夹闭左肾动、静脉45min进行缺血，然后再灌注8周，余同假手术组；青蒿琥酯组：再灌注24h后，开始青蒿琥酯（每天25ms／kg）灌胃处理，假手术组和缺血再灌注组等体积蒸馏水灌胃，余同缺血再灌注组。再灌注8周后，检测各组大鼠血清肌酐、尿素氮水平，苏木素一伊红（HE）染色、Masson染色观察肾组织结构变化和纤维化改变，免疫组织化学观察纤维连接蛋白（Fibronectin）表达，实时荧光定量聚合酶链反应（Real-timePCR）检测α-平滑肌肌动蛋白（α-5MA）水平，Westernblot检测结缔组织生长因子（CTGF）和转化生长因子-β1（TGF-β1）的表达水平。结果在假手术组、缺血再灌注组和青蒿琥酯组中，血清肌酐（μmol／L）分别为30．13±1．52、32．36±2．67、31．29±1．36，血清尿素氮（mmol／L）分别为6．86±0．79、7．43±1．42、7．27±0．61，假手术组的血清肌酐、尿素氮水平明显比缺血再灌注组和青蒿琥酯组低（P〈0．05），而缺血再灌注组的血清肌酐、尿素氮水平显著高于青蒿琥酯组（P〈0．05）。HE染色可见缺血再灌注组中的肾小管上皮细胞明显变性坏死，有大量炎性细胞浸润，而青蒿琥酯可以明显减轻损伤。Masson染色结果可见缺血再灌注组纤维化改变明显，而青蒿琥酯可以明显减轻纤维化改变。与假手术组比较，缺血再灌注组和青蒿琥酯组Fibronectin、α-SMA、CTGF和TGF-β1水平均显著升高，而青蒿琥酯组的上述指标明显低于缺血再灌注组。结论青蒿琥酯可以减轻大鼠肾脏缺血再灌注损伤导致的纤维化改变，其机制可能与抑制相关蛋白的表达有关。

Objective To investigate the effect of artesunate on fibrosis induced by renal ischemia and reperfusion injury （IRI） in rats. Methods Eighteen SD rats were randomly divided into three groups： sham operated group, IRI group and artesunate group. Each group had 6 rats. In sham operated group, the right kidneys of rats were excised. In IRI group, left renal arteries and veins of rats were clamped for 45 min, then vascular clamp was removed and the rats were subjected to reperfusion for 8 weeks. In artesunate group, operation steps were the same as those in IRI group, but 24 h after reperfusion, artesunate was giv- en by intragastric administration [ 25 mg/（ kg· d） ]. In sham operated group and IRI group, equal volume of distilled water was given. The parameters （blood urea nitrogen and creatine） of renal function were de- termined by auto - biochemieal analyzer. The expression of α- smooth muscle aetin （ α - SMA） was de- tected by real - time quantitative polymerase chain reaction （ Real - time PCR） and the changes of renal tissue were examined by hematoxylin and eosin （HE） staining and Masson staining. Immunohistochemistry was performed to detect fibronectin. The expression of connective tissue growth factor （CTGF） and trans- forming growth factor - β1 （ TGF - β1 ） was detected by Western blotting. Results Among sham operated group, IRI group and artesunate group, creatine levels （μmol/L） were 30. 13 ±1.52, 32. 36 ± 2. 67 and 31.29 ± 1.36, and blood urea nitrogen levels （mmol/L） were 6. 86± 0. 79, 7. 43 ± 1.42 and 7.27 ± 0. 61, respectively. There was no significant difference in renal function between sham operated group and IRI group or artesunate group. HE staining and Masson staining indicated that artesunate could reduce the injury and fibrosis caused by renal !RI. As compared with sham operated group, fibronectin, α - SMA, CTGF and TGF -β1 were significantly increased in IRI group and artesunate group. However, these targets in artesunate group were significantly decreased as compared with those in IRI group. Conclusion Artesu- nate can alleviate fibrosis induced by renal renal IRI probably by inhibiting the expression of fibroneetin, α - SMA, CTGF and TGF -β1.