线粒体靶向多肽SS-31对小鼠后肢缺血再灌注损伤的保护作用

Protective effects of mitochondria- targeted peptides SS-31 against posterior limb ischemia reperfusion injury

目的观察线粒体靶向多肽SS-31对小鼠急性后肢缺血／再灌注损伤的保护作用。方法将60只C57BL／6小鼠随机分为5组（n=8）：对照组（C组）、生理盐水预防组（S＋IR组）、SS-31预防组（SS-31＋IR组）、生理盐水治疗组（IR＋S组）、SS-31治疗组（IR＋SS-31组）。空白组小鼠仅麻醉处理，实验组小鼠进行左后肢缺血90min以及再灌注8h，预防组在缺血前30min给药，治疗组在再灌注前30min给药，取小鼠左后肢肌肉测定腺嘌呤核苷三磷酸（ATP）、线粒体膜电位（MMP）、丙二醛（MDA）、细胞色素C（CytochromeC）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、活化型半胱天冬酶3（Cleaved—Caspase3）的含量以及超氧化物歧化酶（SOD）和过氧化氢酶（Catalase）活性和蛋白含量变化；观察小鼠左后肢肌肉组织的病理学变化。结果再灌注8h后SS-31两组与生理盐水两组比较，线粒体功能相关指标如MMP等显著升高（0．80±0．03、0．64±0．03比0．19±0．01、0．17±0．01，P预防=0．001，P治疗=0．003）。抗氧化相关指标如SOD酶活等明显升高[（49．13±0．94）、（41．98±1．20）U／mg比（25．88±1．11）、（25．52±1．51）U／mg，P预防=0．000，P治疗=0．001]，MDA含量明显下降[（0．81±0．04）、（1．08±0．05）nm／mg比（2.06±0．04）、（1．98±0．06）nm／mg，P预防=0．000，P治疗=0．000]，在Western blot结果中SS-31两组在TNF-α、IL—1β、胞质Cytochrome C及Cleaved—Caspase3的蛋白表达量均小于生理盐水两组。结论小鼠后肢缺血再灌注能引起肌肉的炎性反应、氧化应激及线粒体功能的损伤并诱导后肢骨骼肌细胞凋亡；线粒体靶向多肽SS-31的处理能够保护线粒体功能，抑制组织炎性反应及氧化应激，对小鼠后肢骨骼肌的缺血再灌注损伤有预防和治疗作用。

Objective To investigate whether the treatment of mitochondrion -targeted peptides SS -31 could protect posterior limb from ischemia -reperfusion injury in a mouse model. Methods Sixty C57BL/6 mice were randomly divided into five groups ： control group （ group C ） ; saline premedication group （group S ＋ IR） and SS- 31 premedication group （group SS- 31 ＋ IR） underwent intraperitonealin- jection of saline or SS -31 （5 mg/kg） 30 rain before ischemia; saline therapy group （group IR ＋ S） and SS - 31 therapy group （ group IR ＋ SS - 31 ） underwent intraperitoneafinjection of saline or SS - 31 （5 mg/kg） 30 rain before reperfusion. Orthodontic rubber was applied to the left greater trochanter for 1.5 h and followed by an 8 h of reperfusion to build isehemia reperfusion model. Rats were sacrificed at the end of the reperfusion and skeletal muscle samples were collected todetect the level of adenosinetriphosphate （ ATP ） , mitoehondrial membrane potential （ MMP ） , Malondialdehyde （ MDA ） , Cytoehrome C, tumor necrosis factor - α （ TNF - α）, interleukin - 1β （ IL - 6β）, Cleaved - Caspase 3, Superoxide Dismutase （SOD） catalase and the activity of SOD and catalase. The muscle sample will be examined through light microscope for histological assessment. Results The histological changes were milder in both SS - 31 ＋ IR group and IR ＋ SS - 31 group than in S ＋ IR and IR ＋ S group. Compared with S ＋ IR group and IR ＋ S group, the mitochondrial function related indicators like MMP level （ 0. 80 ± 0.03,0. 64 ± 0. 03 vs. 0. 19 ±0.01, 0. 17 ±0. 01, P =0. 001, P =0. 003） and antioxidant related indicator like the activity of SOD [（49.13 ± 0.94）, （41.98 ±1.20） vs. （25.88±1.11）, （25.52 ± 1.51） U/nag, P=0.000, P=0.001] were higher and the MDA level [（0.81 ±0.04）,（1.08 ±0.05） vs. （2.06 ±0.04）,（ 1.98 ±0. 06） nm/mg, Ppre =0. 000, Pthero =0. 0003 was lower in SS - 31 ＋ IR group and IR ＋ SS - 31 group. The protein levels of SOD, catalase and mitoehondria] cytoehrome C in SS - 31 ＋ IR group and IR ＋ SS - 31 group were significantly higher than in S ＋ IR group and IR ＋ S group and the levels of TNF - α, IL- 1β and cytoplasmic eytochrome C were significantly lower in SS -31 ＋ IR group and IR ＋ SS -31 group than in S ＋ IR group and IR ＋ S group. Conclusion The findings suggest that SS - 31 can remark- ably alleviate the posterior limb ischemia reperfusion injury.