虾青素对大强度运动致大鼠骨骼肌氧化应激损伤及细胞凋亡的影响

EFFECTS OF ASTAXANTHIN ON OXIDATIVE STRESS INJURY AND APOPTOSIS OF SKELETAL MUSCLE INDUCED BY HIGH-INTENSITY EXERCISE IN RATS

目的探讨虾青素(astaxanthin)调控核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)通路相关蛋白质表达改善大强度运动大鼠骨骼肌氧化应激及细胞凋亡水平的作用机制。方法40只Wistar大鼠随机分为安静组(C,n=10)、大强度运动组(HI,n=15)和虾青素+大强度运动组(AHI,n=15)。C组不进行运动,HI组、AHI组进行6 w递增负荷跑台训练(起始:速度10 m/min,时间10 min,坡度10°;终止:速度40 m/min,时间180 min,坡度10°)。训练期间AHI组以20 mg/kg、5 ml/kg虾青素灌胃,C组、HI组灌以等体积大豆油。6 w训练结束24 h后取材(血液及比目鱼肌),放射免疫法检测血清睾酮(testosterone,T)与皮质酮(corticosterone,Cor)含量,全自动生化分析仪检测血清肌酸激酶(creatine kinase,CK)与乳酸脱氢酶(lactic dehydrogenase,LDH)活性;HE染色观察骨骼肌形态,Tunel染色检测骨骼肌细胞凋亡水平,免疫组织化学法检测骨骼肌B淋巴细胞瘤-2(B-celllymphoma-2,Bcl-2)、Bcl-2关联X蛋白(bcl-2 associated X protein,Bax)、Nrf2与血红素氧合酶1(heme oxygenase-1,HO-1)蛋白质表达,Elisa法检测骨骼肌超氧化物歧化酶(superoxide dismutase,SOD)活性及丙二醛(malondialdehyde,MDA)含量。结果与C组相比,HI组骨骼肌产生炎性改变,血清T/Cor显著降低(P<0.01),CK、LDH显著升高(P<0.01),骨骼肌Bcl-2、Bcl-2/Bax、SOD、Nrf2、HO-1显著降低(P<0.01),细胞凋亡水平及Bax、MDA显著升高(P<0.01);与HI组相比,AHI组骨骼肌炎性细胞浸润较轻,血清T/Cor显著升高(P<0.01),CK、LDH显著降低(P<0.01),骨骼肌Bcl-2、Bcl-2/Bax、SOD、Nrf2、HO-1显著升高(P<0.01或P<0.05),细胞凋亡水平及Bax、MDA显著降低(P<0.01)。结论6w递增负荷训练诱发大鼠出现过度训练综合症,骨骼肌氧化应激及凋亡加剧,功能/结构出现损伤性变化。虾青素可通过调控Nrf2信号通路相关蛋白质表达,增强下游抗氧化酶活性,缓解氧化应激及细胞凋亡,保护骨骼肌功能/结构。

Objective To explore the mechanism of action by astaxanthin in regulating nuclear factor erythroid 2-related factor 2(Nrf2)pathway related protein expression to improve oxidative stress and apoptosis of skeletal muscle induced by high-intensity exercise in rats.Methods 40 Wistar rats were randomly divided into the control group(C,n=10),high-intensity exercise group(HI,n=15),and astaxanthin plus high-intensity exercise group(AHI,n=15).Rats in Group C did not exercise,and rats in group HI and AHI received increasing load treadmill training for 6 weeks(initialization:velocity 10 m/min,time 10 min,grade 10°;termination:velocity 40 m/min,time 180 min,grade 10°).During the training,rats in group AHI was given astaxanthin(20 mg/kg,5 ml/kg),while rats in group C and group HI were given equal volume of soybean oil.Samples(blood and soleus)were taken from rats 24 h after 6-week training,serum testosterone(T)and corticosterone(Cor)were detected by radioimmunoassay,and serum creatine kinase(CK)and lactic dehydrogenase(LDH)by automatic biochemical analyzer;the morphology of the skeletal muscle was observed by HE staining,apoptosis of skeletal muscle cells was detected by TUNEL assay,the expressions of B-cell lymphoma-2(Bcl-2),bcl-2 associated X protein(Bax),Nrf2 and heme oxygenase-1(HO-1)in skeletal muscle were measured by immunohistochemistry,and superoxide dismutase(SOD)and malondialdehyde(MDA)by Elisa.Results Compared with rats in group C,inflammatory changes occurred in skeletal muscle in rats of group HI,serum T/Cor significantly decreased(P<0.01),CK and LDH significantly increased(P<0.01),skeletal muscle Bcl-2,Bcl-2/Bax,SOD and Nrf2,HO-1 significantly decreased(P<0.01),apoptosis,Bax and MDA significantly increased(P<0.01).Compared with those in group HI,rats in group AHI had less infiltration of skeletal muscle inflammatory cells,serum T/Cor significantly increased(P<0.01),CK and LDH significantly decreased(P<0.01),skeletal muscle Bcl-2,Bcl-2/Bax,SOD,Nrf2 and HO-1 significantly increased(P<0.01 or P<0.05),apoptosis,Bax and MDA significantly decreased(P<0.01).Conclusion Rats developed overtraining syndrome,oxidative stress and apoptosis,and functional/morphological damage of skeletal muscle after 6-week increasing load training.Astaxanthin enhanced the activity of antioxidant enzymes,alleviated oxidative stress and apoptosis,and protected skeletal muscle function/morphology by regulating the expression of proteins related to Nrf2 signaling pathway.