冬凌草甲素对小鼠下肢缺血再灌注损伤的保护作用

Protective effects of Oridonin on hind limb ischemia-reperfusion injury mice

目的探讨冬凌草甲素对小鼠下肢缺血再灌注损伤的保护作用及其机制。方法采用随机分组方式将48只8周龄C57BL/6小鼠分成空白组、模型组、低剂量组和高剂量组,每组12只。模型组、低剂量组和高剂量组进行缺血再灌注处理,低剂量组和高剂量组术前连续7 d腹腔注射冬凌草甲素(10 mg/kg和20 mg/kg)。观察小鼠下肢肌肉形态学和组织学变化;检测小鼠血液指标血清肌酸激酶、血清乳酸脱氢酶、血清白细胞介素-6(IL-6)和全血白细胞的变化;用蛋白印迹法对炎症相关蛋白、凋亡相关蛋白和核苷酸结合寡聚化结构域样受体蛋白3炎症小体(NLRP3)相关蛋白进行检测;原位末端标记法(TUNEL)检测细胞凋亡水平。两组间比较采用t检验,多组间比较采取单因素方差分析。结果在下肢缺血再灌注小鼠中,低剂量组和高剂量组与模型组比较,(1)血清肌酸激酶等组织损伤指标明显低于模型组(1995.67±129.25、1753.00±152.86比2268.67±116.20,F=22.360,P<0.01);(2)血清IL-6等组织炎症指标明显低于模型组(10.89±2.06、8.71±0.86比16.56±2.62,F=25.409,P<0.01);(3)凋亡指数等细胞凋亡指标明显低于模型组[(38.06±4.48)%、(22.31±3.55)%比(52.97±1.91)%,F=58.262,P<0.01];(4)检测肌肉组织NLRP3等炎症小体相关蛋白的表达水平,低剂量组和高剂量组中NLRP3蛋白表达均低于模型组(2.60±0.20、1.51±0.26比4.95±0.14,F=215.416,P<0.01)。结论冬凌草甲素能够抑制NLRP3炎症小体的激活,减少炎性反应和细胞凋亡,是发挥对小鼠下肢缺血再灌注损伤保护作用的重要机制之一。

Objective To investigate the protective role of Oridonin on hindlimb IR injury mice and its pathophysiological mechanisms.Methods Forty-eight adult male C57BL/6 mice were randomly divided into four groups:Control group,Model group,Low dose group and High dose group.Model group,Low dose group and High dose group were subjected to hindlimb IR injury.Oridonin was intraperitoneally injected daily for 7 days prior to hindlimb IR injury in Low dose group(10 mg/kg)and High dose group(20 mg/kg).Serum creatine kinase(CK),lactate dehydrogenase(LDH),IL-6,hematoxylin and eosin(HE)staining,terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining,Western blotting analysis were employed to explore the mechanisms by which Ori exerts a protective effect on a classical hindlimb IR model in mice.The comparison between two groups was done by the non-paired t test,and the comparison among multiple groups was carried out by the multi-factor variance analysis.Results Compare with Control group,the mice in Low dose group and High dose group have these changes:(1)Muscle damage has been alleviated significantly proved by the results such as serum CK(1995.67±129.25,1753.00±152.86 vs.2268.67±116.20,F=22.360,P<0.01);(2)The level of inflammation has been reduced significantly proved by the results such as serum IL-6(10.89±2.06,8.71±0.86 vs.16.56±2.62,F=25.409,P<0.01);(3)Apoptosis has been inhibited significantly proved by the results such as apoptosis index[(38.06±4.48)%,(22.31±3.55)%vs.(52.97±1.91)%,F=58.262,P<0.01];(4)The activation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)inflammasome has been blocked significantly proved by Western blotting(2.60±0.20,1.51±0.26 vs.4.95±0.14,F=215.416,P<0.01).Conclusion Oridonin can block the activation of NLRP3 inflammasome and reduce inflammatory response and apoptosis caused by hind limb ischemia-reperfusion injury,thus playing a protective role in mice.