Klotho蛋白联合红花注射液对小鼠心肌损伤的保护作用

Protective effect of Klotho protein combined with safflower injection on myocardial injury in mice

目的分析Klotho蛋白联合红花注射液(H)对小鼠心肌损伤的保护作用。方法采用异丙肾上腺(ISO)制备小鼠心肌缺血损伤模型。将60只6-7周龄小鼠分成生理盐水(NS)组、ISO组、Klotho(KL)组、丹红(H)组及KL+H组。分别计算心脏质量指数(HW/BW),测定血清乳酸脱氢酶(LDH)活性、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力、血清Klotho浓度,心肌组织石蜡包埋行HE染色、Massion染色、Caspase3染色。结果 ISO实验模型符合要求。与NS组比较,ISO组HW/BW、乳酸脱氢酶(LDH)活性、丙二醛(MDA)含量均显著增加(P<0.01),SOD活力显著减低(P<0.01)。KL组、H组、KL+H组HW/BW%、LDH活性、MDA含量均显著低于ISO组(P<0.01),SOD活力、Klotho浓度显著高于ISO组(P<0.01)。KL+H组HW/BW%、LDH活性显著低于H组(P<0.01),MDA含量显著低于H组(P<0.05),SOD活力显著高于H组(P<0.01),Klotho浓度显著高于H组(P<0.05)。HE染色结果显示:ISO组小鼠心肌细胞横径明显大于NS组,伴随着肌纤维紊乱和心肌间质成纤维细胞增生。治疗组小鼠心肌细胞横径明显小于ISO组;Masson染色结果显示,ISO组小鼠心肌间质及血管壁有大量胶原纤维分布,而治疗组小鼠的心肌间质及血管壁胶原纤维数量较ISO组明显减少;Caspase 3染色结果显示,各治疗组棕黄色染色颗粒分布较ISO组明显减少。染色阳性结果表现为胞质中有大量棕黄色染色颗粒分布。结论 Klotho蛋白联合红花注射液可升高SOD活力,降低LDH活性、MDA含量和HW/BW%;两者联合可能通过抑制LDH活性和降低MDA水平、增加SOD活力和Klotho浓度对心肌损伤起到协同保护的作用。

Objective To explore the protective effect of Klotho protein combined with safflower injection on isoproterenol-induced my- ocardial injury in mice. Methods The myocardial ischemia injury model was induced by injecting isoproterenol （ISO） in mice. Sixty 6-7-week-old mice were randomly divided into five groups as follow： 0.9% sodium chloride solution（NS） group, ISO group, Klotho （KL） group, safflower（H） group and KL ＋ H group. The cardiac index（HW/BW%） was calculated, serum lactate dehydrogenase （LDH） activity was measured by enzyme standard method, the level of malondialdehyde （MDA） was detected by thiobarbituric acid method（TBA） , superoxide dismutase（SOD） activity was determined by WST-1 method, and the concentration of serum Klotho was measured by ELSIA. The cardiac muscular tissue was imbedded with paraffin and stained with HE, Massion and Caspase 3, respec- tively. Results Compared with /~S group, HW/BW%, LDH activity, MDA level were significantly increased in ISO group （P 〈 0.01 ）, and SOD activity was significantly decreased（P 〈 0.01 ）. HW/BW%, LDH activity and MDA level in KL group, H group and in KL ＋ H group were significantly lower than in ISO group （ P 〈 0.01 ）, while concentrations of SOD and Klotho were significantly higher（P 〈0.01 ）. HW/BW, LDH activity in KL ＋ H group were significantly lower than in H group（P 〈0.01 ）, MDA level was also significantly lower（ P 〈 0.05 ） , while SOD activity was significantly higher than in H group（ P 〈 O. 01 ）, and the concentration of Klotho was also significantly higher（ P 〈 O. 05 ）. HE staining results showed that the myocardial cell diameter in ISO group was bigger than in NS group, accompanied by disordered muscle fibers and hyperplastic myocardial interstitial fibroblasts. The cardiomyocytes diameters in treatment groups were much smaller than in ISO group. Masson staining results showed that there was a lot of collagen fiber distribu- tion in the myocardial interstitial and hemal walls in ISO group, but they were significantly decreased in treatment groups. Caspase 3 dyeing results showed that the tan dyeing numbers in treatment group were significantly less than in ISO group. Staining positive results were large brownish yellow dye in kytoplasm. Conclusion Klotho protein combined with safflower injection may protect from myocar- dial injury by inhibiting LDH activity and decreasing MDA level, increasing SOD activity and concentration of Klotho.