Research on the role and mechanism of trimetazidine in ICU-acquired weakness
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目的 研究曲美他嗪(trimetazidine,TMZ)在重症加强治疗病房获得性衰弱(intensive care unit-acquired weakness,ICU-AW)中的作用及机制.方法 选取野生型雄性C57BL/6小鼠70只并采用不同浓度脂多糖(lipopolysaccharide,LPS)腹腔注射构建ICU-AW小鼠模型,观察各组小鼠体重、抓力、96h存活率,筛选出最佳LPS浓度和取材时间,进一步检测小鼠腓肠肌萎缩蛋白Atrogin-1和肌肉细胞特异性泛素蛋白连接酶1(muscle-specific RING finger protein 1,MuRFl)mRNA和蛋白表达验证造模是否成功,根据前期结果以LPS(12 mg/kg)为后续造模浓度.另取70只小鼠随机分为正常对照组(Normal组)、LPS溶剂(Vehicle)组、LPS组、LPS+TMZ 溶剂组、LPS+TMZ 组、LPS+TMZ+AC-YVAD-CMK(AC)溶剂组、LPS+TMZ+AC 组,每组 10 只.Normal组不做任何干预,Vehicle组腹腔注射LPS等体积生理盐水,剩余各组均腹腔注射LPS(12 mg/kg);LPS注射完成后,LPS+TMZ组、LPS+TMZ+AC溶剂组、LPS+TMZ+AC组予以TMZ(5 mg/kg)灌胃,每天1次,持续4 d;LPS+TMZ溶剂组予以TMZ等量生理盐水灌胃,每天1次,持续4 d;其中,LPS+TMZ+AC组在LPS注射前 1 h 腹腔注射半胱氨酸天冬氨酸蛋白酶 1(cysteinyl aspartate specific proteinase 1,Caspase-1)抑制剂 AC-YVAD-CMK(AC,6.5 mg/kg),LPS+TMZ+AC溶剂组注射等量AC溶剂磷酸盐缓冲液.TMZ治疗结束后,检测各组小鼠体重、抓力、96 h 存活率,腓肠肌 MuRF1、Atrogin-1、Caspase-1、消皮素 D(gasdermin D,GSDMD)的 mRNA 和蛋白表达,以及小鼠血清IL-1β和IL-18的浓度,腓肠肌HE染色观察组织病理变化.结果 与Normal组相比,LPS(12 mg/kg)组和LPS(14 mg/kg)组小鼠体重、抓力均显著下降且在3～5d时衰弱最明显(P＜0.05),但LPS(12 mg/kg)组存活率高于LPS(14 mg/kg)组(P＜0.05),LPS(12 mg/kg)组小鼠腓肠肌较Normal组骨骼肌明显萎缩,且MuRF1和Atrogin-1的基因及蛋白表达均显著升高(P＜0.05),最终选取注射LPS(12 mg/kg)4 d(96 h)为后续实验造模和取材条件.LPS组骨骼肌中Caspase-1、GSDMD的mRNA和蛋白表达较Normal组和Vehicle组明显升高(P＜0.01),血清IL-1β和IL-18的浓度明显升高(P＜0.01);TMZ组小鼠体重、抓力、存活率、肌肉萎缩程度均较LPS组及TMZ溶剂组明显改善(P＜0.05),腓肠肌MuRF1、Atrogin-1、Caspase-1、GSDMD的基因和蛋白含量明显降低(P＜0.05),血清IL-1β和IL-18的浓度明显降低(P＜0.05);LPS+TMZ+AC组小鼠体重、抓力、存活率、肌肉萎缩程度较 LPS+TMZ 组、LPS+TMZ+AC 溶剂组明显改善(P＜0.05),腓肠肌 MuRF1、Atrogin-1、Caspase-1、GSDMD的基因和蛋白含量降低(P＜0.05),血清IL-1β和IL-18的浓度降低(P＜0.05).结论 TMZ能够通过抑制Caspase-1/GSDMD介导的细胞焦亡发挥骨骼肌保护作用,对ICU-AW的防治具有重要参考价值.
Objective To investigate the role and mechanisms of trimetazidine(TMZ)in intensive care unit-acquired weakness(ICU-AW).Methods Seventy wild-type male C57BL/6 mice were selected and the ICU-AW mouse model was constructed by intraperitoneal injection of different concentrations of lipopolysaccharide(LPS).The body weights,grip strengths,and 96-hour survival rates of each group were observed,and the optimal concentration of LPS and time of sampling were screened out,the mRNA and protein expression of the gastrocnemius muscle atrophic proteins Atrogin-1 and muscle-specific RING finger protein 1(MuRF1)were further detected to verify the success of modelling,and LPS(12 mg/kg)was used as the subsequent modelling concentration according to the preliminary results.After successful modelling,another 70 mice were randomly divided into normal control group(Normal group),LPS solvent(Vehicle)group,LPS group,LPS+TMZ solvent group,LPS+TMZ group,LPS+TMZ+AC-YVAD-CMK(AC)solvent group,and LPS+TMZ+AC group,with 10 mice in each group.The Normal group did not have any intervention;the Vehicle group was injected intraperitoneally with an equal volume of saline with LPS;the remaining groups were injected intraperitoneally with LPS(12 mg/kg);after the completion of the LPS injection,the LPS+TMZ group,the LPS+TMZ+AC solvent group,and the LPS+TMZ+AC group were given TMZ(5 mg/kg)by gastric gavage once a day for 4 days.The LPS+TMZ solvent group was given TMZ equivalent saline gavage once a day for 4 days.The LPS+TMZ+AC group was injected intraperitoneally with the cysteinyl aspartate specific proteinase 1(Caspase-1)inhibitor AC-YVAD-CMK(AC,6.5 mg/kg)1 h before LPS injection,and the LPS+TMZ+AC solvent group was injected with an equal amount of AC solvent phosphate buffer.At the end of TMZ treatment,body weight,grip strength,96-hour survival rate,mRNA and protein expression of MuRFl,Atrogin-1,Caspase-1,and gasdermin D(GSDMD)in gastrocnemius muscle,as well as serum IL-1 β and IL-18 concentrations in mice were detected in each group,and the gastrocnemius muscle was stained with HE to observe histopathological changes.Results Compared with the Normal group,mice in the LPS(12 mg/kg)and LPS(14 mg/kg)groups showed significant decreases in body weight and grasping strength and the weakening was most obvious at 3-5 d(P＜0.05),but the survival rate of the LPS(12 mg/kg)group was higher than that of the LPS(14 mg/kg)group(P＜0.05),the HE staining of gastrocnemius muscle showed that the mice in the LPS(12 mg/kg)group was significantly atrophied compared with that of the Normal group,and the gene and protein expression of MuRFl and Atrogin-1 were significantly elevated(P＜0.05),and the mice injected with LPS(12 mg/kg)for 4 days(96 h)were finally selected as the conditions for subsequent experimental modelling and sampling.The mRNA and protein expression of Caspase-1 and GSDMD in skeletal muscle was significantly higher in the LPS group compared with the Normal and Vehicle groups(P＜0.01),and the concentrations of serum IL-1 β and IL-18 were significantly higher(P＜0.01).Mice in the TMZ group showed significant improvement in body weight,grip strength,survival rate,and degree of muscle atrophy compared with the LPS and TMZ solvent groups(P＜0.05);gene and protein levels of MuRF1,Atrogin-1,Caspase-1,and GSDMD in the gastrocnemius muscle were significantly reduced(P＜0.05);and levels of serum IL-1β and IL-18 were significantly reduced(P＜0.05));the mice in the LPS+TMZ+AC group had significantly improved body weight,grip strength,survival rate,and muscle atrophy compared with the LPS+TMZ group and the LPS+TMZ+AC solvent group(P＜0.05),and the gene and protein contents of MuRF1,Atrogin-1,Caspase-1,and GSDMD in the gastrocnemius muscle were reduced(P＜0.05),and the serum IL-1β and IL-18 concentrations were reduced(P＜0.05).Conclusion TMZ is able to exert a skeletal muscle protective effect by inhibiting Caspase-1/GSDMD-mediated pyroptosis,which is an important reference for the prevention and treatment of ICU-AW.
Intensive care unit-acquired weaknesstrimetazidinesepsispyroptosiscysteinyl aspartate specific proteinase 1gasdermin D
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