血红素加氧酶通过抑制NLRP3炎性小体的活化减轻小鼠骨骼肌萎缩

Induction of heme oxygenase alleviates skeletal muscle atrophy in mice by inhibiting the activation of NLRP3 inflammasome

目的探讨诱导血红素加氧酶1(heme oxygenase-1,HO-1)是否通过抑制NLRP3炎性小体的活化减轻小鼠骨骼肌萎缩。方法32只8周龄雄性C57BL/6小鼠按照简单随机抽样法分为正常组、尾悬吊组(采用尾悬吊法建立下肢肌萎缩模型)、氯化血红素组(在尾悬吊组建模基础上腹腔注射10 mg/μL的氯化血红素hemin诱导HO-1表达)和锌原卟啉组(在氯化血红素组基础上腹腔注射10 mg/μL的ZnPP拮抗HO-1表达),每组8只;另取8只同龄同种系小鼠进行胫前肌慢病毒注射并同步建立下肢肌萎缩模型,左侧为慢病毒干预组(胫前肌注射NLRP3-RNAi慢病毒抑制NLRP3的表达),右侧为慢病毒对照组(胫前肌注射慢病毒空载体)。各组处理2周后,通过HE染色、肌肉体质量比观察肌肉大小的变化情况;免疫印迹法检测胫前肌NLRP3、ASC、Caspase-1等蛋白表达;ELISA法检测胫前肌IL-1β、IL-18含量及氧化应激分子改变。结果通过尾悬吊建立了典型的小鼠下肢肌萎缩模型,在诱导HO-1的实验中,与尾悬吊组相比,氯化血红素组肌萎缩明显减轻,炎性小体NLRP3及相关蛋白ASC、Caspase-1表达降低(P<0.05);锌原卟啉组NLRP3相关蛋白的表达与尾悬吊组无明显差异。通过慢病毒干预NLRP3表达,与慢病毒对照组相比,慢病毒干预组抑制NLRP3表达后肌萎缩明显减轻,同时NLRP3炎性小体相关蛋白表达降低(P<0.05),下游IL-1β、IL-18含量降低(P<0.05);肌组织氧化应激状态减轻(P<0.05)。结论诱导HO-1能通过抑制NLRP3炎性小体的活化减轻尾悬吊小鼠骨骼肌萎缩。

Objective To investigate whether induction of heme oxygenase-1(HO-1)can reduce skeletal muscle atrophy in mice by inhibiting the activation of NLRP3 inflammasome.Methods Thirty-two 8-week-old male C57BL/6 mice were randomly divided into normal group,model group(the model of lower limb muscular atrophy was established by tail suspension),heme chloride group(HO-1 expression was induced by intraperitoneal injection of hemin in the model mice),and zinc protoporphyrin group(on the basis of hemin chloride group,ZnPP was injected intraperitoneally to antagonize HO-1 expression),with 8 mice in each group.Another 8 mice at the same age were injected with lentivirus into tibialis anterior muscle and the model of lower limb muscle atrophy was established simultaneously,with the left side as lentivirus intervention subgroup(injection of lentiviral vector of NLRP3 RNAi to inhibit the expression of NLRP3),and the right side as lentivirus control subgroup(injection of blank lentiviral vector).The changes of muscle volume were observed by HE staining and muscle/body weight ratio.The protein levels of NLRP3,ASC and Caspase-1 in the tibialis anterior muscle were determined by Western blotting.The levels of IL-1βand IL-18 and oxidative stress molecules in the muscle tissues were determined by ELISA.Results The typical mouse model of lower extremity muscular atrophy was successfully established by tail suspension.HO-1 induction resulted in significantly reduced muscular atrophy and decreased expression of NLRP3 inflammasome related proteins ASC and Caspase-1 in the heme chloride group when compared with the model group(P<0.05).There were no significant differences in the protein levels of above 2 molecules between the zinc protoporphyrin group and the model group.Knockdown of NLRP3 by lentiviral vector led to significantly reduced muscle atrophy,decreased expression of NLRP3 inflammasome related proteins(P<0.05),and lower levels of downstream proteins IL-1βand IL-18(P<0.05)when compared with the lentivirus control subgroup;The oxidative stress of muscle tissue was alleviated(P<0.05).Conclusion Induction of HO-1 can reduce skeletal muscle atrophy in tail suspension mice by inhibiting the activation of NLRP3 inflammasome.