木犀草素调控AMPK/NLRP3轴介导的细胞焦亡对病毒性心肌炎的保护作用

Protective effect of luteolin on viral myocarditis by regulating AMPK/NLRP3 axis-mediated pyroptosis

目的探讨木犀草素调控AMP活化激酶(AMPK)/NOD样受体蛋白3(NLRP3)轴介导的细胞焦亡途径对病毒性心肌炎(VM)小鼠的保护作用。方法将300只BALB/c雄性小鼠随机分为对照组、CVB3病毒组(0.1 mL 1×10^(-5)CVB3)、木犀草素低剂量组(5 mg/kg木犀草素)、木犀草素高剂量组(20 mg/kg木犀草素)、AMPK激活剂组(200 mg/kg AICAR)和木犀草素高剂量+AMPK抑制剂组(20 mg/kg木犀草素+20 mg/kg Compound C)。除对照组外,各组小鼠均用0.1 mL 1×10^(-5)CVB3处理,并按照要求饲养。采用试剂盒法检测小鼠心肌匀浆中LDH、CK-MB、IL-1β、IL-6水平;HE染色观测小鼠心肌组织病理变化,并参照rezkalla法对小鼠心肌病理学进行评分;采用TUNEL试剂盒检测心肌组织凋亡情况;采用Western blot法检测心肌组织Bax、Bcl-2、p-AMPK、AMPK、NLRP3、ASC、Caspase-1水平。结果与对照组相比,CVB3病毒组小鼠心肌匀浆LDH、CK-MB、IL-1β、IL-6、病理积分、心肌凋亡率、Bax蛋白及NLRP3、ASC、Caspase-1蛋白水平均显著升高(均P<0.05,Bcl-2蛋白和p-AMPK/AMPK水平显著降低(均P<0.05);与CVB3病毒组相比,木犀草素低剂量组、木犀草素高剂量组、AMPK激活剂组小鼠心肌匀浆LDH、CK-MB、IL-1β、IL-6、病理积分、心肌凋亡率、Bax蛋白及NLRP3、ASC、Caspase-1蛋白水平均显著降低(均P<0.05,Bcl-2蛋白和p-AMPK/AMPK水平显著升高(均P<0.05);与木犀草素高剂量组相比,木犀草素高剂量+AMPK抑制剂组小鼠心肌匀浆LDH、CK-MB、IL-1β、IL-6、病理积分、心肌凋亡率、Bax蛋白和NLRP3、ASC、Caspase-1蛋白均显著升高(均P<0.05),Bcl-2蛋白和p-AMPK/AMPK水平显著降低(均P<0.05)。结论木犀草素通过激活AMPK/NLRP3介导的细胞焦亡发挥对VM小鼠心肌的保护作用,为VM的临床治疗提供了参考依据。

Objective To investigate the protective effect of luteolin on AMP-activated kinase(AMPK)/NOD-like receptor protein 3(NLRP3)axis-mediated pyroptosis pathway in mice with viral myocarditis(VM).Methods Three-hundred BALB/c male mice were randomly separated into control group,CVB3 virus group(0.1 mL 110^(-5)CVB3),low-dose luteolin group(5 mg/kg luteolin),high-dose luteolin group(20 mg/kg luteolin),AMPK activator group(200 mg/kg AICAR),and high dose luteolin+AMPK inhibitor group(20 mg/kg luteolin+20 mg/kg Compound C),except the control group,all were treated with 0.1 mL 110^(-5)CVB3,and each group was raiseed as required.The kit method was performed to measure the levels of LDH,CK-MB,IL-1βand IL-6 in myocardial homogenate;HE staining was performed to observe the pathological changes of mouse myocardium,and the pathology of mouse myocardium was scored according to the rezkalla method;TUNEL kit was performed to measure myocardial tissue apoptosis;Western blot was performed to measure the levels of Bax,Bcl-2,p-AMPK,AMPK,NLRP3,ASC and Caspase-1 in myocardial tissue.Results Compared with the control group,LDH(8.02±1.01 vs 66.53±8.30),CK-MB(130.51±16.31 vs 358.24±44.78)and IL-1 in myocardial homogenate of mice in CVB3 virus groupβ(30.50±3.88 vs 135.27±19.84),IL-6(55.27±3.87 vs 341.53±42.66),pathological score(0 vs 3.42±0.41),myocardial apoptosis rate(4.87±0.66 vs 23.71±2.86),Bax protein(0.35±0.04 vs 1.77±0.21),NLRP3(0.24±0.03 vs 1.32±0.21),ASC(0.70±0.08 vs 2.41±0.30),caspase-1(0.40±0.05 vs 2.57±0.32)protein levels were significantly increased(all P<0.05),The levels of Bcl-2 protein(2.03±0.24 vs 0.34±0.04)and p-ampk/AMPK(0.94±0.12 vs 0.12±0.04)decreased significantly(all P<0.05);Compared with CVB3 virus group,LDH(66.53±8.30 vs 58.72±7.34,38.54±4.81,41.02±5.13),CK-MB(358.24±44.78 vs 299.46±37.33,195.76±24.40,224.38±27.96)and IL-1 in myocardial homogenate of mice in luteolin low-dose group,luteolin high-dose group and AMPK activator groupβ(135.27±19.84 vs 100.57±12.50,72.69±9.02,81.06±10.12),IL-6(341.53±42.66 vs 299.74±37.46,201.91±25.38,223.40±27.87),pathological score(3.42±0.41 vs 2.03±0.23,1.37±0.16,1.52±0.18),myocardial apoptosis rate(23.71±2.86 vs 18.79±2.33,13.50±1.63,14.06±1.71)The levels of Bax protein(1.77±0.21 vs 1.06±0.13,0.70±0.08,0.74±0.09),NLRP3(1.32±0.21 vs 1.01±0.12,0.72±0.09,0.74±0.09),ASC(2.41±0.30 vs 2.01±0.24,1.60±0.21,1.64±0.21),caspase-1(2.57±0.32 vs 1.83±0.23,1.24±0.15,1.42±0.17)decreased significantly(all P<0.05),Bcl-2 protein(0.34±0.04 vs 0.72±0.09,1.50±0.15,1.42±0.17)and p-ampk/AMPK level(0.12±0.04 vs 0.31±0.04,0.54±0.06,0.46±0.05)increased significantly(all P<0.05);Compared with luteolin high-dose group,luteolin high-dose+AMPK inhibitor group had LDH(38.54±4.81 vs 41.02±5.13),CK-MB(195.76±24.40 vs 224.38±27.96)and IL-1 in myocardial homogenateβ(72.69±9.02 vs 81.06±10.12),IL-6(201.91±25.38 vs 223.40±27.87),pathological score(1.37±0.16 vs 1.52±0.18),myocardial apoptosis rate(13.50±1.63 vs 14.06±1.71),Bax protein(0.70±0.08 vs 0.74±0.09),NLRP3(0.72±0.09 vs 0.74±0.09),ASC(1.60±0.21 vs 1.64±0.21),caspase-1(1.24±0.15 vs 1.42±0.17)protein were significantly increased(all P<0.05),The levels of Bcl-2 protein(1.50±0.15 vs 1.42±0.17)and p-ampk/AMPK(0.54±0.06 vs 0.46±0.05)decreased significantly(all P<0.05).Conclusion Luteolin can protect the myocardium of VM mice by activating AMPK/NLRP3 mediated cell death,which provides a reference basis for the clinical treatment of VM.