5-羟色胺在电针预处理脓毒症小鼠心肌损伤中的作用机制研究

The mechanism of 5-hydroxytryptamine in electroacupuncture pre-treatment for myocardial injury in sepsis mice

目的观察电针足三里穴预处理脓毒症心肌损伤小鼠心肌组织中5-羟色胺(5-HT)水平的变化,探讨电针在脓毒症心肌损伤中发挥的保护效应及可能机制。方法按随机数字表法将20只雄性C57BL/6小鼠分为对照组(NC组)、脓毒症模型组(LPS组)、电针组(EA组)、电针加氟西汀组(EA+FLU组),每组5只。采用腹腔注射脂多糖(LPS)10 g/L的方法建立脓毒症心肌损伤小鼠模型;NC组腹腔注射等量生理盐水。制模前3 d,EA组和EA+FLU组电针双侧足三里穴15 min,每日1次,连续3 d;EA+FLU组在电针前腹腔注射氟西汀1.4 g/L。制模后,苏木素-伊红(HE)染色观察心肌组织病理学改变;检测血清中炎症因子白细胞介素(IL-6、IL-1β)、肿瘤坏死因子-α(TNF-α)水平,心肌内5-HT含量,心肌损伤标志物肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)水平,以及心肌组织中三磷酸腺苷(ATP)、乳酸含量;定量聚合酶链反应(qPCR)检测心肌组织5-羟色胺转运体(5-HTT)、己糖激酶2(HK2)、葡萄糖转运体4(GLUT4)的mRNA表达;免疫荧光法检测心肌组织中GLUT4的表达。结果与NC组相比,LPS组和EA+FLU组血清IL-6、IL-1β、TNF-α含量,心肌内5-HT含量,心肌组织损伤标志物CK-MB、cTnI含量均明显升高。与LPS组相比,EA组上述指标均明显下降〔IL-6(ng/L):443.03±156.16比19843.75±0.00,IL-1β(ng/L):75.72±10.60比894.66±350.88,TNF-α(ng/L):46.17±4.71比533.01±170.58,5-HT(μg/L):161.19±5.96比244.74±14.38,CK-MB(ng/L):468.21±12.46比662.02±22.54,cTnI(ng/L):0.83±0.05比0.99±0.08,均P<0.05〕。与NC组相比,LPS组、EA组和EA+FLU组心肌组织ATP水平均明显下降,乳酸水平均明显升高。与LPS组相比,EA组心肌组织ATP水平明显上升,乳酸水平明显下降〔ATP(mmol/L):0.10±0.01比0.08±0.01,乳酸(mmol/L):56.03±1.07比72.45±4.32,均P<0.05〕。与NC组相比,LPS组心肌组织HK2 mRNA表达明显上升,GLUT4、5-HTT mRNA表达明显下降。与LPS组相比,EA组心肌组织HK2 mRNA表达明显下降,GLUT4、5-HTT mRNA表达明显上升〔HK2 mRNA(相对表达量):0.73±0.19比1.82±0.57,GLUT4 mRNA(相对表达量):1.00±0.33比0.47±0.18,5-HTT mRNA(相对表达量):1.18±0.31比0.38±0.15,均P<0.05〕。与NC组相比,LPS组和EA+FLU组GLUT4荧光强度明显减弱。与LPS组相比,EA组GLUT4荧光强度明显增强。结论电针足三里穴预处理可减少脓毒症小鼠心肌组织中5-HT含量,其调控机制可能与调节5-HTT和GLUT4有关。

Objective To observe the changes of 5-hydroxytryptamine(5-HT)level in myocardial tissue of pre-treatment mice with sepsis myocardial injury by electroacupuncture at Zusanli point,and to explore the protective effect and possible mechanism of electroacupuncture on myocardial injury in sepsis.Methods Twenty male C57BL/6 mice were divided into control group(NC group),sepsis model group(LPS group),electroacupuncture group(EA group)and electroacupuncture+fluoxetine group(EA+FLU group)by random number table method,with 5 mice in each group.The myocardial injury model of sepsis was established by intraperitoneal injection of lipopolysaccharide(LPS)10 g/L.The NC group was intraperitoneally injected with the same amount of normal saline.3 days before mold making,EA group and EA+FLU group were electrocuted at Zusanli point on both sides for 15 minutes,once a day for 3 days.The EA+FLU group was intraperitoneally injected fluoxetine 1.4 g/L before electroacupuncture.After modeling,the cardiac histopathological changes were observed by hematoxylin-eosin(HE)staining.The serum levels of inflammatory cytokines interleukins(IL-6,IL-8),and tumor necrosis factor-α(TNF-α),the content of 5-HT in myocardial tissue,myocardial injury markers MB isoenzyme of creatine kinase(CK-MB),and cardiac troponin I(cTnI),and the levels of adenosine triphosphate(ATP)and lactic acid in myocardial tissue were detected.Quantitative polymerase chain reaction(qPCR)was used to detect the mRNA expressions of 5-hydroxytryptamine transporter(5-HTT),hexokinase 2(HK2)and glucose transporter 4(GLUT4)in myocardial tissue.GLUT4 expression in myocardial tissue was detected by immunofluorescence assay.Results Compared with NC group,the serum levels of IL-6,IL-1βand TNF-α,myocardial 5-HT content,myocardial tissue injury markers CK-MB,cTnI in LPS group and EA+FLU group were significantly increased.Compared with LPS group,the above indexes in EA group were significantly decreased[IL-6(ng/L):443.03±156.16 vs.19843.75±0.00,IL-1β(ng/L):75.72±10.60 vs.894.66±350.88,TNF-α(ng/L):46.17±4.71 vs.533.01±170.58,5-HT(μg/L):161.19±5.96 vs.244.74±14.38,CK-MB(ng/L):468.21±12.46 vs.662.02±22.54,cTnI(ng/L):0.83±0.05 vs.0.99±0.08,all P<0.05].Compared with NC group,the levels of ATP in myocardium of LPS group,EA group and EA+FLU group were significantly decreased,the levels of lactic acid in myocardium were significantly increased.Compared with LPS group,the level of ATP in myocardium of EA group was significantly increased,the level of lactic acid in myocardium was significantly decreased[ATP(mmol/L):0.10±0.01 vs.0.08±0.01,lactic acid(mmol/L):56.03±1.07 vs.72.45±4.32,both P<0.05].Compared with NC group,the mRNA expression of HK2 in myocardium of LPS group was significantly increased,and the mRNA expressions of GLUT4 and 5-HTT were significantly decreased.Compared with LPS group,the mRNA expression of HK2 in myocardium of EA group was significantly decreased,the mRNA expressions of GLUT4 and 5-HTT were significantly increased[HK2 mRNA(relative expression level):0.73±0.19 vs.1.82±0.57,GLUT4 mRNA(relative expression level):1.00±0.33 vs.0.47±0.18,5-HTT mRNA(relative expression level):1.18±0.31 vs.0.38±0.15,all P<0.05].Compared with NC group,the fluorescence intensity of GLUT4 in LPS group and EA+FLU group were significantly decreased.Compared with LPS group,the fluorescence intensity of GLUT4 in EA group was significantly enhanced.Conclusions Electroacupunctureat Zusanli can reduce the content of 5-HT in myocardial tissue of sepsis mice,and its regulatory mechanism may be related to the regulation of 5-HTT and GLUT4.