丹曲林对心房颤动大鼠心肌纤维化及TGF-β_(1)/Smad2信号通路的影响

Effects of Dantrolene on Myocardial Fibrosis and TGF-β_(1)/Smad2 Signaling Pathway in Rats with Atrial Fibrillation

目的:探讨丹曲林对心房颤动大鼠心肌纤维化及转化生长因子-β_(1)(TGF-β_(1))/Smad2信号通路的影响。方法:72只SD大鼠采用随机数字表法分为空白对照组、模型组、丹曲林低剂量组、丹曲林高剂量组、阳性对照组、丹曲林高剂量+SRI-011381(TGF-β激动剂)组,每组12只。丹曲林低剂量组、丹曲林高剂量组大鼠分别腹腔注射5、10 mg/kg丹曲林;阳性对照组大鼠灌胃20 mg/kg维拉帕米;丹曲林高剂量+SRI-011381组大鼠腹腔注射10 mg/kg丹曲林和30 mg/kg SRI-011381;空白对照组、模型组大鼠腹腔注射等体积生理盐水,每日1次,持续4周。4周后,除空白对照组外,其余各组大鼠均按1 mL/kg尾静脉注射乙酰胆碱-氯化钙混合液(乙酰胆碱66μg/mL+氯化钙10 mg/mL)构建心房颤动模型,空白对照组大鼠尾静脉注射等体积生理盐水,每日1次,持续1周,1周后采集心电图数据并记录心房颤动诱发时间;彩色多普勒超声仪检测左室舒张末期内径(LVEDD)、左室缩短分数(LVFS)、左室射血分数(LVEF)、左室收缩末期内径(LVESD)的变化;苏木精-伊红(HE)染色检测大鼠心肌组织病理损伤;Masson染色检测大鼠心肌纤维化;免疫组化法检测心肌组织中Ⅰ型胶原蛋白(COL-Ⅰ)、Ⅲ型胶原蛋白(COL-Ⅲ)表达;酶联免疫吸附测定法(ELISA)法检测心肌组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;蛋白质免疫印迹法(Western Blot)检测TGF-β_(1)、p-Smad2、α平滑肌肌动蛋白(α-SMA)蛋白表达。结果:与空白对照组比较,模型组大鼠心律不齐,心肌组织病理损伤及纤维化严重,LVEDD、LVESD、COL-Ⅰ、COL-Ⅲ阳性表达及IL-1β、TNF-α水平以及TGF-β_(1)、p-Smad2、α-SMA蛋白表达升高,LVFS、LVEF降低(P<0.05);与模型组比较,丹曲林低剂量组、丹曲林高剂量组、阳性对照组对应指标变化趋势与上述相反,且丹曲林浓度越高,对应的变化趋势越明显(P<0.05);SRI-011381减弱了高剂量丹曲林对心房颤动大鼠心肌纤维化及炎症反应的抑制作用。结论:丹曲林可能通过抑制TGF-β_(1)/Smad2信号通路减轻心房颤动大鼠心肌纤维化及炎症反应。

Objective:To investigate the effects of dantrolene on myocardial fibrosis and transforming growth factor-β_(1)(TGF-β_(1))/Smad2 signaling pathway in atrial fibrillation rats.Methods:Seventy-two SD rats were randomly divided into blank control group,model group,dantrolene low-dose group,dantrolene high-dose group,positive control group,dantrolene high-dose+SRI-011381(TGF-βagonist)group,with 12 rats in each group.Dantrolene low-dose group and dantrolene high-dose group were intraperitoneally injected with 5 and 10 mg/kg dantrolene,respectively;rats in positive control group were given 20 mg/kg verapamil;rats in dantrolene high-dose+SRI-011381 group were intraperitoneally injected with 10 mg/kg dantrolene and 30 mg/kg SRI-011381,respectively;rats in blank control group and model group were intraperitoneally injected with equal volume of normal saline once a day for 4 weeks.After 4 weeks,except for the blank control group,the rats in the other groups were injected 1 mL/kg of acetylcholine-calcium chloride mixed solution(acetylcholine 66μg/mL+calcium chloride 10 mg/mL)through the tail vein to construct the atrial fibrillation model.The rats in the blank control group were injected with equal volume of normal saline through the tail vein once a day for 1 week.One week later,ECG data were collected and the onset time of atrial fibrillation was recorded.The changes of left ventricular end-diastolic diameter(LVEDD),left ventricular shortening fraction(LVFS),left ventricular ejection fraction(LVEF),and left ventricular end-systolic diameter(LVESD)were detected by color Doppler ultrasound.Hematoxylin-eosin(HE)staining was used to detect pathological injury of myocardium in rats.Masson staining was used to detect myocardial fibrosis in rats.The expressions of typeⅠcollagen(COL-Ⅰ)and typeⅢcollagen(COL-Ⅲ)were detected by immunohistochemistry.The levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in myocardial tissue were detected by enzyme-linked immunosorbent assay(ELISA).The protein expression of TGF-β_(1),p-Smad2 andα-smooth muscle actin(α-SMA)were detected by Western Blot.Results:Compared with the blank control group,the model group rats showed arrhythmia,serious myocardial tissue pathological damage and fibrosis,positive expression of LVEDD,LVESD,COL-Ⅰ,COL-Ⅲ,IL-1β,TNF-α,and TGF-β_(1),p-Smad2,α-SMA protein expression increased,LVFS,LVEF decreased(P<0.05).Compared with model group,the change trend of corresponding indicators in dantrolene low-dose group,dantrolene high-dose group and positive control group were opposite to the above data,and the higher the concentration of dantrolene,the more obvious the corresponding change trend(P<0.05).SRI-011381 attenuated the inhibitory effect of high-dose dantrolene on myocardial fibrosis and inflammation in rats with atrial fibrillation.Conclusion:Dantrolene may reduce myocardial fibrosis and inflammation in rats with atrial fibrillation by inhibiting TGF-β_(1)/Smad2 signaling pathway.