桂枝汤桂枝-白芍不同比例配伍通过调节TGF-β1/Smads信号通路及慢性炎症改善盐敏感高血压大鼠心肌纤维化

Effect of Guizhitang with Different Proportions of Cinnamomi Ramulus and Paeoniae Alba Radix in Regulating TGF-β1/Smads Signaling Pathway and Chronic Inflammation and Alleviating Myocardial Fibrosis in Salt-sensitive Hypertensive Rats

目的:观察桂枝汤不同桂枝-白芍比例配伍对盐敏感高血压大鼠转化生长因子-β1(TGF-β1)/Smads信号通路以及白细胞介素-10(IL-10),IL-6,肿瘤坏死因子-α(TNF-α)相关炎症因子表达的影响,探讨桂枝汤改善盐敏感高血压大鼠心肌纤维化具体的机制。方法:将40只雄性6周龄盐敏感大鼠随机分为5组,分别为正常组、模型组、桂枝汤桂枝-白芍1∶1组(简称桂芍1∶1组)、桂枝汤桂枝-白芍1∶2组(简称桂芍1∶2组)、桂枝汤桂枝-白芍2∶1组(简称桂芍2∶1组),每组8只。正常组给予低盐饲料喂养,其余4组给予高盐饲料喂养,喂养4周后开始灌胃,正常组、模型组每天给予等体积的生理盐水灌胃,桂芍1∶1组、桂芍1∶2组和桂芍2∶1组分别按4.0,5.5,5.5 g·kg^-1给予桂枝汤水溶液灌胃,连续灌胃4周。每周测量1次血压;采用超声心动图检测左室收缩末期内径(LVESD),左室舒张末期内径(LVEDD),左室射血分数(LVEF),左室短轴缩短分数(LVFS);采用苏木素-伊红(HE)及马松(Masson)染色观察各组大鼠心肌病理形态变化;采用免疫组化检测各组大鼠心肌组织中Ⅰ,Ⅲ型胶原蛋白的表达;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测各组大鼠心肌组织中IL-10,IL-6,TNF-αmRNA的表达;采用蛋白免疫印迹法(Western blot)检测各组大鼠心肌组织中TGF-β1,α-平滑肌肌动蛋白(α-SMA),Smad2,Smad3,Smad7蛋白的表达。结果:与正常组比较,模型组在8~15周的血压升高,LVESD,LVEDD升高,LVFS,LVEF降低,胶原容积分数升高,免疫组化显示Ⅰ,Ⅲ型胶原蛋白的表达增加,IL-10,IL-6,TNF-αmRNA的表达均升高,TGF-β1,Smad2,Smad3,α-SMA蛋白的表达升高,Smad7蛋白的表达降低(P<0.01)。与模型组比较,桂枝汤各组在12~15周血压上升延缓,LVESD,LVEDD降低(P<0.01),LVFS,LVEF升高(P<0.01),胶原容积分数降低(P<0.01),Ⅰ,Ⅲ型胶原蛋白的表达降低(P<0.01),同时,IL-10 mRNA的表达升高(P<0.05,P<0.01),IL-6,TNF-αmRNA的表达降低(P<0.01),TGF-β1,Smad2,Smad3,α-SMA蛋白的表达降低(P<0.05,P<0.01),Smad7蛋白的表达升高(P<0.01);且与桂芍2∶1组相比,桂芍1∶1组改善上述指标的效果更明显(P<0.01)。结论:桂枝汤不同桂芍比例配伍可以改善盐敏感高血压大鼠的心肌纤维化程度,其机制可能与调节TGF-β1/Smads信号通路及减轻炎症反应有关,且桂枝汤桂芍1∶1组减轻炎症反应,改善心肌纤维化效果最好。

Objective:To observe the effect of Guizhitang with different proportions of Cinnamomi Ramulus and Paeoniae Alba Radix on the expressions of transforming growth factor-β1(TGF-β1)/Smads signaling pathway and interleukin-10(IL-10),IL-6 and tumour necrosis factor-α(TNF-α)related inflammatory cytokines in salt-sensitive hypertensive rats,in order to explore the mechanism of Guizhitang in improving myocardial fibrosis in salt-sensitive hypertensive rats.Method:Totally 40 male 6-week-old salt-sensitive rats were randomly divided into 5 groups:the normal control group,the model group,the 1∶1(RC/peony)Guishao group,the 1∶2 Guishao group,and the 2∶1 Guishao group,with 8 in each group.The normal control group was fed with normal salt diet,while the other four groups were fed with high-salt diet.After 4 weeks of feeding,the rats were given intragastric administration,the normal control group and the model group were given the same amount of normal saline,and the1∶1 Guishao group,the 1∶2 Guishao group and the 2∶1 Guishao group were given 4.0,5.5,5.5 g·kg^-1 of Guizhitang by gavage for 4 weeks.Blood pressure was measured once a week,left ventricular end systolic diameter(LVESD),left ventricular end diastolic diameter(LVEDD),left ventricular ejection fraction(LVEF)and left ventricular short axis shortening fraction(LVFS)were detected by using echocardiogram.The pathological changes of myocardial morphology were observed by htoxylin eosin(HE)and Masson staining.The expressions of typeⅠandⅢcollagen in myocardial tissue of each group was detected by immunohistochemistry.The mRNA expression levels of IL-10,IL-6 and TNF-αin myocardial tissue of each group were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR).The protein expression levels of TGF-β1,α-smooth muscle actin(α-SMA),Smad2,Smad3 and Smad7 in myocardial tissue of each group were detected by Western blot.Result:Compared with the normal control group,the blood pressure was increased in the model group at 8-15 weeks,LVESD,LVEDD were increased in the model group,while LVFS,LVEF were decreased in the model group.The collagen volume fraction was increased,immunohistochemistry showed the expression levels of typeⅠandⅢcollagen were increased,mRNA expression levels of IL-10,IL-6 and TNF-αwere increased,the protein expression levels of TGF-β1,Smad2,Smad3 andα-SMA were increased,whereas the protein expression of Smad7 was decreased(P<0.01).Compared with the model group,the blood pressure rise of each group of Guizhitang was delayed in 12-15 weeks,LVESD and LVEDD were decreased in Guizhitang group(P<0.01),LVFS,LVEF were increased in Guizhitang group(P<0.01),the collagen volume fraction was decreased in Guizhitang group(P<0.01),and the expressions of typeⅠandⅢcollagen were decreased in Guizhitang group(P<0.01).At the same time,the mRNA expression of IL-10 was increased in Guizhitang group(P<0.05,P<0.01),the mRNA expressions of IL-6 and TNF-αwere decreased in Guizhitang group(P<0.01),and the protein expressions of TGF-β1,Smad2,Smad3 andα-SMA were decreased in Guizhitang group(P<0.05,P<0.01),and the protein expression of Smad7 was increased in Guizhitang group(P<0.01).Compared with the 2∶1 Guishao group,the effect of the 1∶1 Guishao group in improving the above indicators was more obvious(P<0.05,P<0.01).Conclusion:Guizhitang with different proportions of Ramulus Cinnamomi and Poeny can alleviate the degree of myocardial fibrosis in salt-sensitive hypertensive rats.The mechanism may be related to the regulation of TGF-β1/Smads signaling pathway and the reduction of inflammatory response.Besides,the 1∶1 Guishao group showed the optimal effect in reducing inflammation and improving myocardial fibrosis.