BRL37344对大鼠心肌成纤维细胞磷脂酰肌醇(-3)激酶-蛋白激酶B信号通路的影响

目的 探讨β3-肾上腺素能受体(β3-adrenergic receptor,β3-AR)激动剂BRL37344是否通过细胞内磷脂酰肌醇(-3)激酶-蛋白激酶B(phosphatidylinositol 3 kinase-protein kinase B,PI3K-Akt)信号传导途径促进大鼠心肌成纤维细胞(cardiac fibroblasts,CFbs)增殖及胶原纤维增生.方法 哈尔滨医科大学附属第一医院中心实验室无菌条件下剪取新生1～3 d的Wistar大鼠心脏,采用酶消化法、差速贴壁法获取CFbs.随机数字法分为5组:①空白组:不给予任何药物干预.②BRL组:给予BRL37344孵育.③LY组:给予LY294002(PI3K抑制剂)预孵育1 h后联合BRL37344共同孵育.④Akt-Ⅰ组:给予Akt-Ⅰ(Akt抑制剂)预孵育1 h后联合BRL37344共同孵育.⑤L-A组:LY294002和Akt-Ⅰ预孵育1 h后联合BRL37344共同孵育.通过MTT比色法观察细胞的增殖情况,RT-PCR法观察Ⅰ、Ⅲ型胶原表达情况.组间进行单因素方差分析,组间比较采用Tukey检验.以P＜0.05为差异具有统计学意义.结果 ①CFbs中存在β3-AR.②与BRL组比较,LY组和Akt-Ⅰ组增殖减低(P＜0.01);L-A组较LY组和Akt-Ⅰ组增殖减低更明显(P＜0.01).③与空白组比较,BRL组Ⅰ、Ⅲ型胶原mRNA表达均明显增加,给药48 h增加最显著.选取48 h点为作用时间点,与BRL组比较,LY组、Akt-Ⅰ组Ⅰ、Ⅲ型胶原mRNA表达减低,L-A组较LY组和Akt-Ⅰ组表达减低更显著(P＜0.01).结论 BRL37344通过PI3K-Akt信号传导途径促进心肌成纤维细胞增殖和Ⅰ,Ⅲ型胶原表达增加.

Effect of BRL37344 on cardiac fibroblast phosphatidylinositol 3-kinase-protein kinase B signal transduction pathway

Objective To explore the effects of the β3 adrenergic receptor (β3-AR) agonist BRL37344 on cardiac fibroblast proliferation and collagen fiber hyperplasia in Wistar rats by promoting the phosphaticlylinositol 3 kinase-protein kinase B(PI3K-Akt) signal transduction pathway. Method Cardiac fibroblasts(CFbs) were isolated from 1 - 3 day-old Wistar rats under the sterile environment in the laboratory of the First Affiliated Hospital of Hasbin Medical University, by using enzyme digestion and an modified technique of differential anchoring velocity.The cultured myocardial cells were randomly (random number) divided into five groups. ① In blank group, rats were not treated with drug; ②in BRL group, rats were treated with BRL37344; ③in LY group, rats were treated with LY294002(PI3K antagonist) for one hour before treated with BRL37344;④in Akt-Ⅰ group,rats were treated with Akt-Ⅰ (Akt antagonist) for one hour before treated with BRL37344;⑤in L-A group, rats were treated with LY294002 and Akt-Ⅰ for one hour before treated with BRL37344. MTT colorimetric method and RT-PCR were used to observe the role of β3-AR agonist with or without PI3K antagonist and/or Akt antagonist in cardiac fibroblast proliferation (CFP) and collagen fiber hyperplasia (CFH). Comparisons between groups were made by one-way ANOVA and Tukey test. Results ①β3-AR was present in the CFbs. ②Compared with BRL group, the CFP and CFH in LY and Akt-Ⅰ groups were lower (P ＜0.01) and those in L-A group were lowest (P ＜ 0.01). ③Compared with blank group,the expressions of type Ⅰ and type Ⅲ fiber mRNA obliviously increased in BRL group (P ＜ 0.01),and at 48 hours,the expressionrs reached peak. At 48 hours,compared with BRL group,the expressions in LY and Akt-Ⅰ groups were lower, and were lowest in L-A group ( P ＜0.01). Conclusions BRL37344 promotes cardiac fibroblast proliferation and expressions of type Ⅰ and Ⅲ collagen fiber mRNA by activating the PI3K-Akt signal transduction pathway.