文章摘要
丹参酚酸B通过抑制PI3K/AKT/mTOR通路促进自噬减轻大鼠心肌纤维化的研究
A study of reducing rat myocardial fibrosis by promoting the autophagy resulting from the#$NBSinhibition of PI3K/AKT/mTOR pathway by salvianolic acid B (SA-B)
投稿时间:2019-02-28  修订日期:2019-02-28
DOI:
中文关键词: 丹参酚酸B  自噬  心肌纤维化  PI3K/AKT/mTOR  Beclin1  LC3-II  Western blot
英文关键词: Salvianolic acid B  Autophagy  Myocardial fibrosis  PI3K/AKT/ mTOR  Beclin1  LC3-II  Western blot
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作者单位E-mail
杜琎 南京中医药大学附属中西医结合医院 心胸外科 dujin810106@163.com 
石开虎△ 南京中医药大学附属中西医结合医院 心胸外科 aytcs@vip.163.com 
赵扬 南京中医药大学附属中西医结合医院 心胸外科  
胡定辉 南京中医药大学附属中西医结合医院 心胸外科  
李航 南京中医药大学附属中西医结合医院 心胸外科  
刘尊涛 南京中医药大学附属中西医结合医院 心胸外科  
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中文摘要:
      目的 研究丹参酚酸B(SA-B)能否通过抑制PI3K/AKT/mTOR通路促进自噬,从而减轻大鼠心肌纤维化。方法 选用SD大鼠40只,完全随机化分为对照组、模型组、低剂量SA-B治疗组和高剂量SA-B治疗组。模型组及低、高剂量SA-B治疗组为注射异丙肾上腺素(ISO)大鼠,每天背部皮下注射ISO,连续21d,构建大鼠心肌纤维化模型,对照组皮下注射同等量生理盐水。低、高剂量SA-B治疗组在造模同时灌喂丹参酚酸B水溶液, 低剂量组灌胃量为15mg/kg,高剂量组灌胃量为30mg/kg,灌胃体积均为10ml/kg,对照组和模型组分别灌胃等体积0.9%生理盐水。末次给药后24h处死大鼠,测定心重指数(HW/BW)和左心室重指数(LVW/BW);ELISA法测定心肌中I型、III型胶原水平;Western blot检测自噬相关蛋白PI3K、AKT、p-AKT、mTOR、Beclin1、LC3-II水平;大鼠心肌HE染色评估心肌纤维化程度。结果 与对照组比较,模型组中大鼠的心重指数、左心室重指数和心肌中I型、III型胶原的水平升高(P<0.05),HE染色结果提示心肌组织发生明显的纤维化。模型组大鼠心肌细胞中的自噬相关蛋白PI3K、AKT、p-AKT 、mTOR表达升高,Beclin1、LC3-II表达较对照组明显降低(P<0.05)。SA-B组中心重指数、左心室重指数和心肌中I型、III型胶原的水平明显降低,与模型组比较差异显著(P<0.05),HE染色未见明显纤维化病灶,其自噬相关蛋白PI3K、AKT、p-AKT 、mTOR表达降低,Beclin1、LC3-II表达较模型组明显升高(P<0.05)。随着丹参酚酸B剂量的增加,自噬相关蛋白PI3K、AKT、p-AKT 、mTOR表达逐渐降低,Beclin1、LC3-II表达逐渐升高,具有剂量依耐性。结论 通过皮下注射异丙肾上腺素建立心肌纤维化模型的方法可行,心肌细胞自噬水平下调与心肌纤维化密切相关,丹参酚酸B能够抑制ISO所致的大鼠心肌纤维化,且具有剂量依耐性,其机制与抑制PI3K/AKT/mTOR传导通路促进细胞自噬密切相关。
英文摘要:
      Objective To investigative whether the salvianolic acid B (SA-B) could inhibit the PI3K/AKT/mTOR pathway to promote the autophagy, thereby reducing rat myocardial fibrosis. Methods A total of 40 SD rats underwent the complete randomization and were assigned to four groups: the control group, model group, low-dose and high-dose SA-B group. The rats in the model, low-dose and high-dose SA-B groups received the subcutaneous injection with isoprenaline (ISO) in their backs every day for 21 consecutive days to build the rat myocardial fibrosis model, and the subcutaneous injection with the same dose of normal saline was administrated in the control group. For treatment groups with low- and high-dose SA-B, the modeling was performed while feeding rats with SA-B aqueous solution was conducted, and the dose of intragastric administration in the low- and high-dose group was 15mg/kg and 30mg/kg, respectively, and the volume of gavage for both groups was 10ml/kg, and 0.9% of normal saline with the same volume was administrated in the control and model groups. Rats were killed 24h after the last administration, and the heart weight and body weight index (HW/BW) and left ventricular weight and body weight index (LVW/BW) were measured; the ELISA method was used to detect the type I and III collagen levels in myocardia; the levels of autophagy-related proteins, PI3K, AKT, p-AKT, mTOR, Beclin1, LC3-II were detected by using Western blot; the HE staining in rat myocardia was used for the assessment on the degree of myocardial fibrosis. Results As compared with the control group, it showed an increase for the model group in HW/BW, LVW/BW and type I and III collagen levels in myocardia (P<0.05), and the results of HE staining suggested the significant fibrosis for myocardia. In the model group, it had an increase in expression levels of autophagy-related proteins, PI3K, AKT, p-AKT, mTOR and a significant decrease in expression levels of Beclin1 and LC3-II as compared with the control group (P<0.05). In the SA-B group, it showed an obvious decrease in HW/BW, LVW/BW and type I and III collagen levels in myocardia, which had significant difference as compared with the model group (P<0.05), and the HE staining suggested no significant fibrosis, and it showed a decrease for expression levels of autophagy-related proteins, PI3K, AKT, p-AKT, mTOR and significant increase for expression levels of Beclin1 and LC3-II as compared with the model group (P<0.05). As the increase of SA-B dose, it gradually decreased in expression levels of autophagy-related proteins, PI3K, AKT, p-AKT, mTOR and gradually increased in expression levels of Beclin1 and LC3-II, which showed the dose-dependent relation. Conclusions The subcutaneous injection with ISO could successfully build the myocardial fibrosis model in a short time, and the downregulation in autophagy level of myocardia was closely related to the myocardial fibrosis, moreover, the SA-B could inhibit rat myocardial fibrosis caused by ISO in a dose-dependent manner, and its mechanism was closely associated with autophagy promoting by the inhibition of PI3K/AKT/mTOR pathway.
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