Research Center for High Altitude Medicine, Qinghai University, Xining, 810001, China; Key Laboratory of Application and Foundation for High Altitude Medicine Research in Qinghai Province (Qinghai-Utah Joint Research Key Lab for High Altitude Medicine), Xining, 810001, China; Medical College, Qinghai University, Xining, 810001, China.
Technical Center of Xining Customs District, Key Laboratory of Food Safety Research in Qinghai Province, Xining, 810003, China.
J Ethnopharmacol. 2020 Mar 25;250:112470. doi: 10.1016/j.jep.2019.112470. Epub 2019 Dec 17.
Tsantan Sumtang, which consists of Choerospondias axillaris (Roxb.) Burtt et Hill, Myristica fragrans Houtt and Santalum album L, is a traditional and common prescription of Tibetan medicine. Tsantan Sumtang originates from Four Tantra with properties of nourishing heart and has been used as a folk medicine for cardiovascular diseases and heart failure in Qinghai, Tibet and Inner Mongolia. Our previous studies found that Tsantan Sumtang showed beneficial effects on right ventricular structure in hypoxia rats, while the underling mechanism remains unclear.
To elucidate the underlying mechanisms of Tsantan Sumtang attenuated right ventricular (RV) remodeling and fibrosis of chronic hypoxia-induced pulmonary arterial hypertension (HPAH) rats.
Fifty male Sprague Dawley (SD) rats (170 ± 20 g) were randomly divided into control group, hypoxia group, and hypoxia + Tsantan Sumtang groups (1.0 g· kg·day, 1.25 g· kg·day, 1.5 g ·kg·day). Rats in the hypoxia group and hypoxia + Tsantan Sumtang groups were maintained in a hypobaric chamber by adjusting the inner pressure and oxygen content to simulate an altitude of 4500 m for 28 days. The mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), the ratio of RV weight to tibia length (TL) (RV/TL), heart rate (HR) and RV systolic pressure (RVSP) was determined. Histomorphological assay of RV structure was evaluated by hematoxylin and eosin (HE) staining. RV tissue fibrosis was assessed by collagen proportion area (CPA), collagen I, collagen III and hydroxyproline content. CPA was obtained by picro-sirius red staining (PSR). The expression of collagen I and collagen III were detected by immunohistochemistry and western blotting. The hydroxyproline content was detected by alkaline hydrolysis. In addition, the level of angiotensin II (AngII) and angiotensin 1-7 (Ang1-7) in RV tissue was tested by enzyme-linked immune sorbent assay (ELISA). Protein expression of angiotensin-converting enzyme (ACE), AngII, AngII type 1 receptor (AT1R), angiotensin-converting enzyme 2 (ACE2), Mas receptor (Mas) were determined by immunohistochemistry and western blotting. mRNA level of ACE, AT1R, ACE2, Mas were tested by qPCR. The chemical profile of Tsantan Sumtang was revealed by UHPLC-Q-Exactive hybrid quadrupole-orbitrap mass analysis.
Our results showed that RVHI, RV/TL and RVSP were significantly increased in HPAH rat. Furthermore, levels of collagen I, collagen III and hydroxyproline were up-regulated in RV tissue under hypoxia. We found that RV hypertrophy and fibrosis were associated with increased expression of ACE, AngII, AT1R as well as decreased expression of ACE2, Ang1-7 and Mas. RV remodeling and fibrosis were attenuated after Tsantan Sumtang administration by up-regulating ACE2 and Mas level as well as down-regulating ACE, AngII and AT1R levels in RV tissue. 35 constituents in Tsantan Sumtang were identified.
Tsantan Sumtang attenuated RV remodeling and fibrosis in rat exposed to chronic hypoxia. The pharmacological effect of Tsantan Sumtang was based on equilibrating ACE-AngII-AT1R and ACE2-Ang1-7-Mas axis of RV tissue in HPAH rat.
复方藏药二十五味珍珠丸由诃子、肉豆蔻、肉桂、丁香、降香、沉香、荜茇、红花、炉甘石、木香、马钱子、麝香、乳香、牛黄、松石、珍珠等 25 味藏药材组成，是藏医的常用经典方剂。二十五味珍珠丸来源于藏医“五元学说”中的“五元”，具有养心安神之功效，被用于治疗青海、西藏、内蒙古等地区的心血管疾病和心力衰竭的民间药物。本课题组前期研究发现二十五味珍珠丸对低氧诱导的大鼠右心室结构具有有益作用，但作用机制尚不清楚。
阐明二十五味珍珠丸减轻慢性低氧诱导肺动脉高压（HPAH）大鼠右心室（RV）重构和纤维化的潜在机制。
将 50 只雄性 SD 大鼠（170±20g）随机分为对照组、低氧组和低氧+二十五味珍珠丸组（1.0g·kg·day、1.25g·kg·day、1.5g·kg·day）。低氧组和低氧+二十五味珍珠丸组大鼠通过调节内压和氧含量在低压舱中维持低氧环境，模拟海拔 4500m 的条件 28 天。检测平均肺动脉压（mPAP）、右心室肥厚指数（RVHI）、右心室重量与胫骨长度比值（RV/TL）、心率（HR）和 RV 收缩压（RVSP）。采用苏木精和伊红（HE）染色评估 RV 结构的组织形态学改变。通过胶原比例面积（CPA）、Ⅰ型和Ⅲ型胶原及羟脯氨酸含量评估 RV 组织纤维化。CPA 通过苦味酸-天狼猩红染色（PSR）获得。通过免疫组化和蛋白质印迹法检测Ⅰ型和Ⅲ型胶原的表达。通过碱性水解法检测羟脯氨酸含量。此外，通过酶联免疫吸附试验（ELISA）检测 RV 组织中血管紧张素 II（AngII）和血管紧张素 1-7（Ang1-7）的水平。通过免疫组化和蛋白质印迹法检测血管紧张素转换酶（ACE）、AngII、AngII 型 1 受体（AT1R）、血管紧张素转换酶 2（ACE2）、Mas 受体（Mas）的蛋白表达。通过 qPCR 检测 ACE、AT1R、ACE2、Mas 的 mRNA 水平。采用 UHPLC-Q-Exactive 混合四极杆轨道阱质谱分析揭示二十五味珍珠丸的化学成分特征。
研究结果表明，HPAH 大鼠的 RVHI、RV/TL 和 RVSP 均显著升高。此外，低氧条件下 RV 组织中Ⅰ型和Ⅲ型胶原及羟脯氨酸含量增加。我们发现 RV 肥大和纤维化与 ACE、AngII、AT1R 表达增加以及 ACE2、Ang1-7 和 Mas 表达减少有关。二十五味珍珠丸治疗后，RV 重构和纤维化减轻，RV 组织中 ACE2 和 Mas 水平升高，ACE、AngII 和 AT1R 水平降低。在二十五味珍珠丸中鉴定出 35 种成分。
二十五味珍珠丸减轻了慢性低氧暴露大鼠的 RV 重构和纤维化。二十五味珍珠丸的药理作用基于调节 HPAH 大鼠 RV 组织中的 ACE-AngII-AT1R 和 ACE2-Ang1-7-Mas 轴。
