小檗碱对力竭运动诱导的大鼠心肌损伤具有保护作用

Protective effects of berberine against exhaustion exercise induced myocardial injury in rats

目的探讨小檗碱对力竭运动诱导的大鼠心肌损伤的影响及其机制。方法健康雄性SPF级Sprague-Dawley大鼠40只,采用随机区组设计法将大鼠分为5组,即对照组、力竭运动组、低剂量小檗碱组(50 mg·kg-1·d-1)、中剂量小檗碱组(100 mg·kg-1·d-1)和高剂量小檗碱组(150 mg·kg-1·d-1)。不同剂量小檗碱组大鼠分别灌胃相应浓度的小檗碱溶液1.5 ml·d-1·只-1,对照组和力竭运动组大鼠灌胃同体积的生理盐水;灌胃30 min后各组大鼠分别进行力竭游泳训练(对照组大鼠不运动)。共灌胃14 d,每天进行1次力竭游泳训练。采用小动物超声成像系统检测大鼠心功能指标[左心室短轴缩短率(LVFS)、左心室射血分数(LVEF)、左心室收缩期直径(LVIDs)、左心室舒张期直径(LVIDd)]。苏木素伊红(HE)和Masson染色检测大鼠心肌形态结构。酶联免疫吸附法检测大鼠心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。TUNEL染色法检测大鼠心肌细胞凋亡情况。Western blot法检测大鼠心肌组织中心肌肥厚标志蛋白B型利钠肽(BNP)和凋亡标志蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平。结果(1)各组大鼠的心功能:力竭运动组大鼠LVFS和LVEF均明显低于对照组,LVIDs和LVIDd均明显大于对照组(P均<0.01)。而中剂量小檗碱组和高剂量小檗碱大鼠的LVFS和LVEF则均明显高于力竭运动组,LVIDs和LVIDd均明显小于力竭运动组(P均<0.01)。(2)各组大鼠的心肌结构:HE染色结果显示,对照组大鼠心肌细胞排列紧密、规则,形态正常,结构清晰,染色均匀。力竭运动组大鼠心肌细胞排列紊乱,细胞染色不均匀,细胞质中出现空泡。中剂量小檗碱组大鼠心肌细胞排列紊乱及染色不均等情况均较力竭运动组轻。Masson染色结果显示,对照组大鼠心肌细胞排列紧密、规则,形态正常,结构清晰,出现很少蓝染(纤维化)。力竭运动组大鼠心肌细胞出现明显纤维化。中剂量小檗碱组大鼠心肌细胞纤维化程度则明显较力竭运动组轻。(3)各组大鼠心肌组织中MDA含量和SOD活性:力竭运动组大鼠心肌组织中MDA含量明显高于对照组,中剂量小檗碱组大鼠心肌组织中MDA含量则明显低于力竭运动组(P均<0.01)。力竭运动组大鼠心肌组织SOD活性明显低于对照组,中剂量小檗碱组大鼠心肌组织SOD活性则明显高于力竭运动组(P均<0.01)。(4)各组大鼠心肌细胞凋亡情况:TUNEL染色结果显示,对照组大鼠仅可见少量凋亡心肌细胞,力竭运动组大鼠则可见大量凋亡心肌细胞,而中剂量小檗碱组大鼠凋亡心肌细胞数明显少于力竭运动组。力竭运动组大鼠心肌细胞凋亡指数为39.02%明显高于对照组的2.76%(P<0.01),而中剂量小檗碱组大鼠心肌细胞凋亡指数为20.23%明显低于力竭运动组(P<0.01)。(5)各组大鼠心肌组织中BNP、Bcl-2和Bax的蛋白表达水平:力竭运动组大鼠心肌组织中BNP蛋白表达水平明显高于对照组(P<0.01),中剂量小檗碱组则明显低于力竭运动组(P<0.01)。力竭运动组大鼠心肌组织中Bax蛋白表达水平明显高于对照组,Bcl-2蛋白表达水平明显低于对照组(P均<0.01)。中剂量小檗碱组大鼠心肌组织中Bax蛋白表达水平则明显低于力竭运动组,Bcl-2蛋白表达水平明显高于力竭运动组(P均<0.01)。结论小檗碱可抑制力竭运动诱导的大鼠心肌损伤,其机制可能与抑制力竭运动诱导的自由基释放和凋亡有关。

Objective To investigate the effect and possible mechanism of berberine (Ber) on myocardial injury induced by exhaustion exercise (Ee). Methods Forty healthy male SPF Sprague-Dawley rats were randomly divided into 5 groups using the random unit group design method: control group, Ee group and Ee plus Ber group (low: 50 mg·kg-1·d-1, medium: 100 mg·kg-1·d-1 and high dose: 150 mg·kg-1·d-1, n=8 each). Ber (1.5 ml) or equal volume saline was given per gavage for 14 days. Rats assigned to Ee groups underwent Ee swimming once daily and rats in control group remain sedentary. After 14 days, echocardiographic measurements were performed and left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), left ventricular diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs) were obtained. The morphological structure of heart was detected by HE and Masson staining. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Cardiomyocytes apoptosis was detected by TUNEL method. The protein expression of myocardial hypertrophy marker protein B-type natriuretic peptide (BNP) and apoptotic marker protein (Bcl-2, Bax) in rat myocardial tissue was detected by Western blot. Results (1) Both LVFS and LVEF were significantly lower, and LVIDs and LVIDd were significantly larger in Ee group than those in control group (all P<0.01). The LVFS and LVEF in medium dose of Ber and high-dose Ber groups were significantly higher, and the LVIDs and LVIDd were significantly smaller than those in Ee group (all P<0.01).(2) The results of HE staining showed that the myocardial cells in control group were closely arranged, regular, normal in morphology, clear in structure, and uniform in staining. The myocardial cells of rats in Ee group were disarranged, cell staining was uneven, and vacuoles appeared in the cytoplasm. The disorder of myocardial cell arrangement and unequal staining in the medium dose of Ber were attenuated than in Ee group. The Masson staining results showed that the myocardial cells in control group were closely arranged, regular, normal in shape, clear in structure, and rarely blue-stained (fibrosis). Myocardial cells in rats in Ee group showed obvious fibrosis. The myocardial cell fibrosis in rats with medium dose of Ber was significantly reduced than exercise group.(3) MDA content in myocardial tissue of rats in Ee group was significantly higher than that of control group, and MDA content in myocardial tissue of rats in medium dose of Ber group was significantly lower than in Ee group (P<0.01). The SOD activity of myocardial tissue in rats was significantly lower than that of control group, while that of rats with medium dose of Ber was significantly higher than that of rats in Ee group (P<0.01).(4) TUNEL staining results showed that only a small amount of apoptosis myocardial cells were seen in control group, and a large number of apoptosis myocardial cells were seen in rats in Ee group. However, the number of apoptotic cardiomyocytes in medium dose of Ber was significantly lower than that in Ee group. The AI of rat cardiomyocytes was significantly higher than that of control group (P<0.01), and the AI of rat cardiomyocytes in median dose of Ber group was significantly lower than in Ee group (P<0.01).(5) BNP and Bax protein expression in the myocardial tissues of rats in Ee group were significantly higher than in control group (P<0.01). BNP and Bax protein expression in the myocardial tissues in median dose of Ber group were significantly lower than that of Ee group (P<0.01). The myocardial protein expression level of Bax was significantly higher, and the myocardial protein level of Bcl-2 was significantly lower in Ee group than in control group (both P<0.01), treatment with median dose of Ber could partly reverse above changes (both P<0.01). Conclusion Ber can attenuate exhaustion exercise induced myocardial injury and remodeling in rats, and the beneficial effects of Ber might possibly be mediated by reducing free radical release and cardiomyocytes apoptosis.