黄连素通过B细胞淋巴瘤-2/自噬相关基因复合体抑制自噬减轻脑缺血再灌注损伤的研究

Study of berberine on reducing cerebral ischemia reperfusion injury by inhibiting autophagy through B cell lymphoma 2/ Beclin-1 complex

目的探讨黄连素保护脑缺血再灌注损伤的可能作用机制。方法用线栓法建立大鼠大脑中动脉栓塞模型,并随机分为模型组、对照组、实验组和联合组,每组12只;另取12只正常大鼠仅分离颈总、颈外、颈内动脉,作为假手术组。对照组给予腹腔注射100 mg·kg^(-1)黄连素(10 mg·mL^(-1));实验组给予腹腔注射25 mg·kg^(-1) ABT-199(0.1 mol·L^(-1));联合组给予腹腔注射100 mg·kg^(-1)黄连素(10 mg·mL^(-1))+25 mg·kg^(-1) ABT-199(0.1 mol·L^(-1));假手术组和模型组均给予腹腔注射等量0.9%NaCl。5组大鼠每天给药1次,连续给药2周。用Western Blot法检测脑组织中微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)和自噬相关基因(Beclin-1)蛋白的表达水平,用免疫共沉淀检测Beclin-1和Bcl-2蛋白结合情况。结果实验组、联合组、对照组、模型组和假手术组的LC3-Ⅱ/LC3-Ⅰ比值分别为1.95±0.25,1.16±0.19,0.51±0.16,1.84±0.21和0.31±0.09,Beclin-1蛋白表达量分别为0.92±0.19,0.63±0.14,0.56±0.13,0.79±0.17和0.43±0.11,Beclin-1和Bcl-2结合程度分别为0.23±0.02,0.40±0.02,0.50±0.03,0.37±0.03和0.64±0.04,模型组的上述指标均较假手术组、对照组和联合组显著降低,差异均有统计学意义(均P<0.05),实验组的上述指标均较模型组和联合组显著升高,差异均有统计学意义(均P<0.05)。结论黄连素可减轻脑缺血再灌注损伤大鼠的脑损伤,其保护作用可能与促进Bcl-2与Beclin-1结合抑制自噬有关。

Objective To explore the possible mechanism of Berberine in protecting cerebral ischemia-reperfusion injury. Methods The middle cerebral artery occlusion model was established by suture method and the rats were randomly divided into model group, control group, experimental group and combined group, with 12 rats in each group;the other 12 normal rats as sham operation group were only separated from common carotid artery, external carotid artery and internal carotid artery. The control group was given intraperitoneal injection of 100 mg·kg^(-1) berberine(10 mg·mL^(-1));the experimental group was given intraperitoneal injection of 25 mg·kg^(-1) ABT-199(0.1 mol·L^(-1));the combined group was given intraperitoneal injection of 100 mg·kg^(-1) Berberine(10 mg·mL^(-1)) + 25 mg·kg^(-1) ABT-199(0.1 mol·L^(-1));the sham operation group and model group were given intraperitoneal injection of 0. 9% NaCl. Rats in 5 groups were given once a day for 2 weeks. Western Blot method was used to detect brain tissue microtubule-associated protein 1 light chain3( LC3-Ⅱ) and autophagy related gene( Beclin-1) protein expression;immunoprecipitation was used to detect the binding of Beclin-1 and Bcl-2 protein.Results The LC3-Ⅱ/LC3-Ⅰ ratios of experimental group,combined group,control group,model group and sham operation group were 1. 95 ± 0. 25,1. 16 ± 0. 19,0. 51 ± 0. 16,1. 84 ± 0. 21 and 0. 31 ± 0. 09,Beclin-1 protein expression were 0. 92 ± 0. 19,0. 63 ± 0. 14,0. 56 ± 0. 13,0. 79 ± 0. 17 and 0. 43 ± 0. 11,the binding degree of Beclin-1 and Bcl-2 were 0. 23 ± 0. 02,0. 40 ± 0. 02,0. 50 ± 0. 03,0. 37 ± 0. 03,0. 64 ± 0. 04,the above indexes in model group were significantly lower than those in sham operation group,the control group and the combined group,and the differences were statistically significant( all P < 0. 05),the above indexes in experimental group were significantly higher than those in model group and the combined group,and the differences were statistically significant( all P < 0. 05). Conclusion Berberine can alleviate cerebral in rats with cerebral ischemia-reperfusion injury,which might be related to promoting the binding of Bcl-2 with Beclin-1 and inhibiting autophagy.