补阳还五汤通过激活脂联素通路减轻大鼠脑缺血再灌注后炎症损伤

Buyang Huanwutang Alleviates Inflammatory Injury After Cerebral Ischemia-reperfusion in Rats via Activating Adiponectin Pathway

目的:研究补阳还五汤通过激活脂联素(APN)通路减轻大鼠脑缺血再灌注后炎症损伤、改善神经功能。方法:将雄性SD大鼠随机分为假手术组、模型组、补阳还五汤组和补阳还五汤联合APN抑制剂(GW9662)组。假手术组仅分离组织剥离血管,模型组建立大鼠大脑中动脉闭塞(MCAO)模型,补阳还五汤组给予补阳还五汤灌胃16 g·kg^(-1),2次/d。补阳还五汤+GW9662组于造模术前30 min给予GW9662腹腔注射4 mg·kg^(-1),术后给予补阳还五汤灌胃16 g·kg^(-1),2次/d。脑缺血再灌注24 h后,采用免疫荧光(IF)法观察大鼠脑组织中脂联素受体1(AdipoR1)的表达,以及AdipoR1与神经元标记物(NeuN)、小胶质细胞标记物(Iba1)的共定位;采用酶联免疫吸附测定法(ELISA)检测大鼠血清和脑组织APN含量;行为学检测中,横木行走测试观察大鼠平衡能力,抓力测试观察大鼠肢体力量恢复情况。免疫荧光法检验髓过氧化物酶(MPO)的表达;蛋白免疫印迹法(Western blot)检测脑组织肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)表达水平。结果:与假手术组比较,模型组AdipoR1表达升高(P<0.01),血清和脑组织APN含量降低(P<0.05,P<0.01),横木行走测试评分增高(P<0.01),前肢抓力减弱(P<0.01),MPO、TNF-α、IL-1β和IL-6表达升高(P<0.01),IL-10表达降低(P<0.01)。与模型组比较,补阳还五汤组AdipoR1表达升高(P<0.01),血清和脑组织APN含量升高(P<0.05,P<0.01),横木行走评分降低无统计学意义,前肢抓力显著提升(P<0.01),MPO、TNF-α、IL-1β、IL-6表达降低(P<0.01),IL-10表达升高(P<0.01)。补阳还五汤联合GW9662使用后部分作用被抑制。结论:补阳还五汤可减轻脑缺血再灌注大鼠炎症损伤,改善神经功能,其作用机制可能与APN通路的激活有关。

Objective:To investigate the effects of Buyang Huanwutang(BYWHT)on reducing inflammatory injury and improving neurological function after cerebral ischemia-reperfusion in rats via activating the adiponectin(APN)pathway.Method:Male SD rats were randomized into sham,model,BYHWT(16 g·kg^(-1),twice a day),and BYHWT+APN inhibitor(GW9662)groups.In the sham group,blood vessels were isolated.The rat model of middle cerebral artery occlusion(MCAO)was established.The rats in the BYHWT+GW9662 group was treated with subcutaneous injection of GW9662 at 4 m·kg^(-1)30 min before MCAO surgery and BYHWT at 16 g·kg^(-1) by gavage after MCAO surgery,once in the morning and once in the evening.The immunofluorescence(IF)assay was employed to observe the expression of adiponectin receptor 1(AdipoR1)and the colocalization of AdipoR1 with neuronal nuclei(NeuN)and ionized calcium binding adaptor molecule 1(Iba1)in the brain.Enzyme-linked immunosorbent assay(ELISA)was employed to detect the expression of APN in the serum and brain.The balance beam test was carried out to examine the balance ability,and the grasping test to assess the recovery of limb strength.The immunofluorescence assay was used to detect the expression of myeloperoxidase(MPO).Western blot was employed to determine the expression of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-6,and IL-10 and in the brain.Result:Compared with the sham group,the modeling promoted the expression of AdipoR1(P<0.01),lowered the APN levels in the serum and brain(P<0.05,P<0.01),increased the score in the balance beam test(P<0.01),and decreased the grasping strength of forepaw(P<0.01),which were accompanied with increased MPO,TNF-α,IL-1β,and IL-6 levels(P<0.01)and decreased IL-10 level(P<0.01).Compared with the model group,BYHWT promoted the expression of AdipoR1(P<0.01),elevated APN levels in the serum and brain(P<0.05,P<0.01),and increased the grasping strength of forepaw(P<0.01),which were accompanied with lowered MPO,TNF-α,IL-1β,and IL-6 levels(P<0.01)and elevated IL-10 level(P<0.01).All the above effects were partially blocked by GW9662.Conclusion:BYHWT can reduce inflammatory injury and improve neurological function in the rat model of cerebral ischemia-reperfusion by activating the APN pathway.