填精补髓中药汤剂对脑出血模型小鼠Keap1-Nrf2-ARE信号通路的影响

Effect of Traditional Chinese Medicine Decoction for Filling Essence and Nourishing Marrow on the Keap1-Nrf2-ARE Signaling Pathway in Model Mice with Cerebral Hemorrhage

目的探讨填精补髓中药汤剂对脑出血模型小鼠Keap1-Nrf2-ARE信号通路的影响。方法将48只Wistar雄性小鼠分为假手术组、模型组(均予等体积生理盐水灌胃),阳性药组(每100 g体质量灌胃脑血康30 mg)、中药汤剂组(每100 g体质量灌胃填精补髓中药2 mL),各12只。采用Rynkowski法建立脑出血小鼠模型,各组均于麻醉清醒后给药,每天1次,比较各组小鼠的神经功能(Longa)评分、脑含水量、血肿周围脑组织病理变化情况。应用Western blot法检测Nrf2信号通路相关蛋白(Nrf2,Keap1,HO-1)的表达水平,采用实时荧光定量聚合酶链式反应(RT-PCR)法检测Nrf2,Keap1,HO-1 mRNA的表达水平。结果术后1,3 d,模型组小鼠的Longa评分、脑含水量均较假手术组高,阳性药组、中药汤剂组小鼠的Longa评分、脑含水量均低于模型组,差异均有统计学意义(P<0.05);模型组小鼠的血肿区被大量红细胞浸润,伴随神经细胞数量减少,存在明显的间质水肿,神经细胞排列稀疏,中药汤剂组、阳性药组小鼠的红细胞浸润程度、间质水肿程度均轻于模型组;中药汤剂组、阳性药组小鼠术后3 d的脑组织Nrf2,Keap1,HO-1 mRNA及蛋白表达水平均显著低于模型组(P<0.05)。结论填精补髓中药汤剂可通过激活Keap1-Nrf2-ARE信号通路而发挥抗氧化作用,改善脑出血模型小鼠的神经功能障碍及脑水肿,预防脑出血后继发损伤。

Objective To investigate the effect of traditional Chinese medicine decoction for filling essence and nourishing marrow on the Keap1-Nrf2-ARE signaling pathway in model mice with cerebral hemorrhage.Methods A total of 48 Wistar male mice were divided into the sham surgery group,the model group(all given equal volume of physiological saline by gavage),the positive drug group(30 mg of Naoxuekang per 100 g body weight by gavage),and the traditional Chinese medicine decoction group(2 mL of essence and marrow filling traditional Chinese medicine per 100 g body weight by gavage),with 12 mice in each group.The Rynkowski method was used to establish a mouse model of cerebral hemorrhage.Each group was administered after anesthesia and awakening once a day.The neurological function(Longa)score,brain water content,and pathological changes in the surrounding brain tissue in each group were compared.Western blot was used to detect the expression level of Nrf2 signaling pathway related protein(Nrf2,Keap1,HO-1),and quantitative Real-time polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of Nrf2,Keap1 and HO-1.Results On the 1st and 3rd day after surgery,the Longa score and brain water content in the model group were higher than those in the sham surgery group.The Longa score and brain water content in the positive drug group and the traditional Chinese medicine decoction group were lower than those in the model group,and the differences were statistically significant(P<0.05).The hematoma area in the model group was infiltrated by a large number of red blood cells,accompanied by a decrease in the number of nerve cells and obvious interstitial edema.The arrangement of nerve cells was sparse,and the degree of red blood cell infiltration and interstitial edema in the traditional Chinese medicine decoction group and positive drug group were lighter than those in the model group.The protein and mRNA expression levels of Nrf2,Keap1,and HO-1 in the brain tissue in the traditional Chinese medicine decoction group and positive drug group were significantly lower than those in the model group(P<0.05).Conclusion Tradional Chinese Medicing Decoction at for filling essence and nourishing marrow can exert antioxidant effects by activating the Keap1-Nrf2-ARE signaling pathway,improve neurological dysfunction and brain edema in mice with cerebral hemorrhage,and prevent secondary damage after cerebral hemorrhage.