Effect of Wenyang Zhuyu Decoction on Expressions of Wnt3a and β-catenin after Acute Cerebral Ischemic Injury in Rats with Kidney-Yang Deficiency Syndrome
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目的:观察温阳逐瘀汤对肾阳虚证大鼠急性脑缺血损伤后缺血脑组织无翅型MMTV整合位点家族成员3A(Wnt3a)、β-连环蛋白(β-catenin)表达的影响,并探讨温阳逐瘀汤防治肾阳虚证大鼠脑缺血损伤的作用机制.方法:将176只雄性SD大鼠分为空白组(Blank组,44只)和肾阳虚组(SYX组,132只);成功建立肾阳虚证大鼠模型后随机分为假手术组(Sham组,44只)、脑缺血模型组(MCA O组,44只)、温阳逐瘀汤组(WYZ Y组,44只).WYZ Y组大鼠灌胃温阳逐瘀汤,MCA O组与Sham组灌胃等体积蒸馏水.各组在术后1 d,运用Zea Longa评分标准进行大鼠神经行为学评分,于术后3、7、14、21 d取材,每个时间选11只大鼠.苏木精-伊红(HE)染色观察神经细胞形态变化;运用酶联免疫吸附法(ELISA)检测各组大鼠血浆环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)含量;免疫蛋白印迹法(Western Blot)检测缺血侧脑组织Wnt3a、β-catenin表达;实时荧光定量聚合酶链式反应(RT-PCR)检测Wnt3a、β-catenin mRNA表达.结果:与Blank组比较,SYX组cAMP含量下降(P＜0.05)、cGMP含量升高(P＜0.05).HE染色结果显示,MCAO组细胞排列紊乱、大量细胞坏死、细胞膜消失、细胞核固缩,呈明显的脑缺血损伤表现;WYZY组前期细胞排列紊乱、部分细胞坏死,随着时间的延长,细胞损伤减轻,细胞排列逐渐密集,坏死细胞减少,脑缺血损伤显著改善.与MCAO组、Sham组比较,WYZY组cAMP含量升高,cGMP含量降低(P＜0.05).与MCA O组比较,WYZ Y组1、3、7、14、21 d神经行为学评分均降低(P＜0.05).与Blank组、Sham组比较,MCAO组、WYZY组缺血脑组织3、7、14、21 d Wnt3a、β-catenin表达均有增加(P＜0.05);与MCAO组比较,WYZY组Wnt3a、β-catenin 表达均增加(P＜0.05),7 d Wnt3a 达到 峰值,14 d β-catenin 达到 峰值.与 Blank 组、Sham 组比较,MCAO 组、WYZY 组 3、7、14、21 d Wnt3a、β-catenin mRNA 表达均有增加(P＜0.05);与 MCAO 组比较,WYZY组 Wnt3a、β-catenin mRNA 表达增加(P＜0.05),7 d Wnt3a mRNA达到峰值,14 d β-catenin mRNA达到峰值.结论:温阳逐瘀汤可改善肾阳虚证大鼠cAMP、cGMP水平及肾阳虚病理状态,可能通过上调Wnt3a、β-catenin表达减轻急性脑缺血损伤,减少神经功能缺损.
Objective:To observe the effect of Wenyang Zhuyu decoction on the expression of wingless MMTV integration site family member 3A(Wnt3a)and β-catenin in the ischemic brain tissue of rats with kidney-Yang deficiency syndrome after acute cerebral ischemia injury,and to explore the mechanism of Wenyang Zhuyu decoction in preventing and treating cerebral ischemic injury in rats with kidney-Yang deficiency syndrome.Methods:One hundred and seventy-six male SD rats were divided into Blank group(44 rats)and kidney-Yang deficiency group(132 rats).After the successful establishment of kidney-Yang deficiency syndrome model,the rats were randomly divided into Sham operation group(Sham group,44 rats),cerebral ischemia model group(MCAO group,44 rats)and Wenyang Zhuyu decoction group(WYZY group,44 rats).WYZY group was gavaged with Wenyang Zhuyu decoction,MCAO group and Sham group were gavaged with equal volume of distilled water.One day after operation,neurobehavioral scores of rats in each group were collected by Zea Longa score criteria.Materials were collected on 3 day,7 day,14 day and 21 day after operation,and 11 rats were selected at each time.The morphological changes of nerve cells were observed by hematoxylin-eosin(HE)staining.The contents of cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)in the plasma were detected by enzyme-linked immunoadsorbent assay(ELISA),and the expressions of Wnt3a and β-catenin in ischemic cerebral tissue were detected by Western Blot.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the mRNA expressions of Wnt3a and β-catenin mRNA.Results:Compared with the Blank group,cAMP content in the WSYX group decreased(P＜0.05),and cGMP content increased(P＜0.05).The results of HE staining showed that the MCAO group showed disordered cell arrangement,necrosis,disappearance of cell membrane and contraction of nuclei,and showed obvious ischemic injury.The WYZY group showed disordered cell arrangement and partial cell necrosis in the early stage.With the extension of time,the cell damage was alleviated,the cell arrangement was gradually dense,the necrotic cells reduced,and the ischemic state improved significantly.Compared with MCAO group and Sham group,cAMP content increased and cGMP content decreased in the WYZY group(P＜0.05).Compared with MCAO group,the neuroethological scores of WYZY group on 1 day,3 day,7 day,14 day and 21 day were decreased(P＜0.05).Compared with Blank group and Sham group,the expressions of Wnt3a and β-catenin in ischemic brain tissue of MCAO group and WYZY group increased on 3 day,7 day,14 day and 21 day(P＜0.05).Compared with MCAO group,the expressions of Wnt3a and β-catenin in the WYZY group significantly increased(P＜0.05),and Wnt3a and β-catenin in the WYZY group reached their peak value on 3 day,7 day,respectively.Compared with Blank group and Sham group,the mRNA expressions of Wnt3a and β-catenin in MCAO group and WYZY group were increased on 1 day,3 day,7 day,14 day and 21 day(P＜0.05).Compared with MCAO group,the mRNA expressions of Wnt3a and β-catenin in the WYZY group were increased(P＜0.05),and the mRNA expression of Wnt3a reached its peak value on 7 day and the mRNA expression of β-catenin reached its peak value on 14 day.Conclusion:Wenyang Zhuyu decoction could improve the levels of cAMP,cGMP and the pathological state in rats with kidney-Yang deficiency.Wenyang Zhuyu decoction might relieve acute cerebral ischemia and reduce neurological function injury in rats with kidney-Yang deficiency by up-regulating the expressions of Wnt3a and β-catenin.
acute cerebral ischemiaWenyang Zhuyu decoctionkidney-Yang deficiency syndromewingless MMTV integration site family member 3Aβ-cateninratsexperimental study
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