脊柱推拿介导miRNA-7a/b靶向调控PARP的表达重建脑瘫后胶质细胞凋亡/增殖平衡的机制研究

As a CNS lesion or dysfunction with a high morbidity, cerebral palsy (CP) is difficult to be prevented and treated, and the current studies have not yet revealed its pathogenesis, it is imperative to elucidate its effective prevention and control measures. From our previous studies of animal experiments, we found that spinal massage has a noticeable clinical curative effect for CP by altering the level of miRNA-7a/7b and Poly ADP-Ribose Polymerase (PARP) in glial cells so that brain injury can be modified. Besides,based on some findings that the variation of PARP’s expression could affect the balance of glial cell’s proliferation and apoptosis, we hypothesize that there is a regulatory mechanism for the imbalance between proliferation and apoptosis of glial cells through changing the level of miRNA-7a/7b that may target to regulate the expression of PARP in CP by spinal massage. This study includes three parts. Firstly,we are to confirm the expression alteration of miRNA-7a/b and its effect on cell apoptosis in ischemia/reperfusion injury in glial cells in vitro. Secondly, we apply the methods of the dual-luciferase reporter assay to prove that miRNA-7a/b targets to reduce the level of PARP， and then the cell apoptosis in ischemia/reperfusion injury decreased.Finally,using the neonatal rat hypoxia-ischemia model, we verify the regulatory effect of spinal massage on the balance between apoptosis and proliferation in glail cells in CP through the way that miRNA-7a/7b inhibits PARP. Then we can uncover that spinal massage regulates the apoptosis and proliferation of gilal cells in CP by the mechanism above, which will provide the research foundation for finding a new strategy of preventing and treating CP.

脑瘫(CP)是一种致残率高、治疗手段有限的脑部病变，因发病机制不明，导致对其缺乏安全有效的防治方法。传统医学中的脊柱推拿在CP治疗上有显著疗效，我们前期研究发现脊柱推拿对CP动物模型也有明确疗效，并引起miRNA-7a/7b和多聚ADP核糖聚合酶(PARP)的改变以及凋亡的改善。因此提出：脊柱推拿可通过改变miRNA-7a/7b水平靶向调控PARP，重建胶质细胞凋亡/增殖平衡。本研究先观测原代培养的缺血再灌注(I/RI)损伤胶质细胞中miRNA-7a/7b和PARP的变化及细胞增殖/凋亡情况；再用双荧光素酶报告基因检测方法验证miRNA-7a/7b靶向下调PARP，并抑制I/RI损伤诱导的细胞凋亡；最后在新生鼠缺血缺氧模型上证实脊柱推拿重建CP胶质细胞增殖/凋亡平衡是通过miRNA -7a/7b靶向抑制PARP来实现的。从而阐明脊柱推拿治疗脑瘫的分子机制，为推拿的临床推广提供科学依据。

脑瘫作为一种致残率高、严重威胁婴幼儿身心健康的脑部疾病，其发病机制尚不明确，且缺乏安全有效的治疗方法。目前脑瘫的治疗以药物、手术和物理疗法为主，但效果并不理想。小儿推拿可以通过疏通经络、行气活血、调节脏腑治疗脑瘫，并取得了显著的临床疗效，但因其治病机制不明，限制了小儿推拿的应用和推广。我们前期研究结果提示脊柱推拿可以调节脑瘫幼鼠脑内的miRNA-7a/7b表达和胶质细胞凋亡水平。因此提出：脊柱推拿可通过改变miRNA-7a/7b水平靶向调控PARP，重建胶质细胞凋亡/增殖平衡。我们通过培养原代大鼠星形胶质细胞，进行糖氧剥夺处理引起星形胶质细胞损伤，观察到星形胶质细胞的增殖速度显著减慢、细胞活性下降以及细胞凋亡率明显增加，并且出现星形胶质细胞的miRNA-7a水平降低和PARP1和线粒体凋亡通路的激活。接着通过双荧光素酶报告基因检测方法证实miRNA-7a-5p可以通过降解PARP1的3’UTR区反向调控该基因，并且在新生鼠的缺血缺氧脑损伤的急性、亚急性期脑白质中均观察到线粒体凋亡通路的激活。最后在脑瘫幼鼠上证实脊柱推拿可明显改善脑瘫幼鼠的生长发育和运动平衡功能，并观察到脊柱推拿可以上调脑瘫幼鼠脑内miRNA-7a表达水平，同时抑制脑瘫幼鼠脑白质中PARP1以及线粒体凋亡相关蛋白的表达，因此推测脊柱推拿可能通过上调miRNA-7a抑制PARP1和线粒体凋亡通路的Caspase-9、Caspase-3等表达，从而改善脑瘫幼鼠的脑白质的病理损伤。此外，脑瘫幼鼠经过脊柱推拿干预后，还可以引起海马中NLRP3的表达下调，导致脑瘫后海马中GSDMD、IL-1β和IL-18表达降低进而抑制脑瘫后海马细胞的焦亡。本研究从miRNA对PARP1基因表达的调控入手，针对脑瘫后PARP1引起的细胞凋亡/增殖的分子机制展开研究，解析推拿治疗小儿脑瘫的分子机制，为推拿的临床推广提供科学依据。