聪耳通窍剂经NEFM/MAPK通路对抗小鼠老年性耳蜗感音神经细胞凋亡机制的探讨

Investigation on mechanism of Cong’er-Tong’qiao agent against age-related cochlear sensorineural apoptosis in mice via NEFM/MAPK pathway

摘要:

目的 探讨聪耳通窍剂通过调控C57BL/6J小鼠耳蜗神经丝中链多肽(NEFM)/丝裂原活化蛋白激酶14(MAPK14)信号转导对抗老年性耳蜗感音神经细胞凋亡的机制。方法 实验选取刚断奶1月龄C57BL/6J小鼠54只，随机分为3组，其中10只小鼠饮用自来水至出生后2个月作为幼龄对照组(2月龄对照组)；22只小鼠饮用自来水至出生后7个月作为老年性耳蜗感音神经细胞凋亡模型对照组(7月龄对照组)；22只小鼠饮用聪耳通窍剂至出生后7个月作为中药治疗组(7月龄中药组)。各组小鼠月龄期满即终止实验，每组分别取5只小鼠进行全耳蜗基底膜铺片观察内/外毛细胞形态学，5只小鼠进行耳蜗环氧树脂包埋切片观察螺旋神经节神经元(SGNs)形态学。另从7月龄对照组和7月龄中药组中每组各取6只小鼠耳蜗通过蛋白质组学技术检测NEFM丰度值，6只小鼠耳蜗通过RT-PCR技术检测MAPK14表达量。将数据结果进行统计学分析，并结合String和KEGG数据库分析分子信号转导机制。结果 各组耳蜗钩端附近区域基底膜转弯处内/外毛细胞及相应区域耳蜗SGNs比较显示，2月龄对照组耳蜗内/外毛细胞及SGNs完好无缺，7月龄对照组内/外毛细胞及SGNs丧失殆尽，7月龄中药组内/外毛细胞及SGNs损失明显减少(P<0.001)。蛋白质组学研究发现众多富集差异蛋白之一且表达强度排序前列的耳蜗NEFM在中药干预下，与7月龄对照组比较，丰度值显著增加(P<0.001)。RT-PCR研究显示，与7月龄对照组比较，7月龄中药组耳蜗MAPK14表达量明显下调(P<0.001)。NEFM与MAPK14介导的MAPK信号转导通路和超氧化物歧化酶1(SOD1)介导的抗氧化信号转导通路密切关联，进而和活性氧(ROS)/Bcl-2/Bax/caspase-9等与细胞凋亡或抗凋亡因子形成上下信号转导调控相互关系。结论 聪耳通窍剂具有对抗老年性耳蜗感音神经细胞凋亡的作用，其机制可能通过NEFM/MAPK14调控SOD1/ROS介导的抗细胞调亡信号转导通路从而达到药物效果，提示NEFM和MAPK14可能是聪耳通窍剂有效对抗老年性感音神经细胞凋亡作用的重要靶点之一。

Abstract:

Objective To investigate the mechanism of Cong’er-Tong’qiao agent (CTA) against age-related cochlear sensorineural apoptosis in the C57BL/6J mice by regulating neurofilament medium polypeptide (NEFM)/mitogen-activated protein kinase (MAPK) signaling pathway.Methods Fifty-four C57BL/6J mice at 1 month of age were selected for the experiment. Among them, 10 mice drank tap water until two months after birth as the young control group (2M control group). Twenty-two mice drank tap water until 7 months after birth as the control group of the age-related cochlear sensorineural apoptotic model (7M control group). Twenty-two mice were given the CTA until 7 months after birth as the CTA treating group (7M CTA group). After the expiration of the animal experiments in each group, five animals in each group were used for whole cochlear basilar membrane spread preparations to observe the morphology of inner and outer hair cells, and five animals were used for cochlear epoxy resin embedding and sectioning to observe the morphology of spiral ganglion neurons (SGNs). In addition, from the 7M control group and the 7M CTA group, the cochlear samples of 6 animals were selected from each group to detect the abundance value of NEFM through proteomics technology, and the expression level of MAPK14 was detected in 6 animals’ cochleae through real time PCR (RT-PCR) technology. The data were subjected to statistical analysis, and the molecular signal transduction mechanism was analyzed in combination with the String and Kegg databases.Results By comparison of inner and outer hair cells at basal turn near hook region or SGNs at corresponding region in each group, it was shown that all the inner and outer hair cells and SGNs were intact in the 2M control group, while almost all the inner and outer hair cells or SGNs were missing in the 7M control group, but the losses of the inner and outer hair cells and SGNs were significantly reduced in the 7M CTA group (P<0.001). Proteomic studies revealed that NEFM, one of the abundant enrichment proteins with significant differences and at the top in rank of expression intensity, was significantly increased in abundance after intervention with CTA in the 7M CTA group, compared to the 7M control group (P<0.001). The RT-PCR study showed that, compared with the 7M control group, the expression level of cochlear MAPK14 in the 7M CTA group was significantly decreased (P<0.001). The MAPK signaling pathway mediated by NEFM and MAPK14 was closely related to the antioxidant signaling pathway mediated by superoxide dismutase 1 (SOD1), and subsequently formed an up-down signal transduction regulatory interrelationship with reactive oxygen species (ROS)/Bcl-2/Bax/caspase-9 and other factors related to apoptosis or anti-apoptosis.Conclusions The CTA has the effect of combating age-related sensorineural apoptosis. The mechanism may be that NEFM/MAPK14 regulate the SOD1/ROS-mediated signaling pathway of anti-apoptosis to achieve pharmacodynamic effect. It suggests that NEFM and MAPK14 may be one of the important targets of CTA on effectively resisting age-related sensorineural apoptosis.