橄榄苦苷调节PRAS40/mTORC1信号通路对噪声性耳聋大鼠耳蜗组织损伤的影响

Effect of oleuropein on cochlear tissue damage in noise-induced hearing loss rats by regulating the PRAS40/mTORC1 signaling pathway

摘要:

目的 研究橄榄苦苷调节脯氨酸蛋白激酶底物蛋白(PRAS40)/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号通路对噪声性耳聋(NIHL)大鼠耳蜗组织损伤的影响。方法 总共选取48只大鼠，随机选出12只大鼠作为对照组，剩余大鼠采用白噪声来构建NIHL大鼠模型。随后将NIHL模型大鼠随机分为模型组、橄榄苦苷组、橄榄苦苷+NV-5138组，每组12只。各组给予相应干预7 d。检测大鼠的听性脑干反应(ABR)阈值；HE染色检测大鼠耳蜗组织病理损伤；原位末端标记法染色观察大鼠耳蜗组织细胞凋亡情况；共聚焦显微镜观察耳蜗组织基底膜毛细胞序列变化；蛋白印迹法检测大鼠耳蜗组织磷酸化PRAS40(p-PRAS40)、PRAS40、磷酸化mTORC1(p-mTORC1)、mTORC1、B淋巴细胞瘤-2相关X蛋白(Bax)蛋白表达。结果 与对照组比较，模型组耳蜗组织螺旋神经节细胞数目明显减少，形态异常，基底膜毛细胞结构损坏严重，未见明显的界限，细胞排列不整齐，ABR阈值、耳蜗组织凋亡细胞数目、p-mTORC1/mTORC1蛋白比值、Bax蛋白表达显著升高，p-PRAS40/PRAS40蛋白比值显著降低(P＜0.05)。与模型组比较，橄榄苦苷组耳蜗组织螺旋神经节细胞数目增加，且形态有所恢复，基底膜毛细胞损伤减轻，细胞排列逐渐恢复正常，界限逐渐清晰，ABR阈值、耳蜗组织凋亡细胞数目、p-mTORC1/mTORC1蛋白比值、Bax蛋白表达显著降低，p-PRAS40/PRAS40蛋白比值显著升高(P＜0.05)。NV-5138干预后减弱了橄榄苦苷对NIHL大鼠耳蜗组织病理损伤、基底膜毛细胞序列和上述指标的改善作用(P＜0.05)。结论 橄榄苦苷可能通过调控PRAS40/mTORC1信号通路，促进PRAS40磷酸化，抑制mTORC1磷酸化，从而减轻NIHL大鼠耳蜗组织损伤。

Abstract:

Objective To investigate the effect of oleuropein on cochlear tissue damage in noise-induced hearing loss (NIHL) rats by regulating the proline-rich Akt substrate of 40 kDa (PRAS40)/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Methods A total of 48 rats were selected, 12 rats were randomly regarded as the control group, and the remaining rats were used to construct NIHL rat model using white noise. Subsequently, the NIHL model rats were randomly grouped into model group, oleuropein group, and oleuropein+NV-5138 group, each group consisted of 12 rats and was given corresponding intervention for 7 days. The threshold of auditory brainstem response (ABR) was evaluated. Hematoxylin-eosin staining was applied to detect pathological damage in cochlear tissue. The apoptosis of cochlear tissue was detected by TdT-mediated dUTP nick end labeling staining. The sequences of hair cells in the cochlear basement membrane were observed by confocal microscopy. Western blot was applied to detect the expression of phosphorylated PRAS40 (p-PRAS40), PRAS40, phosphorylated mTORC1 (p-mTORC1), mTORC1, and B-lymphoblastoma-2 related X protein (Bax) in rat cochlear tissue. Results Compared with control group, the number of spiral ganglion cells in the cochlear tissue in the model group was significantly reduced, the morphology was abnormal, the structure of basal membrane hair cells was severely damaged, no obvious boundary could be seen, the cell arrangement was not orderly, the ABR threshold, the number of apoptotic cells in cochlear tissue, the p-mTORC1/mTORC1 protein ratio, and the expression of Bax protein were significantly increased, the p-PRAS40/PRAS40 protein ratio was significantly decreased (P<0.05). Compared with model group, the number of spiral ganglion cells in the cochlear tissue of oleuropein group increased and their shape recovered, the damage of basement membrane hair cells was reduced, the cell arrangement gradually returned to normal, the boundary became clear, and the ABR threshold, the number of apoptotic cells in cochlear tissue, the p-mTORC1/mTORC1 protein ratio and the expression of Bax protein were significantly decreased, and the p-PRAS40/PRAS40 protein ratio was significantly increased (P<0.05). After the intervention of NV-5138, the improvement effects of oleuropein on the pathological injury of cochlear tissue, basal membrane hair cell sequence and the above indexes in NIHL rats were weakened (P<0.05). Conclusion Oleuropein may promote PRAS40 phosphorylation and inhibit mTORC1 phosphorylation by regulating PRAS40/mTORC1 signaling pathway, thereby alleviating cochlear tissue injury in NIHL rats.