人参皂苷Rg_1通过Akt-Nrf2信号通路拮抗顺铂诱导的豚鼠耳毒性作用研究

Ginsenoside Rg_1 attenuates cisplatin-induced ototoxicity in guinea pigs via Akt/Nrf2 pathway

目的探讨人参皂苷Rg_1(Rg_1)对顺铂(CDDP)所致豚鼠听觉损伤的保护作用及其机制。方法将80只豚鼠随机分为5组,对照组连续7 d ip生理盐水3 mL/kg;CDDP组ip CDDP 3 mg/kg,连续7 d;CDDP+Rg_1(5、10、20 mg/kg)组连续10 d ip Rg_1(5、10、20 mg/kg),第4~10天ip Rg_1 1 h后对侧腹腔ip CDDP 3 mg/kg。各组动物于给药前及停药后均行听觉脑干诱发电位(ABR)检测。实验中每天检测豚鼠体质量并观察饮食、毛发脱落及活动状况等一般情况的变化。实验结束后迅速取出豚鼠双侧耳蜗,分别用耳蜗基底膜铺片的方法观察外毛细胞缺失情况;用HE染色方法检测耳蜗内螺旋神经节损伤;用黄嘌呤氧化酶法和硫代巴比妥酸法检测耳蜗组织中总超氧化物歧化酶(SOD)活力和丙二醛(MDA)水平;用Western blotting法检测耳蜗组织内促凋亡蛋白(Caspase-3和p53)及与氧化应激密切相关的信号分子(p-Akt和Nrf2)的表达。结果与CDDP组相比,Rg_1能剂量依赖性降低CDDP诱导的ABR阈值升高,对豚鼠听觉功能具有显著保护作用。形态学观察发现Rg_1可明显减轻CDDP导致的豚鼠内耳外毛细胞大片缺失及螺旋神经节的损伤。耳蜗内组织化学检测则显示CDDP可导致耳蜗组织中MDA水平明显升高(P<0.05)及SOD活性的明显降低(P<0.01);同时应用Rg_1则可显著拮抗CDDP诱导的上述改变。Western blotting结果发现Rg_1可明显抑制CDDP诱导的耳蜗组织内Caspase-3及p53表达的增加(P<0.05);并显著促进耳蜗内p-Akt及Nrf的表达(P<0.05)。结论 Rg_1可通过抗氧化及抗凋亡作用保护CDDP所致豚鼠听觉损伤,该作用可能与激活Akt-Nrf2信号通路有关。

Objective To investigate the protective effects of Ginsenoside Rg1 against cisplatin（CDDP）-induced ototoxicity and its mechanisms. Methods Eighty guinea pigs were randomly divided into five groups with ten rats in each group, namely control group, cisplatin group, 5 mg/kg Rg1 ＋ CDDP group, 10 mg/kg Rg1 ＋ CDDP group, and 20 mg/kg Rg1 ＋ CDDP group. Animals in the control group were administered with normal saline（NS, 3 mL/kg） via intraperitoneal（ip） route for 7 consecutive days. Cisplatin group were injected with cisplatin alone（3 mg/kg, ip） for 7 consecutive days. Rg1 ＋ CDDP groups were given 3 consecutive days of Rg1（5 mg/kg, 10 mg/kg, and 20 mg/kg ip respectively for each group） ahead of the application of Rg1 ＋ cisplatin（3 mg/kg, ip） for 7 consecutive days. For the 7 consecutive days of co-treatment of Rg1 with cisplatin, Rg1 was injected peritoneally 1 hour before the injection of CDDP at the contralateral side. Auditory brainstem response（ABR） test was used to evaluate the auditory function of rats in each group before and after administration of CDDP. Stretched preparation of cochlear basilar membrane was examined for morphological changes of outer hair cells（OHC）. The general case of diet, loss of hair, and activity status of guinea pigs were observed every day. HE staining techniques and optical microscopy were used to evaluate the lesions of spiral ganglion neurons（SGN）. The levels of malondiadehyde（MDA） and superoxidedismutase（SOD） activities in cochlear tissues were detected by biochemical assay kits. The protein expressions of Caspase-3, p53, p-Akt, and Nrf2 were determined by western blotting. Results Compared with CDDP group, Rg1 co-treatment significantly lowered the CDDP-induced ABR threshold elevation（P〈0.05） in a dose dependent manner. The severe damages of cochlear OHCs and SGNs exhibited in CDDP group were also markedly attenuated by co-treatment with Rg1 in the Rg1 ＋ CDDP groups. In CDDP group, the MDA levels were significantly increased while the SOD activity was decreased in cochlear tissue after CDDP injection. Rg1 treatment evidently reduced the level of MDA and enhanced the SOD activity（P〈0.05）. Western blot results indicated that Rg1 treatment significantly attenuated CDDP-induced pro-apoptotic protein expressions（Capase-3 and p53）. Furthermore, treatment with Rg1 resulted in an increased expression of p-Akt and intranuclear Nrf2 in cochlear tissue compared to the CDDP group. Conclusion Ginsenoside Rg1 protects auditory functions from cisplatin-induced ototoxicity via the Akt/Nrf-2 pathway in guinea pigs.