川芎嗪激活Keap1/Nrf2通路抗顺铂致聋模型大鼠氧化应激损伤的作用研究

Effects of Tetramethylpyrazine-activated Keap1/Nrf2 Pathway on Oxidative Stress Injury in Rat with Cisplatin-induced Deafness

目的探讨川芎嗪基于Keap1/Nrf2通路对顺铂致聋模型大鼠氧化应激损伤的作用及机制。方法80只Wistar大鼠随机均分为空白组(B组)与模型组(M组),M组大鼠腹腔注射顺铂4 mg·kg^(-1)·d^(-1)构建药物性聋模型后,将B组与M组各自再随机均分为2组,分别是:空白对照组(BC组),空白+川芎嗪组(BC+TMP组),模型对照组(MC组),模型+川芎嗪组(MC+TMP组)。BC+TMP组和MC+TMP组腹腔注射川芎嗪140 mg·kg^(-1)·d^(-1),BC组和MC组腹腔注射等量生理盐水,均每日1次,连续2周。行ABR检测后用比色法检测各组大鼠血清中丙二醛(MDA)和超氧化物歧化酶(SOD),运用蛋白质印迹法检测各组大鼠耳蜗组织中Keap1、Nrf2、p-Nrf2的蛋白表达水平,运用免疫荧光双标技术检测各组大鼠耳蜗组织中Keap1、Nrf2、Keap1/Nrf2共表达水平。结果M组大鼠ABR阈值明显高于B组(P<0.05),M组大鼠耳蜗毛细胞出现大量溶解破坏等病理改变;MC+TMP组ABR阈值低于MC组(P<0.05),其耳蜗毛细胞损伤程度低于MC组。与BC组比较,MC组大鼠血清中MDA含量明显升高(P<0.05),SOD活性水平明显降低(P<0.05)。与MC组比较,MC+TMP组大鼠MDA含量明显更低(P<0.05),SOD活性水平明显更高(P<0.05)。MC组Nrf2与p-Nrf2蛋白表达水平较BC组明显升高(P<0.05),MC+TMP组的Nrf2与p-Nrf2蛋白表达水平明显高于MC组(P<0.05);MC组Keap1蛋白表达水平较BC组明显升高(P<0.05),MC+TMP组Keap1蛋白表达水平明显低于MC组(P<0.05)。免疫荧光双标结果显示,MC组Keap1、Nrf2、Keap1与Nrf2共表达水平较BC组明显升高(P<0.05),MC+TMP组的Keap1表达水平明显低于MC组(P<0.05),Nrf2、Keap1与Nrf2共表达水平明显高于MC组(P<0.05)。结论顺铂可导致大鼠耳蜗毛细胞损伤,川芎嗪可通过激活Keap1/Nrf2通路减轻顺铂所致大鼠耳蜗组织的氧化应激损伤,从而发挥对耳蜗毛细胞的保护作用。

Objective Based on the Keap1/Nrf2 pathway to investigate the effect and mechanism of tetramethylpyrazin on oxidative stress injury in cisplatin-induced deafness rat.Methods A total of 80 SPF Wistar rats were randomly divided into blank group(group B)and model group(group M),the rats in group M were induced ototoxic deafness model.After the model was successfully replicated,group B and group M were randomly divided into two groups:blank control group(BC group),blank+tetramethylpyrazine group(BC+TMP group),model control group(MC group),and model+tetramethylpyrazine group(MC+TMP group).Rats in BC+TMP group and MC+TMP group were intraperitoneally injected with tetramethylpyrazin injection(140 mg·kg^(-1)·d^(-1)),and rats in BC group and MC group received intraperitoneal injection of the same amount of normal saline,once a day,for continuous intervention two weeks.After ABR was assessed,serum malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by colorimetric method.The protein expression levels of Keap1,Nrf2 and P-Nrf2 in cochlear tissues of rats in each group were determined by Western blot.The levels of Keap1,Nrf2 and co-expression Keap1/Nrf2 in cochlear tissues of rats in each group were detected by immunofluorescence double labeling.Results[KG1]The ABR threshold of rats in group M was significantly higher than that in group B(P<0.05),simultaneously,the cochlear hair cells in the M group showed pathological changes such as massive dissolution and destruction.The ABR threshold of rats in MC+TMP group was lower than that in MC group(P<0.05),and the damage degree of cochlear hair cells was lower than that in MC group.Compared with the BC group,the serum MDA content of the MC group was significantly increased(P<0.05),and the SOD activity level was significantly decreased(P<0.05).Compared with the MC group,the MDA content of the rats in the MC+TMP group was significantly lower(P<0.05),and the SOD activity level was significantly higher(P<0.05).The protein expression levels of Nrf2 and p-Nrf2 in the MC group were significantly higher than those in the BC group(P<0.05).The protein expression levels of Nrf2 and p-Nrf2 in the MC+TMP group were significantly higher than those in the MC group(P<0.05).The protein expression level of Keap1 in the MC group was significantly higher than that in the BC group(P<0.05),and the protein expression level of Keap1 in the MC+TMP group was significantly lower than that in the MC group(P<0.05).Immunofluorescence double-labeling results showed that the levels of Keap1,Nrf2,co-expression Keap1/Nrf2 in the MC group were significantly higher than those in the BC group(P<0.05).The expression level of Keap1 in MC+TMP group was significantly lower than that in MC group(P<0.05).The levels of Nrf2,co-expression Keap1/Nrf2 were significantly higher than those in the MC group(P<0.05).Conclusion Cisplatin is ototoxic and can cause damage to cochlear hair cells in rats.Tetramethylpyrazin can activate Keap1/Nrf2 pathway to reduce oxidative stress injury of cochlear tissue in rats with ototoxic deafness,thus playing a protective role in cochlear hair cells.