Objective: To investigate whether tanshinone ⅡA can protect the apoptosis of mice cochlear pericytes induced by high glucose and its specific protective mechanism, so as to provide experimental evidence for the prevention and treatment of diabetic hearing loss. Methods: C57BL/6J male mice were used to prepare type 2 diabetes model, which were divided into normal (NG) group, diabetic (DM) group, diabetic+tanshinone ⅡA (HG+tanshinone ⅡA) group and tanshinone ⅡA group. Each group had 10 animals. Primary cochlear pericytes were divided into NG group, HG group (high glucose 35 mmol/L), HG+tanshinone ⅡA (1, 3, 5 μmol/L) group, HG+Tanshinone ⅡA+LY294002 (PI3K/AKT pathway inhibitor) group, LY294002 group, tanshinone ⅡA group and DMSO group. Auditory brainstem response (ABR) was used to measure hearing threshold. Evans blue was used to detect the permeability of blood labyrinth barrier in each group. TBA methods were used to detect oxidative stress levels in various organs of mice. Morphological changes of stria vascularis were observed by hematoxylin-eosin staining (HE). Evans blue was used to detect the vascular labyrinth barrier permeability in cochlea. The expression of apoptosis protein in stria vascularis pericytes was observed by immunofluorescence. Pericytes apoptosis rate was observed by flow cytometry. DCFH-DA was combined with flow cytometry to detect intracellular ROS content, and Western blot was used to detect the expression of apoptotic proteins (Cleaved-caspase3, Bax), anti-apoptotic proteins (BCL-2) and pathway proteins (PI3K, p-PI3K, AKT, p-AKT). SPSS software was used for statistical analysis. Independent sample t test was performed, and P<0.05 was considered statistically significant. Results: Animal experiments: Tanshinone ⅡA decreased the hearing threshold of DM group [(35.0±3.5) dB SPL vs. (55.3±8.1) dB SPL] (t=4.899, P<0.01), decreased the oxidative stress level in cochlea (t=4.384, P<0.05), improved the structure disorder, atrophy of cochlea vascular lines, vacuole increased phenomenon. Tanshinone ⅡA alleviated the increased permeability of the blood labyrinth barrier [Evans blue leakage (6.84±0.27) AU vs. (8.59±0.85) AU] in the cochlea of DM mice (t=2.770, P<0.05), reversed the apoptotic protein: Caspase3 (t=4.956, P<0.01) and Bax (t=4.388, P<0.05) in cochlear vascularis. Cell experiments: Tanshinone ⅡA decreased intracellular ROS content in a concentration-dependent way (t=3.569, P<0.05; t=4.772, P<0.01; t=7.494, P<0.01); Tanshinone ⅡA decreased apoptosis rate and apoptotic protein, and increased the expression of anti-apoptotic protein, p-PI3K/PI3K and p-AKT/AKT in concentration-dependent manner (all P values<0.05); LY294002 reversed the protective effect of tanshinone ⅡA on pericytes apoptosis (all P values<0.05). Conclusion: Tanshinone ⅡA can inhibit the apoptosis of cochlear pericytes induced by high glucose by reducing oxidative stress level and activating PI3K/AKT signaling pathway under high glucose environment, thus playing a protective role in diabetic hearing loss.
目的： 探讨丹参酮ⅡA是否能抑制高糖诱导的小鼠耳蜗血管纹周细胞的凋亡及可能机制，为治疗糖尿病性听力损失提供参考。 方法： C57BL/6J雄性小鼠40只，分为对照组、糖尿病（DM）组、糖尿病+丹参酮ⅡA组、丹参酮ⅡA组，每组10只。制备2型糖尿病模型。将原代周细胞分为对照组、高糖（HG）组、HG+丹参酮ⅡA（1、3、5 μmol/L）组、HG+丹参酮ⅡA+LY294002（PI3K/AKT通路抑制剂）组、LY294002组、丹参酮ⅡA组、二甲基亚砜（DMSO）组。ABR检测听力阈值；硫代巴比妥酸试验（thiobarbituric acid，TBA）检测耳蜗氧化应激水平；苏木精-伊红染色（HE）观察耳蜗血管纹形态变化；Evans blue检测耳蜗血管纹血迷路屏障通透性；免疫荧光观察耳蜗血管纹周细胞凋亡蛋白的表达；流式细胞术检测周细胞凋亡率；DCFH-DA结合流式细胞术检测周细胞内活性氧（reactive oxygen species，ROS）含量，免疫蛋白印记（Western Blot，WB）法检测周细胞凋亡蛋白（Cleaved-caspase3、Bax）、抗凋亡蛋白（BCL-2）以及通路蛋白（PI3K、p-PI3K、AKT、p-AKT）的表达。采用 SPSS软件进行统计分析，行独立样本t检验，以P<0.05为差异有统计学意义。 结果： 动物实验：丹参酮ⅡA可降低DM组［（35.0±3.5）dB SPL比（55.3±8.1）dB SPL］听力阈值（t=4.899，P<0.01），降低耳蜗内氧化应激水平（t=4.384，P<0.05），改善耳蜗血管纹结构紊乱、萎缩，空泡增多的现象。丹参酮ⅡA可缓解DM小鼠耳蜗血管纹血迷路屏障通透性［Evans blue渗漏（6.84±0.27）AU比（8.59±0.85）AU］增加的现象（t=2.770，P<0.05），可逆转周细胞凋亡蛋白Cleaved-caspase3（t=4.956，P<0.01）和 Bax（t=4.388，P<0.05）的表达。细胞实验：丹参酮ⅡA可降低高糖干预下的周细胞内ROS含量，且呈浓度依赖性（t=3.569，P<0.05；t=4.772，P<0.01；t=7.494，P<0.01）；丹参酮ⅡA可降低高糖干预下的周细胞凋亡率和凋亡蛋白，增加抗凋亡蛋白、p-PI3K/PI3K和p-AKT/AKT的表达，且均呈浓度依赖性（P值均<0.05）；LY294002可逆转丹参酮ⅡA对周细胞凋亡的保护作用（P值均<0.05）。 结论： 丹参酮ⅡA可通过降低高糖环境下的氧化应激水平以及激活PI3K/AKT信号通路，抑制高糖诱导的小鼠耳蜗血管纹周细胞凋亡，从而发挥对糖尿病性听力损失的保护作用。.