Interventional Effect and Mechanism of Saikosaponin A on Noise-induced Tinnitus in Mice
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摘要:
目的 探究柴胡皂苷A对噪音诱发耳鸣（Tinnitus）小鼠模型的干预作用及机制。 方法 将50只小鼠随机分为对照组（Control组）、噪音诱发耳鸣小鼠模型组（BBN组）、BBN组基础上分别灌胃10 mg/kg、20 mg/kg、40 mg/kg柴胡皂苷A（SSA）的SSA-L组、SSA-M组和SSA-H组，每组10只。检测小鼠耳鸣行为；通过旷场实验、高架十字迷宫实验和强迫游泳实验评估小鼠的抑郁样行为；ABR阈值测定小鼠听力；RT-qPCR和Western blot检测小鼠听觉皮层中IL-6、TNF-α、IL-1β mRNA和蛋白表达。 结果 和Control组相比，BBN组小鼠在噪声暴露2 h后3 d、14 d和28 d时GPIAS%值降低（P < 0.05）；和BBN组相比，SSA-L组、SSA-M组和SSA-H组小鼠在噪声暴露2 h后3 d、14 d和28 d时GPIAS%值升高，且呈剂量依赖（ P < 0.05）。和Control组相比，BBN组小鼠进入中央区时间和距离、进入开放臂次数和时间均减少，不动时间及Click和8、16、32 kHz声刺激下ABR阈值均升高（ P < 0.05）；和BBN组相比，SSA-L组、SSA-M组和SSA-H组小鼠进入中央区时间和距离、进入开放臂次数和时间均增加，不动时间及Click和8、16、32 kHz声刺激下ABR阈值均降低（ P < 0.05）。和Control组相比，BBN组小鼠听觉皮层中IL-6、TNF-α、IL-1β mRNA和蛋白表达均升高（ P < 0.05）；和BBN组相比，SSA-L组、SSA-M组和SSA-H组小鼠听觉皮层中IL-6、TNF-α、IL-1β mRNA和蛋白表达均降低（ P < 0.05）。 结论 柴胡皂苷A可改善噪声诱导耳鸣小鼠抑郁行为和听力，其可能和抑制耳鸣小鼠听觉皮层炎症因子密切相关。 Abstract:Objective To investigate the intervention effect and mechanism of Saikosaponin A on noise-induced tinnitus in a mouse model. Methods Fifty mice were randomly divided into control group（Control group）, noise-induced tinnitus mouse model group（BBN group）, and SSA-L group, SSA-M group and SSA-H group based on BBN group with 10 mg/kg, 20 mg/kg and 40 mg/kg of Caihu saponin A by gavage, respectively, 10 mice in each group. The tinnitus behavior of mice was detected; the depression-like behavior of mice was assessed by the open field experiment, elevated cross-maze experiment and forced swimming experiment; the hearing of mice was measured by ABR threshold; and IL-6, TNF-α, IL-1β mRNA and protein expression in the auditory cortex of mice were detected by RT-qPCR and Western blotting. Results Compared with the Control group, mice in the BBN group had significantly lower GPIAS% values at 3 d, 14 d and 28 d after 2 h of noise exposure（P < 0.05）; compared with the BBN group, mice in the SSA-L, SSA-M and SSA-H groups had significantly higher GPIAS% values at 3 d, 14 d and 28 d after 2 h of noise exposure in a dose-dependent manner（ P < 0.05）. Compared with the Control group, mice in the BBN group had significantly reduced time and distance to the central zone, number and time to the open arm, and significantly higher immobility time and ABR thresholds under Click and 8, 16 and 32 kHz acoustic stimulation（ P < 0.05）; compared with the BBN group, mice in the SSA-L, SSA-M and SSA-H groups had significantly higher time and distance to the central zone, and The number and time of entering the open arm were significantly increased, and the immobility time and Click and ABR thresholds under 8, 16 and 32 kHz acoustic stimulation were significantly decreased（ P < 0.05）. Compared with the Control group, IL-6, TNF-α, IL-1β mRNA and protein expression were significantly increased in the auditory cortex of mice in the BBN group（ P < 0.05）; compared with the BBN group, IL-6, TNF-α, IL-1β mRNA and protein expression were significantly decreased in the auditory cortex of mice in the SSA-L, SSA-M and SSA-H groups（ P < 0.05）. Conclusion Saikosaponin A can improve depressive behavior and hearing in noise-induced tinnitus mice, which may be closely related to the inhibition of inflammatory factors in the auditory cortex of tinnitus mice. -
Key words:
- Saikosaponin A /
- Tinnitus /
- Depressive behavior /
- Inflammatory response
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图 2 各组小鼠进入中央区时间和进入中央区距离比较
A：小鼠运动活动的代表性痕迹；B：小鼠进入中央区时间比较；C：小鼠进入中央区距离比较。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 2. Comparison of the time and distance of mice entering the central region in each group
图 3 各组小鼠进入开放臂次数及进入开放臂的时间比较
A：小鼠迷宫架行为记录；B：小鼠进入开放臂次数比较；C：小鼠进入开放臂时间比较。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 3. Comparison of the number of times and time of mice entering the open arm in each group
图 4 各组小鼠强迫游泳实验不动时间比较
A：小鼠强迫游泳实验装置示意图；B：小鼠不动时间比较。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 4. Comparison of immobility time in forced swimming experiments of mice in each group
图 5 各组小鼠在Click和8、16、32 kHz声刺激下ABR阈值改变
A：小鼠8、16、32 kHz声刺激下ABR阈值改；B：小鼠Click声刺激下ABR阈值改变。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 5. Changes in ABR threshold of mice in each group under Click and 8，16，and 32 kHz sound stimulation
图 6 各组小鼠听觉皮层中IL-6、TNF-α和IL-1β mRNA表达比较
A：小鼠听觉皮层中IL-6 mRNA表达比较；B：小鼠听觉皮层中TNF-α mRNA表达比较；C：小鼠听觉皮层中IL-1β mRNA表达比较。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 6. IL-6 and TNF in the auditory cortex of mice in each group- α And IL-1 β MRNA expression comparison
图 7 各组小鼠听觉皮层中IL-6、TNF-α、IL-1β蛋白表达比较
A：各组小鼠IL-6、TNF-α和IL-1β蛋白电泳图；B：小鼠听觉皮层中IL-6蛋白表达比较；C：小鼠听觉皮层中TNF-α蛋白表达比较；D：小鼠听觉皮层中IL-1β蛋白表达比较。与Control组相比，*P < 0.05；与BBN组相比，#P < 0.05；与SSA-L组相比，△P < 0.05；与SSA-M组相比，☆P < 0.05。
Figure 7. IL-6 and TNF in the auditory cortex of mice in each group- α、 IL-1 β Comparison of protein expression
表 1 引物序列
Table 1. Primer sequence
基因 引物序列 扩增长度 IL-6 F：5'-ATTGTATGAACAGCGATGATGCAC-3' 400 bp R：5'-CCAGGTAGAAACGGAACTCCAGA-3' TNF-α F：5'-TCAGTTCCATGGCCCAGAC-3' 150 bp R：5'-GTTGTCTTTGAGATCCATGCCATT-3' IL-1β F：5'-CCAGCTGGAGAGTGGATCC-3' 300 bp R：5'-AGCTGTCGGAGATTCGTAGC-3' GAPDH F：5'-GGCACAGTCAAGGCTGAGAATG-3' 150 bp R：5'-ATGGTGGTGAAGACGCCAGTA-3' 
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表 2 各组小鼠噪声暴露2 h后不同时间节点中GPIAS%值比较（ $ \bar x \pm s $，n = 10）
Table 2. Comparison of GPIAS% values at different time points after 2-hour noise exposure in each group of mice（ $ \bar x \pm s $，n = 10）
组别 3 d 14 d 28 d Control组 28.69 ± 2.31 37.58 ± 3.15 40.31 ± 3.20 BBN组 19.34 ± 1.52 18.17 ± 1.28 17.23 ± 1.35 SSA-L组 21.53 ± 1.45▲# 22.62 ± 1.31▲# 20.65 ± 1.69▲# SSA-M组 24.09 ± 1.33▲#△ 28.39 ± 1.57▲#△ 27.19 ± 2.01▲#△ SSA-H组 27.15 ± 1.24#△☆ 32.66 ± 2.09▲#△☆ 33.87 ± 5.64▲#△☆ F 57.080 148.700 87.790 P < 0.001 *< 0.001 *< 0.001 **P < 0.05；与Control组相比， ▲P < 0.05；与BBN组相比， #P < 0.05；与SSA-L组相比， △P < 0.05；与SSA-M组相比， ☆P < 0.05。
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