基于PINK1/Parkin及cGAS/STING通路探讨滋水清肝饮的抗抑郁机制

Discussion on the anti-depression mechanism of Zishui Qinggan Decoction based on PINK1/Parkin and cGAS/STING signaling pathways

目的研究滋水清肝饮对抑郁大鼠脑内PTEN诱导激酶1(PINK1)/Parkin及环磷酸鸟苷-腺苷磷酸合成酶(cGAS)/干扰素基因刺激因子(STING)信号通路的影响,揭示滋水清肝饮治疗抑郁症的抗炎机制。方法将60只大鼠按随机数字表法分为对照组、模型组及滋水清肝饮低、中、高剂量组,每组12只。除对照组外,其余各组制备慢性不可预知应激模型(CUMS)抑郁模型。滋水清肝饮低、中、高剂量组分别灌胃12、24、48 g/kg滋水清肝饮水煎剂,对照组、模型组灌胃等体积的蒸馏水,1次/d,持续4周。采用强迫游泳、旷场实验及糖水消耗实验检测各组大鼠行为学变化;采用HE染色观察内侧前额叶皮层细胞结构;采用ELISA法检测IL-1β、IL-6、TNF-α及干扰素-γ(IFN-γ)水平;采用Western blot检测前额叶皮层PINK1、Parkin、cGAS、STING蛋白表达。结果行为学检测结果显示,与模型组比较,滋水清肝饮各剂量组大鼠在强迫游泳实验中进入第1次不动状态潜伏期延长(P<0.05),不动时间缩短(P<0.05);在中央区停留时间增加(P<0.05),进入中央区的潜伏期缩短(P<0.05);糖水消耗百分比明显升高(P<0.05)。HE染色结果显示,滋水清肝饮各剂量组大鼠前额叶皮层细胞核固缩现象减少,神经元数量增加,分布均匀。滋水清肝饮各剂量组大鼠前额叶皮层IL-1β、IL-6、TNF-α、IFN-γ水平降低(P<0.05),前额叶皮层PINK1、Parkin蛋白表达增加(P<0.05),cGAS及STING蛋白表达降低(P<0.05)。结论滋水清肝饮可改善CUMS大鼠的抑郁行为,改善神经元损伤及神经炎症反应,其机制可能与上调PINK1/Parkin信号通路蛋白表达,抑制cGAS/STING通路信号蛋白表达有关。

Objective To explore the effects of Zishui Qinggan Decoction on the PTEN-induced putative kinase protein 1(PINK1)/Parkin and cyclic GMP-AMP(cGAS)/stimulator of interferon genes(STING)signaling pathways;To reveal the anti-inflammatory mechanism of Zishui Qinggan Decoction in treating depression.Methods Totally 60 rats were randomly divided into control group,model group,and Zishui Qinggan Decoction low-,medium-,and high-dosage groups using a random number table method(n=12 in each group).All rats except for the rats in control group were prepared with CUMS induced depression models.The rats in the Zishui Qinggan Decoction low-,medium-,and high-dosage groups were orally administered with 12,24,and 48 g/kg of Zishui Qinggan Decoction for gavage,respectively.The control group and model group were orally administered with distilled water of equal volume for gavage,once a day for 4 weeks.Forced swimming test(FST),the open field test(OFT)and the sucrose preference test(SPT)were used to detect behavioral changes in rats in each group.Hematoxylin eosin(HE)staining was used to observe the cell structure of the medial prefrontal cortex.The levels of IL-1β,IL-6,TNF-αand Interferon-γ(IFN-γ)were detected using ELISA.Western blot was used to detect the expressions of Pink1,Parkin,cGAS and STING.Results Behavioral testing results showed that,compared with the model group,the incubation period for rats in Zishui Qinggan Decoction low-,medium-,and high-dosage groups to enter the first immobility state in FST was significantly prolonged(P<0.05),and the immobility time was significantly shortened(P<0.05);the time spent in the central area was significantly increased(P<0.05),and the incubation period for entering the central area was significantly shortened in(P<0.05);the percentage of sugar water consumption significantly increased in(P<0.05).HE staining revealed that the aggregation of prefrontal cortex nuclei decreased,the number of neurons increased,and the distribution of neurons was uniform in Zishui Qinggan Decoction low-,medium-,and high-dosage groups.Compared with the model group,the levels of IL-1β,IL-6,TNF-αand IFN-γin the Zishui Qinggan Decoction groups significantly decreased(P<0.05).The protein expressions of PINK1 and Parkin in the prefrontal cortex in Zishui Qinggan Decoction groups significantly increased(P<0.05),while the protein expression levels of cGAS and STING significantly decreased(P<0.05).Conclusion Zishui Qinggan Decoction can significantly improve the depressive behavior,neuronal damage,and neuroinflammatory response in CUMS rats.Its mechanism may be related to up-regulating the PINK1/Parkin signaling pathway and inhibiting the cGAS/STING signaling pathway.