基于海马小胶质细胞M1型极化研究逍遥散对OVX联合CUS焦虑抑郁模型大鼠的影响

Effect of Xiaoyao San on OVX combined with CUS anxiety and depression model rats based on hippocampal microglia M1 polarization

研究逍遥散对卵巢切除(ovariectomized,OVX)联合慢性不可预知应激(chronic unpredictable stress,CUS)模型大鼠的抗焦虑抑郁作用及机制。采用OVX联合CUS建立围绝经期抑郁症模型,通过旷场试验评价大鼠的焦虑抑郁水平;Real-time qPCR法检测大鼠海马中白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)mRNA的水平;免疫荧光双染法检测大鼠海马齿状回(dentate gyrus,DG)区小胶质细胞离子钙接头蛋白分子-1(ionized calcium binding adaptor molecule-1,Iba-1)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的荧光表达;Western blot法检测大鼠海马DG区小胶质细胞Iba-1和iNOS的蛋白表达。结果显示,模型组大鼠水平穿格运动次数、垂直运动次数、中心停留时间明显减少,且梳理时间显著延长(P<0.05或P<0.01);海马炎症因子IL-1β和IL-6水平显著升高(P<0.05);大鼠海马DG区小胶质细胞Iba-1/iNOS共表达的阳性细胞增多;海马Iba-1和iNOS蛋白表达水平显著升高(P<0.01),提示应激状态大鼠海马DG区小胶质细胞被激活并向M1型极化。逍遥散高剂量组能显著增加模型大鼠水平穿格运动次数、垂直运动次数和中心停留时间(P<0.05或P<0.01),且可显著下调炎症因子IL-1β和IL-6水平(P<0.05),同时逆转海马小胶质细胞的激活及数量变化;逍遥散低剂量组虽对旷场试验行为学指标和IL-1β和IL-6水平影响不显著,但均表现出改善趋势;低剂量逍遥散显著下调iNOS蛋白水平(P<0.05),高剂量逍遥散对海马小胶质细胞Iba-1和iNOS蛋白表达水平均有显著下调(P<0.05或P<0.01)。因此,逍遥散可以改善OVX联合CUS模型大鼠的焦虑抑郁样行为,其机制与其抑制海马小胶质细胞M1极化而发挥抗炎作用有关。

To investigate the anti-anxiety and anti-depression effect and mechanism of Xiaoyao San on rats with ovariectomy(OVX) combined with chronic unpredictable stress(CUS) model. The model of perimenopausal depression was established by OVX and CUS;the level of anxiety and depression was evaluated by open field test;the levels of interleukin-1β(IL-1β) and interleukin-6(IL-6) mRNA in rat hippocampus were detected by Real-time qPCR;double staining immunofluorescence was used to detect the expression of ionized calcium binding adaptor molecule-1(Iba-1) and inducible nitric oxide synthase(iNOS) in microglia of rat dentate gyrus(DG);Western blot was used to detect the protein expression of Iba-1 and iNOS of microglia in DG region of rat hippocampus. The results showed that in the model group, the number of horizontal movement, the number of vertical movement and central residence time were significantly reduced, and the grooming time was significantly prolonged(P<0.05 or P<0.01);the levels of IL-1β and IL-6 in hippocampus increased significantly(P<0.05);the number of positive cells with co-expression of Iba-1/iNOS of microglia cells in DG region of hippocampus increased;the expression levels of Iba-1 and iNOS protein in hippocampus were significantly increased(P<0.01), suggesting that microglia in DG region of hippocampus was activated and polarized toward M1 type in rats with stress. The high dose group of Xiaoyao San significantly increased the number of horizontal movement, vertical movement and central residence time of model rats(P<0.05 or P<0.01), and significantly down-regulated the levels of inflammatory factors IL-1β and IL-6(P<0.05). Meanwhile, it reversed the activation and quantity change of microglia in hippocampus. Although the Xiaoyao San low dose group had no significant effect on the behavioral indicators in the open field test and the levels of IL-1β and IL-6, they all showed a trend of improvement. Low dose Xiaoyao San significantly decreased iNOS protein level(P<0.05), and high dose Xiaoyao San significantly down-regulated the protein expression of Iba-1 and iNOS in hippocampus microglia(P<0.05 or P<0.01). In conclusion, Xiaoyao San can improve anxiety and depression-like behavior in OVX combined with CUS model rats, and its mechanism is related to its anti-inflammatory effect by inhibiting M1 polarization of hippocampal microglia.