基于Rorα/Bmal1信号轴探讨半夏秫米汤对慢性失眠大鼠睡眠稳态的影响

Investigation of the effect of Banxia Shumi decoction on sleep homeostasis in rats with chronic insomnia based on the Rorα/Bmal1 signaling axis

摘要:

目的 探讨“引阳入阴”代表性方剂半夏秫米汤对慢性失眠（CI）大鼠生物钟基因Rorα/Bmal1信号轴的影响。方法 SPF级大鼠36只，随机分为空白组、模型组、半夏秫米汤低剂量组（BXSM-L组）、半夏秫米汤中剂量组（BXSM-M组）、半夏秫米汤高剂量组（BXSM-H组）、地西泮组（DZ组），每组6只。除空白组外，其余各组均采用小平台水环境法复制CI模型大鼠。BXSM-L组、BXSM-M组、BXSM-H组大鼠分别灌胃4.69、9.38、18.75 g/kg半夏秫米汤水煎剂，DZ组大鼠灌胃0.52 mg/kg地西泮水溶液，空白组和模型组大鼠均给予等体积生理盐水灌胃，连续14 d。给药结束后，用16道生理记录仪监测脑电波，实时荧光定量聚合酶链反应、Western blotting、免疫组织化学法分别检测视交叉上核（SCN）、大脑皮层（CC）的Rorα和Bmal1基因及蛋白表达，酶联免疫吸附试验检测脑干与血清中γ氨基丁酸（GABA）、谷氨酸（Glu）、多巴胺（DA）、去甲肾上腺素（NE）含量。结果 模型复制成功后的大鼠皮毛暗淡无光泽，精神萎靡，易激惹，对周围声、光刺激敏感，昼夜节律消失。空白组α波、β波频率均低于造模组（除空白组外其余各组统称为造模组）、δ波频率高于造模组（P <0.05）。空白组β波波幅低于造模组，δ波波幅高于造模组（P <0.05）。模型组的α波、δ波频率较空白组均下降（P <0.05），β波频率较空白组上升（P <0.05），半夏秫米汤不同剂量组及DZ组α波频率较模型组均上升（P <0.05），β波频率较模型组均下降（P <0.05），BXSM-M组、BXSM-H组及DZ组δ波频率较模型组均上升（P <0.05）。模型组的β波波幅较空白组上升（P <0.05），δ波波幅较空白组下降（P <0.05），半夏秫米汤不同剂量组及DZ组β波波幅较模型组均下降（P <0.05），δ波幅较模型组均上升（P <0.05），BXSM-L组α波波幅较模型组上升（P <0.05）。模型组血清、脑干Glu、GABA含量较空白组均升高（P <0.05），半夏秫米汤不同剂量组及DZ组血清Glu含量较模型组均降低（P <0.05），BXSM-M组、BXSM-H组及DZ组脑干Glu含量较模型组均降低（P <0.05），半夏秫米汤不同剂量组及DZ组血清GABA较模型组均降低（P <0.05），BXSM-L组、BXSM-H组及DZ组脑干GABA含量较模型组均降低（P <0.05）。模型组血清、脑干NE、DA含量较空白组均升高（P <0.05），半夏秫米汤不同剂量组及DZ组血清、脑干NE含量较模型组均降低（P <0.05），BXSM-M组、BXSM-H组及DZ组血清DA含量较模型组均降低（P <0.05），BXSM-H组和DZ组脑干DA较模型组降低（P <0.05）。模型组SCN、CC的Rorα与Bmal1基因和蛋白相对表达量较空白组下降（P <0.05），BXSM-M组、BXSM-H组及DZ组SCN、CC的Rorα与Bmal1基因和蛋白相对表达量较模型组均上升（P <0.05）。结论 首次从生物钟基因角度揭示了半夏秫米汤“引阳入阴”改善CI的机制，可能是通过上调Rorα/Bmal1信号轴调控睡眠稳态。

Abstract:

Objective To investigate the effect of Banxia Shumi decoction, a representative formula for "guiding Yang into Yin," on the Rorα/Bmal1 signaling axis in rats with chronic insomnia (CI).Methods Thirty-six SPF-grade rats were randomly divided into six groups: blank, model, low-dose Banxia Shumi decoction (BXSM-L), medium-dose Banxia Shumi decoction (BXSM-M), high-dose Banxia Shumi decoction (BXSM-H), and diazepam (DZ) groups, with six rats in each group. Except for the blank group, CI models were established using the small platform water environment method. The BXSM-L, BXSM-M, and BXSM-H groups received Banxia Shumi decoction decoction at doses of 4.69, 9.38 and 18.75 g/kg, respectively. The DZ group was administered diazepam solution at 0.52 mg/kg, while the blank and model groups received the same volume of normal saline for 14 days. After treatment, electroencephalogram (EEG) was monitored using a 16-channel physiological recorder. The expression of Rorα and Bmal1 genes and proteins in the suprachiasmatic nucleus (SCN) and cerebral cortex (CC) was assessed using real-time fluorescent quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. Enzyme-linked immunosorbent assay was used to measure levels of γ-aminobutyric acid (GABA), glutamate (Glu), dopamine (DA), and norepinephrine (NE) in the brainstem and serum.Results Rats in the model group exhibited dull, lackluster fur, lethargy, irritability, sensitivity to environmental stimuli (such as sound and light), and loss of circadian rhythm. The blank group had lower α and β wave frequencies and higher δ wave frequency than the model group (P < 0.05). The model group had decreased α and δ wave frequencies and increased β wave frequency compared to the blank group (P < 0.05). The BXSM-L, BXSM-M, BXSM-H, and DZ groups showed increased α wave frequencies and decreased β wave frequencies compared to the model group (P < 0.05). The BXSM-M, BXSM-H, and DZ groups had increased δ wave frequencies compared to the model group (P < 0.05). The model group had higher β wave amplitude and lower δ wave amplitude compared to the blank group (P < 0.05). The BXSM-L, BXSM-M, BXSM-H, and DZ groups had lower β wave amplitudes and higher δ wave amplitudes compared to the model group (P < 0.05). The model group showed elevated levels of serum and brainstem Glu, GABA, NE, and DA compared to the blank group (P < 0.05). The BXSM-L, BXSM-M, BXSM-H, and DZ groups had lower serum Glu levels compared to the model group (P < 0.05). The BXSM-M, BXSM-H, and DZ groups had lower brainstem Glu levels compared to the model group (P < 0.05). The BXSM-L, BXSM-M, BXSM-H, and DZ groups had lower serum GABA levels compared to the model group (P < 0.05). The BXSM-L and BXSM-H groups had lower brainstem GABA levels compared to the model group (P < 0.05). The model group had elevated levels of NE and DA in serum and brainstem compared to the blank group (P < 0.05). The BXSM-L, BXSM-M, BXSM-H, and DZ groups had lower serum NE levels compared to the model group (P < 0.05). The BXSM-M, BXSM-H, and DZ groups had lower serum DA levels compared to the model group (P < 0.05). The BXSM-H and DZ groups had lower brainstem DA levels compared to the model group (P < 0.05). The model group had decreased relative expression of Rorα and Bmal1 genes and proteins in SCN and CC compared to the blank group (P < 0.05). The BXSM-M, BXSM-H, and DZ groups had increased relative expression of Rorα and Bmal1 genes and proteins in SCN and CC compared to the model group (P < 0.05).Conclusion This study reveals, for the first time, that Banxia Shumi decoction may ameliorate CI by upregulating the Rorα/Bmal1 signaling axis, thereby improving sleep homeostasis.