中药材

目的：探讨桂枝芍药知母汤含药血清对大鼠椎间盘髓核细胞退变的影响及其作用机制。方法：20只雄性SD大鼠分别灌胃生理盐水、桂枝芍药知母汤(12.6 g/kg),连续给药7 d后收集血清。另取10只雄性大鼠，分离髓核细胞。(1)采用IL-1β处理来构建髓核细胞退变模型，分别加入不同浓度桂枝芍药知母汤含药血清和铁死亡抑制剂去铁胺(DFO),探究桂枝芍药知母汤含药血清对退变髓核细胞存活率的影响。(2)加入DFO,研究铁死亡在IL-1β诱导髓核细胞退变中的作用。(3)采用IL-1β和20%桂枝芍药知母汤含药血清处理，研究桂枝芍药知母汤含药血清对IL-1β处理髓核细胞PI3K/AKT/FoxO_3a信号通路的影响。(4)在IL-1β和20%桂枝芍药知母汤含药血清处理的同时，加入PI3K/AKT激活剂740Y-P进行干预，研究PI3K/AKT/FoxO_3a通路在桂枝芍药知母汤含药血清调控髓核细胞铁死亡中的作用。CCK-8法检测细胞存活率，比色法检测细胞Fe²⁺、MDA、GSH水平；细胞与胶原的黏附实验检测CollagenⅡ与大鼠髓核细胞结合能力；免疫荧光检测p-PI3K、p-AKT、p-FoxO_3a蛋白表达；Western Blot检测p-PI3K、p-AKT、p-FoxO_3a、SLC7A11、GPX4、MMP-13、CollagenⅡ蛋白表达。结果：与模型组比较，桂枝芍药知母汤含药血清组和DFO组细胞存活率和CollagenⅡ结合能力显著升高，Fe²⁺、MDA水平和p-PI3K、p-AKT、p-FoxO_3a、MMP-13蛋白表达显著降低，SLC7A11、GPX4、CollagenⅡ蛋白表达显著升高，桂枝芍药知母汤含药血清组FoxO_3a蛋白表达显著升高(P<0.05)。740Y-P能逆转桂枝芍药知母汤含药血清的作用(P<0.05)。结论：桂枝芍药知母汤含药血清能激活PI3K/AKT/FoxO_3a信号通路，抑制IL-1β诱导的大鼠髓核细胞铁死亡，延缓椎间盘髓核细胞的退变。

Objective: To explore the effects of the drug-containing serum of Guizhi Shaoyao Zhimu Decoction on the degeneration of nucleus pulposus cells(NCPs) in rat intervertebral discs and its mechanism of action.Methods: 20 male SD rats were gavaged with saline and Guizhi Shaoyao Zhimu Decoction(12.6 g/kg),and serum was collected after 7 days of continuous administration.Another 10 male rats were taken and NCPs were isolated.The model of NCPs degeneration was constructed by the treatment of IL-1β,different concentrations of drug-containing serum of Guizhi Shaoyao Zhimu Decoction and ferroptosis inhibitor deferoxamine(DFO) were added to investigate the effects of drug-containing serum of Guizhi Shaoyao Zhimu Decoction on the cell viability of degenerated NCPs, respectively.The DFO was added to study the role of ferroptosis in IL-1β-induced degeneration of nucleus pulposus cells.IL-1β and 20% of the drug-containing serum of Guizhi Shaoyao Zhimu Decoction were used for treatment to study the effect of the drug-containing serum of Guizhi Shaoyao Zhimu Decoction on the PI3K/AKT/FoxO_3a signaling pathway in IL-1β treated nucleus pulposus cells.While treating with IL-1β and 20% of the drug-containing serum of Guizhi Shaoyao Zhimu Decoction, the PI3K/AKT activator 740Y-P was added for intervention to study the role of the PI3K/AKT/FoxO_3a pathway in the regulation of ferroptosis in nucleus posus cells by the drug-containing serum of Guizhi Shaoyao Zhimu Decoction.The cell survival rate was detected by the CCK-8 method, and the levels of Fe²⁺,MDA and GSH in cells were detected by the colorimetric method.The adhesion experiment of cells to Collagen was used to detect the binding ability of CollagenⅡ to rat nucleus pulposus cells.Immunofluorescence was used to detect the protein expressions of p-PI3K,p-AKT and p-FoxO_3a;The protein expressions of p-PI3K,p-AKT,p-FoxO_3a,SLC7A11,GPX4,MMP-13 and CollagenⅡ were detected by Western Blot.Results: Compared with the model group, the cell survival rate and the binding ability of Collagen Ⅱ in the drug-containing serum of Guizhi Shaoyao Zhimu Decoction group and the DFO group were significantly increased, while the levels of Fe²⁺ and MDA and the protein expressions of p-PI3K,p-AKT,p-FoxO_3a,MMP-13 were significantly decreased.The protein expressions of SLC7A11,GPX4 and CollagenⅡ were significantly increased, and the protein expression of FoxO_3a in the the drug-containing serum of Guizhi Shaoyao Zhimu Decoction group was significantly increased(P<0.05).740Y-P could reverse the effect of the drug-containing serum of Guizhi Shaoyao Zhimu Decoction(P<0.05).Conclusion: The drug-containing serum of Guizhi Shaoyao Zhimu Decoction can activate the PI3K/AKT/FoxO_3a signaling pathway, inhibit IL-1β-induced ferroptosis of rat nucleus pulposus cells, and delay the degeneration of intervertebral disc nucleus pulposus cells.