目的 基于 c-Jun 氨基末端激酶（JNK）信号通路探讨加味通络方含药血清对大鼠原代皮层神经元氧糖剥 夺/复氧（OGD/R）损伤的作用机制。方法 利用胎鼠分离、培养大鼠原代皮层神经元；利用大鼠制备加味通络 方含药血清及空白血清；神经元 OGD 处理 6 h 后，进行复氧 12 h 或 24 h，复制 OGD/R 细胞模型。将大鼠原 代皮层神经元随机分为：空白对照组、含药血清组、OGD6 h/R12 h 组、OGD6 h/R12 h+含药血清组、OGD6 h/ R12 h+抑制剂组、OGD6 h/R12 h+联合组、OGD6 h/R24 h 组、OGD6 h/R24 h+含药血清组。OGD 处理前 1 h， 空白对照组加入 10% 空白血清；含药血清组加入 10% 加味通络方含药血清；抑制剂组加入 20 µmol·L-1 SP600125（JNK 通路抑制剂，溶于 DMSO，终浓度＜0.1%）；联合组加入 10% 含药血清+20 µmol·L-1 SP600125。 采用免疫细胞化学/免疫荧光法（ICC-IF）检测细胞巢蛋白（Nestin）表达水平；CCK-8 法测定细胞增殖率；Annexin V/PI 双染法检测细胞凋亡率；qRT-PCR 法检测细胞 MAPK8、JUN、Bcl-2、Fas、Caspase-3 基因表达水平； Western Blot 法检测细胞 JNK、p-JNK、c-Jun、Fas、Cleaved-Caspase-3、Bcl-2 蛋白表达水平。结果 （1）与 空白对照组比较，含药血清组细胞的 Nestin 荧光表达强度及细胞增殖率显著升高（P＜0.05，P＜0.01），细胞凋 亡率无明显变化（P＞0.05）；OGD6 h/R12 h 组、OGD6 h/R24 h 组细胞的 Nestin 荧光表达强度及细胞增殖率显 著下降（P＜0.01），细胞凋亡率显著升高（P＜0.01）；OGD6 h/R12 h 组细胞 p-JNK、c-Jun、Fas、CleavedCaspase-3 蛋白表达均显著上调（P＜0.01），Bcl-2 蛋白表达显著下调（P＜0.01），MAPK8、JUN、Fas、Caspase-3 mRNA 表达均显著上调（P＜0.01），Bcl-2 mRNA 表达显著下调（P＜0.01）。（2）与相同时间点模型组比较， OGD6 h/R12 h+含药血清组、OGD6 h/R12 h+抑制剂组、OGD6 h/R24 h+含药血清组细胞的 Nestin 荧光表达强度 及细胞增殖率均显著升高（P＜0.01），细胞凋亡率显著降低（P＜0.01）；OGD6 h/R12 h+含药血清组、OGD6 h/ R12 h+抑制剂组细胞 p-JNK、c-Jun、Fas、Cleaved-Caspase-3 蛋白表达均显著下调（P＜0.01），Bcl-2 蛋白表 达显著上调（P＜0.01），MAPK8、JUN、Fas mRNA 表达均显著下调（P＜0.01），Bcl-2 mRNA 表达显著上调 （P＜0.01）；OGD6 h/R12 h+含药血清组细胞 Caspase-3 mRNA 表达明显上调（P＜0.05），OGD6 h/R12 h+抑制剂 组细胞 Caspase-3 mRNA 表达显著下调（P＜0.01）。（3）与 OGD6 h/R12 h+含药血清组或 OGD6 h/R12 h+抑制剂组 比较，OGD6 h/R12 h+联合组细胞的 Nestin 荧光表达强度及细胞增殖率均显著升高（P＜0.01），细胞凋亡率显 著降低（P＜0.01）；细胞 p-JNK、c-Jun、Fas、Cleaved-Caspase-3 蛋白表达均显著下调（P＜0.01），Bcl-2 蛋白 表达显著上调（P＜0.01），MAPK8、JUN、Fas、Caspase-3 mRNA 表达均显著下调（P＜0.01），Bcl-2 mRNA 表 达显著上调（P＜0.01）。（4）与 OGD6 h/R12 h 组比较，OGD6 h/R24 h 组细胞的 Nestin 荧光表达强度及细胞增殖 率均无明显变化（P＞0.05），然而与 OGD6 h/R12 h+含药血清组比较，OGD6 h/R24 h+含药血清组的细胞增殖率 显著下降（P＜0.01）。结论 加味通络方含药血清对原代皮层神经元氧糖剥夺/复氧（OGD/R）损伤具有保护作 用，其作用机制可能与抑制 JNK 信号通路的激活，调控凋亡相关蛋白及基因表达有关。

Objective To investigate the mechanism of Jiawei Tongluo Formula-containing serum on oxygen-glucose deprivation/reoxygenation （OGD/R）-induced injury in primary rat cortical neurons based on the c-Jun N-terminal kinase （JNK） signaling pathway. Methods Primary cortical neurons were isolated and cultured from fetal rats. Jiawei Tongluo Formula-containing serum and blank serum were prepared from rats. The OGD/R cell model was established by subjecting neurons to OGD for 6 hours followed by reoxygenation for 12 hours or 24 hours. Primary rat cortical neurons were randomly divided into the following groups: blank control，medicated serum，OGD6 h/R12 h，OGD6 h/R12 h + medicated serum，OGD6 h/R12 h + inhibitor，OGD6 h/R12 h + combination，OGD6 h/R24 h，and OGD6 h/R24 h + medicated serum. One hour prior to OGD treatment，the blank control group received 10% blank serum；the medicated serum group received 10% Jiawei Tongluo Formula-containing serum； the inhibitor group received 20 µmol·L-1 SP600125（JNK pathway inhibitor， dissolved in DMSO， final concentration ＜0.1%）； and the combination group received 10% medicated serum + 20 µmol·L-1 SP600125. Immunocytochemistry/immunofluorescence （ICC-IF） was used to detect Nestin expression levels；CCK-8 assay was used to measure cell proliferation rate；Annexin V/PI double staining was used to detect apoptosis rate；qRT-PCR was used to detect the mRNA expression levels of MAPK8，JUN， Bcl-2，Fas，and Caspase-3；Western Blot was used to detect the protein expression levels of JNK，p-JNK，c-Jun， Fas， Cleaved-Caspase-3， and Bcl-2. Results （1） Compared with the blank control group， the medicated serum group showed significantly increased Nestin fluorescence intensity and cell proliferation rate （P＜0.05， P＜0.01）， with no significant change in apoptosis rate （P＞0.05）； the OGD6 h/R12 h and OGD6 h/R24 h groups exhibited significantly decreased Nestin fluorescence intensity and cell proliferation rate （P＜0.01），and significantly increased apoptosis rate （P＜0.01）；the OGD6 h/R12 h group showed significantly upregulated protein expression of p-JNK， c-Jun，Fas，and Cleaved-Caspase-3（P＜0.01），significantly downregulated Bcl-2 protein expression （P＜0.01）， and significantly upregulated mRNA expression of MAPK8， JUN， Fas， and Caspase-3（P＜0.01）， along with significantly downregulated Bcl-2 mRNA expression （P＜0.01）.（2） Compared with the model group at the same time point，the OGD6 h/R12 h + medicated serum，OGD6 h/R12 h + inhibitor，and OGD6 h/R24 h + medicated serum groups showed significantly increased Nestin fluorescence intensity and cell proliferation rate （P＜0.01）， and significantly decreased apoptosis rate （P＜0.01）；the OGD6 h/R12 h + medicated serum and OGD6 h/R12 h + inhibitor groups exhibited significantly downregulated protein expression of p-JNK，c-Jun，Fas，and Cleaved-Caspase-3（P＜ 0.01），significantly upregulated Bcl-2 protein expression （P＜0.01），and significantly downregulated mRNA expression of MAPK8，JUN，and Fas （P＜0.01），along with significantly upregulated Bcl-2 mRNA expression （P＜0.01）；the OGD6 h/R12 h + medicated serum group showed significantly upregulated Caspase-3 mRNA expression （P＜0.05）， while the OGD6 h/R12 h + inhibitor group exhibited significantly downregulated Caspase-3 mRNA expression （P＜ 0.01）.（3） Compared with either the OGD6 h/R12 h + medicated serum group or the OGD6 h/R12 h + inhibitor group， the OGD6 h/R12 h + combination group showed significantly increased Nestin fluorescence intensity and cell proliferation rate （P＜0.01），and significantly decreased apoptosis rate （P＜0.01）；protein expression of p-JNK， c-Jun， Fas， and Cleaved-Caspase-3 was significantly downregulated （P＜0.01）， Bcl-2 protein expression was significantly upregulated （P＜0.01），and mRNA expression of MAPK8，JUN，Fas，and Caspase-3 was significantly downregulated （P＜0.01）， while Bcl-2 mRNA expression was significantly upregulated （P＜0.01）.（4） Compared with the OGD6 h/R12 h group， the OGD6 h/R24 h group showed no significant differences in Nestin fluorescence intensity or cell proliferation rate （P＞0.05）；however，compared with the OGD6 h/R12 h + medicated serum group， the OGD6 h/R24 h + medicated serum group exhibited a significantly decreased cell proliferation rate （P＜0.01）. Conclusion Jiawei Tongluo Formula-containing serum exerts a protective effect against oxygen-glucose deprivation/ reoxygenation （OGD/R）-induced injury in primary cortical neurons， and its mechanism may be associated with inhibiting the activation of the JNK signaling pathway and regulating the expression of apoptosis-related proteins and genes.

R285.5

国家自然科学基金青年项目（82305176）；广州市基础研究计划 2023 年度市校（院）企联合资助项目 ［重大项目（2023A03J0778）， 面上项目（2023A03J0309）］；广州市中医药和中西医结合科技项目（20232A011004）。