Zhou Chang, Qiu Shi-Wei, Wang Feng-Ming, Liu Yu-Chen, Hu Wei, Yang Mei-Lan, Liu Wang-Hua, Li Hua
Hunan University of Chinese Medicine, Changsha, Hunan, China.
Provincial Key Laboratory of TCM Diagnostics, Hunan University of Chinese Medicine, Changsha, Hunan, China.
Front Pharmacol. 2024 Jul 29;15:1449452. doi: 10.3389/fphar.2024.1449452. eCollection 2024.
The Chinese ethnic medicine Jie-Du-Huo-Xue Decoction (JDHXD) is used to alleviate neuroinflammation in cerebral ischemia (CI). Our previous studies have confirmed that JDHXD can inhibit microglial pyroptosis in CI. However, the pharmacological mechanism of JDHXD in alleviating neuroinflammation and pyroptosis needs to be further elucidated. New research points out that there is an interaction between autophagy and inflammasome NLRP3, and autophagy can help clear NLRP3. The NLRP3 is a key initiator of pyroptosis and autophagy. The effect of JDHXD promoting autophagy to clear NLRP3 to inhibit pyroptosis on cerebral ischemia-reperfusion inflammatory injury is currently unknown. We speculate that JDHXD can inhibit pyroptosis in CI by promoting autophagy to clear NLRP3.
Chemical characterization of JDHXD was performed using LC-MS. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in SD rats. Neurological deficits, neuron damage, and cerebral infarct volume were evaluated. Western Blot and immunofluorescence were used to detect neuronal pyroptosis and autophagy.
30 possible substance metabolites in JDHXD medicated serum were analyzed by LC-MS (Composite Score > 0.98). Furthermore, JDHXD protects rat neurological function and cerebral infarct size after CI. JDHXD inhibited the expression of pyroptosis and autophagy after CI. Our western blot and immunofluorescence results showed that JDHXD treatment can reduce the expression of autophagy-related factors ULK1, beclin1, and LC3-Ⅱ. The expression of NLRP3 protein was lower in the JDHXD group than in the I/R group. Compared with the I/R group, the expressions of pyroptosis-related factors caspase-1 P 10, GSDMD-NT, IL-18, and IL-1β decreased in the JDHXD group. Furthermore, we observed an unexpected result: immunofluorescence demonstrated that Gasdermin D (GSDMD) was significantly absent in the infarct core, and highly expressed in the peri-infarct and contralateral cerebral hemispheres. This finding challenges the prevailing view that GSDMD is elevated in the ischemic cerebral hemisphere.
JDHXD inhibited pyroptosis and autophagy after MCAO/R. JDHXD suppressed pyroptosis and autophagy by inhibiting NLRP3, thereby alleviating CI. In addition, we present a different observation from previous studies that the expression of GSDMD in the infarct core was lower than that in the peri-infarct and contralateral non-ischemic hemispheres on day 3 of CI.
中药解毒活血汤（JDHXD）用于减轻脑缺血（CI）中的神经炎症。我们之前的研究证实，JDHXD可抑制CI中的小胶质细胞焦亡。然而，JDHXD减轻神经炎症和焦亡的药理机制仍需进一步阐明。新的研究指出，自噬与炎性小体NLRP3之间存在相互作用，且自噬有助于清除NLRP3。NLRP3是焦亡和自噬的关键启动因子。目前尚不清楚JDHXD促进自噬清除NLRP3以抑制焦亡对脑缺血再灌注炎性损伤的影响。我们推测JDHXD可通过促进自噬清除NLRP3来抑制CI中的焦亡。
采用液相色谱-质谱联用（LC-MS）对JDHXD进行化学表征。在SD大鼠中建立大脑中动脉闭塞/再灌注（MCAO/R）模型。评估神经功能缺损、神经元损伤和脑梗死体积。采用蛋白质免疫印迹法（Western Blot）和免疫荧光法检测神经元焦亡和自噬。
通过LC-MS分析了JDHXD含药血清中30种可能的物质代谢产物（综合评分>0.98）。此外，JDHXD可保护CI后大鼠的神经功能并减小脑梗死面积。JDHXD抑制了CI后的焦亡和自噬表达。我们的蛋白质免疫印迹法和免疫荧光结果显示，JDHXD处理可降低自噬相关因子ULK1、beclin1和LC3-Ⅱ的表达。JDHXD组中NLRP3蛋白的表达低于缺血再灌注（I/R）组。与I/R组相比，JDHXD组中焦亡相关因子caspase-1 P10、GSDMD-NT、IL-18和IL-1β的表达降低。此外，我们观察到一个意外结果：免疫荧光显示梗死核心中明显缺乏Gasdermin D（GSDMD），而在梗死周边和对侧脑半球中高表达。这一发现挑战了目前普遍认为缺血性脑半球中GSDMD升高的观点。
JDHXD在MCAO/R后抑制焦亡和自噬。JDHXD通过抑制NLRP3抑制焦亡和自噬，从而减轻CI。此外，我们呈现了与以往研究不同的观察结果，即CI第3天时梗死核心中GSDMD的表达低于梗死周边和对侧非缺血半球。
