益气活血化浊解毒方调控KAT3B/STING轴介导的小胶质细胞极化对缺血性脑卒中大鼠神经功能的影响

Study on the effect of Yiqi Huoxue Huazhuo Jiedu decoction regulating microglial polarization via KAT3B/STING axis on neurological function in rats with ischemic stroke

摘要:

目的 探讨益气活血化浊解毒方调控KAT3B/STING轴介导的小胶质细胞极化对缺血性脑卒中大鼠神经功能的影响。方法 复制缺血性脑卒中大鼠模型。将复制成功的60只大鼠分为缺血性脑卒中组、益气活血化浊解毒方低剂量组（低剂量组）、益气活血化浊解毒方高剂量组（高剂量组）、益气活血化浊解毒方高剂量+2，5-己酮可可碱（DMXAA）组（高剂量+DMXAA组），每组15只。另取15只正常大鼠为Sham组。低剂量组大鼠灌胃0.2 mL 27.5 g/mL的益气活血化浊解毒方药液，高剂量组大鼠灌胃0.2 mL 55.0 g/mL的益气活血化浊解毒方药液，高剂量+DMXAA组大鼠灌胃0.2 mL 55.0 g/mL的益气活血化浊解毒方药液和25 mg/kg的DMXAA，Sham组和缺血性脑卒中组给予等体积生理盐水代替药物；除高剂量+DMXAA组外，其余组大鼠再给予等体积二甲基亚砜溶液灌胃，1 d/次，连续4周。Zea-Longa评分和网屏试验评分评估大鼠神经功能和神经行为学；检测大鼠脑组织含水量；TTC染色法检测大鼠脑梗死面积；酶联免疫吸附试验检测大鼠血清LDH和NGF水平和缺血侧皮层组织TNF-α、IL-6、IL-10水平；HE染色观察大鼠缺血侧皮层组织病理变化；Western blotting检测大鼠缺血侧皮层组织iNOS、Iba1、CD68、CD40、CD206、Arg-1、Cleaved caspase-3、KAT3B、STING蛋白表达。结果 缺血性脑卒中组大鼠Zea-Longa评分和网屏试验评分、脑组织含水量、脑梗死面积百分比均高于Sham组（P <0.05），低剂量组和高剂量组大鼠Zea-Longa评分和网屏试验评分、脑组织含水量、脑梗死面积百分比均低于缺血性脑卒中组（P <0.05），高剂量+DMXAA组大鼠Zea-Longa评分和网屏试验评分、脑组织含水量、脑梗死面积百分比均高于高剂量组（P <0.05）。缺血性脑卒中组大鼠NGF和IL-10水平低于Sham组，LDH、TNF-α和IL-6水平高于Sham组（P <0.05）；低剂量组和高剂量组大鼠NGF和IL-10水平高于缺血性脑卒中组，LDH、TNF-α和IL-6水平低于缺血性脑卒中组（P <0.05）；高剂量+DMXAA组大鼠NGF和IL-10水平低于高剂量组，LDH、TNF-α和IL-6高于高剂量组（P <0.05）。与缺血性脑卒中组比较，低剂量组和高剂量组缺血侧皮层神经元形态有明显改善。缺血性脑卒中组大鼠缺血侧皮层组织中iNOS、Iba1、CD68和CD40蛋白相对表达量比Sham组高，CD206和Arg-1蛋白相对表达量比Sham组低（P <0.05）；低剂量组和高剂量组大鼠缺血侧皮层组织中iNOS、Iba1、CD68和CD40蛋白相对表达量比缺血性脑卒中组低，CD206和Arg-1蛋白相对表达量比缺血性脑卒中组高（P <0.05）；高剂量+ DMXAA组大鼠缺血侧皮层组织中iNOS、Iba1、CD68和CD40蛋白相对表达量比高剂量组高，CD206和Arg-1蛋白相对表达量比高剂量组低（P <0.05）。结论 益气活血化浊解毒方可调节缺血性脑卒中大鼠小胶质细胞极化，减轻神经炎症和神经损伤，改善神经功能，可能与抑制KAT3B/STING轴有关。

Abstract:

Objective To investigate the effects of Yiqi Huoxue Huazhuo Jiedu Decoction (YHHJD) on microglial polarization mediated by the KAT3B/STING axis and its impact on neurological function in rats with ischemic stroke (IS).Methods An ischemic stroke rat model was established. Sixty successfully modeled rats were divided into the ischemic stroke group (IS group), YHHJD low-dose group (Low-dose group), YHHJD high-dose group (High-dose group), and YHHJD high-dose + DMXAA group (High-dose + DMXAA group), with 15 rats in each group. Another 15 normal rats were included as the Sham group. Rats in the Low-dose group were intragastrically administered 0.2 mL of 27.5 g/mL YHHJD, those in the High-dose group were given 0.2 mL of 55.0 g/mL YHHJD, and the High-dose + DMXAA group received 0.2 mL of 55.0 g/mL YHHJD combined with 25 mg/kg DMXAA. Sham and IS groups received equivalent volumes of saline as controls. All groups except the High-dose + DMXAA group were additionally administered dimethyl sulfoxide intragastrically, once daily for four weeks. Neurological and behavioral assessments were performed using the Zea-Longa score and grid-walking test. Brain water content was measured, infarct size assessed via TTC staining, and serum LDH and NGF levels, as well as TNF-α, IL-6, and IL-10 levels in the ischemic cortex, were determined by ELISA. HE staining was used to observe cortical pathology. Western blotting was employed to detect protein expression levels of iNOS, Iba1, CD68, CD40, CD206, Arg-1, Cleaved caspase-3, KAT3B, and STING in the ischemic cortex.Results Zea-Longa scores, grid-walking scores, brain water content, and infarct size percentages were higher in the IS group than in the Sham group (P < 0.05). These indicators were significantly reduced in the Low-dose and High-dose groups compared to the IS group (P < 0.05) but increased in the High-dose + DMXAA group compared to the High-dose group (P < 0.05). NGF and IL-10 levels were lower in the IS group, while LDH, TNF-α, and IL-6 levels were higher compared to the Sham group (P < 0.05). NGF and IL-10 levels were higher, and LDH, TNF-α, and IL-6 levels were lower in the Low-dose and High-dose groups compared to the IS group (P < 0.05). In the High-dose + DMXAA group, NGF and IL-10 levels were lower, while LDH, TNF-α, and IL-6 levels were higher compared to the High-dose group (P < 0.05). Histological observations showed improved neuronal morphology in the ischemic cortex in the Low-dose and High-dose groups compared to the IS group. The expression of iNOS, Iba1, CD68, and CD40 was higher, while CD206 and Arg-1 levels were lower in the IS group than in the Sham group (P < 0.05). These trends were reversed in the Low-dose and High-dose groups (P < 0.05). The High-dose + DMXAA group showed higher iNOS, Iba1, CD68, and CD40 and lower CD206 and Arg-1 expression than the High-dose group (P < 0.05).Conclusion YHHJD decoction can modulate microglial polarization in IS rats, alleviate neuroinflammation and neuronal injury, and improve neurological function, potentially by inhibiting the KAT3B/STING axis.