黄连解毒汤已在临床上用于治疗缺血性中风。然而,HLJD对缺血性卒中的详细保护机制尚未阐明.本研究的目的是基于抑制神经炎症和改善神经细胞损伤阐明HLJD的潜在药理机制。建立大鼠大脑中动脉阻塞再灌注(MCAO/R)模型,并接受HLJD治疗。根据Bederson评分评估HLJD对神经功能的影响,姿势反射测试和不对称评分。2,3,5-氯化三苯基四唑(TTC)染色,用血红素和伊红(HE)和Nissl染色观察脑病理变化。然后,在HLJD干预后,使用转录组学方法筛选MCAO/R模型大鼠脑组织中的差异基因。随后,HLJD对中性粒细胞胞外诱捕网(NET)形成相关神经炎症的影响,γ-氨基丁酸(GABA)能突触激活,使用免疫荧光验证神经细胞损伤和增殖,蛋白质印迹和酶联免疫吸附测定(ELISA)。我们的结果表明,HLJD干预降低了Bederson的得分,MCAO/R模型大鼠的姿势反射测试评分和不对称评分。病理染色显示HLJD治疗减少了脑梗死面积,减轻神经元损伤并增加Nissl体的数量。转录组学表明HLJD影响了MCAO/R大鼠的435个基因。其中,涉及NET形成和GABA能突触途径的几个基因失调。随后的实验验证表明,HLJD减少了MPO+CitH3+阳性表达面积,PAD4、p-P38/P38、p-ERK/ERK蛋白表达降低,IL-1β水平降低,IL-6和TNF-α,逆转了Iba1+TLR4+的增加,Iba1+p65+和Iba1+NLRP3+在脑内阳性表达区域。此外,HLJD增加GABA水平,GABRG1和GAT3蛋白表达升高,TUNEL阳性表达面积减少,Ki67阳性表达面积增加。HLJD干预对MCAO/R大鼠缺血诱发的脑损伤具有多方面的积极影响。这种干预通过减轻NET形成有效抑制神经炎症,同时通过激活GABA能突触改善神经细胞损伤并促进神经细胞增殖。
Huanglian Jiedu decoction (HLJD) has been used to treat ischemic stroke in clinic. However, the detailed protective mechanisms of HLJD on ischemic stroke have yet to be elucidated. The aim of this study is to elucidate the underlying pharmacological mechanisms of HLJD based on the inhibition of neuroinflammation and the amelioration of nerve cell damage. A middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats and received HLJD treatment. Effects of HLJD on neurological function was assessed based on Bederson\'s score, postural reflex test and asymmetry score. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, Hematein and eosin (HE) and Nissl staining were used to observe the pathological changes in brain. Then, transcriptomics was used to screen the differential genes in brain tissue in MCAO/R model rats following HLJD intervention. Subsequently, the effects of HLJD on neutrophil extracellular trap (NET) formation-related neuroinflammation, gamma-aminobutyric acid (GABA)ergic synapse activation, nerve cell damage and proliferation were validated using immunofluorescence, western blot and enzyme-linked immunosorbent assay (ELISA). Our results showed that HLJD intervention reduced the Bederson\'s score, postural reflex test score and asymmetry score in MCAO/R model rats. Pathological staining indicated that HLJD treatment decreased the cerebral infarction area, mitigated neuronal damage and increased the numbers of Nissl bodies. Transcriptomics suggested that HLJD affected 435 genes in MCAO/R rats. Among them, several genes involving in NET formation and GABAergic synapses pathways were dysregulated. Subsequent experimental validation showed that HLJD reduced the MPO+CitH3+ positive expression area, reduced the protein expression of PAD4, p-P38/P38, p-ERK/ERK and decreased the levels of IL-1β, IL-6 and TNF-α, reversed the increase of Iba1+TLR4+, Iba1+p65+ and Iba1+NLRP3+ positive expression area in brain. Moreover, HLJD increased GABA levels, elevated the protein expression of GABRG1 and GAT3, decreased the TUNEL positive expression area and increased the Ki67 positive expression area in brain. HLJD intervention exerts a multifaceted positive impact on ischemia-induced cerebral injury in MCAO/R rats. This intervention effectively inhibits neuroinflammation by mitigating NET formation, and concurrently improves nerve cell damage and fosters nerve cell proliferation through activating GABAergic synapses.