Table1_Zhilong Huoxue Tongyu capsule attenuates intracerebral hemorrhage induced redox imbalance by modulation of Nrf2 signaling pathway.XLS

Background: One of the severely debilitating and fatal subtypes of hemorrhagic stroke is intracerebral hemorrhage (ICH), which lacks an adequate cure at present. The Zhilong Huoxue Tongyu (ZLHXTY) capsule has been utilized effectively since last decade to treat ICH, in some provinces of China but the scientific basis for its mechanism is lacking. Purpose: To investigate the neuroprotective role of ZLHXTY capsules for ICH-induced oxidative injury through the regulation of redox imbalance with the Nrf2 signaling pathway.
Methods: Autologous blood injection model of ICH in C57BL/6J mice was employed. Three treatment groups received ZLHXTY once daily through oral gavage at doses 0.35 g/kg, 0.7 g/kg, and 1.4 g/kg, started after 2 h and continued for 72 h of ICH induction. The neurological outcome was measured using a balance beam test. Serum was tested for inflammatory markers IL-1β, IL-6, and TNF-α through ELISA, oxidative stress through hydrogen peroxide content assay, and antioxidant status by total antioxidant capacity (T-AOC) assay. Nuclear extract from brain tissue was assayed for Nrf2 transcriptional factor activity. RT-qPCR was performed for Nfe2l2, Sod1, Hmox1, Nqo1, and Mgst1; and Western blotting for determination of protein expression of Nrf2, p62, Pp62, Keap, HO1, and NQO1. Fluoro-jade C staining was also used to examine neuronal damage.
Results: ZLHXTY capsule treatment following ICH demonstrated a protective effect against oxidative brain injury. Neurological scoring showed improvement in behavioral outcomes. ELISA-based identification demonstrated a significant decline in the expression of serum inflammatory markers. Hydrogen peroxide content in serum was found to be reduced. The total antioxidant capacity was also reduced in serum, but the ZLHXTY extract showed a concentration-dependent increase in T-AOC speculating at its intrinsic antioxidant potential. Nrf2 transcriptional factor activity, mRNA and protein expression analyses revealed normalization of Nrf2 and its downstream targets, which were previously elevated as a result of oxidative stress induced by ICH. Neuronal damage was also reduced markedly after ZLHXTY treatment as revealed by Fluoro-jade C staining. Conclusion: ZLHXTY capsules possess an intrinsic antioxidant potential that can modulate the ICH-induced redox imbalance in the brain as revealed by the normalization of Nrf2 and its downstream antioxidant targets.

背景：出血性卒中的严重致残且致命的亚型之一为脑出血（intracerebral hemorrhage, ICH），目前尚无有效的治疗手段。蛭龙活血通瘀（Zhilong Huoxue Tongyu, ZLHXTY）胶囊近十年来已在中国部分省份用于治疗ICH并取得良好疗效，但其发挥作用的科学机制尚不明确。
目的：本研究旨在探讨ZLHXTY胶囊通过调控核因子E2相关因子2（Nrf2）信号通路改善氧化还原失衡，从而发挥抗ICH诱导脑氧化损伤的神经保护作用。
方法：采用自体血注射法构建C57BL/6J小鼠ICH模型。设置3个给药组，分别以0.35 g/kg、0.7 g/kg及1.4 g/kg的剂量每日灌胃给予ZLHXTY，于ICH造模后2小时开始给药，连续给药72小时。采用平衡木实验评估神经功能结局。通过酶联免疫吸附试验（ELISA）检测血清炎症因子IL-1β、IL-6及TNF-α水平，以过氧化氢含量检测法评估氧化应激水平，以总抗氧化能力（total antioxidant capacity, T-AOC）检测法评估机体抗氧化状态。提取脑组织核蛋白，检测Nrf2转录因子活性。通过实时荧光定量聚合酶链式反应（RT-qPCR）检测Nfe2l2、Sod1、Hmox1、Nqo1及Mgst1的mRNA表达水平；采用蛋白质免疫印迹（Western blotting）检测Nrf2、p62、Pp62、Keap、HO1及NQO1的蛋白表达水平。此外，采用氟罗朱红C（Fluoro-jade C）染色观察神经元损伤情况。
结果：ICH后给予ZLHXTY胶囊治疗可对脑氧化损伤起到保护作用。神经功能评分结果显示小鼠行为学结局得到改善。ELISA检测结果表明，血清炎症因子表达水平显著降低。血清过氧化氢含量有所下降。血清总抗氧化能力本因ICH出现降低，但ZLHXTY提取物可呈浓度依赖性提升T-AOC水平，提示其具备内源性抗氧化潜能。Nrf2转录因子活性、mRNA及蛋白表达分析结果显示，ICH诱导氧化应激后异常升高的Nrf2及其下游靶标表达恢复至正常水平。氟罗朱红C染色结果显示，ZLHXTY治疗后神经元损伤显著减轻。
结论：综上，ZLHXTY胶囊具有内源性抗氧化潜能，可通过调控Nrf2及其下游抗氧化靶标表达恢复氧化还原稳态，从而改善ICH诱导的脑内氧化还原失衡。