Objectives: Because intracerebral hemorrhage (ICH) has high morbidity, disability and mortality, it is significant to find new and effective treatments for ICH. This study aims to explore the effect of butyphthalide (NBP) on neuroinflammation secondary to ICH and microglia polarization.
Methods: A total of 48 healthy male SD rats were randomly divided into 6 groups: a sham 24 h group, a sham 72 h group, an ICH 24 h group, an ICH 72 h group, an ICH+NBP 24 h group, and an ICH+NBP 72 h group (8 rats per group). After operation, the neurological deficiencies were assessed based on improved Garcia scores and corner test. The expressions of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), aquaporin-4 (AQP4), zonula occludens-1 (ZO-1), occludin, CD68, CD86, and CD206 were observed by Western blotting. Inflammatory cytokines were detected by ELISA. The immunofluorescence was to detect the polarization of microglia.
Results: 1) Compared with the sham groups, the expression of TLR4 (24 h: P<0.05; 72 h: P<0.01), NF-κB (both P<0.01) and Nrf2 (both P<0.01) in the perihematoma of the ICH group was increased, leading to microglia activation (P<0.01). The expressions of IL-6 (24 h: P<0.05; 72 h: P<0.01) and TNF-α (both P<0.01), the pro-inflammatory cytokines were up-regulated, and the expression of anti-inflammatory cytokine IL-4 was down-regulated (both P<0.01). Besides, the expression of AQP4 was enhanced (both P<0.01). The protein level of tightly connected proteins (including ZO-1, occludin) was decreased (all P<0.01). The neurological function of the rats in the ICH group was impaired in the 2 time points (both P<0.01). 2) Compared with the sham group at 24 h and 72 h after the intervention of NBP, the expressions of TLR4 (both P<0.05) and NF-κB (both P<0.01) were significantly declined, and the expression of Nrf2 was further enhanced (both P<0.05) in the perihematoma of the ICH+NBP group. Furthermore, the expression of M1 microglia marker was inhibited (P<0.05), and the polarization of microglia to the M2 phenotype was promoted (P<0.01). 3) In terms of inflammation after ICH, the IL-4 expression in the ICH+NBP group was increased compared with the ICH group (24 h: P<0.05; 72 h: P<0.01); the expression of IL-6 was decreased significantly in the ICH+NBP 72 h group (P<0.01); the level of AQP4 was declined significantly in the ICH+NBP 24 h group (P<0.05), there was a downward trend in the 72-hour intervention group but without significant statistical difference. 4) Compared with the ICH group, the ZO-1 protein levels were increased (24 h: P<0.05; 72 h: P<0.01), and the symptoms of nerve defect were improved eventually (both P<0.05) in the ICH+NBP groups.
Conclusions: After ICH, the TLR4/NF-κB pathway is activated. The M1 microglia is up-regulated along with the release of detrimental cytokines, while the anti-inflammatory cytokines are down-regulated. The expression of AQP4 is increased, the tight junction proteins from the blood-brain barrier (BBB) is damaged, and the neurological function of rats is impaired. On the contrary, NBP may regulate microglia polarization to M2 phenotype and play a role in the neuroprotective effect mediated via inhibiting TLR4/NF-κB and enhancing Nrf2 pathways, which relieves the neuroinflammation, inhibits the expression of AQP4, repairs BBB, and improves neurological functional defects.
目的: 脑出血(intracerebral hemorrhage，ICH)具有较高的发病率、致残率及病死率，寻找新的有效的ICH治疗方法具有重要意义。本文旨在探讨丁苯酞(butyphthalide，NBP)对ICH后继发性损伤和小胶质细胞极化的作用和机制。方法: 将48只健康雄性Sprague-Dawley大鼠随机分为6组，分别为假手术24 h组、假手术72 h组、ICH 24 h组、ICH 72 h组、ICH+NBP 24 h组、ICH+NBP 72 h组。术后评估神经功能缺损，采用蛋白质印迹法检测各组Toll样受体4(Toll-like receptor 4，TLR4)/核因子-κB(nuclear factor-kappa B，NF-κB)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)、水通道蛋白4(aquaporin-4，AQP4)、紧密连接蛋白、小胶质细胞不同表型的表达；采用ELISA检测炎症因子；采用免疫荧光染色检测小胶质细胞的极化情况。结果: 1)与假手术组相比，ICH组中血肿周围脑组织TLR4(24 h：P<0.05；72 h：P<0.01)、NF-κB(均P<0.01)、Nrf2(均P<0.01)表达均增加，小胶质细胞激活增加(均P<0.01)，血肿周围组织的促炎症因子IL-6(24 h：P<0.05；72 h：P<0.01)、TNF-α(均P<0.01)表达均增加，而抗炎因子IL-4表达均减少(均P<0.01)，同时AQP4表达均增加(均P<0.01)，紧密连接蛋白闭锁小带蛋白1(zonula occludens-1，ZO-1)与闭合蛋白(occludin)表达均下降(均P<0.01)，大鼠的神经功能均受损(均P<0.01)。2)与ICH组相比，ICH+NBP组的血肿周围组织TLR4(均P<0.05)及NF-κB(均P<0.01)表达均下降，Nrf2表达上升(均P<0.05)，M1型小胶质细胞标志物CD86表达均下降(均P<0.05)，M2型小胶质细胞标志物CD206表达均上升(均P<0.01)。3)在炎症反应方面，与ICH组同时间点相比，ICH+NBP组IL-4表达均增加(24 h：P<0.05；72 h：P<0.01)；ICH+NBP 72 h组IL-6较ICH 72 h组下降(P<0.01)；ICH+NBP 24 h组的AQP4表达较ICH 24 h组明显下降(P<0.05)，ICH+NBP 72 h组其表达与ICH 72 h组相比有下降趋势，但差异无明显统计学意义(P>0.05)。4)ICH+NBP组ZO-1表达较ICH组上升(24 h：P<0.05；72 h：P<0.01)；与ICH组相比，ICH+NBP组大鼠神经缺损症状最终得到了改善(均P<0.05)。结论: ICH后TLR4/NF-κB通路活化，M1型小胶质细胞表达及促炎因子释放增加，抗炎因子表达减少，AQP4表达上调，血脑屏障紧密连接蛋白破坏，大鼠神经功能受损。NBP能够通过抑制TLR4/NF-κB、增加Nrf2表达而逆转ICH后小胶质细胞极化、神经炎症、血脑屏障的变化，从而起到神经保护的作用。.
Keywords: Toll-like receptor 4; intracerebral hemorrhage; microglia polarization; nuclear factor erythroid 2-related factor 2; nuclear factor-kappa B.