Effects of butyphthalide on microglia polarization after intracerebral hemorrhage and the underlying mechanisms.

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: <0.05; 72 h: <0.01), NF-κB (both <0.01) and Nrf2 (both <0.01) in the perihematoma of the ICH group was increased, leading to microglia activation (<0.01). The expressions of IL-6 (24 h: <0.05; 72 h: <0.01) and TNF-α (both <0.01), the pro-inflammatory cytokines were up-regulated, and the expression of anti-inflammatory cytokine IL-4 was down-regulated (both <0.01). Besides, the expression of AQP4 was enhanced (both <0.01). The protein level of tightly connected proteins (including ZO-1, occludin) was decreased (all <0.01). The neurological function of the rats in the ICH group was impaired in the 2 time points (both <0.01). 2) Compared with the sham group at 24 h and 72 h after the intervention of NBP, the expressions of TLR4 (both <0.05) and NF-κB (both <0.01) were significantly declined, and the expression of Nrf2 was further enhanced (both <0.05) in the perihematoma of the ICH+NBP group. Furthermore, the expression of M1 microglia marker was inhibited (<0.05), and the polarization of microglia to the M2 phenotype was promoted (<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: <0.05; 72 h: <0.01); the expression of IL-6 was decreased significantly in the ICH+NBP 72 h group (<0.01); the level of AQP4 was declined significantly in the ICH+NBP 24 h group (<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: <0.05; 72 h: <0.01), and the symptoms of nerve defect were improved eventually (both <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.