Buyang Huanwu Decoction prevents hemorrhagic transformation after delayed t-PA infusion via inhibiting NLRP3 inflammasome/pyroptosis associated with microglial PGC-1α

Ethnopharmacological relevance: Delayed tissue-type plasminogen activator (t-PA) thrombolysis, which has a restrictive therapeutic time window within 4.5 h following ischemic stroke (IS), increases the risk of hemorrhagic transformation (HT) and subsequent neurotoxicity. Studies have shown that the NLRP3 inflammasome activation reversely regulated by the PGC-1α leads to microglial polarization and pyroptosis to cause damage to nerve cells and the blood-brain barrier. The effect of Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription widely used in the recovery of IS, on HT injury after delayed t-PA treatment had been found with clinical studies, while the underlying mechanisms are reminded to be further clarified.

Aim of the study: This study sought to investigate the therapeutic effect and the underlying mechanisms of BHD in ischemic rat brains with delayed t-PA treatment.

Materials and methods: The components of BHD extracts were identified by High Performance Liquid Chromatography (HPLC) and the effective components in the rat brains from BHD were analyzed by liquid chromatography-mass spectrometry (LC-MS). In vivo experiment was carried out by 5 h of middle cerebral artery occlusion (MCAO) following by t-PA infusion for 0.5 h plus reperfusion 19 h, while the in vitro BV2 cells were stimulated by lipopolysaccharide (LPS)-adenosine triphosphate (ATP) to activate microglia pyroptosis, of which the MCC950 (NLRP3 inhibitor) and NSA (GSDMD inhibitor) were adopted as reverse validation. PGC-1α siRNA was utilized to study the mechanisms of BHD against microglial polarization and pyroptosis in BV2 cells.

Results: HPLC analysis demonstrated the fingerprint of BHD with six reference standards (Hydroxysafflor yellow A, Calycosin-7-glucoside, Paeoniflorin, Formononetin, Ferulic acid and Amygdalin), the last two of which can be found in rat brains by LC-MS analysis. In the following experiments, we found the major discoveries as follow: (1) BHD improved the neurological outcomes, the structural integrity of the blood-brain barrier and the neuronal structure in HT rats with MCAO following by delayed t-PA infusion; (2) the presence of t-PA promoted the suppression of PGC-1α and the activation of microglial NLRP3 inflammasome and pyroptosis in the HT rats; (3) BHD promoted the transformation of microglia from M1 to M2 type for inhibiting inflammatory response; (4) BHD restrained NLRP3 inflammasome/pyroptosis activation in microglia, prevented the translocations of NF-κB into the nucleus, as well as enhanced microglia-specific PGC-1α in ischemic rats following t-PA delayed thrombolysis; (5) BHD suppressed NLRP3 inflammasome assembly and increased PGC-1α expression in the LPS-ATP-induced BV2 cells; (6) PGC-1α silencing withdrew the protective role of BHD against NLRP3 inflammasome/pyroptosis.

Conclusion: Mechanistically, BHD existed the protective effect against HT injury after delayed t-PA treatment through up-regulating microglial PGC-1α to inhibit NLRP3 inflammasome and pyroptosis, and serves as a potential adjuvant therapy for HT injury.