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.