Shuxuetong injection inhibits pyroptosis in acute ischemic stroke via CD44/NLRP3/GSDMD signal

Ethnopharmacological relevance: Acute ischemic stroke (AIS) is an important cause of death and disability in the world. Based on the blood stasis syndrome of stroke, Shuxuetong Injection (SXT) is a representative prescription for the treatment of AIS, which extracted by modern technology from Whitmania pigra Whitman (Shuizhi) and Pheretima aspergillum E.Perrier (Dilong).

Aim of the study: This study is in order to examine whether SXT regulates Pyroptosis in AIS via Cluster of Differentiation 44 (CD44)/NOD-like Receptor thermal protein domain associated protein 3 (NLRP3)/gasdermin D (GSDMD) signal.

Materials and methods: The rats were randomly divided into sham group, model (transient middle cerebral artery occlusion, 24 h) group, SXT low-dose group (0.27 mL/kg), SXT medium-dose group (0.54 mL/kg), SXT high-dose group (1.08 mL/kg) and positive drug group (edaravone injection, 1.35 mL/kg). Transient middle cerebral artery occlusion (tMCAO, 24 h) model of rats was set up. Neurological deficit score, tetrazolium red staining, hematoxylin-eosin staining, and Nissl staining were used to observe and screen out the optimal dosage for improving AIS. Mechanism research indicators included transmission electron microscopy and Western blot. Adeno-associated virus (AAV)-CD44 and small interfering RNA (siRNA) of CD44 were used for knocking down the CD44 expression level to verify whether SXT could resist Pyroptosis through CD44. The oxygen and glucose deprivation/re-oxygenation (OGD/R, 24 h) model of PC12 cells was used for in vitro pharmacological validation. Molecular docking, cellular thermal shift assay and drug affinity responsive target stability were employed to assess the binding affinity of critical components for the CD44 protein.

Results: SXT conspicuously mitigated the neurological function scores and cerebral infarct volume in tMCAO rats, thereby safeguarding nerve cells. In vitro, SXT not only enhanced the viability of PC12 cells subjected to OGD/R but also mitigated cellular swelling and inflammatory infiltration. The optimal dose was 1.08 mL/kg (rats) or 72.56 mg/mL (PC12 cells). SXT reduced Pyroptosis and inflammation in tMCAO rats and OGD/R cells by decreasing the expression levels of GSDMD-N, NLRP3 and CD44. In addition, after knocking down the expression level of CD44 by using AAV-CD44 and siRNA-CD44, it was found that the Pyroptosis of AIS intervened by SXT was closely related to the CD44/NLRP3/GSDMD signal. The pivotal constituent of SXT, xanthine, exhibited pronounced binding affinity towards the CD44 protein, thereby demonstrating the capacity to stabilize this molecular target.

Conclusion: This study demonstrates that Shuxuetong Injection inhibits Pyroptosis in acute ischemic stroke via CD44/NLRP3/GSDMD signal.

Acute ischemic stroke; CD44/NLRP3/GSDMD signal; Inflammation; Pyroptosis; Shuxuetong injection.