Monoclonal Antibody to Podoplanin SZ-168 Attenuates Microglia Pyroptosis and Neuroinflammation in Experimental Cerebral Haemorrhage

Abstract Background Neuroinflammation is an important factor in the development of secondary brain injury after intracerebral haemorrhage (ICH), involving the interaction of haemorrhage formation and inflammatory pathways. Podoplanin is a small transmembrane mucin-like glycoprotein, and it has been demonstrated that the PDPN/CLEC-2 axis is involved in inflammatory diseases. This study aims to investigate the therapeutic effect of a monoclonal antibody against podoplanin, SZ168, on neuroinflammation in a mouse model of cerebral haemorrhage. Methods Male C57BL/6 mice (weight 22–25 g, 6–8 weeks old, n = 108) were randomly divided into sham-operated, ICH-operated and SZ168-treated groups. Brain haemorrhage was induced by collagenase injection into the striatum, and SZ168 IgG was administered intraperitoneally 6 h after ICH. Neurological regression was assessed at the molecular, cellular, histological and functional levels by short-term neurobehavioural tests, cerebral oedema, Evans blue staining, Western blotting, ELISA and immunofluorescence staining. In vitro, human-derived microglial HMC3 cells were pretreated with SZ168 IgG and washed platelets. Then hemin treatment was added to construct an in vitro model of cerebral haemorrhage, which was detected by wound healing assay, transwell migration assay, cell counting kit-8 reagent, colony formation on solid media and western blot to detect the functional changes of microglia after PDPN inhibition. Results PDPN expression in mouse brains was elevated 48 h after ICH, and inhibition of PDPN function with SZ168 improved short-term neurological function after ICH and reduced cerebral oedema and cerebral haemorrhage. In vitro, SZ168 treatment inhibited microglial invasion, migration and proliferation functions. Mechanistically, inhibition of PDPN function with SZ168 in vivo and in vitro was able to attenuate ICH-induced expression of nlrp3, caspase-1 and gasdermin D as well as inflammatory factors IL-1β, TNF-α and MPO. Conclusions Blocking PDPN can reduce microglial invasion, migration and proliferation functions by inhibiting the microglial NLRP3/Caspase-1/GSDMD inflammatory pathway, which may play a role in reducing the inflammatory situation at the site of cerebral haemorrhage and ameliorating secondary brain injury.