In vitro cultured calculus bovis alleviates cerebral ischemia-reperfusion injury in rats through regulating microglial polarization and inhibiting NLRP3.

Abstract

Objectives: To investigate the effect of in vitro cultured calculus bovis (ICCB) on cerebral ischemia/reperfusion injury (CIRI) and its mechanism.

Methods: The CIRI animal model and cell model were induced by middle cerebral artery occlusion (MCAO) in Sprague Dawley rats and oxygen glucose deprivation/reperfusion (OGD/R) in BV2 cells, respectively. The CIRI of rat model was evaluated using modified neurological severity score (mNSS), brain water content, and cerebral infarction volume after 1.5 h of ischemia and 72 h of reperfusion. The histopathological changes in the cortex and hippocampal CA1 region were observed with hematoxylin-eosin staining, the microglial polarization and NOD-like receptor thermal protein domain associated protein (NLRP) 3 inflammasome expression in the cortex were examined with IF assay. The viability of the BV2 cells was assessed with MTT assay after treatment with ICCB and Nigericin. The expressions of NLRP3, ASC, caspase-1 pathway proteins and inflammatory factors were detected with Western blotting in BV2 cells after OGD for 0.5 h and reperfusion for 24 h; and also in BV2 cells after 24 h pretreatment with the NLRP3-specific agonist Nigericin.

Results: ICCB treatment markedly enhanced neurological function, decreased cerebral infarct volume and brain water content, and mitigated pathological damage in the cortical and hippocampal CA1 regions of rats subjected to CIRI (all P<0.05). ICCB promoted the transition of cortical microglia from M1 to M2 phenotypes and suppressed NLRP3 activation in microglial cells (all P<0.01). ICCB significantly down-regulated the expression of NLRP3, ASC, and caspase-1 proteins, and reduced the secretion of IL-18 and IL-1β in BV2 cells of OGD/R model (all P<0.01). In addition, the NLRP3 agonist Nigericin significantly reversed the salvage effect of ICCB on model cells (both P<0.01) and the modulation of inflammatory factors (P<0.05).

Conclusions: ICCB exerts a protective effect against cerebral ischemia-reperfusion injury by mitigating neuroinflammation, through the reduction of M1 microglial polarization, promotion of M2 conversion, and suppression of the NLRP3/ASC/caspase-1 signaling pathway.