Cathepsin B Regulates the Activation of NLRP3 Inflammasome by Calcium Signaling

Author(s): Qifang Zhang, Juanjuan Duan, Bo Yang, Dejun Yu, Lijie Liu, Hua Bai

Cathepsin B (CatB) is a proteolytic enzyme present in the cytosol which catalyzes the hydrolysis of benzoyl-L-argininamide. Whether CatB can regulate the activation of NLRP3 inflammasome by affecting calcium signal is worth studying. The specific siRNA targeting CatB gene was designed. CatB gene silencing cell line (CB-si-BV2) was established in microglia BV2, and BV2 cells were treated with hydrogen peroxide. GdCl3, Cat-B inhibitor CA-074Me, calcium ion chelating agent BAPTA-AM were used to treat cells simultaneously or separately. NLRP3 protein was detected by Western Blot. NLRP3 mRNA was detected by qPCR. IL-1β and Caspase-1 were detected by ELISA. Cell viability was detected by MTT assay. Cell apoptosis was detected by Hoechst33258 staining. After treatment with hydrogen peroxide, the expression of NLRP3 protein and caspase-1 mRNA increased in BV2 cells. After adding GdCl3, the expression of NLRP3, NLRP3 mRNA, Caspase-1 and IL-1β were significantly increased. After treated with GdCl3, some NLRP3 protein was detected in CB-si-BV2 cells, NLRP3mRNA and Caspase-1 increased, compared with the control group, the difference was statistically significant. After treated with BAPTA-AM, NLRP3, NLRP3mRNA and Caspase-1 were significantly reduced. The growth rate of some CB-si-BV2 cells was significantly slowed down. There was more apoptosis of CB-si-BV2 cells in H2O2 group and (H2O2+GdCl3) group. In the microglial oxidative stress model, CatB plays a role in regulating the activation of NLRP3 inflammasome, which is probably mediated by calcium signaling.

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