·The gingipain extracts were obtained from Porphyromonas gingivalis ATCC33277 by sonication method combined with low temperature ultracentrifugation, followed by ultrafiltration and concentration. Gingipain extracts were identified by Western blotting after protein quantification. SDS polyacrylamide gel electrophoresis was performed and then transferred the proteins to the polyvinylidene fluoride (PVDF) membrane. After blockage, the proteins were probed with lysine-specific gingipain (Kgp) antibody and arginine-gingipain (Rgp) antibody, respectively, to detect the immunogenicity of gingipain in the extracts. The acute infection model was established by intraperitoneal gingipain extracts in C57BL/6N mice. The specific fluorescent substrates Z-His-Glu-Lys-MCA for Kgp and Boc-Phe-Ser-Arg-MCA for Rgp were used to detect the activities of two gingipains in plasma at different time points after intraperitoneal injection of gingipain extracts in mice. C57BL/6N mice were randomly divided into 4 groups, which were recorded as the control group, the gingipain group, the gingipain＋Kgp inhibitor (COR388) group and the COR388 group, respectively. Mice in the gingipain＋COR388 group were pre-treated with COR388 for 1 h, and then intraperitoneally injected with gingipain extracts. Twenty-four hours later, the samples were collected under anesthesia and the brains were processed sequentially by procedures such as fixing, dehydrating, embedding and dissecting. The microglia and astrocytes in the mouse cortex were labeled with ionized calcium binding adapter molecule 1 antibody (anti-Iba1) and glial fibrillary acidic protein antibody (anti-GFAP), respectively, and the activation of microglia and astrocytes in the cortex of the above four groups of mice were observed through immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were evaluated by enzyme linked immunosorbent assay (ELISA).

·There were relatively clear bands around 50 000 and 70 000 in the Western blotting analysis of the gingipain extracts, suggesting that the extracts contained gingipain immunogenicity. Meanwhile, the gingipain extracts were biologically active, and the activity of gingipain in the plasma of mice showed a trend of first increasing and then decreasing after intraperitoneal injection of the gingipain extracts. The activity of plasma gingipain in mice reached a peak 4?8 h after intraperitoneal injection of gingipain extracts, and then gradually decreased. Immunohistochemistry results showed that the microglia and astrocytes in the cortex had increased volume of cell bodies and irregular protrusions, suggesting that intraperitoneal injection of gingipain extracts could activate microglia and astrocytes in the mouse brain. ELISA results showed that the expression of inflammatory factors (TNF-α and IL-1β) in cortex increased.

·Acute infection of gingipain extracts can induce neuroinflammation in mice.