ABSTRACT
Objective:To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16, 19 (L16 and L19) on the expression of immune regulatory factors in human monocytic leukemia cell line (THP-1 cells). Methods:THP-1 cells activated with phorbol ester (PMA) were used as an in vitro experimental cell model, and a group design was used to co-culture L16, L19 and THP-1 cells (L16 stimulated group, L19 stimulated group), respectively. THP-1 cells activated with PMA were used as the control group. When co-cultured for 4 hours, immunofluorescence staining (IFS) and Western blotting were used to detect whether L16 and L19 entered the cells, respectively; when co-cultured for 12, 24 hours, real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1 (IRF-1) and trans activator protein of major histocompatibility complex class Ⅱ (CⅡTA); Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3 (Tim-3) and γ interferon receptor 1 (IFNGR1). Results:When co-cultured for 4 hours, L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group, respectively. When co-cultured for 12 hours, the expression level of IRF-1 mRNA in the L16 stimulated group (0.16 ± 0.15) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 24 hours, the expression level of CⅡTA mRNA in the L16 stimulated group (0.17 ± 0.10) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 12 and 24 hours, there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group ( P > 0.05). Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group, L16 stimulated group, and L19 stimulated group after co-cultured for 12 and 24 hours ( F = 50.92, 6.80, 148.73, 156.57, P < 0.05). Among them, when co-cultured for 12 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were significantly lower than that in the control group, and the L19 stimulated group was higher than the L16 stimulated group ( P < 0.05), and the expression level of Tim-3 protein in the L19 stimulation group was higher than that in the control group ( P < 0.05). When co-cultured for 24 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were lower than that in the control group, and the L19 stimulated group was higher than that in the L16 stimulated group ( P < 0.05); and the expression level of Tim-3 protein in the L16 stimulated group was higher than that in the control group and L19 stimulated group ( P < 0.05). Conclusions:Brucella L16 can downregulate the expression levels of IRF-1 and CⅡTA mRNA in THP-1 cells. Both L16 and L19 can downregulate IFNGR1 and upregulate Tim-3 protein expression levels.
ABSTRACT
Objective:This study is designed to investigate the toxicity of lipoprotein (L16) and non-lipoprotein (U16) of Brucella outer membrane protein (OMP) 16 on osteoblasts. Methods:Recombinant L16 and U16 proteins were prepared by using prokaryotic expression system of Escherichia coli ( E. coli) BL21 (DE3) and purified by Ni column. Using group design, mouse osteoblasts (MC3T3 cells) were co-incubated with L16 and U16, respectively. Brucella lipopolysaccharide (LPS) stimulus was used as the positive control, and cells without any stimulation were used as the negative control. Incubation time was 24 h. The activity of co-incubated MC3T3 cells were detected by CCK-8; the supernatant of cultured cells was collected and the release rate of lactate dehydrogenase (LDH) in the supernatant was detected by bioluminescence, and the virulence of L16 and U16 on MC3T3 cells was evaluated. Annexin Ⅴ-PE/7-AAD double staining flow cytometry was further used to analyze the apoptosis rate of MC3T3 cells, and the activation level of apoptosis executive protein Caspase-3 was detected by Western blotting (WB). Results:The activity of MC3T3 cells in L16 group [(56.16±1.63)%] was significantly lower than that in U16 and LPS groups [(97.02±1.44)%, (98.64±0.90)%, P < 0.01], the LDH release rate [(84.64±0.96)%] was significantly higher than that in U16 and LPS groups [(34.82±3.41)%, (26.75±1.95)%, P < 0.01]. Annexin Ⅴ-PE/7-AAD double staining results showed that the apoptosis rate was (46.45±2.19)% in L16 group, while the remaining groups were all less than 1%. WB results showed that activated Caspase-3 (cleaved-Caspase-3) existed in L16 stimulated cells, but not in U16 stimulated cells and LPS control cells. Conclusion:L16 can induce the apoptosis of osteoblasts and inhibit the proliferation of osteoblasts, but U16 has no obvious effect indicating that Brucella L16 with complete lipid structure is necessary for virulence effect.