The expression levels and effects of TNF-α and IL-1β after Brucella.ovis and Mycobacterium tuberculosis infecting osteoblasts

English Keywords:Brucella.ovis  Mycobacterium tuberculosis  Osteoblasts  Tumor necrosis factor-α  Interleukin-1β

English Abstract:

  【Abstract】 Objectives: To measure the expression levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in osteoblasts infected by Brucella.ovis and Mycobacterium tuberculosis and to explore the reasons of different degrees of bone injury in Brucella spondylitis and spinal tuberculosis. Methods: Primary osteoblasts were extracted from the skulls of SD rats at 2-5 days after birth and cultured to the third generation, and alkaline phosphatase(ALP) staining was applied to identify osteoblasts. Brucella.ovis and Mycobacterium tuberculosis were cultured with prepared bacterial culture medium and counted to prepare the transfection medium. The osteoblasts of fluorescent Mycobacterium tuberculosis were observed and recorded under fluorescence microscope when the multiplicity of infection(MOI) was 1∶10 and 1∶20. The lysates of osteoblasts infected with Brucella.ovis with MOI of 1:100 were extracted at different time, and the plate streak method was used for counting to determine whether Mycobacterium tuberculosis and Brucella.ovis can infect osteoblasts. The osteoblasts were infected with Brucella ovis and Mycobacterium tuberculosis at different MOI and time, and CCK-8 was used to detect the cell survival rate after infection by the two kinds of bacteria to choose the best MOI and time. The osteoblasts were divided into three groups: Mycobacterium tuberculosis infection group(infected with the best MOI Mycobacterium tuberculosis), Brucella.ovis infection group (infected with the best MOI Brucella.ovis) and negative control group. Cell immunohistochemistry staining was used to observe the TNF-α and IL-1β expression levels on the cells′ surface of the three groups; Enzyme-linked immunosorbent assay(ELISA) was used to measure TNF-α and IL-1β expression levels on the cell supernatant of the three groups; Western Blot was used to detect the TNF-α and IL-1β protein expression levels in the three groups of cells. One-way ANOVA and T test were used to compare the differences among the three groups, P<0.05, the difference was statistically significant. Results: After ALP staining, the cytoplasm of osteoblasts was blue and the nucleus was green, which conformed to the characteristics of osteoblasts. Both Brucella.ovis and Mycobacterium tuberculosis can infect osteoblasts. The MOI of Mycobacterium tuberculosis group was 1∶20 and 24h, and that of Brucella ovis group was 1∶100 and 24h, respectively, and the cell survival rate was 50%, thus the MOI and time were the best comparison. Cellular immunohistochemistry, Western Blot and ELISA showed that after Mycobacterium tuberculosis and Brucella.ovis infected osteoblasts, the expression of TNF-α and IL-1β increased, and the expression levels of TNF-α and IL-1β in the Mycobacterium tuberculosis group were significantly higher than those in the Brucella.ovis group(P<0.05). Conclusions: The expression levels of TNF-α and IL-1β in osteoblasts infected with Mycobacterium tuberculosis were higher than those of Brucella.ovis, which may be one of the reasons for the different degrees of bone damage between spinal tuberculosis and Brucella spondylitis.