Research On PI3K/AKT Signal Pathway In Bone Destruction Mechanism Of Osteoarticular Tuberculosis

Objective: to simulate the bone resorption during the pathogenic process of osteoarticular tuberculosis in vitro, and explore the action mechanism of P13K/AKT signal pathway in the bone resorption during the pathogenic process of osteoarticular tuberculosis. Through the experimental study, explain the influence of P13K/AKT signal pathway on the bone resorption mechanism during the pathogenic process of osteoarticular tuberculosis, providing the new reference for the diagnosis and treatment in the future. Methods:(1) induce the RAW264.7 osteoclast culture using the mycobacterium tuberculosis ultrasonic splitting product with different concentrations, stain and count using TRAP staining method(tartrate-resistant acid phosphatase staining method), then observe the changes of differentiation capacity of osteoclast.(2) induce the RAW264.7 osteoclast culture using the mycobacterium tuberculosis ultrasonic splitting product, add specific inhibitors with different concentrations LY294002(5u M, 10 u M, 20 u M), detect the expression of related signaling proteins, AKT, p-AKT, using western-bolt; stain and count using the tartrate-resistant acid phosphatase(TRAP), then observe the changes of differentiation capacity of osteoclast. Results:(1) after 7d of culture, the cell growth adhering to the wall could be seen under the microscope; the mycobacterium tuberculosis ultrasonic splitting product acted on RAW264.7 cells, and a lot of single long shuttle or round-like cells could be seen, and with the increase of culture time, the volume was increased. Under the TRAP staining, a large number of TRAP positive cells were formed. The resorption lacunae was formed in the cortical bone, proving that the mycobacterium tuberculosis ultrasonic splitting product could induce the RAW264.7 cells to differentiate towards the osteoclast, leading to bone destruction, which could be used as the research model for the in-vitro simulation of osteoarticular tuberculosis bone resorption mechanism.(2) the mycobacterium tuberculosis ultrasonic splitting product could act on RAW264.7 cells, and the expression of AKT and p-AKT, the downstream molecule of P13K/AKT signal pathway, was increased significantly(P<0.05). After the blocking of specific inhibitor LY294002, p-AKT became less dose-dependent(P<0.05), and there was no difference in the AKT protein expression level. The results confirmed that LY294002 successfully blocked the P13K/AKT signal pathway, and inhibited the mycobacterium tuberculosis ultrasonic splitting product from inducing the RAW264.7 cell to differentiate towards osteoclast, and inhibited the bone absorption. Conclusion:(1) the mycobacterium tuberculosis ultrasonic splitting product(MTUS) within certain concentration range can induce RAW264.7 cells to differentiate towards osteoclast(OC), thus successfully building the research model for the in-vitro simulation of osteoarticular tuberculosis bone resorption mechanism;(2) the high-concentration mycobacterium tuberculosis ultrasonic splitting product(MTUS) can lead to the RAW264.7 cell death directly, and cannot induce it towards osteoclast, maybe caused by the too much bacterial virulence;(3) during the process that the mycobacterium tuberculosis ultrasonic splitting product(MTUS) induces RAW264.7 cells to differentiate towards osteoclast(OC)(in-vitro simulation of osteoarticular tuberculosis bone resorption mechanism), the PI3K/AKT signal pathway participated in it;(4) LY294002 can inhibit the mycobacterium tuberculosis ultrasonic splitting product(MTUS) from inducing RAW264.7 cells to differentiate towards osteoclast(OC), whish is related to the concentration of LY294002.