Effect Of Tongluo Shenggu Capsule Serum On Osteoclast And TLR4/NF-kappa B Signaling Pathway

Objective:Effects Research Tong Lu Sheng Gu capsule during monocyte differentiation into osteoclasts to the Toll-like receptor 4(Toll-like Receptor 4,TLR4)signaling pathway,for Tong Lu Sheng Gu capsule on osteoclast differentiation molecular mechanisms of inhibition of the process,provide a theoretical basis for the further capsule Tong Lu Sheng Gu clinical application.Methods:(1)Twenty-eight 12-month-old C57BL/6 mice were randomly divided into Tongluoshenggu capsule high-dose,medium-dose,low-dose gavage group and normal saline gavage group.The drug-containing serum was prepared by continuous gavage for 14 days.Control serogroup;(2)RAW264.7 cell line was induced by RANKL and M-CSF inducing factor,and drug-containing serum and control serum were added on the4 th day after induction.Each group was observed at 24 h,48h,72 h and96h after induction.The growth morphology and degree of fusion of osteoblast precursors were observed by TRAP on the 10 th day,and the expression of key proteins and phosphorylation levels were detected by western blot.Results(1)Morphological observation of RAW264.7 cells In the RAW264.7 cell suspension just inoculated,the cells grew round,adherent and fast.After 2 hours of adherence,the cells were round,elliptic,a little spindle-shaped,mostly monocytes.RAW264.7 cells were incubated with30ng/ml M-CSF and 60ng/RANKL for 3 days.All cells became osteoclast precursors(cells grew in colonies,mostly spindle-shaped,with a few pseudopods).Then they were incubated with 30ng/ml M-CSF and200ng/RANKL continuously.During the incubation period,the number of osteoclast precursors increased rapidly and expanded.On the 8th day,90% of cells fused into polynuclear cells.(2)TRAP staining After 8 days of TRAP staining,it was found that there were many polynuclear cells(nuclei(> 3)in the high,middle and low dose groups and the control group,with nuclear purple staining,large cells,vacuole-like cytoplasm,light red staining,irregular cell membrane boundaries and pseudopodia stretching around them.However,the number of osteoclasts in the high,middle and low dose groups of Tongluo Shenggu Capsule was less than that in the control group.The higher the concentration of serum containing drugs,the less the number of osteoclasts,the difference between the groups was statistically significant.(3)The content of TNF-alpha in cell supernatant determined by ELISA was lower in Tongluo Shenggu Capsule group than that in blank group(P<0.01),but no significant difference was found between Tongluo Shenggu Capsule group and normal saline group.The expression of TNF-alpha in Tongluo Shenggu Capsule group was similar to that in low dose group and Tongluo Shenggu Capsule group,and there was no significant difference between Tongluo Shenggu Capsule group and Tongluo Shenggu Capsule group.The significance of accounting.(4)Detection of TLR4 and NF-kappa Bp65 protein by Western blot Compared with the control group,the protein expressions of TLR4 and NF-kappa Bp65 in Tongluo Shenggu Capsule at high,middle and low doses were significantly different(P <0.01).The expression of TLR4 and NF-kappa Bp65 decreased with the increase of serum concentration of Tongluo Shenggu Capsule.Conclusion:Tongluo Shenggu Capsule has a stable inhibitory effect on osteoclast differentiation of monocytes,and the inhibitory effect increases with the increase of Tongluo Shenggu Capsule concentration.Tongluo Shenggu Capsule has an effect on the expression of TLR4 and NF-kappa Bp65,which are key proteins in TLR4 signaling pathway during osteoclast differentiation of monocytes.The inhibitory effect is positively correlated with the concentration.