Pilose Antler Peptide Blocks Osteoclasts Ogenesis And Bone Resorption By TLR4 Signaling

Objective:At present,the prevalence of osteoporosis is extremely high,but the existing commonly used drugs have serious complications.The treatment of diseases with traditional Chinese medicine has the characteristics of less complications,and the use of Chinese medicine has fewer complications.With the development of this technology,the extraction of effective ingredients from Chinese medicine to treat diseases is a hot spot.Pilose antler peptide is the main active compenent of velvet antler,and PAP has been found to inhibit osteoclasts in vitro.This study clarifies the way of action of PAP in the treatment of menopausal osteoporosis,and provides a new method for the treatment of postmenopausal osteoporosis.Methods:In order to determine the role of PAP in vitro,four-week-old C57BL/6 mouse bone marrow macrophages were isolated,and the effects of PAP on the cytotoxicity,proliferation and apoptosis of bone marrow macrophages were detected,and the expression of TRAP were detected by q RT-PCR.TRAP staining was used to determine the effect of PAP on the differentiation of bone marrow macrophages into osteoclasts.In order to determine the role of PAP in vivo,12-week-old SPF female C57BL/6 mice were ovariectomized or sham-operated to establish an animal model of postmenopausal osteoporosis,and CTX-1 and pathological characteristics were used to determine the role of PAP.In order to determine how PAP inhibits the differentiation of bone marrow macrophages into osteoclasts,experiments were performed with 4-week-old C57BL/6 mice conditionally knocked out of the TLR4gene,Western blotting was used to determine whether the knockout was successfu.Experimental detection of TRAP expression and TRAP staining to determine whether PAP inhibits the differentiation of bone marrow macrophages into osteoclasts through the TLR4 signaling pathway.In order to determine whether PAP also inhibits bone resorption through the TLR4 signaling pathway in vivo,we performed ovariectomy or sham operation on 12-week-old SPF female TLR4^f/fmice and TLR4^Ctskmice to establish postmenopausal osteoporosis animal models and use Micro CT to determine whether PAP inhibits bone resorption through the TLR4 signaling pathway in vivo.Results:PAP had no significant effect on proliferation,apoptosis and cell activity of bone marrow macrophages in vitro（P>0.05）,but had significant effect on inhibiting differentiation of bone marrow macrophages into osteoclasts（P<0.05）.In vivo,PAP inhibited the differentiation of bone marrow macrophages into osteoclasts（P<0.05）.For TLR4 conditional knockout bone marrow macrophages,PAP inhibited the differentiation of bone marrow macrophages into osteoclasts（P>0.05）.For TLR4conditional knockout mice,the effect of PAP on bone volume fraction and trabecular thickness was not statistically significant（P>0.05）.Conclusion:PAP can inhibit the differentiation of bone marrow macrophages into osteoclasts in vitro,and inhibit bone resorption by inhibiting the differentiation of bone marrow macrophages into osteoclasts in vivo.For menopausal osteoporosis,PAP can inhibit the differentiation of bone marrow macrophages into osteoclasts and inhibit bone resorption through the TLR4 signaling pathway.