The Effect Of Cryptotanshinone On Alveolar Bone Loss In Postmenopausal Osteoporosis Associated With Osteoclast Formation And Its Molecular Mechanisms

Objective Postmenopausal osteoporosis is a common metabolic disease of bone in the elderly people.An increasing number of studies have provided evidence that postmenopausal osteoporosis also occurs in the alveolar bone.Salvia miltiorrhiza is one of the most commonly used traditional Chinese medicine in traditional Chinese medicine.Cryptotanshinone is one of the characteristic components of Salvia miltiorrhiza.Our research aims to explore the effect of cryptotanshinone on postmenopausal osteoporosis associated with osteoclast formation and its molecular mechanisms,basing on the ovariectomized mice model.Methods In vitro,we cultured mouse bone marrow mononuclear cells(bmms)and monocyte macrophage cell line(RAW264.7 cells)into osteoclasts.CCK-8 and flow cytometry were used to observe the effects of cryptotanshinone on osteoclast proliferation and apoptosis.Trap staining and pieces of bone absorption were used to clear the effects of cryptotanshinone on osteoclast differentiation and bone resorption.Immunofluorescence,Western blot and PCR techniques were used to observation the effect of cryptotanshinone on osteoclast differentiation process by gene and protein level.In vivo,3-month C57BL/6J mice were used to investigate the effect of cryptotanshinone on alveolar bone.Bilateral OVX operation was taken to make osteoporosis mice model.During the cryptotanshinone treatment,polychrome sequential labeling was used to observe the mineral apposition rate.Finally,the maxillae,serum were collected for further examination including Micro-CT,polychrome sequential labeling observation,hematoxylin and eosin(H&E)staining,tartrate-resistant acid phosphatase(TRAP)staining and enzyme-linked immunosorbent assays(ELISA).Results In vitro,it was found that the concentration of the cryptotanshinone was6.25?M,which showed a significant inhibition effect on the cell proliferation,and IC50 was 16.54?M.In the safe concentration range of 0.8?M,1.57?M and 3.125?M,the number and area of the osteoclasts(p<0.01)were obviously inhibited,and the area of bone absorption was reduced(p<0.01).Cryptotanshinone down regulate the expression of osteoclast specific gene,such as TRAP?Cathepsin K?Calcitonin receptor?DC-STAMP?c-fos?NFATC1?V-ATPase a3?V-ATPase d2,and block the JNK/MAPKs signaling pathway.In the animal model establishment,comparing with Sham group,the percent bone volume(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Tb.N)decrease(p<0.01),and promote the trabecular separation(Tb.Sp)increase(p<0.05)in OVX group.Compared with OVX group,Low and High groups could aggravate BV/TV,Tb.Th and Tb.N(p<0.01 or p<0.05),and could decrease Tb.Sp(p<0.01 or p<0.05).The administration of cryptotanshinone prevented local alveolar bone loss by inhibiting osteoclast formation in a dose-dependent manner.Conclusion(1)Cryptotanshinone could inhibit the formation of osteoclast by blocking the JNK/MAPKs signaling pathway;(2)Cryptotanshinone has the potential to block the bone loss of alveolar bone in mice with postmenopausal osteoporosis,so as to provide new ideas and methods for the treatment of osteoporotic bone resorption of alveolar bone in clinic.