Effects Of Ginkgetin On The Survival Of Bone Marrow Derived Macrophages And Expression Of Related Genes After Osteoclast Induction

Osteoporosis is a common disease that seriously endangers human health,often leading to long-term bed rest followed by serious complications such as crushing pneumonia,pressure sores,and lower limb thrombosis,resulting in increased socioeconomic burden,shorter life expectancy,and reduced quality of life.Drug therapy plays an important role in the prevention and treatment of osteoporosis,but the effectiveness of treatment is affected by the side effects of commonly used drugs,such as gastrointestinal inflammation and liver and kidney function damage.In order to find drugs with better efficacy,the search for new anti-osteoporosis treatment mechanisms or targets in TCM has become a hot research topic.A large number of in vivo and ex vivo experiments have confirmed that TCM has various biological activities such as anti-inflammatory and anti-tumor.ginkgetin(GK)is extracted from Ginkgo biloba leaves.Previous studies have identified that GK can affect nuclear factor kappa B(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways,The above signaling pathways are important for osteoclast differentiation and maturation,so it is speculated that GK may have considerable anti-osteoporosis activity.Objectives:1.To explore the effect of GK on the survival of bone marrowderived macrophages(BMMs);2.To explore the effect of GK on specific gene expression after osteoclast differentiation induced by BMMs.Methods: BMMs were divided into the control group and GK(0,0.001,0.01,0.1,1,10,25,and 50 μM)groups.cell counting kit 8(CCK8)was used to measure cell proliferation,tartrate-resistant acid phosphatase(TRAP)staining was used to detect osteoclast generation capacity,Expression of cathepsin k(CTSK),and activated t cell nuclear factor c1(NFATc1)were detected by real-time PCR(RT-PCR).Results:1.GK inhibited the proliferation of BMMs in a concentrationdependent manner.In the concentration primary screening assay,GK at concentrations greater than 1 μM inhibited the proliferation of BMMs;in the concentration fine screening assay,GK at concentrations greater than 12.5 μM inhibited the proliferation of BMMs in the group with an effect duration of 24 h and at concentrations greater than 6.25 μM in the groups with an effect duration of 48 h and 72 h,all three groups inhibited the proliferation of BMMs differentiation to osteoblasts in all three groups(P<0.05).2.GK inhibited the expression of specific genes after osteoclast induction in BMMs in a concentration-dependent manner.RT-PCR results showed that after 72 h of GK action,the expression of CTSK and NFATc1 in the experimental group was lower than that in the control group,and the expression of CTSK and NFATc1,specific genes for differentiation of BMMs into osteoclasts,was inhibited at GK concentrations greater than 12.5 μM,and in a concentration dependence(P< 0.05).Conclusions: In vitro experiments,GK at concentrations greater than 12.5μM inhibited the proliferation and osteoclastic differentiation of BMMs,as well as the expression of the specific genes CTSK and NFATc1 after osteoclastic induction.