Effect of Kidney-tonifying Method on OPG/RANK/RANKL Signaling Pathway in Postmenopausal Osteoporosis Animal Model： A Meta-analysis / 中国实验方剂学杂志

Objective:As the problem of global aging intensifies，postmenopausal osteoporosis （PMOP） has become a global health problem among females. At present，the commonly used biological agents have been proved not suitable for long-term use due to multiple adverse reactions. Several Meta-analyses have confirmed the good safety and effectiveness of kidney-tonifying method against PMOP，but its therapeutic mechanism remains unclear. The purpose of this Meta-analysis was to evaluate the effect of kidney-tonifying method on osteoclastogenesis inhibitory factor（OPG）/receptor activator of nuclear transcription factor （NF）-<italic>κ</italic>B （RANK）/receptor activator of NF-<italic>κ</italic>B ligand （RANKL） signaling pathway in PMOP animal model，so as to provide an experimental basis for the treatment of PMOP with kidney-tonifying method. Method:The related articles were retrieved from PubMed，Ovid Medline，Embase，China National Knowledge Infrastructure （CNKI），Chongqing Weipu Database for Chinese Technical Periodicals （VIP），and Wanfang Data Knowledge Service Platform with the retrieval time set from their inception to January 2020. The quality of each included article was evaluated using the SYRCLE's risk of bias tool. Then RevMan 5.3 was utilized for Meta-analysis according to the Cochrane systematic review methodology. Result:Thirty-two studies involving 619 rats were included. The quality score of these studies ranged from 3 to 5 points. The results of the Meta-analysis indicated obvious advantages of kidney-tonifying method in increasing bone mineral density （BMD）［standardized mean difference （SMD）=2.01，95% confidence interval（CI）=1.50-2.52，<italic>P</italic><0.000 01］），serum OPG level （SMD=3.33，95% CI=2.59-4.07，<italic>P</italic><0.000 01），and OPG mRNA expression （SMD=11.81，95% CI=7.49-16.13，<italic>P</italic><0.000 01），promoting OPG protein production （SMD=4.95，95% CI=3.09-6.81，<italic>P</italic><0.000 01），reducing serum RANKL（SMD=-4.88，95% CI=-6.01--3.75，<italic>P</italic><0.000 01） and RANK levels （SMD=-7.30，95% CI=-9.53--5.07，<italic>P</italic><0.000 01），and down-regulating RANKL （SMD=-6.22，95%CI=-8.95--3.49，<italic>P</italic><0.000 01） and RANK mRNA （SMD=-3.18，95% CI=-6.19--0.18，<italic>P</italic><0.05） expression and RANKL protein expression in bone tissue （SMD=-3.99，95% CI=-5.47--2.50，<italic>P</italic><0.000 01）. Conclusion:The kidney-tonifying method has been proved to possess potential advantages in regulating the balance of OPG/RANK/RANKL signaling pathway in PMOP animal model. Nevertheless，more large-sample sized，properly designed，and high-quality animal experiments are still needed for further verification.

Investigation of doxorubicin inhibiting osteogenesis and promoting osteoclastogenesis in vitro and its mechanism / 中华内分泌代谢杂志

Objective:To investigate the effects of doxorubicin(DOX) on osteoblast differentiation of rat bone marrow mesenchymal stem cells(BMSCs) and osteoclast differentiation of bone marrow monocytes(BMMs) in vitro.Methods:Rat BMSCs were treated with various concentrations of DOX in osteogenic medium. The cell viability was detected by CCK-8 assay. Alizarin red staining and alkaline phosphatase(ALP) activity were used to detect the effect of DOX on osteogenic differentiation. Expressions of osteogenic differentiation-related genes were detected by real-time quantitative PCR, Western blot, and immunofluorescence. Similarly, BMMs were treated with various concentrations of DOX and its effects on cell viability and osteoclast differentiation were measured. Finally, the expressions of osteoclast-related genes were detected.Results:DOX treatment inhibited the ALP activity during BMSCs differentiation into osteoblasts and reduced the number of calcium nodules, along with decreased expressions of osteogenic-related genes(ALP, collagen-Ⅰ, and osteocalcin, P<0.05). DOX suppressed the expressions of Smad 1/5/9, bone morphogenetic protein 2(BMP-2), Osterix, and core binding factor α1(Runx2). BMP-2 supplement antagonized the effect of DOX on ALP activity. DOX promoted receptor activator of NF-κB ligand(RANKL) expression and inhibited osteoprotegerin expression. DOX promoted the osteoclast formation and expressions of osteoclast-related genes such as tartrate-resistant acid phosphatase, nuclear factor of activated T cells c1(NFATc1), and c-Fos in a direct and indirect manner. Conclusion:DOX inhibits BMSCs differentiation into osteoblasts through BMP-2/Smads signaling pathway while promotes RANKL-induced BMMs differentiation into osteoclasts in vitro.

Icaritin exerted effect in anti-postmenopausal osteoporosis by binding to RANKL protein target and inhibiting bone resorption / 中草药

Objective To demonstrate that icaritin (IT) inhibits bone resorption against osteoporosis by binding to RANKL protein targets. Methods The effects of RANKL and IT on the osteoporosis of ovariectomized rats were established by molecular docking technique. The effects of IT on the body weight, bone resorption serum index (ALP and TRACP-5b), bone mineral density and bone morphology of OVX rats were evaluated. Results IT could be stably docked with target protein RANKL. The body weight of IT group was significantly lower than that of OVX group (P andlt; 0.05). IT could significantly decrease the levels of serum ALP and TRACP-5b (P andlt; 0.01), and the value of femur BS/BV and Tb.Sp (P andlt; 0.01) in OVX rats. Moreover, IT significantly increased BMD, BV/TV, Tb.ThN, Tb.N values (P andlt; 0.01). Conclusion IT can inhibit osteoclast differentiation and play anti-osteoporosis by binding with RANKL protein target.

The function and meaning of receptor activator of NF-κB ligand in arterial calcification / 华中科技大学学报（医学）（英德文版）

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.

Clinical Significance of Serum and Urine RANKL Test for Diagnosis of Osteoporosis / 中国医科大学学报

Objective To investigate the significance of test of receptor activator of nuclear factor?kappa B(NF?κB)ligand(RANKL)in serum and urine for the diagnosis of osteoporosis. Methods A total of 53 patients with osteoporosis(the experimental group)and 45 healthy controls(the normal control group)were recruited in this study. The expression levels of RANKL in serum and urine was measured and compared by enzyme?linked immunosorbent assay. Results The serum and urine levels of RANKL in the experimental group were significantly higher than those in the normal control group(P<0.01). The areas under receiver operating characteristic(ROC)curve of serum and urine RANKL were 0.898 and 0.734, respectively. The combined detection of serum and urine RANKL and Ca2+reached a high sensitivity of 89.5%and a specificity of 86.1%for diagno?sis of osteoporosis. Conclusion RANKL may be closely associated with the progression of osteoporosis. Serum and urine RANKL test may be help?ful in the diagnosis of osteoporosis.

The effects of fluorine on expression of osteoprotegerin and receptor activator of NF-κB ligand in cultured rat osteoblasts / 中华地方病学杂志

Objective To study the influence of fluorine on signaling pathway of osteoprotegerin(OPG)/ receptor activator of NF-κB ligand(RANKL) in cultured rat osteoblasts.Methods Osteoblasts were isolated from skull of neonatal rats(＜ 24 hours) by enzyme digestion,and fluorine of different concentrations [0 (control),1 × 10-3,1 × 10-4,1 × 10-5,1 × l0-6 and 1 × 10-7 mol/L] were added into the culture medium of second generation of osteoblasts.The expressions of OPG and RANKL mRNA were determined using real-time PCR 24 and 48 hours after culturing.The expressions of OPG and RANKL protein were measured by Western blotting.Results ① After exposed to fluorine for 24 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =30.95,22.62,all P ＜ 0.01),the expression of RANKL mRNA(5.99 ± 0.39) in the 1 × 10-5 mol/L group and the expressions of OPG mRNA(3.52 ± 0.09,4.81 ± 0.15,3.68 ± 0.04) in the 1 × 10-4,1 × 10-5 and 1 × 10-6 mol/L groups were higher than those of the control group(3.20 ± 0.19,3.09 ± 0.58,all P ＜ 0.05),but in the 1 × 10-3 mol/L group,RANKL mRNA(2.29 ± 0.18) was lower than that of the control group(P ＜ 0.05).After exposed to fluorine for 48 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =26.62,5.72,all P ＜ 0.01),the expressions of RANKL and OPG mRNA(6.67 ± 0.49 and 5.05 ± 0.51) in the 1 × 10-5 mol/L group were higher than those of the control group(4.29 ± 0.07 and 4.34 ± 0.12,all P ＜ 0.05),and in the 1 × 10-3 mol/L group the expression of OPG mRNA(3.63 ± 0.49) was lower than that of the control group(P ＜ 0.05).② The expression of RANKL protein was not statistically significant between 24 hours and 48 hours groups (F =0.07,0.49,all P ＞ 0.05) ; the differences of OPG protein expression had statistical significance between groups(F =3.26,P ＜ 0.05),the expression of OPG protein in the 1 × 10-5 mol/L group(1.45 ± 0.10) was higher than that of the control group(1.05 ± 0.06,P ＜ 0.05) at the 24 hours.After 48 hours,the expression of OPG protein was not statistically significant(F =0.44,P ＞ 0.05).Conclusions At lower fluorine concentrations,bone formation is the main activity.But when fluorine concentration increased and time prolonged,the osteoclast differentiation and maturation are promoted,and the bone resorption is the main thing.