Traditional Chinese Medicine in Prevention and Treatment of Osteonecrosis of Femoral Head Based on OPG/RANK/RANKL Signaling Pathway： A Review / 中国实验方剂学杂志

Osteonecrosis of the femoral head （ONFH） is a painful and debilitating disease caused by impaired blood supply to the femoral head and cellular and tissue degeneration， leading to gradual destruction of the bone structure and progressive collapse of the femoral head. The main pathological mechanism of ONFH is the disruption of the balance between bone absorption and the reconstruction of new bone， resulting from microcirculation damage and decreased cellular tissue ability. This imbalance leads to biomechanical changes and accelerates the pathological progression of ONFH. In the early stages， clinical manifestations may not be obvious， mainly presenting as pain or discomfort in the hip or groin area， which can be relieved after rest. In the later stage of the disease， pain intensifies， and limb shortening， lower limb weakness， difficulty walking， or limping may occur. Currently， western medicine commonly uses osteogenic agents， anticoagulants， and artificial joint replacement for treatment， but there are also many issues such as prosthesis loosening and infection. Research has shown that traditional Chinese medicine （TCM） treatment of ONFH takes a holistic approach and employs multi-functional， multi-target， and multi-system Chinese medicine therapies， ensuring the safety and effectiveness of the treatment. The osteoprotegerin （OPG）/receptor activator of nuclear factor-κB （RANK）/RANK ligand （RANKL） signaling pathway plays a crucial role in maintaining the dynamic balance of bone remodeling. TCM treatments utilize this pathway to promote apoptosis of osteoclasts， reduce bone resorption， and accelerate bone formation， thereby playing an important role in the prevention and treatment of ONFH. This paper reviewed the role of OPG/RANK/RANKL signaling pathway and related cytokine expression in ONFH by reviewing relevant literature in China and abroad and research status of Chinese medicinal monomers， Chinese medicinal formulations， and combinations with physical therapy in increasing osteoblast secretion， promoting OPG expression， enhancing cytokine expression levels， and inhibiting osteoclast activity for the prevention and treatment of ONFH. This paper is expected to provide new ideas and directions for TCM in the prevention and treatment of ONFH.

Effect of Jingui Shenqiwan on Diabetic Osteoporosis in Mice via AGEs/RANKL/NF-κB Pathway Based on Theory of "Kidneys Governing Bones" / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Jingui Shenqiwan on diabetic osteoporosis （DOP） in mice by regulating the advanced glycation end products （AGEs）/receptor activator of nuclear factor-κB ligand （RANKL）/nuclear factor-κB （NF-κB） signaling pathway based on the theory of "kidneys governing bones". MethodForty 6-week-old male and female skeletal-muscle-specific， dominant negative insulin-like growth factor-1 receptor （MKR） mice were selected and fed on a high-fat diet for eight weeks to establish the DOP model. The model mice were randomly divided into a model group， low- and high-dose Jingui Shenqiwan group （1.3， 2.6 g·kg-1）， and an alendronate sodium group （0.01 g·kg-1）， with 10 mice in each group. Additionally， 10 FVB/N mice of the same age were assigned to the normal group. The corresponding drugs were administered orally to each group once a day for four weeks. After the administration period， fasting blood glucose （FBG） measurement and oral glucose tolerance test （OGTT） were conducted. Kidney function and kidney index were measured. Renal tissue pathological changes were observed through hematoxylin-eosin （HE） and Masson staining. Immunohistochemistry was performed to assess the protein expression levels of AGEs， phosphorylated NF-κB （p-NF-κB）， and RANKL in renal tissues. Western blot analysis was conducted to measure the expression of proteins related to the AGEs/RANKL/NF-κB signaling pathway， osteoprotegerin （OPG）， and Runt-related transcription factor 2 （RUNX2） proteins in femoral bone tissues. ResultCompared with the normal group， mice in the model group exhibited significantly increased FBG （P<0.01）， trabecular bone degeneration， abnormal bone morphological parameters， significantly increased area under the curve （AUC） of OGTT （P<0.01）， enlarged kidney volume， significantly increased kidney function indicators and kidney index （P<0.01）， disrupted renal glomeruli and renal tubule structures， significantly increased expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues （P<0.05）， and significantly decreased expression of OPG and RUNX2 in femoral bone tissues （P<0.01）. Compared with the model group， mice in the Jingui Shenqiwan groups showed a significant decrease in OGTT AUC （P<0.01）. Histopathological analysis revealed alleviated structural lesions in renal glomeruli and renal tubules. Furthermore， the expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues was significantly reduced （P<0.05， P<0.01）， and the expression of RUNX2 and OPG in femoral bone tissues was significantly increased （P<0.05， P<0.01）. ConclusionJingui Shenqiwan can improve kidney function and downregulate the AGEs/RANKL/NF-κB signaling pathway to inhibit inflammatory reactions， thereby alleviating the symptoms of DOP in mice， demonstrating a therapeutic effect on DOP from the perspective of the kidney.

Traditional Chinese Medicine Intervenes in Signaling Pathways Associated with CKD-MBD： A Review / 中国实验方剂学杂志

Traditional Chinese medicine （TCM） has certain advantages in the treatment of chronic kidney disease-mineral and bone disorder （CKD-MBD）. In recent years， there have been many studies on the treatment of CKD-MBD by Chinese medicinal compounds and monomers. As revealed by literature retrieval， the research on the mechanism of Chinese medicine in intervening in signaling pathways related to CKD-MBD was mainly based on self-made Chinese medicinal compounds， and the action pathways involved fibroblast growth factor 23/Klotho （FGF23/Klotho） signaling pathway， Wnt/β-catenin signaling pathway， receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin （RANK/RANKL/OPG） system， and other signaling pathways. TCM can improve calcium and phosphorus metabolism and bone metabolism disorder， and regulate inflammatory reaction， oxidative stress， apoptosis， and autophagy by regulating this series of signaling pathways for the treatment of CKD-MBD. This paper introduced the research results of these signaling pathways and the mechanism of TCM in the treatment of CKD-MBD in order to provide ideas and references for the related research of Chinese medicine in the treatment of CKD-MBD.

The osteoprotective effects and mechanisms of RA model mice by total saponins of Chaenomeles speciosa / 中国药房

OBJECTIVE To study the osteoprotective effects and possible mechanism of total saponins of Chaenomeles speciosa on rheumatoid arthritis （RA） model mice， and to provide reference for further development of anti-RA drugs. METHODS Seventy male DBA/1 mice were randomly divided into normal group， model group， low-dose and high-dose groups of C. speciose total saponins （60， 240 mg/kg）， Tripterygium wilfordii polyglycoside tablets group （positive control， 30 mg/kg）， with 14 mice in each group. In addition to the normal group， the other groups of mice were induced by glucose-6-phosphate isomerase mixed polypeptide to prepare RA model. The body weight， rear toes thickness and arthritis scores of each group were recorded； the synovial inflammation， bone and cartilage destruction of ankle joint tissues were observed by hematoxylin-eosin staining， tartrate- resistant acid phosphatase staining and safranin O-fast green staining； the contents of interleukin-6 （IL-6） in serum and tumor necrosis factor α （TNF-α）， IL-4 and IL-10 in ankle joint tissues were detected by ELISA； the expression levels of receptor activator of nuclear factor-κB ligand （RANKL）， receptor activator of nuclear factor-κB （RANK）， osteoprotegerin （OPG）， tumor necrosis factor receptor-associated protein 6 （TRAF6） and nuclear factor of activated T cells 1 （NFATC1） protein in ankle joint tissues were detected by Western blot assay. RESULTS At the end of administration， compared with normal group， the body mass of mice in the model group was significantly reduced （P＜0.05）， and the arthritis score and the thickness of the left and right rear toes were significantly increased （P＜0.05）； the ankle joint tissues of mice in the model group showed significant synovial proliferation and inflammatory infiltration， the number of osteoclasts increased significantly and significant destruction of cartilage tissue. The content of IL-6 in serum， the content of TNF-α， the protein expression levels of RANKL， RANK， TRAF6 and NFATC1 in the ankle joint tissues were increased significantly （P＜0.05）， while the contents of IL- 4 and IL-10， the protein expression level of OPG in the ankle joint tissues were decreased significantly （P＜0.05）. Compared with model group， above pathomorphological changes and the content/level of indicators of mice in each administration group were significantly improved （P＜0.05）. CONCLUSIONS Total saponins of C. speciosa may exert osteoprotective effects on RA model mice， the mechanism of which may be associated with reducing the contents of IL-6 and TNF-α， increasing the contents of IL-4 and IL-10， inhibiting the activation of RANKL/RANK/OPG signal pathway， thus inhibiting the proliferation of osteoclasts and promoting the repair of cartilage and bone tissue.

Association of serum sRANKL and Omentin-1 levels with bone mineral density and bone metabolism in postmenopausal osteoporosis patients / 中华内分泌外科杂志

Objective:To investigate the relationship between serum soluble receptor activator of nuclear factor-κB ligand (sRANKL), Omentin-1 levels and postmenopausal osteoporosis (PMOP) .Methods:A total of 310 menopausal patients admitted to Qingdao Municipal Hospital from Jun. 2017 to Jul. 2021 were selected, including 165 patients with PMOP and 145 women with simple menopause as the control group. Serum sRANKL and Omentin-1 levels were detected by ELISA. Bone mineral density and bone metabolism indexes [N-terminal propeptide of typeⅠprecollagen (PINP), bone alkaline phosphatase (BALP), β isomer of the C-terminal telopeptide of type Ⅰ collagen (β-CTX) and osteocalcin (OC) ] were compared between the two groups. The correlation between serum sRANKL and Omentin-1 levels and bone mineral density and bone metabolism indexes in PMOP patients was analyzed by Pearson. The predictive value of sRANKL and Omentin-1 to PMOP was analyzed by ROC curve. Logistic regression analysis of the influence of multiple factors on PMOP.Results:Compared with the control group (15.62±4.41) (42.56±8.53), the serum sRANKL level (26.63±8.12) was increased and Omentin-1 level (32.32±5.52) was decreased in PMOP group ( t=14.55, P<0.001; t=12.69, P<0.001). The serum sRANKL in PMOP group was positively correlated with PINP, β-CTX and OC, while the serum Omentin-1 level was negatively correlated with the above indexes by Pearson analysis. ROC curve showed that serum sRANKL and Omentin-1 had important reference significance in predicting PMOP. Logistic regression suggested that increased sRANKL and decreased Omentin-1 were risk factors for PMOP. Conclusion:Serum sRANKL and Omentin-1 in patients with PMOP are correlated with bone mineral density and bone metabolism, and have potential as diagnostic targets of PMOP.

Effect of Extracts from Veronica peregrine on Breast Cancer Cell-induced Osteoclastic Bone Metastasis via Inhibiting Runx2 Activation / 中国实验方剂学杂志

ObjectiveTo investigate inhibitory effect of extracts from Veronica peregrina （EVP） on the osteoclastic bone metastasis induced by breast cancer cells. MethodBone metastasis model was established by injection of MDA-MB-231 cells， a human breast cancer cell line， into the left ventricle of BALB/c nude mice. The expression of human cytokeratin-19 （Ck-19） gene in mouse bone marrow was determined by nested polymerase chain reaction（PCR） to assess the bone metastasis of MDA-MB-231 cells. To assess the effects of EVP on the activation of bone marrow macrophages （BMMs）， we counted the multinuclear cells and measured the secretion of Cathepsin K. Western blot was adopted to assess the effects of EVP on receptor activator of nuclear factor-κB （RANK）， Runt-related transcription factor 2 （ Runx2 ）， phosphorylated Runx2 （p-Runx2）， and matrix metalloproteinase-9 （MMP-9） in BMMs. Gelatin zymography was employed to determine the activities of matrix metalloproteinases （MMPs）. ResultCompared with that in the blank group， Ck-19 expression was down-regulated in EVP groups （P<0.05）. The multinucleated cells increased when the BMMs were induced by soluble receptor activator of nuclear factor-κB ligand （sRANKL）， which was inhibited by EVP （P<0.05）. The level of cathepsin K in the supernatant of sRANKL group increased compared with that of the blank group， while EVP groups had lower cathepsin K levels than sRANKL group （P<0.05）. Compared with the blank group， the sRANKL group showed up-regulated RANK expression， Runx2 phosphorylation， and MMP-9 expression （P<0.05）， while the expression levels of RANK， p-Runx2， and MMP-9 were down-regulated when the cells were incubated with EVP （P<0.05）. Furthermore， exposure of BMMs to sRANKL resulted in an increase in gelatin hydrolyzation compared with the blank group （P<0.01）， which， however， was reversed in EVP groups （P<0.05）. ConclusionEVP significantly inhibits bone marrow metastasis of MDA-MB-231 cells， which may be associated with the suppression of osteoclast activation by inhibiting Runx2 phosphorylation.

Differences of B cells, plasma cells, and related cytokines expression in gingival tissues between periodontitis and periodontal healthy subjects / 华西口腔医学杂志

OBJECTIVE@#This study aimed to compare the differences of B cells, plasma cells, and related cytokines expression in gingival tissues between periodontitis and periodontal healthy subjects.@*METHODS@#Gingival tissues were collected from periodontal healthy subjects (periodontal healthy group, n=12) and periodontitis patients (periodontitis group, n=15). Hematoxylin-eosin (HE) staining was used for histopathological examination. Immunohistochemical staining (CD19, CD38, and CD138) was applied to detect the expression of B cells and plasma cells. B cell-activating factor (BAFF) and soluble receptor activator of nuclear factor-κB ligand (sRANKL) were detected by enzyme-linked immunosorbent assay.@*RESULTS@#Extensive inflam-matory cell infiltration was found in the gingival tissues of the periodontitis group. The number of CD19(+), CD38(+), and CD138(+) cells of the periodontitis group was significantly higher than that of the periodontal healthy group (P<0.000 1). BAFF and sRANKL levels of the periodontitis group were higher than those of the periodontal healthy group (P<0.01, P<
0.001, respectively).@*CONCLUSIONS@#The expression of B cells, plasma cells, and their related BAFF and sRANKL cytokines were significantly higher in periodon-titis patients than those in the periodontal healthy subjects, sug-gesting that B cells and plasma cells may be involved in the development of periodontitis.

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.

Mechanism of participation of osteocytes in the formation of osteoclasts under hypoxia / 华西口腔医学杂志

OBJECTIVE@#To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia.@*METHODS@#The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected.@*RESULTS@#DFO (100 μmol·L⁻¹) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 μmol·L⁻¹ DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01).@*CONCLUSIONS@#Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upre-gulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.

Risedronate inhibits rat bone marrow adipogenesis and reduces RANKL expression in adipocytes / 南方医科大学学报

OBJECTIVE@#To investigate the effects of risedronate on bone marrow adipogenesis and the expression of the receptor activator of nuclear factor κB ligand (RANKL) in adipocytes in the bone marrow micro-environment.@*METHODS@#Primary cultured rat mesenchymal stem cells (BMSCs) with or without adipogenic induction for 14 days were treated with 1, 5, 10, and 25 μmol/L risedronate. The droplets of the differentiated adipocytes were analyzed, and Western blotting was performed to detect the expression level of RANKL. Female SD rats (24-week-old) were randomly divided into sham-operated group and ovariectomy (OVX) group, and 12 weeks after the operation, the OVX rats were further divided into control group and risedronate group (2.4 μg/kg, injected subcutaneously for 3 times a week). Eight weeks later, the bone mineral density (BMD) of the rats and bone marrow histopathology of the femurs was examined to evaluate the effect of risedronate on the fat fraction in the bone marrow.@*RESULTS@#Risdronate significantly inhibited adipogenic differentiation of rat BMSCs and suppressed RANKL expression in the adipocytes derived from the BMSCs in a concentration-dependent manner. In OVX rats, risdronate treatment significantly increased the BMD and decreased the fat content in the bone marrow.@*CONCLUSIONS@#Risdronate can effectively inhibit the adipogenic differentiation of rat BMSCs, decrease fat content in the bone marrow, and suppress the generation and function of osteoclasts by down-regulating the expression of RANKL, which can be an important mechanism underlying the therapeutic effect of risedronate against osteoporosis.

Clinical significance of transcription factor SOX5 expression in patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the expression and clinical significance of transcription factor SOX5 in peripheral blood mononuclear cells (PBMCs) and serum in patients with rheumatoid arthritis (RA). Methods The relative expression of representative genes of the SOX gene family in the PBMCs from RA patients were detected by Real-Time Polymerase Chain Reaction (RT-PCR), and serum levels of SOX5 expression were measured by enzyme-linked immunosorbent assay (ELISA) in 30 RA patients, 27 osteoarthritis (OA) patients and 30 healthy controls (HC). The expression levels of SOX5 in PBMCs were detected by RT-PCR after stimulated with IL-6, TNF-α, IL-1β and IL-17 for 24 hours. The relationship between SOX5 and receptor activator of nuclear factor κB ligand (RANKL) was detected by co-Immunoprecipitation (co-IP). The formation of TRAP-positive cells after silence SOX5 in osteoclast precursor cell treated with RANKL was observed by Tartrate-resistant acid phasphate stain (TRAP). The differences were tested using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis Correlations were analyzed using Pearson's analysis. Results SOX5 was predominantly expressed in the PBMCs of RA as compared with other SOX family genes in PBMC. PBMC levels of SOX5 in RA patients (21±19) were higher than the OA patients (10±8) and healthy control group (5±4)(F=8.343, P<0.01). While, Serum levels of SOX5 in RA patients [(19132±12054) pg/ml were higher than the OA patients [(9065±15172) pg/ml] and healthy control group [(3242±1251) pg/ml] (F=15.31, P<0.01). IL-6, TNF-α, IL-1β and IL-17 led to the up-regulation of SOX5 expression in PBMCs. IL-6, TNF-α, IL-1β promoted the interaction of SOX5 and RANKL in PBMCs. Silencing SOX5 reduced the formation of TRAP-positive cells in osteoclast precursor cell treated with RANKL. Conclusion Our results have proven that transcriptional factor SOX5 regulates the expression of RANKL and participates in the process of RA bone erosion. Inhibition of SOX5 expression may be a new therapy target of RA.

Expression profiles of CaMKⅡγ during osteoclast differentiation / 中南大学学报(医学版)

Objective:To study the expression profiles and the role of Ca2+/calmodulin-dependent protein kinase Ⅱγ (CaMKⅡγ) during osteoclast differentiation.Methods:Mouse RAW264.7 cells were induced for osteoclastogenesis with 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and the cells were harvested at 0,1,3 and 5 days after induction.Tartrate-resistant acid phosphotase staining was performed to verify osteoclasts formation.RT-PCR,Western blot and immunofluorescent cytochemistry were used to detect the CaMKⅡγ gene expression during osteoclastogenesis.Results:The osteodasts were formed at day 3 under RANKL induction and more osteoclasts were observed at day 5.At day 0,1,3 and 5,the relative level of CaMKⅡγ mRNA were (1.067±0.179),(1.840±0.070),(9.493±0.453) and (30.767±0.573),respectively,and the relative protein level were (0.454±0.065),(0.613±0.021),(0.858±0.019) and (0.980±0.023),respectively.CaMKⅡγ expression was increased in a time-dependent manner except relative protein level at day 1 (P＜0.01),which showed no significant difference at day 0 (P＞0.05).Immunofluorescence assay showed that CaMKⅡγ protein was also increased with differentiation of osteoclasts.Conclusion:The CaMKⅡγ expression was increased in a time-depended manner during osteoclast differentiation and it might play a vital role during osteoclastogenesis.

The effects of Xuling-Jiangu formula on the mRNA expression of osteoprotegerin and receptor activator of nuclear factor κB ligand in osteoporosis model rats / 国际中医中药杂志

Objective To study the effects and mechanism of Xuling-Jiangu formula on bone mineral density in the osteoporosis model rats.Methods According to the random number table method,50 SD female rats were randomly divided into the sham group,the model group,the low dose group of Xuling-Jiangu formula group,the medium dose group and the high dose of group.In addition to the sham group,the other groups were osteoporosis model.After 30 days,low dose group received intragastric Xuling-Jiangu formula solution 7.5 g/kg;medium dose group and high dose group received 15 and 30 g/kg,respectively.And the sham group and the model group received normal saline 10 ml/kg.After 12 weeks treatment,the bone mineral density of the left tibia was measured by double energy X ray.Estrone,osteocalcin in serum had been detected by Enzyme linked immunosorbent assay (ELISA).The mRNA expression of osteoprotegerin (OPG),receptor activator of nuclear factor κB ligand (RANKL) in lumbar were measured by quantitative real-time PCR.Results The bone mineral density (0.215 ± 0.010 g/cm2,0.222 ± 0.013 g/cm2 vs.0.196 ± 0.016 g/cm2) of the Xuling-Jiangu formula group were significantly higher than that of the model group (P＜0.01 or P＜0.05).The estrone in low,middle and high dose groups showed an upward trend,but there was no significant difference compared with the model group.The OC (87.0 ± 8.9 ng/L vs.100.5 ± 16.8 ng/L) of high dose group was significantly lower than that of the model group (P＜0.05).Compare with the model group,OPG mRNA level (0.97 ± 0.23 vs.0.78 ± 0.17) in high dose group increased (P＜0.05),the RANKL mRNA level decreased significantly (1.12 ± 0.17,0.97 ± 0.38,1.04 ± 0.29 vs.1.31 ± 0.18) in three Xuling-Jiangu formula groups (P＜0.05).Conclusions The bone mineral density of rats with osteoporosis can be improved by treating Xuling-Jiangu formula.It may be related to the increase of mRNA expression of OPG,and the reduction of RANKL.

OPG and RANKL expression in alveolar bone of type 2 diabetic rats with periodontitis / 实用口腔医学杂志

Objective:To investigate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in type 2 diabetic rats with periodontitis.Methods:46 male Wistar rats were randomly divided into the healthy group(n =10),the periodontitis group(n =12),the type 2 diabetes mellitus group(n =12) and the type 2 diabetic periodontitis group(n =12).Animal models were prepared respectively.The expression of OPG and RANKL protein in alveolar bone was detected by immunohistochemistry(IHC).Results:Compared with the healthy group,the expression of OPG in the type 2 diabetes mellitus group,the periodontitis group,the type 2 diabetes mellitus with periodontitis group decreased in turn,however the expression of RANKL increased in turn.The expression of OPG and RANKL had no significant difference between periodontitis group and type 2 diabetes periodontitis group,while there was statistically significant difference among the other groups (P ＜ 0.05).Conclusion:Inflammation may lcad to upregulation of RANKL in osteoclasts and immune cells,and downregulation of OPG in osteoblasts.

Clinical research of interleukin-34 in psoriatic arthritis patients / 中华风湿病学杂志

Objective To investigate the relationship between interleukin (IL)-34 and bone erosions in psoriatic arthritis (PsA) patients.Methods Forty PsA patients,20 psoriasis (Ps) patients and 20 healthy volunteers were recruited into this study.The levels of IL-34 and osteoclast related cytokines [including tumor necrosis factor (TNF)-α,receptor activator of nuclear factor-κB ligand (RANKL),osteoclast precursors (OPG)] were detected in the serum samples of all subjects.The correlations among IL-34,the number of osteoclast precursors (OCP),disease activity and imaging scores were analyzed.All data were analyzed by graphpad prism 6.Differences between groups was analyzed with One-way analysis of variance,q tests,and Spearman's correlation was used to explore the relation between disease activity/radiographic scores and laboratory results and followed by linear regressions.Results The serum level of IL-34 in patients with PsA [(328±476) pg/ml] was higher than that in Ps [(33±52) pg/ml,q =3.92,P＜0.01] and healthy controls [(32±32) pg/ml,q =3.93,P＜0.01],the erosive PsA group were higher than the non-erosive PsA group [(449±527) pg/ml and (47±24) pg/ml,q=4.04,P＜0.01].The levels of TNF-α,RANKL and OCP in patients with PsA [(125±79) pg/ml,(488± 475) pg/ml and (17.7±4.8) 5 sigh views] were higher than those in PS [(40±22) pg/ml,(26±3) pg/ml and (5.2± 0.8),q=7.32,6.14 and 2.94,P＜0.01] and healthy controls [(41±19) pg/ml,(65±8) pg/ml and (6.2±1.8),q=6.67,5.62 and 2.71,P＜0.01],whereas the OPG/RANKL ratio in PsA patients (0.5±0.4) was significantly lower than Ps patients (4.3±2.7,q=-3.30,P＜0.01) and healthy controls (1.8±0.6,q=-1.72,P＜0.01).IL-34,TNF-α and RANKL levels were all positively correlated with OCP (r=0.10,P＜0.05;r=0.12,P＜0.05;r=0.13,P＜0.(5,respectively).Conclusion The level of IL-34 is not only high in patients with PsA but also positively correlates with the number of OCP.In PsA,IL-34 is probably related to the OCP and osteoclast differentiation,and further participates in the process of bone destruction.Therefore,IL-34 is promising to become a new target or alternative choice for the treatment of PsA.

Expression profiles of CaMKIIδat different stages of osteoclast differenti-ation / 中国病理生理杂志

[ ABSTRACT] AIM:To study the expression profiles and the role of Ca 2+/calmodulin-dependent kinase II delta ( CaMKIIδ) during osteoclast differentiation .METHODS:Mouse RAW264.7 cells were induced by receptor activator of nuclear factor κB ligand ( RANKL) at 50μg/L for osteoclastogenesis .Tartrate-resistant acid phosphatase ( TRAP) staining and bone resorption lacunae examination were performed to verify osteoclast formation .The expression of CaMKIIδat mR-NA and protein levels was also determined by immunofluorescent cytochemistry , RT-qPCR and Western blot at days 0, 1, 3 and 5.RESULTS:TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKIIδdetected by RT-qPCR were 1.028 ±0.041, 2.478 ±0.087, 10.524 ±1.284 and 42.914 ± 2.667 at days 0, 1, 3 and 5, respectively, while the protein levels of CaMKIIδ detected by Western blot were 0.762, 0.963, 1.802 and 3.136, respectively.The changes of protein level were also verified by immunofluorescence cytochemis -try, in which the fluorescence intensity increased in a time-dependent manner (P<0.05).CONCLUSION:The expres-sion of CaMKIIδincreases with the differentiation of osteoclasts .CaMKIIδmay play a key role in the osteoclastogenesis .

Serum Dickkopf-1 levels in patients with ankylosing spondylitis not affected by using tumor necrosis factor-α inhibitor / 中华风湿病学杂志

Objective Dickkopf-1 (DKK-1) is an antagonist of the Wnt signaling pathway of bone formation and it is the target gene of TNF.When DKK-1 is inhibited,osteoprotegerin (OPG) would increase,OPG can antagonize bone erosion caused by receptor activator of NF-κB ligand (RANKL).Detection the levelsof serum DKK-1,OPG,RANKL and TNF-α of patients with AS before and after applying TNF-α antagonist,the effects of anti-TNF-α treatment on signaling pathway of bone formation was explored.Methods Medical records and serum samples of 43 AS patients receiving TNF-α receptor-Ⅱ IgG Fc fusion protein for 12 weeks were collected and the levels of serum DKK-1,OPG,RANKL and TNF-α were detected by enzyme-linked immunosorbent assay.Paired t-test and Person's correlation analysis were used for data analysis.Results Disease activity indexes in AS patients with anti-TNF-α treatment,including VAS score,time of morning stiffness,BASDAI,BASFI,ESR and CRP,which were [(2.7±2.0) cm,(7±4) min,1.1±1.1,0.8±1.2,(10±9) mm/1 h and (16±26) mg/L] respectively,were reduced when compared with those before treatment,[(7.6±1.9) cm,(46±33) min,6.0±1.3,4.4±2.0,(39±29) mm/1 h and (76±43) mg/L] respectively,and the differences were statistically significant (P＜0.05).Serum levels of DKK-1,(3.3±1.5) μg/L vs (3.2±1.3) μg/L,OPG,(144±71) pg/ml vs (136±62) pg/ml,and RANKL,(71±37) pg/ml vs (68±30) pg/ml,were not changed (t=14.043,8.031,20.166,13.521,7.679,9.563,all P＞0.05).The levels of TNF-α increased from (1.9±1.3) pg/ml to (55.0±50.6) pg/ml (t=6.951,P＜0.05).There were no relations between DKK-1 and TNF-α,RANKUOPG,patient VAS,duration of morning stiffness,BASDAI,BASFI,ESR and CRP,allP＞0.05.Conclusion Clinical indicators are improved significantly by anti-TNF-α therapy; however,it should not be withdrawal suddenly.There is no relationship between serum levels of DKK-1 and level of inflammation in AS patients.

The study on the expression of interleukin-17 and receptor activator of nuclear factors κB-ligand in serum of patients with ankylosing spondylitis / 中华风湿病学杂志

Objective To investigate the role and value of interleukin (IL)-17,receptor activator of nuclear factors κB-ligand (RANKL),osteoprotegerin (OPG) in the pathogenesis of AS by detecting the expression of IL-17,RANKL,OPG.Methods IL-17,RANKL,OPG in 44 AS and 15 healthy controls were measured by ELISA.HLA-B27 was measured by flow cytometry.T-test,Spearman's correlation analysis were used for statisical analysis.Results ① Serum IL-17 [(45±12) pg/ml],RANKL [(354±96) pg/ml] levels and RANKL/OPG [(6.5±3.4) pg/ml] ratio were significantly higher in AS patients than those in normal controls (t=11.18,P＜0.05; t=18.66,P＜0.05; t=15.48,P＜0.05),and the serum RANKL levels was higher in AS patients in middle and late period than that in AS patients in early stage (t=47.02,P＜0.05).The serum OPG levels was higher in AS patients in middle and late period than that in AS patients in early stage (t=15.48,P＜0.05).② There were positive linea correletion,respectively,between the serum levels of IL-17 and RANKL (r=0.532,P=O.021),and between serum levels of IL-17 and disease activities of BASDAI (r=0.625,P=0.023),between serum levels of RAN KL and disease activities of BASDAI (r=0.431,P=0.016).Conclusion The surem levels of IL-17 and RANKL are increased in AS patient.

Osteoclasts and T-helper cells / 国际儿科学杂志

The interaction between T cell and osteoclasts is important in osteoimmunology.T-helper cells,which produce interleukin-17,induce the expression of receptor activator of nuclear factor κB ligand in synovial cells.Companied by with inflammatory cytokines,they stimulate the differentiation and activation of osteoclasts.Osteoimmunology helps us understand how antirheumatic drugs work,as well as developing new therapeutic strategies for rheumatic diseases.

Measurement and clinical significance of serum DKK-1 and sRANKL levels in multiple myeloma / 中华内科杂志

Objective To detect serum concentrations of Dickkopf-1(DKK-1) and soluble receptor activator of nuclear factor-κB ligand (sRANKL) in patients with multiple myeloma (MM) and to investigate its clinical significance. Methods Serum DKK-1, sRANKL, osteoporotegerin(OPG) and tartrate-resistant acid phosphatase 5b (TRACP-5b) levels were quantified in 30 newly diagnosed MM patients and 20 healthy control subjects by using sandwich ELISA. Results The serum DKK-1, sRANKL,OPG and TRACP-5b levels were significantly higher than those in the healthy controls (42.96 μg/L vs 5.33 μg/L, 1.83 pmol/Lvs 0. 79 pmol/L, 1799. 30 pmol/L vs 822.40 pmol/L, 5.81 U/L vs 0. 28 U/L, respectively; all P＜0. 05). Serum levels of DKK-1 were positively correlated with sRANKL and TRACP-5b, respectively.Serum concentrations of DKK-1 and sRANKL were significantly elevated in stage Ⅲvs stages Ⅰ and Ⅱaccording to International Staging System (ISS) (46. 33 μg/L vs 37.91 μg/L, 2.26 pmol/L vs 1.19pmol/L, respectively, all P ＜0.05). Serum concentrations of DKK-1 , sRANKL and TRACP-5b were significantly higher in patients with more than 3 lytic bone lesions than those with only 1-3 lytic bone lesions (46. 30 μg/L vs 31.98 μg/L, 2. 18 pmol/L vs 0. 69 pmol/L, 6.02 U/L vs 5. 13 U/L, all P ＜ 0.05).Conclusions MM patients have increased serum DKK-1, sRANKL, OPG and TRACP-5b levels as compared with the healthy controls. Serum concentrations of DKK-1 and sRANKL have close relationship with MM stage and lytic bone disease.