[Effects of isopsoralen on tibial fracture and vascular healing in mice].

OBJECTIVE: To explore effects of isopsoralen (ISO) with different doses on fracture and vascular healing in mice. METHODS: Sixty 2-month-old male C57BL/6 mices with body mass of (20±2) g were selected and divided into 4 groups by random number table method:model group (model), low dose group (isopsoralen-low dose, ISO-L), medium dose group (isopsoralen-medium dose, ISO-M) and high dose group (isopsoralen-high dose, ISO-H), with 15 animals in each group. The right tibial fracture model was established. After operation, ISO-L group, ISO-M group and ISO-H group were given ISO concentration of 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1, respectively. Model group was given same volume of normal saline once a day for 28 days. Weighed once a week. X-ray was performed on 7, 14, 21 and 28 days, respectively, and modified I.R. Garrett scoring method was used to evaluate callus growth. After 28 days, the main organs were stripped and weighed, and organ coefficients were calculated. Hematoxylin eosin staining (HE staining) was performed on the organs to observe whether there were pathological structural changes. Micro-computed tomography (Micro-CT) was used to scan fracture area and conduct three-dimensional reconstruction to obtain the effect map, and quantify bone volume fraction (bone volume/total volume, BV/TV). After decalcification, the tibia was embedded in paraffin wax and sectioned. The healing and shape of fracture end were observed by HE staining and ferruxin solid green staining. The right tibia was removed and decalcified after intravascular infusion of Microfil contrast agent. Micro-CT was used to scan the callus microvessels in the fracture area, and the vascular volume fraction and vessel diameter were quantified. RESULTS: After 28 days of administration, there was no significant difference in body mass and organ coefficient among all groups (P>0.05), and no significant pathological changes were found in HE staining of organs. The results of X-ray and improved I.R. Garrett score showed that ISO-M group was higher than that of Model group at 28 days (P<0.05). Scores of ISO-H group at 14, 21 and 28 days were higher than those of the other 3 groups (P<0.05). Micro-CT results showed intracavitary callus in ISO-M group was significantly reduced, which was lower than that in Model group (P<0.05), most of the callus in ISO-H group were subsided, and BV/TV in ISO-H group was lower than that in the other 3 groups (P<0.05). The results of HE staining and ferrubens solid green staining showed fracture area of ISO-H group was closed, continuous laminar bone had appeared, and the fracture healing process was higher than that of other groups. Angiographic results showed vascular volume fraction in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05), and the vascular diameter in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05). CONCLUSION: In the concentration range of 10-40 mg·kg-1, ISO has no obvious toxic and side effects, and could improve bone microstructure, promote formation of callus microvessels, and accelerate healing of fracture ends in a concentration-dependent manner.

Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

[Correlation between macrophage chemotaxis and disease severity in patients with knee osteoarthritis].

OBJECTIVE: To investigate the enhancement of macrophage chemotaxis in patients with knee osteoarthritis (KOA) and its correlation with the disease severity. METHODS: Eighty patients with KOA admitted from July 2019 to June 2022 were enrolled as the observation group and divided into 29 cases of moderate group, 30 cases of severe group and 21 cases of extremely severe group. At the same time, 30 healthy subjects were included as the control group. The gene expressions of NF-κB, CXC chemokine receptor 7 (CXCR7) and CXC chemokine ligand 12 (CXCL12) in macrophages of each group were analyzed. Visual analogue scale(VAS) was used to evaluate the degree of joint pain. Joint function was evaluated by knee Joint Society Scoring system(KSS). Finally, data analysis was carried out. RESULTS: The expression levels of NF-κB, CXCR7 and CXCL12 in moderate group, severe group and extreme recombination group were higher than those in control group. The VAS, the expression of NF-κB, CXCR7 and CXCL12 in the severe group and the extreme recombination group were higher than those in the moderate group, whereas KSS was lower than that in the moderate group. The VAS, expression levels of NF-κB, CXCR7 and CXCL12 in the extremely severe group were higher than those in the severe group, and KSS was lower than that in the severe group (all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with VAS score, but negatively correlated with KSS(all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with the severity of disease. After excluding the influence of traditional factors (gender, age and disease duration), multiple linear regression analysis further showed that the expression levels of NF-κB, CXCR7 and CXCL12 were still positively correlated with the severity of disease(all P<0.01). CONCLUSION: The chemotaxis of macrophages in patients with KOA increased with the aggravation of the disease, and was related to the degree of pain and function impairment.

Protection of primary cilia is an effective countermeasure against the impairment of osteoblast function induced by simulated microgravity.

The molecular mechanism for the microgravity-induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)-simulated microgravity and that the microgravity-induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 µM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP-2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP-2, BMPRII, Smad1/5/8 and p-Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA-129-3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA-129-3p in ROBs significantly attenuated microgravity-induced inhibition of BMP-2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA-129-3p in microgravity-induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA-129-3p as an effective countermeasure against microgravity-induced loss of primary cilia and impairment of osteoblast function.

Evaluation efficacy and safety of epidural analgesia in second-trimester induced labor: A single-center, prospective, non-randomized, controlled study.

BACKGROUND: Second-trimester induced labor in pregnant women was often more likely to suffer from psychological and physiological double pain. However, the analgesic management received less attention, and the optimal analgesic mode for second-trimester induced labor had not been determined. Our objective was to evaluate the feasible of epidural analgesia (EA) in second-trimester induced labor. METHODS: From January 2020 to December 2021, Primipara who planned to undergo second-trimester induced labor in the First Affiliated Hospital of Yangtze University were collected. The method of labor induction was oral mifepristone + amniotic cavity injection of Ethacridine Lactate. Based on whether or not patients received epidural analgesia, which were divided into EA group (30 cases) and non-EA (NEA) group (30 cases). The primary outcome were visual analog scale (VAS) score of pain and result of follow-up, the secondary outcomes included relative clinical parameter and labor duration. RESULTS: Vaginal induction of labor was successful in both groups. There was no statistically significant difference in VAS of pain between the two groups before analgesia (P > .05), but the VAS of pain in the EA group was significantly lower than the NEA group (P < .05) after analgesia or at delivery. The following outcomes showed no statistical difference between two groups: labor duration, postpartum hemorrhage, hemorrhage ≥ 500 mL, intrapartum injury, second days hemoglobin, C-reactive protein, antibiotic therapy days, hospitalizations days, and placenta residue (P > .05). The median hospitalization costs of EA group was 4697.5 yuan, and NEA group was 3673 yuan, the difference was statistically significant (P < .001). No adverse events related to EA occurred during hospitalization, only 3 patients showed mild lumbago and back pain after follow-up to three months postpartum, which was significantly relieved after proper rest. CONCLUSION: EA can significantly reduce the pain of parturients, which may be effective and safe in the second-trimester induced labor.

A Real-World Study on the Effect of Imrecoxib for Patients with Axial Spondyloarthritis.

Purpose: Non-steroidal anti-inflammatory drugs (NSAIDs) have generally been viewed as first-line therapy for axial spondyloarthritis (axSpA). Imrecoxib is a selective COX-2 inhibitor developed independently in China. At present, only one single-center RCT trial has shown that imrecoxib is equally effective as celecoxib in treating axSpA. Based on real-world data, our study aims to explore the efficiency of imrecoxib and TNF inhibitor (TNFi) combined with imrecoxib in treating axSpA. Patients and Methods: A total of 163 patients with axSpA who had more than two follow-up records in 6 months and treated with imrecoxib/celecoxib/TNFi combined with imrecoxib/TNFi combined with celecoxib from the First Affiliated Hospital of Anhui Medical University SpA Real World Database (AHSpA) were selected for analysis of our study. The linear mixed model was used to compare efficacy indexes before and after treatment and between different groups, adjust baseline measurement value and follow-up time. The Kaplan-Meier survival analysis was used to identify the differences in cumulative clinical remission rates between groups with different treatment at the follow-up period. Results: Results showed that after treatment ASDAScrp was slightly improved in imrecoxib group and celecoxib group within 6 months (p < 0.05). CRP, ESR, BASDAI, ASDAScrp, BASFI, occiput to wall distance and finger floor distance all significantly improved in TNFi combined with imrecoxib group and TNFi combined with celecoxib group within 6 months (all p < 0.05). According to the Kaplan-Meier survival curve and Log rank test analysis, the clinical remission rate was not significantly different between different treatment during 24-month follow-up (all p > 0.05). Conclusion: ASDAScrp improved slightly within 6 months after treatment with imrecoxib, and TNFi combined with imrecoxib significantly improved multiple effect indexes in axSpA patients. The efficacy of imrecoxib and celecoxib in the treatment of axSpA is equivalent. Also, they have the same efficacy after being combined with TNFi.

The interdependent relationship between the nitric oxide signaling pathway and primary cilia in pulse electromagnetic field-stimulated osteoblastic differentiation.

Pulsed electromagnetic fields (PEMFs) have long been recognized being safe and effective in treating bone fracture nonunion and osteoporosis. However, the mechanism of osteogenic action of PEMFs is still unclear. While primary cilia are reported to be a sensory organelle for PEMFs, and nitric oxide (NO) plays an indispensable role in osteogenic effect of PEMFs, the relationship between NO and primary cilia is unknown. In this study, effects of treatment with 50 Hz 0.6 mT PEMFs on osteogenic differentiation and mineralization, NO secretion, and ciliary location of specific proteins were examined in rat calvarial osteoblasts (ROBs) with normal or abrogated primary cilia. It was found that PEMFs stimulated the osteogenic differentiation by activating the NOS/NO/sGC/cGMP/PKG signaling pathway, which need the existence of primary cilia. All components of the signaling pathway including iNOS, eNOS, sGC, PKG-1, and PKG-2 were localized to primary cilia, and eNOS was phosphorylated inside the primary cilia. Besides, primary cilia were elongated significantly by PEMF treatment and changed dynamically with the activation NO/cGMP pathway. When the pathway was blocked by L-NAME, PEMFs could no longer elongate the primary cilia and stimulate the osteoblastic differentiation. Thus, this study for the first time observed activation of the NO/cGMP signaling pathway in ciliary compartment of osteoblasts, and PEMFs could not stimulate the osteoblastic differentiation if the NO signaling pathway was blocked or the ciliogenesis was inhibited. Our findings indicate the interdependent relationship between NO and primary cilia in the PEMF-promoted osteogenesis.

Synergy of sarcopenia and vitamin D deficiency in vertebral osteoporotic fractures in rheumatoid arthritis.

OBJECTIVES: To explore the synergistic effect of vitamin D deficiency and sarcopenia on vertebral osteoporostic fracture (VF) in patients with rheumatoid arthritis (RA). METHODS: A total of 188 patients with RA and 158 control subjects were enrolled. Bone mineral density (BMD) at the total hip, neck of femur, lumbar vertebra 1-4, and skeletal muscle mass was measured by dual energy X-ray absorptiometry (DXA) and biological electrical impedance, respectively. Serum 25(OH)D was tested by electrochemiluminescence. The prevalence of VF and osteoporosis (OP) were compared between RA and controls. The synergism of sarcopenia and vitamin D deficiency on VF in patients with RA was tested by χ2 test and logistic regression. RESULTS: The prevalence of OP at all measured sites and VF in RA patients were all higher than those in controls (P < 0.0001). The incidence of VF in RA either with sarcopenia or with vitamin D deficiency was higher than for those without sarcopenia or without vitamin D deficiency (χ2 = 5.069, P = 0.027, χ2 = 8.822, P = 0.001). Age, disease duration, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), DAS28, health assessment questionnaire (HAQ), Sharp score, and body mass index (BMI) were significantly different between RA with sarcopenia or not (P < 0.05). Logistic regression analysis found that age (OR = 1.095, 95%, CI: 1.044-1.150, P < 0.0001) was a significant risk factor for VF in patients with RA, while high skeletal muscle mass (SMI) (OR = 0.513, 95% CI: 0.327-0.804, P = 0.004) was a protective factor for VF in RA patients. CONCLUSIONS: VF, sarcopenia, and vitamin D deficiency are common in patients with RA. Sarcopenia and vitamin D deficiency may be risk factors for the incidence of VF in RA patients. KEY POINTS: • RA patients had a higher incidence of OP and VF, also a high prevalence of sarcopenia and vitamin D deficiency. • Vitamin D deficiency and sarcopenia may might have a synergistic effect on VF in RA. • Aging and sarcopenia are risk factors for VF in RA patients, and sarcopenia were associated with disease activity and structural damage.

Presence of subclinical inflammation in axial spondyloarthritis patients with NSAID/anti-TNF-α drug-induced clinical remission.

OBJECTIVE: To investigate the rate of subclinical inflammation in patients with axial spondyloarthritis (axSpA) with nonsteroidal anti-inflammatory drug (NSAID)/anti-tumor necrosis factor (TNF)-α drug-induced clinical remission and to explore factors influencing clinical and imaging remission. METHODS: One hundred twenty-five patients with axSpA followed up for at least 6 months were enrolled in this prospective study and randomly divided into two groups. Ninety patients were treated with anti-tumor necrosis factor (TNF)-α or anti-TNF-α combined with nonsteroidal anti-inflammatory drugs (NSAIDs) (anti-TNF-α treatment group), and thirty-five patients were treated with only NSAIDs (non anti-TNF-α treatment group). The improvements in the clinical remission rate, imaging remission rate, and disease parameters before and after the different treatments were compared. Risk factors for clinical and imaging remission were analyzed by multivariate logistic regression analysis. RESULTS: The clinical and imaging remission rate was increased after treatment especially in the anti-TNF-α group (P < 0.001). The remission rate of imaging in the group with clinical remission was higher than that in the group with clinical non-remission (P < 0.05). After treatment, the remission rates of imaging in the clinical remission and non-remission group were significantly higher than those before treatment (P < 0.0001). The results of multivariate logistic regression analysis showed that higher CRP was a risk factor for failure of clinical remission in axSpA (OR = 2.034, 95% CI:1.595 ~ 2.617, P < 0.001), while higher ASDAScrp was a risk factor for failure of imaging remission (OR = 1.306, 95% CI:1.026 ~ 1.688, P < 0.05). Anti-TNF-α treatment was a protective factor for both clinical (OR = 0.234, 95% CI:0.091 ~ 0.605, P < 0.05) and imaging remission (OR = 0.511, 95% CI:0.286 ~ 0.914, P < 0.05). CONCLUSION: Even after regular treatment, some clinical remission patients continued to have evidence of subclinical inflammation. Higher CRP and ASDAScrp are risk factors for clinical and imaging non-remission in axSpA respectively, Continuous NSAID treatment (more than 1 year) can effectively improve clinical and MRI inflammation in patients, but anti-TNF-α treatment is more beneficial for clinical and imaging remission. Key Points • Some patients achieving ASDAScrp remission status continue to have inflammation when assessed with objective imaging techniques. • MRI can sensitively measure bone marrow inflammation and may provide a more accurate assessment of remission. • Controlling inflammation, especially reducing CRP and ASDAScrp levels, is a key factor for achieving clinical and imaging remission in patients with axSpA.

The Potential Regulatory Mechanism of lncRNA 122K13.12 and lncRNA 326C3.7 in Ankylosing Spondylitis.

This work aims to analyze and construct a novel competing endogenous RNA (ceRNA) network in ankylosing spondylitis (AS) with bone bridge formation, lncRNA. Using RNA sequencing and bioinformatics, we analyzed expression profiles of long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in whole blood cells from 5 AS patients and 3 healthy individuals. Next, we verified the expression levels of candidate lncRNAs in 97 samples using the ΔΔCt value of real-time quantitative polymerase chain reaction (qRT-PCR). We used multivariate logistic regression analysis to screen lncRNAs and clinical indicators for use in the prediction model. Both SPSS 24.0 and R software were used for data analysis and prediction model construction. The results showed that compared with the normal controls, 205 long noncoding RNAs (lncRNAs), 961 microRNAs (miRNAs), and 200 mRNAs (DEmRNAs) were differentially expressed in the AS patients. We identified lncRNA 122K13.12 and lncRNA 326C3.7 among 205 lncRNAs differentially expressed between AS patients and healthy humans. Then, we noted that 30 miRNAs and five mRNAs formed a ceRNA network together with these two lncRNAs. These ceRNA networks might regulate the tumor necrosis factor (TNF) signaling pathway in AS development. In addition, the expression level of lncRNA 122K13.12 and lncRNA 326C3.7 correlated with various structural damage indicators in AS. Specifically, the lncRNA 326C3.7 expression level was an independent risk factor in bone bridge formation [area under the ROC curve (AUC) = 0.739 (0.609-0.870) and p = 0.003], and the best Youden Index was 0.405 (sensitivity = 0.800 and specificity = 0.605). Moreover, we constructed a lncRNA-based nomogram that could effectively predict bone bridge formation [AUC = 0.870 (0.780-0.959) and p < 0.001, and the best Youden Index was 0.637 (sensitivity = 0.900 and specificity = 0.737)]. In conclusion, we uncovered a unique ceRNA signaling network in AS with bone bridge formation and identified novel biomarkers and prediction models with the potential for clinical applications.

Interactive effect of sarcopenia and falls on vertebral osteoporotic fracture in patients with rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) had higher incidences of sarcopenia, falls, osteoporosis, and vertebral osteoporotic fractures (VOPF). Sarcopenia was associated with longer disease duration, higher disease activity, and more severe RA. The interactive effect of sarcopenia and falls was associated with a higher risk of VOPF in patients with RA. PURPOSE: Whether sarcopenia and falls are a risk factor for vertebral fracture in RA patients has not been demonstrated. This study aimed to explore the incidence of vertebral osteoporotic fracture (VOPF) and its relationship with sarcopenia and falls in RA patients. METHODS: A total of 474 RA patients and 156 controls were enrolled in this study. Anteroposterior and lateral X-ray examinations of the vertebral column (T4-L4) were used for the semiquantitative assessment of VOPF. Bone mineral density was measured by dual-energy X-ray absorptiometry. Skeletal muscle mass was measured by direct segmental multifrequency bioelectrical impedance analysis (DSM-BIA method). RESULTS: RA patients had an increased risk of sarcopenia (62.4% vs 9.0%, x2 = 47.478, P < 0.001), falls (30.2% vs 3.2%), osteoporosis (OP) (33.5% vs 12.8%, x2 = 134.276, P < 0.001), and VOPF (20.3% vs 3.8%, x2 = 47.478, P < 0.001) than controls. Patients with sarcopenia were more likely to have VOPF than RA without sarcopenia (24.0% vs 14.0%, x2 = 6.802, P = 0.009). RA with sarcopenia and prior falls had the highest incidences of VOPF (36.7%). Older age (OR = 1.056, P < 0.001, 95% CI 1.030-1.083), falls (OR = 2.043, P = 0.003, 95% CI 1.238-3.371), OP (OR = 1.819, P = 0.034, 95% CI 1.046-3.163), and usage of glucocorticoids (GCs) (OR = 1.862, P = 0.022, 95% CI 1.093-3.172) were risk factors for VOPF in RA patients, while a higher skeletal muscle index (SMI) was a protective factor (OR = 0.754, P = 0.038, 95% CI 0.578-0.984) for VOPF in RA patients. CONCLUSIONS: The interactive effect of sarcopenia and falls is associated with a higher risk of VOPF in patients with RA.

The frequency window effect of sinusoidal electromagnetic fields in promoting osteogenic differentiation and bone formation involves extension of osteoblastic primary cilia and activation of protein kinase A.

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation.

Synergistic effect of sarcopenia and poor balance on osteoporotic vertebral fracture in Chinese patients with rheumatoid arthritis.

OBJECTIVES: This study aimed to investigate the synergistic effect of sarcopenia and poor balance on osteoporotic vertebral fracture (VOPF) in Chinese patients with rheumatoid arthritis (RA). METHODS: A total of 238 RA patients and 158 normal subjects were enrolled in the case-control study. Poor balance capability (Berg balance scale (BBS) score < 40) and sarcopenia (skeletal muscle mass index (SMI) <7.0 (male)/5.7 (female)) between RA patients and normal subjects were compared. Associations of poor balance capability or sarcopenia with disease activity, structural damage, and joint function in different groups were also investigated. RESULTS: The incidence of sarcopenia in RA was 58.4%, significantly higher than that in controls (P<0.0001). Moreover, the percentages of low balance capacity (BBS<40) in RA were 43.7%, which was higher than that in controls (P<0.0001). The prevalence of VOPF in the case group was 19.3%, which was higher than that in the controls (P<0.0001). In the RA group, compared to RA patients without VOPF, RA patients with VOPF had higher percentages of poor balance and sarcopenia (P<0.05). Compared with RA patients without sarcopenia or good balance, RA patients with sarcopenia or poor balance had a higher incidence of VOPF, higher disease activity, severer structural damage, and worse joint function (P<0.05). The incidence of VOPF in patients combined with good balance and non-sarcopenia (4.8%) was significantly lower than that in patients combined with poor balance and sarcopenia (38.2%) (P<0.0001). Logistic regression indicated that higher SMI and higher BBS scores were protective factors for VOPF in RA patients, while age was a risk factor for VOPF in RA patients (P<0.0001). CONCLUSION: Sarcopenia and poor balance are popular in Chinese patients with RA, and they are associated with disease activity and structural damage. There is a synergistic effect of sarcopenia and poor balance on VOPF in RA. Key Points • Sarcopenia and balance capability were popular (about a half) in patients with RA. • Sarcopenia and poor balance had a synergistic effect on VOPF in RA.

[Effect of low frequency pulsed electromagnetic field on peak bone mass in young rats].

OBJECTIVE: To compare effects of low frequency pulsed electromagnetic fields on bone quality in growing rats between 1 h and 1.5 h. METHODS: Thirty male SPF SD rats aged 4 weeks selected, which with the average body weight (115.8± 1.5) g, were divided into three groups according to random number table, 10 rats in each group. Control groups put rats into electromagnetic field device with 1.5 h every day, the other two groups put rats in electromagnetic field for 1 h and 1.5 h with a 50 Hz 0.6 mT intensity pulsed. The body weight of rats was weighed every 2 weeks and detected bone mineral density. Rats were sacrificed after 6 weeks to measure bone mineral density and biomechanical value of the right femur and lumbar vertebrae. Serum osteocalcin (OC) and tartrate-resistant acid phosphatase 5b (TRACP 5b) concentrations were determined by ELISA methods. After the magenta-picric acid staining, the micro tissue structure of the right tibia was observed, and the parameters of trabecular bone were analyzed by IPP 6.0 software. RESULTS: There were no statistical difference in body weight and organ coefficient among each groups at different times. Bone mineral density results showed that the body thickness of the 1.5 h group was significantly increased compared with control group at 6 weeks, and bone mineral density of femoral and vertebra in 1.5 h group were higher than that of in 1 h group. The results of three point femoral bending and vertebral compression test showed that the maximum load value of femur and vertebrae in 1.5 h group increased significantly compared with control group, and the maximum femoral load value in 1.5 h group was significantly higher than that of 1 h group, while there was no difference in elastic modulus values among each groups. Results of serum biochemical indicators showed that level of OC in 1.5 h group was significantly increased compared with control group, and significantly higher than that of 1 h group, while no significant difference in TRACP 5b values among each groups. Bone histomorphometry analysis results showed that there was no statistical difference in trabecular thickness, number and resolution between 1 h group and control group, trabecular bone thickness and number in 1.5 h group were increased, and trabecular bone resolution was decreased; The thickness and number of trabecular bone in 1.5 h group were also significantly increased compared with 1 h group, and the degree of resolution was reduced, and had significant difference between two groups. CONCLUSION: Intervention of 50 Hz 0.6 mT low frequency pulsed electromagnetic field for 1.5 h could effectively increase peak bone mineral density and bone microstructure in young rats, enhance biomechanical properties of bone, promote concentration of bone formation markers in rat blood. The results indicating that pulsed electromagnetic field could be used as a good way to prevent and treat osteoporosis.

Inflammatory anemia may be an indicator for predicting disease activity and structural damage in Chinese patients with rheumatoid arthritis.

OBJECTIVES: This study aimed to investigate the relationship of serum hemoglobin (HB) level with disease activity and structural damage in Chinese patients with rheumatoid arthritis (RA). METHODS: A total of 890 RA patients and 890 normal subjects were enrolled in the case-control study. A HB threshold of< 110 g/L (women) and < 120 g/L (men) was used to determine anemia. All the patients were divided into three groups: non-anemia group (HB ≥ 120 g/L (male) or 110 g/L (female)), mild anemia group ((90 g/L < HB < lower limit of normal), and medium to severe anemia group (HB ≤ 90 g/L). Serum HB level and anemia prevalence between RA patients and normal subjects were compared. Associations of HB level with disease activity, structural damage, and function of joint in different groups were also investigated. RESULTS: The average of HB level in RA was (109.08 ± 17.96)g/l, which was lower than that in controls (136.75 ± 14.57)g/l (P < 0.001). Anemia was observed in 47% of the RA patients, while prevalence of anemia in control group was only 4.4%. In RA group, percentages of non-anemia, mild anemia, and medium to severe anemia were 47%, 38%, and 15%. Compared with non-anemia RA patients, RA patients with anemia had higher disease activity, severer structural damage and worse function of joint (P < 0.001). With the increase of anemia, the disease activity, structural damage, and dysfunction of joints increased significantly (P < 0.05-0.001). Linear regression analysis showed that HB level was negatively correlated with disease activity parameters, degree of joint destruction, and function (P < 0.05-0.001). Logistic regression indicated that serum HB level was protective factors for disease activity and structural damage in RA (P < 0.001). CONCLUSION: HB level was significantly related to disease activity and structural damage in RA patients.Key Points• Inflammatory anemia was popular (about a half) in patients with RA.• HB level was related to disease activity and structural damage in RA patients.

[Preventive effect and mechanism of puerarin on rat models of disuse osteoporosis].

To investigate the preventive effect and possible mechanism of puerarin(Pur) in rat model of disuse osteoporosis(DOP),thirty healthy Wistar female rats of 2 months old were randomly divided into control group(Control), hindlimb suspension group(HLS), and puerarin group(HLS+Pur) in hindlimb suspension, with 10 rats in each group. A disuse osteoporosis model was established by tail suspension method, and 15.4 mg·kg~(-1) puerarin suspension was administered to HLS+Pur group every day, and the same volume of distilled water was administered to Control group and HLS group respectively. After 28 days, the rats were sacrificed by abdominal aorta blood collection, the main organs of the rats were removed, and the bone tissues of the rats were dissected. The organ index of the rats was calculated and the histopathology of the organs was observed under microscope. Bone mineral density test and bone biomechanical experiment were performed. Bone histomorphometry results were observed after bone tissue sectioning, and serum biochemical markers of bone metabolism were determined. There was no significant difference in organ index between the groups. There was no obvious abnormality in the pathological examination of the organs. The results of bone mineral density showed that puerarin could significantly increase the bone density of the tibia and vertebrae caused by hindlimb suspension. The mechanical parameters experiments showed that puerarin could effectively increase the maximum load and elastic modulus of the tibia and vertebrae. Fluorescence labeling showed that the fluorosis interval increased and the bone formation increased during puerarin treatment. The VG staining results showed that compared with the HLS group, in the puerarin group, the number of trabecular bone increased, the thickness of the trabecular bone became thicker, and the bone separation became smaller, which greatly improved the bone microstructure after hindlinb suspension. In addition, serum biochemical indicators showed that puerarin could promote bone formation index bone calcium. The content of osteocalcin(OC) increased and inhibited the formation of tartrate-resistant acid phosphatase 5 b(TRACP 5 b). Puerarin has a preventive effect in the rat model of disuse osteoporosis and its effect is good, and its mechanism may be related to promoting bone formation and inhibiting bone resorption.

[Different Types of Low-frequency Electromagnetic Fields Resist Bone Loss Caused by Weightlessness].

Objective To compare the effects of 50-Hz 0.6-mT low-frequency pulsed electromagnetic fields(PEMFs) and 50-Hz 1.8-mT sinusoidal alternating electromagnetic fields(SEMFs) in preventing bone loss in tail-suspended rats,with an attempt to improve the prevention and treatment of bone loss caused by weightlessness.Methods Tail-suspension rat models were used to simulate microgravity on the ground. Forty rats were randomly divided into four groups[control group,hindlimb-suspended(HLS) group,HLS+PEMFs group,and HLS+SEMFs group],with 10 rats in each group. In the PEMFs treatment group and SEMFs treatment group,the intervention was 90 min per day. Rats were sacrificed after four weeks. Bone mineral density(BMD) of femur and vertebra was measured by dual-energy X-ray absorptiometry and biomechanical strength by AG-IS biomechanical instrument. Serum osteocalcin(OC),tartrate-resistant acid phosphatase 5b(Tracp 5b),parathyroid hormone(PTH),and cyclic adenosine monophosphate(cAMP) were detected by ELISA. The microstructure of bone tissue was observed by Micro-CT and HE staining.Results The BMD of the femur(P=0.000) and vertebrae(P=0.001) in the HLS group was significantly lower than in the control group;the BMD of the femurs(P=0.001) and vertebrae(P=0.039) in the HLS+PEMFs group was significantly higher than in the HLS group;the BMD of the femurs in the HLS+SEMFs group was significantly higher than in the HLS group(P=0.003),but the BMD of the vertebrae showed no significant difference(P=0.130). There was no significant difference in the BMD of the femur(P=0.818) and vertebrae(P=0.614) between the HLS+PEMFs group and the HLS+SEMFs group. The maximum load(P=0.000,P=0.009) and elastic modulus(P=0.015,P=0.009) of the femurs and vertebrae in the HLS group were significantly lower than those in the control group;the maximum load of the femur(P=0.038) and vertebrae(P=0.087) in the HLS+PEMFs group was significantly higher than that in the HLS group,but the elastic modulus was not significantly different from that in the HLS group(P=0.324,P=0.091). The maximum load(P=0.190,P=0.222) and elastic modulus(P=0.512,P=0.437) of femurs and vertebrae in the HLS+SEMFs group were not significantly different from those in the HLS group. There were no significant differences in the maximum load and elastic modulus of femurs(P=0.585,P=0.948) and vertebrae(P=0.668,P=0.349) between the HLS+PEMFs group and the HLS+SEMFs group. The serum OC level in the HLS group was significantly lower than that in the control group(P=0.000),and the OC level in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.006) were significantly higher than that in the HLS group. The serum Tracp 5b concentration in the HLS group was significantly higher than that in the control group(P=0.011). There was no significant difference between the HLS+PEMFs group(P=0.459) and the HLS+SEMFs group(P=0.469) compared with the control group.Serum Tracp 5b concentrations in the HLS+PEMFs group(P=0.056) and the HLS+SEMFs group(P=0.054) were not significantly different from those in the HLS group. The PTH(P=0.000) and cAMP concentrations(P=0.000) in the HLS group were significantly lower than those in the control group. The PTH(P=0.000,P=0.000) and cAMP concentrations(P=0.000,P=0.000) in the HLS+PEMFs group and the HLS+SEMFs group were significantly higher than in the HLS group. The femoral cancellous bone of the HLS group was very sparse and small compared with the control group. The density and volume of the cancellous bone were similar among the control group,HLS+PEMFs group,and HLS+SEMFs group. Compared with the control group,the HLS group had lower BMD(P=0.000),bone volume (BV)/tissue volume(TV)(P=0.000),number of trabecular bone (Tb.N)(P=0.000),and trabecular thickness(Tb.Th)(P=0.000) and higher trabecular bone dispersion(Tb.Sp)(P=0.000) and bone surface area(BS)/BV(P=0.000). Compared with the HLS group,the HLS+PEMFs group and the HLS+SEMFs group had significantly lower Tb.Sp(P=0.000,P=0.000) and BS/BV(P=0.000,P=0.000) and significantly increased BMD(P=0.000,P=0.000),BV/TV(P=0.001,P=0.004),Tb.Th(P=0.000,P=0.001),and Tb.N(P=0.000,P=0.001). The trabecular thickness significantly differed between the HLS+PEMFs group and the HLS+SEMFs group(P=0.024). The HLS group(P=0.000),HLS+PEMFs group(P=0.000),and HLS+SEMFs group(P=0.000) had the significantly lower osteoblast density on the trabecular bone surface than the control group;however,it was significantly higher in the HLS+SEMFs group(P=0.000) and the HLS+PEMFs group(P=0.000) than in the HLS group. The HLS group had significantly lower density of osteoblasts in the endothelium than the control group(P=0.000);however,the density of osteoblasts was significantly higher in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than HLS group and was significantly higher in HLS+PEMFs group than in HLS+SEMFs group(P=0.041). Compared with the control group,a large number of fatty cavities were produced in the bone marrow cavity in the HLS group,but the fat globules remarkably decreased in the treatment groups,showing no significant difference from the control group. The number of adipose cells per mm 2 bone marrow in the HLS group was 4 times that of the control group(P=0.000);it was significantly smaller in the HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than in the HLS group,whereas the difference between the HLS+PEMFs group and the HLS+SEMFs group was not statistically significant(P=0.086). Conclusions 50-Hz 0.6-mT PEMFs and 50-Hz 1.8-mT SEMFs can effectively increase bone mineral density and biomechanical values in tail-suspended rats,increase the concentration of bone formation markers in rat blood,activate the cAMP pathway by affecting PTH levels,and thus further increase the content of osteoblasts to prevent the deterioration of bone micro-structure. In particular,PEMFs can prevent the reduction of bone mineral density and maximum load value by about 50% and increase the bone mass of tail-suspended rats by promoting bone formation.

[Effect of Low-frequency Pulsed Electromagnetic Fields on Bone Formation in Rat Osteoblasts and Its Mechanism].

Objective To observe the effect of low-frequency pulsed electromagnetic fields(PEMFs) on bone formation in rat osteoblasts(ROBs) and explore the mechanism of action of the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cyclic adenosine effect binding protein(CREB) signaling pathway.Methods The skulls of newborn Wistar rats were harvested,and the ROBs were obtained by multiple enzymatic digestion methods for subculture. After treatment with 50 Hz 0.6 mT PEMFs for 3,6,and 9 days,the alkaline phosphatase(ALP) concentration in ROBs was detected;after 0,15,30,60,90,and 120 min,the expression of bone formation-related factor(RUNX2),the protein expression of osteogenesis-associated transcription factor(OSX),the cAMP concentration,as well as the protein expressions of p-PKA,p-CREB,and CREB were detected. The p-CREB nuclear translocation was observed. After interference with IFT88 by RNA interference,the expressions of RUNX2,OSX,p-PKA,and p-CREB protein in ROBs were detected.Results After treatment with PEMFs for 3,6,and 9 days,the ALP activity values in ROBs were 24.356±4.911,37.688±2.151,and 39.922±5.486,respectively,which were significantly higher than 18.531±2.401(P=0.0121),33.675±4.366(P=0.0324),and 36.574±1.339(P=0.0134) in the control groups. RUNX2 and OSX activities in ROBs were significantly higher than untreated group after PEMFs treatment for 30(P=0.0042 and P=0.0058),60(P=0.0097 and P=0.0079),and 90 min(P=0.0083 and P=0.0098). After PEMFs treatment for 30(P=0.0012) and 60 min(P=0.0035),the cAMP concentrations in ROBs were significantly higher than that in untreated group. After PEMFs treatment for 15(P=0.0018),30(P=0.0087),90(P=0.0250),and 120 min(P=0.0350),the p-PKA levels in ROBs were significantly higher than that in the untreated group. After PEMFs treatment for 15(P=0.0075),30(P=0.0017),60(P=0.0074),and 90 min(P=0.0096),the level of p-CREB in the ROBs was significantly higher than in the untreated group. After PEMFs treatment of ROBs for 15 min,CREB phosphorylated and accumulated in the nuclei. PKA and p-PKA were co-localized with primary cilia and stained,and it was found that p-PKA was localized on the primary cilia. After the primary cilia was removed by RNA interference,the protein expression levels of p-PKA(F=78.602,P=0.0270),p-CREB(F=76.082,P=0.0089),RUNX2(F=41.064,P=0.0230) and OSX(F=57.524,P=0.0310) were significantly lower than those of the non-interfered group.Conclusion PEMFs promote bone formation in ROBs by activating the primary cilia-associated cAMP/PKA/CREB signaling pathway.

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats.

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.