Electroacupuncture suppresses neuronal ferroptosis to relieve chronic neuropathic pain.

Growing evidence supports the analgesic efficacy of electroacupuncture (EA) in managing chronic neuropathic pain (NP) in both patients and NP models induced by peripheral nerve injury. However, the underlying mechanisms remain incompletely understood. Ferroptosis, a novel form of programmed cell death, has been found to be activated during NP development, while EA has shown potential in promoting neurological recovery following acute cerebral injury by targeting ferroptosis. In this study, to investigate the detailed mechanism underlying EA intervention on NP, male Sprague-Dawley rats with chronic constriction injury (CCI)-induced NP model received EA treatment at acupoints ST36 and GV20 for 14 days. Results demonstrated that EA effectively attenuated CCI-induced pain hypersensitivity and mitigated neuron damage and loss in the spinal cord of NP rats. Moreover, EA reversed the oxidative stress-mediated spinal ferroptosis phenotype by upregulating reduced expression of xCT, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH1) and superoxide dismutase (SOD) levels, and downregulating increased expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), malondialdehyde levels and iron overload. Furthermore, EA increased the immunofluorescence co-staining of GPX4 in neurons cells of the spinal cord of CCI rats. Mechanistic analysis unveiled that the inhibition of antioxidant pathway of Nrf2 signalling via its specific inhibitor, ML385, significantly countered EA's protective effect against neuronal ferroptosis in NP rats while marginally diminishing its analgesic effect. These findings suggest that EA treatment at acupoints ST36 and GV20 may protect against NP by inhibiting neuronal ferroptosis in the spinal cord, partially through the activation of Nrf2 signalling.

Changes in macrophage and inflammatory cytokine expressions during fracture healing in an ovariectomized mice model.

BACKGROUND: Macrophages and inflammatory cytokines play important roles in bone fracture healing. However, the expression patterns of macrophages and inflammatory cytokines during fracture healing under the condition of postmenopausal osteoporosis have not been fully revealed. METHODS: Tibia transverse fracture was established 12 weeks after ovariectomy or sham operation in 16-week old female mice. Tibias were harvested before fracture or 1, 3, 5, 7, 14, 21, 28 days after fracture for radiological and histological examinations. M1/M2 inflammatory macrophages, osteal macrophages and gene expressions of tumor necrosis factor-α, interleukin-6, interleukin-1ß and macrophage conversion related molecules in the fracture haematoma or callus were also detected. RESULTS: The processes of fracture healing, especially the phases of endochondral ossification and callus remodeling, were delayed in ovariectomized mice. The expressions of tumor necrosis factor-α and interleukin-6, but not interleukin-1ß, in the fracture haematoma or callus were disturbed. Expressions of tumor necrosis factor-α were decreased at 1, 14 and 21 days post-fracture (DPF), and were increased at 3, 5 and 7 DPF. Interleukin-6 expressions at 1, 3 and 21 DPF were significantly increased. We found the decreases in M1 and M2 macrophages at 1 DPF of the initial inflammatory stage. M2 macrophages at 14 DPF of the middle stage and osteal macrophages at 14, 21 and 28 DPF of the middle and late stages of fracture healing were also reduced in ovariectomized mice. CONCLUSIONS: The expressions of macrophages and inflammatory cytokines were impaired in ovariectomized mice, which might contribute partially to poor fracture healing.

Oleanolic acid exerts bone protective effects in ovariectomized mice by inhibiting osteoclastogenesis.

Postmenopausal osteoporosis (POP) is quite prevalent and many new drugs are under development to obtain better therapeutic outcomes. Oleanolic acid (OA) has been reported to prevent bone loss in ovariectomized (OVX) rats by stimulating osteoblastogenesis. One previous study has demonstrated that acetate of OA suppressed lipopolysaccharides (LPS)-induced bone loss in mice. However, the role of OA in the receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclastogenesis is still not elucidated. Here we show that OA dose-dependently inhibits RANKL-mediated osteoclastogenesis and the formation of functional osteoclasts without impairing the viability and osteoclastic potential in bone marrow macrophages (BMMs). Moreover, OA administration attenuates bone loss in OVX mice by inhibiting osteoclast's densities. Mechanistically, OA does not affect RANKL-induced activation of the NF-кB, JNK, p38, ERK and Akt pathways, but inhibits the expression of the nuclear factor of activated T-cells c1(NFATc1) and c-Fos. Moreover, OA significantly suppresses the expression of RANKL-activated osteoclast genes encoding matrix metalloproteinase 9 (MMP9), Cathepsin K(Ctsk), tartrate-resistant acid phosphatase (TRAP) and carbonic anhydrase II (Car2). This work has elucidated the molecular mechanism of OA in RANKL-mediated osteoclastogenesis and revealed the promising potential of OA to be further developed as a new drug to prevent and treat POP.

Efficacy and safety of bloodletting puncture and cupping in postherpetic neuralgia: A systematic review and meta­analysis.

The present study aimed to evaluate the efficacy and safety of bloodletting puncture and cupping (BLP-C) in postherpetic neuralgia (PHN) and to provide guidance for clinical treatment. Randomized controlled trials (RCTs) of BLP-C therapy in PHN were systematically searched in eight databases from inception to September 2022. Literature screening, data extraction and quality assessment were performed by two independent researchers. Dichotomous and continuous variables were pooled using the risk ratio (RR) and weighted mean difference (WMD), respectively. A total of 13 studies involving 1,129 patients with PHN (571 in the experimental group and 558 in the control group) were included in the present meta-analysis. Overall efficacy (RR=1.21, 95% CI: 1.15 to 1.28, P<0.00001), VAS score (WMD=-1.10, 95% CI: -1.31 to -0.90, P<0.00001) and PSQI score (WMD=-2.42, 95% CI: -2.87 to -1.96, P<0.0001) were significantly different between the BLP-C group and Western medicine group. Furthermore, subgroup analysis demonstrated that BLP-C alone or combined with other traditional Chinese medicines was more effective than Western medicine in PHN. A total of four RCTs mentioned adverse reactions, most of which were in the Western medicine group and were relieved after treatment discontinuation. In conclusion, BLP-C is superior to Western medicine in relieving pain and improving the sleep quality of patients with PHN with a lower incidence of adverse effects.

Efficacy of manual therapy on shoulder pain and function in patients with rotator cuff injury: A systematic review and meta­analysis.

To critically evaluate the effects of manual therapy (MT) on pain and functional improvement in patients with rotator cuff injury (RCI), a systematic review of all randomized controlled trials (RCTs) on MT for RCI was conducted in the following databases: PubMed, Cochrane Central Register of Controlled Trials, Embase, Web of Science, Physiotherapy Evidence Database, Chinese National Knowledge Infrastructure, Wan-fang Data, Chinese Scientific Journal Database, and Chinese Biomedical Literature database from inception to March 28, 2023. A total of 1,110 participants from 24 eligible RCTs were included in the analysis. Compared with placebo, MT could not effectively relieve pain [standardized mean difference (SMD)=-0.25; 95% CI: -0.51 to 0.01; P=0.06], although its impact on functional improvement appears limited (SMD=0.20; 95% CI: -0.09 to 0.49; P=0.18). Combining MT with exercise had significant advantages over exercise alone, as combined therapy contributed to both pain reduction (SMD=0.36; 95% CI: 0.08 to 0.64; P=0.01) and functional enhancement (SMD=0.32; 95% CI: 0.11 to 0.52; P=0.002). Furthermore, MT combined with multimodal physiotherapy showed additional benefits in pain reduction (mean difference=1.57; 95% CI: 0.18 to 2.96; P=0.03) and functional improvement (SMD=0.77; 95% CI: 0.43 to 1.12; P<0.0001) compared with multimodal physiotherapy alone. These findings highlight the superior pain alleviation and functional improvement provided by MT when combined with exercise or physiotherapy. Consequently, MT has emerged as a pivotal component of therapeutic intervention for RCI.

Reactive oxygen species (ROS)-mediated M1 macrophage-dependent nanomedicine remodels inflammatory microenvironment for osteoarthritis recession.

Osteoarthritis (OA) is a common chronic inflammatory disorder. Effective remodeling of inflammatory microenvironment in the joint is a promising strategy to prevent OA. However, current drugs remain unsatisfactory due to a lack of targeted and effective ways for relieving inflammatory conditions in OA joints. Bortezomib (BTZ), a proteasome inhibitor, could effectively inhibit proinflammatory cytokines but with poor accumulation in the inflammatory tissues. To overcome the shortcomings of BTZ delivery and to improve the efficacy of OA therapy, herein, we designed a novel nanomedicine (denoted as BTZ@PTK) by the co-assembly of BTZ and an amphiphilic copolymer (denoted as PTK) with ROS-cleaved thioketal (TK) linkages. The TK units in BTZ@PTK are first cleaved by the excessive ROS at OA sites, and then triggered the controlled release of BTZ, resulting in the accurate delivery and the inflammatory microenvironment remodeling. Accordingly, BTZ@PTK suppressed ROS generation and proinflammatory cytokines while promoting M1 macrophage apoptosis in lipopolysaccharide (LPS)-activated RAW264.7 macrophages or LPS/IFN-γ-treated primary macrophages, which leads to a better effect than BTZ. In OA mice, BTZ@PTK passively accumulates into inflamed joints to attenuate pain sensitivity and gait abnormality. Importantly, BTZ@PTK treatment successfully ameliorates synovitis with the reduction of synovial hyperplasia and synovitis scores by suppressing M1 macrophage polarization and promoting M1 macrophage apoptosis in the synovium, thereby delaying cartilage damage. Collectively, BTZ@PTK can effectively modulate inflammatory microenvironment for OA recession by activating M1 macrophage apoptosis and inhibiting M1macrophage-mediated inflammatory response.

Efficacy of Combining Traditional Chinese Manual Therapy (Tuina) and Specific Therapeutic Neck Exercise in Young Adults with Non-Specific Chronic Neck Pain: Study Protocol for a Randomized Controlled Trial.

Purpose: Non-specific chronic neck pain (NSCNP) is an increasingly common musculoskeletal disease and an important issue in the global healthcare system. Some studies have shown that the combination of manual therapy and exercise is effective in treating NSCNP but still with several limitations. Traditional Chinese manual therapy (tuina) is a Chinese manual therapy that consists of soft tissue manipulation and spinal manipulation. This study aims to design a randomized controlled trial to assess the effect of a tuina combined with specific therapeutic neck exercise modified protocol for NSCNP patients. Patients and Methods: This is a study protocol for a randomized, participant-, assessor- and analyst-blinded controlled trial. Eighty-eight eligible NSCNP patients will be randomly allocated into tuina combined with specific therapeutic neck exercise group (TSTE group) and tuina combined with sham therapeutic neck exercise group (TS group) in a ratio of 1:1. All participants will receive 8 treatment sessions applied in 4 weeks and then be followed up for another 12 weeks. Clinical data will be collected at baseline, during treatment phase (at the 2- and 4-week) and at the 8-, 12-, 16-week follow-ups. The primary outcome is the changes in neck pain intensity (visual analogue scale). The secondary outcomes include neck disability (Neck Disability Index), cervical range of motion (ROM), neck muscle endurance, cervical muscle cross-sectional area, cervical curvature and analgesic consumption. Adverse events will be collected and recorded throughout the study. Conclusion: We will discuss whether our tuina combined with specific therapeutic neck exercise modified protocol is more effective at improving pericervical muscle endurance, ROM, cervical muscle cross-sectional area and cervical curvature than tuina alone, thereby decreases neck pain and disability in individuals with NSCNP more effectively. Trial Registration: Chinese Clinical Trials Registry, ChiCTR2300067903. Registered on 31 January 2023.

Efficacy of five-step shoulder manipulation for rotator cuff-related shoulder pain: protocol for a multicenter randomized controlled trial.

BACKGROUND: Rotator cuff-related shoulder pain (RCRSP) is the most common cause of shoulder disorders. In China, manipulation has been used extensively for the treatment of patients with RCRSP. However, high-quality clinical evidence to support the therapeutic effect of manipulation is still limited. METHODS: A multicenter, participant-, outcome assessor-, and data analyst-blinded, randomized, placebo-controlled trial will be conducted. A total of 280 participants with RCRSP will be recruited from three hospitals and randomly assigned to a five-step shoulder manipulation (FSM) group or a sham manipulation (SM) group. Each group will receive four weekly treatment sessions, with all participants performing exercises at home for 12 weeks. Assessments, namely the Constant-Murley score, visual analog scale, range of motion, and 36-Item Short Form Survey, will be made at baseline, 4, 12, 18, and 24 weeks. Adverse events during the study will also be recorded. DISCUSSION: This is a pragmatic clinical trial to evaluate the efficacy and safety of FSM in patients with RCRSP. The findings of this study will provide worthy clinical evidence for manual therapy for RCRSP. TRIAL REGISTRATION: China Registered Clinical Trial Registration Center ChiCTR2000037577. Registered on 29 August 2020.

Aconine attenuates osteoclast-mediated bone resorption and ferroptosis to improve osteoporosis via inhibiting NF-κB signaling.

Osteoporosis (OP), a prevalent public health concern primarily caused by osteoclast-induced bone resorption, requires potential therapeutic interventions. Natural compounds show potential as therapeutics for postmenopausal OP. Emerging evidence from in vitro osteoclastogenesis assay suggests that aconine (AC) serves as an osteoclast differentiation regulator without causing cytotoxicity. However, the in vivo functions of AC in various OP models need clarification. To address this, we administered intraperitoneal injections of AC to ovariectomy (OVX)-induced OP mice for 8 weeks and found that AC effectively reversed the OP phenotype of OVX mice, leading to a reduction in vertebral bone loss and restoration of high bone turnover markers. Specifically, AC significantly suppressed osteoclastogenesis in vivo and in vitro by decreasing the expression of osteoclast-specific genes such as NFATc1, c-Fos, Cathepsin K, and Mmp9. Importantly, AC can regulate osteoclast ferroptosis by suppressing Gpx4 and upregulating Acsl4, which is achieved through inhibition of the phosphorylation of I-κB and p65 in the NF-κB signaling pathway. These findings suggest that AC is a potential therapeutic option for managing OP by suppressing NF-κB signaling-mediated osteoclast ferroptosis and formation.

Clinical research for whether the Traditional Chinese medicine could promote the resorption of lumbar disc herniation: a randomized controlled trial.

Lumbar disc herniation (LDH) is a common, disabling musculoskeletal disorder. Magnetic resonance imaging has clarified the natural history of lumbar disc lesions and has documented that disc lesions can become smaller and can even be completely resorbed. Previous studies have confirmed that some traditional Chinese medicine (TCM) therapies can promote resorption of the protrusion. However, high-quality research evidence is needed to support the effectiveness of the protocol. OBJECTIVE: This clinical trial aims to establish whether TCM can promote the resorption of LDH and to assess the efficacy of such therapy for LDH, thereby evaluating its clinical effect. METHODS: The present study design is for a single-center, 2-arm, open-label randomized controlled trial. A total of 150 eligible LDH patients will be randomly assigned to either a TCM treatment group or a control group in a 1:1 ratio. Patients in the TCM group will be administered a TCM decoction for 4 weeks. Patients in the conventional drug control group will be instructed to take a specific daily dose of celecoxib. The primary outcome measure is the change from baseline in the volume of the protrusion, as assessed using MR images. Secondary outcome measures include visual analog scale pain scores and Japanese Orthopaedic Association scores assessed at 3 and 6 months. DISCUSSION: The design and methodological rigor of this trial will allow evaluation of the basic clinical efficacy and safety data for TCM in the treatment of patients with LDH. The trial will also assess whether TCM can promote the resorption of LDH. This research will therefore help provide a solid foundation for the clinical treatment of LDH and for future research in TCM therapy. TRIAL REGISTRATION: ChiCTR1900022377.

The efficacy and safety of 'antianxiety granule' for anxiety disorder: a multicentre, randomized, double-blind, placebo-controlled, parallel-group trial.

BACKGROUND: Anxiety disorders are the most prevalent class of lifetime disorders in China, and generalized anxiety disorder (GAD) is one of the most common but frequently overlooked anxiety disorders. Conventional pharmacological treatments for GAD have varying degrees of side effects, dependency, and/or withdrawal syndromes. Traditional Chinese medicine (TCM) is considered a valuable therapeutic option for anxiety disorders and a potentially effective technique to reduce the side effects associated with antipsychotic drugs. This trial aimed to evaluate the clinical efficacy and safety of Antianxiety Granule, a granular Chinese medicine compound, for treatment of GAD. METHODS/DESIGN: The current work is a multicentre, randomized, double-blind, placebo-controlled, parallel-group clinical trial with a 6-week treatment schedule. The study consists of three periods: a 1-7-day screening period, a 6-week primary treatment period, and a 1-week follow-up period. Follow-up assessments will be conducted 1 week after the last visit with a face-to-face interview or by telephone. The clinical efficacy of Antianxiety Granule for the treatment of GAD will be evaluated by examining the change in the Hamilton anxiety scale (HAMA) score, state-trait anxiety inventory (STAI) score, and TCM symptom scale in patients with GAD who receive daily TCM treatment. Moreover, an intention-to-treat (ITT) analysis will also be used in this randomized controlled trial (RCT). DISCUSSION: Our study is a multicentre, randomized, double-blind, placebo-controlled, parallel-group trial to evaluate the safety and efficacy of Antianxiety Granule for the treatment of GAD. The results of this trial will provide valuable clinical evidence for the treatment of GAD. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800016039. Registered on 8 May 2018.

Inhibition of Axin1 in osteoblast precursor cells leads to defects in postnatal bone growth through suppressing osteoclast formation.

Axin1 is a negative regulator of ß-catenin signaling and its role in osteoblast precursor cells remains undefined. In the present studies, we determined changes in postnatal bone growth by deletion of Axin1 in osteoblast precursor cells and analyzed bone growth in newborn and postnatal Axin1Osx mice and found that hypertrophic cartilage area was largely expanded in Axin1Osx KO mice. A larger number of chondrocytes and unabsorbed cartilage matrix were found in the bone marrow cavity of Axin1Osx KO mice. Osteoclast formation in metaphyseal and subchondral bone areas was significantly decreased, demonstrated by decreased TRAP-positive cell numbers, associated with reduction of MMP9- and cathepsin K-positive cell numbers in Axin1Osx KO mice. OPG expression and the ratio of Opg to Rankl were significantly increased in osteoblasts of Axin1Osx KO mice. Osteoclast formation in primary bone marrow derived microphage (BMM) cells was significantly decreased when BMM cells were cultured with conditioned media (CM) collected from osteoblasts derived from Axin1Osx mice compared with BMM cells cultured with CM derived from WT mice. Thus, the loss of Axin1 in osteoblast precursor cells caused increased OPG and the decrease in osteoclast formation, leading to delayed bone growth in postnatal Axin1Osx KO mice.

The Prognostic and Clinical Value of CD44 in Colorectal Cancer: A Meta-Analysis.

Background: CD44 is widely used as a putative cancer stem cells (CSCs) marker for colorectal cancer (CRC). However, the prognostic role of CD44 in CRC remains controversial. Methods: We performed a systematic review and meta-analysis to evaluate the association of various CD44 isoforms and overall survival (OS) and clinicopathological features of CRC patients. Results: A total of 48 studies were included in the meta-analysis. Total CD44 isoforms overexpression was significantly correlated with worse OS of patients with CRC (HR = 1.32, 95% CI = 1.08-1.61, P = 0.007). In a stratified analysis, a higher level of either CD44v6 or CD44v2 had an unfavorable impact on OS (HRCD44v6 = 1.50, 95% CI = 1.10-2.14, P = 0.010; HRCD44v2 = 2.93, 95% CI = 1.49-5.77, P = 0.002). Additionally, CD44 was shown to be associated with some clinicopathological features, such as lymph node metastasis (ORCD44 = 1.56, 95% CI = 1.01-2.41, P = 0.044; ORCD44v6 = 1.97, 95% CI = 1.19-3.26, P = 0.008; ORTotal CD44 isoforms = 1.57, 95% CI = 1.15-2.14, P = 0.004), distant metastasis (ORCD44 = 2.90, 95% CI = 1.08-7.83, P = 0.035; ORTotal CD44 isoforms = 1.89, 95% CI = 1.02-3.53, P = 0.044). Moreover, a high level of CD44 showed a possible correlation with poor differentiation (ORTotal CD44 isoforms = 1.44, 95% CI = 1.00-2.08, P = 0.051), elevated level of CD44v6 tend to be correlated with tumor size (OR = 1.71, 95% CI = 0.99-2.96, P = 0.056). Conclusions: This meta-analysis demonstrated that CD44 overexpression might be an unfavorable prognostic factor for CRC patients and could be used to predict poor differentiation, lymph node metastasis and distant metastasis.

Deletion of Opg Leads to Increased Neovascularization and Expression of Inflammatory Cytokines in the Lumbar Intervertebral Disc of Mice.

STUDY DESIGN: Neovascularization and expression of inflammatory cytokines were examined in Osteoprotegerin (Opg) knockout (KO) mice that show intervertebral disc (IVD) degeneration. OBJECTIVE: The aim of this study was to clarify the pathological changes in lumbar IVD degeneration in Opg KO mice. SUMMARY OF BACKGROUND DATA: Osteoporosis is a controversial risk factor for IVD degeneration. Deletion of Opg resulted in IVD degeneration in mice. Neovascularization and inflammatory cytokines are key factors in IVD degeneration. METHODS: Opg KO mice and their wild-type (WT) littermates were euthanized. Lumbar IVDs were harvested. Safranin O/Fast Green staining was performed to examine the pathological changes. Microcomputed tomographic (micro-CT) analysis was performed to determine the structural changes at the junction of lumbar IVD cartilage and vertebrae. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast formation. Protein expression of vascular endothelial growth factor A (VEGF-A), CD31, VE-cadherin, CD 34, interleukin-1ß (IL-1ß), and tumor necrosis factors α (TNF-α) were analyzed by immunohistochemistry (IHC) assays. Gene expressions of IL-1ß, IL-6, and TNF-α were analyzed by real-time polymerase chain reaction (RT-PCR). RESULTS: In 12-week-old Opg KO mice, new bone was formed in the endplate cartilage of lumbar IVDs and this became more obvious in 24-week-old Opg KO mice. Three-dimensional (3D) µCT reconstruction analyses showed that the edges of the L4 and L5 vertebrae were rugged with bone marrow cavities in it. Protein expression of VEGF-A, CD31, VE-cadherin, and CD34 was increased in the endplate and growth plate of lumbar IVDs of Opg KO mice. Gene expression of IL-1ß, IL-6, and TNF-α as well as protein expression of IL-1ß and TNF-α were highly expressed in the lumbar IVDs of Opg KO mice. CONCLUSION: Deletion of Opg leads to increased neovascularization and expression of inflammatory cytokines in the lumbar disc in Opg KO mice, which may play important roles in IVD degeneration. LEVEL OF EVIDENCE: N/A.

Biomechanical Characteristics of Osteoporotic Fracture Healing in Ovariectomized Rats: A Systematic Review.

Biomechanical tests are widely used in animal studies on osteoporotic fracture healing. However, the biomechanical recovery process is still unknown, leading to difficulty in choosing time points for biomechanical tests and in correctly assessing osteoporotic fracture healing. To determine the biomechanical recovery process during osteoporotic fracture healing, studies on osteoporotic femur fracture healing with biomechanical tests in ovariectomized rat (OVX) models were collected from PUBMED, EMBASE, and Chinese databases. Quadratic curves of fracture healing time and maximum load were fitted with data from the analyzed studies. In the fitted curve for normal fractures, the predicted maximum load was 145.56 N, and the fracture healing time was 88.0 d. In the fitted curve for osteoporotic fractures, the predicted maximum load was 122.30 N, and the fracture healing time was 95.2 d. The maximum load of fractured femurs in OVX rats was also lower than that in sham rats at day 84 post-fracture (D84 PF). The fracture healing time was prolonged and maximum load at D84 PF decreased in OVX rats with closed fractures. The maximum load of Wister rats was higher than that of Sprague-Dawley (SD) rats, but the fracture healing time of SD and Wister rats was similar. Osteoporotic fracture healing was delayed in rats that were < = 12 weeks old when ovariectomized, and at D84 PF, the maximum load of rats < = 12 weeks old at ovariectomy was lower than that of rats >12 weeks old at ovariectomy. There was no significant difference in maximum load at D84 PF between rats with an osteoporosis modeling time <12 weeks and > = 12 weeks. In conclusion, fracture healing was delayed and biomechanical property decreased by osteoporosis. Time points around D95.2 PF should be considered for biomechanical tests of osteoporotic femur fracture healing in OVX rat models. Osteoporotic fracture healing in OVX rats was affected by the fracture type but not by the strain of the rat.

Osteoprotective effects of osthole in a mouse model of 5/6 nephrectomy through inhibiting osteoclast formation.

The present study aimed to investigate the effects of osthole on osteoclast formation and bone loss in a mouse model of 5/6 nephrectomy. The mice in control and osthole groups were treated 1 month following 5/6 nephrectomy with either a placebo or osthole, respectively. At 2 months post­nephrectomy, the L4 vertebrae were harvested. The bone mineral density (BMD) of cancellous bone was measured using micro­CT and tartrate­resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast formation. Immunohistochemistry staining and reverse transcription­quantitative polymerase chain reaction were performed to detect the expression of nuclear factor of activated T­cells, cytoplasmic­1 (NFATc­1), c­Fos, cathepsin K, Trap, matrix metalloproteinase 9 (Mmp9), osteoprotegerin (Opg) and receptor activator for nuclear factor­κB ligand (Rankl). Bone marrow cells were cultured with osthole, and osteoclast formation was shown by TRAP staining. Primary calvaria osteoblasts were cultured with osthole, and expression levels of Opg and Rankl were detected. Compared with the sham group, the BMD of mice in model group was significantly reduced. The numbers of osteoclasts and the expression levels of NFATc­1, c­Fos, cathepsin K and Mmp9 were significantly increased. Compared with the control group, the mice in the osthole group exhibited increased BMD of the L4 vertebrae, a reduction in osteoclast numbers and decreased expression levels of NFATc­1, c­Fos, cathepsin K and Mmp9. In vitro experiments also showed that osteoclast formation was decreased following treatment with osthole. Osteoprotegerin (Opg)/receptor activator for nuclear factor­κB ligand (Rankl) was upregulated by osthole treatment in the L4 vertebrae and in primary cultures of calvarial osteoblasts. Osthole inhibited osteoclast formation and partially reversed the bone loss induced by 5/6 nephrectomy in mice through the upregulation of OPG/RANKL.

Analgesic mechanism of electroacupuncture in a rat L5 spinal nerve ligation model.

The aim of this study was to investigate the analgesic mechanism of electroacupuncture (EA) in the treatment of neuropathological pain. A total of 60 Sprague-Dawley rats were randomly divided into three groups, namely the spinal nerve ligation (SNL), electroacupuncture (SNL + EA) and normal control groups, with 20 rats in each group. The up-down method was used to determine the bipedal 50% mechanical paw withdrawal threshold (PWT). The ultrastructure of the injured-side L5 nerve root (n=6) was observed by electron microscopy. The mRNA levels of brain-derived neurotrophic factor (BDNF) and purinergic receptor P2X, ligand-gated ion channel 4 (P2X4) in the spinal cord (n=14) were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The postoperative PWT of the injured-side hindpaw in the SNL group at each time point was lower than that in the control group (P<0.01); there were differences of statistical significance between the PWT values of the SNL + EA and SNL groups on postoperative days 14 and 21 (P<0.05). Postoperatively, the PWT of the hindpaw on the uninjured-side was significantly lower in the SNL group when compared with that of the control group on days 10, 14 and 21 (P<0.05). Following the EA treatment, axonal demyelination was reduced and vascular proliferation was observed within the visual field. In addition, following the EA treatment, BDNF expression levels in the spinal dorsal horn increased (P<0.05), while the expression of P2X4 was not different from that in the SNL group. EA exerted an analgesic effect on the SNL model in a time-dependent manner, and improved the blood supply to the nerve root. Following the EA treatment, the expression of P2X4 did not change significantly compared with that in the SNL group, whereas the spinal secretion of BDNF increased. However, the exact mechanism requires further study.