TSP-1 increases autophagy level in cartilage by upregulating HSP27 which delays progression of osteoarthritis.

This study aimed to determine whether Thrombospondin-1 (TSP-1) can be used as a biomarker to diagnose early osteoarthritis (OA) and whether it has a chondroprotective effect against OA. We examined TSP-1 expression in cartilage, synovial fluid, and serum at different time points after anterior cruciate ligament transection (ACLT) surgery in rats. Subsequently, TSP-1 was overexpressed or silenced to detect its effects on extracellular matrix (ECM) homeostasis, autophagy level, proliferation and apoptosis in chondrocytes. Adenovirus encoding TSP-1 was injected into the knee joints of ACLT rats to test its effect against OA. Combined with proteomic analysis, the molecular mechanism of TSP-1 in cartilage degeneration was explored. Intra-articular injection of an adenovirus carrying the TSP-1 sequence showed chondroprotective effects against OA. Moreover, TSP-1 expression decreases with OA progression and can effectively promote cartilage proliferation, inhibit apoptosis, and helps to sustain the balance between ECM anabolism and catabolism. Overexpression of TSP-1 also can increase autophagy by upregulating Heat Shock Protein 27 (HSP27, hspb1), thereby enhancing its effect as a stimulator of autophagy. TSP-1 is a hopeful strategy for OA treatment.

Effect of Moderate Exercise on the Superficial Zone of Articular Cartilage in Age-Related Osteoarthritis.

This study aimed to evaluate the effect of exercise on the superficial zone of the osteoarticular cartilage during osteoarthritis progression. Three-month-old, nine-month-old, and eighteen-month-old Sprague Dawley rats were randomly divided into two groups, moderate exercise and no exercise, for 10 weeks. Histological staining, immunostaining, and nanoindentation measurements were conducted to detect changes in the superficial zone. X-ray and micro-CT were quantitated to detect alterations in the microarchitecture of the tibial subchondral bone. Cells were extracted from the superficial zone of the cartilage under fluid-flow shear stress conditions to further verify changes in vitro. The number of cells and proteoglycan content in the superficial zone increased more in the exercise group than in the control group. Exercise can change the content and distribution of collagen types I and III in the superficial layer. In addition, TGFß/pSmad2/3 and Prg4 expression levels increased under the intervention of exercise on the superficial zone. Exercise can improve the Young's modulus of the cartilage and reduce the abnormal subchondral bone remodeling which occurs after superficial zone changes. Moderate exercise delays the degeneration of the articular cartilage by its effect on the superficial zone, and the TGFß/pSmad2/3 signaling pathways and Prg4 play an important role.

Network Pharmacology and in vitro Experimental Verification on Intervention of Quercetin, Present in Chinese Medicine Yishen Qutong Granules, on Esophageal Cancer.

OBJECTIVE: To explore the potential mechanism of Yishen Qutong Granules (YSQTG) for the treatment of esophageal cancer using network pharmacology and experimental research. METHODS: The effective components and molecular mechanism of YSQTG in treating esophageal cancer were expounded based on network pharmacology and molecular docking. The key compound was identified by high-performance liquid chromatography and mass spectrometry (HPLC-MS) to verify the malignant phenotype of the key compounds in the treatment of esophageal cancer. Then, the interaction proteins of key compounds were screened by pull-down assay combined with mass spectrometry. RNA-seq was used to screen the differential genes in the treatment of esophageal cancer by key compounds, and the potential mechanism of key compounds on the main therapeutic targets was verified. RESULTS: Totally 76 effective compounds of YSQTG were found, as well as 309 related targets, and 102 drug and disease interaction targets. The drug-compound-target network of YSQTG was constructed, suggesting that quercetin, luteolin, wogonin, kaempferol and baicalein may be the most important compounds, while quercetin had higher degree value and degree centrality, which might be the key compound in YSQTG. The HPLC-MS results also showed the stable presence of quercetin in YSQTG. By establishing a protein interaction network, the main therapeutic targets of YSQTG in treating esophageal cancer were Jun proto-oncogene, interleukin-6, tumor necrosis factor, and RELA proto-oncogene. The results of cell function experiments in vitro showed that quercetin could inhibit proliferation, invasion, and clonal formation of esophageal carcinoma cells. Quercetin mainly affected the biological processes of esophageal cancer cells, such as proliferation, cell cycle, and cell metastasis. A total of 357 quercetin interacting proteins were screened, and 531 genes were significantly changed. Further pathway enrichment analysis showed that quercetin mainly affects the metabolic pathway, MAPK signaling pathway, and nuclear factor kappa B (NF- κ B) signaling pathway, etc. Quercetin, the key compound of YSQTG, had stronger binding activity by molecular docking. Pull-down assay confirmed that NF- κ B was a quercetin-specific interaction protein, and quercetin could significantly reduce the protein level of NF- κ B, the main therapeutic target. CONCLUSION: YSQTG can be multi-component, multi-target, multi-channel treatment of esophageal cancer, it is a potential drug for the treatment of esophageal cancer.

Moderate Nrf2 Activation by Genetic Disruption of Keap1 Has Sex-Specific Effects on Bone Mass in Mice.

Keap1 is a negative controller of the transcription factor Nrf2 for its activity. The Keap1/Nrf2 signaling pathway has been considered as a master regulator of cytoprotective genes, and exists in many cell types including osteoblasts and osteoclasts. Our previous study shows Nrf2 deletion decreases bone formation. Recent studies show hyperactivation of Nrf2 causes osteopenia in Keap1-/- mice, and Keap1-/- osteoblasts have significantly less proliferative potential than Keap1+/- osteoblasts. We aimed to examine if moderate Nrf2 activation by disruption of Keap1 impacts bone metabolism. We examined bone phenotype of Keap1 heterozygotic mice (Ht) in comparison with Keap1 wild type (WT) mice. Deletion or knockdown of Keap1 enhanced the gene expression of Nrf2, ALP and wnt5a in cultured primary osteoblasts compared to WT control. In male mice, compared with their age-matched littermate WT controls, Keap1 Ht mice showed significant increase in bone formation rate (+30.7%, P = 0.0029), but did not change the ultimate force (P < 0.01). The osteoclast cell numbers (-32.45%, P = 0.01) and surface (-32.58%, P = 0.03) were significantly reduced by Keap1 deficiency in male mice. Compared to male WT mice, serum bone resorption marker in male Keap1 Ht mice was significantly decreased. Our data suggest that moderate Nrf2 activation by disruption of Keap1 improved bone mass by regulating bone remodeling in male mice.

microRNA-214-3p Suppresses Ankylosing Spondylitis Fibroblast Osteogenesis via BMP-TGFß Axis and BMP2.

Recent investigations suggest microRNAs (miRs) exert functions in fibroblast osteogenesis in ankylosing spondylitis (AS), an inflammatory rheumatic disease. But the mechanism of miR-214-3p in osteogenic differentiation in AS is not clearly understood yet. In this study, fibroblasts were obtained from the capsular ligament of patients with AS and femoral neck fracture and cultured for osteogenic induction and identified. The roles of miR-214-3p and bone morphogenic protein 2 (BMP2) in AS fibroblast osteogenesis were assessed via gain- and loss-of-function, alizarin red S staining, and alkaline phosphatase (ALP) detection. Levels of miR-214-3p, BMP2, osteogenic differentiation-related proteins, and BMP-TGFß axis-related proteins were further measured. Consequently, miR-214-3p was downregulated in AS fibroblasts, with enhanced ALP activity and calcium nodules, which were reversed by miR-214-3p overexpression. BMP2 was a target gene of miR-214-3p and promoted AS fibroblast osteogenesis by activating BMP-TGFß axis, while miR-214-3p inhibited AS fibroblast osteogenesis by targeting BMP2. Together, miR-214-3p could prevent AS fibroblast osteogenic differentiation by targeting BMP2 and blocking BMP-TGFß axis. This study may offer a novel insight for AS treatment.

Low-Frequency Sonophoresis of Chinese Medicine Formula Improves Efficacy of Malignant Pleural Effusion Treatment.

OBJECTIVE: To evaluate whether low-frequency ultrasound-facilitated transdermal delivery of a Chinese medicine (CM) formula could improve the efficacy of intrapleural administration of interleukin-2 (IL-2) in treatment of malignant pleural effusion (MPE). METHODS: A total of 110 eligible participants were randomized into the low-frequency sonophoresis (LFS) of CM (LSF/CM) group (55 cases) and the control group (55 cases) by simple randomization using a random number table. The control group was treated with an intrapleural administration of IL-2; and the LFS/CM group was treated with LFS of a CM gel formulation, combined with the same IL-2 injection as in the control group. The CM formula consisted of Semen Lepidii, Semen Sinapis, Ramulus Cinnamomi, Poriacocos, Herba Lycopi, and Radix Paeoniae Rubra. After 2-week treatment, the therapeutic outcome was determined by the change of the amount of MPE, which was evaluated by B-scan ultrasound and/or chest X-ray, and the change of quality of life (QOL) scores, which were evaluated by the Eastern Cooperative Oncology Group (ECOG) performance status. RESULTS: A significantly higher objective remission rate (ORR) was obtained with intrapleural IL-2 plus LFS/CM than IL-2 treatment alone (P=0.049). In addition, more patients in the LFS/CM group than in the control group had an improved QOL score (P=0.048), and no patients in the LFS/CM group had a reduced QOL. CONCLUSION: LFS of CM formulation could effectively alleviate MPE and improve the QOL of cancer patients.

Topical Delivery of Modified Da-Cheng-Qi Decoction () Using Low-Frequency Ultrasound Sonophoresis for Refractory Metastatic Malignant Bowel Obstruction: An Open-Label Single-Arm Clinical Trial.

OBJECTIVE: To evaluate the efficacy and safety of topical delivery of modified Da-Cheng- Qi Decoction (, MDCQD) by low-frequency ultrasound sonophoresis (LFUS) in patients with refractory metastatic malignant bowel obstruction (MBO) using an objective performance criteria (OPC) design. METHODS: Fifty patients with refractory metastatic MBO were enrolled in this open-label single-arm clinical trial. Alongside fasting, gastrointestinal decompression, glycerol enema, intravenous nutrition and antisecretory therapy, a 50 g dose of MDCQD (prepared as a hydrogel) was applied through topical delivery at the site of abodminal pain or Tianshu (S 25) using LFUS for 30 min, twice daily for 5 consecutive days. The overall outcome was the remission of intestinal obstruction, and improvement on abdominal pain, abdominal distention, nausea and vomiting scores. Indicators of safety evaluation included liver and renal function as well as blood coagulation indicators. RESULTS: Among 50 patients, 5 patients (10%) showed complete remission of intestinal obstruction and 21 patients (42%) showed improvement of intestinal obstruction. The overall remission rate of bowel obstruction was 52%. The results of the symptom score, based on the severity and frequency of the episode, are as follows: 26 patients (52%) showed improvment on symptom scores, 20 patients (40%) did not respond to treatment, and 4 patients (8%) discontinued treatment due to intolerance. No serious adverse effects or abnormal changes on liver and renal function or blood coagulation were observed. CONCLUSION: Topical delivery of MDCQD at 100 g/day using LFUS can improve the treatment response in patients with refractory metastatic MBO.

Upregulating MicroRNA-410 or Downregulating Wnt-11 Increases Osteoblasts and Reduces Osteoclasts to Alleviate Osteonecrosis of the Femoral Head.

BACKGROUND: Little is known regarding the functional role of microRNA-410 (miR-410) in osteonecrosis of the femoral head (ONFH); hence, the aim of the present study was to investigate miR-410 targeting Wnt-11 to modulate the osteogenic and osteoclastic mechanism in the prevention of ONFH. METHODS: Fifteen ONFH samples and 15 normal samples were gathered. The pathological changes of the femoral head, osteoblasts, and osteoclasts in the clinical samples were observed. The rat model of ONFH was injected with agomir-miR-410, Wnt-11-siRNA, or oe-Wnt-11. MiR-410; Wnt-11; osteoblast-related factors alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (BGLAP), and Collα1 expression; and osteoclast-related factors acid phosphatase 5 (ACP5), cathepsin K (CTSK), and MMP9, as well as Bcl-2 and Bax expression, were tested by RT-qPCR and western blot analysis. The osteogenic function index ALP and OCN together with osteoclast function index NTX-1 and CTX-1 in serum was tested by ELISA. RESULTS: MiR-410, ALP, BGLAP, and Collα1 degraded as well as Wnt-11, ACP5, CTSK, and MMP9 enhanced in ONFH tissues of the clinical samples. Upregulated miR-410 and downregulated Wnt-11 enhanced bone mineral density (BMD) and BV/TV of rats, heightened the BMD level of the femoral shaft, femoral head, and spinal column, and also raised the serum calcium and phosphorus levels of rats, while restrained apoptosis of osteocytes, elevated OCN, ALP, BGLAP, and Collα1 expression and declined ACP5, CTSK, NTX-1, CTX-1, and MMP9 expression in rats. CONCLUSION: This study suggested that upregulating miR-410 or downregulating Wnt-11 increases osteoblasts and reduces osteoclasts to alleviate the occurrence of ONFH. Thus, miR-410 may serve as a potential target for the treatment of ONFH.

Cancer Survivors Could Get Survival Benefits from Postdiagnosis Physical Activity: A Meta-Analysis.

BACKGROUND: Physical activity presents significant protection against death from cancer in the general population, so the global recommendations on physical activity for health are recommended by the WHO. While the recommendation is a guideline for general population, whether all cancer patients could get benefits from physical activity and whether the cancer patients who did not meet the requirement of the recommendation could get benefits from the physical activity, compared with the cancer patients with no physical activity, are unclear. Accordingly, we conducted a meta-analysis to identify whether the physical activity, even if low level of physical activity, could reduce the mortality of various cancer patients. METHOD: We conducted a systematic search of PubMed, Embase, and Cochrane Library for published cohorts and case-control studies of cancer survivors with physical activity comparing with no physical activity and reported outcomes of mortality through October 15, 2018. Two investigators independently reviewed the included studies and extracted relevant data. The effect estimate of interest was the hazard ratios (HRs). RESULTS: There are 21811 participants in total in the nine studies, and 2386 cancer deaths in this meta-analysis. Among them, 1 was a case-control study and 8 were cohort studies. The meta-analysis results showed that physical activity was associated with a significantly reduced risk of mortality in cancer survivors, with a pooled HR and 95% CI of 0.66 (0.58∼0.73), reducing mortality by 34% and also suggested that low level of physical activity could reduce the mortality with an HR and 95% CI of 0.60 (0.50∼0.69). CONCLUSION: The results of this meta-analysis demonstrated that postdiagnosis physical activity, no matter the level of physical activity, could significantly reduce the mortality by 34%, compared with the no physical activity. At the same time, the results also suggested that cancer survivors undergoing low level of physical activity had a 40% reduction in mortality, which means that the cancer patients with poor ECOG need to do physical activity as much as they can, even if the amount of physical activity was low.

Low­frequency pulsed electromagnetic field inhibits RANKL­induced osteoclastic differentiation in RAW264.7 cells by scavenging reactive oxygen species.

Bone homeostasis is a dynamic balance maintained by bone formation and resorption. An increase in the number and activity of osteoclasts leads to excessive bone resorption, which in turn results in bone disease, including osteoporosis. Therefore, inhibiting the differentiation and activity of osteoclasts is important for maintaining bone mass. Several studies have revealed that the use of a low­frequency pulsed electromagnetic field (PEMF) is an effective method to treat osteoporosis. However, its exact mechanism remains to be fully clarified. Therefore, the present study was designed to examine the effects that PEMF exerts on receptor activator of nuclear factor­κB ligand (RANKL)­induced osteoclastogenesis and intracellular reactive oxygen species (ROS) production in RAW264.7 cells. The viability of cells was determined using a Cell Counting Kit­8 assay, and gene and protein expression were investigated via reverse transcription­quantitative polymerase chain reaction and western blot analyses. Furthermore, microscopy was performed to detect the levels of intracellular ROS and tartrate­resistant acid phosphatase (TRAP). Following the culture of RAW264.7 cells with RANKL (50 ng/ml) for 4 days (3 h/day) under PEMF (75 Hz, 1 mt) exposure, it was observed that PEMF had an inhibitory effect on RANKL­induced osteoclastic differentiation. Multinucleated osteoclast formation, the activity of TRAP and the expression of osteoclastogenesis­associated genes, including cathepsin K, nuclear factor of activated T cells cytoplasmic 1 and TRAP, were significantly reduced by PEMF. Furthermore, PEMF effectively decreased the generation of intracellular ROS during osteoclastic differentiation. In addition, the results demonstrated that ROS are the key factor in osteoclast differentiation and formation. Reducing intracellular ROS with diphenylene­iodonium chloride significantly inhibited RANKL­induced osteoclast differentiation. Taken together, the results of the present study demonstrated that PEMF may inhibit RANKL­induced osteoclastogenesis by scavenging intracellular ROS. These results may provide the groundwork for future PEMF clinical applications in osteoclast­associated bone disease.

3D-Printed ß-Tricalcium Phosphate Scaffold Combined with a Pulse Electromagnetic Field Promotes the Repair of Skull Defects in Rats.

Trauma, infection, cancer, and congenital diseases can lead to bone defects. The combination of 3D printing with biomaterials is of great significance in the treatment of bone defects. In addition, pulse electromagnetic fields (PEMFs) can promote bone regeneration. The main purpose of this study was to evaluate the effects of 3D-printed scaffolds using ß-tricalcium phosphate (ß-TCP) as the raw material combined with a PEMF on the proliferation and differentiation of rat adipose stem cells (rADSCs) and on the repair of critical defects of the rat skull. The Cell Counting Kit-8 assay was performed to assess the proliferation of rADSCs. Alkaline phosphatase (ALP) activity, ALP staining, and the detection of osteogenic-related gene expression were performed to assess the differentiation of rADSCs. Micro-computed tomography and hematoxylin-eosin staining were used to assess the repair of rat skull defects. The results showed that the combination of the scaffold and PEMF could significantly promote the proliferation and differentiation of rADSCs and the repair of a critical defect in the rat skull. Therefore, the combination of ß-TCP and PEMF with 3D printing technology can provide better treatment of clinical bone defect patients.

The Effects of a Pulsed Electromagnetic Field on the Proliferation and Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

BACKGROUND A low frequency pulsed electromagnetic field (PEMF) has been confirmed to play an important role in promoting the osteogenic differentiation of human bone marrow stem cells (BMSCs). Adipose-derived stem cells (ASCs) possess some attractive characteristics for clinical application compared to BMSCs, such as abundant stem cells from lipoaspirates, faster growth, less discomfort and morbidity during surgery. ASCs can become adipocytes, osteoblasts, chondrocytes, myocytes, neurocytes, and other cell types. Thus, ASCs might be a good alternative in clinical work involving treatment with PEMF. MATERIAL AND METHODS Human ASCs (hASCs)were divided into a control group (without PEMF exposure) and an experimental group (PEMF for two hours per day). We examined the effect of PEMF on promoting cell proliferation and osteogenic differentiation from several aspects: CCK-8 proliferation assay, RNA extraction, qRT-PCR detection, western blotting, and immunofluorescence staining experiments. RESULTS PEMF could promote cell proliferation of human ASCs (hASCs) at an early stage as determined by CCK-8 assay. A specific intensity (1 mT) and frequency (50 Hz) of PEMF promoted osteogenic differentiation in hASCs in alkaline phosphatase (ALP) staining experiments. In addition, bone-related gene expression increased after two weeks of PEMF exposure, the protein expression of OPN, OCN, and RUNX-2 also increased after a longer period (three weeks) of PEMF treatment as determined by western blotting and immunofluorescence staining. CONCLUSIONS We found for the first time that PMEF has a role in stimulating cell proliferation of hASCs at an early period, subsequently promoting bone-related gene expression and inducing the expression of related proteins to stimulate osteogenic differentiation.

Inhibiting checkpoint kinase 1 protects bone from bone resorption by mammary tumor in a mouse model.

DNA damage response plays a critical role in tumor growth, but little is known about its potential role in bone metabolism. We employed selective inhibitors of Chk1 and examined their effects on the proliferation and migration of mammary tumor cells as well as the development of osteoblasts and osteoclasts. Further, using a mouse model of bone metastasis we evaluated the effects of Chk1 inhibitors on bone quality. Chk1 inhibitors blocked the proliferation, survival, and migration of tumor cells in vitro and suppressed the development of bone-resorbing osteoclasts by downregulating NFATc1. In the mouse model, Chk1 inhibitor reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Analysis of RNA-seq expression data indicated that the observed effects were mediated through the regulation of eukaryotic translation initiation factor 2 alpha, stress to the endoplasmic reticulum, S100 proteins, and bone remodeling-linked genes. Our findings suggest that targeting Chk1 signaling without adding DNA damaging agents may protect bone from degradation while suppressing tumor growth and migration.

The role of curcumae rhizoma-sparganii rhizoma medicated serum in epithelial-mesenchymal transition in the triple negative breast cancer: Pharmacological role of CR-SR in the TBNC.

The aim of this study was to investigate the effect of Curcumae Rhizoma-Sparganii Rhizoma (CR-SR) medicated serum on the changes of epithelial-mesenchymal transition (EMT) in the triple-negative breast cancer (TNBC). The EMT model was developed by using the TNBC MDA-MB-468 cells, which were treated with TGF-ß1. The migration and invasion abilities of TGF-ß1-treated MDA-MB-468 cells were detected by wound healing assay and transwell assay. Protein expression levels of E-cadherin and vimentin were determined by western blot. CR-SR medicated serum repressed the phenotypic transition in the TGF-ß1-induced MDA-MB-468 cells. Moreover, CR-SR medicated serum inhibited TGF-ß1-induced cell proliferation, migration and invasion. Besides, CR-SR medicated serum could reduce TGF-ß1-induced up-regulation of the phosphorylation levels of Smad3, and reduce the expression of several transcription factors (Snail1, Snail2 and Twist1). CR-SR medicated serum might suppress TGF-ß1-induced EMT in TNBC by decreasing the phosphorylated Smad3 (p-Smad3) pathway in vitro.

Elemene inhibits osteosarcoma growth by suppressing the renin­angiotensin system signaling pathway.

Osteosarcoma remains the most prevalent primary malignant bone tumor in children and young adults globally. Therefore, novel and highly effective antitumor agents are urgently required. Elemene is a natural plant compound extracted from the medicinal Chinese herb, Rhizomazedoariae, which has been employed as an antitumor agent for the treatment of a number of tumors, including osteosarcoma. However, the mechanisms underlying its antitumor effect are currently unclear. The human osteosarcoma cell lines, MG­63 and U2OS, were employed in the present study. MTT, migration, transwell invasion and terminal deoxynucleotidyltransferase­mediated deoxy­UTP­fluorescein nick end­labeling assays were performed to evaluate cell viability, migration, invasion and apoptosis, respectively. Western blotting and immunohistochemistry analyses were performed to measure the levels of renin­angiotensin system (RAS) components. In order to evaluate the effect of elemene on tumor weight and volume, MG­63 and U2OS cells were injected into mice. Treatment of osteosarcoma cell lines, MG­63 and U2OS, with elemene led to the inhibition of cell viability, migration and invasion, as well as induction of cell apoptosis. In addition, elemene treatment downregulated the expression of a number of RAS components. The growth of osteosarcoma cell­transplanted tumors in nude mice and angiotensin II expression were inhibited by elemene treatment. The results of the present study indicate that the antitumor effects of elemene may partly be due to downregulation of the RAS signaling pathway, and that RAS may be a putative pharmacological target for osteosarcoma therapy.

Inhibitory Effects of Dopamine Receptor D1 Agonist on Mammary Tumor and Bone Metastasis.

Dopaminergic signaling plays a critical role in the nervous system, but little is known about its potential role in breast cancer and bone metabolism. A screening of ~1,000 biologically active compounds revealed that a selective agonist of dopamine receptor D1 (DRD1), A77636, inhibited proliferation of 4T1.2 mammary tumor cells as well as MDA-MB-231 breast cancer cells. Herein, we examined the effect of A77636 on bone quality using a mouse model of bone metastasis from mammary tumor. A77636 inhibited migration of cancer cells in a DRD1-dependent fashion and suppressed development of bone-resorbing osteoclasts by downregulating NFATc1 through the elevation of phosphorylation of eIF2α. In the mouse model of bone metastasis, A77636 reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Collectively, we expect that dopaminergic signaling might provide a novel therapeutic target for breast cancer and bone metastasis.