OBJECTIVES:Osteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by a high potential for proliferation and metastasis. Patients with osteosarcoma who have distant metastases generally have a poor prognosis. Challenges in treatment include incomplete resection of tumor and chemotherapy resistance, with no effective cure currently available. Recent studies suggest that β-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) plays a role in the progression of various malignant tumors. However, the function of B4GALNT1 in osteosarcoma cells has not been reported. This study aims to investigate the expression of B4GALNT1 in osteosarcoma tissues compared to normal tissues and to explore its effects on the proliferation, migration, and invasion of osteosarcoma cells, thereby providing new theoretical foundations and directions for the treatment of osteosarcoma patients. METHODS:Tumor tissues and corresponding normal tissue samples were collected from 16 osteosarcoma patients who underwent tumor resection at the Second Xiangya Hospital of Central South University. The patients' ages ranged from 8 to 17 years (median age 12 years). The expression of B4GALNT1 mRNA in osteosarcoma tissues, corresponding normal tissues, 3 osteosarcoma cell lines (MG63, Saos-2, and U2OS), and human fetal osteoblastic cells (hFOB) was detected using real-time reverse transcription PCR (real-time RT-PCR). The effects of B4GALNT1 knockdown on the proliferation of osteosarcoma cells Saos-2 and U2OS were analyzed using cell counting kit-8 (CCK-8) assays and colony formation assays. The effects of B4GALNT1 knockdown on the migration and invasion abilities of Saos-2 and U2OS cells were evaluated using Transwell migration and invasion assays. Western blotting analysis was performed to assess the impact of B4GALNT1 knockdown on the expression of epithelial-mesenchymal transition (EMT) and invasion-related proteins in Saos-2 and U2OS cells. RESULTS:Real-time RT-PCR results showed that B4GALNT1 mRNA expression levels were significantly higher in osteosarcoma tissues and the 3 osteosarcoma cell lines compared to normal tissues and hFOB cells (all P<0.01). CCK-8 and colony formation assays indicated that B4GALNT1 knockdown significantly reduced the proliferation rate of osteosarcoma cells compared to the control group (all P<0.05). Transwell migration and invasion assays demonstrated that B4GALNT1 knockdown significantly decreased the number of migrating and invading osteosarcoma cells (all P<0.01). Western blotting analysis revealed that B4GALNT1 knockdown inhibited the expression of N-cadherin, Snail, Vimentin, and matrix metalloproteinase 9 (MMP9) compared to the control group (all P<0.01). CONCLUSIONS:B4GALNT1 is upregulated in osteosarcoma tissues and cell lines, and its knockdown suppresses the malignant phenotype of osteosarcoma cells. B4GALNT1 may function as an oncogene in the proliferation and metastasis of osteosarcoma cells.

BackgroundRemimazolam is a new ultrashort-acting benzodiazepine for sedation and anesthesia. The effects of remimazolam and the mechanism by which it functions in cancer cells have not been determined. This research aimed to explore the mechanism of remimazolam action in colon cancer treatment, using bioinformatics analysis and in vitro experiments.MethodsCell cycle progression, colony formation, self-renewal capacity, and apoptosis detection were performed in HCT8 cells treated with or without remimazolam. Transcriptome sequencing, Gene Ontology, Kyoto Encyclopedia of Genes and Genome, Protein–Protein Interaction, Gene Set Enrichment Analysis, Western blotting, and qPCR were performed to investigate the mechanism of action of remimazolam in HCT8 colon cancer cells.ResultsRemimazolam promoted proliferation and cell-cycle progression of HCT8 cells. After remimazolam treatment, a total of 1,096 differentially expressed genes (DEGs) were identified: 673 genes were downregulated, and 423 genes were upregulated. The DEGs were enriched mainly in “DNA replication“, “cell cycle”, and “G1/S transition” related pathways. There were 15 DEGs verified by qPCR, and representative biomarkers were detected by Western Bloting. The remimazolam-mediated promotion of cell proliferation and cell cycle was reversed by G1T28, a CDK4/6 inhibitor.ConclusionRemimazolam promoted cell-cycle progression and proliferation in HCT8 colon cancer cells, indicating that the long-term use of remimazolam has potential adverse effects in the anesthesia of patients with colon cancer.

AIM:Repeated exposure to ketamine during the neonatal period in mice leads to cognitive impairments in adulthood. These impairments are likely caused by synaptic plasticity and excitability damage. We investigated the precise role of brain-derived neurotrophic factor (BDNF) in the cognitive impairments induced by repeated ketamine exposure during the neonatal period.METHODS:We evaluated the cognitive function of mice using the Morris water maze test and novel object recognition test. Western blotting and immunofluorescence were used to detect the protein levels of BDNF. Western blotting, Golgi-Cox staining, transmission electron microscopy, and long-term potentiation (LTP) recordings were used to assess synaptic plasticity in the hippocampus. The excitability of neurons was evaluated using c-Fos. In the intervention experiment, pAdeno-CaMKIIα-BDNF-mNeuronGreen was injected into the hippocampal CA1 region of mice to increase the level of BDNF. The excitability of neurons was enhanced using a chemogenetic approach.RESULTS:Our findings suggest that cognitive impairments in mice repeatedly exposed to ketamine during the neonatal period are associated with downregulated BDNF protein level, synaptic plasticity damage, and decreased excitability of glutamatergic neurons in the hippocampal CA1 region. Furthermore, the specific upregulation of BDNF in glutamatergic neurons of the hippocampal CA1 region and the enhancement of excitability can improve impaired synaptic plasticity and cognitive function in mice.CONCLUSION:BDNF downregulation mediates synaptic plasticity and excitability damage, leading to cognitive impairments in adulthood following repeated ketamine exposure during the neonatal period.

The basolateral amygdala (BLA) is the subregion of the amygdala located in the medial of the temporal lobe, which is connected with a wide range of brain regions to achieve diverse functions. Recently, an increasing number of studies have focused on the participation of the BLA in many neuropsychiatric disorders from the neural circuit perspective, aided by the rapid development of viral tracing methods and increasingly specific neural modulation technologies. However, how to translate this circuit-level preclinical intervention into clinical treatment using noninvasive or minor invasive manipulations to benefit patients struggling with neuropsychiatric disorders is still an inevitable question to be considered. In this review, we summarized the role of BLA-involved circuits in neuropsychiatric disorders including Alzheimer's disease, perioperative neurocognitive disorders, schizophrenia, anxiety disorders, depressive disorders, posttraumatic stress disorders, autism spectrum disorders, and pain-associative affective states and cognitive dysfunctions. Additionally, we provide insights into future directions and challenges for clinical translation.

Opioids are the most effective and widely used treatments for acute and chronic pain in patients with cancer. This review focuses on the impact of opioids and mu-opioid receptors (MORs) on the stages of oncologic metastasis. Studies have shown that opioids can facilitate tumor progression and are related to a poor prognosis in patients with cancer. As the primary receptor for opioids, MORs play a significant role in regulating malignant tumor transformation and are involved in processes, such as proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), circulating tumor cells (CTCs) and the tumor microenvironment (TME). While clinical trials have investigated the relationship between opioids and patient prognosis, further research is needed to clarify the relationship between opioids, MORs and metastasis.

With the ageing of the population, the health problems of elderly individuals have become particularly important. Through a large number of clinical studies and trials, it has been confirmed that elderly patients can experience postoperative cognitive dysfunction after general anesthesia/surgery. However, the mechanism of postoperative cognitive dysfunction is still unknown. In recent years, the role of epigenetics in postoperative cognitive dysfunction has been widely studied and reported. Epigenetics includes the genetic structure and biochemical changes of chromatin not involving changes in the DNA sequence. This article summarizes the epigenetic mechanism of cognitive impairment after general anesthesia/surgery and analyses the broad prospects of epigenetics as a therapeutic target for postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a serious and common complication induced by anesthesia and surgery. Neuronal apoptosis induced by general anesthetic neurotoxicity is a high-risk factor. However, a comprehensive analysis of general anesthesia-regulated gene expression patterns and further research on molecular mechanisms are lacking. Here, we performed bioinformatics analysis of gene expression in the hippocampus of aged rats that received sevoflurane anesthesia in GSE139220 from the GEO database, found a total of 226 differentially expressed genes (DEGs) and investigated hub genes according to the number of biological processes in which the genes were enriched and performed screening by 12 algorithms with cytoHubba in Cytoscape. Among the screened hub genes, Agt, Cdkn1a, Ddit4, and Rhob are related to the neuronal death process. We further confirmed that these genes, especially Ddit4, were upregulated in the hippocampus of aged mice that received sevoflurane anesthesia. NMDAR, the core target receptor of sevoflurane, rather than GABAAR, mediates the sevoflurane regulation of DDIT4 expression. Our study screened sevoflurane-regulated DEGs and focused on the neuronal death process to reveal DDIT4 as a potential target mediated by NMDAR, which may provide a new target for the treatment of sevoflurane neurotoxicity.

AIMS:Cognitive dysfunction associated with chronic pain may be caused by impaired synaptic plasticity. Considering the impact of silent information regulator 1 (SIRT1) on synaptic plasticity, we explored the exact role of SIRT1 in cognitive impairment caused by chronic pain.METHODS:We evaluated the memory ability of mice with the fear conditioning test (FCT) after spared nerve injury (SNI) model. Western blotting and immunofluorescence were used to analyze the expression levels of SIRT1. Hippocampal synaptic plasticity was detected with Golgi staining, transmission electron microscopy, and long-term potentiation (LTP). In the intervention study, AAV9-CaMKIIα-Cre-EGFP was injected to SIRT1flox/flox mice to knockdown the expression levels of SIRT1. Besides, SNI mice were injected with AAV2/9-CaMKIIα-SIRT1-3*Flag-GFP or SRT1720 to increase the expression levels or enzymatic activity of SIRT1.RESULTS:Our current results indicated that cognitive function in SNI mice was impaired, SIRT1 expression in glutaminergic neurons in the hippocampal CA1 area was downregulated, and synaptic plasticity was altered. Selective knockdown of SIRT1 in hippocampus damaged synaptic plasticity and cognitive function of healthy mice. In addition, the impaired synaptic plasticity and cognitive dysfunction of SNI mice could be improved by the upregulation of SIRT1 expression or enzyme activity.CONCLUSIONS:Reduced SIRT1 expression in hippocampus of SNI mice may induce cognitive impairment associated with chronic pain by mediating the impaired synaptic plasticity.

The impact of anesthetic management on the prognosis of patients with cancer undergoing surgery is controversial. Circulating tumor cells (CTCs) play critical roles during cancer metastasis and can be released in large quantities during surgery. The ferroptosis of CTCs is related to metastasis. Whether anesthetics affect distant metastasis by increasing the survival of CTCs is unknown. To test this hypothesis, mice were inoculated with cancer cells via tail vein injection before treatment with propofol or sevoflurane for 2 h. After 2 weeks, more metastases were observed in the propofol group compared with the sevoflurane and vehicle groups. Then, we used the ferroptosis inhibitor ferrostatin-1 to explore the effect of ferroptosis on metastasis. Similar to propofol, pretreatment with ferrostatin-1 significantly increased CTC survival in mouse lungs at 24 h and the tumor burden at 10 weeks post-inoculation. Moreover, propofol protected cancer cells from RSL3-induced ferroptosis in vitro, as evidenced by decreases in intracellular levels of reactive oxygen species (ROS), lipid peroxide, and ferroptosis markers. Further studies showed that propofol treatment upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target genes, including HO-1, NQO1, and SLC7A11. Finally, the targeted knockdown of Nrf2 abolished the anti-ferroptosis effect of propofol. Collectively, we demonstrated the risk of a specific type of anesthetic, propofol, in promoting cancer cell metastasis through Nrf2-mediated ferroptosis inhibition. These findings may guide the choice of anesthetic for surgical removal of tumors.

Circular RNAs are highly stable single-stranded circular RNAs and enriched in the brain. Previous studies showed that circRNAs, as part of competing endogenous RNAs (ceRNAs) network, play an important role in neurodegenerative and psychiatric diseases. However, the mechanism of circRNA-related ceRNA networks in postoperative cognitive dysfunction (POCD) has not been elucidated yet. POCD usually occurs in elderly patients and is characterized by hippocampal dysfunction. Here, aged C57BL/6 mice were subjected to exploratory laparotomy under sevoflurane anesthesia, and this POCD model was verified by Morris water maze test. Whole-transcriptome sequencing was performed on the hippocampus of control group (Con) and surgery group. One hundred and seventy-seven DEcircRNAs, 221 DEmiRNAs and 2,052 DEmRNAs were identified between two groups. A ceRNA network was established with 92 DEcircRNAs having binding sites with 76 DEmiRNAs and 549 target DEmRNAs. In functional enrichment analysis, a pathological pattern of POCD was highlighted in the ceRNA network: Abnormal metabolic process in neural cells, including oxygen metabolism, could promote apoptosis and then affect the synaptic function, which may undermine the neural plasticity and eventually lead to changes in cognitive function and other behavioral patterns. In conclusion, this specific ceRNA network of circRNAs-miRNAs-mRNAs has provided novel insights into the regulatory mechanisms of POCD and revealed potential therapeutic gene targets.

The induction of anesthesia in children makes its safety one of the most important global health problems. Neuroinflammation contributes to anesthesia-induced neurotoxicity in young individuals. However, the mechanisms underlying anesthesia-induced neurotoxicity have not been established. In this study, the level of interleukin (IL)-6 in the hippocampus of mice and N2A cells treated with sevoflurane was increased, and long noncoding RNA (LncRNA) Riken was sufficient to decrease sevoflurane-induced neurotoxicity, and the level of inflammatory cytokine IL-6. The RNA pull-down assay verified that miR-101a was bound to lncRNA Riken in N2A cells. In addition, miR-101a blocked the protective effect of lncRNA Riken on anesthesia-induced neuroinflammation. These data suggest that lncRNA Riken attenuated anesthesia-induced neuroinflammation by interacting with microRNA-101a. Finally, we also demonstrated that MAPK phosphatase 1 (MKP-1) was a downstream target of miR-101a, and lncRNA Riken can regulate the expression of MKP-1; the JNK signal transduction pathway has been implicated in sevoflurane-induced IL-6 secretion. Our findings demonstrated that lncRNA Riken alleviated the sevoflurane-induced neurotoxic effects, and the lncRNA Riken/miR-101a/MKP-1/JNK axis plays an important role in the cognitive disorder.

BackgroundAseptic prosthetic loosening is one of the main factors causing poor prognosis of limb function after joint replacement and requires troublesome revisional surgery. It is featured by wear particle-induced periprosthetic osteolysis mediated by excessive osteoclasts activated in inflammatory cell context. Some natural compounds show antiosteoclast traits with high cost-efficiency and few side effects. Tussilagone (TUS), which is the main functional extract from Tussilago farfara generally used for relieving cough, asthma, and eliminating phlegm in traditional medicine has been proven to appease several RAW264.7-mediated inflammatory diseases via suppressing osteoclast-related signaling cascades. However, whether and how TUS can improve aseptic prosthetic loosening via modulating osteoclast-mediated bone resorption still needs to be answered.MethodsWe established a murine calvarial osteolysis model to detect the preventative effect of TUS on osteolysis in vivo. Micro-CT scanning and histomorphometric analysis were used to determine the variation of bone resorption and osteoclastogenesis. The anti–osteoclast-differentiation and anti–bone-resorption bioactivities of TUS in vitro were investigated using bone slice resorption pit evaluation, and interference caused by cytotoxicity of TUS was excluded according to the CCK-8 assay results. Quantitative polymerase chain reaction (qPCR) analysis was applied to prove the decreased expression of osteoclast-specific genes after TUS treatment. The inhibitory effect of TUS on NF-κB and p38 MAPK signaling pathways was testified by Western blot and NF-κB-linked luciferase reporter gene assay.ResultsTUS better protected bones against osteolysis in murine calvarial osteolysis model with reduced osteoclasts than those in the control group. In vitro studies also showed that TUS exerted antiosteoclastogenesis and anti–bone-resorption effects in both bone marrow macrophages (BMMs) and RAW264.7 cells, as evidenced by the decline of osteoclast-specific genes according to qPCR. Western blotting revealed that TUS treatment inhibited IκBα degradation and p38 phosphorylation.ConclusionsCollectively, our studies proved for the first time that TUS inhibits osteoclastogenesis by suppressing the NF-κB and p38 MAPK signaling pathways, therefore serving as a potential natural compound to treat periprosthetic osteolysis-induced aseptic prosthetic loosening.

BACKGROUNDAnesthesia in pregnant rodents causes neurotoxicity in fetal and offspring rodents. However, the underlying mechanisms and targeted treatments remain largely to be determined. Isoflurane and propofol are among commonly used anesthetics. Thus, we set out to investigate whether propofol can mitigate the isoflurane-induced neurotoxicity in mice.METHODSPregnant C57BL/6 mice at gestational day 15 (G15) were randomly assigned to 4 groups: control, isoflurane, propofol, and isoflurane plus propofol. Levels of interleukin (IL)-6 and poly-ADP ribose polymerase (PARP) fragment were measured in the brains of G15 embryos, and levels of postsynaptic density (PSD)-95 and synaptophysin were determined in the hippocampal tissues of postnatal day 31 (P31) offspring using Western blotting and immunohistochemical staining. Learning and memory functions in P31 offspring were determined using a Morris water maze test.RESULTSIsoflurane anesthesia in pregnant mice at G15 significantly increased brain IL-6 (222.6% ± 36.45% vs 100.5% ± 3.43%, P < .0001) and PARP fragment (384.2% ± 50.87% vs 99.59% ± 3.25%, P < .0001) levels in fetal mice and reduced brain PSD-95 (30.76% ± 2.03% vs 100.8% ± 2.25%, P < .0001) and synaptophysin levels in cornu ammonis (CA) 1 region (57.08% ± 4.90% vs 100.6% ± 2.20%, P < .0001) and dentate gyrus (DG; 56.47% ± 3.76% vs 99.76% ± 1.09%, P < .0001) in P31 offspring. Isoflurane anesthesia also impaired cognitive function in offspring at P31. Propofol significantly mitigated isoflurane-induced increases in brain IL-6 (117.5% ± 10.37% vs 222.6% ± 36.45%, P < .0001) and PARP fragment (205.1% ± 35.99% vs 384.2% ± 50.87%, P < .0001) levels in fetal mice, as well as reductions in PSD-95 (49.79% ± 3.43% vs 30.76% ± 2.03%, P < .0001) and synaptophysin levels in CA1 region (85.57% ± 2.97% vs 57.08% ± 4.90%, P < .0001) and DG (85.05% ± 1.87% vs 56.47% ± 3.76%, P < .0001) in hippocampus of P31 offspring. Finally, propofol attenuated isoflurane-induced cognitive impairment in offspring.CONCLUSIONSThese findings suggest that gestational isoflurane exposure in mice induces neuroinflammation and apoptosis in embryos and causes cognitive impairment in offspring. Propofol can attenuate these isoflurane-induced detrimental effects.

DNA methylation is an essential epigenetic mechanism involving in gene transcription modulation. An age-related increase in promoter methylation has been observed for neuronal activity and memory genes, and participates in neurological disorders. However, the position and precise mechanism of DNA methylation for memory gene modulation in anesthesia related cognitive impairment remained to be determined. Here, we studied the effects of sevoflurane anesthesia on the transcription of memory genes in the aged rat hippocampus. Then, we investigated changes in DNA methylation of involved genes and verified whether dysregulated DNA methylation would contribute to anesthesia induced cognitive impairment. The results indicated that sevoflurane anesthesia down-regulated the mRNA and protein levels of three memory genes, Arc, Bdnf, and Reln, which were accompanied with promoter hypermethylation and increased Dnmt1, Dnmt3a, and Mecp2 expression, and finally impaired hippocampus dependent memory. Furthermore, inhibition of DNA hypermethylation by 5-Aza rescued sevoflurane induced memory gene expression decrease and cognitive impairment. These findings provide an epigenetic understanding for the pathophysiology of cognitive impairment induced by general anesthesia in aged brain.

基于1960-2017年甘肃河西及其周边23个气象台站6-7月逐日气象资料和小麦干热风灾害等级国家气象行业标准,分析了河西地区干热风发生时段内气候资源变化特征及其对干热风的影响.研究表明:(1)气候资源变化特征上,干热风发生时段内气温明显升高,相对湿度先升高后降低;中部地区升温幅度和相对湿度变化最大,受气候变化影响最为敏感.(2)发生特征上,干热风发生日数先缓慢减少后迅速增多;2000年后干热风影响范围扩大,大面积干热风事件明显增多.(3)与各要素对应关系上,干热风日数的小波系数与气温和潜在蒸散量的小波系数多为正对应,与相对湿度多为负对应,此对应关系在21世纪后较为明显,日数与潜在蒸散量的对应关系最好.(4)发生条件上,日最高气温达到32 ℃时,河西西北部发生干热风的可能性最大,风险概率由北向南逐渐降低;14:00(北京时)相对湿度≤30％时,河西安敦盆地及鼎新、民勤发生干热风的可能性最大,风险概率由低海拔向高海拔站点逐渐降低.

Differentiated embryonic chondrocyte-expressed gene 1 (DEC1) is associated with various types of human cancer; however, there is limited data regarding the functions of DEC1 in osteosarcoma. The present study aimed to examine the expression of DEC1 in human osteosarcoma tissues and cell lines. Furthermore, the effects of DEC1 on the proliferation, adhesion, invasion and epithelial-mesenchymal transition (EMT) of osteosarcoma cells were investigated. Using reverse transcription-quantitative PCR and western blot analysis, it was found that the expression levels of DEC1 were higher in human osteosarcoma tissues and osteosarcoma cell lines than in the controls. Both gain- and loss-of-function experiments suggested that DEC1 promotes the proliferation, adhesion and invasion of osteosarcoma cells in vitro, as determined by MTT, cell adhesion and cell invasion assays, respectively. Additionally, DEC1 was found to upregulate the mesenchymal markers N-cadherin and vimentin, whilst downregulating the epithelial marker E-cadherin. In conclusion, this present study showed increased expression levels of DEC1 in human osteosarcoma tissues and cell lines, and identified that DEC1 may exert its effect on osteosarcoma progression by promoting cell proliferation, adhesion and invasion. Furthermore, DEC1 was shown to have an inducible effect on EMT in osteosarcoma cell lines, thus contributing to the aggressiveness of osteosarcoma cells. This initial study indicated that DEC1 may serve as a novel molecular target for the treatment of osteosarcoma.

Osteoblastic bone formation and osteoclastic bone resorption dynamically maintain the bone homeostasis; in the present study, we attempt to investigate the mechanism of the excessive activation of osteoclasts inducing the deregulation of bone homeostasis from the perspective of non-coding RNA regulation. Differentially expressed patterns of circRNAs were examined in non-treated and RANKL + CSF1-treated bone marrow monocyte/macrophage (BMM) cells and differentially-expressed miRNAs during osteoclast differentiation were analyzed and identified. We found that circRNA_28313 was significantly induced by RANKL + CSF1 treatment. circRNA_28313 knockdown significantly inhibited RANKL + CSF1-induced differentiation of osteoclasts within BMM cells in vitro, while suppressed ovariectomized (OVX)-induced bone resorption in mice in vivo. Via bioinformatics analyses, it has been demonstrated that miR-195a might bind to circRNA_28313 and CSF1 and together form a circRNA-miRNA-mRNA network. circRNA_28313 relieves miR-195a-mediated suppression on CSF1 via acting as a ceRNA, therefore modulating the osteoclast differentiation in BMM cells. In conclusion, circRNA_28313, miR-195a, and CSF1 form a ceRNA network to function in RANKL + CSF1-induced osteoclast differentiation, thus affecting OVX-induced bone absorption in mice.

Postmenopausal osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a global public health burden. Natural compounds are emerging as potential therapeutics for postmenopausal osteoporosis. In vitro osteoclastogenesis assay was conducted to investigate the effect of Glycyrrhizic acid (Gly) on osteoclast differentiation without cytotoxity. We applied bone resorption pit assessment and F-actin immunofluoresence to explore the effect of Gly on osteoclasts function in vitro. RT-qPCR was used to evaluate the expression level of RANKL-induced osteoclast-specific gene. Western blotting was conducted for analyzing potential mechanisms of inhibitory influence of Gly on the formation and function of osteoclasts in vitro. Ovariectomized (OVX) mice model, micro-CT and histomorphometry was used to survey the potential effect of Gly on the resorption of bone in vivo. Gly inhibited osteoclast differentiation and function without significant cytotoxicity at a dose of no more than 8 mM in vitro. Gly attenuated mRNA expression of osteoclast-specific genes including NFATc1, c-fos, TRAP, CTR, cathepsin K, and V-ATPase d2 in vitro. Gly inhibited degradation of IκBα, phosphorylation of ERK and JNK without disturbing phosphorylation of p38 after treating osteoclasts in vitro. In OVX mice, Gly attenuates osteoclast formation and preserves bone mass and trabecular structure. Gly can effectively inhibit osteoclast maturation and bone resorption by suppressing NF-κB, ERK, and JNK pathway in vitro and exhibits an osteoprotective effect in OVX mice.

Osteoporosis is a metabolic bone lesion in which the bone mass is reduced per unit volume due to increased bone resorption. Its main characteristics are bone pain and increasing danger of fragility fracture. Excessive osteoclast activation is known to be responsible for extensive bone resorption. Thus, inhibition of osteoclastic bone resorption and regulation of the bone microenvironment are vital treatment strategies for osteoporosis. For the first time, we investigated the effect of proanthocyanidins (PACs) extracted from grape seed, which significantly inhibited osteoclast formation and differentiation from bone marrow macrophages (BMMs) and the RAW264.7 cell line and efficiently attenuated osteoclastic bone resorption without toxicity. These findings were confirmed by changes in the NF-κB and JNK/mitogen-activated protein kinase (MAPK) signaling pathways, which are major and classical signaling pathways involved in RANKL-mediated osteoclastogenesis in vitro. The PACs inhibited osteoclast formation and differentiation by inhibiting the NF-κB and JNK signaling pathways and might be useful for the treatment of osteoporosis.