Supplementing Glucose Intake Reverses the Inflammation Induced by a High-Fat Diet by Increasing the Expression of Siglec-E Ligands on Erythrocytes.

Siglec-9/E is a cell surface receptor expressed on immune cells and can be activated by sialoglycan ligands to play an immunosuppressive role. Our previous study showed that increasing the expression of Siglec-9 (the human paralog of mouse Siglec-E) ligands maintains functionally quiescent immune cells in the bloodstream, but the biological effects of Siglec-9 ligand alteration on atherogenesis were not further explored. In the present study, we demonstrated that the atherosclerosis risk factor ox-LDL or a high-fat diet could decrease the expression of Siglec-9/E ligands on erythrocytes. Increased expression of Siglec-E ligands on erythrocytes caused by dietary supplementation with glucose (20% glucose) had anti-inflammatory effects, and the mechanism was associated with glucose intake. In high-fat diet-fed apoE-/- mice, glucose supplementation decreased the area of atherosclerotic lesions and peripheral inflammation. These data suggested that increased systemic inflammation is attenuated by increasing the expression of Siglec-9/E ligands on erythrocytes. Therefore, Siglec-9/E ligands might be valuable targets for atherosclerosis therapy.

Application of biomaterials in treating early osteonecrosis of the femoral head: Research progress and future perspectives.

Osteonecrosis of the femoral head (ONFH), a progressive pathological process of femoral head ischemia and osteocyte necrosis, is a refractory orthopedic disease caused by multiple etiologies and there is no complete cure at present. With the extension of ONFH duration, osteocyte apoptosis and trabecular bone loss can decrease the load-bearing capacity of the femoral head, which leads to the collapse of the articular cartilage and subchondral bone. Therefore, an urgent clinical need exists to develop effective treatment strategies of early-stage ONFH for maintaining the hip joint function and preventing femoral head collapse. In recent years, extensive attention has been paid to the application of diverse biomaterials in treating early ONFH for sustaining the normal morphology and function of the autologous femoral head, and slowing disease progression. Herein, we review the research progress of bone grafts, metallic materials, bioceramics, bioglasses and polymer materials for early ONFH treatment, and discuss the biological mechanisms of bone repair and regeneration in the femoral-head necrotic area. We propose suggestions for future research directions, from a special perspective of improving the local microenvironment in femoral head by facilitating vessel-associated osteoclasts (VAOs) generation and coupling of bone-specific angiogenesis and osteogenesis, as well as inhibiting bone-associated osteoclasts (BAOs) and BAO-mediated bone resorption. This review can provide ideas for the research, development, and clinical application of biomaterials for treating early ONFH. STATEMENT OF SIGNIFICANCE: We believe that at least three aspects of this manuscript make it interesting to readers of the Acta Biomaterialia. First, we briefly summarize the incidence, pathogenesis, risk factors, classification criteria and treatment of early osteonecrosis of the femoral head (ONFH). Second, we review the research progress in biomaterials for early ONFH treatment and the biological mechanisms of bone repair and regeneration in femoral-head necrotic area. Third, we propose future research progress on improving the local microenvironment in femoral head by facilitating vessel-associated osteoclasts generation and coupling of bone-specific angiogenesis and osteogenesis, as well as inhibiting bone-associated osteoclasts and bone resorption. We hope this review can provide ideas for the research, development, and clinical application of biomaterials for treating early ONFH.

Development of novel oridonin analogs as specifically targeted NLRP3 inflammasome inhibitors for the treatment of dextran sulfate sodium-induced colitis.

Abnormal activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is closely associated with a variety of inflammatory diseases. Herein, we describe the discovery and optimization of a series of NLRP3 inflammasome inhibitors based on the oridonin skeleton. These inhibitors exhibited moderate to potent inhibitory activity against interleukin 1ß (IL-1ß) release. Compound E6 showed the strongest inhibitory activity and better safety range against IL-1ß (IC50 = 0.45 ± 0.02 µM, selectivity index = 36.49). Compared with oridonin, the activity and selectivity index of compound E6 increased 11.5 and 7.2 times, respectively. Compound E6 also exhibited broad-spectrum activity and specificity. Compound E6 mainly reduced the release of IL-1ß by targeting the NLRP3 protein, thereby inhibiting the NLRP3-caspase 1-gasdermin D (GSDMD), as well as inhibiting the caspase 4-GSDMD signaling pathway. Further studies revealed an important therapeutic effect of E6 on dextran sulfate sodium-induced colitis. Compound E6 may be promising for the treatment of NLRP3-related diseases including inflammatory bowel disease.

LncRNA AK077216 promotes RANKL-induced osteoclastogenesis and bone resorption via NFATc1 by inhibition of NIP45.

Osteoclasts derived from the monocyte/macrophage hematopoietic lineage regulate bone resorption, a process balanced by bone formation in the continual renewal of the skeletal system. As dysfunctions of these cells result in bone metabolic diseases such as osteoporosis and osteopetrosis, the exploration of the mechanisms regulating their differentiation is a priority. A potential mechanism may involve long noncoding RNAs (lncRNAs), which are known to regulate various cell biology activities, including proliferation, differentiation, and apoptosis. The expression of the lncRNA AK077216 (Lnc-AK077216) is significantly upregulated during osteoclastogenesis identified by microarray and verified by qPCR. Up- and downregulation of Lnc-AK077216, respectively promotes and inhibits osteoclast differentiation, bone resorption, and the expression of related genes on the basis of tartrate-resistant acid phosphatase staining, qPCR, and western blot results. In addition, Lnc-AK077216 suppresses NIP45 expression and promotes the expression of NFATc1, an essential transcription factor during osteoclastogenesis. Besides, it was found that the expression of Lnc-AK077216 and Nfatc1 is upregulated, whereas Nip45 expression is downregulated in bone marrow and spleen tissues of ovariectomized mice. The results suggest that Lnc-AK077216 regulates NFATc1 expression and promotes osteoclast formation and function, providing a novel mechanism of osteoclastogenesis and a potential biomarker or a new drug target for osteoporosis.

Chemical Self-Assembly of Multifunctional Hydroxyapatite with a Coral-like Nanostructure for Osteoporotic Bone Reconstruction.

Bone defects/fractures are common in older people suffering from osteoporosis. Traditional hydroxyapatite (HA) materials for osteoporotic bone repair face many challenges, including limited bone formation and aseptic loosening of orthopedic implants. In this study, a new multifunctional HA is synthesized by spontaneous assembly of alendronate (AL) and Fe3O4 onto HA nanocrystals for osteoporotic bone regeneration. The chemical coordination of AL and Fe3O4 with HA does not induce lattice deformation, resulting in a functionalized HA (Func-HA) with proper magnetic property and controlled release manner. The Func-HA nanocrystals have been encapsulated in polymer substrates to further investigate their osteogenic capability. In vitro and in vivo evaluations reveal that both AL and Fe3O4, especially the combination of two functional groups on HA, can inhibit osteoclastic activity and promote osteoblast proliferation and differentiation, as well as enhance implant osseointegration and accelerate bone remodeling under osteoporotic condition. The as-developed Func-HA with coordinating antiresorptive ability, magnetic property, and osteoconductivity might be a desirable biomaterial for osteoporotic bone defect/fracture treatment.

Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway.

Endochondral ossification is crucial for bone formation in both adult bone repair process and embryo long-bone development. In endochondral ossification, bone marrow-derived mesenchymal stem cells (BMSCs) first differentiate to chondrocytes, then BMSC-derived chondrocytes endure a hypertrophic process to generate new bone. Endochondral ossification-based bone repair is a promising strategy to cure massive bone defect, which is a major clinical issue in orthopedics. However, challenges still remain for this novel strategy. One challenge is to ensure the sufficient hypertrophic differentiation. Another is to maintain the survival of the above hypertrophic chondrocytes under the hypoxic environment of massive bone defect. To solve this issue, mangiferin (MAG) was introduced to endochondral ossification-based bone repair. In this report, we proved MAG to be a novel autophagy inducer, which promoted BMSC-derived hypertrophic chondrocyte survival against hypoxia-induced injury through inducing autophagy. Furthermore, MAG enhances hypertrophic differentiation of BMSC-derived chondrocytes via upregulating key hypertrophic markers. Mechanistically, MAG induced autophagy in BMSC-derived chondrocytes by promoting AMPKα phosphorylation. Additionally, MAG balanced the expression of sex-determining region Y-box 9 and runt-related transcription factor 2 to facilitate hypertrophic differentiation. These results indicated that MAG was a potential drug to improve the efficacy of endochondral ossification-based bone repair in massive bone defects.-Bai, Y., Liu, C., Fu, L., Gong, X., Dou, C., Cao, Z., Quan, H., Li, J., Kang, F., Dai, J., Zhao, C., Dong, S. Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway.

LncRNA-AK131850 Sponges MiR-93-5p in Newborn and Mature Osteoclasts to Enhance the Secretion of Vascular Endothelial Growth Factor a Promoting Vasculogenesis of Endothelial Progenitor Cells.

BACKGROUND/AIMS: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. METHODS: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs), respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH) and luciferase reporter assay were carried out to dissect molecular mechanisms. RESULTS: In this study, we found that newborn OCs (N-OC) and mature OCs (M-OC) during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs). Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa), were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. CONCLUSION: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in N-OC and M-OC during osteoclastogenesis to enhance the secretion of VEGFa, thus promoting vasculogenesis of EPCs.

Involvement of microRNA-135a-5p in the Protective Effects of Hydrogen Sulfide Against Parkinson's Disease.

BACKGROUND/AIMS: Development of effective therapeutic drugs for Parkinson's disease is in great need. During the progression of Parkinson's disease, Rho-associated protein kinase 2 (ROCK2) is activated to promote neurodegeneration. Hydrogen sulfide (H2S) has a neuroprotective effect during the neural injury of Parkinson's disease. However, the mechanisms that underlie the effects of ROCK2 and H2S remain ill-defined. In the current study, we addressed these questions. METHODS: We used a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse subacute model of Parkinson's disease to study the effects of H2S on astrocytic activation in the mouse striatum, on the levels of tyrosine-hydroxylase (TH)-positive neuron loss, on the apomorphine-induced rotational behavior of the mice, and on the changes in ROCK2 and miR-135a-5p expression. Plasmid transfection was applied to modify miR-135a-5p levels in a neuronal cell line HCN-1A. Bioinformatics analysis was performed to predict the relationship between ROCK2 and miR-135a-5p in neuronal cells, and then was confirmed by luciferase reporter assay. RESULTS: H2S alleviated MPTP-induced astrocytic activation in the mouse striatum, alleviated the increases in TH-positive neuron loss, and improved the apomorphine-induced rotational behavior of the mice. H2S significantly attenuated the increases in ROCK2 and the decreases in miR-135a-5p by MPTP. MiR-135a-5p targeted the 3'-UTR of ROCK2 mRNA to inhibit its translation in neuronal cells. CONCLUSION: MiR-135a-5p-regulated ROCK2 may play a role in the protective effects of hydrogen sulfide against Parkinson's disease.

HDAC2 regulates FoxO1 during RANKL-induced osteoclastogenesis.

The bone-resorbing osteoclast (OC) is essential for bone homeostasis, yet deregulation of OCs contributes to diseases such as osteoporosis, osteopetrosis, and rheumatoid arthritis. Here we show that histone deacetylase 2 (HDAC2) is a key positive regulator during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Bone marrow macrophages (BMMs) showed increased HDAC2 expression during osteoclastogenesis. HDAC2 overexpression enhanced, whereas HDAC2 deletion suppressed osteoclastogenesis and bone resorption using lentivirus infection. Mechanistically, upon RANKL activation, HDAC2 activated Akt; Akt directly phosphorylates and abrogates Forkhead box protein O1 (FoxO1), which is a negative regulator during osteoclastogenesis through reducing reactive oxygen species. HDAC2 deletion in BMMs resulted in decreased Akt activation and increased FoxO1 activity during osteoclastogenesis. In conclusion, HDAC2 activates Akt thus suppresses FoxO1 transcription results in enhanced osteoclastogenesis. Our data imply the potential value of HDAC2 as a new target in regulating osteoclast differentiation and function.

Changing expression profiles of lncRNAs, mRNAs, circRNAs and miRNAs during osteoclastogenesis.

Bone is a dynamic organ continuously undergoing shaping, repairing and remodeling. The homeostasis of bone is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts (OCs) are specialized multinucleated cells derived from hematopoietic stem cells (HSCs) or monocytes/macrophage progenitor cells. There are different stages during osteoclastogenesis, and one of the most important steps to form functional osteoclasts is realized by cell-cell fusion. In our study, microarray was performed to detect the expression profiles of lncRNA, mRNA, circRNA and miRNA at different stages during osteoclastogenesis of RAW264.7 cells. Often changed RNAs were selected and clustered among the four groups with Venn analysis. The results revealed that expressions of 518 lncRNAs, 207 mRNAs, 24 circRNAs and 37 miRNAs were often altered at each stage during OC differentiation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on the correlation analysis between the differentially expressed RNAs. The present study provided a systematic perspective on the potential function of non-coding RNAs (ncRNAs) during osteoclastogenesis.

4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2.

Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl-) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs.

MiR-183 promotes growth of non-small cell lung cancer cells through FoxO1 inhibition.

Non-small cell lung cancer (NSCLC) is a prevalent cancer in lung of high incidence. NSCLCs often appear to be fast growing, which renders comprehension of the mechanisms underlying the growth of NSCLC extremely critical. Previous study has addressed a role of microRNA (miR) family member, miR-183, in the regulation of the invasiveness of NSCLC, whereas the role of miR-183 in the growth control of NSCLC is not clear. Here, we analyzed the regulation of FoxO1 by miR-183 in vitro using luciferase-reporter assay. We also analyzed the effects of miR-183 on NSCLC cell growth in vitro using a microculture tetrazolium (MTT) assay and in vivo by visualizing tumor growth using bioluminescence assay. We found that overexpression of miR-183 in NSCLC cells decreased FoxO1 protein levels, whereas inhibition of miR-183 increased FoxO1 protein levels without affecting FoxO1 transcripts. Moreover, miR-183 bound to FoxO1 mRNA to prevent its translation through its 3'untranslated region (UTR). Furthermore, administration of miR-183 suppressed FoxO1 levels in NSCLC, resulting in a significant increase in NSCLC growth in vitro and in vivo, while administration of antisense of miR-183 significantly increased FoxO1 levels in NSCLC resulting in a significant decrease in NSCLC growth. Taken together, our data demonstrate that miR-183/FoxO1 axis may be a novel therapeutic target for regulating the growth of NSCLC.

Busulfan inhibits growth of human osteosarcoma through miR-200 family microRNAs in vitro and in vivo.

Osteosarcoma typically arises in tissues of mesenchymal origin, and is the most malignant bone tumor characterized by high local aggressiveness, with poor therapeutic outcome. Busulfan has been widely used to treat CML. So far, there are no reports on the therapeutic effect of busulfan on osteosarcoma. Here, we showed that busulfan dose-dependently reduced the cell viability and proliferation, and induced cell apoptosis, senescence, and reactive oxygen species levels in two osteosarcoma cell lines. Moreover, a series of loss-of-function and gain-of-function experiments further indicated that busulfan may have its anti-osteosarcoma effect by upregulating the microRNA-200 (miR-200) family which subsequently downregulated its target genes ZEB1 and ZEB2. Furthermore, treatment with busulfan potentially inhibited the growth of implanted osteosarcoma in nude mice. Taken together, our data suggest that busulfan may have an anti-osteosarcoma effect through downregulating ZEB1 and ZEB2 through activating the miR-200 family, highlighting a possibility of using busulfan as a novel therapy for osteosarcoma.

Vinyl chloride monomer (VCM) induces high occurrence of neural tube defects in embryonic mouse brain during neurulation.

The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.

Overexpression of CDK5 in neural stem cells facilitates maturation of embryonic neurocytes derived from rats in vitro.

To further understand the effects of cyclin-dependent kinase 5 (CDK5) on the differentiation of neural stem cells, which were cultured and transfected with CDK5-EGFP recombinant overexpression vector (OV-CDK5 Group), successful transfection was confirmed by RT-PCR and Western blot. Our results showed that the CDK5 mRNA expression significantly increased in 6 h after transfection. Increase in the levels of the CDK5 protein expression was observed in 72 h, compared with Empty Vector Control Group (EV-CTL Group) (P < 0.01). Furthermore, in OV-CDK5 Group, the percentage of S-phase cells was significantly higher than in EV-CTL Group (P < 0.01). Differentiated cells were showed with short processes in 24 h and with obviously enlarged cell body, and extended cellular processes in 72 h, in comparison to those in EV-CTL Group (P < 0.01). In 72 h under treatment with 10 µmol/L all-trans retinoic acid (ATRA), in OV-CDK5 Group, processes of the GFP-positive cells were reduced slightly and little GFP-positive debris was found. However, in the EV-CTL Group, processes of the GFP-positive cells were obviously shortened and deformed and much GFP-positive debris were found. Moreover, the percentage of G0/G1-phase cells was lesser, while the percentage of S-phase cells was higher than that in EV-CTL Group (P < 0.01, P < 0.01). In conclusion, our experiment suggested that CDK5 might promote proliferation and differentiation of neural stem cells, lengthen the processes of differentiated neurocytes, and accelerate morphological maturation of such cells. Furthermore, CDK5 might antagonize ATRA-induced inhibition against proliferation and differentiation in differentiated neurocytes.

IGF1(CA)19 and IGFBP-3-202A/C gene polymorphism and cancer risk: a meta-analysis.

Insulin-like growth factor 1 (IGF1)(CA)19 and insulin-like growth factor-binding protein-3 (IGFBP-3)-202A/C gene polymorphisms had been focused by many epidemiological studies recently, which were associated with common cancer risk including colorectal, breast, prostate, and lung cancer. However, the findings of epidemiological investigations are not coincident. We did a systematic review and meta-analysis of case-control studies, including studies nested in cohorts, of the association between IGF1(CA)19 and IGFBP-3-202A/C gene polymorphism and prostate, colorectal, premenopausal and postmenopausal breast cancer. We identified 17 eligible studies (24 datasets), which included 9,744 cases and 11,332 controls. The result displays that individuals carrying (CA)19 allele had a subtly decreased risk of all cancer sites [OR(95% CI) 0.92(0.87,0.97); 0.882(0.809,0.962); 0.902(0.849,0.958)] and postmenopausal breast cancer [OR(95% CI) 0.893(0.832,0.959); 0.834(0.719,0.968); 0.862(0.776,0.958)] in allele contrast model, CA19/CA19 vs. non-CA19/non-CA19 model, and recessive genetic model. In subgroup analysis according to ethnicities, (CA)19 repeat polymorphism had an increased risk of common cancers in Asian [OR (95% CI) of allele contrast model: 1.105(1.000,1.224); additive model: 1.103(0.844,1.441), 1.197(1.013,1.413); recessive model: 1.039(0.831,1.300); and dominant model: 1.191(1.030,1.376)]. On the other hand, IGFBP-3-202A/C gene polymorphism did not seem to be associated with all the cancer sites in any genetic model and ethnicity. In conclusion, the result of this meta-analysis indicates that the IGF1(CA)19 polymorphism is a candidate gene polymorphism for cancer susceptibility regardless of environmental factors, especially in Asian.