Pulsed electromagnetic fields regulate osteocyte apoptosis,RANKL/OPG expression,and its control of osteoclastogenesis depending on the presence of primary cilia

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).     

Licochalcone A attenuates abdominal aortic aneurysm induced by angiotensin II via regulating the miR-181b/SIRT1/HO-1 signaling

Licochalcone A (LA), a chalcone derived from liquorice, exhibits multiple biological activities, including anti-oxidation and anti-inflammation. This study aimed to investigate the role and underlying mechanism of LA in the abdominal aortic aneurysm (AAA). AAA model was established by continuous infusion of 1000 ng/kg/min of angiotensin II (AngII) in ApoE ^-/- mice for 4 weeks. At 7 days before AngII administration, 5 mg/kg/day or 10 mg/kg/day of LA was intraperitoneally administered to mice and continued for 4 weeks. The characteristics and quantification of AAAs were determined in situ. Real-time PCR or western blot was used to measure mRNA or protein levels of matrix metalloproteinase 2 and matrix metalloproteinase 9; pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6; apoptosis-related proteins Bax, Bcl-2, and active caspase-3; miR-181b; Sirtuin 1 (SIRT1); and heme oxygenase-1 (HO-1). Mouse-aorta-origin vascular smooth muscle (MOVAS) cells were used to confirm the involved pathways in vitro. We found LA administration dose-dependently reduced the incidence of AngII-induced AAA, aneurysm diameter enlargement, elastin degradation, matrix metalloproteinase production, pro-inflammatory cytokines and miR-181b expression, and vascular smooth muscle cell apoptosis. It elevated SIRT1 and HO-1 expression that was suppressed by AngII. AngII enhanced miR-181b but reduced SIRT1 and HO-1 expression in MOVAS cells. In AngII-stimulated MOVAS cells, downregulation of miR-181b significantly upregulated the expression of SIRT1 and HO-1, the effect of which was abrogated by SIRT1 siRNA. Collectively, LA could attenuate AngII-induced AAA by modulating the miR-181b/SIRT1/HO-1 signaling. LA might be a potential medical therapy for small AAA.     

A study on the protective effects of CpG oligodeoxynucleotide-induced mucosal immunity against lung injury in a mouse acute respiratory distress syndrome model

This study aims to determine the feasibility of using oligodeoxynucleotides with unmethylated cytosine-guanine dinucleotide sequences (CpG ODN) as an immunity protection strategy for a mouse model of acute respiratory distress syndrome (ARDS). This is a prospective laboratory animal investigation. Twenty-week-old BALB/c mice in Animal research laboratory were randomized into groups. An ARDS model was induced in mice using lipopolysaccharides (LPSs). CpG ODN was intranasally and transrectally immunized before or after the 3rd and 7th days of establishing the ARDS model. Mice were euthanized on Day 7 after the second immunization. Then, retroorbital bleeding was carried out and the chest was rapidly opened to collect the trachea and tissues from both lungs for testing. CpG ODN significantly improved the pathologic impairment in mice lung, especially after the intranasal administration of 50 μg. This resulted in the least severe lung tissue injury. Furthermore, interleukin-6 (IL-6) and IL-8 concentrations were lower, which was second to mice treated with the rectal administration of 20 µg CpG ODN. In contrast, the nasal and rectal administration of CpG ODN in BALB/c mice before LPS immunization did not appear to exhibit any significant protective effects. The intranasal administration of CpG ODN may be a potential treatment approach to ARDS. More studies are needed to further determine the protective mechanism of CpG ODN.     

Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment

Nucleotide oligomerization domain protein-1 (NOD1), a cytosolic pattern recognition receptor for the γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) is associated with the inflammatory diseases. Very little is known how bovine hepatocytes respond to specific ligands of NOD1 and sodium butyrate (SB). Therefore, the aim of our study was to investigate the role of bovine hepatocytes in NOD1-mediated inflammation during iE-DAP or LPS treatment or SB pretreatment. To achieve this aim, hepatocytes separated from cows at ∼160 days in milk (DIM) were divided into six groups: The nontreated control group (CON), the iE-DAP-treated group (DAP), the lipopolysaccharide-treated group (LPS), iE-DAP with SB group (DSB), LPS with SB group (LSB), and the SB group. Both iE-DAP and LPS highly increased the expression of both NOD1 and RIPK2, the two key factors for the immune response in hepatocytes. IκBα, NF-κB/p65, and MAP kinases (ERK, JNK, and p38) were activated through phosphorylation. The activation of NF-κB and MAPK pathway consequently increased the proinflammatory cytokines, IL-6, TNF-α, IL-8, and IFN-γ and the chemokines CCL5, CCL20, and CXCL-10. Both treatments improved iNOS/NOS2 expression. However, iE-DAP was failed to express acute phase protein SAA3, but HP and LPS HP but SAA3. These ligands also increased LRRK2, TAK1, TAB1, and β-defensins expression. The SB pretreatment at lower dose restored the function of hepatocytes by suppressing these increased molecules, as HDAC3 was inhibited. The activated NOD1 negatively regulated the expression of FOXA2. Altogether these data suggest an important role of bovine hepatocytes to promote immune responses via NOD1 expression during infection in the liver and a key role of SB to attenuate inflammation.     

MiR-455-5p ameliorates HG-induced apoptosis,oxidative stress and inflammatory via targeting SOCS3 in retinal pigment epithelial cells

Diabetic retinopathy (DR) remains the leading cause of blindness in adults with diabetes mellitus. Numerous microRNAs (miRNAs) have been identified to modulate the pathogenesis of DR. The main purpose of this study was to evaluate the potential roles of miR-455-5p in high glucose (HG)-treated retinal pigment epithelial (RPE) cells and underlying mechanisms. Our present investigation discovered that the expression of miR-455-5p was apparently downregulated in ARPE-19 cells stimulated with HG. In addition, forced expression of miR-455-5p markedly enhanced cell viability and restrained HG-induced apoptosis accompanied by decreased BCL2-associated X protein (Bax)/B-cell leukemia/lymphoma 2 (Bcl-2) ratio and expression of apoptotic marker cleaved caspase-3 during HG challenged. Subsequently, augmentation of miR-455-5p remarkably alleviated HG-triggered oxidative stress injury as reflected by decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) content as well as NADPH oxidase 4 expression, concomitant with enhanced the activities of superoxide dismutase, catalase, and GPX stimulated with HG. Furthermore, enforced expression of miR-455-5p effectively ameliorated HG-stimulated inflammatory response as exemplified by repressing the secretion of inflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumour necrosis factor-α in ARPE-19 cells challenged by HG. Most importantly, we successfully identified suppressor of cytokine signaling 3 (SOCS3) as a direct target gene of miR-455-5p, and miR-455-5p negatively regulated the expression of SOCS3. Mechanistically, restoration of SOCS3 abrogated the beneficial effects of miR-455-5p on apoptosis, accumulation of ROS, and inflammatory factors production in response to HG. Taken together, these findings demonstrated that miR-455-5p relieved HG-induced damage through repressing apoptosis, oxidant stress, and inflammatory response by targeting SOCS3. The study gives evidence that miR-455-5p may serve as a new potential therapeutic agent for DR treatment.     

KPC1 alleviates hypoxia/reoxygenation-induced apoptosis in rat cardiomyocyte cells though BAX degradation

Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia-reperfusion (I/R)-induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R-myocardium in vivo and in hypoxia and reoxygenation (H/R)-induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short-hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress-induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R-induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.     

Long noncoding RNA AC073284.4 suppresses epithelial–mesenchymal transition by sponging miR-18b-5p in paclitaxel-resistant breast cancer cells

Breast cancer (BC) is the most prevalent malignant cancer in the world, is the leading cause of cancer-related death female. Recently, there is accumulating evidence that long noncoding RNAs (lncRNAs) might as an important role in the progression of BC. (epithelial-mesenchymal transition (EMT) is considered to play a vital role in tumor cells migration and invasion. Nevertheless, the entire biological mechanisms and functions of lncRNAs in tumor migration, invasion, and EMT remain uncertain. In the present research, we observed that the expression of lncRNA AC073284.4 was downregulated in BC paclitaxel-resistant (PR) cells (MCF-7/PR) and tissues. Bioinformatics analysis predicted that miR-18b-5p was a direct target of AC073284.4, which has been validated by dual-luciferase reporter gene assay. We further proved that AC073284.4 could directly bind to miR-18b-5p and relieve the suppression for dedicator of cytokinesis protein 4 (DOCK4). Furthermore, the underlying functional experiments demonstrated that AC073284.4 might sponge miR-18b-5p to attenuate the invasion, metastasis, and EMT of BC cell through upregulating DOCK4 expression. In summary, AC073284.4 might serve as a competing endogenous RNA (ceRNA) in BC progression via modulating miR-18b-5p/DOCK4 axis, which weakens EMT and migration of BC. These results suggesting that AC073284.4 might function as a potential novel diagnostic biomarker in the progression of BC.     

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin β1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinβ1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKβ induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1β), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinβ1-siRNA or the JNK inhibitor. We also found that integrinβ1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation.