Neuroligin-3 activates Akt-dependent Nrf2 cascade to protect osteoblasts from oxidative stress.

Excessive oxidative stress will cause significant injury to osteoblasts, serving as one major pathological mechanism of osteoporosis. Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein and is expressed in the bone. We here explored its potential activity against hydrogen peroxide (H2O2)-induced oxidative injury in cultured osteoblasts. In primary murine and human osteoblasts, NLGN3 stimulation dose-dependently induced Akt, Erk1/2 and S6K activation. NLGN3 pretreatment ameliorated H2O2-induced cytotoxicity and death in osteoblasts. Moreover, H2O2-induced reactive oxygen species (ROS) production and oxidative injury were alleviated with NLGN3 pretreatment in cultured osteoblasts. Further studies showed that NLGN3 activated Nrf2 signaling cascade and induced Nrf2 protein Serine-40 phosphorylation, Keap1-Nrf2 dissociation, Nrf2 protein stabilization and nuclear translocation in osteoblasts. NLGN3 also increased antioxidant response element (ARE) activity and induced expression of Nrf2-ARE-dependent genes (HO1, GCLC and NQO1) in osteoblasts. Moreover NLGN3 mitigated osteoblast oxidative injury by dexamethasone or sodium fluoride (NaF). Nrf2 cascade activation is essential for NLGN3-induced cytoprotective activity in osteoblasts. Nrf2 shRNA or knockout (KO) abolished NLGN3-induced osteoblast cytoprotection against H2O2. Contrarily forced Nrf2 cascade activation by Keap1 KO mimicked NLGN3-induced anti-oxidative activity in murine osteoblasts. Importantly, NLGN3-induced Serine-40 phosphorylation and Nrf2 cascade activation were blocked by an Akt inhibitor MK-2206 or by Akt1 shRNA. Importantly, Akt inhibition, Akt1 silencing or Nrf2 S40T mutation largely inhibited NLGN3-induced osteoblast cytoprotection against H2O2. At last, we showed that NLGN3 mRNA and protein expression was significantly downregulated in necrotic bone tissues of dexamethasone-taken patients. Taken together, NLGN3 activated Akt-dependent Nrf2 cascade to protect osteoblasts from oxidative stress.

Identification and validation of key long non-coding RNAs in resveratrol protect against IL-1ß-treated chondrocytes via integrated bioinformatic analysis.

BACKGROUND: Long non-coding RNAs (lncRNAs) participate in regulation of gene transcription, but little is known about the correlation among resveratrol and lncRNAs. This study aimed to identify and validate the key lncRNAs in resveratrol protect against IL-1ß-treated chondrocytes. METHODS: In this experiment, high-throughput sequencing technique was performed to identify the differentially expressed lncRNAs, miRNAs, and mRNAs between IL-1ß-treated chondrocytes with or not resveratrol. Moreover, gene ontology and KEGG pathway of the differentially expressed genes were carried out by R software. Then, lncRNA-miRNA-mRNA network was constructed by Cytoscape software. Venn diagram was performed to identify the potentially target miRNAs of LINC00654. Then, real-time polymerase chain reaction (RT-PCR) was performed to validate the most significantly differentially expressed lncRNAs. RESULTS: Totally, 1016 differentially expressed lncRNAs were identified (493 downregulated) between control and resveratrol-treated chondrocytes. Totally, 75 differentially expressed miRNAs were identified (downregulated = 54, upregulated = 21). Totally, 3308 differentially expressed miRNAs were identified (downregulated = 1715, upregulated = 1593). GO (up) were as follows: skin development, response to organophosphorus. GO (down) mainly included visual perception, single fertilization, and sensory perception of smell. KEGG (up) were as follows: TNF signaling pathway and TGF-beta signaling pathway. KEGG (down) were as follows: viral protein interaction with cytokine and cytokine receptor. We identified that LINC00654 and OGFRL1 were upregulated in resveratrol-treated chondrocytes. However, miR-210-5p was downregulated in resveratrol-treated chondrocytes. CONCLUSION: In sum, the present study for the first time detected the differential expressed lncRNAs involved in resveratrol-treated chondrocytes via employing bioinformatic methods.

Knockdown of long non-coding RNA LEF1-AS1 attenuates apoptosis and inflammatory injury of microglia cells following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is associated with health burden both at personal and societal levels. Recent assessments on the role of lncRNAs in SCI regulation have matured. Therefore, to comprehensively explore the function of lncRNA LEF1-AS1 in SCI, there is an urgent need to understand its occurrence and development. METHODS: Using in vitro experiments, we used lipopolysaccharide (LPS) to treat and establish the SCI model primarily on microglial cells. Gain- and loss of function assays of LEF1-AS1 and miR-222-5p were conducted. Cell viability and apoptosis of microglial cells were assessed via CCK8 assay and flow cytometry, respectively. Adult Sprague-Dawley (SD) rats were randomly divided into four groups: Control, SCI, sh-NC, and sh-LEF-AS1 groups. ELISA test was used to determine the expression of TNF-α and IL-6, whereas the protein level of apoptotic-related markers (Bcl-2, Bax, and cleaved caspase-3) was assessed using Western blot technique. RESULTS: We revealed that LncRNA LEF1-AS1 was distinctly upregulated, whereas miR-222-5p was significantly downregulated in LPS-treated SCI and microglial cells. However, LEF1-AS1 knockdown enhanced cell viability, inhibited apoptosis, as well as inflammation of LPS-mediated microglial cells. On the contrary, miR-222-5p upregulation decreased cell viability, promoted apoptosis, and inflammation of microglial cells. Mechanistically, LEF1-AS1 served as a competitive endogenous RNA (ceRNA) by sponging miR-222-5p, targeting RAMP3. RAMP3 overexpression attenuated LEF1-AS1-mediated protective effects on LPS-mediated microglial cells from apoptosis and inflammation. CONCLUSION: In summary, these findings ascertain that knockdown of LEF1-AS1 impedes SCI progression via the miR-222-5p/RAMP3 axis.

Resveratrol alleviates the interleukin-1ß-induced chondrocytes injury through the NF-κB signaling pathway.

BACKGROUND: Osteoarthritis (OA) is a regular age-related disease that affects millions of people. Resveratrol (RSV) is a flavonoid with a stilbene structure with different pharmacological effects. The purpose of the experiment was to evaluate the protective role of RSV against the human OA chondrocyte injury induced by interleukin-1ß (IL-1ß). METHODS: Chondrocytes were isolated from OA patients and identified by type II collagen, safranin O staining, and toluidine blue staining. Differentially expressed genes in chondrocytes treated RSV were identified by RNA sequencing. Kyoto encyclopedia of genes and genomes (KEGG) pathway as well as gene ontology (GO) were further conducted through Metascape online tool. A cell counting kit-8 (CCK-8) assay was applied to discover the viability of chondrocytes (6, 12, 24, and 48 µM). Many genes associated with inflammation and matrix degradation are evaluated by real-time PCR (RT-PCR) as well as western blot (WB). The mechanism of RSV for protecting IL-1ß induced chondrocytes injury was further measured through immunofluorescence and WB assays. RESULTS: A total of 845 differentially expressed genes (upregulated = 499, downregulated = 346) were found. These differentially expressed genes mainly enriched into negative regulation of catabolic process, autophagy, and cellular catabolic process, intrinsic apoptotic, apoptotic, and regulation of apoptotic signaling pathway, cellular response to abiotic stimulus, external stimuli, stress, and radiation. These differentially expressed genes were obviously enriched in NF-kB signaling pathway. RSV at the concentration of 48 µM markedly weakened the viability of the cells after 24 h of treatment (87% vs 100%, P < 0.05). No obvious difference was observed between the 6, 12, and 24 µM groups (106% vs 100%, 104% vs 100%, 103% vs 100%, P > 0.05). RSV (24 µM) also markedly depressed the levels of PGE2 and NO induced by IL-1ß by 25% and 29% respectively (P < 0.05). Our experiment pointed out that RSV could dramatically inhibit the inflammatory response induced by IL-1ß, including the MMP-13, MMP-3, and MMP-1 in human OA chondrocytes by 50%, 35%, and 33% respectively. On the other hand, RSV inhibited cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and inducible nitric oxide synthase (iNOs) expression (P < 0.05), while increased collagen-II and aggrecan levels (P < 0.05). From a mechanistic perspective, RSV inhibited the degradation of IκB-α as well as the activation of nuclear factor-kappa B (NF-κB) induced by IL-1ß. CONCLUSION: In summary, RSV regulates the signaling pathway of NF-κB, thus inhibiting inflammation and matrix degradation in chondrocytes. More studies should be focused on the treatment efficacy of RSV for OA in vivo.

MiR-708 inhibits MC3T3-E1 cells against H2O2-induced apoptosis through targeting PTEN.

BACKGROUND: The dysregulation of proliferation and apoptosis plays a significant role in the pathogenesis of postmenopausal osteoporosis (PO). MicroRNAs play an important role in regulating apoptosis of MC3T3-E1 cells. However, the role and potential mechanism of miR-708 for regulating H2O2-induced apoptosis is unknown. This study aimed to investigate the protective function of miR-708 in H2O2-induced apoptosis of MC3T3-E1 osteoblasts. METHODS: MC3T3-E1 was co-cultured with H2O2 for 8 h, then, flow cytometry, malondialdehyde (MDA), and glutathione peroxidase (Gpx) levels were measured to establish the oxidative model. MiRNA microarray was performed to assess differentially expressed miRNAs between control and H2O2-treated MC3T3-E1 cells. We then performed RT-PCR to identify the relative expression of miR-708 and PTEN. After transfected MC3T3-E1 with miR-708 mimics, flow cytometry, MDA, and Gpx level were performed to identify the apoptosis rate and oxidative stress in these groups. Furthermore, we small interfering RNA of PTEN to identify the role of PTEN in H2O2-induced apoptosis of MC3T3-E1 cells. RESULTS: H2O2 (100 nM) could significantly induce the apoptosis of MC3T3-E1 cells. Moreover, H2O2 could significantly increase the MDA level and downregulated Gpx level. RT-PCR found that H2O2 significantly decrease the level of miR-708. Compared with H2O2 group, H2O2 + miR-708 mimic significantly decreased the apoptosis rate. CONCLUSIONS: miR-708 plays a protective role in H2O2-induced MC3T3-E1 osteoblasts apoptosis and its protective effect is proceeded by regulating ROS level and PTEN expression level.

microRNA-7 inhibition protects human osteoblasts from dexamethasone via activation of epidermal growth factor receptor signaling.

Sustained dexamethasone (Dex) treatment could induce secondary osteoporosis, osteonecrosis, or even bone fractures. Dex can induce potent cytotoxicity in cultured human osteoblasts. The aim of this study was to test the potential role of microRNA-7 (miR-7), which targets the epidermal growth factor receptor (EGFR), in Dex-treated human osteoblasts. In OB-6, hFOB1.19, and primary human osteoblasts, miR-7 depletion by a lentiviral antagomiR-7 construct (LV-antagomiR-7) increased EGFR expression and downstream Akt activation, protecting cells from Dex-induced viability reduction, cell death, and apoptosis. In contrast, forced overexpression of miR-7 by a lentiviral miR-7 construct (LV-miR-7) inhibited EGFR expression and Akt activation, potentiating Dex-induced cytotoxicity in OB-6, hFOB1.19, and primary human osteoblasts. EGFR is the primary target of miR-7 in human osteoblasts. Luciferase activity of the EGFR 3-untranslated region was enhanced by LV-antagomiR-7, but decreased by LV-miR-7 in OB-6 cells. Further, LV-antagomiR-7-induced osteoblast cytoprotection against Dex was abolished by the EGFR inhibitors AG1478 and PD153035. Moreover, neither LV-antagomiR-7 nor LV-miR-7 was functional in EGFR-KO OB-6 cells. We also show that miR-7 is upregulated in the necrotic femoral head tissues of Dex-administered patients, correlating with EGFR downregulation. Together, we conclude that miR-7 inhibition protects human osteoblasts from Dex via activation of EGFR signaling.