Microenvironment-responsive Cu-phenolic networks coated nanofibrous dressing with timely macrophage phenotype transition for chronic MRSA infected wound healing.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a pressing clinical issue that impedes wound healing. Pro-inflammatory M1 macrophages is required to clear bacteria and recruit various cell types during the initial phase of wound healing, but timing of this process is crucial. Herein, a microenvironment-responsive nanofibrous dressing capable of timely macrophage phenotype transition in vivo is constructed by coating copper ions (Cu2+)-polydopamine (PDA) networks on poly (ε-caprolactone) fiber (PCL-fiber) membrane. During the initial post-implantation period, the nanofibrous dressing show pH-sensitive Cu2+ release in the acidic infection microenvironment. The release Cu2+ have a direct killing effect on MRSA, and promote the proinflammatory M1 phenotype of macrophages to enhance the antibacterial macrophage response. Later, PDA to become a reactive oxygen species (ROS) scavenger when in microenvironments with elevated ROS levels, which conferred the dressing with an immunomodulatory activity that convert M1 macrophages into M2 macrophages. In vivo examination in an MRSA infected full-thickness skin wounds of rat model demonstrates that this dressing significantly facilitated infection eradication and wound healing through modulating local inflammatory phenotype. Overall, this study offers a simple and effective approach for timely manipulation of inflammation progression to promote infected wound healing.

Melatonin inhibits RANKL­induced osteoclastogenesis through the miR­882/Rev­erbα axis in Raw264.7 cells.

Melatonin, secreted in a typical diurnal rhythm pattern, has been reported to prevent osteoporosis; however, its role in osteoclastogenesis remains unclear. In the present study, the ability of melatonin to inhibit receptor activator of nuclear factor­κB ligand (RANKL)­induced osteoclastogenesis and the associated mechanism were investigated. Raw264.7 cells were cultured with RANKL (100 ng/ml) and macrophage colony­stimulating factor (M­CSF; 30 ng/ml) for 7 days, and tartrate­resistant acid phosphatase (TRAP) staining was used to detect osteoclastogenesis following treatment with melatonin. In addition, the effect of melatonin on cathepsin K and microRNA (miR)­882 expression was investigated via western blotting and reverse transcription­quantitative PCR. Melatonin significantly inhibited RANKL­induced osteoclastogenesis in Raw264.7 cells. From bioinformatics analysis, it was inferred that nuclear receptor subfamily 1 group D member 1 (NR1D1/Rev­erbα) may be a target of miR­882. In vitro, melatonin upregulated Rev­erbα expression and downregulated miR­882 expression in the osteoclastogenesis model. Rev­erbα overexpression boosted the anti­osteoclastogenesis effects of melatonin, whereas miR­882 partially diminished these effects. The present results indicated that the miR­882/Rev­erbα axis may serve a vital role in inhibiting osteoclastogenesis following RANKL and M­CSF treatment, indicating that Rev­erbα agonism or miR­882 inhibition may represent mechanisms through which melatonin prevents osteoporosis.

Metformin promotes cell proliferation and osteogenesis under high glucose condition by regulating the ROS­AKT­mTOR axis.

Metformin, a cost­effective and safe orally administered antidiabetic drug used by millions of patients, has exhibited great interest for its potential osteogenic­promoting properties in different types of cells, including mesenchymal stem cells (MSCs). Diabetic osteopathy is a common comorbidity of diabetes mellitus; however, the underlying molecular mechanisms of metformin on the physiological processes of MSCs, under high glucose condition, remain unknown. To determine the effects of metformin on the regulatory roles of proliferation and differentiation in MSCs, under high glucose conditions, osteogenesis after metformin treatment was detected with Alizarin Red S and ALP staining. The results demonstrated that high glucose levels significantly inhibited cell proliferation and osteogenic differentiation under high glucose conditions. Notably, addition of metformin reversed the inhibitory effects induced by high glucose levels on cell proliferation and osteogenesis. Furthermore, high glucose levels significantly decreased mitochondrial membrane potential (MMP), whereas treatment with metformin helped maintain MMP. Further analysis of mitochondrial function revealed that metformin significantly promoted ATP synthesis, mitochondrial DNA mass and mitochondrial transcriptional activity, which were inhibited by high glucose culture. Furthermore, metformin significantly scavenged reactive oxygen species (ROS) induced by high glucose levels, and regulated the ROS­AKT­mTOR axis inhibited by high glucose levels, suggesting the protective effects of metformin against high glucose levels via regulation of the ROS­AKT­mTOR axis. Taken together, the results of the present study demonstrated the protective role of metformin on the physiological processes of MSCs, under high glucose condition and highlighted the potential molecular mechanism underlying the effect of metformin in promoting cell proliferation and osteogenesis under high glucose condition.

Melatonin rescues glucocorticoid-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways.

AIMS: High-dose glucocorticoid (GC) administration causes osteoporosis. Many previous studies from our group and other groups have shown that melatonin participates in the regulation of osteoblast proliferation and differentiation, especially low concentrations of melatonin, which enhance osteoblast osteogenesis. However, the role of melatonin in glucocorticoid-induced osteoblast differentiation remains unknown. MATERIALS AND METHODS: An examination of the expression of osteoblast differentiation markers (ALP, OCN, COLL-1), as well as alkaline phosphatase staining and alkaline phosphatase enzymatic activity assay to measure osteoblast differentiation and quantifying Alizarin red S staining to measure mineralization, were performed to determine the effects of dexamethasone (Dex) and melatonin on the differentiation of MC3T3-E1 cells. We used immunofluorescence staining to detect the expression of Runx2 in melatonin-treated MC3T3-E1 cells. The expression of mRNA was determined by qRT-PCR, and protein levels were measured by western blotting. KEY FINDINGS: In the present study, we found that 100 µM Dex significantly reduced osteoblast differentiation and mineralization in MC3T3-E1 cells and that 1 µM melatonin attenuated these inhibitory effects. We found that only inhibition of PI3K/AKT (MK2206) and BMP/Smad (LDN193189) signalling abolished melatonin-induced differentiation and mineralization. Meanwhile, MK2206 decreased the expression of P-AKT and P-Smad1/5/9 and LDN193189 decreased the expression of P-Smad1/5/9 but had no obvious effect on P-AKT expression in melatonin-treated and Dex-induced MC3T3-E1 cells. SIGNIFICANCE: These findings suggest that melatonin rescues Dex-induced inhibition of osteoblast differentiation in MC3T3-E1 cells via the PI3K/AKT and BMP/Smad signalling pathways and that PI3K/AKT signalling may be the upstream signal of BMP/Smad signalling.

Relationship between autophagy, apoptosis and endoplasmic reticulum stress induced by melatonin in osteoblasts by septin7 expression.

Melatonin secreted by the pineal body is associated with the occurrence and development of idiopathic scoliosis. Melatonin has a concentration­dependent dual effect on osteoblast proliferation, in which higher concentrations can inhibit osteoblast proliferation and induce apoptosis; however, the underlying mechanism remains unclear. In the present study, flow cytometry was used to demonstrate that osteoblast cells treated with melatonin exhibited significantly increased early and late stage apoptotic rates as the concentration increased. Chromatin condensation in the nucleus and apoptotic body formation could be observed using fluorescent microscopy in osteoblast cells treated with 2 mM melatonin. Western blotting results showed that there was an upregulation in the expression of apoptosis marker proteins [poly (ADP­ribose) polymerase 1 (PARP­1)], endoplasmic reticulum stress [ERS; C/EBP homologous protein (CHOP) and glucose­regulated protein, 78 kDa (GRP78)] and autophagy [microtubule­associated protein 1 light chain 3ß (LC3)­I/LC3II]. PARP­1 expression was not altered when treated with ERS inhibitor 4PBA and autophagy inhibitor 3MA, whereas 4PBA or 3MA in combination with 2 mM melatonin (or the three together) significantly increased PARP­1 expression. Furthermore, the use of septin7 small interfering RNA confirmed that increased expression of GRP78 and CHOP was related to septin7, and melatonin­â€‹mediated ERS was necessary for septin7 activation. These findings suggest that ERS and autophagy might occur in the early stage of treatment with a high concentration of melatonin, and each might play a protective role in promoting survival; in a later stage, ERS and autophagy might interact and contribute to the induction of apoptosis. Overall, the results indicated that septin7 may be a target protein of melatonin­induced ERS.

Melatonin Inhibits Glucose-Induced Apoptosis in Osteoblastic Cell Line Through PERK-eIF2α-ATF4 Pathway.

Osteoporosis is a common disease resulting in deteriorated microarchitecture and decreased bone mass. In type 2 diabetes patients, the incidence of osteoporosis is significantly higher accompanied by increased apoptosis of osteoblasts. In this study, using the osteoblastic cell line MC3T3-E1, we show that high glucose reduces cell viability and induces apoptosis. Also, high glucose leads to endoplasmic reticulum (ER) stress (ERS) via an increase in calcium flux and upregulation of the ER chaperone binding immunoglobulin protein (BiP). Moreover, it induces post-translational activation of eukaryotic initiation factor 2 alpha (eIF2α) which functions downstream of PKR-like ER kinase (PERK). This subsequently leads to post-translational activation of the transcription factor 4 (ATF4) and upregulation of C/EBP-homologous protein (CHOP) which is an ER stress-induced regulator of apoptosis, as well as downstream effectors DNAJC3, HYOU1, and CALR. Interestingly, melatonin treatment significantly alleviates the high-glucose induced changes in cell growth, apoptosis, and calcium influx by inhibiting the PERK-eIF2α-ATF4-CHOP signaling pathway. Additionally, the MC3T3-E1 cells engineered to express a phosphodead eIF2α mutant did not show high glucose induced ER stress, confirming that melatonin protects osteoblasts against high-glucose induced changes by decreasing ER-stress induced apoptosis by impacting the PERK-eIF2α-ATF4-CHOP signaling pathway. The protective of melatonin against high glucose-induced ER stress and apoptosis was attenuated when the cells were pre-treated with a melatonin receptor antagonist, indicating that the effect of melatonin was mediated via the melatonin receptors in this context. These findings lay the provide mechanistic insights of melatonin's protective action on osteoblasts and will be potentially be useful in ongoing pre-clinical and clinical studies to evaluate melatonin as a therapeutic option for diabetic osteoporosis.