Silent Information Regulator Type Research Articles

Melatonin ameliorates retinal ganglion cell senescence and apoptosis in a SIRT1-dependent manner in an optic nerve injury model

Melatonin is involved in exerting protective effects in aged-related and neurodegenerative diseases through a silent information regulator type 1 (SIRT1)-dependent pathway. However, little was known about the impact of melatonin on retinal ganglion cell (RGC) senescence and apoptosis following optic nerve crush (ONC). Thus, this study aimed to examine the effects of melatonin on RGC senescence and apoptosis after ONC and investigate the involvement of SIRT1 in this process. To study this, an ONC model was established. EX-527, an inhibitor of SIRT1, was injected intraperitoneally into mice. And melatonin was administrated abdominally into mice after ONC every day. Hematoxylin & eosin staining, retina flat-mounts and optical coherence tomography were used to evaluate the loss of retina cells/neurons. Pattern electroretinogram (p-ERG) was performed to evaluate the function of RGCs. Immunofluorescence and western blot were used to evaluate protein expression. SA-β-gal staining was employed to detect senescent cells. The results demonstrated that melatonin partially rescued the expression of SIRT1 in RGC 3 days after ONC. Additionally, melatonin administration partly rescued the decreased RGC number and ganglion cell complex thickness observed 14 days after ONC. Melatonin also suppressed ONC-induced senescence and apoptosis index. Furthermore, p-ERG showed that melatonin improved the amplitude of P50, N95 and N95/P50 following ONC. Importantly, the protective effects of melatonin were reversed when EX-527 was administered. In summary, this study revealed that melatonin attenuated RGC senescence and apoptosis through a SIRT1-dependent pathway after ONC. These findings provide valuable insights for the treatment of RGC senescence and apoptosis.

Niacin treatment prevents bone loss in iron overload osteoporotic rats via activation of SIRT1 signaling pathway

Recently, more and more studies have revealed that iron overload can lead to osteoporosis by inducing oxidative stress. Niacin (NAN), also known as nicotinate or vitamin B3, has been confirmed to possess potent antioxidative effects. In addition, very little is currently known about the protective effects of NAN on iron overload in osteoporotic bone tissue. Therefore, we aimed to evaluate the protective effect of niacin on iron overload-induced bone injury and to investigate the effect and underlying mechanisms of the niacin and iron overload on intracellular antioxidant properties. When MC3T3-E1 and RAW264.7 cells were cultured in the presence of ammonium ferric citrate(FAC), NAN therapy could increase the matrix mineralization and promote expression of osteogenic markers in MC3T3-E1, inhibit osteoclastic differentiation of RAW264.7 cells, while increasing intracellular reactive oxygen species (ROS) levels and strengthening mitochondrial membrane potential (MMP). In the ovariectomized (OVX) rat model, NAN had an obvious protective effect against iron-overloaded injury. Meanwhile, superoxide dismutase 2 (SOD2), intracellular antioxidant enzymes and silent information regulator type 1 (SIRT1), were up-regulated in response to NAN exposures in MC3T3-E1. Furthermore, SIRT1 inhibitor EX527 attenuated the protective effects of NAN. Results revealed that NAN could stimulate osteogenic differentiation, inhibit osteoclastic differentiation and markedly increased antioxidant properties in cells through the induction of SIRT1. Studies suggest that niacin is a promising agent for preventing bone loss in iron overload conditions.

Melatonin rescues the mitochondrial function of bone marrow-derived mesenchymal stem cells and improves the repair of osteoporotic bone defect in ovariectomized rats.

Osteoporotic bone defects, a severe complication of osteoporosis, are distinguished by a delayed bone healing process and poor repair quality. While bone marrow-derived mesenchymal stem cells (BMMSCs) are the primary origin of bone-forming osteoblasts, their mitochondrial function is impaired, leading to inadequate bone regeneration in osteoporotic patients. Melatonin is well-known for its antioxidant properties and regulation on bone metabolism. The present study postulated that melatonin has the potential to enhance the repair of osteoporotic bone defects by restoring the mitochondrial function of BMMSCs. In vitro administration of melatonin at varying concentrations (0.01, 1, and 100 μM) demonstrated a significant dose-dependent improvement in the mitochondrial function of BMMSCs obtained from ovariectomized rats (OVX-BMMSCs), as indicated by an elevation in mitochondrial membrane potential, adenosine triphosphate synthesisand expression of mitochondrial respiratory chain factors. Melatonin reduced the level of mitochondrial superoxide by activating the silent information regulator type 1 (SIRT1) and its downstream antioxidant enzymes, particularly superoxide dismutase 2 (SOD2). The protective effects of melatonin were found to be nullified upon silencing of Sirt1 or Sod2, underscoring the crucial role of the SIRT1-SOD2 axis in the melatonin-induced enhancement of mitochondrial energy metabolism in OVX-BMMSCs. To achieve a sustained and localized release of melatonin, silk fibroin scaffolds loaded with melatonin (SF@MT) were fabricated. The study involved the surgical creation of bilateral femur defects in OVX rats, followed by the implantation of SF@MT scaffolds. The results indicated that the application of melatonin partially restored the mitochondrial energy metabolism and osteogenic differentiation of OVX-BMMSCs by reinstating mitochondrial redox homeostasis. These findings suggest that the localized administration of melatonin through bone implants holds potential as a therapeutic approach for addressing osteoporotic bone defects.

Impact of protocatechuic acid on alleviation of pulmonary damage induced by cyclophosphamide targeting peroxisome proliferator activator receptor, silent information regulator type-1, and fork head box protein in rats

Cyclophosphamide (CP) is a chemotherapeutic agent that causes pulmonary damage by generating free radicals and pro-inflammatory cytokines. Pulmonary damage has a high mortality rate due to the severe inflammation and edema occurred in lung. PPARγ/Sirt 1 signaling has been shown to be cytoprotective effect against cellular inflammatory stress and oxidative injury. Protocatechuic acid (PCA) is a potent Sirt1 activator and exhibits antioxidant as well as anti-inflammatory properties. The current study aims to investigate the therapeutic impacts of PCA against CP-induced pulmonary damage in rats. Rats were assigned randomly into 4 experimental groups. The control group was injected with a single i.p injection of saline. CP group was injected with a single i.p injection of CP (200 mg/kg). PCA groups were administered orally with PCA (50 and 100 mg/kg; p.o.) once daily for 10 consecutive days after CP injection. PCA treatment resulted in a significant decrease in the protein levels of MDA, a marker of lipid peroxidation, NO and MPO along with a significant increase in GSH and catalase protein levels. Moreover, PCA downregulated anti-inflammatory markers as IL-17, NF-κB, IKBKB, COX-2, TNF-α, and PKC and upregulated cytoprotective defenses as PPARγ, and SIRT1. In addition, PCA administration ameliorated FoxO-1 elevation, increased Nrf2 gene expression, and reduced air alveoli emphysema, bronchiolar epithelium hyperplasia and inflammatory cell infiltration induced by CP. PCA might represent a promising adjuvant to prevent pulmonary damage in patients receiving CP due to its antioxidant and anti-inflammatory effects with cytoprotective defenses.

As a Modulator, Multitasking Roles of SIRT1 in Respiratory Diseases

As far the current severe coronavirus disease 2019 (COVID-19), respiratory disease is still the biggest threat to human health. In addition, infectious respiratory diseases are particularly prominent. In addition to killing and clearing the infection pathogen directly, regulating the immune responses against the pathogens is also an important therapeutic modality. Sirtuins belong to NAD+-dependent class III histone deacetylases. Among 7 types of sirtuins, silent information regulator type-1 (SIRT1) played a multitasking role in modulating a wide range of physiological processes, including oxidative stress, inflammation, cell apoptosis, autophagy, antibacterial and antiviral functions. It showed a critical effect in regulating immune responses by deacetylation modification, especially through high-mobility group box 1 (HMGB1), a core molecule regulating the immune system. SIRT1 was associated with many respiratory diseases, including COVID-19 infection, bacterial pneumonia, tuberculosis, and so on. Here, we reviewed the latest research progress regarding the effects of SIRT1 on immune system in respiratory diseases. First, the structure and catalytic characteristics of SIRT1 were introduced. Next, the roles of SIRT1, and the mechanisms underlying the immune regulatory effect through HMGB1, as well as the specific activators/inhibitors of SIRT1, were elaborated. Finally, the multitasking roles of SIRT1 in several respiratory diseases were discussed separately. Taken together, this review implied that SIRT1 could serve as a promising specific therapeutic target for the treatment of respiratory diseases.

Iprodione and/or chlorpyrifos exposure induced testicular toxicity in adult rats by suppression of steroidogenic genes and SIRT1/TERT/PGC-1α pathway.

There is cumulative evidence that iprodione (IPR) fungicide and chlorpyrifos (CPF) insecticide are endocrine disruptors that can evoke reproductive toxicity. Yet, the underlying mechanisms are still unclear. Besides, the outcomes of their co-exposure to male sexual behavior and male fertility are still unknown. The effects of IPR (200 mg/kg b.wt) and CPF (7.45 mg/kg b.wt) single or mutual exposure for 65 days on sexual behavior, sex hormones, testicular enzymes, testis, and accessory sex gland histomorphometric measurements, apoptosis, and oxidative stress biomarkers were investigated. In addition, expression of nuclear receptor subfamily group A (NR5A1), 17β-hydroxysteroid dehydrogenase (HSD17B3), silent information regulator type-1 (SIRT1), telomerase reverse transcriptase (TERT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) genes has been assessed. Our results revealed that the individual or concurrent IPR and CPF exposure significantly disturb the sexual behavior, semen characteristics, testicular enzymes, and male hormones level. Oxidative stress caused by IPR and CPF activates apoptosis by inducing Caspase-3 and reducing Bcl-2. Downregulation of HSD17B3, NR5A1, and SIRT1/TERT/PGC-1α pathway was evident. Of note, most of these disturbances were exaggerated in rats co-exposed to IPR and CPF compared to IPR or CPF alone. Conclusively, our findings verified that IPR and CPF possibly damage the male reproductive system, and concurrent exposure should be avoided.

A Pathophysiological and Pharmacological review on Alzheimer’s disease: A Current Need

Developing countries including India faces major setback in medicine and public health due to the neurodegenerative disorders. Among various neurodegenerative diseases like Parkinsonism, Hunting ton's disorder, Amyotrophic lateral syndrome, Alzheimer's is a usual subtype of dementia which has affected about 25 million people globally in 2000 and this statisticis believed to increase to 114 million in 2050. Aging has been found as one of the factors associated with Alzheimer's disease. Their association was confirmed with an increase in the incidence of this disease. A measure of the main constituent of plaque, cerebrospinal fluid levels of Aβ, and constituent of a neurofibrillary tangle, tau protein are the in-vivo biological markers of Alzheimer's disease patients. From ancient times various herbal plants were used for the treatment of Alzheimer’s. The Pharmacological drugs used were Anticholinesterase, Muscarinic receptor agonist, Glutamate receptor antagonist. The newer monoclonal antibodies were introduced for the treatment but the success rate was merge. Resveratrol, an activator of silent information regulator type1(SIRT1) was the latest drug in treating this neurodegenerative disorder. The multifactorial aetiologies leading to neurodegeneration in Alzheimer's made the treatment more complex. At present, the introduction of novel therapy mainly targeting on the pathophysiology of neuroinflammation mediated by microglia and astrocytes gave a newer insight on Alzheimer's. The determination of biomarkers and newer detection techniques can help in the future for early detection in elderly patients and better pharmacotherapy in this complicated disease.

Silent information regulator type-1 mediates amelioration of inflammatory response and oxidative stress in lipopolysaccharide-induced acute respiratory distress syndrome

Silent information regulator type-1 (SIRT1) is crucial during the development of acute respiratory distress syndrome (ARDS). We aimed to explore whether SIRT1 activation could protect against ARDS. SIRT1 was activated by its agonist SRT1720. ARDS was induced by intraperitoneal injection of 5 mg/kg lipopolysaccharide (LPS). Lung injuries were determined by the lung wet/dry ratio, inflammatory cells in the broncho-alveolar lavage fluid (BALF) and histological analysis. Inflammatory cytokine release was detected by enzyme-linked immunosorbent assay. The accumulation of neutrophils was detected by myeloperoxidase activity. Oxidative stress was evaluated by malondialdehyde, reduced glutathione, superoxide dismutaseand catalase activities. The protein expression levels were detected using Western blot. SIRT1 activation, either by SRT1720 administration or recombinant SIRT1 expression eliminated high dose LPS-induced mortality in mice, attenuated lung injury, influenced cytokine release in BALF, and decreased oxidative stress in the lung tissues of ARDS mice. Mechanically, SRT1720 administration inhibited p65 phosphorylation in the lung tissues of ARDS mice. SIRT1 ameliorates inflammatory response and oxidative stress in LPS-induced ARDS.

Melatonin Rescues the Ti Particle-Impaired Osteogenic Potential of Bone Marrow Mesenchymal Stem Cells via the SIRT1/SOD2 Signaling Pathway.

Wear particles released by joint implants are a major cause of osteolysis around the prosthesis by negatively affecting bone reconstruction. Bone marrow mesenchymal stem cells (BMMSCs) stimulated by wear particles showed an impaired osteogenic potential. Melatonin has been shown beneficial effects on intracellular antioxidant functions and bone formation; however, whether it could restore the osteogenic potential of BMMSCs inhibited by wear particles was unknown. This study aimed to evaluate the protective effect of melatonin on the osteogenic capacity of BMMSCs exposed to titanium (Ti) wear particles and to investigated the underlying mechanisms involving intracellular antioxidant properties. When BMMSCs were exposed to Ti particles in vitro, melatonin treatment successfully improved the matrix mineralization and expression of osteogenic markers in BMMSCs, while decreasing the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide. The protective effect of melatonin on osteolysis was validated in a Ti particle-exposed murine calvarial model. Meanwhile, silent information regulator type 1 (SIRT1) and intracellular antioxidant enzymes were significantly up-regulated, particularly superoxide dismutase 2 (SOD2), in melatonin-treated BMMSCs. Furthermore, inhibition of SIRT1 by EX527 completely counteracted the protective effect of melatonin on Ti particle-treated BMMSCs, evidenced by the reduced expression of SOD2, increased ROS and superoxide, and decreased osteogenic differentiation. These results demonstrated that melatonin restored the osteogenic potential and improved the antioxidant properties of BMMSCs through the SIRT1 signaling pathway. Our findings suggest that melatonin is a promising candidate for treating osteolysis induced by wear particles.

β-mercaptoethanol promotes osteogenesis of human mesenchymal stem cells via sirt1-ERK pathway.

Human umbilical cord-derived mesenchymal stem cells (hUMSCs) hold strong self-renewal capacity and low immunogenicity, which have attracted attention as potential candidates for bone repair and regeneration. However, insufficient osteogenic differentiation markedly hinders the clinical applications of hUMSCs. In the present study, the effect of β-mercaptoethanol (BME), a small molecule antioxidant which has been identified to regulate cell proliferation and differentiation, on osteogenic differentiation of hUMSCs and underlying signaling mechanism were investigated. The results indicated that under osteogenic induction conditions, BME treatment increased the alkaline phosphatase (ALP) activity and promoted calcium mineralization in hUMSCs. The gene and protein expression of osteogenesis-related markers such as ALP, osteopontin (OPN), osteocalcin (OCN) and collagen type I (COLI) were also significantly up-regulated. Besides, BME promoted the protein expression of silent information regulator type 1 (sirt1) and stimulated the activation of extracellular signal-related kinase (ERK), contributing to increased Runx2 expression. Furthermore, blocking the expression of sirt1 attenuated BME-enhanced ERK phosphorylation and osteogenic differentiation of hUMSCs. These results indicated that BME accelerated osteogenic differentiation of hUMSCs by activating the sirt1-ERK signaling pathway, thereby providing insights into the development of MSCs-based bone regeneration strategies.

Elderberry Extracts increase Rat L6 Myocyte proliferation, reduce Apoptosis and act as HDAC1/SIRT1 Agonists in vitro

Sarcopenia is a geriatric syndrome that is characterized by low muscle mass, strength, and function, that increases morbidity and mortality, and the rates of hospitalization and nursing home admissions in geriatric populations. Poor nutritional intake and alterations in the muscle response to anabolic stimuli from meals in aging populations contribute to the overall pathogenesis of sarcopenia. Research has suggested that there is an increase in skeletal myocyte apoptosis in aging populations. The extent of apoptosis in muscle increases with increasing age, which parallels the loss of both muscle mass and strength. Myocyte apoptosis may be reduced by caloric restriction, exercise training, hormone supplements, drugs and various nutritional supplements. Sambucus nigra L. (Adoxaceae), also known as elderberry is a deciduous tree with blue‐black fruits and cream‐white flowers. Elderberry has been used in traditional systems of medicine to for its antiviral activities, to reduce inflammation and diabetic symptoms. The chemical constituents of elderberry include the flavonoids, such as flavonols, proanthocyanidins and anthocyanins, and simpler phenolic acids. This study investigated the effects of elderberry extracts in rat L6 myoblasts to determine the effects on cell proliferation and apoptosis. L6 cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s medium, with 2.5 mM L‐glutamine (without phenol red). Elderberry extracts were prepared in ethanol and partitions containing anthocyanins were prepared in acetone (SNA) and ethanol (SNE). In L6 cells, ethanol partitions of elderberry increased the proliferation of rat L‐6 myoblasts by 242% and prevented cellular apoptosis induced by serum starvation. The acetone partition increased cell growth by 121% in serum starved L6 cells, the extracts also prevented myoblast apoptosis. Gene expression analysis an increase in the anti‐apoptotic Bcl‐2 mRNA expression and a reduction in Bax expression, thereby reducing the Bax/Bcl‐2 ratio to reduce apoptosis. SNE also increased the expression of PPARγ mRNA by 6.5 fold. Peroxisome proliferator‐activated receptors (PPARs) are ligand‐activated transcription factors belonging to the nuclear receptor superfamily that regulate lipid metabolism, glucose homeostasis, cell proliferation, and differentiation and modulate inflammatory responses. SNE also increased the expression of HDAC1 by 6 fold and SIRT1 by 12 fold. The human Silent Information Regulator Type 1 (SIRT1) is an NAD(+)‐dependent deacetylase that is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence and cellular apoptosis. The data suggest that SNE reduces myocyte apoptosis and that the extracts and partitions act as HDAC1/SIRT1 agonists in myocytes.Support or Funding InformationThis work was supported in part by a research grant from the Regenstein Foundation (GBM); a Postdoctoral fellowship award from the Schlumberger Foundation (TOL/GBM); and a Raman Post‐Doctoral Fellowship by the University Grants Commission, Govt. of India to NAR.

Melatonin restores the osteoporosis-impaired osteogenic potential of bone marrow mesenchymal stem cells by preserving SIRT1-mediated intracellular antioxidant properties

Postmenopausal osteoporosis (OP) is one of the most common bone diseases that affects millions of aging women. Reduced osteogenesis and increased oxidative stress have been implicated in bone marrow mesenchymal stem cells (BMMSCs) derived from OP patients. Melatonin has shown positive effects on osteoblast differentiation and bone formation; however, it was unknown whether melatonin could restore OP-impaired osteogenic potential of BMMSCs and what the underlying mechanisms entailed. The objective of this study is to investigate (1) whether melatonin can restore the impaired osteogenic potential of OP BMMSCs by preserving their antioxidant functions, and if so, (2) whether intravenous administration of melatonin can prevent OP-induced bone loss in ovariectomized (OVX) rats. Ovariectomies were performed in female rats and BMMSCs were isolated from the osteoporotic rats 3 months later. In vitro treatment with melatonin successfully improved the osteogenic differentiation of OP BMMSCs, as evidenced by increased levels of matrix mineralization and osteoblast-specific genes. In melatonin-treated OP BMMSCs, intracellular oxidative stress was significantly attenuated, while levels of intracellular antioxidant enzymes were noticeably up-regulated – particularly superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1). Silent information regulator type 1 (SIRT1) was involved in the melatonin-mediated recovery of osteogenesis and antioxidant functions. Meanwhile, in vivo injections of melatonin via the tail vein successfully ameliorated the bone micro-architecture in ovariectomized rat femurs. Further experiments confirmed that BMMSCs derived from melatonin-treated OVX rats exerted well-preserved antioxidant properties and osteogenic potential. Our findings demonstrate that the administration of melatonin is a promising strategy for treating patients with postmenopausal OP by preserving the antioxidant properties and osteogenic potential of their BMMSCs.

Palmitate-induced IL6 expression ameliorated by chicoric acid through AMPK and SIRT1-mediated pathway in the PBMCs of newly diagnosed type 2 diabetes patients and healthy subjects

Inhibition of inflammation is one of the possible therapeutic approaches for Insulin resistance (IR) during type 2 diabetes mellitus (T2DM). In the current study we investigated the effects of palmitate and chicoric acid (CA) on inflammation in peripheral blood mononuclear cells (PBMCs) of newly diagnosed T2DM patients and healthy subjects and explored the mechanism by which palmitate and CA influence inflammation.20 newly diagnosed T2DM patients and 20 healthy subjects were recruited in our study. Blood sample were collected and PBMCs were isolated. Interleukin 6 (IL6), silent information regulator type 1 (SIRT1), AMP-activated protein kinase (AMPK) and phospho-AMPK (pAMPK) were evaluated both in vivo and in vitro. PBMCs were treated with palmitate and CA to investigate their effects on inflammation. IL6 and SIRT1 genes expression were evaluated by real-time PCR. The levels of IL6 in culture medium were measured by ELISA. Proteins levels of AMPK and pAMPK in PBMCs were detected by western blotting.IL6 expression was higher and SIRT1 expression and pAMPK levels were lower in PBMCs of diabetic patients and obese subjects compared to healthy subjects and non-obese subjects, respectively. CA significantly prevented against increased IL6 levels as well as its gene expression in PBMCs induced by palmitate. Also, CA returned reduction in SIRT1 expression and pAMPK levels mediated via palmitate to near control level.These findings reveal that CA reduces inflammation in PBMCs probably through upregulation of SIRT1 and pAMPK. Therefore, CA would be suggested as a novel agent for the treatment of T2DM.

Mechanical stretch induces antioxidant responses and osteogenic differentiation in human mesenchymal stem cells through activation of the AMPK-SIRT1 signaling pathway

Mesenchymal stem cells (MSCs) are promising cell sources for regenerative medicine. Growing evidence has indicated that mechanical stimuli are crucial for their lineage-specific differentiation. However, the effect of mechanical loading on redox balance and the intracellular antioxidant system in MSCs was unknown. In this study, human bone marrow-derived MSCs (BM-MSCs) were subjected to cyclic stretch at the magnitude of 2.5%, 5%, and 10%. Cell proliferation, intracellular reactive oxygen species (ROS), expression of antioxidant enzymes, and osteogenic differentiation were evaluated. RNA was extracted and subjected to DNA microarray analysis. Sirtinol and compound C were used to investigate the underlying mechanisms involved silent information regulator type 1 (SIRT1) and AMP-activated protein kinase (AMPK). Our results showed that mechanical stretch at appropriate magnitudes increased cell proliferation, up-regulated extracellular matrix organization, and down-regulated matrix disassembly. After 3 days of stretch, intracellular ROS in BM-MSCs were decreased but the levels of antioxidant enzymes, especially superoxide dismutase 1 (SOD1), were up-regulated. Osteogenesis was improved by 5% stretch rather than 10% stretch, as evidenced by increased matrix mineralization and osteogenic marker gene expression. The expression of SIRT1 and phosphorylation of AMPK were enhanced by mechanical stretch; however, inhibition of SIRT1 or AMPK abrogated the stretch-induced antioxidant effect on BM-MSCs and inhibited the stretch-mediated osteogenic differentiation. Our findings reveal that mechanical stretch induced antioxidant responses, attenuated intracellular ROS, and improved osteogenesis of BM-MSCs. The stretch-induced antioxidant effect was through activation of the AMPK-SIRT1 signaling pathway. Our findings demonstrated that appropriate mechanical stimulation can improve MSC antioxidant functions and benefit bone regeneration.

Upregulation of SIRT1 by Kartogenin Enhances Antioxidant Functions and Promotes Osteogenesis in Human Mesenchymal Stem Cells.

Osteoarthritis is a chronic degenerative joint disease involving both articular cartilage and subchondral bone. Kartogenin (KGN) was recently identified to improve in vivo cartilage repair; however, its effect on bone formation is unknown. The aim of this study was to investigate the effect of KGN on antioxidant properties and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). Human BM-MSCs were treated with KGN at concentrations ranging from 10−8 M to 10−6 M. Our results indicated that KGN improved cell proliferation and attenuated intracellular reactive oxygen species. The levels of antioxidant enzymes and osteogenic differentiation of BM-MSCs were enhanced by KGN in a dose-dependent manner. Furthermore, KGN-treated BM-MSCs showed upregulation of silent information regulator type 1 (SIRT1) and increased phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK), indicating that KGN activated the AMPK-SIRT1 signaling pathway in BM-MSCs. Inhibition of SIRT1 by nicotinamide reversed the antioxidant effect of KGN on BM-MSCs and suppressed osteogenic differentiation. In conclusion, our results demonstrated that KGN improved intracellular antioxidant properties and promoted osteogenic differentiation of BM-MSCs by activating the AMPK-SIRT1 signaling pathway. Thus, KGN may have the potential for not only articular cartilage repair but also the clinical application of MSCs in bone tissue engineering.

Spontaneous up-regulation of SIRT1 during osteogenesis contributes to stem cells' resistance to oxidative stress.

Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is a central event in bone formation. However, oxidative stress has a deleterious impact on BM-MSC osteogenesis. In this study, we hypothesized that oxidative stress influenced BM-MSC osteogenesis differently in the early or late stages, in which silent information regulator type 1 (SIRT1) played a critical role. A continuous exposure to sublethal concentrations of hydrogen peroxide (H2 O2 ), ranging from 25 to 100 µM for 21 days, resulted in the complete inhibition of BM-MSC osteogenesis. We found that a 7-day treatment with H2 O2 inhibited the lineage commitment of BM-MSCs toward osteoblasts, as evidenced by a significant reduction of alkaline phosphatase activity (a typical marker for early osteogenesis). However, moderate oxidative stress did not affect late-differentiated BM-MSCs, as there were comparable levels of matrix mineralization (a typical marker for late osteogenesis). In addition, we observed a spontaneous up-regulation of SIRT1 and intracellular antioxidant enzymes such as superoxide dismutase 2, catalase, and glutathione peroxidase 1, which accounted for the enhanced resistance to oxidative stress upon osteogenic differentiation. Activation of SIRT1 by resveratrol rescued the effect of H2 O2 on early-differentiated BM-MSCs and inhibition of SIRT1 by nicotinamide intensified the effect of H2 O2 on late-differentiated BM-MSCs, indicating that the SIRT1-mediated pathway was actively involved in MSC osteogenesis and antioxidant mechanisms. Our findings uncovered the relationship between SIRT1 and resistance to H2 O2 -induced oxidative stress during BM-MSC osteogenesis, which could provide a new strategy for protecting MSCs from extracellular oxidative stress.

Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS.

Osteoporosis is a bone disorder linked to impaired bone formation and excessive bone resorption. Melatonin has been suggested to treat osteoporosis due to its beneficial actions on osteoblast differentiation. However, the direct effects of melatonin on osteoclastogenesis in bone marrow monocytes (BMMs) remain unknown. This study was to investigate whether melatonin at either physiological or pharmacological concentrations could affect osteoclast differentiation. Primary BMMs were isolated from the femurs and tibias of C57BL/6 mice and were induced toward multinucleated osteoclasts, in the presence of melatonin at either physiological (0.01 to 10nM) or pharmacological (1 to 100μM) concentrations. Tartrate-resistant acid phosphatase (TRAP) staining was used to label multinucleated osteoclasts and the levels of osteoclast-specific genes were evaluated. To further explore the underlying mechanisms, the roles of silent information regulator type 1 (SIRT1) and reactive oxygen species (ROS) were evaluated. We found that melatonin at pharmacological concentrations, rather than at physiological concentrations, significantly inhibited osteoclast formation in a dose-dependent manner. The number of TRAP-positive cells and the gene expression of osteoclast-specific markers were significantly downregulated in melatonin-treated BMMs. The melatonin-mediated repression of osteoclast differentiation involved the inhibition of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. The treatment with SIRT1 inhibitors did not affect osteoclast differentiation but, when supplemented with exogenous hydrogen peroxide, a partial rescue of melatonin-suppressed osteoclastogenesis was observed. Melatonin at pharmacological doses directly inhibited osteoclastogenesis of BMMs by a ROS-mediated but not a SIRT1-independent pathway.

SIRT1-dependent anti-senescence effects of cell-deposited matrix on human umbilical cord mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi-lineage differentiation. This investigation evaluated the effect of cell-deposited decellularized ECM (DECM) on oxidative stress-induced premature senescence in UC-MSCs. Sublethal dosages of H2 O2 , ranging from 50 μm to 200 μm, were used to induce senescence in MSCs. We found that DECM protected UC-MSCs from oxidative stress-induced premature senescence. When treated with H2 O2 at the same concentration, cell proliferation of DECM-cultured UC-MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H2 O2 , fewer senescence-associated β-galactosidase-positive cells were observed on DECM than those on TCPS (17.6 ± 4.0% vs. 60.4 ± 6.2%). UC-MSCs cultured on DECM also showed significantly lower levels of senescence-related regulators, such as p16INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC-MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)-dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM-mediated anti-senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell-deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Activation and overexpression of Sirt1 attenuates lung fibrosis via P300

Persistent fibroblast activation is a predominant feature of idiopathic pulmonary fibrosis (IPF), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. Silent information regulator type-1 (Sirt1) is a member of class Ⅲ histone deacetylase with important regulatory roles in a variety of pathophysiologic processes, but its role in fibrotic lung diseases is not clearly elucidated. Sirt1 expression in lung tissues of IPF patients and in a mouse model of bleomycin (BLM)-induced lung fibrosis were evaluated by immunofluorescence. The function of Sirt1 in BLM-induced lung fibrosis in the mouse model or transforming growth factor β1 (TGF-β1)-mediated lung fibroblast cellular model was investigated by Sirt1 activation, overexpression and knockdown of Sirt1. Finally, the involvement of p300 signaling pathways was assessed. In this study, we found up-regulation of Sirt1 in BLM-induced lung fibrosis, as well as in the lungs of IPF patients, including in the aggregated pulmonary fibroblasts of fibrotic foci. Activation or overexpression of Sirt1 attenuated TGF-β1-mediated lung fibroblast differentiation and activation and diminished the severity of experimental lung fibrosis in mice. Whereas knockdown of Sirt1 promoted the pro-fibrogenic activity of TGF-β1 in lung fibroblasts. A potential mechanism for the role of Sirt1 in lung fibrosis was through regulating the expression of p300. Thus, we characterized Sirt1 as an important regulator of lung fibrosis and provides a proof of principle for activation or overexpression of Sirt1 as a potential novel therapeutic strategy for IPF.

Alcohol Induces Cellular Senescence and Impairs Osteogenic Potential in Bone Marrow-Derived Mesenchymal Stem Cells.

Chronic and excessive alcohol consumption is a high-risk factor for osteoporosis. Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in bone formation; however, they are vulnerable to ethanol (EtOH). The purpose of this research was to investigate whether EtOH could induce premature senescence in BM-MSCs and subsequently impair their osteogenic potential. Human BM-MSCs were exposed to EtOH ranging from 10 to 250 mM. Senescence-associated β-galactosidase (SA-β-gal) activity, cell cycle distribution, cell proliferation and reactive oxygen species (ROS) were evaluated. Mineralization and osteoblast-specific gene expression were evaluated during osteogenesis in EtOH-treated BM-MSCs. To investigate the role of silent information regulator Type 1 (SIRT1) in EtOH-induced senescence, resveratrol (ResV) was used to activate SIRT1 in EtOH-treated BM-MSCs. EtOH treatments resulted in senescence-associated phenotypes in BM-MSCs, such as decreased cell proliferation, increased SA-β-gal activity and G0/G1 cell cycle arrest. EtOH also increased the intracellular ROS and the expression of senescence-related genes, such as p16INK4α and p21. The down-regulated levels of SIRT1 accompanied with suppressed osteogenic differentiation were confirmed in EtOH-treated BM-MSCs. Activation of SIRT1 by ResV partially counteracted the effects of EtOH by decreasing senescence markers and rescuing the inhibited osteogenesis. EtOH treatments induced premature senescence in BM-MSCs in a dose-dependent manner that was responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 was effective in ameliorating EtOH-induced senescence phenotypes in BMSCs and could potentially lead to a new strategy for clinically preventing or treating alcohol-induced osteoporosis. Ethanol (EtOH) treatments induce premature senescence in marrow-derived mesenchymal stem cells in a dose-dependent manner that is responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 is effective in ameliorating EtOH-induced senescence phenotypes, which potentially leads to a new strategy for clinically treating alcohol-induced osteoporosis.