The Role Of Melatonin In The Treatment Of Osteoporosis And Its Molecular Mechanism

Part I Alcohol induces cellular senescenceand impairs osteogenic potential in bone marrow-derived mesenchymal stem cellsObjective: 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(Et OH).The purpose of this research was to investigate whether Et OH could inducfe premature senescence in BM-MSCs and subsequently impair their osteogenic potential.Methods: To examine whether Et OH would induce premature senescence in Human BM-MSCs,cells were exposed to 10,50 and 250 m M Et OH for 7 days,untreated cells served as the control group.Cell proliferation was evaluated using a cell counting kit-8.Cell morphology was evaluated by FDA staining.The distribution of the cell cycle was analyzed by Annexin V/PI staining.Senescence associated β-galactosidase(SA-β-gal)activity were performed by SA-β-gal staining.Intracellular levels of reactive oxygen species(ROS)were evaluated by flow cytometry analysis of DCF-DA fluorescence intensity.BM-MSCs were cultured in osteogenic differentiation medium for 21 days.Mineralization of the matrix was determined by Alizarin Red S staining.Osteoblast-specific genes and protein expression during osteogenesis in Et OH-treated BM-MSCs were analyzed by RT-PCR.To investigate the role of silent information regulator Type 1(SIRT1)in Et OH-induced senescence,BM-MSCs were treated with 10 μM Res V(a SIRT1 agonist)in the presence of 250 m M Et OH.Results: Et OH treatments resulted in senescence-associated phenotypes in BM-MSCs.FDA staining suggested that BM-MSCs lost their spindle-like morphology and became a flattened cell shape following Et OH treatment.Et OH-treated BM-MSCs also showed a decreased cell density compared to untreated cells.The CCK-8 assay confirmed that Et OH treatment suppressed cell proliferation.The results of cell cycle phase distribution suggested that Et OH at 250 m M induced BM-MSCs into a G0/G1 cell cycle arrest.Both the treatment with 50 and 250 m M Et OH significantly increased the transcript levels of P16INK4 A and P21.Western blot analysis confirmed that Et OH treatment up-regulated the protein levels of p16INK4α and p21.Et OH also increased the intracellular ROS.The m RNA levels of SIRT1 in BM-MSCs decreased upon treatment with Et OH and the protein levels were confirmed by western blot analysis.We found that exposure to Et OH enhanced phosphorylation of p38 in BM-MSCs in a dose-dependent manner.In Et OH-treated cells,the level of matrix mineralization decreased by 35.2% at 50 m M and 91.7% at 250 m M compared to untreated cells compared to untreated cells.RT-PCR data showed that treatments with 50 and 250 m M Et OH down-regulated the m RNA levels of COL1A1,RUNX2 and BGLAP.Activation of SIRT1 by Res V partially counteracted the effects of Et OH by decreasing senescence markers and rescuing the inhibited osteogenesis.Conclusion: Et OH treatments induced premature senescence in BM-MSCs in a dose-dependent manner that was responsible for Et OH-impaired osteogenic differentiation.Activation of SIRT1 was effective in ameliorating Et OH-induced senescence phenotypes in BMSCs and could potentially lead to a new strategy for clinically preventing or treating alcohol-induced osteoporosis.Part II Melatonin prevents premature senescence in mesenchymal stem cells via the SIRT1-dependent pathwayObjective: Mesenchymal stem cells(MSCs),originally identified in bone marrow stroma,have attracted great interest for cell-based strategies for tissue engineering and regenerative medicine due to their capacity for self-renewal and multilineage differentiation.However,it has been reported that MSCs subjected to oxidative stress may undergo a process of premature senescence in pathological conditions.Premature senescence irreversibly inhibits BM-MSCs proliferative ability,and suppresses their regenerative potentials.Our previous studies demonstrated that melatonin protects MSCs from proinflammatory cytokines by decreasing ROS generation and enhancing superoxide dismutase activities.In this study,MSCs were induce oxidative stress-induced premature senescence in vitro with H2O2,and the protective effects of melatonin on reversing MSCs senescent phenotypes in a dose-dependent manner would be studied.We would focus on the critical role of SIRT1-dependent pathway in protective effects of melatonin.Methods: For induction of premature senescence,BM-MSCs at approximately 50% confluence were exposed to 100 μM,200 μM,and 400 μM of H2O2 for 2 h.For the experiment investigating the effects of melatonin on reversing premature senescence,BM-MSCs were treated with 200 μM H2O2,the cells were cultured in growth medium and supplemented with 10 n M,1 μM,and 100 μM melatonin for an additional 4 days.In the osteogenic differentiation assay,BM-MSCs were treated with 200 μM H2O2,followed by incubation in osteogenic differentiation medium supplemented with 10 n M,1μM,and 100 μM melatonin for an additional 21 days.For the antagonist studies,after treating with 200 μM H2O2,10 μM luzindole(a melatonin receptor inhibitor)or 40 μM sirtinol(a SIRT1 inhibitor)was added in combination with 100 μM melatonin for an additional 4 days.Senescence associated β-galactosidase(SA-β-gal)activity,cell cycle distribution,cell proliferation and reactive oxygen species(ROS)were evaluated.Mineralization of the matrix was determined by Alizarin Red S staining.Transcript levels of P16INK4 A,SIRT1,and osteogenic marker genes,including COL1A1,RUNX2,SPP1 and BGLAP were evaluated by RT-PCR.P16INK4 A,SIRT1,p38,phosphorylation of p38 and Osteoblast-specific protein expression were analyzed by Western blot.Results: Our results showed that low moderate concentrations of H2O2,such as 100 μM and 200 μM,induced positive staining of SA-β-gal activity and arrested cell cycle progression.Treatment with melatonin before H2O2 exposure cannot significantly prevent premature senescence;however,treatment with melatonin subsequent to H2O2 exposure successfully reversed the senescent phenotypes of BM-MSCs in a dose-dependent manner.This result was made evident by improved cell proliferation,decreased senescence-associated β-galactosidase activity,and the improved entry of proliferating cells into the S phase.In addition,treatment with 100 μM melatonin restored the osteogenic differentiation potential of BM-MSCs that was inhibited by H2O2-induced premature senescence.We also found that melatonin attenuated the H2O2-stimulated phosphorylation of p38 mitogen-activated protein kinase,decreased expression of the senescence-associated protein p16INK4 a,and increased SIRT1.Further molecular experiments revealed that luzindole,a nonselective antagonist of melatonin receptors,blocked melatonin-mediated antisenescence effects.Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin,suggesting that melatonin reversed the senescence in cells through the SIRT1-dependent pathway.Conclusion: Melatonin protects MSCs from H2O2-induced premature senescence,increasing cell proliferation,and restoring osteogenic differentiation potentials of BM-MSCs in a dose-dependent manner.These findings lay the groundwork for understanding oxidative stress-induced premature senescence and promoting therapeutic utilization of melatonin in stem cell-based regenerative medicine.Part III Melatonin suppresses the differentiation of monocytes into osteoclasts via the attenuation of intracellular ROSObjective: Osteoporosis is a common bone disease prevalent in the aging population.The pathogenesis of osteoporosis has been linked to impaired bone formation and excessive bone resorption.The potential applications of melatonin in treating osteoporosis have been investigated in previous clinical trials.However,the direct effects of melatonin on osteoclast differentiation in bone marrow monocytes(BMMs)and bone resorption remain unknown.This study was to investigate whether melatonin at either physiological or pharmacological concentrations could affect osteoclastogenesis.Methods: To prepare primary cell cultures,BMMs were isolated from the femurs and tibias of C57BL/6 mice.To investigate the effect of melatonin on osteoclastogenesis,BMMs were incubated with osteoclastogenic differentiation media in addition to melatonin at physiological(0.1,1,and 10 n M)or pharmacological(1,10,and 100 μM)concentrations.Tartrate-resistant acid phosphatase(TRAP)staining was used to label multinucleated osteoclasts.The formation of F-actin rings were assessed by immunofluorescence staining.The levels of osteoclast-specific genes were evaluated by RT-PCR.To investigate if melatonin inhibit osteoclast differentiation through blocking NF-κB pathway,immunofluorescence staining was conducted to trace p65.Western blot was conducted to show p65 and inhibitory κB protein(IκB)protein levels.To further explore the underlying mechanisms,the roles of silent information regulator type 1(SIRT1)and reactive oxygen species(ROS)were evaluated.For the SIRT1 antagonist studies,10 m M NAM or 40 μM sirtinol was added in osteoclastogenic differentiation media in the presence of 100 μM melatonin.For the experiment investigating the role of ROS on melatonin-mediated anti-osteoclastogenesis,20 μM H2O2 was added in osteoclast differentiation media in the presence of 100 μM melatonin.Osteoclast differentiation was evaluated by TRAP staining,F-actin ring staining and RT-PCR.Results: We found that melatonin at pharmacological concentrations,rather than at physiological concentrations,significantly inhibited osteoclast formation in a dose-dependent manner.The formation of TRAP-positive multinucleated osteoclasts,the actin rings in multinucleated osteoclasts and the gene expression of osteoclast-specific markers were significantly downregulated in melatonin-treated BMMs.We found the level of ROS in BMMs significantly decreased in the presence of 100 μM melatonin compared with the RANKL-treated group.In the presence of 100 μM melatonin,nuclear translocation of p65 in RANKL-treated BMMs was inhibited,suggesting that the NF-κB signaling pathway was blocked by melatonin at pharmacological concentrations.The western blot results confirmed that the phosphorylation of IκB in BMMs was enhanced by treatment with RANKL,but it was inhibited by treatment with 100 μM melatonin.The protein levels of SIRT1 were not significantly different in all groups and the transcript levels of SIRT1 showed a similar tendency.The TRAP staining assay showed that,in the absence of melatonin,the formation of giant and multinucleated osteoclasts was not affected by treatment with SIRT1 inhibitors and,in the presence of melatonin,the addition of sirtinol and NAM did not improve OCG either.RT-PCR suggested that,in the presence of melatonin,the transcript levels of TRAP,CTSK,and OSCAR were not upregulated in RANKL-stimulated BMMs by treatment with SIRT1 inhibitors.When supplemented with exogenous hydrogen peroxide,a partial rescue of melatonin-suppressed osteoclastogenesis was observed.Conclusion: Melatonin at pharmacological concentrations,rather than at physiological concentrations,significantly suppressed osteoclast differentiation in a dose-dependent manner.Melatonin mediated anti-osteoclastogenesis employed SIRT1-independent but ROS-dependent inhibition of the NF-κB signaling pathway.These results suggested that melatonin could be used as a potential treatment against age-related osteoporosis by inhibiting deleterious bone resorption.