| Introduction: Melatonin is a sort of neuroendocrine hormone secreted mainly by the pineal body,which plays an important part in regulating the physiological and pathological causes of the human body such as the bone growth and the vertebrae body moulding.However,it is still unclear about the melatonin and the occurrence and development of the idiopathic scoliosis.High concentration of melatonin could inhibit the progression of the experimentally induced spinal scoliosis was reported recently.And a prospective study also demonstrated that melatonin deficiency played a key role in the prognosis of idiopathic scoliosis and that melatonin supplements may prevent the progression of idiopathic scoliosis.During our early experiments,we demonstrated that the melatonin plays a biphasic effect on the osteoblast cells,and depends on the concentration: low concentration of melatonin can stimulate the cell proliferation,but high concentration of melatonin can inhibit the cell proliferation.We also demonstrated that low concentration of melatonin act on the melatonin receptor of the osteoblast cells,combine with the G-protein,activate the IP3,upgrade the concentration of Ca2+,via the Ca MKs/MEK/ERK pathway,to stimulate the cell proliferation;as the concentration of the melatonin increased,when the concentration reached a high point,the mechanism above was inhibit.And the calcium pathway was activated,the extracellular calcium flowed into the cell,imbalanced the homeostasis of the intracellular calcium concentration with the calcium overload,and inhibit the cell proliferation.Our early experiments demonstrated that high concentration of melatonin can cause calcium overload and inhibit the osteoblast cell proliferation.We infer its cause could be correlated to ERS,excessive stress could cause the apoptosis,and autophagy could take a part during the whole process.So in this study,we will discuss the inhibit process of the osteoblast cell proliferation induced by the melatonin,whether apoptosis is induced,whether ERS and autophagy take a part in the whole process,and the correlations between them to provide a theoretical basis and a new idea for the therapy of using melatonin on the idiopathic scoliosis patients.Materials and methods:1.Cell proliferation assay2.Apoptosis was detected by flow cytometry with the fluorescent dye Annexin V-FITC/PI Apoptosis Detection Kit3.Chromatin condensation in nucleus was observed by the fluorescent microscopy with the fluorescent dye Hoechst333424.Autophagy body was observed by the fluorescent microscopy with the fluorescent dye Monodansylcadaverine(MDC)5.Septin7 small interfering(si)RNA transfection.6.Overexpression plasmid construction and rescue experiment.7.Protein expression is assayed by western blottingResults:1.Proliferation of the osteoblast cells was inhibited by high concentration of melatonin The results of the MTT assay indicated that the proliferation rates decreased significantly when the osteoblast cells were treated with high concentration of melatonin(1m M,2m M,4m M,8m M)for 24,48 and 72 h.And when the concentration of melatonin went higher(2m M,4m M,8m M)for 6h,the cell proliferation rate decreased significantly,too.We noticed that when the concentration of melatonin came to 2m M for 24 h,the cell proliferation rate decreased extremely significantly.2.Apoptosis of the osteoblast cells induced by high concentration of melatonin was detected by flow cytometry with the fluorescent dye Annexin V-FITC/PI Apoptosis Detection Kit The results of the fluorescent dye Annexin V-FITC/PI Apoptosis Detection Kit detected by flow cytometry indicated that when the osteoblast cells were treated with melatonin for 24 h,and the concentration reached 2m M and more,the early and late apoptotic rates of the osteoblast cells increased significantly compard to the control group,and they increased as the concentration increased.When treated with endoplastic reticulum stress inhibitor 4PBA or autophagy inhibitor3 MA alone,the apoptotic rate changed without significance.But when treated a combination with endoplastic reticulum stress inhibitor 4PBA or autophagy inhibitor3 MA and 2m M concentration of melatonin,or three of all together on osteoblast cells for24 h,the induced apoptotic rate increased significantly.3.Chromatin condensation in nucleus and the apoptotic body were observed by the fluorescent microscopy with the fluorescent dye Hoechst33342 The results of the fluorescent dye Hoechst33342 indicated that when the osteoblast cells were treated with melatonin for 24 h,and the concentration of melatonin reached 1m M,the nuclei became dense and hyperchromatic.When the concentration of melatonin reached 2m M,nucleus fragmentation and chromaic condensation were observed,and the apoptotic body which was the sign of apoptosis was observed under high power field.4.Autophagy body was observed by the fluorescent microscopy with the fluorescent dye Monodansylcadaverine(MDC)The results of the fluorescent dye monodansylcadaverine(MDC)indicated that when the osteoblast cells were treated with melatonin of 2m M concentration for 24 h,the autophagy body which was the sign of autophagy was observed by monodansylcadaverine(MDC)dye with the fluorescent microscope.5.The expression of the marker protein of apoptosis,autophagy and ERS was observed by western blotting The results of the western blotting indicated that when the osteoblast cells were treated with melatonin of 2m M concentration for 24 h and 48 h,the marker protein of apoptosis PARP-1 expressed significantly.When the osteoblast cells were treated with melatonin of2 m M concentration for 6h,24 h and 48 h,the marker protein of autophagy LC3-II accumulated significantly and the marker protein of ERS GRP78 and CHOP expressed significantly,too.When the osteoblast cells were treated with endoplastic reticulum stress inhibitor 4PBA or autophagy inhibitor 3MA alone,the marker protein of apoptosis PARP-1 expressed without significance.But when treated a combination with endoplastic reticulum stress inhibitor 4PBA or autophagy inhibitor 3MA and 2m M concentration of melatonin,or three of all together on osteoblast cells for 24 h,the marker protein of apoptosis PARP-1expressed significantly.6.Relationship between septin7 and melatonin induced ER stress To better understand the relationship between septin7 and ER stress in melatonin-induced osteoblasts,septin7 si RNA designed to knock down septin7 expression led to significantly decrease GRP78 and CHOP expression,and the changes were not significant even combined with melatonin.These data confirmed that melatonin-mediated ER stress induces osteoblast proliferation via regulating septin7 expression.We also determined whether exogenous expression of septin7 could rescue the inhibition of GRP78 and CHOP expression caused by septin7 knockdown using septin7 knockdown osteoblasts transduced with septin7 overexpression plasmid.The levels of septin7,GRP78 and CHOP proteins were evaluated by Western blot.As shown in figure 8A,B,exogenous expression of septin7 restored cellular septin7 levels in septin7 knockdown cells induced by melatonin,and we also found that when transfected with septin7 overexpression plasmid,the osteoblasts showed higher GRP78 and CHOP expression compare with the control.An interesting finding was that septin7 expression did not change.Conclusions:When osteoblast cells were treated with high concentration of melatonin,the proliferation of the cells was inhibited,the endoplasmic reticulum stress was triggered,autophagy was triggered,and apoptosis was induced.The endoplasmic reticulum stress and autophagy occurred in the early stage when treated with high concentration of melatonin,both of them played a protective effect to promote the survival,and in the later stage they interacted with each other which contributed to the induction of apoptosis,and septin7 is the target protein of melatonin induced-endoplasmic reticulum 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