N-(3-Oxododecanoyl)-L-Homoserine Lactone Affects Osteoblast Apoptosis And Differentiation By Regulating Intracellular Calcium Release Patterns

Objective: Quorum Sensing?QS?refers to the group behavior that bacteria regulate the expression of their important genes by sensing changes in bacterial population density.N-?3-oxododecanoyl?-L-homoserine lactone?AHL?is the most representative quorum sensing molecule,which is secreted by Pseudomonas aeruginosa?P.a?.Studies indicates that AHL has a very wide range of biological regulatory effects on host cells.For example,AHL is a powerful trigger of cell apoptosis by disrupting mitochondria or activating the death receptor.The immunomodulatory effects of AHL involve the destruction of epithelial cell junctions,chemotaxis,Nuclear factor-kappa B?NF-?B?-dependent pro-inflammatory responses,which show that AHL has a wide range of biological regulatory activities.On the other hand,there are many kinds of bacteria in the oral cavity,but usually the symbiotic relationship between the flora is not pathogenic.However,in some severe periodontitis and persistent periapical disease which the symptom is bone defects,opportunistic pathogens P.a is characteristically detected.Some researchers have proposed that periodontal pathogens combined with P.a infection significantly increased the degree of inflammation and bone destruction in periodontal disease.Therefore,there may be a causal relationship between P.a and bone damage.In addition,P.a often survive in the biofilm.Studies have shown that AHL accumulated in large quantities in the biofilm produced by P.a,with concentrations as high as 300–600 ?M.Therefore,given the strong biological regulatory activity of AHL,whether AHL plays an important role in diseases related to odontogenic bone destruction has attracted our attention.Osteoblast metabolism is a well-coordinated physiological process that includes differentiation from mesenchymal progenitors and death by apoptosis,which is critical for the maintenance of the quality and quantity of bone.Osteoblast differentiation promotes bone formation by enhancing the expression of osteogenesis markers,including Runx2,Osterix,alkaline phosphatase?ALP?,osteocalcin?OCN?,and bone sialoprotein?BSP?.Apoptosis occurs,in part,through the activation of mitochondria-mediated-intrinsic signaling,which offsets osteoblast differentiation to maintain the stability of osteoblast metabolism.Osteoblast differentiation and apoptosis affect each other and are co-regulated by certain upstream molecules,such as calcium ion(Ca²⁺).Ca²⁺ is a pivotal osteoblast regulator,which is associated with the regulation of osteoblast function.Elevation of intracellular Ca²⁺ levels and activation of downstream signaling pathways promote osteoblast proliferation and differentiation.However,an overload of intracellular Ca²⁺ paradoxically induces reactive oxygen species and triggers apoptosis in osteoblasts.Under normal physiological conditions,cellular differentiation and apoptosis are coordinated to maintain the metabolic balance of osteoblasts.Ca²⁺ signals can be disturbed by some bacterial secretions,leading to abnormal osteoblast metabolism.Coincidentally,AHL can induce the release of reactive oxygen species in human platelets and intestinal goblet cells,causing premature loss of the acrosome in sperm cells.This leads to pro-inflammatory cytokine production or decrease in cell gap junctional intercellular communication in airway epithelial cells,which are Ca²⁺-dependent processes.However,it is still unknown whether AHL can regulate osteoblast metabolism by disrupting intracellular Ca²⁺.In this study,we examined the effects of AHL on the proliferation,apoptosis and differentiation of mouse pre-osteoblasts MC3T3-E1 cells;and explored the co-regulatory effect of AHLinduced intracellular Ca²⁺ release patterns on osteoblast apoptosis and differentiation;We try to reveal the potential influence mechanism of AHL on osteoblast metabolism in order to provide more theoretical support for the treatment of odontogenic bone defects.Methods: 1.Experimental methods 1.1 The Effect of AHL on osteoblast apoptosis 1.1.1 MC3T3-E1 cells were treated with different concentrations of AHL?10,20,30,40,50 ?M?for 1,3,and 5 d.Microscope photographs were used to observe the cell phasecontrast.CCK-8 analysis was used to detect cell activity.1.1.2 MC3T3-E1 cells were treated with different concentrations of AHL for 24 h.Apoptotic cells were fluorescently labeled by TUNEL?Terminal Deoxynucleotidyl Transferase-mediated d UTP Nick-End Labeling?method.Apoptotic cells were detected by fluorescence microscopy and the number of apoptotic cells?per mm2?was counted.1.1.3 MC3T-E1 cells were treated with AHL?50 ?M?for 1,3,6,12,24 h or treated with different concentrations of AHL for 3 h.Western blotting was used to detect the protein expression of Cleaved-Caspase-3 and Cleaved-PARP.1.1.4 The mitochondria of MC3T3-E1 cells were pre-stained with red mitochondrial fluorescent dye,and green fluorescent labeled AHL?30,50 ?M?was added for 15 min.The confocal laser scanning microscope system was used to observe the colocalization of AHL and mitochondria.1.1.5 MC3T3-E1 cells were treated with AHL?30,50 ?M?for 15 min,and the mitochondrial membrane potential was detected using the JC-1 fluorescent probe.1.1.6 The mitochondria of MC3T3-E1 cells were pre-stained with red mitochondrial fluorescent dye,and then treated with AHL?30,50 ?M?for 3 h.After that,the immunofluorescence staining of cytochrome c was performed.The confocal laser scanning microscope system was used to observe the redistribution of cytochrome c from mitochondria to cytoplasm.1.1.7 MC3T-E1 cells were treated with AHL?30,50 ?M?for 3 h,and mitochondrial and cytoplasmic proteins were separated.Western blotting was used to detect the expression of Cytochrome c protein in mitochondria and cytoplasm.1.2 The effect of AHL on osteoblast differentiation 1.2.1 MC3T-E1 cells were treated with osteogenic differentiation medium containing series concentration of AHL?10,20,30,40,50 ?M?for 7,10,14 and 17 days.The state of the cells was observed after 24 h under microscope.ALP staining was detected.1.2.2 MC3T-E1 cells were treated with osteogenic differentiation medium containing series concentration of AHL for 3,7,10,14 and 17 days.The ALP activity was detected.1.2.3 MC3T-E1 cells were treated with osteogenic differentiation medium containing series concentration of AHL for 21 d.Alizarin red S staining was used to detect mineralized nodules.1.2.4 MC3T-E1 cells were treated with osteogenic differentiation medium containing series concentration of AHL for 7,10,14 and 17 days.Real-time PCR was used to detected the m RNA expression of Runx2,Osterix,Bsp and OCN.1.3 AHL regulated intracellular calcium release patterns and affected osteoblast apoptosis and differentiation 1.3.1 MC3T3-E1 cells were treated with green fluorescent-labeled AHL?30,50 ?M?for 15 min.Then the immunofluorescent staining of the endoplasmic reticulum?ER?membrane protein Calreticulin was performed.The confocal laser scanning microscope system was used to observe the colocalization of AHL and ER.1.3.2 Pre-treatment of MC3T3-E1 cells with dual-fluorescence Ca²⁺ probes.AHL?30,50 ?M?was added under the monitoring of a confocal laser scanning microscope.The change of intracellular Ca²⁺ fluorescence intensity was checked.The ratio analysis of fluorescence intensity was performed using Image J to draw the curve of intracellular Ca²⁺ levels.1.3.3 The method was the same as 1.3.2.The AHL?30,50 ?M?with or without calmodulin antagonist W7 was added under the monitoring of a confocal laser scanning microscope.The effect of W7 on AHL-induced intracellular Ca²⁺ release pattern was analyzed.1.3.4 MC3T3-E1 cells were treated with AHL?30,50 ?M?in the present or absent of calmodulin antagonist W7 for 3 h.Western blotting was used to detect the protein expressions of Cleaved-Caspase-3 and Cleaved-PARP.1.3.5 MC3T3-E1 cells were treated with osteogenic differentiation medium containing AHL?30,50 ?M?in the present or absent of W7 for 10 d.ALP staining and ALP activity were detected.Real-time PCR was used to detect Runx2,Osterix,Bsp and OCN m RNA expression.2.Statistical analysis Each experiment was repeated at least 3 times.All data were presented as the mean ± SD.One-way analysis of variance?ANOVA?and Dunnett's t test were used for comparisons between multiple groups.P < 0.05?*?was considered statistically significant.P < 0.01?**?was considered extremely statistically significant.Results: 1.AHL induced mitochondrial-dependent intrinsic apoptosis in osteoblasts. Compared with the control,AHL?20-50 ?M?gradually reduced the osteoblast CCK-8 OD value in a dose-dependent manner,but the OD value gradually increased with time.The numbers of green apoptotic cells marked by green fluorescence gradually increased with the increased concentration of AHL.Western detection of apoptotic marker proteins showed that the protein expression of Cleaved-Caspase-3 and Cleaved-PARP reached a peak at 3 h and were in a dose-dependent manner.Fluorescent detection of AHL localization in osteoblasts,it can be seen that green fluorescently labeled AHL overlapped with red fluorescently labeled osteoblastic mitochondria.AHL dose-dependently reduced mitochondrial membrane potential by using JC-1 detection.Cytochrome c was labeled by green immunofluorescence staining and osteoblastic mitochondria were labeled with red fluorescent dye.It can be seen that cytochrome c in the AHL group was released from the mitochondria and spread throughout the cytoplasmic region.Mitochondria and cytoplasmic proteins were separated.The expression of cytochrome c protein was decreased in mitochondria but increased in cytoplasm.2.Bidirectional regulation of osteoblast differentiation by different concentrations of AHL.50 ?M AHL inhibited osteoblastic ALP staining,ALP activity,and mineralized nodules,and down-regulated m RNA expression of the osteoblastic functional genes Runx2,Osterix,Bsp,and OCN.30 ?M AHL inhibited ALP activity at day 3,but promoted ALP activity,the production of mineralized nodules,and up-regulated the m RNA expression of Runx2,Osterix,Bsp and OCN at day 7-17.In addition,in the remaining concentrations of AHL group,osteoblastic ALP activity and osteogenic differentiation function genes fluctuated within a certain range.3.AHL mediated osteoblast apoptosis and differentiation by regulating intracellular Ca²⁺ release pattern.Fluorescence detection of AHL localization in osteoblasts,visible green fluorescent labeled AHL and red fluorescent labeled osteoblastic ER images was overlapped.Dual fluorescent probes labeled intracellular Ca²⁺ in osteoblasts showed that AHL can rapidly burst intracellular Ca²⁺ levels release.Calmodulin antagonist W7 completely blocked the release pattern of AHL-induced transient bursts of intracellular Ca²⁺,which was changed to a sustained gentle rise pattern.The final intracellular Ca²⁺ level maintained at a higher level?compared to cells treated with only AHL?.In addition,W7 effectively inhibited the expression of AHL-induced osteoblast apoptotic proteins Cleaved-Caspase-3 and Cleaved-PARP,and partially restored?50 ?M AHL?or promoted?30 ?M AHL?the ALP staining or ALP activity of in osteoblasts treated with AHL.Conclusions: AHL affects the osteoblastic apoptosis and differentiation by regulating the intracellular Ca²⁺ release pattern.50 ?M AHL induces an instant burst of intracellular Ca²⁺,causing an irreversible mitochondrial-dependent apoptosis in osteoblasts.A large number of cell deaths disrupts the metabolic balance of osteoblasts and severely inhibits osteoblastic differentiation.The Ca²⁺ burst induced by 30 ?M AHL decreased,and the apoptotic response was relatively mild.After the Ca²⁺ level return to the resting level,it began to rise slowly,which was helpful to activate the signal of osteoblast differentiation.Moreover,the proapoptotic effect of AHL decreased gradually with the prolonging of cell culture time,and the activity of osteoblasts was restored gradually,thus osteogenic differentiation of some certain stages was enhanced.The AHL concentration used in this experiment could induce osteoblast apoptosis in early stage,and the inhibitory effect of 50 ?M AHL on osteoblast differentiation was observed throughout the experiment.However,the AHL concentration used in this experiment was still relatively low.By comparison,in the infections that P.a is detected,P.a is often present in the form of biofilms to evade host immunity.With the influence of quorum sensing,high concentrations of AHL can be produced in biofilms.Meanwhile,osteoblasts have been damaged by the main pathogen and are in an inflammatory or immune state,which may result in an imbalance of strength between osteoblasts and AHL.Accordingly,AHL disrupts the metabolic balance of osteoblasts by interfering with the Ca²⁺ signal,which aggravate the destruction of bone or obstruct the regeneration of bone.Therefore,for the treatment of severe periodontitis or persistent periapical periodontitis,it is helpful to re-examine the negative regulation of opportunistic pathogens and their quorum sensing molecules on bone.