Osteoblast Autophagy Induced By Titanium Ion Through SIRT3/SOD2/mROS Signal Pathway And The Reversal Mechanism Of Melatonin

BackgroundDental implant has become the first choice for patients with dentition defect and dentition loss.At present,implant metal materials used in clinic are mainly pure titanium(grade 1-4),Ti-6Al-4V,ti-5al-25fe,Ti-6Al-7Nb,etc.These metals and their alloys are widely used in the dental implant field because of their excellent corrosion resistance,mechanical properties and biocompatibility.However,after implanting,it is immediately surrounded by blood,extracellular fluid,protein and other substances.In the complex environment of human body,the plant will be affected by stress corrosion,electrochemical corrosion and chemical corrosion,resulting in the release of titanium ions from titanium and its alloy implants,which will directly or indirectly cause osteoblast damage or death and result in bone resorption.In 1960,Ferguson et al.reported for the first time that titanium ions were released from the titanium implant,the concentration of titanium ions in the tissue around the implant was 20 times higher than that in the normal tissue 4-6 months after implanting.It is reported that the concentration of titanium ion in serum can reach 20.89?mol/L in the successful cases and 83.56?mol/L in the failed cases.Although titanium and its alloy have been proved to be relatively safe by a large number of studies,the above results suggest that the released titanium ions may play an important role in bone absorption at the interface of implant and bone.Studies have shown that the occurrence of peri-implant inflammation is accompanied by the increased oxidative stress in the patients saliva.In addition,studies have revealed that the expression and activity of antioxidant enzymes in saliva and gingival fluid of patients with peri implant inflammation are significantly reduced in pathological state,which suggests that oxidative stress plays an important role in the occurrence and development of peri-implant inflammation.However,in this process,specific regulatory mechanism has not been fully elucidated.Mitochondrion is the main site of oxidative phosphorylation and ATP formation in cells,and the center of energy metabolism of cells,which is also called "power plant"of the cell.The oxidative phosphorylation of mitochondria is often accompanied by the production of reactive oxygen species(ROS).Mitochondria is the main source of ROS.so mitochondrial dysfunction is the main cause of ROS imbalance in vivo.Appropriate ROS plays an important physiological role,but excessive ROS will bring about many hazards.At present,a large number of studies have shown that under oxidative stress autophagy can remove oxidative damaged mitochondria,endoplasmic reticulum,proteins and lipids to prevent cell from death.Autophagy,originally discovered and named in lower organisms,is composed of substances in the cytoplasm that are surrounded by a double membrane structure to form autophagosomes,which then combined with lysosomes to form autophagosomes,is a hot topic in recent studies.Proper autophagy is necessary to maintain its physiological homeostasis,but excessive autophagy may lead to cell death.The previous studies showed that when osteoblasts were exposed to metal ions,the activity of osteoblasts decreased,and the change of activity was in direct proportion to the concentration of metal ions.In our preliminary study,we found that the activity of osteoblasts decreased and autophagy occurred after titanium ion treatment.At the same time,the content of active oxygen in mitochondria increased,while the antioxidant NAC(n-acyl-l-cystein)could not alleviate the damage of titanium ion on osteoblasts.While MitoQ(mitoquinone)was added,the activity of osteoblasts was restored,the content of active oxygen in mitochondria decreased,and the function of mitochondria was also restored.These results suggest that oxidative stress and dysfunction of mitochondria play an important role in autophagy of osteoblasts induced by titanium ions.Therefore,it is very important to clarify the exact mechanism and potential treatment of osteoblast injury induced by oxidative stress after titanium ion treatment.Cells regulate mitochondrial derived oxygen free radicals through a rigorous mechanism.Mitochondria have a specific function to scavenge oxygen free radicals from mitochondria to maintain cell homeostasis.ROS scavenging in mitochondria was mainly degraded by SOD2.SOD2 is the only antioxidant enzyme in mitochondria that can convert superoxide anions into hydrogen peroxide,and plays an important role in regulating the concentration of mitochondrial reactive oxygen species.The activity of SOD2 enzyme is mainly affected by its acetylation level.In recent years,it has been found that SIRT3 can deacetylate the acetylated mitochondrial protein,thus increasing the activity of ROS scavenging enzyme and stabilizing the function of mitochondria to inhibit the accumulation of ROS in mitochondria.thus maintaining and regulating the normal physiological function of mitochondria,which is of great significance to alleviate and reduce the damage caused by oxidative stress.At present,there are three deacetylases in mitochondria,namely SIRT3,SIRT 4 and SIRT 5.SIRT3 is a class ? deacetylase dependent on nicotinamide adenine dinucleotide(NAD),which is the strongest deacetylase in mitochondria.It is directly involved in the synthesis of mitochondrial energy and the control of mitochondrial oxygen free radical level.It has been reported that more than 20%of mitochondrial proteins are acetylated.SIRT3 regulates mitochondrial ROS clearance by changing the acetylation level of SOD2.What's more,SIRT3 can directly combine with SOD2,which leads to the deacetylation of SOD2,enhances the activity of SOD2,and plays an important role in the regulation of ROS homeostasis in mitochondria.Our previous study found that titanium ions can lead to an obvious increase of ROS in the mitochondria of osteoblasts,so how will the antioxidant mechanism,SOD2,etc.in mitochondria change under this condition?We hypothesized that the activity of mitochondrial SOD2 decreased through SIRT3 pathway,which led to the increase of ROS and injured the osteoblasts.Melatonin(MT)is a kind of neuroendocrine hormone which is secreted by the pineal gland of mammalian at night and has a wide range of physiological and pharmacological effects.The most significant effects of melatonin and its metabolites are scavenging free radicals and antioxidation.Melatonin can effectively eliminate the accumulation of excess reactive oxygen species in cells,improve cell proliferation.Melatonin can improve the expression of CuZn-SOD and Mn-SOD through the superoxide dismutase(SOD)signal pathway,so as to eliminate excess free radicals.A series of recent studies have focused on the role of melatonin in regulating autophagy in both healthy and disease states.A search of domestic and foreign literatures revealed that there was no study on the molecular mechanism of titanium ion causing autophagy in osteoblasts and the effect of titanium ion on the SIRT3/SOD2 signaling pathway in mitochondria of osteoblasts.Based on our previous research,we intend to use melatonin to treat osteoblasts injury caused by titanium ions,to study its effect on inhibiting the generation of mROS,reducing the autophagy level of osteoblasts and improving their functions,and to evaluate its role in the treatment of peri-implant inflammation.Methods1.Establish the model of osteoblasts injured by titanium ion.Evaluate the toxic effect of titanium ion on osteoblasts by means of cell activity test and so on.Detect the mROS level of cells by ELISA.The expression of autophagy marker proteins LC3II and P62 in osteoblasts were detected by Western Blot.Autophagosomes of osteoblasts were detected by co-localization of GFP-LC3 and TOMM20 with confocal microscopy.The effect of titanium ions on autophagy flow and cell activity was further detected by lysosomal activity inhibitor barflomycin A.After the intervention of mitochondrial antioxidant Mito-TEMPO(mitochondrial-targeted SOD mimetic),the mROS level were detected,and so as to autophagy level and cell activity to evaluate the effect of titanium-induced mROS on osteoblasts.2.Exposed osteoblasts to different concentrations of titanium ions for 24h.The changes of SIRT3/SOD2/mROS signal regulation axis in osteoblasts were evaluated by detecting the SOD2 activity,SOD2 protein expression,SIRT3 activity,SIRT3 mRNA expression and SOD2 acetylation level in osteoblasts under the action of titanium ions.By constructing LV5-SIRT3 lentivirus,and instantaneously transfect to osteoblasts,lead to SIRT3 overexpression in osteoblasts.The effects of titanium ions on SIRT3 protein level,SIRT3 activity,SOD2 acetylation level,SOD2 activity,mROS level,autophagy level and cell viability of SIRT3-overexpressed osteoblasts were detected.3.To detect the mechanism and reversal effect of melatonin on titanium ion induced osteoblasts injury.The levels of mROS and autophagy in osteoblasts and cell activity between the two groups(melatonin +Ticl4 group and Ticl4 group)were compared to evaluate the therapeutic effect of melatonin.By comparing the SIRT3 activity,SIRT3 protein level,SOD2 acetylation level and SOD2 activity in the osteoblasts between melatonin +Ticl4 group and Ticl4 group,the possible mechanism of melatonin was researched.Results1.With the increase of titanium ion concentration,the activity of osteoblasts decreased progressively,but the apoptosis of osteoblasts was not obvious.With the increase of titanium ion concentration,the expression of autophagy protein LC3II increased and the expression of P62 decreased.The increase of autophagosomes in osteoblasts was observed by confocal microscopy.To detect autophagic flux,we measured the level of LC3-II and GFP-LC3-positive autophagosomes in the absence and presence of Baf Al.We found that a Baf A1 challenge resulted in increased LC3-II expression and GFP-LC3-positive autophagosomes in the cells treated with titanium ion.Thus confirming that titanium ion can increase the autophagy flux of osteoblasts.In the presence of Baf A1,the activity of osteoblasts improved,thus confirming that autophagy caused osteoblasts damage.In the presence of titanium ions,the level of mitochondrial ROS in osteoblasts was significantly increased.The mitochondrial antioxidant Mito-TEMPO suppressed mitochodria-derived ROS generation,and significantly suppressed the titanium-mediated increase in LC3-II expression,and the cell activity was improved,thus confirming that mitochondrial ROS caused cell damage through autophagy.2.With the increase of titanium ion concentration,SOD2 activity decreased progressively.Compared with the control group,SOD2 activity level decreased by 22.5%,43%and 58.5%respectively,but the expression of SOD2 protein was not affected.Titanium ions significantly increased the acetylation levels of SOD2 when compared with controls.Titanium ions treatment resulted in a significant decrease in SIRT3 mRNA and protein levels.Our experimental data also showed that SIRT3 activity was reduced in the titanium group when compared with the control.We overexpressed SIRT3 in osteoblast cells using a transient transfection method.We found that overexpression of SIRT3 attenuated the Titanium ions-induced suppression of SIRT3 protein expression and activity.Furthermore,overexpression of SIRT3 decreased the expression of acetylated SOD2,increased SOD2 activity,and efficiently suppressed mitochondrial-derived ROS production in osteoblast cells exposed to 50?M Ticl4.SIRT3 overexpressing cells also exhibited a marked reduction in LC3-II expression and an elevation in cell viability.3.We tested whether melatonin protects mitochondria against titanium ions induced oxidative stress and autophagy.We found that melatonin blocked mitochondrial derived ROS elevation induced by titanium ions in osteoblast cells.Concordantly,melatonin restored the levels of LC3-II and cell viability in titanium ions treated osteoblast cells compared to that of untreated controls.Melatonin could partially restored the titanium ions mediated reduction in SIRT3 activity,and the expression of SIRT3 protein increased.As expected,melatonin significantly blocked the titanium ions induced expression of acetylated SOD2 and restored SOD2 activity.Conclusion1.The excessive mROS induced by titanium ions triggers autophagy and lead to autophagic cell death in mouse MC3T3-E1 osteoblasts,which indicates that the autophagy induced by titanium ions through mitochondrial ROS is one of the important causes of osteoblast injury.2.Titanium ions interfered with the function of SIRT3/SOD2/mROS pathway in mitochondria in mouse MC3T3-E1 osteoblasts,leading to overproduction of mROS.These results suggest that inhibition of SIRT3 activity is one of the important mechanisms of titanium ion induced osteoblastic injury.3.Melatonin shows a compensatory and protective role in eliminating mitochondrial derived ROS and suppressing autophagy and restore cell activity through the SIRT3/SOD2/mROS pathway in mouse MC3T3-E1 osteoblasts.This result confirmed that autophagy caused by mitochondrial ROS was an important cause of titanium ion induced osteoblasts injury,and melatonin which can recover SIRT3 activity could be used as one of the protective drugs for titanium ion injury.