| Background and ObjectiveType 2 Diabetic Mellitus(T2DM)is a common metabolic disease in clinic,which is mainly characterized by elevated blood glucose and dyslipidemia,often accompanied by a variety of serious complications,and has become a global public problem endangering human health.Studies have pointed out that T2DM is closely related to the occurrence of periodontal disease,which can lead to tooth loss and dentition defect.As the most ideal method to repair lost teeth,oral implant has become the first choice for many patients.Although there is a large demand for implant-supported overdentures in patients with type 2 diabetes,the disease will adversely affect bone metabolism,damage the function of osteoblasts,inhibit new bone regeneration around the implant,and affect the formation of osseointegration.So T2DM is considered to be a relative contraindication of oral implant.In recent years,the exploration of the pathological mechanism for poor osseointegration caused by type 2 diabetes has been one of the difficulties and hotspots in the scientific research.Therefore,elucidating its pathogenic mechanism and developing effective therapeutic preparations will be of great significance to improve the success rate of implant in patients with type 2 diabetes.cGMP-dependent protein kinase G2(PKG2)is a serine/threonine protein kinase in eukaryotic cells.As a downstream signal molecule of cGMP,PKG2 can control bone growth by not only regulate chondrocyte differentiation and endochondral osteogenesis,but also regulate osteoblast function,which has been considered to be the key regulator of bone homeostasis and bone remodeling.In addition,cGMP/PKG2 pathway also plays an important role in maintaining the stability of glucose metabolism.The damage of this pathway in a variety of tissues is considered to be related to the occurrence and development of diabetic complications,and specific activation of PKG2 can effectively improve this situation.Based on the fact that PKG2 plays an important role in both bone metabolism and glucose metabolism,we speculate that it may be a key target for poor implant osseointegration caused by type 2 diabetes,and activating its expression can play a positive role in improving osteoblast dysfunction in diabetic environment.As a new type of PKG2 activator,cinaciguat can directly activate oxidized soluble guanylate cyclase(sGC)without NO,thus efficiently producing cGMP,to enhance the activity of PKG2.It has a definite effect on improving heart disease and nephropathy,and is regarded as the most potential candidate drug for the treatment of osteoporosis.Also,cinaciguat has a good therapeutic effect on diabetes-related diseases,which can protect cardiomyocytes in the environment of glucose toxicity,and increase the bone trabeculae and compact bone of type 1 diabetic mice.However,whether cinaciguat can be used as an activator of PKG2 to alleviate the dysfunction of osteoblasts and improve the osseointegration of implants in the environment of type 2 diabetes and the related molecular mechanisms have not been reported.In this study,the model of T2DM in vivo and the model of high-sugar and high-fat in vitro were firstly established to explore the relationship between the expression of PKG2 and implant osseointegration dysfunction caused by type 2 diabetes mellitus,and to determine its reliability as a therapeutic target;secondly,it was verified that increasing the expression of PKG2 by lentivirus,could improve the osteoblast dysfunction induced by diabetes in vitro;finally,to explore whether the rational use of cinaciguat in vivo and in vitro can reverse the low expression of PKG2 in diabetic environment,protect the function of osteoblasts,improve the osseointegration of implants caused by type 2 diabetes,and explore the effect molecules and related mechanisms by high-throughput proteomics.The purpose of this study is to reveal the molecular basis of the important clinical phenomenon that type 2 diabetes affects implant osseointegration,so as to find a new key target PKG2 and therapeutic drug cinaciguat to improve the success rate of implant denture treatment in patients with type 2 diabetes.Materials and methods1.Effects of T2DM on osseointegration of implants in rats,biological function of osteoblasts and changes of PKG2 expressionThe T2DM rat model was induced by high-fat and high-carbohydrate diet,followed by intraperitoneal injection with low-dose streptozotocin,and the artificial implants were implanted into the femur of rats.After determining the successful modeling,Micro-CT scan reconstruction,hard tissue section staining,hematoxylin-eosin and modified-Masson trichrome staining were used to evaluate the effect of type 2 diabetes on osseointegration of implants in vivo.Moreover,immunohistochemical(IHC)staining was used to detect the expression of PKG2 in the bone area around the implant.Primary rat osteoblasts were extracted by enzymatic digestion of neonatal rat skull tissue,and the isolated and purified osteoblasts were identified by alkaline phosphatase staining and alizarin red staining.25mM glucose and 200μM sodium palmitate were used to simulate the high glucose and high lipid(HGHL)environment of type 2 diabetes in vitro.The experimental group of HGHL medium was established,and the normal medium group and solvent group were used as the control group to observe the changes of osteoblasts biological function under the intervention of each group.It mainly included the comparison of the proliferation ability by 5-acetylethynyldeoxyuridine(EDU)fluorescent staining;the detection of cell apoptosis by flow cytometry;the detection of cells adhesion by rhodamine phalloidin fluorescence staining,and the osteogenic differentiation ability of the three groups was reflected by alkaline phosphatase staining and activity quantitative analysis,alizarin red detection of mineralized nodule formation and detection of osteogenesis-related protein expression.Finally,the expression of PKG2 in different osteoblasts groups were detected by immunofluorescence and western blot.2.Effects of overexpression of PKG2 on osteoblast function damage induced by HGHL in vitroPKG2 was overexpressed in osteoblasts by lentivirus transfection,and the overexpression efficiency was detected by qRT-PCR and western blot.After confirming that PKG2 can be stably overexpressed in osteoblasts,HGHL+OEPKG2 group was established by adding HGHL medium,and the biological functions of osteoblasts were compared with normal medium group,HGHL medium group and HGHL+OENC group(overexpression control group).It mainly included using EDU fluorescence labeling to compare the cell proliferation ability;using rhodamine phalloidin fluorescence staining to detect cell adhesion;using alkaline phosphatase staining,activity quantitative analysis,alizarin red staining and western blot to detect the osteogenic differentiation ability of cells to verify the effect of overexpression of PKG2.3.Research of the improving effect of cinaciguat on osteoblast dysfunction and poor osseointegration caused by T2DMIn vitro experiment,100nmol/L cinaciguat was added to HGHL medium to interfere with osteoblasts,and the solvent DMSO was used as the control group.The activation effect of cinaciguat on PKG2 was observed by immunofluorescence and western blot.At the same time,EDU fluorescence labeling,flow cytometry,rhodamine phalloidin fluorescence staining,alkaline phosphatase staining,activity quantitative analysis,alizarin red staining and western blot detection were used to detect the improvement of cell proliferation,apoptosis,adhesion and osteogenesis after adding cinaciguat.Moreover,the small interference RNA(siRNA)technique was used to knockdown the intracellular PKG2 of osteoblasts,to verify that the beneficial effect of cinaciguat was mediated by PKG2.In vivo experiment,the T2DM rat models and normal rat models were established,and the diabetic rats were treated by intraperitoneal injection of cinaciguat,subcutaneous injection of insulin or combination of the two methods.And calcein double fluorescence labeling,Micro-CT scanning reconstruction,environmental scanning electron microscope,hard tissue section staining,hematoxylin-eosin staining and modified-Masson trichrome staining were used to evaluate the effect of different treatments on osseointegration of implants.4.Proteomic analysis of cinaciguat reversing the damage of osteoblasts under T2DM conditionSamples were collected from two groups of osteoblasts with or without cinaciguat intervention in HGHL medium,and 3 repetitive groups were set up in each group,After all the samples were collected,proteomic analysis was carried out,including protein quantitative and quality evaluation,tandem mass tag(TMT)labeling,classification,mass spectrometry analysis,database comparison and so on.After obtaining the original data of protein expression level from proteomic analysis,the up-and-down differentially expressed proteins(DEPs)between the two groups were determined according to the set standard,and the functional enrichment and biological signal pathway information of DEPs were determined by gene ontology(GO)and signal pathway analysis.Last but not least,construct a protein-protein interaction(PPI)network to predict the potential relationship between the key protein PKG2 and DEPs.5.Study on the mechanism of cinaciguat improving osteoblast dysfunction induced by T2DM through PKG2Firstly,the proteomic results were verified by western blot to determine the interaction between the key protein PKG2 and the differential protein-PLCβ1,which predicted by the protein-protein interaction network.Secondly,calcium fluorescence technique was used to detect calcium overload in osteoblasts.Thirdly,transmission electron microscope and western blot,which detected the protein expression of endoplasmic reticulum stress markers,were used to evaluate the endoplasmic reticulum stress response of osteoblasts.Finally,EDU fluorescence staining,flow cytometry,rhodamine phalloidin fluorescence staining,alkaline phosphatase staining,activity quantitative analysis and western blot were used to detect the effects of activation or inhibition of endoplasmic reticulum stress on osteoblasts proliferation,apoptosis,adhesion and osteogenesis.Results1.T2DM impaired implant osseointegration and osteoblast function in rats,and led to down-regulation of PKG2 expressionThe T2DM rat models were successfully established in vivo,and the fasting blood glucose of the model rats were higher than 11.1 mmol/L and stable for more than one week.12 weeks after the implant implantation,the rats were sacrificed and samples were collected to detect the osseointegration of femoral implants in different groups.The results of Micro-CT reconstruction analysis and hard tissue section staining showed that the percentage of osseointegration and bone related parameters in type 2 diabetic rats were significantly lower than those of normal rats.Hematoxylin-eosin staining and modified-Masson staining showed that massive fibrous tissue around implants could be observed in type 2 diabetic rats,while the normal group was surrounded by a large amount of bone tissue.The result of IHC staining showed that the expression of PKG2 in the peri-implant bone tissue of diabetic rats was significantly lower than that of normal rats.Primary osteoblasts were successfully isolated and identified in vitro,and their characteristics were in accordance with the standard of osteoblasts.In the detection of multiple biological functions between the two groups of osteoblasts,the results showed that HGHL medium could significantly reduce the ability of cell proliferation,but increase the rate of apoptosis,moreover,the adhesion state of the cells on the titanium sheet becomes worse,and easier to fall off.Also,osteogenic ability is also severely impaired in HGHL group.The results of immunofluorescence and western blot showed that the expression of PKG2 in osteoblasts composed with HGHL was significantly lower than that in the control group.2.Overexpression of PKG2 improved osteoblast function damage induced by HGHL in vitroThe overexpression PKG2 model of osteoblasts in vitro was successfully constructed by lentivirus transfection,and the transfection efficiency was good.When detecting the biological function of cells in each group,it was found that overexpression PKG2 could promote the proliferation,improve the adhesion,and promote the osteogenic differentiation of osteoblasts in HGHL environment.3.The application of cinaciguat could alleviate the osteoblast dysfunction and promote the osseointegration of implantsThe results of immunofluorescence and western blot in vitro showed that cinaciguat could up-regulate the expression of PKG2 in osteoblasts in HGHL medium,and could enhance the osteoblasts proliferation,inhibit cell apoptosis,improve cell adhesion,and promote osteogenic differentiation in HGHL environment under the mediation of PKG2.Also,the results of IHC staining in vivo showed that cinaciguat could up-regulate the expression of PKG2 in peri-implant osteoblasts of type 2 diabetic rats,and compared with the single treatment(subcutaneous injection of insulin or intraperitoneal injection of cinaciguat),cinaciguat as an adjuvant combined with insulin could improve the osseointegration of implants in type 2 diabetic rats.The specific manifestations are that the average distance of calcein double fluorescence labeling distance is larger,the percentage of implant osseointegration is significantly higher,the quality of bone ultrastructure on the implant surface is higher,and the number of bone tissue around the implant is more,and more mature.4.Proteomic analysis of cinaciguat reversing the damage of osteoblasts under T2DM conditionThe differences of protein components between osteoblasts with or without cinaciguat intervention in HGHL medium were compared by high throughput proteomics technique,and the proteins were screened according to the two basic principles(p<0.05 and absolute multiple change≥1.2),and a total of 221 DEPs were obtained.The results of bioinformatics analysis showed that the DEPs were mainly enriched in extracellular matrix(ECM)in terms of GO function,and pathological metabolism in diabetes and bone metabolism were related to signal pathway.More importantly,when PKG2 is used as the key protein to analyze the protein-protein interaction network,we predicted that PLCβ1 might be the downstream effector protein.5.Cinaciguat alleviated calcium overload and endoplasmic reticulum in osteoblasts by inhibiting PLCβ1 through PKG2The results of western blot showed that the expression level of PLCβ1 was consistent with that of proteomic analysis,and the inhibitory effect of cinaciguat on PLCβ1 was mediated by PKG2,which further clarified the regulatory effect of PKG2 on PLCβ1.In addition,calcium fluorescence and transmission electron microscopy results displayed that cinaciguat could reduce the calcium overload and endoplasmic reticulum stress caused by the increased expression of PLCβ1 in osteoblasts through PKG2.Moreover,cinaciguat could enhance osteoblast proliferation,inhibit cell apoptosis,improve cell adhesion and promote osteoblast differentiation in type 2 diabetes by inhibiting endoplasmic reticulum stress response.Conclusions1.The osseointegration of femur implants in T2DM rat was poor,and the proliferation,apoptosis,adhesion and osteogenic ability of primary osteoblasts were obviously impaired in HGHL medium.At the same time,the expression of PKG2 in osteoblasts decreased under T2DM condition in vivo and in vitro.2.Overexpression of PKG2 by lentivirus transfection could improve the functional damage of osteoblasts induced by HGHL in vitro,which confirmed the positive role of PKG2 in regulating the biological behavior of osteoblasts in diabetic environment.3.Exogenous addition of cinaciguat had a protective effect on the functional damage of osteoblasts induced by HGHL in vitro,and could be used as an adjuvant combined with insulin to significantly improve the osseointegration of type 2 diabetic rats.4.Through high-throughput proteomics and bioinformatics analysis,it is predicted that PLCβ1 may be the downstream effector protein of PKG2,which was defined as the key node protein.5.Cinaciguat could inhibit the activation of PLCβ1 by up-regulating PKG2,and reduce the intracellular Ca2+overload-endoplasmic reticulum stress,thus protected osteoblasts from T2DM and improve the osseointegration of implants. |
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