Role Of ATP Sensitive Potassium Channels In Hydrogen Sulfide Inhibiting High Glucose Induced Injury In Osteoblast

Background and ObjectiveDiabetes mellitus is an endocrine metabolic syndrome characterized by chronic hyperglycemia.It is often associated with bone metabolism and disorders of calcium and phosphorus metabolism,resulting in secondary osteopenia and osteoporosis and other diabetic osteopathy.Diabetic osteopathy have similar performance in the jaw,need to accept orthodontic treatment in adolescents with type I diabetes patients often due to occlusal problems and jaw dysplasia treatment,patients with type II diabetes in adults with periodontal degeneration and occlusal problems.Orthodontic treatment success depends largely on the bone remodeling,tissue reaction generated by the orthodontic force is not only related to local factors,and effects of systemic factors,bone metabolism hormones and cytokines.How to prevent the bone loss in the orthodontic treatment of diabetic patients and promote the bone repair and reconstruction has been the focus of orthodontic scholars.Diabetes can lead to abnormal bone metabolism mechanism has not been fully elucidated,the present study suggest that hyperglycemia can directly inhibit osteoblast function and bone formation was inhibited,disorders of calcium and vitamin D metabolism of bone turnover level,resulting in bone mineralization disorders caused by abnormal bone metabolism.Central link abnormal bone metabolism is dysfunction of osteoblasts,osteogenic cells as target cells of bone metabolism drugs,to prevent the damage of bone cells under high glucose,promote bone repair and reconstruction is an urgent need for clinical treatment,has also been the focus of basic research.Hydrogen sulfide(H2S)is a new member of gasotransmitter family after nitric oxide and carbon monoxide,it has been demonstrated that endogenous H2S is widely distributed in the human heart,nerve,brain and skeletal system and other tissues and organs,play important physiological functions in the body,it has anti-inflammatory,cell protection and prevention of chemical potential,and has anti-cancer effect.Some scholars further study found that H2S regulates multiple signal pathway in mammalian cells.Defects in bone metabolism are associated with abnormal metabolism of H2S,suggesting that if an effective non-toxic H2S donor is available in animals,it may be possible to treat osteoporosis and other diseases caused by hydrogen sulfide deficiency.ATP sensitive potassium channel(KATP)is the earliest reported molecular biological effects of hydrogen sulfide,hydrogen sulfide by vascular smooth muscle cells,myocardial cells,pancreatic beta cells on ATP sensitive potassium channels,regulating vascular tension,myocardial contractility,insulin secretion,hormone secretion,neurotransmission,gastrointestinal smooth muscle contraction effect.At the same time,H2S can produce anti-inflammatory,analgesic,anti apoptotic and other cell protective effects by KATP.It is found that KATP is closely related to the formation of bone,and KATP influences the osteogenic differentiation of Bone marrow mesenchymal stem cells(BMSCs)and regulates the proliferation of osteoblasts.As an ion channel,KATP maintains the membrane potential of bone cells and may participate in mechanical sensitivity and intercellular communication of bone cells.KATP is also expressed on Periodontal ligament cells(PDL)and acts as an early inflammatory signaling molecule in PDL cells in response to mechanical stress.In the body systems and tissue physiological mechanism of endogenous H2S production function and pathology are revealed,with the study of synthesis,the exogenous H2S donor further use of exogenous H2S or its donor,in the occurrence and development of disease control is to play its positive role,H2S has a broad therapeutic applications.In this experiment,primary cultured mandibular OB of rats as the research object,the application of molecular biology and cell biology technology,firstly detect whether exogenous H2S is involved in the regulation of OB damage induced by HG through its effector molecules KATP,then discuss the exogenous H2S through the KATP channel function influence on OB expression,including cell proliferation,apoptosis and mineralization the analysis and bone differentiation related gene signal molecules.This study focuses on whether exogenous H2S through KATP channels regulate bone cell injury induced by high glucose,and exogenous H2S on whether the protective effect of bone cells,to provide new ideas for promoting the reconstruction of diabetic patients with bone healing,provide direction and strategy for the new bone metabolic disease.Method1、Isolation,culture and identification of primary osteoblasts from the mandible ofratsOsteoblasts were cultured by enzymatic digestion combined with tissue block method to obtain primary osteoblasts from rats.After subculture,the cells were observed morphologically,and the cells were counted on the cell count plate,and the growth curve was drawn.The cultured osteoblasts were identified by alkaline phosphatase,calcium cobalt and alizarin red staining.2、The observation of hydrogen sulfide and high glucose intervention into the expression pressure bone cell KATP channelThe 3-5generation of vigorous and stable osteoblasts were selected for the experiment.The cells at 2×105/ml in 12 well plate culture,to be full of about 80%,according to the group control,HG,NaHS,NaHS+HG,RNA and extraction of total nuclear protein,protein and gene expression of KATP was determined by RT-PCR and Western Blot.3、Detect the changes of the signal molecules related to the proliferation,apoptosis,mineralization and osteogenic differentiation of osteoblasts under the influence of high glucose on hydrogen sulfide and KATP channelsThe 3-5 generation of vigorous and stable osteoblasts were selected for the experiment.cells according to the experimental requirements in the appropriate cell culture plate culture,to be full of about 80%,according to the group control,HG,NaHS,NaHS+HG,Gli,Gli+HG+NaHS,Pin,NaHS+HG+Pin,Pin+HG,group,flow cytometry was used to detect the apoptosis of bone cells,cell proliferation was detected by CCK8 and EDU method,alizarin red staining detection and analysis of cell mineralization,and detected by RT-PCR expression changes in bone differentiation related signal molecules ALP,collal,Runx2 and OSX.Result1、The primary osteoblasts were isolated,cultured and identified successfully.Under the microscope,the cells were triangular,spindle shaped and polygonal,and the osteoblasts were in good shape.The cells were spindle or cubic at the bottom of the bottle and arranged closely.From the observation of cell growth curve;the first 2-3 days began to grow fast,reached peak growth on day 7-8,the number reached the maximum growth rate gradually slowed down,the number of cells into the platform period began to decline,and the emergence of cells stopped proliferation apoptosis.After alkaline phosphatase,calcium cobalt staining and alizarin red staining,the cells were characterized by osteoblasts.2、H2S attenuated the inhibitory effect of HG on KATP channel proteins and genes in OB.Western blot analysis showed that the expression of KATP was significantly inhibited in HG group,NaHS+HG group,KATP expression was significantly higher than that of HG group(P<0.01),the expression of KATP protein decreased in the HG under the influence of NaHS on the expression of HG and decreased the inhibition of KATP.RT-PCR results showed that the expression of KATP gene decreased significantly under the influence of HG,while NaHS attenuated the expression of KATP inhibited by HG.3、H2S regulates HG inhibition of OB proliferation and apoptosis via KATP channels.HG has significant inhibitory effect on the proliferation of OB,KATP and HG blocking agent Gli inhibitory effect on the proliferation of OB on OB proliferation inhibition is similar to that of NaHS and KATP channel opener Pin is similar,on the proliferation of OB increased obviously,the results were statistically significant(P<0.01).When Gli is used to block the KATP channel,the number of apoptotic cells increased significantly(P<0.01),while the KATP agonist Pin effect on cell apoptosis compared with control had no obvious effect on NaHS,and the addition of Pin compared with HG,cell apoptosis was significantly reduced,the results were statistically significant(P<0.01).4、H2S regulates OB differentiation and bone formation related gene expression through KATP,and promotes osteoblastic differentiation;H2S inhibits HG induced reduction of OB mineralization,but the inhibition is independent of KATP channels.HG significantly inhibited the expression of ALP,OPN,Runx2 and OSX in OB(P<0.01).With NaHS treatment,the enhancement of ALP was not obvious,but the expression of OPN,Runx2 and OSX increased markedly compared with control,and the result was statistically significant(P<0.05).The inhibitory effect of HG on Runx2 and OSX was significantly attenuated by NaHS(P<0.01),which led to an increase in OB differentiation and bone formation related genes.The amount of OB mineralized after HG treatment significantly decreased(P<0.01),OB and NaHS mineralization pretreatment on HG lead to a decrease of the inhibitory effect of NaHS pretreatment can increase the amount of mineralization of OB,however,Gli or Pin of OB mineralization compared with contal had no significant effect(P>0.05),show that the KATP channel has nothing to do with the osteoblast mineralization.Conclusions1、Exogenous H2S can antagonize the osteoblast damage induced by high glucose through KATP channel,and has protective effect on osteoblast;2、Exogenous H2S can be used as KATP channel opener for osteoblasts;3、Exogenous H2S antagonized the decrease of proliferation,apoptosis and differentiation of osteoblasts induced by high glucose by KATP channel,but it was not related to the decrease of osteoblast mineralization induced by HG.