Effects Of High Glucose On The Expression And Channel Function Of Connexin43 In Osteocytes And Its Mechanism Research

Back ground and purpurs: Type 2 diabetes mellitus(T2DM)is widely prevalent in the world.In China,the incidence rate of T2 DM is as high as 11.1%.A large number of epidemiological studies have shown that the fracture risk of diabetic patients is significantly higher than that of the general population.Fractures often occur at a relatively high bone mineral density(BMD)level in T2 DM patients.Therefore,the effect of T2 DM on bone structure has become a new research focus.Bone structure mainly consists of cancellous bone and cortical bone.Cortical bone is the main load-bearing part of bone tissue,and subtle structural changes in cortical bone cause greater changes in bone strength than cancellous bone.Fractures in T2 DM often occur in the cortical bone region of the extremities.Therefore,it is of great significance to study cortical bone structure changes in T2 DM patients.High resolution peripheral quantitative computed tomography(hr-pqct)and micro-CT 3D imaging were used to evaluate cortical bone structure in T2 DM patients,and it was found that cortical bone structure damage,including increased porosity,increased micro-crack and decreased bone strength in T2 DM patients and T2 DM rat models.Osteocytes account for more than 90% of the total cells in cortical bone and play a key role in orchestrating bone modeling and remodeling.Osteocyte can form complex network with their dendrites and contact with each other as well as osteoblast,osteoclast,and mesenchymal stem cells.Through the network,osteocyte can exchange sclerosis,and a variety of reactive molecules such as RANKL,regulating the balance of various kinds of bone cell activity and function,so as to maintain steady state of bone.Osteocytes are also the main receptors of mechanical stress,and convert mechanical stimulation into chemical In the observation and study of T2 DM patients and mouse model,it was found that the volume of osteocytes in cortical bone increased,while the number of osteocytes decreased.According to the mathematical model of extension and computer analysis,the network connection of bone cells was disordered,and the difference in the number of connected cells at a single node was larger than that in the non-t2 dm group.However,the mechanism of these changes in bone cells remains unclear.Gap junction(GJ)is an important membrane channel structure located on the cell membrane,which controls the signal communication and material transmission between cells and extracellular matrix,and also plays an important role in the formation of the network structure and material transport of bone cells.Cx43 is the most abundant gap junction protein in bone.Defects in the Cx43 gene can lead to systemic bone dysfunction,delayed bone mineralization,and skull deformities.Osteocyte-specific Cx43 deficiency make the negative effect of mechanical unloading on bone blunted,suggesting that Cx43 was a necessary condition for osteocytes to sense mechanical stress.Mice lacking Cx43 in cortical bone showed senescence related phenotype,while Cx43 overexpressed reduced the senescence of bone tissue.Cx43 also plays an important role in protecting bone cells from oxidative stress.Cx43 knockout osteocytes showed increased sensitivity to oxidative stress-induced cell death.In view of the important role of Cx43 and its gap-junction and hemichannel in osteocyte survival,resistance to oxidative stress,network formation,and mechanical stress perception,we hypothesized that Cx43 and its gap-junction and hemichannel may be involved in the pathological process of bone cell morphology and network structure changes induced by T2 DM.The purpose of this study was to observe the expression of Cx43 and its effect on the function of gap junction channel in bone cells under the treatment of high glucose,and to further explore its mechanism,so as to provide a new pathogenic mechanism and therapeutic target for cortical bone structure damage caused by T2 DM.Methods : Mouse bone cell line MLO-Y4 cells were used.The changes of Cx43 in MLO-Y4 cells treated with different concentration of glucose were detected by western blot and immunofluorescence.Meanwhile,the scrape loading assay and dye uptake assay were used to detect the function of Cx43 gap junction and hemichannel.Meanwhile,according to the inclusion and exclusion criteria formulated,cortical bone specimens of femoral neck were collected from T2 DM and non-T2 DM patients undergoing hip replacement due to osteoporotic femoral neck fracture.HE staining and immunohistochemical analysis were used to observe the bone lacunas in cortical bone of T2 DM patients and the expression of Cx43.Moreover,the activation of the P38MAPK/ERK signaling pathway related to the internalization of Cx43 under the action of high glucose was further detected to explore the possible mechanism of the decreased expression and channel function of Cx43 caused by high glucose.Results.1)Through CCK8 cell activity detection,it was found that MLO-Y4 cell activity decreased slowly and dose dependently with the treatment of high glucose.2)Cx43 protein expression in MLO-Y4 cells treated with high glucose was decreased using Western-blot and immunofluorescence assay.3)The function of Cx43 channel and half channel in MLO-Y4 cells was also decreased.4)Cortical bone specimens were collected from T2 DM patients and non-T2 DM patients of the same range of age at femoral neck.After fixation,decalcification,sectioning,and HE staining,it was observed that the ratio of empty bone lacunae in cortical lacuna increased in T2 DM patients compared with non-T2 DM patients.5)The expression of Cx43 in osteocytes of T2 DM patients' cortical bone was lower than that of non-T2 DM patients by immunohistochemical assay.6)Membrane protein of MLO-Y4 cells treated with high glucose was extracted,and analyzed by western blot.The expression of Cx43 in MLO-Y4 cell membrane protein was also decreased after high glucose treatment.7)The key protein of p38MAPK/ erk1/2signaling pathway in MLO-Y4 cells with high glucose treatment was detected,and increased expression was found,indicating that the pathway was activated.The signal pathway is related to the internalization of Cx43 in the cell membrane.8)inhibitors PD98059 of p38 MAPK and U0126 of ERK can partially restore the Cx43 protein level decreased due to high glucose,and partially reverse the function reduction of Cx43 channel and half channel under high glucose.9)The expression of autophagy marker protein LC3I/II in MLO-Y4 with high glucose treatment increased.Autophagy is closely related to Cx43 degradation.Conclusion.High glucose can reduce the cellular activity of MLO-Y4,as well as the expression of Cx43 protein and its gap junction and hemichannel function by activating the Cx43 internalization pathway p38MAPK/ERK.The internalized Cx43 protein degrades rapidly through the activated autophagy.Inhibition of p38MAPK/ERK pathway can partially reverse the function of Cx43 channel and half channel reduced by high glucose.