| Objective: Diabetic nephropathy is a common complication of diabeties,and its unique pathophysiological changes increase the risk of anesthesia,which has been paid more and more attention by clinical anesthesiologists.Dexmedetomidine,as ?2-adrenergic receptor agonist,has stable sedative and analgesic effects,and protective effects in acute renal injury and renal ischemia-reperfusion injury.Long-term high glucose status in diabetic patients can cause tissues and cells with oxidative stress damage,and then lead renal tubular cells into epithelial-mesenchymal transition,which is closely related to renal interstitial fibrosis and seriously affects renal function.Through cell experiment in vitro and animal experiment in vivo,this study has explored the protective effect of dexmetadimidine on diabetic nephropathy and relevant mechanisms,in order to provide a reliable theoretical basis for the selection of anesthetic drugs and guidance of the clinical anesthesia management for diabetic nephropathy.Methods: 1.A model of high glucose damage to human renal tubular epithelial cells(HK-2 cells)was established in vitro,and HK-2 cells were treated with different concentrations(0 m M,10 m M,20 m M,30 m M,40 m M,50 m M,60 m M)of glucose for 6 h.The survival rate of cells in different groups was performed by CCK-8 method.The morphological changes of cells in different groups were observed by an inverted microscope.The expression of AKT,p-AKT,ERK,p-ERK and EMT-related proteins(E-Cadherin,?-SMA,and Claudin-1)expression in groups of different concentrations was detected by western blotting.2.CCK-8 method was used to detect the cell survival rate of each group of dexmedetomidine at different concentrations to determine its concentration and time.The vitro experiments were divided into three groups: the normal group(Normal group),the high glucose injury group(Glu group)and high glucose injury with Dexmedetomidine group(Glu+Dex group).HE staining was used to detect the morphological changes of the three groups of cells.Fluorescent probe method was used to detect the levels of reactive oxygen species in the cells of the three groups,and flow cytometry with Annexin V/PE was used to detect cell cycle and cell apoptosis of the three groups.Western blot was used to detect the expression of AKT,p-AKT,ERK,pERK and EMT-related proteins(E-Cadherin,?-SMA,and Claudin-1)expression among the three groups.3.A rabbit model of diabetic nephropathy was established in vivo.The vivo experiments were divided into three groups: control group(Control group),diabetic nephropathy group(DN group),and diabetic nephropathy with dexmedetomidine group(DN+Dex group).Dexmedetomidine was administered to diabetic nephropathy rabbits at 50 ?g/kg for 6 h.The general indicators of the three groups were measured including body weight,blood glucose,kidney weight /body weight,urinary protein.Observation under the light microscope and HE staining were used to detect pathological changes in the kidneys of the three groups while western blotting to detect the expression of AKT,p-AKT,ERK,p-ERK,and EMT-related proteins(E-Cadherin,?-SMA,and Claudin-1).Results: 1.Establishment of human renal tubular epithelial cells(HK-2 cells)injury model by high glucose in vitro.(1)As the glucose treatment concentration increased,the survival rate of HK-2 cells gradually decreased.(2)With the increase of glucose treatment concentration,the cell shape gradually changed from an approximately circular shape to a long spindle shape,the intercellular space became larger,the number of adherent cells decreased,and the growth state became worse.(3)As the glucose treatment concentration increased,the expression of AKT in HK-2 cells decreased,the expression of ERK,p-AKT and p-ERK was up-regulated,the ratio of p-AKT/AKT and p-ERK/ERK was significantly increased,and the expression of E-Cadherin and Claudin-1 was significantly down-regulated.2.Dexmedetomidine attenuated epithelial-mesenchymal transition induced by high glucose in human renal tubular epithelial cells.(1)With the decrease of Dex concentration,the survival rate of HK-2 cells increased,and finally selected 0.01 n M for 6 h.(2)The results of HE staining showed that the normal morphology of the cells in the Glu group changed,the cell volume became larger,the number of viable cells decreased,and the cells in the Glu+Dex group and Normal group had good morphology.(3)Fluorescence probe CM-H2 DCFDA was used to detected intracellular ROS level in each group.The fluorescence intensity of the Glu group was significantly higher than that of the Glu+Dex group and Normal groups,and both differences were statistically significant.(4)Apoptosis of each group was detected by flow cytometry Annexin V/PE method.Compared with the Glu group,the apoptosis rate of Glu+Dex group and Normal group was reduced,and the differences were statistically significant.(5)The cell cycle distribution of each group was detected by flow cytometry.the Glu+Dex group and the Normal group had an increase in G1 phase and a decrease in G2 phase,the differences were statistically significant.(6)The expression of related proteins in each group was detected by western blotting.Compared with the Glu group,the expression of PI3 K and AKT in the Glu+Dex group and the Normal group was decreased,the expression of p-AKT and the ratio of p-AKT/AKT decreased while the expression of ERK and p-ERK was decreased,the ratio of p-ERK/ERK was decreased.(7)The PI3K/AKT pathway inhibitor LY294002 and ERK pathway inhibitor U0126 were added to detect the expression of related proteins.The expression of pAKT and p-ERK in the Dex+LY294002 group was decreased significantly,while the expression of p-ERK in the Dex+U0126 group was decreased significantly but p-AKT was not decreased obviously.The expression of E-Cadherin and Claudin-1 was increased while the expression of ?-SMA decreased in the Dex+LY294002 group and the Dex+U0126 group compared with other groups,and the differences were statistically significant.3.Dexmedetomidine had protective effect on epithelial-mesenchymal transition in diabetic nephropathy rabbits.(1)Compared with the Control group,the blood glucose of the DN group and the DN+Dex group was significantly increased,the weight of them was significantly decreased,urinary protein was significantly decreased,and the differences were statistically significant.Compared with the DN group,the blood glucose of the DN+Dex group was decreased,the weight was increased,and urinary protein was increased.(2)Compared with the Control group,the glomerular capillary loop area of the DN group and the DN+Dex group was increased,and the relative area of the glomerular mesangium and basement membrane were increased which there were statistically significant differences.Compared with DN group,it showed a relative decrease of the above two indexes in DN+Dex group.(3)The results of HE staining showed that compared with the control group,the glomerular mesangial matrix in the DN group was significantly increased,and the number of cells in the glomerulus was significantly increased.Compared with DN group,it showed a relative decrease of the above two indexes in DN+Dex group.(4)Compared with the control group and DN+Dex group,the expression of p-AKT and p-ERK in the DN group was increased,the expression of E-Cadherin and Claudin-1 was decreased,and the expression of ?-SMA was increased.The differences were statistically significant.Conclusion: 1.High glucose can increase the expression of p-AKT and p-ERK in human renal tubular epithelial cells,decrease the expression of E-Cadherin and Claudin-1,increase the expression of ?-SMA,and cause epithelial-mesenchymal transition of cells.2.Dexmedetomidine can up-regulate the expression of E-Cadherin and Claudin-1,down-regulate the expression of ?-SMA,and reduce epithelial-mesenchymal transition in human renal tubular epithelial cells by regulating AKT and ERK.3.Dexmedetomidine has the protective effect on epithelial-mesenchymal transition in diabetic nephropathy of the rabbits. |
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