| ObjectiveDiabetic kidney disease(DKD)is regarded as renal microvascular complications caused by diabetes,which is characterized by proteinuria and progressive renal function damage.DKD is one of the main causes of end-stage renal disease.DKD is one of public health issue in the world,which brings substantial financial burden to families and society.Incidence of end-stage renal disease caused by DKD increased year after year.It is of great social significance and economic value in exploring the pathogenesis of DKD and seeking for effective treatment on easing the financial burden and solveing global public health issue.The pathogenesis of DKD is complicated,and remains to be fully elucidated.Hyperglycemia,inflammation,oxidative stress and hemodynamic changes are involved.Hyperglycemia and oxidative stress play important roles in the pathogenesis of DKD.On one hand,they can activate multiple pathways related to hyperplasia and hypertrophy,which lead to renal hypertrophy in the early stage of DKD.it plays an important role in the occurrence and development of DKD.On the other hand,hyperglycemia and oxidative stress can give rise to mitochondria damage and mitophagy,and cause mitochondrial dysfunction and imbalance of redox reaction.Finally,all of this exacerbate the progression of DKD.Artemether(Art)is derived from artemisinin,an extract from the traditional Chinese medicine Artemisia apiacea.In recent years,massive studies have been focused on the pharmacological of Art,which showsed that Art exhibit inhibiting effects on different types of tumor cell.Studies also showed that Art had a certain therapeutic effect on metabolic diseases.However,the effect of artemether on type 1 DKD has not been reported and the mechanism remains unclear.By applying streptozocin(STZ)-induced type 1 diabetic mouse model and in vitro cell experiment,this study focused on exploring the effect of Art on type 1 diabetic mouse model.,The purpose of this study is to explore:(1)Whether artemether has a renal protective effect on STZ-induced type 1 diabetic mice;(2)Whether artemether can improve the symptoms of type 1 diabetes;(3)Whether artemether can improve type 1 diabetic renal hypertrophy;(4)Whether artemether can improve kidney mitochondrial function in type 1 diabetic mice.Method1 Animal experimentStudy on STZ-induced diabetic mice,diabetic mice were randomly divided into three groups,control group(TlD-ctrl group),diabetes model group(STZ group)and artemether treatment group(STZ+Art group).The diabetic mice were induced by STZ intraperitoneal injection.Mice in TID-ctrl group and STZ group were fed with standard food,and mice in STZ+Art group were fed with Art-containing food(0.67g/kg).The beginning of treatment defined as week-O andthe treatment sustained to week-8.24-hour urine of the mice were collected by using metabolic cage at week-O,week-4,week-8.In the meantime,the amount of 24-hour water consumpution,food intake,faeces and urine were recorded.After 8 weeks of Art treatment,the whole blood,serum,pancreas,kidneys and other specimens of all mice were collected.Urinary albumin,NGAL,Kim-1 and serum insulin levels were measured by ELISA.Urine glucose level was detected by biochemical methods.PAS staining was applied to observe the morphological changes of the mouse kidney and measure the glomerular tuft area,the glomerular tuft volume,the mesangial matrix area and the area of renal tubule renal tubular lumen tubular wall.Glomerular and tubular basement membrane thickness,and foot process width were detected by electron microscopy.HE staining was used to measure the area and number of islets in the pancreas.The laser confocal microscope was used to detect the proportion of the positive ?,?,and ? cells in the islets.Amplex UltraRed was used to detect the release rate of H2O2 in kidney mitochondria.Immunohistochemistry was used to detect the distribution of Catalase,SOD2,p-Erkl/2,and p-S6RP in kidney tissue.Real-time quantitative PCR was used to detect the relative mRNA expression of Catalase,SOD2,PDH,MPC1,and PDK1 in kidney tissue.Western blot was used to detect the protein expression of PDK1?PGC-1??catalase?SOD2?p-Erk1/2?p-MEK1/2?p-S6RP?p70S6K?p-Akt?p-mTOR?and p-p27kip in kidney tissues.2 Cell experimentNormal rat kidney epithelial cells(NRK-52E)was applied,the viability of NRK-52E cell was promoted by the stimulation of high concentration glucose,and the cell viability was detected by MTT.Cells were cultured at different time point for 24h,48h,72h after Art was administered.Cell viability was detected by MTT,and the effect of Art on mTOR/p70S6K signaling pathway was detected by western blot.Results1 Animal experiment(1)Art can significantly reduce the urine protein excretion rate and the weight of kidney in STZ mice.(2)Art can alleviate the renal hypertrophy in type 1 diabetic mice.Art can reduce the area and volume of the glomerular turf,the area of glomerular mesangial matrix,the thickness of glomerular basement membrane,the foot process width,the area of renal tubule,tubular lumen and tubular,renal tubular basement membrane thickness.(3)Art can reduce fasting blood glucose and glycated hemoglobin levels in diabetic mice,and it can also reduce urinary glucose level and alleviate polydipsia,polyphagia,polyuria in diabetic mice caused by diabetes;(4)Art can improve the imbalance ratio of ?,? and ? cells in mouse islets and increase serum insulin level.(5)Art can reduce protein expression of p-Erk1/2,p-MEK1/2,p-S6RP,p70S6K,p-Akt,p-mTOR and p-p27kip in mouse kidneys.(6)Art can reduce the protein expression of PDK1 in kidney tissue and in kidney mitochondria.Art also reduce the relative mRNA expression of PDK1 and increase the relative mRNA expression of PDH and MPC1 in kidney tissue.Art can increase the protein level of PGC-1? in kidney tissue.(7)Art can increase the protein expression of Catalase and SOD2 in kidney tissue,and increase the relative mRNA expression of Catalase and SOD2;(8)Art can increase the level of H2O2 release in renal mitochondria.2 Cell experiment(1)NRK-52E cells viability was increased in a time-dependent manner stimulated by different concentrations of glucose.(2)Art can significantly inhibit the proliferation of NRK-52E cells stimulated by high concentration glucose;(3)Art can down-regulate the protein phosphorylation level of S6RP,p70S6K,and mTOR in NRK-52E cells after stimulated by high glucose for 24h,48h,and 72h,obviously at 48h.In ConclusionThis study verified for the first time that Art had a renal protective effect on STZ-induced type 1 diabetic mice,and it can improve the diabetic symptoms,reduce the urinary albumin excretion rate and improve kidney pathological damage.The protective mechanism of Art on diabetic kidney may be associated with the factors as follows.(1)Art can increase the secretion of insulin in STZ mice and reduce blood glucose;(2)Art can regulat oxidative stress,inhibit Akt/mTOR/p70S6K and MAPK/Erk signaling pathway activation in diabetic kidney;(3)Art can improve mitochondrial function by regulating the pyruvate uptake and oxidation capacity of diabetic renal mitochondria. |
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