| BackgroundType 1 Diabetes Mellitus(T1DM)is a chronic systemic inflammatory disease caused by the immune system and eventually leads to dysregulation of autoantig-ens.Acute kidney injury(AKI)is a disease of rapid loss of renal function,with high morbidity and mortality.The most common cause of AKI is ischemia/reperfusion(I/R)injury.AKI proximal renal tubular damage activation of innate immunity and adaptive immunity leads to kidney inflammation is the common pathway.In allogeneic transplantation and autologous kidney,whether transplantation or trauma,which is leading to poor clinical outcomes the most common factor.A large number of clinical studies have shown that diabetes is a high risk factor for acute kidney injury.and patients with diabetic nephropathy have high sensitivity to acute renal injury and poor prognosis.The pathological mechanism of diabetes and ischemic acute kidney injury include inflammatory response.The hypoxia caused by the reperfusion injury of ischemic organs caused a strong inflammatory response.After necrosis,the molecules in the cell may enter the outer space of the cell.Regulate inflammatory infiltration.The inflammatory cytokines and chemokines that are characterized by the release of the inflammatory cytokines and the inflammatory processes also include the expression of adhesion molecule protein expression,and the white blood cells are grouped into ischemic kidneys.Apoptosis and necrotic cells are the main factors that regulate inflammatory response.Toll-like receptor 7,as an important pattern recognition receptor,plays an important role in organism defense against pathogen invasion.Many studies have shown that TLR7 is involved in many diseases including systemic lupus erythematosus,autoimmune nephropathy,coronary ischemia reperfusion injury,etc.However,there was no study on whether the TLR7 mediated TLR7/MyD88/NF-κB signaling pathway was involved in the participation in diabetic model ischemic acute renal injury.Therefore,the mechanism of TLR7 in diabetic acute renal injury is urgent to be studied.Part One:The study of TLR7 in kidney ischemia/reperfusion of type 1 diabetic ratsObjective:To investigate the role of TLR7 in acute ischemic kidney injury in type 1 diabetic rats.To observe the important mechanism of TLR7 through TLR7/MyD88/NF-κB signaling pathway in acute kidney injury.Methods:Male adult Sprague-Dawley(SD)rats were treated with a single intraperitoneal injection of streptozotocin(STZ)to establish type 1 diabetes mellitus models,and age-matched non-diabetic normal rats served as controls.The renal ischemia injury model was established by bilateral renal pedicle clamping for 25 minutes.Some of the diabetic rats were pre-treated with chloroquine 7 days before ischemia reperfusion injury.Diabetic rats and normal rats Randomly divided into 5 groups(n=6):①the non diabetic rats in sham operation group(group ND+Sham);②the diabetic rats in sham operation group(group DM+Sham);③the non diabetic rats with renal ischemia reperfusion group(group ND+I/R);④renal ischemia and reperfusion in diabetic rats(group DM+I/R);⑤ ischemia the reperfusion in diabetic rats+chloroquine pretreatment group(group CD+I/R).The rats were killed 48 hours after reperfusion,and the renal sections were stained with H&E,TUNEL and immunohistochemistry.Serum BUN and Cr levels were measured,and levels of IL-6 and TNF-α in renal were measured.The expression levels of TLR7,MyD88 and NF-κB protein were detected by Western and blot.Results:Compared with non diabetic group,Diabetic rats have a higher blood sugar and weight loss(P<0.05).IHC assay showed/showed that TLR7 was mainly expressed in renal tubules and slightly expressed in glomeruli.Compared with the ND+I/R group,BUN、Cr、IL-6 and TNF-α,renal tubular epithelial cell apoptosis,expression of TLR7,MyD88 and NF-κB,DM+I/R group was significantly increased(P<0.05).All of these changes were further improved by chloroquine inhibition TLR7(P<0.05).Conclusions:Renal ischemia reperfusion can cause acute kidney injury in rats.Inhibition TLR7 expression can protect diabetic kidney ischemia-reperfusion injury,by reducing the levels of inflammatory reaction,reducing apoptosis.Inhibiting TLR7 activation,the sensitivity of diabetic nephropathy to acute renal injury was decreased.Part Two:The mechanism of TLR7 in high glucose and hypoxia reoxygenation injury of human renal tubular epithelial cellsObjective:To research the damage of TLR7 in HK-2 cells under high glucose,hypoxia and reoxygenation.Methods:The HK-2 cells were randomly divided into 8 groups(n=6):①low-glycemic group(LG group);②High sugar group(HG group);③The low sugar+mannitol group(M group);④The hypoxia hypoxia complex oxygen group(LH/R group);⑤The hypoxia complex oxygenation group(HH/R group);⑥High sugar+anoxic complex oxygen+TLR7 gene silencing group(HH/R-siRNA group);⑦Once high sugar+hypoxia reoxygenation,RNA interference in the control group(HH/R-Scrambled siRNA group);⑧A pre-treatment group(HH/R-CQ group)was used to treat high sugar+anoxic+hypoxic+chloroquine.Using sugar stimulates the 72 h first,followed by a lack of oxygen 4 h and 2 h reoxygenation model of human renal tubular epithelial cells in high glucose and hypoxia,respectively using CCK-8 and LDH releasing cell activity assay,ELISA assay of intracellular IL-6 and TNF-α levels change.Cell apoptosis was detected by flow rate method.The expressions of TLR7,MyD88 and NF-κB protein in renal cells were detected by immunofluorescence and Western blotting.Results:Compared with the low glucose group,the activity of CCK-8 decreased in the high glucose group,and the expression of LDH,IL-6,TNF-α,apoptosis rate,immunofluorescence expression,TLR7 protein,MyD88 protein and NF-κB protein increased(P<0.05).Compared with group LH/R,the activity of CCK-8 in HH/R group decreased,LDH,IL-6,TNF-α,apoptosis rate,immunofluorescence expression,TLR7 protein,MyD88 protein and NF-κB protein expression increased(P<0.05),Compared with HH/R group,TLR7 group and si-RNA HK-2 cells increased chloroquine pretreatment group the activity of CCK-8,LDH,IL-6,TNF-α,apoptosis rate,immunofluorescence and the expression of TLR7 protein and reduce the expression of MyD88 protein and NF-κB protein(P<0.05).Conclusions:In HK-2 cells,the inhibition of TLR7 can alleviate the damage to cells by high glucose,hypoxia and reoxygenation. |
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