| Background:Diabetic kidney disease(DKD)is one of the common complications of diabetes.About 30-40% of diabetic patients in the world will have a diabetic kidney disease.It has become a global public health problem affecting the life and quality of life of diabetic patients.At present,there is no cure for DKD,and people with DKD eventually progress to the end stage,requiring dialysis,kidney transplantation and even death.Therefore,it is important to develop new prevention and control strategies for DKD.Studies have shown that abnormal accumulation of lipids in the kidney can cause lipid toxicity,induce apoptosis of renal tubule epithelial cells,and eventually lead to tubular atrophy and tubule interstitial fibrosis,which is one of the main causes of DKD.Adropin is a protein encoded by Enho gene and plays an important role in the regulation of energy balance,glucose and fatty acid metabolism.Low Adropin level has been shown to be associated with the risk of DKD,and supplementation of exogenous Adropin may be a potential prevention and treatment method for DKD.The mechanism of Adropin in the pathophysiology of DKD is still not completely clear.Whether Adropin is involved in the regulation of lipid metabolism is one of the focuses of this study.In addition,protein drugs have poor stability and insufficient targeting ability.It has become a hot topic in biopharmaceutical research to improve the clinical efficacy,reduce the dose and frequency of drug administration,and achieve better targeting and tolerance through targeted protein modification and optimization of drug carrier.Reactive oxygen species(ROS)response drug vectors have the ability of high drug loading,specific targeting and in situ treatment.Reactive oxygen species(ROS)response drug vectors are a promising and efficient drug delivery tool to achieve precise treatment.Excessive accumulation of ROS is one of the main characteristics of DKD,and the synthesis of ROS response drug vectors is a feasible strategy to improve the effect of Adropin and other protein drugs on DKD.Therefore,the synthesis of ROS response Adropin nanocapsules to improve the targeting ability and therapeutic effect of exogenous Adropin is another focus of this study.Objectives:(1)By analyzing the serum Adropin level of clinical patients with DKD and establishing mouse models of DKD in vivo and in vitro,the expression and significance of Adropin in DKD were discussed from the perspectives of molecular biology and histology.(2)Based on the characteristics of high levels of ROS in the DKD microenvironment,a ROS responsive nanocapsular loaded Adropin(Ad@Gel)was synthesized,and the relationship between the therapeutic effect of Ad@Gel and lipid metabolism in vivo and in vitro DKD mouse models was evaluated.(3)To explore whether Ad@Gel plays a therapeutic role through Nnat/TSPO signal axis by knocking down Nnat and overexpressing TSPO,so as to provide a theoretical basis for Ad@Gel to improve DKD.Methods:(1)Serum samples from 112 patients with type 2 diabetes mellitus(T2DM)were collected from the hospital biobank,and Adropin protein levels in serum samples were detected by ELISA,and clinical data were analyzed.HK2 cells were induced by high glucose to establish in vitro model,and mice DKD model was established by combining high glucose and high lipid with streptozotocin to evaluate the therapeutic effect of Adropin.(2)In situ radical copolymerization,Adropin protein was encapsulated in nanocapsules to prepare Ad@Gel.Transmission electron microscopy and Fourier infrared spectroscopy were used to verify the preparation of nanocapsules.The chemical and biological properties of Ad@Gel were evaluated by drug loading,ROS response release assay,cellular and in vivo toxicity assay.(3)In vivo and in vitro models of DKD,the efficacy of Ad@Gel was evaluated compared with Adropin or enalapril alone;Mitochondrial membrane potential was detected by flow cytometry and mtDNA level was detected by qPCR to analyze the influence of Ad@Gel on mitochondrial function.Lipid accumulation in renal tissue was observed by oil red O staining and Bodipy staining,and the role of Ad@Gel on lipid metabolism was determined by immunofluorescence detection of SREBP-1 and ADRP lipid-producing protein.(4)Immunofluorescence was used to analyze Nnat and TSPO protein expression in vitro and in vivo,and lentivirus transfection was used in vivo to observe the effect of Nnat knockdown or TSPO overexpression on Ad@Gel therapeutic efficacy..Results:(1)Compared with non-DKD group,serum Adropin level in DKD group was significantly decreased;Logistic regression analysis showed that low serum Adropin level was associated with increased risk of DKD.The supplementation of exogenous Adropin can significantly improve the damage of HK2 induced by high sugar,and also improve renal function in DKD mice in vivo,but the improvement effect is not as good as the positive control drug enalapril.(2)Transmission electron microscopy and Fourier infrared spectroscopy confirmed that Ad@Gel was successfully prepared,the maximum loading rate was27.05±1.59%,and could be released stably in high ROS microenvironment with no toxic effects in vivo and in vitro.(3)The supplementation of Ad@Gel can significantly reduce the damage of high sugar on HK2 cells,effectively reduce lipid accumulation and restore mitochondrial function,and the therapeutic effect is not significantly different from that of Adropin alone.Ad@Gel treatment can effectively control blood glucose in DKD mice,improve renal function and histological damage,reduce lipid accumulation,inhibit oxidative stress and restore mitochondrial function in DKD mice.In addition,the therapeutic effect of Ad@Gel was significantly exceeded when the loading of Ad@Gel was only half of that of Adropin group.Moreover,the improvement effect of Ad@Gel was even better than enalapril when the dose of Adropin was the same as that of Adropin group.(4)In vivo and in vitro DKD models,Ad@Gel significantly down-regulated TSPO protein expression and up-regulated Nnat protein expression.After Nnat knockdown in vivo,Ad@Gel significantly reversed the effect of inhibiting lipid accumulation,improving mitochondrial function and down-regulating TSPO protein expression.Overexpression of TSPO had no significant effect on Ad@Gel inhibition of renal lipid accumulation and up-regulation of Nnat protein expression in DKD mice,but significantly reversed the effect of Ad@Gel on improving mitochondrial function.Conclusions:Decreased Adropin level is associated with renal dysfunction in patients with T2 DM,which is a valuable biomarker and therapeutic target for early diagnosis of DKD.In vivo and in vitro models,Adropin level is closely related to DKD,and exogenous Adropin supplementation can effectively improve DKD,but in vivo model,the therapeutic effect is not as good as enalapril.Therefore,we synthesized ROS responsive nanocapsules(Ad@Gel)that can improve the renal targeting ability of exogenous Adropin protein,enhance the therapeutic effect of DKD mice,and even better than enalapril when the dose loading is the same as that of Adropin alone.In addition,Ad@Gel improves lipid accumulation and restores mitochondrial function through Nnat/TSPO signaling axis,and finally plays the therapeutic role of DKD. |
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