Preparation Of Iron Sulfide-EGCG Nanomedicine And Study In Alleviating Acute Kidney Injury

Acute kidney injury(AKI)usually presents with sudden increases in serum creatinine(CRE)and urea nitrogen(BUN),decreased urine volume,and decreased glomerular filtration rate(GFR)within a few hours to a few days.AKI is often accompanied by multiple complications such as hyperkalemia,metabolic acidosis,volume overload,and uremic symptoms resulting from decreased GFR.AKI is a complex disease that can be triggered by surgery,sepsis,trauma,and toxic reactions to medications.Reactive oxygen species(ROS)play a crucial role in renal function homeostasis during the occurrence and development of AKI.When the balance between ROS production and elimination is disrupted,renal infiltration and excess ROS production by endogenous cells induce apoptosis through mitochondrial swelling and dysfunction.Unfortunately,there are only mild treatments available,such as fluid rehydration,kidney dialysis and other supportive treatments.Therefore,it is urgent to develop effective drugs to treat AKI.Given the important role of ROS in the occurrence and development of AKI,elimination of ROS seems to be a promising strategy for the prevention and treatment of AKI.With the emergence and rapid development of nanotechnology,nanomedicine with anti-inflammatory and antioxidant therapeutic effects has become an effective treatment for a variety of ROS-related diseases,including AKI,and is expected to have a profound impact on human health.Among them,nanozymes stand out among many drugs because of their high catalytic activity of natural enzymes and the characteristics of simulating enzyme stability and economy.Nanozymes overcome the limitation that natural enzymes can only play a catalytic role under mild conditions,and have the ability to simulate the activities of natural enzymes such as catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GPx),showing a broad spectrum of ROS scavenging ability.Hydrogen sulfide(H2S)is a ubiquitous gaseous signaling molecule that is one of three endogenous gas transmitters that play an important role in many physiological processes such as neuroregulation,vascular tone regulation,cell protection,oxygen sensing,inflammation regulation,and cell growth.Hydrogen polysulfide(H2Sn)has been found to be produced by 3-mercaptopyruvate thiotransferase(3MST)and to regulate the activity of ion channels,tumor suppressors,and protein kinases.Overvulcanization of cysteine residues of proteins is thought to be one way in which H2S acts by adding sulfur atoms to cysteine residues of target proteins to do its job.Since H2S and cysteine residues have the same oxidation state of sulfur,H2S is not easy to vulcanize cysteine residues.In contrast,H2Sn with an oxidation state of 0 or-1 readily vulcanizes cysteine-based mercaptan.Tumor suppressor protein phosphate and tensin congeners(PTEN),protein kinase G1α and glycolytic enzyme glycerol-3-phosphate dehydrogenase(GAPDH)are all targets of H2Sn.Traditional Chinese medicine is a drug that guides collection,processing and preparation,explains the mechanism of action and guides clinical application under the guidance of traditional Chinese medicine theory.Traditional Chinese medicine is mainly derived from natural medicine and its processed products,including plant medicine,animal medicine,mineral medicine and part of chemical and biological products.In addition to the ability of nanomaterials to remove ROS antioxidants,traditional Chinese medicine polyphenols can also interact with ROS to stop the chain reaction before cell viability is seriously affected.Polyphenols are natural compounds in plants,which have anti-tumor,antibacterial,antioxidant and other biological activities.Epigallocatechin-3-gallate(EGCG),the main polyphenol component of green tea,is a potent antioxidant and free radical scavenger that can be used to treat a variety of diseases.EGCG has been shown to have antiinflammatory,anti-arthritis,antibacterial,anti-angiogenesis,anti-aging,antiviral,and neuroprotective effects,and these effects may have therapeutic applications in the treatment of many diseases,including atherosclerosis and cardiovascular and metabolic diseases.EGCG can achieve antioxidant purpose by scavenging free radicals,and iron chelation is an important way of scavenging free radicals.In addition,EGCG can increase the levels of antioxidant enzymes related to oxidative stress,including superoxide dismutase(SOD),glutathione mercaptotransferase(GST),and heme oxygenase-1(HO-1),both in vivo and in vitro.Among many different catechin components,EGCG is the most effective inducer for HO-1 expression.Iron sulfide nanozymes have good free radical scavenging activity and also contain rich ferrous ions.Based on this,we combined EGCG with iron sulfide nanozymes in coordination,and utilized the chelation of EGCG with iron to play a stronger free radical scavenging and cell protection ability,which also had a better therapeutic effect on AKI.This paper is divided into two parts about studies on ROS removal by GFeSNs alone in the treatment of AKI and ROS removal by EGCG-GFeSNs in the treatment of AKI.The main contents are as follows:1.GFeSNs as reactive oxygen scavenger to alleviate acute kidney injuryDuring AKI progression,excessive production of ROS reacts with biomolecules,leading to irreversible and permanent biomolecular damage.Therefore,removal of ROS is the key to the treatment of AKI.In this chapter,we prepared four kinds of iron sulfide nanozymes(CFeSNs,DFeSNs,GFeSNs and TFeSNs)by solvothermal synthesis method.The iron sulfide nanozymes GFeSNs with small-size lamelift structure were screened by scanning electron microscopy,hydrogen polysulfide release and O2·-removal efficiency experiments.Compared with CFeSNs,DFeSNs and TFeSNs,GFeSNs can release the most hydrogen polysulfide and has the highest O2·-scavenging ability at the concentration of 10μg/mL.Therefore,we selected GFeSNs for follow-up experiments.SEM showed that the size of GFeSNs was about 200 nm and it was mainly composed of Fe and S,which was consistent with the element analysis of EDS map.XPS and XRD showed that GFeSNs mainly consisted of FeS.Then,we further tested the release ability of polysulfide and scavenging ability of various free radicals of GFeSNs.The results show that GFeSNs has strong scavenging ability on ·OH,O2·-,ABTS·+,H2O2,DPPH and other free radicals.Cell experiments showed that GFeSNs was non-toxic to NRK-52E cells and HEK293 cells at concentrations ranging from 2.5 to 20 μg/mL,and could promote cell proliferation.The results of SSP4 staining showed that GFeSNs produced a large amount of hydrogen polysulfide and entered the cells.Subsequently,we evaluated the ability of GFeSNs to clear ROS protective cells.The results showed that 10 μg/mL GFeSNs could restore the viability of cells damaged by 300 μmol/L H2O2 to 92%,and about 84%of cells could survive in the presence of 500 μmol/L H2O2.DCFH-DA staining,mitochondrial membrane potential staining and Calcein AM/PI staining also demonstrated the ability of GFeSNs to clear ROS protective cells.The levels of intracellular inflammatory factors and antioxidant levels showed that after GFeSNs treatment,the levels of intracellular inflammatory factors such as TNF-α,IL-6 and IL-1β were significantly decreased,and intracellular MDA and GSH also returned to normal levels.These results strongly suggest that GFeSNs can play a synergistic role in anti-inflammatory and ROS clearance in order to restore intracellular antioxidant capacity and play a protective role in cells.Subsequently,we explored the therapeutic effect of GFeSNs on AKI mice.The results showed that GFeSNs could significantly reduce the levels of CRE and BUN in AKI mice,continuously restore the body weight of AKI mice to normal level after a brief decrease,and significantly prolong the survival time of AKI mice.Then,tissue level factors were detected in each group of mice,and the results showed that the release of TNF-α,IL-6 and IL-1β of AKI mice was reduced by GFeSNs treatment.KIM-1 and HO-1 levels were significantly reduced in GFeSNs-treated AKI mice compared with AKI mice.At the same time,SOD,MDA,LDH,H2O2 and GSH contents at tissue level also recovered to the level of normal mice.H&E,DHE and TUNEL staining of mice showed that the damage of renal tubules in mice treated with GFeSNs was reduced,and the apoptosis of renal tissue cells and the level of intracellular superoxide were reduced,which was close to that of normal mice.Biochemical analysis of blood routine and H&E staining of major organs showed that GFeSNs had no obvious toxicity to major organs and had high biocompatibility.In addition,we analyzed AKI related protein expression by WB and found that the expression of Bax and Cleaved caspase-3 decreased in GFeSNs-treated mice,while the expression of Bcl-2 increased,indicating decreased apoptosis.Increased expression of Nrf-2 and decreased expression of Keap-1,indicating the recovery of intracellular antioxidant level.Together,these results clearly demonstrate GFeSNs’ outstanding therapeutic performance for AKI,making it a potential candidate for ROS-related diseases.2.EGCG-GFeSNs alleviates acute kidney injuryAntioxidants have a protective effect against inflammation and oxidative stress caused by AKI,and EGCG is the strongest antioxidant component of catechins.In Chapter 1,we have demonstrated that GFeSNs has excellent free radical scavenging ability and cell protection,and also has a significant therapeutic effect on AKI.In this chapter,we combined EGCG with GFeSNs.Scanning electron microscopy showed that the size and morphology of EGCG-GFeSNs did not change significantly after the combination of EGCG and GFeSNs,and the structure of EGCG-GFeSNs was still 200 nm.FT-IR spectrometer showed successful combination of EGCG and GFeSNs.Cell experiments showed that EGCGGFeSNs was non-toxic to NRK-52E cells cells at concentrations ranging from 2.5 to 20μg/mL.Subsequently,we evaluated the ability of EGCG-GFeSNs to clear ROS-protected cells.The results showed that 2.5 μg/mL EGCG-GFeSNs could restore more than 80%of the cells damaged by 300 μmol/L H2O2,and 5 μg/mL EGCG-GFeSNs could restore more than 90%of the cells damaged by 300 μmol/L H2O2.DCFH-DA staining,mitochondrial membrane potential staining and Calcein AM/PI staining also showed that EGCG-GFeSNs had better scavenging ability of ROS protective cells than GFeSNs.The hemolysis test results showed that the hemolysis rate of 80 μg/mL EGCG-GFeSNs was lower than 5%,indicating that EGCG-GFeSNs had high biocompatibility.These results all indicated that EGCG-GFeSNs had better scavenging ability of ROS and stronger protective effect on cells than GFeSNs.Next,we explored the in vivo therapeutic effect of EGCG-GFeSNs on glycerol-induced AKI mice.The results showed that 250 μg/kg EGCG-GFeSNs could significantly reduce the levels of CRE and BUN in AKI mice.The body weight of AKI mice began to rise on day 4 and continued to recover to the normal level.The survival time of AKI mice was also significantly prolonged.In contrast,after treatment with 250 μg/kg GFeSNs,CRE and BUN levels remained high in AKI mice and some AKI mice died.Then,tissue level factors were detected in each group of mice,and the results showed that the release of IL-6 and IL-1β,and the levels of KIM-1 and HO-1,markers of kidney injury,were significantly decreased in AKI mice after treatment with EGCG-GFeSNs.At the same time,SOD and GSH content in tissue level also returned to the level of normal mice.However,the renal tissue factor content in the GFeSNs treatment group was still at a higher level.H&E,DHE and TUNEL staining of mice showed that the kidney tissue damage of EGCG-GFeSNs treated mice was reduced,and the apoptosis and intracellular superoxide levels of kidney tissue were reduced,which was close to that of normal mice.H&E staining of major organs showed that EGCGGFeSNs had no obvious toxicity to major organs and had high biocompatibility.These results suggest that the combination of EGCG with GFeSNs significantly improves the scavenging ability of ROS,and also shows better therapeutic effect on AKI.