| BackgroundRadioiodine contrast agent is widely used in clinical invasive imaging examination.Contrast-induced acute kidney injury(CI-AKI)is an important complication occurring after the intravascular administration of radiographic contrast media(CM)in diagnostic and interventional procedures,and is the third most common cause of hospital-acquired acute renal failure in hospitalized patients.It prolongs the patient's hospital stay and affects the long-term prognosis.At the same time,it also increases the hospital mortality and the social medical burden.Although hydration therapy is widely used to prevent CI-AKI in clinic,the prevention and treatment of CI-AKI is still not satisfactory.Especially for elderly patients and patients with hypertension,heart failure or renal insufficiency,the use of hydration therapy is limited.In addition,hydration therapy combined with drug intervention can further reduce the incidence of CI-AKI and improve the long-term prognosis of patients.Therefore,drug research has gradually become a research hotspot of CI-AKI prevention.CI-AKI usually refers to the acute or subacute renal injury within 48 hours after the use of contrast agents,that is,serum creatinine(SCr)increases by more than 25%or more than 44.2?mol/L in the absence of any other diseases.Previous studies have confirmed that various mechanisms such as oxidative stress,apoptosis,and inflammation are involved in the occurrence and development of CI-AKI,but they have not yet been fully elucidated.The direct cytotoxic of radioiodine contrast agent on renal tubular epithelial cells and vascular endothelial cells leads to cell swelling,vacuolation,apoptosis,and finally necrosis,but the exact mechanism is still not fully elucidated.The change of renal hemodynamics and cytokines caused by iodine-CM,which result in renal medullary ischemia and hypoxia,is also one of the important mechanisms of renal injury.Oxidative stress is at the core of these mechanisms,and the role of Reactive oxygen species(ROS)produced by oxidative stress is attracting more and more attention.Excessive ROS can directly damage the renal tubular epithelial cells and adjacent cells.In addition,ROS can cause renal cell apoptosis through the activation of stress kinases and intrinsic pathway,including c-Jun N-terminal kinases,p38 MAPK stress kinases,and caspases.The response of these signal transductions to contrast media stimulation is mainly in the mitochondria,leading to permeabilization of the outer mitochondrial membrane and the release of cytochrome c and other pro-apoptotic molecules.Cytochrome c can bind to apoptotic protease activating factor-1 and promote its binding to caspase-9 to form an apoptotic complex,which could activate caspases and thereby induce apoptosis.ROS and osmotic stress induced directly by CM,as well as the damage-related molecules released by the damaged renal tubular epithelial cells,could active Nod-like receptor pyrin containing 3(NLRP3).NLRP3 recruits caspase-1 and promotes the formation of inflammasome.Activated caspase-1 then cleaves pro-IL-1? to form mature IL-1?,which would activate inflammatory cascades.These evidences suggest that renal apoptosis and inflammation may play an important role in the pathogenesis of CI-AKI.Melatonin(Mel)is a hormone,secreted by the brain's pineal gland,that regulates circadian rhythms,and it was discovered by Lerner in 1958 and isolated from the bovine pineal gland.It belongs to indole heterocyclic compounds and its chemical name is Nacetyl-5 methoxytryptamine.Melatonin is a powerful antioxidant that can scavenge free radicals.In addition,due to its highly lipophilic properties,melatonin can easily cross cell membranes and physiological barriers(such as the blood-brain barrier).Therefore,it can affect the physiological functions of various organs.Published literatures have shown that melatonin can prevent a variety of diseases,including inflammatory diseases,hypertension,ischemia/reperfusion injury,and other cardiovascular diseases.Moreover,recent studies have confirmed that the cardiovascular and renal protective effects of melatonin are mainly related to its antioxidant,anti-apoptotic and antiinflammatory properties.However,the role and mechanism of melatonin in CI-AKI are still not fully understood.Based on the above research results,this study intends to use the CI-AKI mouse model and the renal tubular epithelial cell injury model to elucidate the protective effect of melatonin on CI-AKI and explore its molecular mechanism preliminarily.Therefore,we can provide theoretical foundation for finding new opportunities and drug therapeutic targets for the prevention of acute kidney injury induced by contrast meidia in clinical practice.Objective1.To investigate the effect of melatonin on renal injury in CI-AKI mice;2.To clarify the renoprotection mechanisms of melatonin in CI-AKI mice;3.To explore the molecular mechanisms underlying the protective effect of melatonin on CI-AKI mice.Methods1.Animal modelEight-week-old 129 wild-type mice(Wild Type,WT,129S1/SVIMJ)were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.After adaptive feeding for one week,sixty male mice were randomly divided into the following four groups:(?)Control group(Con);(?)Melatonin group(Mel);(?)Acute kidney injury group(CM);(?)Acute kidney injury+Melatonin group(CM+Mel).The mice with acute kidney injury were deprived of water for 16 h in advance.Subsequently,indomethacin(10mg/kg)and N-nitroL-arginine methyl ester(L-NAME,10mg/kg)were injected intraperitoneally,and lohexol(350mg Iodine/ml)at a dose of 3.0 g lodine/kg was injected intravenously though caudal vein after 15 min.Mice in the control group were given the same volume of saline alone at each time point.30 min before the injection of iohexol through the caudal vein,the mice in melatonin group received the intraperitoneal injection of melatonin(20mg/kg).The mice were euthanized 24 h later,while blood samples from the hearts of mice were used to test renal function,and both kidney tissues were collected for histological and molecular studies.2.The measurement of serumSerum was separated from the blood samples and detected.Serum creatinine(SCr)and urea nitrogen(BUN)were measured to evaluate renal function.3.Histopathological examinationsAt the end of the experiment,the body and kidney weight of the mice were weighed.The kidney tissues of mice in each group were fixed with 4%paraformaldehyde and embedded in paraffin to prepare sections.Hematoxylin-eosin staining(H?E)was used to observe the general morphology of renal tissue,and assess the degree of tubular injury.Immunohistochemical staining was used to evaluate the expression and distribution of neutrophil gelatase-associated lipid(NGAL),Bax,Bcl2,cleave-caspase3 and Nox4 in renal tissue.4.Detection of MDA,SOD and GSH-Px in renal tissueThe levels of malondialdehyde(MDA),and the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in kidney tissues were detected according to the manufacture's instruction,respectively.5.Cell culture and treatmentRat NRK-52E cells(normal rat proximal tubular epithelial cell line)were grown in RPMI-1640 medium supplemented with 10%fetal bovine serum plus 1%streptomycin/penicillin at 37? in a 5%CO2 atmosphere.When the cells grew to about 80%confluence,the cells were pretreated with melatonin(100?mol/L)and iohexol(100 mgI/mL 4h)according to cell protocol in different groups.6.TUNEL assayFrozen sections of renal tissue were made and cell slides were fixed following the standard procedures,and then they were immersed in 0.1%Triton-100.Subsequently,30ul reaction solution was added to each sample and samples were incubated at 37? for 1 h in the dark.When the samples were sealed with antifluorescence quenching agent,the degree of nuclear DNA damage in renal tubular epithelial cells and NRK-52E cells was observed under fluorescence microscope7.Real-time quantitative RT-PCRAfter the kidney tissues and cells were collected,RNA was extracted and the mRNA expression differences were detected by reverse transcription and real-time fluorescence PCR amplification.8.ROS measurementThe ROS level in NRK-52E cells were measured using DCFH-DA.In brief,cells were pretreated with serum-free medium with/without melatonin for 1h and then stimulated with iohexol for another 4h.Subsequently,the wells were added 1 ml serumfree medium containing DCFH-DA with a final concentration of 10?mol/L,and cultured in the incubator for 40 min in the dark.The wells were washed three times with PBS and inverted fluorescence microscopy was used to observe ROS production in renal tubular epithelial cells after iohexol stimulation.9.Western blot analysisTissue and cell in different groups were collected to extract protein.After detecting the protein concentration with BCA kit,SDS-PAGE was performed before being electrotransferred onto PVDF membranes.Each membrane was blocked with 5%fat-free milk for 1 h at room temperature and then incubated overnight at 4? with specific primary antibodies.The next day,the membranes were incubated with appropriate secondary antibodies for 1.5 h at room temperature.The blots were visualized using AI680 images device and then analyzed by the Image J software.10.Statistical analysisThe experimental results were statistically analyzed by GraphPad Prism 8.0.1 software,and the data were expressed as MeanąSEM.Differences between the two groups were evaluated by student's t-test and differences among multiple groups were assessed by one-way ANOVA.P<0.05 was regarded as statistically significant.Results1.Melatonin pretreatment can ameliorate CI-AKI in miceThe renal function results showed that compared with the control group,mice in the CM group had obvious renal function damage.In addition,immunohistochemical staining showed that Neutrophil gelatase-associated lipid(NGAL)protein in the CM group,a marker of kidney injury,was also significantly higher than that in the control group.Furthermore,H?E staining showed that compared with the control group,pathological lesions were more severe in the CM group,including diffuse renal interstitial edema,severe granulation and vacuolation of renal tubular epithelial cells,and destruction of the cytoskeleton structure in renal tubular epithelial cells.Taken together,our founding indicated that the mice had serious kidney damage in CM group.Compared with CM group,when the mice were pre-administrated with melatonin,all the above cases of renal injury were significantly reduced.In brief,our data showed that melatonin has the renoprotective effect on CI-AKI model in mice.2.Melatonin pretreatment can reduce the level of oxidative stress on CI-AKI in miceCompared with control group,western blot indicated that the level of Nox4 significantly increased in CM group,and when the mice were pretreated with melatonin,the level of Nox4 was decreased.In addition,immunohistochemical staining showed that compared with the control group,the expression of Nox4 was increased in the CM group,and when the mice pretreated with melatonin,the expression of Nox4 was significantly decreased.Furthermore,compared with control group,the level of malondialdehyde(MDA)was increased,the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were decreased,and the mRNA level of catalase(CAT)was decreased.Compared with CM group,the level of MDA was decreased,the activities of SOD and GSH-Px were increased,and the mRNA expression of CAT was increased as well in CM+Mel group.These results suggested that melatonin pretreatment could reduce the oxidative stress induced by CM.3.Melatonin pretreatment reduces apoptosis of the contrast-induced renal tubular epithelial cells in miceWestern blot was performed on kidney tissue from four groups of mice.Compared with the control group,the levels of Bax and cleaved caspase-3 were increased in CM group,while the protein level of Bcl2 was significantly decreased.Compared with CM group,when the mice were given melatonin pretreatment,the levels of Bax and cleaved caspase-3 were obviously decreased,and the protein expression of Bcl2 was increased.Meanwhile,the results of immunohistochemical staining were consistent with those of western blot.In addition,TUNEL staining was performed on kidney tissue sections from four groups of mice as well.Compared with the control group,the proportion of TUNEL positive cells in the kidney tissue of the CM group was significantly increased.Compared with the CM group,the proportion of TUNEL positive cells in the kidney tissue of the CM+Mel group was significantly decreased.Our data suggested that melatonin has a renoprotection effect against apoptosis on CI-AKI in mice.4.Melatonin pretreatment alleviates inflammation of CI-AKI in miceTo explore the inflammatory effect of melatonin on CI-AKI,we measured the changes of inflammatory cytokines.Compared with the control group,the mRNA level of Interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?)and transforming growth factor?(TGF?)in the kidney tissue of CM group were significantly increased.Compared with the CM group,when the mice were pretreated with melatonin,the mRNA levels of these inflammatory cytokines were signi ficantly decreased in CM+Mel group.It indicated that melatonin has an anti-inflammatory effect on CI-AKI.5.Melatonin pretreatment mitigates the oxidative stress induced by iohexol in NRK-52E cellsWestern blot showed that compared with the control group,the expression of Nox4 in Iohexol group was significantly increased.Compared with the iohexol group,the expression of Nox4 in NRK-52E cells of Iohexol+Mel group was prominently reduced.Compared with the control group,iohexol stimulation on NRK-52E cells significantly promoted the production of the reactive oxygen species(ROS)level.Compared with the iohexol group,when cells were given melatonin pretreatment,the intracellular ROS level in Iohexol+Mel group was prominently reduced.6.Melatonin pretreatment reduces the apoptosis of NRK-52E cells administrated by iohexolCompared with the control group,western blot showed that the level of Bax and cleaved caspase-3 were significantly increased,while the expression of Bcl2 protein was significantly decreased.Compared with iohexol group,the level of Bax and cleaved caspase-3 were reduced in Iohexol+Mel group,and the expression of Bcl2 protein was increased.In addition,TUNEL staining was performed in NRK-52E cells of four groups.Compared with the control group,the TUNEL proportion of positive cells in Iohexol group was significantly increased.While,compared with the Iohexol group,the TUNEL proportion of positive cells in Iohexol+Mel group was obviously reduced.7.Melatonin pretreatment alleviates the inflammation of NRK-52E cells simulated by iohexolRT-PCR was carried out on NRK-52E cells of four group.Compared with the control group,the mRNA level of IL-1?,TNF? and TGF? in Iohexol group was prominently increased.It meaned that NRK-52E cells had inflammation.However,the mRNA levels of these inflammatory cytokines were significantly decreased after treatment with melatonin.Our founding indicated that melatonin has a renal protective effect against inflammation on NRK-52E cells induced by iohexol.8.The level of Sirt3 protein is increased in CI-AKI model,and melatonin pretreatment could enhance its expression and activityWestern blot showed that the expression of Sirt3 was increased in the CM-treated mice compared with the control group.It suggested that Sirt3 plays an important role in contrast-induced acute kidney injury in mice.In addition,when the mice were pretreated with melatonin,the expression level of Sirt3 showed a higher increasing in the CM+Mel group compared with the CM group.Furthermore,the expression level of Ac-SOD2 K68,which was used to evaluate the activity of Sirt3,was significantly decreased,indicating that the activity of Sirt3 was increased.Cell experiments came to the same conclusion as animal experiments.Our results indicated that Sirt3 plays an important role in mice of CI-AKI model,and melatonin increases its expression and activity.Conclusion1.Melatonin has a protective effect on contrast-induced acute kidney injury in mice;2.Melatonin plays a protective role in CI-AKI of mice through anti-oxidative stress,anti-apoptosis and anti-inflammatory effects;3.The molecular mechanism of melatonin renoprotection on contrast-induced acute kidney injury in mice may be achieved by the activation of Sirt3.BackgroundWith the improvement of social economy and medical conditions,the incidence of cardiovascular disease is increasing year by year.Percutaneous coronary intervention(PCI)is one of the most effective treatment of coronary atherosclerotic heart disease,so more and more patients choose PCI.In recent years,with the rapid development of interventional technology,the incidence of contrast-induced acute kidney injury(CIAKI)has increased.Some studies have reported that the incidence of CI-AKI is more than 10%.Because elderly patients mostly have renal function decline and suffer from diabetes,hypertension or other basic diseases,the incidence of CI-AKI is higher than that of other populations,and severe cases can cause acute renal failure with high mortality and poor prognosis.At present,there is still a lack of effective treatment in clinical practice,so reducing the incidence of CI-AKI has gradually attracted the attention of clinicians.CI-AKI is the most common complication of iodine-containing contrast agent,and its mechanism is very complicated,which has not been fully elucidated.Many studies have confirmed that multiple mechanisms are involved in the occurrence and development of CI-AKI,among which the most important two factors are the hypoxia injury of renal parenchyma(especially renal medulla)and the toxic effect of Contrast media(CM)on renal microvessels and renal tubules.Renal medulla ischemia and hypoxia break the balance between vasoconstrictor and vasodilator in the kidney,which causes the increase of vasoconstrictor and renal tubular necrosis,thus forming a vicious cycle.Reduced blood flow activates the release of Reactive oxygen(ROS),leading to lipid peroxidation and increased cytotoxic damage.In addition,oxidative stress,osmotic necrosis,and vacuolization,occur in renal tubular cells under the action of CM,which eventually lead to cell apoptosis.Moreover,CM can activate endogenous or mitochondrion-mediated apoptosis pathway.Bax is an important pro-apoptotic member of the Bcl2 protein family,which can lead to the release of cytochrome C,thus activating the Caspase3 pathway to promote apoptosis.Therefore,inhibition of oxidative stress and apoptosis may protect against contrast-induced acute kidney injury.Melatonin was originally known to the public as a hormone that regulates circadian rhythms.It is produced by multiple organs in the body,but is mainly synthesized and secreted by the pineal gland.Melatonin has been shown to play an important protective role in kidney disease.Studies have reported that cisplatin can induce severe acute kidney injury after the resection of pineal gland,and melatonin supplementation can reduce kidney injury.The mechanism may be related to melatonin's powerful anti-oxidative stress,anti-apoptosis and anti-inflammation effects.Recent studies have found that melatonin can not only reduces ROS,but also can upregulate the level of antioxidant enzymes and down-regulate the level of oxidationpromoting enzymes.Our previous experiments also proved that melatonin has a protective effect on contrast media-induced acute kidney injury in mice,but the specific mechanism has not been fully elucidated.Silent information adjustment factor protein 3(Sirt3)is a kind of dependence nicotinamide adenine dinucleotide(NAD+)of histone acetylation enzyme,and mainly exists in the mitochondrial matrix and the nucleus.It is related to aging,cancer,glucose metabolism,and energy homeostasis.Studies have shown that Sirt3 can maintain mitochondrial homeostasis,reduce intracellular ROS production,oxidative stress,inflammation and apoptosis.Recent literature has reported that Sirt3 can protect the heart and kidney from ischemia-reperfusion injury by enhancing mitochondrial fusion.These results suggest that SIRT3 may play an important role in CI-AKI.In conclusion,it is still unclear whether melatonin plays a protective role against contrast-induced acute kidney injury in mice by activating the expression and activity of Sirt3.Sirt3-/-and Wild type mice(WT)were used to construct the CI-AKI model to clarify the specific mechanism of melatonin's protective effect on kidney.Objective1.To determine whether Sirt3-/-mice treated with contrast agent had more severe acute kidney injury than wild-type mice;2.To investigate the mechanism of the protective effect of Sirt3 on CI-AKI induced by melatonin;3.To elucidate the protective effect of melatonin on contrast agent acute kidney injury by regulating Sirt3 signaling pathway.Methods1.Animal modelSixty 8-week-old 129 Wild type(WT)mice and Sirt3-/-mice were randomly divided into the following four groups after adaptive feeding for one week:?WT+CM group;?Sirt3-/-+CM group;?WT+CM+Mel group;?Sirt3-/-+CM+Mel group.The mouse model of acute kidney injury was achieved by injecting the contrast agent iohexol into the tail vein,and the specific method was the same as the first part of this paper.The drug intervention group received intraperitoneal injection of melatonin(20mg/kg)30 minutes before the injection of CM.The mice were euthanized 24 hours later.2.The measurement of serumSerum was separated from the blood samples and detected.Serum creatinine(SCr)and urea nitrogen(BUN)were measured to evaluate renal function.3.Histopathological examinationsAt the end of the experiment,the body and kidney weight of the mice were weighed.The kidney tissues of mice in each group were fixed with 4%paraformaldehyde and embedded in paraffin to prepare sections.Hematoxylin-eosin staining(H?E)was used to observe the general morphology of renal tissue,and assess the degree of tubular injury.Immunohistochemical staining was used to evaluate the expression and distribution of neutrophil gelatase-associated lipid(NGAL),Bax,Bcl2,cleave-caspase3 and Nox44.Detection of MDA,SOD and GSH-Px in renal tissueThe levels of malondialdehyde(MDA),and the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in kidney tissues were detected according to the manufacture's instruction,respectively.5.Cell culture and treatmentRat NRK-52E cells(normal rat proximal tubular epithelial cell line)were grown in RPMI-1640 medium supplemented with 10%fetal bovine serum plus 1%streptomycin/penicillin.Sirt3 expression was silenced by small interfering RNA.6.TUNEL assayFrozen sections of renal tissue were made and cell slides were fixed following the standard procedures,and then they were immersed in 0.1%Triton X-100.Subsequently,30?l reaction solution was added to each sample and samples were incubated at 37?for 1 h in the dark.When the samples were sealed with anti-fluorescence quenching agent,the degree of nuclear DNA damage in renal tubular epithelial cells and NRK52E cells was observed under fluorescence microscope.7.Real-time quantitative RT-PCRAfter the kidney tissues and cells were collected,RNA was extracted and the mRNA expression differences were detected by reverse transcription and real-time fluorescence PCR amplification.8.ROS measurementThe ROS level in NRK-52E cells were measured using DCFH-DA.In brief,after cell experiment,the wells were added 1 ml serum-free medium containing DCFH-DA with a final concentration of 10?mol/L,and cultured in the incubator for 40 min in the dark.Then the inverted fluorescence microscopy was used to observe ROS production in renal tubular epithelial cells after iohexol stimulation.9.Western blot analysisTissue and cell in different groups were collected to extract protein.After detecting the protein concentration with BCA kit,SDS-PAGE was performed before being electrotransferred onto PVDF membranes.Each membrane was blocked with 5%fatfree milk for 1 h at room temperature and then incubated overnight at 4? with specific primary antibodies.The next day,the membranes were incubated with appropriate secondary antibodies for 1.5 h at room temperature.The blots were visualized using AI680 images device and then analyzed by the Image J software.10.Statistical analysisThe experimental results were statistically analyzed by GraphPad Prism 8.0.1 software,and the data was expressed as MeanąSEM.Differences between the two groups were evaluated by student's t-test and differences among multiple groups were assessed by one-way ANOVA.P<0.05 was regarded as statistically significant.Results1.Sirt3 knockout can reverse the effect of melatonin on CI-AKI modelThe renal function results showed that compared with the WT group,mice in the Sirt3-/-group had obvious renal function damage.In addition,immunohistochemical staining showed that Neutrophil gelatase-associated lipid(NGAL)protein,a marker of kidney injury,was also significantly higher than that in the control group.Furthermore,H?E staining showed that compared with the WT group,pathological lesions were more severe in the Sirt3-/-group,including diffuse renal interstitial edema,severe granulation and vacuolation of renal tubular epithelial cells,destruction of the cytoskeleton structure in renal tubular epithelial cells.Taken together,our founding indicated that the mice had serious kidney damage in Sirt3-/-group.Although Sirt3-/mice were pretreated with melatonin,the kidney damage in Sirt3-/-mice was not reduced compared with WT mice.In summary,our data showed that Sirt3-/-mice have more severe acute kidney injury than WT mice and the protective effect of melatonin is weakened.2.In CI-AKI model,melatonin plays an anti-oxidative stress role by increasing the expression and activity of Sirt3Western blot analysis showed that Nox4 levels were significantly increased in Sirt3-/-mice compared to wild-type mice,however,there was no decrease in Nox4 levels in Sirt3-/-mice after both groups were treated with melatonin.In addition,compared with WT mice,the expression level of ac-SOD2 K68 in Sirt3-/-mice was significantly increased,indicating that Sirt3 activity was decreased.The decrease of acSOD2 K68 expression was not observed in Sirt3-/-mice after melatonin treatment in both groups.Meanwhile,immunohistochemical staining showed that Nox4 levels in the cortical tissues of Sirt3-/-mice were significantly higher than those of WT mice,but Nox4 levels did not decrease when melatonin was given to both groups.Moreover,the MDA content,the activities of SOD and GSH-Px and the mRNA levels of catalase(CAT)in renal cortex of each group were consistent.These indicate that the antioxidative stress effect of melatonin is absent in Sirt3-/-mice.In combination with the first part,we concluded that melatonin plays an anti-oxidative stress role by increasing the expression and activity of Sirt3.3.Sirt3 knockout can reverse the antiapoptotic effect of melatonin on contrastinduced acute kidney injury miceWestern blot analysis of renal cortical tissues in each group showed that the protein levels of Bax and cleaved caspase3 were significantly increased and Bcl2 was decreased in Sirt3-/-mice compared with WT mice.Compared with mice treated with melatonin alone,Bax and cleaved caspase3 protein levels were significantly increased and Bcl2 protein level was significantly decreased in Sirt3-/-mice.Meanwhile,the results of immunohistochemical staining of apoptosis-related proteins were consistent with those of Western blot.Furthermore,frozen sections of renal cortical tissue were stained.Compared with WT mice,the proportion of TUNEL positive cells in Sirt3-/mice was significantly increased.The proportion of TUNEL positive cells in Sirt3-/mice did not decrease after treatment with melatonin in either group.In conclusion,our data indicated that mice with Sirt3 knockout experiences severe apoptosis and the antiapoptotic effect of melatonin is weakened.4.In CI-AKI model,Sirt3 knockout can block the anti-inflammatory protective effect of melatoninWe also examined inflammatory factors in renal cortical tissues in each group after Sirt3 knockout and melatonin treatment.Compared with WT mice,mRNA levels of IL1?,TNF? and TGF? in renal cortical tissues of Sirt3-/-mice were significantly increased.Levels of these inflammatory factors did not decrease in Sirt3-/-mice after melatonin treatment in both groups.These results suggested that Sirt3 knockout increases inflammation and the anti-inflammatory protective effect of melatonin is weakened.5.Melatonin regulates the oxidative stress of NRK-52E cells induced by iohexol by increasing the expression and activity of Sirt3Western blot analysis showed that Nox4 protein level was significantly increased after Sirt3 silence in NRK-52E cells compared with that in Iohexol group.However,when the cells in both groups were pretreated with melatonin,Nox4 levels did not decrease in the Sirt3 silence group.In addition,compared with the Iohexol group,the expression level of ac-SOD2 K68 was significantly increased in the Sirt3 silence group,and the activity of Sirt3 was decreased.The expression level of ac-SOD2 K68 in Sirt3 silence group did not decrease after melatonin treatment in both groups.At the same time,ROS detection showed that the ROS level in Sirt3 silence group was significantly higher than that in Iohexol group,but when the cells in both groups were treated with melatonin,the ROS level did not decrease in the Sirt3 silence group.This indicated that Sirt3 silence weaken the anti-oxidative stress effect of melatonin.Combined with the first part of cell experiments,we concluded that melatonin plays an anti-oxidative stress role by increasing the expression and activity of Sirt3 in cells.6.Sirt3 mediates the protective effect of melatonin on iohexol-induced NRK-52E cells apoptosisWestern blot analysis showed that Bax and cleaved Caspase3 protein levels were significantly increased and Bcl2 protein levels were significantly decreased in the Sirt3 silence group compared with the Iohexol group.Compared with melatonin treatment alone,the protein levels of Bax and cleaved Caspase3 were significantly increased and the protein levels of Bcl2 were significantly decreased after Sirt3 silence.In addition,TUNEL staining showed that the proportion of TUNEL-positive cells significantly increased after Sirt3 gene silencing compared with the Iohexol group.This trend did not change after treatment with melatonin.In conclusion,the apoptosis of NRK-52E cells is increased after Sirt3 silence and the antiapoptotic effect of melatonin is wenakened on the iohexol induced NRK-52E cells injury.7.Sirt3 is involved in the anti-inflammatory effect of melatonin on iohexol-induced NRK-52E cellsRT-PCR assay showed that the mRNA levels of IL-1?,TNFa and TGF? in Sirt3 silence group were significantly increased compared with those in the Iohexol group.When the cells in both groups were treated with melatonin,Sirt3 silence did not reduce the levels of these inflammatory cytokines.These results suggested that Sirt3 silence increases the level of inflammation in mice and Sirt3 is involved in the anti-apoptosis effect of melatonin on cells.Conclusion1.Sirt3 knockout aggravates the contrast-induced acute kidney injury in mice;2.Sirt3 mediates the anti-oxidative stress,anti-apoptosis and anti-inflammatory effects of melatonin on CI-AKI model;3.The protective effect of melatonin on contrast-induced acute kidney injury is partly realized by increasing the expression and activity of Sirt3. |
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