maresin 1 mitigates high glucose-induced mouse glomerular...

Research ArticleMaresin 1 Mitigates High Glucose-Induced Mouse GlomerularMesangial Cell Injury by Inhibiting Inflammation and Fibrosis

Shi Tang12 Chenlin Gao3 Yang Long1 Wei Huang1 Jiao Chen2 Fang Fan2

Chunxia Jiang2 and Yong Xu1

1Endocrinology Department The Affiliated Hospital of Southwest Medical University Luzhou Sichuan 646000 China2The Graduate School of Southwest Medical University Luzhou Sichuan 646000 China3State Key Laboratory of Quality Research in Chinese Medicine Faculty of Chinese MedicineMacau University of Science and Technology Avenida Wai Long Taipa Macau

Correspondence should be addressed to Yong Xu xywyllaliyuncom

Received 9 October 2016 Revised 6 December 2016 Accepted 19 December 2016 Published 15 January 2017

Academic Editor Dah-Yuu Lu

Copyright copy 2017 Shi Tang et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Inflammation and fibrosis are the important pathophysiologic processes in diabetic nephropathy (DN) Maresin 1 isa potential anti-inflammatory lipid mediator which has displayed powerful proresolving activities Aim We determine whethermaresin 1 has protective effect on mouse glomerular mesangial cells (GMCs) induced by high glucose Methods We culturedGMCs stimulated by high glucose and categorized as follows normal glucose group (56mmolL) high glucose group (30mmolL)mannitol groupmaresin 1 intervention group (1 10 and 100 nmolL)maresin 1 and normal glucose group and theN-acetylcysteine(NAC) intervention group (10120583molLNAC) After 24 h the expression of ROS NLRP3 caspase-1 procaspase-1 IL-1120573 and pro-IL-1120573 was detected by western-blot RT-PCR and immunofluorescence After 48 h the expression of TGF-1205731 and FN was detectedby RT-PCR and ELISA Results Compared with normal glucose group the expression of ROS NLRP3 caspase-1 IL-1120573 TGF-1205731and FN increased in high glucose group (119875 lt 005) but it decreased after the treatment of maresin 1 in different concentrationsOn the contrary the expression of procaspase-1 and pro-IL-1120573 protein was restrained by high glucose and enhanced by maresin 1in a dose-dependent manner (119875 lt 005) Conclusion Maresin 1 can inhibit NLRP3 inflammasome TGF-1205731 and FN in GMCs itmay have protective effect on DN by mitigating the inflammation and early fibrosis

1 Introduction

Diabetic nephropathy (DN) is unique and serious diabeticmicrovascular complications which also is the leading causeof end-stage renal disease (ESRD) in the United States andother developed countries [1]Theprimary pathologicalman-ifestations are glomerular mesangial lesions and extracellularmatrix synthesis increase which would lead to glomerularsclerosis and interstitial fibrosis ultimately [2] The etiologyand pathogenesis of DN are complex and have not beencompletely clarified It has been well known that DN is a low-grade inflammatory disease Metabolic disorder and hemo-dynamics changes caused by hyperglycemia persistently cantrigger renal inflammation [3 4] Therefore how to preventDN effectively is the focus and difficulties in clinical work [5]

Reactive oxygen species (ROS) induced by sustainedhyperglycemia in renal tissues may directly or indirectly acti-vate nucleotide binding and oligomerization domainmdashlikereceptor family pyrin domain-containing 3 (NLRP3) inflam-masome NLRP3 inflammasome activates caspase-1 whichcan promote the maturation and secretion of inflammatorycytokine interleukin 1120573 (IL-1120573) and trigger a powerful andendogenous inflammatory cascade reaction which wouldcause the occurrence and development of DN [6 7] Theactivation of NLRP3 inflammasome includes two charac-teristics the upregulation of NLRP3 and the production ofcaspase-1 and IL-1120573 In macrophages NF-PB can promoteNLRP3 upregulation [8 9] The latest studies have shownthat careful targeting of NLRP3 signaling pathways could bebeneficial for the treatment of diabetes mellitus and diabetic

HindawiMediators of InflammationVolume 2017 Article ID 2438247 11 pageshttpsdoiorg10115520172438247

2 Mediators of Inflammation

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Figure 1 The mRNA expression of NLRP3 caspase-1 and IL-1120573 in mesangial cells treated with 30mmolL glucose for different time (a)RT-PCR strip chart for different time NLRP3 caspase-1 and IL-1120573 mRNA increased significantly after 6 h 12 h 24 h and 48 h of exposureto 30mmolL glucose (b) The corresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005 versus NC group

nephropathy so the NLRP3 inflammasome should be a newpotential target for the treatment of diabetic nephropathy[10ndash12] Additionally researchers have found intraperitonealinjection stephania and piperine can reduce the expression ofNLRP3 inflammasome which would inhibit the progressionof DN in diabetic rats [13]

Epithelial-mesenchymal transition (EMT) plays animportant role in the early progression of renal fibrosis inDN [14] Transforming growth factor-1205731 (TGF-1205731) which ishighly expressed in DN renal tissue is a key regulator of EMTand is one of the important marks of renal fibrosis [15] TGF-1205731 can increase the expression of fibronectin (FN) Studieshave shown that oxymatrine can prevent high glucose-induced renal EMT by inhibiting TGF-1205731Smad signalingpathway thereby alleviating early DN renal fibrosis [16]

Numerous clinical studies have shown that n-3 polyunsat-urated fatty acid can resist acute or chronic inflammation [17]Maresin 1 is one of the latest families of anti-inflammatorylipid mediator which derives from n-3 polyunsaturatedfatty acids and has displayed both anti-inflammatory andproresolving activities in zymosan-induced peritonitis [18]Researches in inflammation-related disease have proved thatmaresin 1 plays a protective role in the body by limitingneutrophil infiltration enhancing macrophage phagocytosisdecreasing the production of proinflammatory cytokinesinhibiting NF-PB activation and so on [19 20] Recentstudies show that maresin 1 not only had a significant role interms of anti-inflammatory but also restrained the progres-sion of pulmonary fibrosis by inhibiting EMT that is inducedby TGF-1205731 and by reducing the expression of FN [21] How-ever the relationship between maresin 1 and pathogenesis ofDN is unclear In this study we explored something new weobserved anti-inflammatory and antifibrosis effect ofmaresin1which is produced by inhibitingNLRP3 inflammasome acti-vation and by lessening the TGF-1205731-induced EMT on glom-erular mesangial cells (GMCs) stimulated by high glucose

2 Materials and Methods

21 Cell Culture and Stimulation Mouse glomerular mesan-gial cells (GMCs) were purchased from Chinese Academyof Sciences and cultured in low glucose Dulbeccorsquos ModifiedEagleMedium (DMEMHycloneUSA)with 10 fetal bovineserum (FBS BOVOGEN Australia) at 37∘C and 5 CO

2

GMCs were used between the 2nd and 5th passages for allexperiments To observe the effect of maresin 1 we dividedthe cells into 8 groupsA normal control group (NC group)56mmolL glucose B osmotic pressure control group (OPgroup) 56mmolL glucose + 244mmolLmannitolC highglucose group (HG group) 30mmolL glucose D differentconcentrations of maresin 1 + high glucose group (M + HGgroup) the GMCs being treated with different concentrationof maresin 1 (1 10 and 100 nmolL Cayman Chemical USA)for 30min before being exposed to high glucose (30mmolL)[22 23] 1 nmolL maresin 1 + 30mmolL glucose (M1 + HGgroup) 10 nmolL maresin 1 + 30mmolL glucose (M2 + HGgroup) 100 nmolL maresin 1 + 30mmolL glucose (M3 +HG group) E maresin 1 + normal control group (M + NCgroup) the most obvious effect of maresin 1 concentration +56mmolL glucoseFN-acetylcysteine (NAC) interventiongroup as positive control (10 120583molL NAC + HG group) [2425] Each test was independently repeated more than 3 times

22 Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from GMCs using an RNAextraction kit (Beijing Tiangen Biotech China) Total RNAwas reverse transcribed with the reverse transcriptase kit(TOYOBO Japan) All kits were used according to themanufacturerrsquos instructions RT-PCR was performed as pre-viously described [26]The annealing temperature of NLRP3caspase-1 IL-1120573 TGF-1205731 and 120573-actin was 589∘C 60∘C645∘C 598∘C and 54∘C The primer sequences (Shanghaibiological Engineering Co Ltd China) were as follows

Mediators of Inflammation 3

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Figure 2 The mRNA and protein level of NLRP3 caspase-1 procaspase-1 IL-1120573 and pro-IL-1120573 in each group (a) RT-PCR detected theexpression of NLRP3 caspase-1 and IL-1120573mRNAafter intervened by high glucose andmaresin 1 for 24 h inGMCs and themRNA expressionof NLRP3 caspase-1 and IL-1120573 increased in HG group significantly but compared with HG group it decreased obviously in M + HG groupas a concentration-dependent manner of maresin 1 (b) The corresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005versus NC group 119875 lt 005 versus HG group (c) Western-blot detected the expression NLRP3 caspase-1 procaspase-1 IL-1120573 and pro-IL-1120573 protein after intervened by high glucose and maresin 1 for 24 h in GMCs and the protein expression of NLRP3 caspase-1 and IL-1120573increased and procaspase-1 and pro-IL-1120573 decreased in HG group significantly but compared with HG group the protein expression ofNLRP3 caspase-1 and IL-1120573 decreased and procaspase-1 and pro-IL-1120573 increased obviously in M + HG group as a concentration-dependentmanner of maresin 1 (d) The corresponding relative gray value statistics graph of the protein level lowast119875 lt 005 versus NC group 119875 lt 005versus HG group

NLRP3 (Forward 51015840-AGAAGAGACCACGGCAGAAAG-31015840 Reverse 51015840-CTT GGA ACC AGG TTG AGT GT-31015840)caspase-1 (Forward 51015840-TGG AAG GTA GGC AAG ACT-31015840 Reverse 51015840-ATA GTG GGC ATC TGG GTC-31015840) IL-1120573(Forward 51015840-GTCTTTCCCGTGACCTTC-31015840 Reverse 51015840-ATC TCG GAG CCT GTT AGT GC-31015840) TGF-1205731 (Forward51015840-CTG TCC AAA CTA AGG CTC GC-31015840 Reverse 51015840-AGACAG CCA CTC AGG CGT AT-31015840) and 120573-actin (Forward51015840-ACC TCT ATG CCA ACA CAG TG-31015840 Reverse 51015840-GGACTC ATC GTA CTC CTG CT-31015840)

23 Western Blotting Total protein was extracted fromGMCs using a protein extraction kit (Roche USA) Proteinconcentrations were assessed by using BCAProtein Assay Kit(Bioworld Technology USA) The OD value was measuredat a wavelength of 562 nm the standard curve (119903 gt 099)calculated protein sample concentration Western blottingwas performed as previously described [27] Immunoblottingwas performed using NLRP3 antibody (rabbit 1 800 CSTUSA) caspase-1 antibody (rabbit 1 1000 Abcam USA)procaspase-1 antibody (rabbit 1 1000 Abcam USA) IL-1120573

4 Mediators of InflammationN

LRP3

NC HG M3 + NC

NC HG M3 + HG

M3 + HG

M3 + NC

NC HG M3 + HG M3 + NC

Casp

ase-

1IL

-1120573

Figure 3The level of NLRP3 caspase-1 and IL-1120573 in each group detected by immunofluorescenceThe expression of NLRP3 caspase-1 andIL-1120573 increased in HG group significantly but it decreased obviously in M3 + HG group and there was no difference between NC group andNC + M3 group

(rabbit 1 800 Abcam USA) pro-IL-1120573 (rabbit 1 1500Abcam USA) and GAPDH (rabbit 1 10000 BioworldTechnology USA) The second antibody of NLRP3 caspase-1 procaspase-1 IL-1120573 and pro-IL-1120573 (1 2000 anti-rabbit)was obtained from the Beyotime Institute of BiotechnologyShanghai China The proteins were detected with HRPchemiluminescence reagent (Millipore USA) and imageswere capturedwith theUVP imaging system (Bio-RadUSA)

24 ROS Assay In order to detect the ROS we used ROSdetection kit (Nanjing Jiancheng Institute of BiotechnologyChina) GMCs were grown in 6-well and 24-well platesand then were incubated with 10 120583molL DCFH-DA for 30minutes at 37∘C and 5 CO

2 After washing with PBS for

three times 6-well cells were observed and imaged by fluores-cencemicroscope In addition in order to obtain comparabledata 24-well cells were collected to detect the expressionof ROS using ROS detection kit (Nanjing Jiancheng Insti-tute of Biotechnology China) by spectrophotometer withexcitation wavelength at 488 nm and emission wavelength at525 nm

25 Enzyme-Linked Immunosorbent Assay (ELISA) GMCswere grown in 24-well plates and were cultured with different

medium at 37∘C and 5 CO2 After 48 h the levels of FN

in supernatant of GMCs for each group were determinedby using FN ELISA kits (Beijing Cheng Lin BiotechnologyChina) according to manufacturerrsquos instruction

26 Immunofluorescence GMCswere grown on coverslips in6-well plates After overnight adherence cells were incubatedwith different compounds for 24 h as described above andthen were fixed in 4 paraformaldehyde and then blockedwith 5 rabbit serum The cells were incubated overnightwith the anti-NLRP3 caspase-1 and IL-1120573 primary anti-bodies (dilution 1 50) and incubated for 60min with sec-ondary antibody conjugated to the fluorescein isothiocyanatefluorescent dye (dilution 1 100) DAPI (4101584061015840-diamino-2-phenylindole) was used to stain the nucleus in the cellsImages were taken with a laser scanning confocal microscope(Leica Germany)

27 Statistical Analysis All data are indicated as mean plusmnstandard deviation (SD) and analyzed using one-way analysisof variance (ANOVA) followed by the LSD post hoc test formultiple comparisons (SPSS 200 statistical software) 119875 lt005 was considered significant


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Figure 4The level of NLRP3 caspase-1 P-caspase-1 IL-1120573 and P-IL-1120573 in each group (a) RT-PCRdetected the expressionNLRP3 caspase-1and IL-1120573mRNA after intervened by high glucose and 100 nmolL maresin 1 in GMCs and in HG group the mRNA expression of NLRP3caspase-1 and IL-1120573 was higher than that in NC group but for M3 + HG group the expression reduced visibly compared to the HG group(b) The corresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005 versus NC group

119875 lt 005 versus HG group(c) Western-blot detected the expression of NLRP3 caspase-1 P-caspase-1 IL-1120573 and P-IL-1120573 protein after intervened by high glucose and100 nmolL maresin 1 in GMCs and the protein expression of NLRP3 caspase-1 and IL-1120573 was higher than that in NC group but for M3 +HG group the expression reduced visibly compared to the HG groupThe expression of P-caspase-1 and P-IL-1120573 was just the reverse (d)Thecorresponding relative gray value statistics graph of the protein level lowast119875 lt 005 versus NC group 119875 lt 005 versus HG group

3 Results

31 Anti-Inflammatory Effect of Maresin 1 on GMCs Inducedby High Glucose

311 The Expression of NLRP3 Caspase-1 Procaspase-1 IL-1120573 and Pro-IL-1120573 in GMCs Induced by 30mmolL Glucose forEach Group Compared with NC group the mRNA expres-sion of NLRP3 caspase-1 and IL-1120573 increased significantlyafter exposing to 30mmolL glucose for 6 h 12 h 24 h and48 h (119875 lt 005) and the expression was positively correlated

with time changes (Figures 1(a) and 1(b)) Moreover in M+ HG group for 24 h the protein and mRNA expressionof NLRP3 caspase-1 and IL-1120573 decreased but procaspase-1and pro-IL-1120573 were enhanced obviously as a concentration-dependent manner of maresin 1 compared with HG group(119875 lt 005) (Figures 2(a) 2(b) 2(c) and 2(d)) And we usedimmunofluorescence to detect the level of NLRP3 caspase-1and IL-1120573 in each group and we observed the same change(Figure 3) There were no apparent differences among NCgroup OP group and M + NC group (119875 gt 005) (Figures4(a) 4(b) 4(c) and 4(d))


NC

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Figure 5 (a) DCFH-DA detected the expression of ROS in GMC after the treatment of NAC andmaresin 1 lowast119875 lt 005 versus NC group 119875 lt005 versus HG group (b) GMCs were stained with DCFH-DA probe and images were captured using fluorescence microscope Comparedwith NC group the ROS generation increased in HG group but it decreased in NAC +HG andM +HG group in a concentration-dependentmanner

312 The ROS Level of GMCs Which Were Treated withNAC and Different Doses of Maresin 1 for 30min and WereExposed to High Glucose (30mmolL) for 24 h by Using Spec-trophotometer and Fluorescence Microscope As ROS plays animportant role in high glucose-induced damage we exploredROS level in GMCs using DCFH-DA In spectrophotometeranalysis we observed that high glucose caused a significantROS generation in GMCs (119875 lt 005) compared with NCgroup Notably ROS generationwas significantly inhibited byNACandmaresin 1 (1 10 and 100 nmolL) (119875 lt 005) and theROS level decreased in a concentration-dependentmanner ofmaresin 1 (Figure 5(a)) Meanwhile we got a similar result byusing fluorescence microscopy (Figure 5(b))

32 Antifibrosis Effect of Maresin 1 on GMCs Induced byHigh Glucose Compared with NC group the expression ofTGF-1205731 and FN upregulated in HG group for different time(119875 lt 005) especially for 48 h (Figures 6(a) 6(b) and 6(c))However it downregulated significantly in M + HG group

by a negative correlation with concentration of maresin 1(119875 lt 005) (Figures 7(a) 7(b) and 7(c)) It seemed that10 nmolL maresin 1 was effective enough for counteractingthe expression of TGF-1205731 and FN enhanced by high glucoseTherefore we chose 10 nmolL as a protective concentrationin the following experiments And then we detected that themRNA level of TGF-1205731 and the expression of FN decreasedsignificantly in M2 + HG group compared with HG group(119875 lt 005) and there was no significant difference betweenNC group and other groups (Figures 8(a) 8(b) and 8(c))

4 Discussion

Diabetic nephropathy (DN) is the common and seriousmicrovascular complications of diabetes it can be dividedinto early stage kidney disease and clinical kidney diseaseaccording to the course of the disease Once in clinical kidneydisease it will be irreversible and there is little effectivetreatment for DN Finally the patient who was tortured by


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Figure 6 The expression of TGF-1205731 and FN in mesangial cells treated with 30mmolL glucose for different time (a) RT-PCR strip chart fordifferent time Compared with NC group the expression of TGF-1205731 upregulated in HG group for different time especially for 48 h (b) Thecorresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005 versus NC group (c) FN ELISA kit detected the level of FNin supernatant of GMCs treated with 30mmolL glucose for different time Compared with NC group the expression of FN upregulated inHG group for different time especially for 48 h lowast119875 lt 005 versus NC group

DN can be kept alive only by blood purification treatmentIn western country more than 50 of all patients whoneed blood purification treatment because of renal failurewere caused by DN Therefore how to effectively delay orprevent the development of DN is a hot and difficult topicthat the researchers explored now but the mechanism ofDN is unclear Recent studies suggested that the chroniclow-level inflammation mediated by natural immune plays acentral role in the occurrence and development of DN [4]The increasing secretion of cytokine and proinflammatorymedia would activate inflammatory response in local accom-panied with mesangial extracellular matrix accumulationglomerular sclerosis and tubular interstitial fibrosis [3] Itshows that inflammatory reaction would lead to injury bythe pathological process of collagen deposition and fibrosis[3] And the persistent inflammation and fibrosis in renaltissues are an important pathophysiological basis for DN inchronic hyperglycemia condition So if we can resist thismetabolic inflammation and early renal fibrosis the renaldamage process may be delayed significantly [13 28]

Increasing evidence supported that proresolving medi-ators such as LXs resolvins and protectins can attenuatediabetes-related pathologies including kidney disease andadipose inflammation [29] Maresin 1 (7R 14S-dihydroxy-docosa-4Z 8E 10E 12Z 16Z 19Z-hexaenoic acids) producedby macrophages from docosahexaenoic acid (DHA) exertspotent proresolving and tissue homeostatic actions [30] Itis synthesised by lipoxygenase enzyme oxidation pathwayin inflammation subsidising period and conjugates triene

double bond [31] Studies have confirmed that maresin 1can suppress neutrophil infiltration and adhesion and reduceproinflammatory mediators such as TNF-120572 IL-1120573 and IL-6in mice induced by acute lung injury [32] Gong et al haveconfirmed that maresin 1 and resolvin D2 can successfullyprevent atheroprogression suggesting that resolving lipidmediators potentially represent an innovative strategy toresolve arterial inflammation [33] Meanwhile maresin 1 alsoinhibits TGF-1205731-induced EMT in alveolar type II epithelialcells by restoring epithelial marker (E-Cadherin) and inhibit-ing fibroblast phenotypes (fibronectin and a-SMA) whichalleviate progress of lung fibrosis [21] Additionally maresin1 has antioxidative and anti-inflammatory properties in CCl4induced liver injury whose possible mechanisms are partlyimplicated in its abilities to inhibit ROS generation activationof NF-PB and MAPK pathway [34] Furthermore Hong etal found maresin-like mediators (1422-dihydroxy-docosa-4Z 7Z 10Z 12E 16Z 19Z-hexaenoic acids) were producedby leukocytes and PLT and involved in wound healing byrestoring reparative functions to diabeticmacrophages (Mfs)additionally it could ameliorateMfs inflammatory activationand had the potential to suppress the chronic inflammationin diabetic wounds caused by activation of Mfs [35]

Inflammation is an innate immune response which isconsidered a common basis for diabetes Inflammasomes aremultiprotein platforms which act as cytoplasmatic sensorsfor stimulation with Pathogen Associated Molecular Pat-tern (PAMPs) and Damage-Associated Molecular Patterns(DAMPs) leading to an infectiouspathogenic or sterile


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Figure 7 The expression of TGF-1205731 and FN in each group (a) Expression of TGF-1205731 in GMCs which were treated with different does ofmaresin 1 for 30min andwere exposed to high glucose (30mmolL) for 48 hComparedwithNCgroup expression ofTGF-1205731was significantlyincreased in HG group and compared with HG group expression of TGF-1205731 was significantly lower in M + HG group and there wereeffective changes in M2 + HG group (b) The corresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005 versus NCgroup 119875 lt 005 versus HG group (c) FN ELISA kit detected the level of FN in each supernatant of GMCs we detected the similar changewith TGF-1205731 lowast119875 lt 005 versus NC group 119875 lt 005 versus HG group

inflammation [36] So far different inflammasomes havebeen identified including nucleotide-binding oligomeriza-tion domain-like receptors (NLRs) and absent in melanoma2 (AIM2) [37] NLRP3 inflammasome is the most distinctivemember in NLR family which comprises NLRP3 apoptosisassociated speck like protein (ASC) and the serine proteasecaspase-1 [37] In metabolic diseases the expression ofNLRP3 inflammasome will increase and now more andmore evidence suggests that NLRP3 inflammasome plays animportant role inmany noninfectious inflammatory diseasessuch as gout atherosclerosis and diabetes [38] The mecha-nismofNLRP3 activation and IL-1120573 secretion is not clearThesecretion of IL-1120573 needs to activate caspase-1 and dead cellreleases endogenousDAMPs activating caspase-1 to promotethe secretion of IL-1120573 by NLRP3 [33] Researches show thatin this process reactive oxygen species (ROS) formation wasalso thought to be related to the oligomerization reactionof NLRP3 and the endoplasmic reticulum was one of theimportant sources of ROS ROS can cause NLRP3 activationby resolving the interaction of protein (TXNIP) from thiore-doxin [39] Previous studies have found that sustained hyper-glycemia triggers the production of ROS which directly orby stimulating the release of thioredoxin interacting protein(TXNIP) indirectly activate NLRP3 inflammasome forma-tion Then procaspase-1 will form an active caspase-1 p1020

tetramer by self-splicing easily The activated caspase-1 canprocess pro-IL-1120573 hydrolyze into its mature form IL-1120573 andinduce inflammation cascade effect which could mediatedthe occurrence of DN [40 41] Our research demonstratedthat high glucose can increase mRNA expression of NLRP3caspase-1 and IL-1120573 inGMCs and can cause a significant ROSgeneration in GMCs (119875 lt 005) compared with NC groupThese suggest that ROS-NLRP3-IL-1120573 plays an importantrole in high glucose-induced damage in GMCs Besidesour results firstly detected that maresin 1 can inhibit theROS generation and the activation of NLRP3 inflammasomepathway that is induced by high glucose in GMCs in a dose-dependent manner These provided an important molecularand cellular foundation for elucidating the inflammatoryresponse mechanisms of DN and suggested a new target andstrategy for the prevention and treatment of DN

TGF-1205731 is a key regulator of EMT in pathological pro-cesses of glomerular sclerosis in DN which participate inthe renal fibrosis of DN commonly [14 15] Extracellularhigh glucose can be used as profibrotic factors in GMCsto promote the synthesis and secretion of TGF-1205731 andincrease the expression of FN that would prompt depositionof extracellular matrix and destruct the complete basementmembrane Finally EMT and stablemesenchymal phenotypeoccurred [42] This study in vitro found that high glucose


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Figure 8 The level of TGF-1205731 and FN in each group (a) RT-PCR detected the expression of TGF-1205731 mRNA after the treatment of maresin 1for 48 h compared with HG group TGF-1205731 mRNA was significantly decreased in M2 + HG group (b)The corresponding relative gray valuestatistics graph of the mRNA level lowast119875 lt 005 versus NC group 119875 lt 005 versus HG group (c) FN ELISA kit detected the level of FN insupernatant of GMCs after the treatment of maresin 1 lowast119875 lt 005 versus NC group 119875 lt 005 versus HG group

could upregulate the expression of TGF-1205731 and FN in GMCsin time dependence suggesting that the high glucose maypromote glomerular sclerosis and renal fibrosis in DN Whatis more maresin 1 could restrain the expression of TGF-1205731 and FN induced by high glucose in GMCs in a dose-dependent manner indicating that maresin 1 may inhibitEMT inGMCs and reduceDNglomerular fibrosisThereforewe hypothesized that maresin 1 potentially offers a thera-peutic option for better treatment of DN through its anti-inflammatory effect and inhibition of renal fibrosis Howeverthis experiment is not enough since the limit experimentaltime we failed to investigate the specific mechanism ofaction which needs the further basis experiments to prove

In conclusion maresin 1 can reduce the expression ofNLRP3 caspase-1 IL-1120573 and ROS generation that is inducedby high glucose in GMCs furthermore the increased expres-sion of TGF-1205731 and FN following with high glucose in GMCscan also be reversed And the mechanism may be relatedto the inhibition of NLRP3 inflammasome and early renalfibrosis growth factor TGF-1205731 and FN induced by EMT

So maresin 1 may have protective effect on diabeticnephropathy by mitigating the inflammation and early fibro-sis

Disclosure

Chenlin Gao is co-first author

Competing Interests

The authors declare that they have no competing interestsregarding the publication of this paper

Acknowledgments

The authors gratefully acknowledge Clinical Center Labora-tory for technical assistance The authors also thank BioMedProofreading for English expression polished

References

[1] M Y Donath ldquoTargeting inflammation in the treatment of type2 diabetes time to startrdquoNature Reviews DrugDiscovery vol 13no 6 pp 465ndash476 2014

[2] Y S Kanwar L Sun P Xie F Liu and S Chen ldquoA glimpseof various pathogenetic mechanisms of diabetic nephropathyrdquoAnnual Review of Pathology Mechanisms of Disease vol 6 pp395ndash423 2011

[3] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[4] P M Garcıa-Garcıa M A Getino-Melian V Domınguez-Pimentel and J FNavarro-Gonzalez ldquoInflammation in diabetickidney diseaserdquoWorld Journal of Diabetes vol 5 no 4 pp 431ndash443 2014


[5] S C W Tang G C W Chan and K N Lai ldquoRecent advan-ces in managing and understanding diabetic nephropathyrdquoF1000Research vol 5 Article ID F1000 Faculty Rev-1044 2016

[6] D De Nardo and E Latz ldquoNLRP3 inflammasomes link inflam-mation and metabolic diseaserdquo Trends in Immunology vol 32no 8 pp 373ndash379 2011

[7] C Wang Y Pan Q-Y Zhang F-M Wang and L-D KongldquoQuercetin and allopurinol ameliorate kidney injury in STZ-treated rats with regulation of renal NLRP3 inflammasomeactivation and lipid accumulationrdquo PLoS ONE vol 7 no 6Article ID e38285 2012

[8] C Bryant and K A Fitzgerald ldquoMolecular mechanismsinvolved in inflammasome activationrdquo Trends in Cell Biologyvol 19 no 9 pp 455ndash464 2009

[9] F G Bauernfeind G Horvath A Stutz et al ldquoCutting edgeNF-120581B activating pattern recognition and cytokine receptorslicense NLRP3 inflammasome activation by regulating NLRP3expressionrdquo Journal of Immunology vol 183 no 2 pp 787ndash7912009

[10] H-M Lee J-J Kim H J Kim M Shong B J Ku and E-K JoldquoUpregulated NLRP3 inflammasome activation in patients withtype 2 diabetesrdquo Diabetes vol 62 no 1 pp 194ndash204 2013

[11] J Wada and H Makino ldquoInnate immunity in diabetes anddiabetic nephropathyrdquo Nature Reviews Nephrology vol 12 no1 pp 13ndash26 2016

[12] Y Qiu and L Tang ldquoRoles of the NLRP3 inflammasome inthe pathogenesis of diabetic nephropathyrdquo PharmacologicalResearch vol 114 no 5 pp 251ndash264 2016

[13] Y A Samra H S Said N M Elsherbiny G I Liou M MEl-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-120581B andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash199 2016

[14] Y Li Y S Kang C Dai L P Kiss X Wen and Y LiuldquoEpithelial-to-mesenchymal transition is a potential pathwayleading to podocyte dysfunction and proteinuriardquo The Ameri-can Journal of Pathology vol 172 no 2 pp 299ndash308 2008

[15] H Y Lan ldquoDiverse roles of TGF-120573Smads in renal fibrosis andinflammationrdquo International Journal of Biological Sciences vol7 no 7 pp 1056ndash1067 2011

[16] L Liu YWang R Yan et al ldquoOxymatrine inhibits renal tubularEMT induced by high glucose via upregulation of SnoN andinhibition of TGF-1205731Smad signaling pathwayrdquo PLOSONE vol11 no 3 Article ID e0151986 2016

[17] A P Simopoulos ldquoOmega-3 fatty acids in inflammation andautoimmune diseasesrdquo Journal of the American College of Nutri-tion vol 21 no 6 pp 495ndash505 2002

[18] C N Serhan ldquoPro-resolving lipid mediators are leads for res-olution physiologyrdquo Nature vol 510 no 7503 pp 92ndash101 2014

[19] J Dalli M Zhu N A Vlasenko et al ldquoThe novel 13S14S-epoxy-maresin is converted by humanmacrophages to maresin1 (MaR1) inhibits leukotriene A

4hydrolase (LTA

4H) and shifts

macrophage phenotyperdquo FASEB Journal vol 27 no 7 pp 2573ndash2583 2013

[20] A Chatterjee A Sharma M Chen R Toy G Mottola and MS Conte ldquoThe pro-resolving lipid mediator maresin 1 (MaR1)attenuates inflammatory signaling pathways in vascular smoothmuscle and endothelial cellsrdquo PLoS ONE vol 9 no 11 ArticleID e113480 2014

[21] Y Wang R Li L Chen et al ldquoMaresin 1 inhibits epithelial-to-mesenchymal transition in vitro and attenuates bleomycininduced lung fibrosis in vivordquo Shock vol 44 no 5 pp 496ndash5022015

[22] T M Nordgren A J Heires T A Wyatt et al ldquoMaresin-1reduces the pro-inflammatory response of bronchial epithelialcells to organic dustrdquo Respiratory Research vol 14 no 1 article51 2013

[23] C N Serhan J Dalli S Karamnov et al ldquoMacrophage prore-solving mediator maresin 1 stimulates tissue regeneration andcontrols painrdquo FASEB Journal vol 26 no 4 pp 1755ndash1765 2012

[24] X Sun X Jiao Y Ma et al ldquoTrimethylamine N-oxide inducesinflammation and endothelial dysfunction in human umbil-ical vein endothelial cells via activating ROS-TXNIP-NLRP3inflammasomerdquo Biochemical and Biophysical Research Commu-nications vol 481 no 1-2 pp 63ndash70 2016

[25] A Eftekhari E Ahmadian Y Azarmi A Parvizpur HHamishehkar and M A Eghbal ldquoIn vitrovivo studies towardsmechanisms of risperidone-induced oxidative stress and theprotective role of coenzyme Q10 and N-acetylcysteinerdquo Toxicol-ogy Mechanisms and Methods vol 26 no 7 pp 1ndash9 2016

[26] L Liu C Gao G Chen et al ldquoNotch signaling moleculesactivate TGF-120573 in rat mesangial cells under high glucoseconditionsrdquo Journal of Diabetes Research vol 2013 Article ID979702 8 pages 2013

[27] C Gao G Chen L Liu et al ldquoImpact of high glucose andproteasome inhibitor MG132 on histone H2A and H2B ubi-quitination in rat glomerular mesangial cellsrdquo Journal of Dia-betes Research vol 2013 Article ID 589474 10 pages 2013

[28] X Chen D-D Wang T Wei S-M He G-Y Zhang and Q-L Wei ldquoEffects of astragalosides from Radix Astragali on highglucose-induced proliferation and extracellularmatrix accumu-lation in glomerular mesangial cellsrdquo Experimental and Thera-peutic Medicine vol 11 no 6 pp 2561ndash2566 2016

[29] E Borgeson and C Godson ldquoResolution of inflammation ther-apeutic potential of pro-resolving lipids in type 2 diabetes mel-litus and associated renal complicationsrdquo Frontiers in Immunol-ogy vol 3 article no 318 2012

[30] C N Serhan R Yang K Martinod et al ldquoMaresins novelmacrophage mediators with potent antiinflammatory andproresolving actionsrdquo Journal of Experimental Medicine vol206 no 1 pp 15ndash23 2009

[31] K Sasaki D Urabe H AraiM Arita andM Inoue ldquoTotal syn-thesis and bioactivities of two proposed structures of maresinrdquoChemistry - An Asian Journal vol 6 no 2 pp 534ndash543 2011

[32] J Viola P Lemnitzer Y Jansen et al ldquoResolving lipidmediatorsmaresin 1 and resolvin D2 prevent atheroprogression in micerdquoCirculation Research vol 119 no 9 pp 1030ndash1038 2016

[33] J Gong Z-YWuHQi et al ldquoMaresin 1mitigates LPS-inducedacute lung injury in micerdquo British Journal of Pharmacology vol171 no 14 pp 3539ndash3550 2014

[34] R Li Y Wang E Zhao et al ldquoMaresin 1 a proresolving lipidmediator mitigates carbon tetrachloride-induced liver injuryin micerdquo Oxidative Medicine and Cellular Longevity vol 2016Article ID 9203716 13 pages 2016

[35] S Hong Y Lu H Tian et al ldquoMaresin-like lipid mediatorsare produced by leukocytes and platelets and rescue reparativefunction of diabetes-impaired macrophagesrdquo Chemistry andBiology vol 21 no 10 pp 1318ndash1329 2014

[36] C Volpe P Anjos and J Nogueira-Machado ldquoInflammasomeas a new therapeutic target for diabetic complicationsrdquo RecentPatents on Endocrine Metabolic amp Immune Drug Discovery vol10 no 1 pp 56ndash62 2016

[37] K Schroder and J Tschopp ldquoThe inflammasomesrdquoCell vol 140no 6 pp 821ndash832 2010


[38] C-S Yang D-M Shin and E-K Jo ldquoThe role of NLR-relatedprotein 3 inflammasome in host defense and inflammatorydiseasesrdquo International Neurourology Journal vol 16 no 1 pp2ndash12 2012

[39] R Zhou A Tardivel B Thorens I Choi and J TschoppldquoThioredoxin-interacting protein links oxidative stress toinflammasome activationrdquo Nature Immunology vol 11 no 2pp 136ndash140 2010

[40] H L Hutton J D Ooi S R Holdsworth and A R KitchingldquoThe NLRP3 inflammasome in kidney disease and autoimmu-nityrdquo Nephrology vol 21 no 9 pp 736ndash744 2016

[41] H Feng J Gu F Gou et al ldquoHigh glucose and lipopolysaccha-ride prime NLRP3 inflammasome via ROSTXNIP pathway inmesangial cellsrdquo Journal of Diabetes Research vol 2016 ArticleID 6973175 11 pages 2016

[42] H W Schnaper T Hayashida S C Hubchak and A-CPoncelet ldquoTGF-120573 signal transduction and mesangial cell fibro-genesisrdquo American Journal of Physiology - Renal Physiology vol284 no 2 pp F243ndashF252 2003

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


NLRP3

Caspase-1

IL-1120573

120573-Actin

6h 12h 24h 48hNC OP

(a)


NCOP

0

05

1

15

Relat

ive m

RNA

expr

essio

n

6h

12h24h48h

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast lowast

(b)

Figure 1 The mRNA expression of NLRP3 caspase-1 and IL-1120573 in mesangial cells treated with 30mmolL glucose for different time (a)RT-PCR strip chart for different time NLRP3 caspase-1 and IL-1120573 mRNA increased significantly after 6 h 12 h 24 h and 48 h of exposureto 30mmolL glucose (b) The corresponding relative gray value statistics graph of the mRNA level lowast119875 lt 005 versus NC group

nephropathy so the NLRP3 inflammasome should be a newpotential target for the treatment of diabetic nephropathy[10ndash12] Additionally researchers have found intraperitonealinjection stephania and piperine can reduce the expression ofNLRP3 inflammasome which would inhibit the progressionof DN in diabetic rats [13]

Epithelial-mesenchymal transition (EMT) plays animportant role in the early progression of renal fibrosis inDN [14] Transforming growth factor-1205731 (TGF-1205731) which ishighly expressed in DN renal tissue is a key regulator of EMTand is one of the important marks of renal fibrosis [15] TGF-1205731 can increase the expression of fibronectin (FN) Studieshave shown that oxymatrine can prevent high glucose-induced renal EMT by inhibiting TGF-1205731Smad signalingpathway thereby alleviating early DN renal fibrosis [16]

Numerous clinical studies have shown that n-3 polyunsat-urated fatty acid can resist acute or chronic inflammation [17]Maresin 1 is one of the latest families of anti-inflammatorylipid mediator which derives from n-3 polyunsaturatedfatty acids and has displayed both anti-inflammatory andproresolving activities in zymosan-induced peritonitis [18]Researches in inflammation-related disease have proved thatmaresin 1 plays a protective role in the body by limitingneutrophil infiltration enhancing macrophage phagocytosisdecreasing the production of proinflammatory cytokinesinhibiting NF-PB activation and so on [19 20] Recentstudies show that maresin 1 not only had a significant role interms of anti-inflammatory but also restrained the progres-sion of pulmonary fibrosis by inhibiting EMT that is inducedby TGF-1205731 and by reducing the expression of FN [21] How-ever the relationship between maresin 1 and pathogenesis ofDN is unclear In this study we explored something new weobserved anti-inflammatory and antifibrosis effect ofmaresin1which is produced by inhibitingNLRP3 inflammasome acti-vation and by lessening the TGF-1205731-induced EMT on glom-erular mesangial cells (GMCs) stimulated by high glucose

2 Materials and Methods

21 Cell Culture and Stimulation Mouse glomerular mesan-gial cells (GMCs) were purchased from Chinese Academyof Sciences and cultured in low glucose Dulbeccorsquos ModifiedEagleMedium (DMEMHycloneUSA)with 10 fetal bovineserum (FBS BOVOGEN Australia) at 37∘C and 5 CO

2

GMCs were used between the 2nd and 5th passages for allexperiments To observe the effect of maresin 1 we dividedthe cells into 8 groupsA normal control group (NC group)56mmolL glucose B osmotic pressure control group (OPgroup) 56mmolL glucose + 244mmolLmannitolC highglucose group (HG group) 30mmolL glucose D differentconcentrations of maresin 1 + high glucose group (M + HGgroup) the GMCs being treated with different concentrationof maresin 1 (1 10 and 100 nmolL Cayman Chemical USA)for 30min before being exposed to high glucose (30mmolL)[22 23] 1 nmolL maresin 1 + 30mmolL glucose (M1 + HGgroup) 10 nmolL maresin 1 + 30mmolL glucose (M2 + HGgroup) 100 nmolL maresin 1 + 30mmolL glucose (M3 +HG group) E maresin 1 + normal control group (M + NCgroup) the most obvious effect of maresin 1 concentration +56mmolL glucoseFN-acetylcysteine (NAC) interventiongroup as positive control (10 120583molL NAC + HG group) [2425] Each test was independently repeated more than 3 times

22 Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from GMCs using an RNAextraction kit (Beijing Tiangen Biotech China) Total RNAwas reverse transcribed with the reverse transcriptase kit(TOYOBO Japan) All kits were used according to themanufacturerrsquos instructions RT-PCR was performed as pre-viously described [26]The annealing temperature of NLRP3caspase-1 IL-1120573 TGF-1205731 and 120573-actin was 589∘C 60∘C645∘C 598∘C and 54∘C The primer sequences (Shanghaibiological Engineering Co Ltd China) were as follows


NLRP3

Caspase-1

IL-1120573

120573-Actin

NC

HG

M1 +

HG

M2 +

HG

M3 +

HGOP

(a)


NCOPHG


lowastlowast

lowast

0

03

06

09

Relat

ive m

RNA

expr

essio

n

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1120573

Pro-IL-1120573

GAPDH

NC

HG

M1 +

HG

M2 +

HG

M3 +

HGOP

(c)

NCOPHG



05

1

15

2

Relat

ive p

rote

in ex

pres

sion

lowast

lowast

lowast

lowastlowast

lowast

(d)





LRP3

NC HG M3 + NC

NC HG M3 + HG

M3 + HG

M3 + NC


Casp

ase-

1IL

-1120573












NLRP3

Caspase-1

IL-1120573

120573-Actin


(a)


NCOPHG

0

03

06

09

Relat

ive m

RNA

expr

essio

n

lowast

lowast

lowast

lowast

M3 + HG

M3 + NC

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1β

Pro-IL-1120573

GAPDH


(c)


NCOPHG

M3 + HGM3 + NC

0

05

1

15

2Re

lativ

e pro

tein

expr

essio

n

lowast

lowast

lowast

lowast

lowastlowastlowast

(d)



3 Results





NC

M3

+ N

C

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NAC

+ H

G

lowast

0

2

4

6

8

10

Relat

ive R

OS

expr

essio

n

(a)

NC Blank control

HG

NAC + HG


M3 + NC

(b)





4 Discussion



TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NLRP3

Caspase-1

IL-1120573

120573-Actin

NC

HG

M1 +

HG

M2 +

HG

M3 +

HGOP

(a)


NCOPHG


lowastlowast

lowast

0

03

06

09

Relat

ive m

RNA

expr

essio

n

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1120573

Pro-IL-1120573

GAPDH

NC

HG

M1 +

HG

M2 +

HG

M3 +

HGOP

(c)

NCOPHG



05

1

15

2

Relat

ive p

rote

in ex

pres

sion

lowast

lowast

lowast

lowastlowast

lowast

(d)





LRP3

NC HG M3 + NC

NC HG M3 + HG

M3 + HG

M3 + NC


Casp

ase-

1IL

-1120573












NLRP3

Caspase-1

IL-1120573

120573-Actin


(a)


NCOPHG

0

03

06

09

Relat

ive m

RNA

expr

essio

n

lowast

lowast

lowast

lowast

M3 + HG

M3 + NC

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1β

Pro-IL-1120573

GAPDH


(c)


NCOPHG

M3 + HGM3 + NC

0

05

1

15

2Re

lativ

e pro

tein

expr

essio

n

lowast

lowast

lowast

lowast

lowastlowastlowast

(d)



3 Results





NC

M3

+ N

C

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NAC

+ H

G

lowast

0

2

4

6

8

10

Relat

ive R

OS

expr

essio

n

(a)

NC Blank control

HG

NAC + HG


M3 + NC

(b)





4 Discussion



TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LRP3

NC HG M3 + NC

NC HG M3 + HG

M3 + HG

M3 + NC


Casp

ase-

1IL

-1120573












NLRP3

Caspase-1

IL-1120573

120573-Actin


(a)


NCOPHG

0

03

06

09

Relat

ive m

RNA

expr

essio

n

lowast

lowast

lowast

lowast

M3 + HG

M3 + NC

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1β

Pro-IL-1120573

GAPDH


(c)


NCOPHG

M3 + HGM3 + NC

0

05

1

15

2Re

lativ

e pro

tein

expr

essio

n

lowast

lowast

lowast

lowast

lowastlowastlowast

(d)



3 Results





NC

M3

+ N

C

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NAC

+ H

G

lowast

0

2

4

6

8

10

Relat

ive R

OS

expr

essio

n

(a)

NC Blank control

HG

NAC + HG


M3 + NC

(b)





4 Discussion



TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NLRP3

Caspase-1

IL-1120573

120573-Actin


(a)


NCOPHG

0

03

06

09

Relat

ive m

RNA

expr

essio

n

lowast

lowast

lowast

lowast

M3 + HG

M3 + NC

(b)

NLRP3

Caspase-1

Procaspase-1

IL-1β

Pro-IL-1120573

GAPDH


(c)


NCOPHG

M3 + HGM3 + NC

0

05

1

15

2Re

lativ

e pro

tein

expr

essio

n

lowast

lowast

lowast

lowast

lowastlowastlowast

(d)



3 Results





NC

M3

+ N

C

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NAC

+ H

G

lowast

0

2

4

6

8

10

Relat

ive R

OS

expr

essio

n

(a)

NC Blank control

HG

NAC + HG


M3 + NC

(b)





4 Discussion



TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC

M3

+ N

C

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NAC

+ H

G

lowast

0

2

4

6

8

10

Relat

ive R

OS

expr

essio

n

(a)

NC Blank control

HG

NAC + HG


M3 + NC

(b)





4 Discussion



TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

rela

tive

mRN

A ex

pres

sion

lowastlowast

lowast lowast


0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN lowast lowast

lowast lowast

(c)







TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TGF-1205731

120573-Actin

OP

HG

M1

+ H

G

M2

+ H

G

M3

+ H

G

NC

(a)


05

1

15

TGF-

1205731

rela

tive m

RNA

expr

essio

n

lowast

(b)

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FV


lowast

(c)






TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TGF-1205731

120573-Actin



05

1

15

TGF-

1205731

relat

ive m

RNA

ex

pres

sion

lowast

(b)


lowast

0

03

06

09

ELIS

A d

etec

ted

the

expr

essio

n of

FN

(c)





Disclosure


Competing Interests


Acknowledgments


References





















4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































4hydrolase (LTA

4H) and shifts































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















maresin 1 mitigates high glucose-induced mouse glomerular...

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