Yayi HuangI, Fang ZhouI, Chen ShenII, Huaxin WangI, Yeda XiaoIII

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling

pathway1

ORIGINAL ARTICLE Experimental Surgery

Acta Cir Bras. 2019;34(1):e20190010000003

Abstract

Purpose: To investigate the influence of lycium barbarum polysaccharides (LBP), a functional derivative from lycium barbarum, on septic kidney injury. Methods: The SD male rats were randomly divided into 8 groups. The concentration of IL-1β, IL-6, IL-8, TNF-α, NF-κB and ROS, in kidney cortex homogenates after 12 h treatments were determined by enzyme-linked immunosorbent assay and ROS test kit, respectively. Morphology observation of kidney tissue was conducted with HE staining. The mRNA and protein expression levels of Nrf2, HO-1, NQO1, NF-κB, and Keap1 in kidney tissues were determined by qRT-PCR and Western blot, respectively. Results: LPS treatment significantly increased the oxidative stress. After LBP treatment, the ROS content reduced significantly in a dose-depend manner. However, the levels of HO-1, NQO1 and Nrf2 as molecular elements that respond to oxidative stress were further increased. Also, administration of LBP increased the levels of NF-κB and Keap1, and decreased the levels of Nrf2 in the Keap 1-Nrf2∕ARE signaling pathway. By administrating the brusatol, the inhibition of Nrf2 enhanced the expression of NF-κB, inhibits the antioxidant responses, and further reverse the protective effect of LBP on the LPS induced septic kidney injury.Conclusion: Lycium barbarum polysaccharides can reduce inflammation and activate the antioxidant responses via regulating the level of pro-inflammatory cytokines and the Keap1-Nrf2/ARE signaling pathway.Key words: Antioxidant Response Elements. Lycium. Kelch-Like ECH-Associated Protein 1. Rats.

DOI: http://dx.doi.org/10.1590/s0102-865020190010000003

IPhD, Department of Anesthesiology, Renmin Hospital of Wuhan University, China. Conception and design of the study, manuscript preparation and writing.IIGraduate student, Department of Anesthesiology, Renmin Hospital of Wuhan University, China. Acquisition of data, technical procedures.IIIPhD, Department of Anesthesiology, Renmin Hospital of Wuhan University, China. Technical procedures, final approval.

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LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway Huang Y et al.

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In this study,we found the protectiveeffect of LBP treatment in septic rat kidney.LBPcanup-regulatethelevelofNrf2andpro-inflammatorycytokines,andfurtherregulatingthe Keap1-Nrf2/ARE signaling pathway,consequently activating the antioxidantresponsesandreducinginflammation.

■ Methods

Sepsis animal model

AllexperimentswereapprovedbytheAnimal Care and Use Committee of RenminHospital of Wuhan University following thePrinciples of Laboratory Animal Care (NIHpublicationno.85-23,revised1985). SDmalerats(weight220-240g;n=48;6 ineachgroup) recruited in this studywereobtained from Shanghai Laboratory AnimalCenter.Afterfeedingoneweek,theratswererandomly divided into 8 groups: (1) Normalcontrolgroup(Con) :normal feeding; (2)LPSmodel group (LPS): intraperitoneal injectionwith LPS (5 mg/kg); (3) Ulinastatin group(ULI): intravenous injection with Ulinastatin(10000 U/kg); (4) Low LBP dose group (LBP-1):givingintragastricadministrationwith200mg/kgLBP1hafterLPS injection;(5)MiddleLBP dose group (LBP-2): giving intragastricadministrationwith400mg/kg LBP1h afterLPS injection; (6) High LBP dose group (LBP-3):givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection;(7)Brusatolgroup:intraperitonealinjectionwithLPS(800mg/kg) together with 2 mg/kg of brusatol,then LBP was administrated 1 h after; (8)DMSOcontrolgroup (DMSO): intraperitonealinjectionwithDMSO.

Specimen collection

The experimental rats after 12h

■ Introduction

Sepsisisakindofsystemicinflammatoryresponsesyndrome(SIRS),mainly infectedbybacteria,fungi,virusesandparasites1.TheSIRScausedbysepsismayincurfunctiondamageofsystemicmultipleorgan,likesepticacutekidneyinjury(AKI)2.Nuclearfactor-erythroid2relatedfactor 2 (Nrf2) is an important transcriptionfactorinoxidativestressresponse.Itregulatesantioxidant response elements (AREs)-mediatedepressionofantioxidantenzymeandcytoprotective proteins. Moreover, multiplestudies have demonstrated that Nrf2-Keap1(Kelch-like ECH-associated protein 1)-AREsignal pathway involves in the endogenousantioxidant defensemechanisms and it is animportantsignalingpathwayinoxidativestresssystem3. Lycium barbarum polysaccharides(LBPs) are functional derivative from Lyciumbarbarum. The therapeutic effects of LBPon human health has been explored in thepast years. For example, LBPs have beenreported to inhibit cell proliferation, induceapoptosis, and interrupt intracellular calciumbalance of cancer cells4. Also, it has beendemonstratedthatLBPsexhibitanti-agingandneuroprotective functions both in vitro andin vivo5,6 In addition, as a potent antioxidant,LBPs have been shown to up-regulate thelevel of antioxidant biomarkers in humanserum7and protect the body from chemical/exercise-induced oxidative stress8,9. Recently,Kawara et al.10reportedthatshikoninamajorcomponentofzicao(aChineseherbalmedicinewith various biological activities) possessesantioxidant effects via activation of Nrf2against lipopolysaccharide (LPS)-induced AKIinamurinemodel.However,whetherLBPhasprotectiveeffectson inflammatory injuryandoxidativestressreactionofsepticratkidneyisstillunclear.

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway

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of treatments were anaesthetized byintraperitoneal injection with 7% chloralhydrate.Anesthesiaratswerethenfixedontheoperating table in supine position. UnilateralkidneysoftheSDratswereseparatedtofixwithformalin,then gradient alcohol dehydrationandembedding.Theothersideofkidneywereusedforthepreparationoftissuehomogenateand the extraction of protein and RNA. Also,bloodwascollectedandheparinized,andthenbloodureanitrogen(BUN)andcreatininewereanalyzedusingReflotronPlusClinicalChemistryAnalyzer(Roche,USA).

Enzyme-linked immunosorbent assay (ELISA)

Inflammatorycytokines(IL-1β, IL-6, IL-8,TNF-αandNF-κB)weredeterminedinkidneycortexhomogenatesafter12htreatmentsbyusing specific kits (Invitrogen,USA) accordingtothemanufacturer’sinstructions.

Determination the ROS content in kidney tissues

ROS test kit (Beyotime, China) wasusedtodeterminatetheROScontentinkidneycortex homogenates. ROS formation wasquantifiedandexpressedaspmolDCF/min/mgprotein.

HE staining and morphology observation of kidney tissue

The unilateral kidney sections of theSD rats were stained with HE (hematoxylin-eosin) staining. The pathological changes ofkidney tissue were observed under opticalmicroscope. The kidney injury scores weredeterminedby lightmicroscopyona scaleof0–5:0=normalhistology;1=degenerationonlywithoutnecrosis;and2 (<25%),3 (<50%),4

(<75%),and5(>75%)=necrosis,degeneration,regeneration,tubulardilatation,proteincasts,andinterstitiallymphocyticinfiltrationlevelsoftheproximalconvolutedtubules.

Immunohistochemical study

The unilateral kidney sections wereincubatedwithanti-Nrf2monoclonalantibody(1 : 100; Santa Cruz, USA), followed by theincubationwithgoatanti-rabbitIgGsecondaryantibody (Santa Cruz, USA). The immunereaction resulted in the oxidation of the3,3′-diaminobenzidine by peroxidase (DAKO,USA) into an insoluble brown precipitate.The positive cellswere visualized as a brownstaining.

Quantitative real-time reverse transcription-PCR (qRT-PCR)

Total RNA of kidney tissue in SD ratswasextractedusingtheTRIzolreagent(Takara,China), according to the manufacturer’sinstructions.cDNAwassynthesizedfrom1μgof total RNA using PrimeScript™ RT MasterMix (Takara, China). RT-PCR was performedin a CFX96TM real-time detection system (ABIPrism 7500) with a total volume of 20 μL,containing 10 μL 2 × TransStart Tip GreenqPCRSuperMix (Trans), 1μLofeachprimer,1 μL cDNA templates and 7 μL ddH2O. Thespecific primer sequences are listed in Table1.Theamplificationconditionsareasfollows:initial denaturation at 94°C for 30s; followedby 40 cycles of denaturation at 94°C for 5s,annealingat56°Cfor15s;andafinalextensionat 72°C for 10s. The relative expression levelwasdeterminedusingthe2−ΔΔCtmethod.Eachtreatmentwasrepeatedintriplicates.Thedatawerepresentedasthemeans±standarderror(S.E).

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Table 1 - Primersequencesofthespecificgenes.Target genes Sequences (5′→3′)HO-1 Forward:CTGGAAGAGGAGATAGAGC Reverse:CTGGTGTGTAAGGGATGGNQO1 Forward:AACGACATCACAGGGGAG Reverse:GCACCCCAAACCAATACANF-κB Forward:ACGATCTGTTTCCCCTCATCT Reverse:TGCTTCTCTCCCCAGGAATAKeap1 Forward:TGCTCAACCGCTTGCTGTATG Reverse:CCAAGTGCTTCAGCAGGTACANrf2 Forward:GCCAGCTGAACTCCTTAGAC Reverse:GATTCGTGCACAGCAGCAGAPDH Forward:TTCAATGGCACAGTCAAGGC Reverse:TCACCCCATTTGATGTTAGCG

Western blot analysis

The total proteins from kidney tissuewere extracted using the Cytoplasmic andNuclearProteinExtractionKit (ProMab,USA),and the the protein concentrations weredetermined using the BCA method (ThermoFisher Scientific,USA). Then, protein samplesweresubjectedtoSDS-PAGEandtransferredtoapolyvinylidenedifluoride(PVDF)membrane.AfterblockingwithPBST(PBScontaining0.1%Tween-20) containing 5% non-fat milk, themembranes were washed for 3 times withPBST,andthenincubatedwithrabbitantiNrf2,HO-1, NQO1, NF-κB, Keap1 of ratantibodies(1:500 dilution; Beyotime, China) for 1 h atroom temperature (RT). After 3 more timesof washing with PBST, the membranes wereincubated with goat anti-rabbit IgG (1:2000dilution; Beyotime, China) for 1 h at RT. Theimmunoblot signalwasdetectedusingPierceECLWesternBlottingSubstrate(ThermoFisherScientific,USA).

Statistical analysis

Data were compared by one-wayanalysis of variance (ANOVA), followed byTukey’stestwithSPSS18.0statisticalsoftware.Alldatawererepresentedas themean±S.E.Differenceswere considered significantwhenP<0.05.

■ Results

Influence of LBP on the expressions of immune factors in sepsis rat kidney

To investigate the inflammatoryresponseoftheLPSinducedsepsisrattoLBPtreatment, inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α and NF-κB) were determinedin kidney cortex homogenates after 12htreatment through the Enzyme-LinkedImmunosorbent Assay (ELISA). The result(Fig.1A-E)showedthattheconcentrationofIL-1β, IL-6, IL-8, TNF-α and NF-κB increasedsignificantlyafterinjectionwithLPS,comparedwith the control (P<0.01). Ulinastatin is aserineproteaseinhibitor,whichcandecreaseinflammatory cytokine levels and mortalityin experimental sepsis11. Therefore, wefurtherconductintravenousadministrationofulinastatintotherat.Theresultshowedthatthe expression level of all the five cytokinessignificantly decreased (P<0.01, versus the LPSgroup).Consistently,afterdifferentdosesof LBP injection, the expression levels of IL-1β, IL-6, IL-8, TNF-α and NF-κB presented asignificant reduction (P<0.01, versus the LPSgroup) ina concentrationdependedmanner.All these results indicate that the LBP exertsaprotectiveeffectonthekidneyofthesepsisinducedrat.

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway

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Figure 1 -InfluenceofLBPontheexpressionsofimmunefactorsinsepsisinducedratkidney.(A)IL-1β;(B)IL-6;(C)IL-8;(D)NF-κB;(E)TNF-α.**,P<0.01,versusthecontrolgroup;##,P<0.01,versustheLPSgroup.Normalcontrolgroup(Con):normalfeeding;LPSmodelgroup(LPS): intraperitoneal injectionwithLPS(5mg/kg);Ulinastatingroup(ULI):intravenousinjectionwithUlinastatin(10000U/kg);LBP-1group:givingintragastricadministrationwith200mg/kgLBP1hafterLPSinjection;LBP-2group:givingintragastricadministrationwith400mg/kgLBP1hafterLPSinjection;LBP-3group:givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection.

LBP has a protective role against LPS induced septic kidney injury

In order to understand the functionalandpathologicalchangesofkidneytissueafterLBPintervention,theserumBUNandCrwereanalyzedat theendof the treatmentperiod,and HE stainingwas utilized to observed theunilateral kidneysectionsof theSD ratsafter12hofintervention.AsshowninFigure2(A,B),theconcentrationofBUNandcreatinineraiseddramatically after LPS treatment (P<0.001,versusthecontrolgroup),indicatingthatkidneyfunction was declined. Consistently, our HEstainingresults(Fig.2C,D)showedthat,inthecontrol group, normal organization structure

was observed in rat kidney tissue and therewerenoobviousabnormalchanges.However,lots of inflammation cells aggregation andcellularswellingandinfiltrationwereobservedin the kidney tissue after 12h post-injectionof LPS. As expected, in the LBP interventiongroups, the concentration of BUN andcreatininereducedsignificantly(P<0.05,versus the LPS group) in a concentrationdependentmanner (Fig. 2 A,B). Also, in the HE stainingresults, cellular edema, structural disorder,and inflammatory cell infiltration can still beobserved,neverthelessmuchlessthantheLPSgroup (Fig.2C,D).These results indicate thatadministration of LBP could improve kidneytissueinjuryofsepticrats.

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway Huang Y et al.

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Figure 2 - Function and Pathological morphology observation of kidney tissue among groups. (A) Bloodureanitrogen(BUN)and(B)creatininelevelsinheparinizedratbloodsamples.(C)Pathologicalmorphologyobservation of kidney tissue. (D) The kidney injury scores determined by lightmicroscopy on a scale of0–5.**,P<0.01,versus thecontrolgroup;##,P<0.01,versus theLPSgroup.Normalcontrolgroup (Con):normalfeeding;LPSmodelgroup(LPS):intraperitonealinjectionwithLPS(5mg/kg);Ulinastatingroup(ULI):intravenousinjectionwithUlinastatin(10000U/kg);LBP-1group:givingintragastricadministrationwith200mg/kgLBP1hafterLPSinjection;LBP-2group:givingintragastricadministrationwith400mg/kgLBP1hafterLPSinjection;LBP-3group:givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection.

Effect of LBP on the antioxidant response in LPS induced septic kidney

To evaluate the role of LBP on theoxidative stress,we evaluated the content ofROSinkidneyhomogenates.TheresultshowedthatthecontentofROSinLPSgroupincreasedsignificantly compared with the control.After different doses of LBP treatments, theROS content reduced significantly in a dose-dependmanner(Fig.3A-a).Tofurtherconfirmthe role of LBP on antioxidant response, weevaluated themRNAandprotein expressionsof HO-1, NQO1, Nrf2 as molecular elementsthat respond to oxidative stress. The resultshowedthatcomparedwiththecontrolgroup,the mRNA (Fig. 3A) and protein (Fig. 3 C,D)expression levels of HO-1, NQO1, Nrf2 afteradministrationofLPSwas increased (P<0.01).

AfterLBPintervention,theexpressionlevelsofHO-1,NQO1andNrf2werefurther increasedwith the increaseof LBPdoses (P<0.01whentreatedwith 400mg/kg and 800mg/kg LBP,versus the LPS group). These results indicatethat LBP treatment can protect the septickidney injury by activating the antioxidantresponses.

The effect of LBP on the Keap1- Nrf2∕ARE signaling pathway

To further investigate the effectmechanism of LBP on the Keap1-Nrf2∕AREsignaling pathway, qRT-PCR andwestern blotwere utilized to determinate the expressionlevelofNF-κBandKeap1inkidneyhomogenatesamong groups. The qRT-PCR results showedthat compared with the control group, the

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway

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mRNAlevelofNF-κBandKeap1afterinjectionLPS significantly increased (P<0.01), whileLBP treatment abolished these increases in a

dose-dependentmanner (Fig. 3B). ConsistentresultswerealsoobservedbytheWesternblot(Fig.3C,D).

Figure 3 -LBPtreatmentprotectstheseptickidneyinjurybyactivatingtheantioxidantresponsesthroughKeap 1-Nrf2∕ARE signaling pathway. (A) Effect of LBP on the antioxidant response in LPS induced septickidney.(B)EffectofLBPontheKeap1-Nrf2∕AREsignalingpathwayrelatedgenes.(C)WesternblotanalysisofantioxidantresponsesandKeap1-Nrf2∕AREsignalingpathwayrelatedproteins.(D)QuantitativeanalysisoftheWesternblotresults.**,P<0.01,versusthecontrolgroup;##,P<0.01,versustheLPSgroup.Normalcontrolgroup(Con):normal feeding;LPSmodelgroup(LPS): intraperitoneal injectionwithLPS(5mg/kg);Ulinastatingroup(ULI):intravenousinjectionwithUlinastatin(10000U/kg);LBP-1group:givingintragastricadministrationwith200mg/kgLBP1hafterLPSinjection;LBP-2group:givingintragastricadministrationwith400mg/kgLBP1hafterLPSinjection;LBP-3group:givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection.

LBP treatment increases the Nrf2 accumulation in nucleus by activating Keap1-Nrf2∕ARE signaling pathway

InordertofurtherconfirmtheroleofLBPontheKeap1-Nrf2∕AREsignalingpathway,

thebrusatol,aninhibitorofNrf2,wasappliedin this study. As shown in Figure 4A, theimmunohistochemical study demonstratedthat the LBP treatment could dramaticallyincrease thepercentageofNrf2positivecellswhencomparedwiththeLPSgroup(P<0.001),

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which can be successfully reversed by thebrusatol treatment. Considering the nuclearaccumulationofNrf2duringKeap1-Nrf2∕AREsignaling pathway activation, we furtherdetermined the protein levels of Nrf2 in the

nucleus.The result (Fig.4B) showedthatLBPtreatment could significantly increase thelevel of Nrf2 in the nucleus compared withthe LPS group (P<0.001), consistently, whichwasreversedbythebrusatoltreatment.

Figure 4 - LBPtreatmentincreasestheNrf2accumulationinnucleusbyactivatingKeap1-Nrf2∕AREsignalingpathway.(A)ImmunohistochemicalstudyofNrf2positivecellsinkidneytissueafterdifferenttreatments.(B)NuclearaccumulationofNrf2amongdifferentgroupsdeterminedbyWesternblot.***,P<0.001,versustheDMSOgroup;###,P<0.001,versustheLPSgroup;ΔΔΔ,P<0.001,versustheLBPgroup.DMSOcontrolgroup(DMSO):intraperitonealinjectionwithDMSO.LPSmodelgroup(LPS):intraperitonealinjectionwithLPS(5mg/kg);LBP-3group:givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection;Brusatolgroup:intraperitonealinjectionwithLPStogetherwith2mg/kgofbrusatol,thenLBPwasadministrated1hafter.

Nrf2 inhibition by brusatol alleviates the antioxidant response induced by LBP

Moreover, the antioxidant responsesinducedbyLBPafterNrf2inhibitionbybrusatolwerefurther investigated.Asshown inFigure5A the content of ROS in brusatol treatedgroup increased significantly compared withtheLBP-3control.Consistently,themRNAandproteinexpressionsofHO-1,NQO1,Nrf2after

administration of brusatol were decreased(P<0.01;Fig.5).Moreover,comparedwiththeLBP-3group,thegeneexpressionlevelsofNrf2and Keap1 after brusatol treatment showednosignificantdifferencewhencomparedwiththe LBP-3 group. In addition, for the proteinexpressionlevelsofNrf2andKeap1,wefoundthat brusatol treatment has no significanteffectontheKeap1expression,whiletheNrf2expressionwassignificantly inhibited(P<0.01,

LBP reduces theinflammatory injuryof kidney in septic rat and regulates the Keap1-Nrf2∕ARE signaling pathway

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versus the LBP-3 group). All these resultsindicate that Nrf2 inhibition by brusatol caninhibittheKeap1-Nrf2∕AREsignalingpathway,

and increase the expression of NF-κB, thenfurtherinhibitstheantioxidantresponses.

Figure 5 -Nrf2inhibitionbybrusatolalleviatestheantioxidantresponseinducedbyLBP(A)Effectofbrusatolon theantioxidant response in LPS induced septickidney. (B) Effectofbrusatolon theKeap1-Nrf2∕AREsignalingpathwayrelatedgenes.(C)WesternblotanalysisofantioxidantresponsesandKeap1-Nrf2∕AREsignalingpathwayrelatedproteins.(D)QuantitativeanalysisoftheWesternblotresults.**,P<0.01,versus theDMSOgroup;##,P<0.01,versus theLPSgroup;ΔΔ,P<0.01,versustheLBPgroup.DMSOcontrolgroup(DMSO):intraperitonealinjectionwithDMSO.LPSmodelgroup(LPS):intraperitonealinjectionwithLPS(5mg/kg);LBP-3group:givingintragastricadministrationwith800mg/kgLBP1hafterLPSinjection;Brusatolgroup:intraperitonealinjectionwithLPStogetherwith2mg/kgofbrusatol,thenLBPwasadministrated1hafter.

Nrf2 inhibition by brusatol reverses the protective effect of LBP on the LPS induced septic kidney injury

The ELISA results showed that theinflammatory cytokines (IL-1β, IL-6, IL-8,

TNF-α and NF-κB) concentrations increasedsignificantly after the treatment of brusatol,compared with the LBP-3 group (P<0.01;Fig. 6 A-E). Also, the concentration of BUNand creatinine raised dramatically afterbrusatol treatment (P<0.01, versus the

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LBP-3group;Fig.6F,G),indicatingthatkidneyfunctionwas declined by brusatol treatment.Consistently,comparedwiththeLBP-3group,HE staining showed that more inflammationcells aggregation and cellular swelling and

infiltrationwereobservedinthekidneytissueafter brusatol treatment (Fig. 6H). The resultindicates thatNrf2 inhibition by brusatol canreversetheprotectiveeffectofLBPontheLPSinducedseptickidneyinjury.

Figure 6 -Nrf2inhibitionbybrusatolreversestheprotectiveeffectofLBPontheLPSinducedseptickidneyinjury. (A-E) Influence of brusatol on the expressions of immune factors in sepsis induced rat kidney. (F)Pathologicalmorphologyobservationandscoringofkidneytissueamonggroups.**,P<0.01,versustheDMSOgroup;##,P<0.01,versus theLPSgroup;ΔΔ,P<0.01,versustheLBPgroup.DMSOcontrolgroup(DMSO):intraperitoneal injectionwithDMSO.LPSmodelgroup(LPS): intraperitoneal injectionwithLPS(5mg/kg);LBP-3group:giving intragastricadministrationwith800mg/kgLBP1hafterLPS injection;Brusatolgroup:intraperitonealinjectionwithLPStogetherwith2mg/kgofbrusatol,thenLBPwasadministrated1hafter.

■ Discussion

Inflammatory injury and oxidative-stressareimportantfactorsofthepathogenesisof sepsis and tissue damage12,13. Our studyfoundthattheconcentrationofIL-1β,IL-6,IL-8,TNF-αandNF-κBinratkidneytissuesincreasedsignificantly at 12h after injection with LPS,however,afterdifferentdosesofLBPinjection,

the concentration of these inflammatorycytokines presented a significant reduction.These findings suggest that LBP treatmentcanplayaprotectiveroleontheinflammationdamageofsepticratkidney. LBPhasbeenshownefficientindiabetickidney disease models; diabetic rats treatedwith LBP (10 mg/kg) for 8 weeks showedincreasedactivityofantioxidantenzymesand

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increased scavenging of oxygen radicals14.Recently,Kawaraetal.reportedthatoxidativedamage caused by LPS induced AKI may beregulated through theNrf2 signal pathway10.Nrf2 is a nuclear transcription factor whichis related to oxidative stress reaction andplays an important role in cell defense andprotection. Nrf2 mainly expresses in lung,kidney, small intestine and other organs,always accompanied by the occurrence ofdetoxification reaction15. Nrf2 regulates theexpressionofdownstreamgenesthatencodeantioxidantproteinandphaseIIdetoxificationenzyme through interaction with the Keap1and antioxidant response element (ARE),therebyplayingaroleincellprotection.Nrf2-Keap1/ARE is an important signal pathwayof oxidative stress system. In physiologicalstate, Nrf2, combination with Keap1 in thecytoplasm,isinarelativeinhibitorystateandwould not activate the downstream signal14.With oxidative stress stimulated by ROS andelectronic,Nrf2andKeap1aredissociatedandNrf2istransferredtothenucleustocombinewith ARE, thus, promoting the transcriptionand expression of ARE downstream genes,such as antioxidant protein or pro-oxidantprotein synthetase, to resist the destructivestimulus15. In this study, we found that thecontent of ROS, as well as the expressionsof HO-1, NQO1, Nrf2 asmolecular elementsthatrespondtooxidativestress,inLPSgroupincreasedobviouslycomparedwiththecontrol.Moreinterestingly,theexpressionlevelofNF-κBandKeap1intheKeap1-Nrf2∕AREsignalingpathwayincreasedsignificantlyafterinjectionLPS. All these indicate that the oxidativestress stimulated by LPS could regulate theNrf2-Keap1 dissociation and then promotethe transcription and expression of thedownstreamantioxidantprotein.With these,we further evaluated the role of LBP on theoxidativestress.AfterLBPtreatment,theROScontent, aswell as the expressions of NF-κB

and Keap1, reduced significantly in a dose-depend manner. However, the expressionlevels ofHO-1,NQO1 andNrf2were furtherincreased,whichindicatesthatLBPtreatmentcan protect the septic kidney injury byactivating the antioxidant responses throughKeap1-Nrf2∕AREsignalingpathway. Brusatol is isolated from the Bruceajavanicaplant. Ithasbeenshowntoserveasan inhibitor ofNrf2 signaling16. The result ofthis study showed that brusatol treatmentsignificantly increasedthecontentofROS,aswell as the expression of NF-κB, comparedwiththeLBPtreatedcontrol.Asexpected,theexpressionsofHO-1,NQO1afteradministrationofbrusatolwerealsodecreased.Unexpectedly,thegeneexpressionlevelsofNrf2andKeap1afterbrusatoltreatmentshowednosignificantdifference when compared with the LBP-3 group. For the protein expression levelsof Nrf2 and Keap1, we found that brusatoltreatment has no significant effect on theKeap1 expression, while the Nrf2 expressionwas significantly inhibited. This findingsuggests that brusatol depletes Nrf2 proteinthroughapost-transcriptionalmechanismandthrough a Keap1-independent mechanism,which is consistent with the report byOlayanjuet al.17.Alltheseresultsindicatethatinhibiting Nrf2 can increase the expressionof NF-κB, and further inhibit the antioxidantresponses.Furthermore,areversedeffectoninflammatory cytokines concentrations wasalsoobservedcomparedwith theLBPgroup.Complemented with the HE staining results,wefoundthatNrf2inhibitionbybrusatolcanreversetheprotectiveeffectofLBPontheLPSinduced septickidneyinjury.

■ Conclusions

Takentogether,ourresultssuggestthatLBPcanup-regulatethelevelofNrf2anddown-regulate the pro-inflammatory cytokines,

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and further regulating the Keap1-Nrf2/AREsignaling pathway, consequently activatingthe antioxidant responses and reducinginflammation.Alongwithin-depthresearchofsepsisandinflammatoryinjury,themechanismbywhichhowtoreducetheoxidativestressinsepsisdamagetothebodyisstillnotveryclear.Therefore, further studies and developmentarestillneeded,whichcouldprovideabetterfoundation for clinical treatment of septickidneyinjury.

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Correspondence:Yeda XiaoDepartmentofAnesthesiology,RenminHospitalofWuhanUniversity99ZiYangRoad,WuhanHubei430060ChinaPhone:86+02788041911Xiaoyedakaj@163.com

Received:Sep08,2018Review:Nov10,2018Accepted:Dec12,2018

Conflictofinterest:noneFinancialsource:none

1Research performed at Department ofAnesthesiology, Renmin Hospital of WuhanUniversity,China.