Protective Effect Of Hyperoside On LPS-induced Acute Kidney Injury And The Underlying Mechanisms

BackgroundAcute kidney injury(AKI),as a serious disease,suffered about one in five patients in emergency cases.In hospitalized patients,AKI becomes a common and dangerous factor for death progressively.AKI can be caused by variety factors,such as pharmacologic toxins and sepsis.LPS,the outer membrane component of gram-negative bacteria,has been identifid as the major factor that leads to AKI.In the mice model of LPS induced AKI,LPS signifiantly induces the release of inflmmatory cytokines which promote kidney disease.Studies showed that inflmmatory cytokines TNF-?,IL-6,and IL-1? played critical roles in the pathologic process of kidney injury.And inhibition of these inflmmatory cytokines could attenuate the injury of kidney tissues.Thus,early anti-inflmmatory therapy can improve renal function.Hyperin,a flvonoid compound found in Ericaceae.Guttifera,and Celastraceae,has been reported to have anti-inflmmatory effects.Hyperin has been reported to inhibit LPS-induced nitrite production in rat peritoneal macrophages.Hyperin also inhibited LPS-induced acute liver injury in mice.Furthermore,hyperin has been found to protect the heart against ischemia and reperfusion lesions.However,whether hyperin has protective effects against LPS-induced AKI remains unclear.Therefore,in the present study,we investigated the protective effects and mechanism of hyperin on LPSinduced AKI in mice.Objective1 To explore the effect of Hyperoside on TNF-alpha.IL-6 and IL-1 beta in mice with acute renal injury induced by LPS2 To explore the effect of Hyperoside on blood urea nitrogen(BUN)and uric acid in mice with acute renal injury induced by LPS3 To explore the effect of Hyperoside on the expression of TLR4,NF-kappa B and NLRP3 in mice with acute renal injury induced by LPSMaterial and Method1 Animals and groupingSixty mice(BABL/c mice,6-8 weeks)in this study were provided by center of Experimental Animals of Binzhou Medical University and all the experiments complied with Institutional Animal Care and Use Committee of Binzhou Medical University.For the model of AKI,mice were given with 15 mg/kg body weight of LPS in 50 u 1 PBS via intraperitoneal injection.24 h after LPS challenge,the mice were euthanized and the blood and kidney tissues were collected.Sixty mice were randomly separated into fie groups,and each group contained 12 mice.Group 1 was treated as negative control,which received equal amount of PBS.Group 2 was served as LPS group,which received with 15 mg/kg body weight of LPS in 50 ?l PBS via intraperitoneal injection.Group 3-5 were served as hyperin(25,50,100 mg/kg)+LPS groups,which received 25,50,100mg/kg 1 h before LPS treatment.2 H&E staining of kidney tissuesKidney tissues were collected and fied in PBS containing 10%formalin.Then the fied tissues were trimmed in adapted cube and embedded in paraffi.The paraffi sections(slice thickness is 5 microns)were stained with H&E staining.Finally,the sections were examined by microscope.3 Measurement of BUN and creatinineAfter 24 hours of the treatment,the blood of each mouse was collected.In this study,AutoAnalyzer was employed to explore the levels of BUN and creatinine.The protocol was conformed to the instruction of the instrument.4 ELISA assayKidney tissues from different groupwere homogenized with PBS on ice,and then centrifuged at 4? for 40 min at 12,000 rpm.Subsequently,the supernatants were collected to determine the expression of TNF-a,IL-6,and IL-1?.The levels of inflmmatory cytokines were measured by using ELISA kits(R&D)according to the manufacturer's instructions.5 Western blot analysisKidney tissues were homogenized in protein extraction buffer and centrifuged at 10000 g for 10 min.The supernatants were measured using a BCA protein assay kit to obtain protein concentration.The proteins were separated on 10%SDS-PAGE and transferred to PVDF membranes.After being blocked with 5%fatfree dry milk,the membranes were incubated with the primary antibody at 4? for 24 h.After washing three times with TBS/Tween20,the membranes were probed with HRP-conjugated secondary antibodies at room temperature for 1 h.Finally,the membranes were visualized with ECL-chemiluminescent kit(ECL-plus,Thermo Scientifi,USA).6 Statistical analysisThe data are presented as the mean ±SEM.Statistical evaluation of the results were analyzed using one way ANOVA(Dunnett)s t-test)and two-tailed Student's t-test.Statistical signifiance was accepted at P<0.05.RESULTS1 Effects of hyperin on LPS-induced kidney histopathologic changesTo investigate the protective effects of hyperin,we applied H&E staining to detect the histological changes in renal tissues comparing normal tissues to injured tissues.As shown in Figure 1A,the renal tissues exhibited normal morphology in control group.The renal tissues of LPS group showed severe injury on renal tissues,including tubular cells sloughing,loss of brush,and apoptosis of nephron(Figure 1B).However,the injury was signifiantly ameliorated by hyperin(Figure 1C,1D,1E).2 Hyperin relieves the dysfunction of kidney function of AKIThe levels of BUN and creatinine were measured to assess renal function.As shown in Figure 2,compared with the control group,BUN and creatinine levels were found to be dramatically increased in the LPS group.However,the levels of BUN and creatinine induced by LPS were dose-dependently inhibited by hyperin(25,50.100 mg/kg).3 Inhibits the expression levels of cytokinesTo investigate the anti-inflmmatory effects of hyperin,the levels of inflmmatory cytokines TNF-a,IL-6,and IL-1? production were detected by ELISA.As shown in Figure 3,compared with the control group,the levels of TNF-a,IL-6,and IL-1?were found to be dramatically increased in the LPS group.However,treatment of hyperin signifiantly inhibited LPS-induced TNF-?,IL-6,and IL-1? production.4 Hyperin inhibits LPS-induced TLR4 expression and NF-?B activationTLR4 plays an important role in LPS-induced acute kidney injury.To investigate the anti-inflmmatory mechanism of hyperin,we investigated the effects of hyperin on TLR4 signaling pathway.As shown in Figure 4,LPS challenge signifiantly up-regulated the expression of TLR4 and activated NF-?B.However,hyperin signifiantly inhibited LPS-induced TLR4 expression.Furthermore,treatment of hyperin dose-dependently inhibited phosphorylation of NF-?B and I?B?.5 Hyperin inhibits LPS-induced NLRP3 signaling pathwayTo further investigate the anti-inflmmatory mechanism of hyperin,we examined the effects of hyperin on NLRP3 signaling pathway.As shown in Figure 5,compared with the control group,the expression of NLRP3,ASC,and caspase-1 were found to be dramatically increased in the LPS group.However,hyperin signifiantly suppressed LPS-induced NLRP3,ASC,and caspase-1 expression.ConclusionHyperin inhibit LPS-induced TNF-?,IL-6,and IL-1? production.And creatinine were also suppressed by hyperin.Furthermore,LPS-induced TLR4 expression and NF-?B activation were also inhibited by hyperin.In addition,treatment of hyperin dose-dependently inhibited LPS-induced NLRP3 signaling pathway.In conclusion,the results showed that hyperin inhibited LPS-induced inflmmatory response by inhibiting TLR4 and NLRP3 signaling pathways.Hyperin has potential application prospects in the treatment of sepsis-induced AKI.