Effects And Mechanisms Of Protocatechuic Aldehyde On Cisplatin-induced Acute Kidney Injury

Cisplatin,a classic chemotherapeutic agent widely used to treat different types of cancers including ovarian,head and neck,testicular and uterine cervical carcinomas.However,the most common adverse effect of cisplatin is acute kidney injury that has witnessed a serious kidney tubular epithelial injury,a high level of blood urea nitrogen and serum creatinine and so on,all of which may lead to a high mortality rate in patients.As we all known,Traditional Chinese Medicine(TCM)may be a promising and novel avenue to treat human diseases effectively with lower toxicity,especially the successful application of artemisnin against malaria,which strengthen the belief of Chinese traditional medicine research.In our pilot study,we tested ten potential antiinflammatory TCM monomers including aloin,icariin,protocatechuic acid,protocatechuic aldehyde(PA),puerarin and so on,all of which have investigated in enormous literature.Results presented here show that PA,isolated from in the root of S.miltiorrhiza,is one of the most powerful protective TCM monomers.The mechanism of PA was further detected in cisplatin nephropathy,where it prevented decline of renal function and attenuated renal injury.More importantly,this findings provide a novel target to protect against acute kidney injury.The experiment is divided into two sections(in vivo and in vitro),as follows:1.PA attenuated cisplatin-induced acute kidney injury.The effect of PA in concentrations of 0.45,0.9 and 1.8mg/kg was evaluated in vivo in cisplatin nephropathy,including kidney function injury,renal morphological change and level of KIM1 change.Results of PAS,BUN,Cr and urinary NGAL revealed that administration of PA declined kidney function injuy and tubular necrosis,dilation,and cast formation that is induced by cisplatin.The therapeutic effects of PA were further confirmed by detection of KIM1,covering Western-blot and IHC;We used an MTT assay to analyze the impact of PA on cell viability in the human tubular epithelial cell line(HK2)and ensure an effective concentrations of PA on cisplatin induced HK2 cells.In addition,we examined m RNA and protein expression of KIM1.Results of MTT assay show that PA in concentration of 0.25,0.5,1?M significantly restored cell viability after cisplatin treatment.Western blot and real-time PCR results show that cisplatin upregulated KIM1,which was decreased by PA treatment in a time-dependent manner in HK-2 cells.2.PA protected against cisplatin-induced inflammation response;Results form real-time PCR and IHC show that PA reduced relvant inflammatory cytokines and chemokines.Meanwhile,the anti-inflamatory effect of PA were further confirmed by real-time PCR and Elisa in cisplatin exploring cells.3.PA protected against cisplatin-induced progrommed cell death;Western-blot results indicated that PA suppressed activation of RIP1/RIP3/MLKL axis,which is regarded as the key pathway mediating necroptosis and decreased levels of cleaved-caspase-3 in cisplatin nephropathy.And,the effect of attenuating progrommed cell death were further suporrted by flow cytometric analysis and western-blot in cisplatin-induced cells.4.PA decreased cisplatin-induced oxidative stress.The level of oxidative stress was detected by GSH assay,MDA assay,IHC and Western blot in model mice.Likewise,DCF and DHE fluorescent staining was preformed to measure the effect of PA on oxidative stress.To further experiments,APO(NADPH inhibitor),Nox4-KD cells and Nox2-KD cells was used to determine the mechanism of PA in mediating its protective effects.Results indicated that PA suppressed cisplatin-induced both oxidative stress in vitro and in vivo.As we known,APO or PA treatment decreases cisplatin-induced damage of tubular epithelial cells.However,we found that after blocking Nox enzymes with apocynin,PA failed to further reduce the renal damage.This indicated a role for the Nox family in mediating the protective effect of PA.Therefore,we further analyzed the main members of the Nox family in kidney(Nox2 and Nox4),especially in Nox4-KD cells and Nox2-KD cells.Western blot results show that PA further suppressed KIM1 levels in absence of Nox2.But,PA failed to further attenuate renal tubular cell damage when Nox4 was disrupted.This indicates an essential role for Nox4 in mediating protective effects of PA.Interestingly,the target for protection against cisplatin-induced acute kidney injury was further confirmed in lentivirus-mediated knockdown of Nox4 mice.