Study On Lycopene Antagonizing Renal Toxicity Of Aristolochic Acid By Targeting Nrf2 Antioxidant System

Objective: Chinese herbal medicines containing Aristolochic acids(AAs)are included in the previous editions of the Chinese Veterinary Pharmacopoeia.Among Chinese herbal medicines containing AAs,except for Guanmu Tong,which is prohibited from being used due to its high content,most Chinese herbal medicines containing AAs are used in veterinary clinical practice.Chronic use of herbal medicines of the AAs class can lead to nephrotoxicity.It is pathologically characterized by extensive tubulointerstitial fibrosis with proximal tubular atrophy and loss.AAs cause disease in the vivo,and the kidney is the main target organ.The main mechanism is through activation of oxidative stress response,production of excessive ROS,causing tubular epithelial cell injury,tubulointerstitial fibrosis.Lycopene(LYC),as a carotenoid,is antioxidant.LYC ameliorates oxidative stress damage that occurs in the body due to various factors.The aim of this study was to investigate whether LYC attenuated AAs-induced nephrotoxicity by acting on the Nrf2 antioxidant system.It provides a theoretical basis for LYC as a drug to prevent nephrotoxicity in AAs.Methods: LYC,Nrf2 antioxidant system and AAs renal injury were combined in this study to propose the hypothesis whether LYC improves the inflammatory response and apoptosis in tubular epithelial cells.36 weeks old male C57BL/6 mice were divided into five groups randomly: normal group(intragastric administration of 0.5% CMC vehicle 0.2 ml/mouse/day),model group(intragastric administration of AAI 10 mg/kg/day),LYC control group(intragastric administration of LYC 20 mg/kg/day),AAI + LYC low-dose group(intragastric administration of AAI 10mg/kg/day + LYC 5 mg/kg/day),AAI + LYC high-dose group(intragastric administration of AAI10 mg/kg/day + LYC 20 mg/kg/day).The model cycle was 28 days to establish a mouse model of renal injury in AAs.Body weight changes and mental status of mice in each group were observed.Renal tissue structure and function damage(renal coefficient,morphological changes and serological parameters)were detected.In vitro assay,cell viability was tested using CCK-8 kit after simultaneous intervention of NRK52 E cells using AAI and LYC.Optimal AAI and LYC concentrations were selected for subsequent assays.They were divided into 5 groups: normal control group,model group(AAI 40 μM),LYC group(LYC 10 μM),LYC high/low dose treatment group(AAI 40 μM + LYC 5/10 μM),and the drug action time was 24 h.The main mechanism of LYC in improving nephrotoxicity of AAs was verified by ELISA,Immunohistochemistry,immunofluorescence,Western blot and q RT-PCR.Oxidative stress-related parameters(ROS,MDA,GSH-PX,T-AOC,SOD),expression of key factors of the Nrf2 pathway(Keap-1,HO-1,Nrf2,NQO1),and expression of key factors of the apoptosis pathway(BAX,BCL-2,Caspase-3,Caspase-9,Cleaved-Caspase-3,Cleaved-Caspase-9)were detected.In addition,while cells were intervened with AAI(40 μM)and LYC(10 μM),cells were pretreated with Nrf2 inhibitor ML385(5 μM)and Nrf2 activator AKBA(40 μM),respectively.After 24 h,Nrf2 pathway and apoptosis pathway-related parameters were measured in order to investigate whether LYC improves nephrotoxicity of AAI by acting on the Nrf2 antioxidant system and improves AAI-induced renal inflammatory response with renal cell apoptosis.The test results are as follows:(1)Compared with the control group,AAI-exposed mice had significantly lower body weight,and severe renal histopathological damage: significant tubular structural abnormalities,Pathological changes such as epithelial cell detachment and inflammatory cell infiltration occur.The serum BUN and Cr levels significantly increased(P<0.0001).These results suggested abnormal renal function in mice.However,mice treated with LYC had significantly higher body weight,reduced renal histopathological damage,and significantly lower serum levels of Cr and BUN compared with AAI(P<0.0001).(2)The results of in vivo experiments showed that compared with the blank group,AAI exposure could induce an increase in the content of MDA,ROS and IL-1 in the kidneys of mice.The expression of NF-κB,TNF-α,IL-6,IL-1 β,Cleaved-Caspase-9,Cleaved-Caspase-3,BAX was activated.The expression of Nrf2,Keap1,NQO1,HO-1,IκB,BCL-2 and the activity of SOD,TAOC,GSH-PX,IL-12 was significantly inhibited(P<0.0001).These suggested that AAI can induce oxidative stress damage,inflammatory response,and cell apoptosis in the kidney.Compared with the AAI group,the content of MDA,ROS,IL-6,IL-1 and the expression of NF-κB,TNF-α,IL-1 β,Cleaved-Caspase-9,Cleaved-Caspase-3,BAX was significantly reduced.In the LYC intervention group,the expression of Nrf2,Keap1,NQO1,HO-1,IκB,BCL-2 and the content of SOD,T-AOC,GSH-PX,and IL-12 were significantly increased(P<0.0001).These suggested that LYC activated the Nrf2 antioxidant signaling pathway,reduced damage caused by oxidative stress,and inhibited NF-κB signaling pathway to induce renal interstitial inflammatory response and mitochondrial pathway cell apoptosis.(3)The results of in vitro experiments showed that cell viability was significantly inhibited(P<0.0001)and cell morphology was changed when cells were intervened with AAI at a concentration of 40 μM.Compared with the blank group,the content of ROS was significantly increased in NRK52 E cells treated with AAI.It activated the expression of TNF-α,NF-κB,IL-1β,IL-6,Cleaved-Caspase-9,Cleaved-Caspase-3,and BAX,and inhibited the expression of Nrf2,Keap1,NQO1,HO-1,IκB,BCL-2 and the activity of SOD and GSH-PX(P<0.0001).However,LYC increased cell viability and the content of ROS.The expression of TNF-α,NF-κB,IL-1β,IL-6,Cleaved-caspase-9,Cleaved-caspase-3,and BAX were significantly decreased;and the expression of Nrf2,Keap1,NQO1,HO-1,IκB,BCL-2 and the content of SOD and GSH-PX were significantly increased(P<0.0001).The flow cytometry showed that the number of apoptotic cells induced by AAI was significantly increased(P<0.0001).These results suggested that LYC inhibited AAIinduced oxidative stress injury in NRK52 E cells and ameliorated NF-κB signaling pathway-induced inflammatory response and apoptosis in NRK52 E cells.(4)Before NRK52 E cells were treated with AAI and LYC,ML385,an inhibitor of Nrf2,was added and it was found that the activation of Nrf2 antioxidant signaling pathway by LYC was inhibited.The ROS content significantly increased and the expression of Keap1,Nrf2,HO-1 and NQO1 significantly decreased.In addition,the expression of NF-κB,TNF-α,BAX,IL-1β,CleavedCaspase-3 increased and the expression of BCL-2 decreased.However,after adding AKBA,an activator of Nrf2,NF-κB,TNF-α,BAX,IL-1β and Cleaved-Caspase-3 expression decreased,and the expression of BCL-2 increased.These results suggested that LYC reversed the alleviating effect of AAI-induced renal cell inflammatory response and apoptosis after Nrf2 antioxidant pathway was inhibited.Conclusion: Chronic administration of traditional Chinese medicines containing AAI can induce kidney injury.LYC intervention was able to prevent the development and progression of AAI-induced chronic kidney disease.Its mechanism of action is that LYC is able to activate the Nrf2/HO-1 antioxidant system and maintain the homeostasis of oxidative stress in the kidney,which in turn improves AAI-induced activation of the NF-κB signaling pathway and apoptosis of the mitochondrial pathway.Nrf2 can be used as a research target to prevent and treat such CKD.