| The safety of traditional Chinese medicine(TCM)containing aristolochic acids(AAs)is a hot issue of widespread concern in the society.Although some varieties with high aristolochic acid I(AA-Ⅰ)content,such as Aristolochia manshuriensis Kom.,Aristolochia fangchi Y.C.Wu ex L.D.Chou et S.M.Hwang.and Aristolochia debilis Sieb.et Zucc.have been banned,some TCM of Aristolochiaceae such as Asarum are still used in clinic.There has been a call to ban all Aristolochiaceae plants,but due to the lack of in-depth,systematic and comprehensive research,there is a lack of sufficient scientific basis for whether related varieties are banned or not.This makes the clinical use and drug regulation of related varieties of Aristolochiaceae face great challenges.So far,more than 170 kinds of AAs have been found.There are different kinds and contents of AAs in different Aristolochaceae plants.For example,the content of AA-Ⅰ in Aristolochia contorta Bge.is very high,while the content of AA-Ⅰin Asarum is very low or can not be detected,but it mainly contains aristolochic acidⅣa(AA-Ⅳa).Previous studies on the toxicity of AAs mainly focused on AA-Ⅰ,and it has been proved that AA-Ⅰ can cause acute nephrotoxicity and chronic renal interstitial fibrosis.However,there is a lack of research on whether AA-Ⅳa has nephrotoxicity and whether AA-Ⅳa has the same potential nephrotoxicity as AA-Ⅰ,especially renal interstitial fibrosis.Asarum is a widely used TCM in clinic,and it is also the only Aristolochaceae plant included in the 2020 edition of Chinese Pharmacopoeia.According to statistics,there are about 200 kinds of TCM preparations containing Asarum,many of which are very commonly used,including drugs commonly used by children.Clarifying the safety of AA-Ⅳa is of great scientific value and significance for the clinical safe use and scientific supervision of TCM mainly containing AA-Ⅳa,such as Asarum.Objective:Through the short-term and long-term toxicity study of parallel comparison between AA-ⅠVa and AA-Ⅰ,to determine whether AA-Ⅳa has potential nephrotoxicity,especially whether it may cause renal interstitial fibrosis,and objectively evaluate the safety of AA-Ⅳa.To clarify the toxicity difference between AA-Ⅳa and AA-Ⅰ,and to elucidate the mechanism of nephrotoxicity difference between them through proteomic study,so as to provide scientific basis for clinical safe use and supervision of traditional Chinese medicine containing AA-Ⅳa.Methods:1.Analysis of the species and contents of AAs in four commonly used Aristolochiaceae TCM such as AsarumIn this study,4 commonly used Aristolochaceae plants were selected,including 19 batches of Aristolochiae fructus,17 batches of Aristolochiae herba,15 batches of Asarum and 15 batches of Aristolochia cinnabarina.Five aristolocholic acid derivatives(AA-Ⅰ,AA-Ⅱ,AA-Ⅲa,AA-Ⅳa and AL-Ⅰ)were quantitatively analyzed by high performance liquid chromatography-diode array detector.The separation was performed on an Agilent Eclipse XDB C18 column(4.6×150 mm,5 μm)with acetonitrile-0.1%formic acid water as mobile phase and gradient elution at a flow rate of 1.0 mL/min.The above five aristolocholic acid derivatives(AA-Ⅰ,AA-Ⅱ,AA-Ⅲa,AA-Ⅳa and AL-Ⅰ)were quantitatively analyzed by external standard method with the detection wavelength of 254 nm.2.Nephrotoxicity comparison between AA-Ⅰ and AA-Ⅳa(1)Comparison of the toxicity of AA-Ⅰ and AA-Ⅳa in vitro:After HK-2 cells were treated with different concentrations of AA-Ⅰ and AA-Ⅳa for 24 h and 48 h,respectively,the IC50 value of half inhibitory concentration of AA-Ⅰ and AA-Ⅳa on HK-2 cells was detected by CCK-8 proliferative toxicity reagent.Different fluorescence probes were used to investigate the effects of AA-Ⅰ and AA-Ⅳa on mitochondrial function(mitochondrial membrane potential:MMP,mitochondrial weight),intracellular calcium concentration and apoptosis of HK-2 cells through high-content screening system.(2)Comparison of nephrotoxicity of AA-Ⅰ and AA-Ⅳa by single gavage administration:70 C57 mice were randomly divided into 7 groups with 10 mice in each group,5 females and 5 males respectively.They were control group,different AA-Ⅰ dose groups(10,20 and 40 mg/kg),and different AA-Ⅳa dose groups(10,20 and 40 mg/kg).The death of animals in each group was observed for 14 consecutive days after single oral administration.At the end of the 14-day observation period,the eyeballs of mice were removed for blood collection,and the serum,liver and kidney tissues were collected for biochemical index detection and pathological examination,respectively.(3)Comparison of DNA damage induced by AA-Ⅰ and AA-Ⅳa:20 ICR mice were randomly divided into negative control group,ethyl methanesulfonate(EMS),AA-Ⅰ(20 mg/kg)and AA-ⅣA(20 mg/kg)groups,with 5 mice in each group.They were administered orally for 3 consecutive days,and 2 hours after the last administration,the primary cells of kidney were extracted and used to detect the damage of DNA by comet electrophoresis.(4)Comparison of nephrotoxicity between AA-Ⅰ and AA-Ⅳa:190 C57 male mice were randomly divided into 6 groups:control group,AA-Ⅰ different groups(0.1,1 and 10 mg/kg)and AA-Ⅳa different groups(1 and 10 mg/kg).Except 40 animals in AA-Ⅰ10 mg/kg group,30 animals in other groups.The drug was administered by gavage 3 times a week(Monday,Wednesday and Friday)for 22W,and continued to observe 50W after stopping the drug.Observe the general condition of animals every day,and record the death,weekly weight and food intake.Some mice were sacrificed at 22W of administration,29W and 50W of withdrawal,and blood was taken for dissection.Biochemical indexes(BUN,CRE),organ weight and coefficient,organ pathology,and masson staining of kidney sections were detected to evaluate the renal toxicity of AA-Ⅰ and AA-Ⅳa,and to determine whether AA-Ⅳa caused renal interstimal fibrosis.3.Study on nephrotoxicity mechanism of AA-Ⅰ and AA-Ⅳa(1)Screening and validation of key targets and signaling pathways of AA-Ⅰ induced renal interstitial fibrosis:Kidney tissues of control mice and AA-Ⅰ treated mice at the 22th week were collected for TMT and phosphorylated proteomics studies.Original data were obtained through protein extraction-protein quality inspection-protein pretreatment(enzyme digestion,desalting)-TMT labeled peptides-enrichment of phosphorylated peptides-liquid quality testing.After data annotation,filtering,identification and quantitative processing,differentially expressed proteins can be obtained.The protein with significant quantitative difference between the experimental group and the control group satisfies the following conditions:(P<0.05,|log2FC|>*(FC>*or FC<*[fold change,FC]).The differentially expressed proteins were annotated by gene ontology(GO),the subcellular localization,molecular function and biological process of related proteins were summarized,and the enrichment of KEGG pathway was analyzed.For differential expression,volcanic map analysis,cluster heat map analysis and KEGG pathway enrichment analysis were performed.In addition,the kinases of phosphorylated proteins were predicted and analyzed.NetworkIN 3.0 calculation model was used to construct the interaction network between proteins and kinases,and the kinases corresponding to phosphorylated modification sites were found.The relationship between phosphorylated protein substrates and kinases was further determined based on the information of target protein sequence resources and the background information of protein-kinase interaction.The key targets and signal pathways were verified by WB assay in vivo and in vitro,and the relationship between kinases and substrates was verified by inhibitor assay.In addition,STAT3 interfering small RNA,STAT3 overexpression plasmid,wild-type luciferase plasmid of S100A11 promoter and internal reference plasmid were constructed and transferred into HK-2 cells,and the regulation of transcription factor STAT3 on downstream target genes was verified by luciferase reporter gene.(2)Differences in the effects of AA-Ⅰ and AA-Ⅳa on key targets and signaling pathways:WB assay was conducted to detect the effects of AA-Ⅳa administration of 10 mg/kg for 22W on key proteins and signaling pathways activated in the above AA-Ⅰ 10 mg/kg group in kidney tissue,and the mechanism of the difference in nephrotoxicity between AA-Ⅰ and aa-ⅳa was investigated.Results:1.Analysis of the species and contents of AAs in four commonly used Aristolochiaceae TCM such as AsarumThe types and contents of AAs in 4 commonly used Aristolochiaceae plants were significantly different,among which Aristolochiae fructus and Aristolochiae herba contained 5 kinds of AAs(AA-Ⅰ,AA-Ⅱ,AA-Ⅲa,AA-Ⅳa and AL-Ⅰ),and Aristolochia cinnabarina contained 4 kinds of AAs(AA-Ⅰ,AA-Ⅱ,AA-Ⅲa and AA-Ⅳa).Asarum contains only 2 AAs(AA-Ⅳ and AA-Ⅰ).The content of AA-Ⅰ in Aristolochiae fructus and Aristolochia cinnabarina was higher,reaching the level of 0.5 mg/g and 3.7 mg/g crude drug,respectively.The content of AA-Ⅰ in Aristolochiae herba was very low,only at the level of 0.02 mg/g crude drug.Almost no AA-Ⅰ and AA-Ⅱ were detected in Asarum,but the content of AA-Ⅳa in Aristolochiae herba and Asarum was higher.The crude drug levels were 0.26 mg/g and 0.1 mg/g,respectively.It can be seen that AA-Ⅳa is the dominant AAs in Aristolochiae herba and Asarum,while the known toxic AA-Ⅰ levels are minimal.2.Nephrotoxicity comparison between AA-Ⅰ and AA-Ⅳa(1)Comparison of the toxicity of AA-Ⅰ and AA-Ⅳa in vitro:The IC50 values of AA-Ⅰand AA-Ⅳa on HK-2 cells were 251 μM and>1000 μM after 24 h treatment,respectively.When the treatment time extended to 48 h,the IC50 value of AA-Ⅰdecreased to 82.99μM,while the IC50 value of AA-Ⅳa was still higher than 1000 μM.AAⅣa can reduce the number of HK-2 cells,increase the mitochondrial membrane potential,increase calcium ion concentration and increase cell apoptosis,suggesting that AA-Ⅰ is highly cytotoxic and may induce cell apoptosis by affecting mitochondrial function.However,AA-Ⅳa had little effect on the above indexes,suggesting that AA-Ⅳa had little effect on mitochondrial function and apoptosis,and its cytotoxicity was low.(2)Comparison of nephrotoxicity of AA-Ⅰ and AA-Ⅳa by single gavage administration:Death began to occur on the 5th day after single administration of AA-Ⅰ at 40 mg/kg,and 7 out of 10 animals died during the 14-day observation period(♂:4,♀:3).No animal death was observed in AA-Ⅰ 20 and 10 mg/kg groups.The BUN and CRE levels of male mice in 20 mg/kg AA-Ⅰ group were significantly higher than those in control group(P<0.05),while the renal function of female mice was normal.No abnormality was observed in the above indexes of female and male mice in the 10 mg/kg group.Pathological examination showed that the renal tissue damage of mice in AA-Ⅰ group was obvious,including partial renal tubular necrosis,epithelial cell swelling and shedding,etc.No animal death was observed in the 3 AA-Ⅳa dose groups,no significant difference was found in serum BUN and CRE levels compared with the control group,and no abnormality was found in renal pathological examination,The results showed that a single administration of AA-Ⅳa had no significant renal toxicity,but AA-Ⅰ could cause acute renal injury and death.(3)Comparison of DNA damage induced by AA-Ⅰ and AA-Ⅳa:Mice were given 20 mg/kg AA-Ⅰ and AA-Ⅳa for 3 times by intragastric administration,and primary renal cells were extracted for comet electrophoresis.The results showed that the renal cells in the AA-Ⅰ group showed obvious cell tailing,and the olive tail moment was significantly increased compared with the control group,indicating that AA-Ⅰ caused DNA damage in renal cells.However,there was no significant difference in the above comet electrophoresis related indexes between the AA-Ⅳa 20 mg/kg group and the control,suggesting that AA-Ⅳa did not cause DNA damage in renal cells.(4)Comparison of nephrotoxicity between AA-Ⅰ and AA-Ⅳa:Long-term administration of AA-Ⅰ(10,1 mg/kg)showed obvious toxic reactions,and a large number of animals died during the experiment.AA-Ⅰ 10 mg/kg group started at 10th week during the administration period and 1 mg/kg group began at 21th week after withdrawal.The renal toxicity of AA-Ⅰ was obvious.Serum BUN and CRE increased significantly compared with the control group.Renal histopathology showed diffuse fibrosis,atrophy of renal tubular epithelial cells and dilation of lumen.The above lesions were not alleviated or reversed after drug withdrawal.The results showed that AA-Ⅰ could cause severe renal toxicity and irreversible renal damage.At 22W of administration and 29W and 50W of withdrawal,no death was observed in the 10 and 1 mg/kg AA-Ⅳa group,no significant difference was found in renal function indexes BUN and CRE compared with the control group,no obvious pathological changes were observed in renal histopathology,and no obvious renal interstitial fibrosis was observed in Masson staining.It is suggested that long-term administration of AA-Ⅳa does not cause significant renal toxicity.These results suggest that AA-Ⅳa has significant toxicity difference with AA-Ⅰ,and the nephrotoxicity of AA-Ⅳa is not obvious..3.Study on nephrotoxicity mechanism of AA-Ⅰ and AA-ⅣaAccording to the above study on the difference of nephrotoxicity between AA-Ⅰand AA-Ⅳa,AA-Ⅰ caused obvious nephrotoxicity,especially renal interstitial fibrosis,while AA-Ⅳa showed no obvious nephrotoxicity.Therefore,we further studied the key targets and signaling pathways of AA-Ⅰ in renal interstitial fibrosis.On this basis,the effects of AA-Ⅳa on key targets and signaling pathways of renal interstitial fibrosis were further studied to clarify the mechanism of the difference in renal toxicity between AA-Ⅳa and AA-Ⅰ.(1)Screening and validation of key targets and signaling pathways of AA-Ⅰ induced renal interstitial fibrosis:Using TMT and phosphorylation proteomics,we quantified 6475 proteins,of which 4420 proteins were phosphorylated,which corresponded to 14500 phosphorylation events.Proteomic data analysis showed that there were 984 differential proteins in the AA-Ⅰ 10 mg/kg group,including 580 up-regulated proteins and 404 down-regulated proteins.However,AA-Ⅰ caused significant changes in 1,933 phosphorylated proteins,among which 1,490 phosphorylated proteins were significantly up-regulated and 443 phosphorylated proteins were significantly down-regulated,suggesting that AA-Ⅰ was more focused on affecting protein functions.Proteomic results showed that many metabolic pathways were significantly down-regulated in mice with renal interstitial fibrosis induced by AA-Ⅰ,and AA-Ⅰ also activated some signals,including extracellular matrix related pathways,PI3K-AKT signaling pathway,adhesion plaques,and cancer-related pathways.Phosphorylated proteome results showed that many similar metabolic pathways were inhibited in AA-Ⅰ induced renal interstitial fibrosis mice,but their activation was different from that of the proteome,including MAPK signaling,mTOR signaling,TNF signaling and Ras signaling.We found that the number of kinases corresponding to phosphorylated proteins in AA-Ⅰ induced renal interstitial fibrosis mice was higher than that in control mice,suggesting that phosphorylated proteins play an important role in the occurrence and development of renal interstitial fibrosis induced by AA-Ⅰ,and AA-Ⅰ may promote the occurrence and development of renal interstitial fibrosis by activating MAPK signaling pathway.Inhibitor assay indicated that STAT3 may be the substrate of p38MAPK kinase.We verified the activation of p38MAPK-STAT3 signal in renal tissue of mice treated with 10 mg/kg AA-Ⅰ for 22W by WB experiment,and confirmed that STAT3 could regulate the transcription expression of S100A11 by double luciferase reporter gene experiment.Therefore,it was found that the activation of p38MAPK-STAT3-S100A11 signal axis was involved in the occurrence and development of AA-Ⅰ-induced renal interstitial fibrosis.At the same time,it was found that target proteins,such as Bad,a-SMA,BAG3,Bcl-xL and MMP2,which may be regulated by STAT3,were significantly up-regulated in AA-Ⅰ 10 mg/kg group,which may be involved in the occurrence and development of AA-Ⅰ-induced renal interstitial fibrosis and tumor.(2)Differences in the effects of AA-Ⅰ and AA-Ⅳa on key targets and signaling pathways:The same dose of AA-Ⅳa for 22W could only activate p38MAPK,but had no significant effect on STAT3 signal,so it was unable to regulate the target genes of STAT3.In addition,the significantly up-regulated proteins such as Bad and a-SMA in the kidney tissue of mice in the AA-Ⅰ 10 mg/kg group did not change significantly in the AA-Ⅳa 10 mg/kg group.These results suggest that AA-Ⅳa and AA-Ⅰ regulation differences in p38MAPK-STAT3-S100A11 signaling pathway,Bad,a-SMA and other key proteins may be responsible for the differences in renal toxicity between AA-Ⅳa and AA-Ⅰ.Conclusion:1.Different varieties of Aristolochiaceae plants contain different types of AAs.Asarum basically does not contain AA-Ⅰ and AA-Ⅱ,but contains a high level of AA-Ⅳa and Aristolochiae herba also contains a high level of AA-Ⅳa.2.AA-Ⅰ can cause severe renal toxicity.A single administration of high dose can cause acute renal toxicity and death,and even a long-term administration of low dose can also cause renal interstitial fibrosis.No significant renal toxicity was observed in single or long-term administration of AA-Ⅳa.3.AA-Ⅰ induced renal interstitial fibrosis may be related to the activation of p3 8MAPK-STAT3-S100A11 signaling pathway and the increased expression of a-SMA and Bad.Although AA-Ⅳa can activate p38MAPK,it has no significant effect on STAT3 and its potential downstream targets S100A11,a-SMA and Bad,suggesting that the activation of STAT3 may be the reason for the difference in nephrotoxicity between AA-Ⅰ and AA-Ⅳa.4.Different kinds of AAs have different toxicity.The toxicity of TCM containing AAs can not be generalized and should be recognized objectively and treated scientifically.Asarum basically does not contain AA-Ⅰ and AA-Ⅱ,but mainly contains AA-Ⅳa,which may have a low risk of renal toxicity.5.Based on scientific evidence,this study revealed that the toxicity of AA-Ⅳa and AA-Ⅰ is completely different.Therefore,we proposed for the first time the academic viewpoint that "the toxicity of AAs varies with different types,not all AAs have renal toxicity,and the toxicity of TCM containing AAs cannot be generalized". |
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