| Objective:Acute kidney injury(AKI)is a common clinical critical disease and threatens the life safety of patients seriously with high mortality.Currently,there is still a lack of effective drug therapy against AKI,and symptomatic supportive treatment and renal replacement therapies are the main ones.Cordyceps sinensis is an ingredient of traditional Chinese medicine,and clinical applications have confirmed its satisfactory efficacy in treating kidney diseases.Cordycepin(Cor),a major functional component of Cordyceps sinensis exhibits a wide range of biological effects,including anti-inflammatory,antioxidant,and antiapoptotic.A previous study reported cordycepin has a protective effect on AKI model animals,but the mechanisms for the renoprotective effects of cordycepin on AKI have not been clearly elucidated.In the investigation,a comprehensive network pharmacology strategy and molecular docking approach was used in combination with preliminary in vitro and in vivo validations to examine the molecular mechanisms.Methods:1.Pubchem database and PharmMapper database were used to dig out cordycepin-related targets.GeneCards database,OMIM database and Disgenet database were used to dig out AKI-related targets.To construct protein-protein network and mine core targets with Cytoscape software.To analyze related signaling pathways of core targets with DAVID online instrument.Molecular docking verification of the affinity between cordycepin and its core target was performed using AutoDock software.2.In vivo,48 male SD rats were divided to sham operation group(Sham;n=12),ischemia-reperfusion group(I/R;n=18),and cordycepin group(I/R+Cor;n=18).The ischemia-reperfusion induced AKI was established using a bilateral renal artery clamp model.Rats in the I/R+Cor group were injected intravenously with cordycepin after the I/R surgery was performed;rats in the other groups received tail vein injection of 1ml saline.After reperfusion 12h,24h and 48h,blood was collected for the determination of serum creatinine and blood urea nitrogen(BUN),kidneys were harvested for histopathology,and TUNEL assay for apoptosis.Western blot analysis and RT-qPCR were used to detect the protein and mRNA expression levels of PI3K、Akt、mTOR、Beclin-1、p62、LC3B、caspase-3、Bcl-2 and Bax after reperfusion 48h.Autophagy associated morphological changes were tested by transmission electron microscopy.3.In vitro,human renal tubular epithelial cells(HK-2)exposed to hypoxia/reoxygenation(H/R).HK-2 cells were randomly divided into three groups,as follows:the control group(without establishing and H/R model),the H/R model group,the Cor group(H/R model with various concentrations of cordycepin for 24h).The concentration of cordycepin is 15μM,30μM,45μM,60μM,75μM,90μM,105μM,120μM,135μM,150μM.CCK-8 assay was applied to evaluate the cell viability after 24h of reoxygenation.To further examine the role of PI3K/Akt/mTOR signaling pathway in Cor protecting AKI,the H/R HK-2 cell model was reestablished,the cells were divided into four group:control,H/R,Cor,and LY294002 group(PI3K inhibitor,50μM).Cell viabilities were determined by CCK-8 assays,protein expression of p-PI3K、p-Akt、p-mTOR、Beclin-1、p62、LC3B、caspase3、cleaved caspase-3、Bax and Bcl-2 were examined by Western blot analysis.Results:1.234 cordycepin-related targets were dig out by pharmacological database,and we got 761 AKI-related targets.We got 42 core targets shared with cordycepin and AKI with Cystoscape analysis.After analyzing the core targets,we found that the signaling pathway related to cordycepin and AKI involves multiple signaling pathways such as PI3K/Akt、mTOR and apoptosis.Cordycepin was molecularly docked with PI3K/Akt/mTOR signaling pathway targets PIK3CA、AKT1、mTOR、p62(SQSTM1)、LC3B、BECN1、BAX、BCL2、CASP3,and the binding energies were all<-5.0 kJ·mol-1 except p62(SQSTM1).2.After 45 min of ischemia and reperfusion for 12 h,24 h and 48 h,the Pallers score of renal tissue injury and the number of apoptotic cells detected by TUNEL in the I/R group were significantly higher than those in the Sham group(P<0.01,P<0.05).The Pallers score and the number of apoptotic cells in the I/R+Cor group were significantly lower than those in the I/R group at the same period(P<0.01,P<0.05).Transmission electron microscopy demonstrated that the number of autolysosomes and autophagosomes in model group were significantly decreased compared with the sham group.While the number of autolysosomes and autophagosomes increased treated with Cordycepin.Cordycepin can reduce the protein expression of p-PI3K,pAkt,p-mTOR,caspase-3,cleaved caspase-3,increase the protein expression of Beclin-1 and Bcl-2,increase the ratio of LC3Ⅱ/LC3Ⅰ and reduce the ratio of Bax/Bcl2 in the kidney tissue of rats with ischemia for 45 minutes and reperfusion for 48 hours;Cordycepin can reduce PI3K,caspase-3,Bax mRNA expression.3.The viability of H/R HK-2 cells at 24h reoxygenation was significantly decreased by CCK-8,but after intervention with various concentrations of cordycepin,the viability of H/R HK-2 cells could be improved(P<0.001),and the concentration of 90 μM cordycepin was effective in increasing H/R HK-2 cells(P<0.001).H/R HK2 cells had the most obvious viability,so the concentration of 90 μM cordycepin was selected for later experiments.Hypoxia and reoxygenation injury significantly increased the apoptosis rate of HK-2 cells,and the apoptosis rate decreased significantly after the intervention of cordycepin(P<0.01).Cordycepin and LY294002 can inhibited the activation of PI3K,Akt and mTOR,up-regulated Beclin1 protein expression,reduce the protein expression of p62,increase the ratio of LC3II/LC3I,activated autophagy.Cordycepin can inhibit the activation of caspase-3,decreased apoptosis and up-regulated the anti-apoptotic protein Bcl-2 protein expression and inhibit the activation of caspase-3,decreased apoptosis.Conclusion:The present study identified that cordycepin served a role in AKI treatment by modulating the PI3K/Akt/mTOR pathways.Cordycepin could activated renal tubular epithelial cell autophagy and ameliorates apoptosis effectively in I/R AKI by inhibiting PI3K/Akt/mTOR signaling pathways is now well-established from cell and animal studies.This present study provides a theoretical basis for the application of cordycepin in the treatment of ischemia-reperfusion AKI. |
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