Protective Effect And Mechanisms Of Paeoniflorin On Cisplatin-induced Acute Kidney Injury

Background and objective:Acute kidney injury(AKI)is also known as acute kidney failure.The incidence of AKI has increased significantly in recent years.About 13 million people suffer from acute kidney injury every year worldwide(85% of patients live in developing countries),and about 1.7 million people die from acute kidney injury and its complications.Immune inflammation,ischemia,oxidative stress,poisoning,autoimmunity are interrelated,promote or restrict,form a very complex network relationship,many aspects of the formation and progress of AKI.Cisplatin is a commonly used chemotherapy drug in the treatment of solid tumors such as head and neck tumors and ovarian tumors,but its application is limited due to its toxic and side effects,especially renal toxicity.But so far,there are no effective clinical drugs to prevent or treat cisplatin-induced acute kidney injury,so it is important to find new and more effective therapeutic drugs.Paeoniflorin(Pae)is a water-soluble monoterpene glycoside,which is the main active component of Paeoniflorin and has been used in medicine for more than 1200 years.Over the past few decades,the pharmacological activity of Pae has been further studied.The toxicity of Pae is low and it has a wide range of pharmacological effects both in vivo and in vitro.Studies have shown that Pae has anti-inflammatory,antioxidant,anticonvulsive and immunomodulatory effects,and can play a protective role in some renal disease models.In the early stage,our research group also found that some traditional Chinese medicine monomers were effective in kidney disease models.Therefore,this topic further explored the specific molecular mechanism of Pae’s protective effect on cisplatin-induced acute kidney injury,and sought possible therapeutic targets to provide scientific basis for clinical search for effective therapeutic drugs.Methods:In vitro experiment: Human renal tubular epithelial cells(HK2)from the 6th to 15 th generation were selected for the experiment.Firstly,MTT assay was used to detect cell viability,and the toxicity and optimal concentration of Pae were respectively detected.It was found that Pae showed a dose-dependent protective effect,so the experiment was divided into 6 groups: normal group,normal dosing group,model group,and low-medium-high dose group.The protein and mRNA of the cells were extracted,and the renal damage factor(KIM-1)and NGAL were detected by Western blot,immunofluorescence and real-time PCR.Cell RNA was extracted for transcriptomic sequencing,and GO and KEGG were enriched.The expression levels of key pathway proteins Hsp90AA1 and Akt were detected by Western blot.Then Western blot was used to detect the expressions of Caspase3,Bcl-2 and Bax,the key proteins of apoptosis.Cell apoptosis was detected by Tunel and flow cytometry.Finally,Real-time PCR was used to detect the expression levels of cytokines TNF-α,IL-1β and MCP-1.Network pharmacology was used to search for the key proteins of Pae’s protective effect,and molecular docking,CETSA and SPR experiments were used to verify the binding of Pae and Hsp90AA1.Hsp90AA1 expression was knocked down by siRNA transfection,and after confirming the effect of Hsp90AA1 knockdown,Western blot was used.The effects of Pae on HK2 cell damage(KIM-1,NGAL),inflammation(TNF-α,IL-1β,MCP-1)and apoptosis(Caspase3,Bcl-2,Bax)were determined by Real-time PCR.In vivo: Acute kidney injury model was established by intraperitoneal injection of cisplatin(20mg/kg)in mice,and intraperitoneal injection of Pae(12.5,25,50mg/kg).The experiment was divided into six groups: normal control group,single dosing group,cisplatin model group,low dose group,medium dose group and high dose group.After successful modeling,serum creatinine and urea nitrogen were detected to observe the changes of renal function in each group.HE and PAS staining were used to observe the changes of renal tubule morphology,and transmission electron microscopy was used to detect the changes of mitochondria.The expressions of KIM-1 and NGAL in renal tissues were detected by immunohistochemistry and Western blot.Immunohistochemistry and Western blot were also used to detect the expressions of key proteins Hsp90AA1 and Akt,and the apoptosis-related proteins Caspase3,Bcl-2and Bax were detected by Western blot.The apoptosis of renal tubular epithelial cells was observed by Tunel staining.Finally,the inflammatory cytokines TNF-α,IL-1β and MCP-1 were detected by Real-time PCR.Results:In vitro experiments: MTT showed that the toxic and side effects of Pae were low and dose-dependent.Western blot,immunofluorescence and Real-time PCR results all showed that Pae could reduce cisplatin induced renal tubular epithelial cell damage.We found through network pharmacology that the target of Pae is heat shock protein90 Alpha family Class A member 1(Hsp90AA1),which plays an important role in the stability of many customer proteins including Akt.RNA-seq revealed that the KEGG enrichment pathway is PI3K-Akt pathway.The correlation with the protective effect of Pae was the greatest,which was consistent with network pharmacology.Go analysis showed that the main biological processes of Pae against Cis-AKI include cellular regulation of inflammation and apoptosis.Hsp90AA1 was confirmed as a possible target of Pae by cell thermal shift assay(CESTA)and surface plasmon resonance assay(SPR).Co-immunoprecipitation further indicated that Pae preconditioning promoted Hsp90AA1-Akt-protein-protein interactions(PPIs).Thus,Pae accelerates the formation of the Hsp90AA1-Akt complex and leads to significant Akt activation.Western blot and Real-time PCR were used to detect apoptosis-related proteins,Caspase3,Bcl-2 and Bax.The results showed that the apoptosis level was significantly reduced after Pae treatment,and flow cytometry also showed that the apoptosis level was reduced.Real-time PCR detection of inflammation-related factors TNF-α,IL-1βand MCP-1 showed that inflammation levels were also reduced.Then,after Pae treatment on Hsp90AA1 knockdown cells,Western blot and Real-time PCR results showed that Pae could not further play its protective role after Hsp90AA1 knockdown.In vivo experiments: Urea nitrogen and serum creatinine tests showed that Pae significantly reduced cisplatin-induced kidney injury.HE and PAS staining also showed that Pae reduced renal tubule dilatation.Western blot and Real-time PCR also confirmed that Pae reduced the expression of renal damage factor 1(KIM-1)and NGAL,markers of acute kidney injury.Transmission electron microscopy(TEM)showed that Pae could reduce mitochondrial damage induced by cisplatin.Real-time PCR confirmed that both cisplatin-induced inflammation and apoptosis levels could be antagonized by Pae.Conclusion:(1)In vitro studies have shown that Pae has a protective effect on cis-platin-induced renal tubular epithelial cells.The mechanism is through targeting the Hsp90AA1-Akt pathway.(2)In vivo experiments showed that Pae had protective effect on cisplatin-induced AKI mice.It can effectively inhibit the renal function decline,inflammatory reaction and apoptosis of cisplatin-induced acute kidney injury mice.This study provides a new target and strategy for the prevention and treatment of cisplatin-induced acute kidney injury.