Effects And Mechanisms Of Astragaloside II On Apoptosis, Migration And Invasion Of Renal Clear Cell Carcinom

Purpose:Renal cell carcinoma is a common tumor of urinary system,and it was reported that in2022 about 400,000 people worldwide suffered from renal cell carcinoma.Clear cell renal cell carcinoma(cc RCC)is the most common type of renal cell carcinoma,accounting for 75.80%of renal cell carcinoma.The most common clinical manifestations of clear cell renal cell carcinoma are abdominal pain,hematuria,and abdominal mass,often referred to as the triad of renal cell carcinoma.Local renal cell carcinoma can be treated with partial nephrectomy,cryoablation and active surveillance,but there are still about 30% of patients with tumor metastasis,and eventually need systemic treatment.Conventional radiotherapy and chemotherapy are ineffective,with high mortality and poor prognosis.In the past,the treatment of advanced renal cell carcinoma is based on interleukin-2,interferon-α and other immunotherapy,but its effective rate is only about 10%.However,in recent years,there has been a breakthrough in the treatment of advanced renal cell carcinoma.Due to the hypervascular nature of clear cell renal cell carcinoma,inhibitors targeting VEGF and its receptor tyrosine kinase were first recommended as first-and second-line treatments for metastatic renal cell carcinoma in the United States and Europe,such as sunitinib,sorafenib,axitinib,and pazopanib.Immunotherapy has also made breakthroughs in recent years.However,the high price of targeted drugs limits their clinical application.On the other hand,patients with advanced renal cell carcinoma also show drug resistance to new small molecule targeted drugs represented by sunitinib.Traditional Chinese Medicine believes that tumor is pathogenicfactors,and the main idea of anti-tumor is to strengthen the right and expel the evil,that is,to enhance the lethality of the immune system has the effect of strengthen the right,itself also has the effect of directly expel the evil,Astragalus is known as the most important medicine of reinforcing Qi.It is especially suitable for the prevention and treatment of tumor and is also commonly used in tumor prescriptions.Therefore,in order to replace the potential of chemotherapy-induced tumor resistance,we chose astragalus,a completely natural component of traditional Chinese medicine,to study its inhibitory effect on tumor cell activity.In this study,we use network pharmacology,cytology,cell biology,molecular biology and nude mice xenograft model to investigate the effects of Astragaloside II on renal cancer cell migration and invasion in vivo and in vitro at cellular and molecular levels.We intend to explore a new target of Astragaloside II in the treatment of clear cell renal cell carcinoma through studying the role of PI3K/AKT/mTOR pathway and Wnt/β-catenin pathway from the perspective of inducing cell apoptosis and inhibiting cell invasion,and lay a theoretical foundation for future clinical application.Material and method:1.To explore the mechanism of astragalus in treating clear cell renal cell carcinoma based on network pharmacology and bioinformaticsThe active ingredients and action targets of Astragalus membranaceus were screened by TCMSP;and the intersection targets with clear cell renal cell carcinoma were screened by OMIM,Genecards,Pharm Gkb,Drug Bank and TTD databases.The Ingredient-Targets-DrugDisease Network was constructed to screen the core components of Astragalus treatment of clear cell renal cell carcinoma.The protein interaction networks and visualization maps were established and analyzed by Cytoscape 3.8.0 software and STRING database respectively;the obtained targets were analyzed by KEGG and GO enrichment with the help of R language and Bioconductor software package;finally,the action intensity of the core active ingredients and key targets were verified.2.Astragaloside II regulate the oxidative stress-mediated PI3K/AKT/mTOR signaling pathway on the growth of renal clear cell cancer cellsIn the second part of the experiment,786-O human kidney cancer cells were selected for culture,and the effects of Astragaloside II on the proliferation and colony formation of kidney cancer cells were observed by detecting the activity and colony-forming unit assays after adding different concentrations of Astragaloside II.In order to determine the effect of Astragaloside II on apoptosis of kidney cancer cells,the apoptosis of 786-O cells treated with Astragaloside II for 24 h was detected by Annexin V-FITC/PI double staining kit.Q2 and Q3 represent late apoptosis and early apoptosis,respectively,and Q2+Q3 is the total apoptosis rate.The expression of apoptosis-related proteins in 786-O cells treated with Astragaloside II for 24 h were detected by Western blot experiment.Western blot experiment was used to detect changes in the expression levels of PI3K/AKT/mTOR pathway related proteins in 786-O cells treated with Astragaloside II.Western blot experiment was used to detect changes in the expression levels of PI3K/AKT/mTOR pathway related proteins in 786-O cells treated with Astragaloside II,and to explore the effects of Astragaloside on PI3K/AKT/mTOR Ⅱsignaling pathway.Moreover,PI3 K agonist IGF-1 was added to reverse the cytotoxicity of Astragaloside II on 786-O cells,and it was further determined whether the effect of Astragaloside II on apoptosis of renal cancer cells was mediated by PI3K/AKT/mTOR.Finally,DCFH-DA fluorescent probe was used to detect the effects of Astragaloside II on oxidative stress(ROS)levels in renal carcinoma cells.In order to clarify the relationship between oxidative stress and apoptosis of 786-O cells,Astragaloside and the antioxidant ⅡN-acetyl-L-cysteine(NAC)were added to 786-O cells at the same time,and the cell viability was observed.In order to further explore the molecular mechanism of oxidative stressinduced apoptosis of 786-O cells,we detected the changes in the expression levels of PI3K/AKT/mTOR pathway related proteins in 786-O cells treated with AstragalosideⅡ+NAC by Western blot experiment.3.Study on the mechanism of Astragaloside II regulating Wnt/β-catenin signaling pathway to inhibit the invasion and migration of clear cell renal cell carcinoma.In the third part of the experiment,786-O human renal carcinoma cells were selected for culture,and the effects of Astragaloside II on migration and invasion ability of renal carcinoma cells were detected by scratch healing experiment and Transwell experiment.EMT is a process by which epithelial cells acquire mesenchymal properties during tumor progression,which is characterized by the loss of epithelial marker E-cadherin and the upregulation of mesenchymal markers N-cadherin and vimentin.Western blot experiment was used to detect the effects of Astragaloside II on the expression levels of E-cadherin,Ncadherin and vimentin in 786-O cells,and to explore the effects of Astragaloside II on EMT process in 786-O cells.To further explore the role of Wnt/β-catenin signaling pathway in the inhibition of Astragaloside II on the migration of clear cell renal cell carcinoma,the experimental cells were divided intoe Control group,Astragaloside Ⅱgroup,Li Cl group,Astragaloside Ⅱ+Li Cl group.To explore the effect of Astragaloside II on Wnt/β-catenin signaling pathway,the expression of β-catenin,a key protein in Wnt/β-catenin signaling pathway,was detected by Western blot experiment.In order to further verify whether Astragaloside could inhibit EMT of clear cell renal cell carcinoma through Wnt/βⅡ-catenin signaling pathway,the expressions of EMT-related proteins E-cadherin,N-cadherin and vimentin were detected by Western blot.To explore whether the inhibitory effect of Astragaloside on clear cell renal cell carcinoma migration was mediated through Wnt/βⅡ-catenin signaling pathway,the effect of Astragaloside Ⅱcombined with Li Cl on the migration ability of 786-O cells was determined by scratch healing experiment.4.Effect of Astragaloside II on nude mice xenograft model.In the fourth part,male BALB/c nude mice were selected as research animals to establish786-O human renal cancer xenografts in nude mice.The experimental animals were divided into two groupse control group and astragalus saponin intervention group.First,the effect Ⅱof Astragaloside II on inhibiting the proliferation of renal carcinoma cells was observed by the tumor volume.The morphological changes of 786-O cells were observed by HE staining,and the expressions of apoptosis-related proteins,PI3K/AKT/mTOR related proteins,EMTrelated proteins and Wnt/β-catenin related proteins were further detected by immunohistochemical staining to explore the specific mechanism of Astragaloside II on proliferation,migration and invasion ability of nude mice xenograft model.Results:1.To explore the mechanism of astragalus in treating clear cell renal cell carcinoma based on network pharmacology and bioinformaticsAstragalus has 21 active ingredients,395 potential targets,180 drug-disease intersection targets,quercetin,kaempferol,astragaloside,etc.The core targets are AKT1,TP53,MAPK14,JUN,ESR1,etc.The KEGG and GO pathway enrichment analysis showed that Astragalus is involved in over pathways for the treatment of cc RCC with potential mechanism was related to the regulation of PI3K-Akt,MAPK,AGE-RAGE,TNF and other signaling pathways.Molecular docking experiments showed that the core active components of Astragalus had strong binding ability to all five key targets,with Astragaloside binding most tightly to AKT1.2.Astragaloside II regulate the oxidative stress-mediated PI3K/AKT/mTOR signaling pathway on the growth of renal clear cell cancer cells.Astragaloside II inhibited the proliferation and colony formation of 786-O cells.Astragaloside II showed an inhibitory effect at the concentration of 20μM at 24 h and 48 h.The viability of 786-O cells was inhibited by the concentration of 20μM,40μM and 80μM,and the maximum inhibitory effect was observed at the concentration of 80μM.The cell viability of Astragaloside II treated for 48 h was lower than that of Astragaloside II treated for24 h at 20μM,40μM and 80μM(P < 0.05,P < 0.05,P < 0.001).In addition,colony formation assay was performed to evaluate the colony formation ability of the cells,and it was found that 10μM Astragaloside II could inhibit the colony formation of the cells.Annexin V-FITC/PI double staining kit for cell apoptosis showed that the control group,namely the 0μM group Q2+Q3=5.0%,the 10μM group Q2+Q3=15.1%,the 20μM group Q2+Q3=20.1%,and the 30μM group Q2+Q3=28.9%.It can be seen that the apoptosis rate also increased with the increase of concentration.Western blot experiment detected the expression of apoptosis-related proteins,and the results showede pro-caspase3 expression had no significant change,but cleaved caspase3 expression was increased in the 10 and 20μM groups,and cleaved caspase3 expression intensity was increased in the 20μM group compared with the 10μM group(P < 0.05).Western blot experiment detected changes in the expression levels of proteins related to PI3K/AKT/mTOR pathway.The results showed that p-PI3 K,pAKT and p-mTOR in PI3K/AKT/mTOR pathway decreased after Astragaloside treatment Ⅱof 786-O cells(P < 0.05),and the differences were statistically significant.This trend was reversed with the addition of IGF-1.DCFH-DA fluorescent probe was used to detect the effect of Astragaloside Ⅱon oxidative stress(ROS)levels in renal carcinoma cells.The results showed that DCF fluorescence intensity of 786-O cells increased with the increase of Astragaloside concentration,and the differences were statistically significant(Ⅱ P < 0.01).After the addition of antioxidants,the cell activity increased and antagonized the decrease of p-PI3 K,p-AKT and p-mTOR induced by Astragaloside II.The expression of apoptosisrelated proteins in 786-O cells was detected by Western blot experiment.Compared with NC group,Astragaloside Ⅱ increased the expression of cleaved caspase3 protein(P < 0.01).After addition of NAC,the protein expression level of cleaved caspase3 was decreased compared with simple Astragaloside Ⅱ group,and the difference was statistically significant(P < 0.001).3.Study on the mechanism of Astragaloside II regulating Wnt/β-catenin signaling pathway to inhibit the invasion and migration of clear cell renal cell carcinoma.Scratch healing and Transwell experiments demonstrated that Astragaloside II inhibited the migration and invasion of renal carcinoma cells.The effects of Astragaloside II on the expression levels of EMT-related protein and Wnt/β-catenin-related protein in 786-O cells were detected by Western blot experiment.The results showed that Astragaloside Ⅱpromoted the expression of E-cadherin protein,but inhibited the expression of N-cadherin and vimentin protein,and the differences were statistically significant(P < 0.01).In order to explore the effect of Astragaloside Ⅱon Wnt/β-catenin signaling pathway,Western blot experiment was used to detect the expression of β-catenin,a key protein of Wnt/β-catenin signaling pathway in renal cancer cells.The results showed that,compared with NC group,Astragaloside significantly downⅡ-regulated the expression of β-catenin,and Western blot rescue experiment showed that the inhibition effect of Astragaloside Ⅱon Wnt/β-catenin signaling pathway could be antagonized by Li Cl(activator of Wnt/β-catenin signaling pathway).The differences were statistically significant(P < 0.05).In order to further verify whether Astragaloside Ⅱcan inhibit EMT in renal cancer cells through Wnt/β-catenin signaling pathway,Western blot experiment was used to detect the expression levels of EMTrelated proteins E-cadherin,N-cadherin and vimentin.The results showed that,compared with the NC group,Astragaloside Ⅱup-regulated the expression of E-cadherin and downregulated the expression of N-cadherin and vimentin in renal cancer cells,and this trend was reversed after the addition of Li Cl.In order to investigate whether the inhibition of Astragaloside on the migration ability of renal carcinoma cells plays a Ⅱ role through the Wnt/β-catenin signaling pathway,we determined the effect of Astragaloside Ⅱcombined with Li Cl on the migration ability of 786-O cells by the scratch healing experiment.The results showed that,compared with the NC group,Astragaloside Ⅱ inhibited the scratch healing rate of renal cancer cells,while the addition of Wnt/β-catenin pathway activator Li Cl increased the scratch healing rate of renal cancer cells,with statistical significance(P < 0.01).4.Effect of Astragaloside II on nude mice xenograft model.The nude mice xenograft model was successfully created.Astragaloside II inhibited the growth of nude mice xenograft model.Compared with model group,the volume of transplanted tumor in Astragaloside intervention group was significanⅡ tly decreased,with statistical significance(P < 0.01).HE staining method was used to observe the morphological changes of 786-O cells.Compared with the model group,Astragaloside intervention group Ⅱshowed cell shrinkage,smaller volume,deep staining and different sizes of nuclei,and pyknosis.Immunohistochemical staining showed that compared with the model group,Ki67 protein expression in Astragaloside group was decreased(P < 0.05),Cleaved caspase3 and ⅡBax protein expression were increased,and Bcl-2 protein expression was decreased.The results showed that Astragaloside Ⅱ could promote apoptosis of renal carcinoma cells.Immunohistochemical detection of PI3K/AKT/mTOR related proteins in 786-O cells showed that compared with control group,the expression levels of p-PI3 K,p-AKT and p-mTOR in Astragaloside Ⅱgroup were significantly decreased,and the difference was statistically significant(P < 0.05).These results suggested that Astragaloside Ⅱ can down-regulate the phosphorylation level of PI3K/AKT/mTOR related proteins.Immunohistochemistry was also used to detect the expression of EMT-related proteins in786-O cells.The results showed that,compared with NC group,E-cadherin protein expression was increased in Astragaloside Ⅱ group,while N-cadherin and vimentin protein expression was decreased in Astragaloside Ⅱ group.The difference was statistically significant(P < 0.05).These data suggest that Astragaloside Ⅱ can inhibit the EMT process of 786-O cells.In order to determine the effect of Astragaloside Ⅱ on the expression of β-catenin,a key protein of Wnt/β-catenin signaling pathway in renal carcinoma cells,we detected the expression of β-catenin protein by immunohistochemistry experiment.The results showed that Astragaloside Ⅱ significantly down-regulated the expression of β-catenin compared with NC group(P < 0.05).Combined with the third part of the study,we suggest that Astragaloside Ⅱ may inhibit EMT of renal carcinoma cells through Wnt/β-catenin signaling pathway.Conclusion:1.This study initially elucidated that the active ingredients in Astragalus may play a role in regulating the immune function of the body through the key target AKT1,participating in signaling pathways such as PI3K-AKT,and treating clear cell renal cell carcinoma by affecting cell proliferation,differentiation,apoptosis and migration.2.Astragaloside II inhibited proliferation and colony formation of 780-O cells,and induced expression of cleaved caspase3,an executive protein of apoptosis,and promoted apoptosis by inhibiting phosphorylation of PI3K/AKT/mTOR signaling pathway.Oxidative stress induced by Astragaloside Ⅱmay play a role in promoting apoptosis through PI3K/AKT/mTOR signaling pathway.3.Astragaloside II can significantly inhibit the invasion and migration of clear cell renal clear carcinoma.From a mechanistic perspective,our results suggest that Astragaloside II can regulate the invasion and migration of renal cancer cells by inhibiting EMT via Wnt/β-catenin signaling pathway.4.Based on the previous network pharmacology and cell experiments,this part of the study further proved that Astragaloside II could inhibit the growth,migration and invasion of transplanted tumor in nude mice.Astragaloside II induced oxidative stress may play a role in promoting cell apoptosis through PI3K/AKT/mTOR signaling pathway.Astragaloside II may inhibit the migration and invasion of clear cell renal cell carcinoma by inhibiting EMT of renal cell carcinoma cells through Wnt/β-catenin signaling pathway in vivo.