Study On The Role And Mechanism Of HP-1 In Protecting Renal Function And Resisting Renal Cell Carcinoma

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HP-1 attenuates renal toxicity caused by CP and protects renal functionBackgroundWith the continuous improvement of people's living standards and health consciousness,many previously undetected cancer patients are diagnosed through screening health checks.According to data released by the American Cancer Society,there were 18.1 million new cancer patients worldwide in 2018,of which 9.6 million intact patients died.The use of anticancer drugs has become an indispensable means for tumor treatment.Cisplatin(CP),a metal platinum complex,has a wide range of applications in clinical cancer treatment because of its remarkable antitumor effect,broad cancer spectrum,and low price.Cisplatin binds to DNA in the nucleus,causing DNA damage and destroying DNA transcription and replication.It also inhibits RNA and protein synthesis at high concentrations.Cancer cells are more rapidly metabolized than normal cells and are therefore more sensitive to CP.However,the indiscriminate nature of CP treatment often leads to damage to normal cells,causing many adverse reactions.Especially kidney damage,because cisplatin is mainly excreted through the kidney,and accumulated in the kidney,especially the renal tubules,causing acute renal dysfunction.In severe cases,renal tubular necrosis leads to anuria.This may be closely related to the inflammatory response caused by cisplatin and cell mitochondrial damage.Traditional Chinese medicine therapy has attracted more and more attention due to its small toxic and side effects,convenient and easy to accept,and significant effect of adjuvant therapy.Huaier,a medicinal fungus,has been used in China for thousands of years.In recent decades of research,Huaier has been shown to have potential anti-tumor effects and improve systemic immunity,so it has attracted attention and applied to the adjuvant treatment of clinical tumors.It has been reported that Huaier water extract(the main component is Huaier polysaccharide,HP-1)can improve the inflammatory response,reduce the infiltration of inflammatory cells,and protect the mitochondrial function of cells through its specific immune regulation.Therefore,in this study we hypothesized that HP-1 could reduce the level of oxidative stress induced by CP chemotherapy,reduce inflammatory infiltration,and reduce mitochondrial dysfunction,so it could protect renal function and reduce renal damage.For this reason,we have carried out a lot of research,hoping to focus on exploring the mechanism of possible HP-1 antagonistic cisplatin side effects at the molecular level,in order to provide effective auxiliary means for the future clinical application of cisplatin.Objectives1.Evaluate whether HP-1 can reduce kidney damage in vivo.2.To detect the effect of HP-1 on the autophagy activation and mitochondrial dysfunction caused by CP.3.Study the mechanism of HP-1 reducing CP-induced oxidative stress4.To explore the mechanism of renal inflammation and infiltration induced by HP-1 suppression of CP.5.To explore the biological effects of HP-1 on reducing apoptosis and cell cycle arrest caused by CP.6.Find out which signal pathways HP-1 uses to regulate CP-induced adverse reactions.Methods1.A mouse model of acute kidney injury was induced by cisplatin and treated with different concentrations of HP-1.The mouse serum BUN and Cr concentration levels were used to evaluate mouse kidney function.HE staining was used to verify the effect of HP-1 in protecting renal function.The level of KIM-1 protein was measured by western experiments to determine the presence or absence of kidney damage.Secondly,the ultrastructure of mouse kidney cells was observed by an electron transmission microscope,and the results of intuitively the presence or absence of kidney damage were obtained.2.Observe the number of autophagy vesicles and mitochondrial microstructure in mouse kidney cells by electron transmission microscope,and evaluate the effect of HP-1 on antagonizing CP-induced autophagy and mitochondrial damage.The expression of autophagy-related protein LC3-? in RPTEC cells after CP induction was detected by western experiments,and the cell viability changes of cells in vitro after HP-1 application were evaluated by CCK-8 experiments.At the same time,immunofluorescence experiments were used to characterize the level of LC3-? after HP-1 treatment in vitro cells to corroborate the experimental results of LC3-? in vivo.3.Using western experiments to detect the expression of oxidative stress-related proteins,and simultaneously measure the levels of oxidative stress-related indicators MDA,ROS and GSH,and analyze the mechanism of HP-1 reducing CP-induced oxidative stress.4.First,use immunohistochemistry to evaluate the expression level of TNF-?,a representative factor of inflammation in mouse kidney tissues before and after HP-1 treatment.Secondly,the effects of HP-1 treatment on the expression levels of TNF-?,COX-2,IL-8,and p-NF-?B were examined at the molecular level using western experiments.Finally,ELISA was used to detect the levels of TNF-? and IL-1? in mouse serum.Combined with in vivo and in vitro experiments to find out the mechanism of HP-1 reducing renal inflammatory infiltration.5.The level of Bax in mouse kidney tissue was detected by immunohistochemistry to identify the apoptosis of mouse kidney cells;the level of apoptosis and the expression of major cell cycle proteins were determined by western experiment;the cell in HP-1 was detected by TUNEL in vitro The antagonistic effect of CP-1 induced by-1 treatment was corroborated by western and flow cytometry;the results of western experiments were verified by flow cytometry,and the effect of HP-1 on reducing apoptosis induced by CP was demonstrated.6.The expression of major proteins in the PI3K-AKT-mTOR signaling pathway was detected by western experiments,which could support apoptosis and cycle-related protein expression.The simultaneous use of LY294002 specific PI3K signaling inhibitors further proves the hypothesis that HP-1 regulates CP-induced renal damage through the PI3K-AKT signaling pathway.7.Using human renal tubular epithelial normal cells HK-2,it was verified according to the above experimental steps that HP-1 also has the effect of preventing CP-induced renal damage in human cells.Results1.After the application of HP-1,renal function was significantly improved Although the levels of BUN and Cr did not reach normal levels,they had fallen compared to the CP group.HE showed that compared with the CP group,renal tubular and glomerular damage were reduced,protein casts were reduced,and KIM-1 protein expression levels were also reduced after HP-1 treatment,indicating that renal damage was reduced.Transmission electron microscopy also observed that after HP-1 treatment,the cells tended to be normal and the villi structure improved.At the same time,no toxic side effects of HP-1 treatment on important organs were found.2.Observation of reduction of autophagosomes,improvement of mitochondrial plasma membrane structure,and reduction of autophagy-related protein expression under electron microscope,indicating that HP-1 treatment reduced CP-induced autophagy and mitochondrial dysfunction.In vitro CCK-8 experiments,select appropriate CP induction and HP-1 treatment concentrations,and obtain cell viability data.It was also proved by immunofluorescence experiments that HP-1 treatment reduced the autophagy of extracellular cells.3.Compared with the CP group,western experiments confirmed that the expression level of oxidative stress-related proteins after HP-1 treatment was reduced.The levels of oxidative stress factor ROS and MDA were also reduced after HP-1 treatment,while the level of antioxidant stress factor GSH increased.This indicates that oxidative stress is effectively controlled and cell function is improved.4.Immunohistochemistry confirmed that HP-1 can significantly reduce the expression of inflammatory factors in tissues,and western also verified that related inflammatory proteins were reduced after using HP-1.At the same time,the ELISA results are the same as the western results.HP-1 is thought to reduce inflammatory infiltration by reducing the expression of inflammatory proteins and inflammatory factors.5.From the in vivo and in vitro perspectives:1).Reduced apoptotic proteins in mouse kidney tissue;2).Flow cytometry and TUNEL have demonstrated that the number of in vitro apoptosis is reduced after using HP-1;3).Western results showed that the expression of apoptosis-initiating proteins and major apoptosis-involving proteins was reduced,and the expression of proteins related to cell cycle progression was restored.This shows that after the use of HP-1,both the number of apoptosis in vivo and in vitro is reduced,and the cell cycle is restored,and individual functions are updated and improved.6.HP-1 reduces the onset of apoptosis by reducing the phosphorylation of PI3K,AKT,and mTOR proteins.And by increasing the expression of GSK-3?,the inhibition of the cell cycle is lifted.LY294002 was used to further verify the role of PI3K-AKT-mTOR signaling pathway in HP-1 treatment of CP-induced injury.7.From the perspective of human cells,it has been proved that HP-1 also plays a role in reducing the oxidative stress,reducing the inflammatory response,and reducing apoptosis in the human cell environment.Conclusion1.HP-1 can reduce CP-induced acute renal damage and restore renal function.2.HP-1 has no organ toxicity.3.HP-1 reduces cell autophagy induced by CP.4.HP-1 weakens mitochondrial function damage caused by CP.5.HP-1 restored cell viability without significant cytotoxicity in vitro.6.HP-1 reduces the expression of oxidative stress-related proteins and factors,and attenuates the oxidative stress response caused by CP.7.HP-1 reduces the infiltration of inflammatory factors and the expression of inflammatory proteins,thereby improving the cell survival environment.8.HP-1 reduces apoptosis and restores the cell cycle,thereby improving tissue and organ function.9.HP-1 attenuates renal damage caused by CP chemotherapy through the PI3K-AKT-mTOR signaling pathway.10.The therapeutic effect of HP-1 achieved the same results in human cells HK-2.Part ? Biological effect and mechanism of HP-1 against renal cell carcinomaBackgroundWith the development and progress of the material society,the quality of the population and the level of education have been continuously improved,the consciousness of citizens' health has gradually increased.Some tumors were found as a result of normal examination.Kidney cancer is a common tumor of the urinary system and the main research object of urologists.Worldwide,kidney cancer accounts for approximately 3%of all malignancies,and the incidence of kidney cancer is increasing at a rate of 2%per year.At the same time,renal cancer is also the most common solid lesion in the kidney,accounting for approximately 90%of the total number of renal malignancies.In terms of gender differences,the male to female ratio is 1.5:1.There are more men than women,and the age of onset tends to be aging,with the most in the 60-70 age group.In pathology and genetics,there are different tissue types and genetic variations.Among them,clear cell carcinoma of the kidney(ccRCC)accounts for the highest proportion,accounting for about 80%of renal cancer.Closely related item in the biological basis of inducing renal clear cell carcinoma is the mutation of the VHL gene.The abnormal function of the VHL gene has led to a large accumulation of the transcriptional regulator HIF-a(hypoxia-inducible factor),transcription up-regulates vascular endothelial growth factor(VEGF)and activates a number of kinase-dependent signaling pathways,such as the PI3K-AKT-mTOR signaling pathway.At present,the preferred treatment method for ccRCC is surgical resection.For the patients with advanced stage losing surgical significance,it is necessary to choose the appropriate treatment plan according to their own conditions.Targeting certain target genes and proteins,the development of targeted drugs is a new hope for patients with end-stage renal cancer,such as humanized anti-VEGF factor monoclonal antibodies,small molecule tyrosine kinase inhibitors and mTOR inhibitors.Sunitinib is an oral,small-molecule,multi-target receptor tyrosine kinase inhibitor that has multiple effects of inhibiting tumor angiogenesis and anti-tumor cell growth.Studies have shown that in the treatment of renal cell carcinoma,the drug mainly blocks several receptors downstream of the VEGF and PDGF signaling pathways to exert anticancer effects,including:PDGFR,VEGFR.However,with the application of the drug,its side effects and drug resistance of the tumor gradually appear,which further limits its clinical application.Huaier is a medicinal fungus that has been proven to play an important role in anti-tumor and enhance the body's immunity,can inhibit tumor cell growth,proliferation,metastasis and interfere with tumor angiogenesis.Enhance the sensitivity of tumor cells to therapeutic drugs,activate the autoimmune system and promote the apoptosis of tumor cells.The results of the CCK-8 experiment,colony formation experiment,scratch test,and transwell experiment that we used show that Huaier polysaccharide(HP-1)can effectively slow the tumor progression.And when HP-1 is used in combination with sunitinib,it can enhance the antitumor properties of sunitinib by inducing apoptosis and cell cycle arrest.A series of effects of HP-1 are achieved by relying on down-regulation of CIP2A expression and suppression of epithelial-mesenchymal transition(EMT).Moreover,real-time quantitative PCR and western blot experiments showed that CIP2A downregulation was particularly significant after HP-1 combined with sunitinib,and it was thought to be related to the suppression of EMT.In addition,the combined application of HP-1 and sunitinib inhibited the PI3K/Akt/VEGFR signaling pathway,resulting in a decrease in the expression of proteins associated with this pathway.It was also observed in a mouse model of in vivo xenograft that the combination of HP-1 with sunitinib enhanced the antitumor effect of sunitinib.In summary,it is suggested that HP-1 has a certain anti-renal cell carcinoma(RCC)effect and can enhance the therapeutic effect of sunitinib.Objectives1.To evaluate whether HP-1 can inhibit the cell viability of tumor cells and enhance the sensitivity of renal cancer cells to sunitinib.2.To evaluate the effect of HP-1 and HP-1 combined with sunitinib on tumor cell proliferation.3.Observe the effect of HP-1 and HP-1 combined with sunitinib on the activity of renal cancer cells.4.Observe the ability of HP-1 and HP-1 combined with sunitinib to suppress the metastasis and invasion of renal cancer cells.5.Through the detection of renal cancer cell cycle,explore the mechanism of HP-1,HP-1 combined with sunitinib affecting the cycle.6.Through the detection of renal cancer cell apoptosis,explore how HP-1 and HP-1 combined with sunitinib induce apoptosis.7.Find out whether HP-1 and drug combination affect the viability and metastatic ability of renal cancer cells by regulating the expression of CIP2A gene and its corresponding protein.8.Investigate the molecular mechanism of HP-1 and combination drug inhibition of tumor cell growth through EMT signaling pathway and PI3K-AKT-VEGFR signaling pathway.9.In vivo experiments explore the inhibitory effect of HP-1 and its combination with sunitinib on tumor growth and its underlying mechanism.Methods1.CCK-8 determination of cell viability of tumor cells when different concentrations of HP-1,HP-1 and Sunitnib were used in combination.Colony formation experiments determine the ability of renal cancer cells to form colonies after drug treatment.Observe the morphological changes of renal cancer cells after drug treatment under a microscope.2.Scratch test to determine the activity of renal cancer cells after drug treatment.Transwell assay was used to determine the proliferation and invasion ability of renal cancer cells after application of HP-1 and combined Sunitnib.3.Flow cytometry was used to detect the cell cycle distribution of renal cancer cells after drug treatment.Western assays determine the mechanism by which drugs affect the cancer cell cycle's intrinsic protein expression.4.Flow cytometry was used to detect the apoptosis of renal cancer cells before and after drug treatment.Western detection of apoptosis-related protein expression to determine the intrinsic mechanism of HP-1 induced apoptosis.5.The expression of CIP2A gene was measured by PCR,and the expression of CIP2A protein was detected by western detection.Qualitative analysis was performed by immunofluorescence.Overexpression of CIP2A was used to determine the effects of HP-1,HP-1 and Sunitinib on cell viability and metastatic invasion.6.After the application of HP-1,HP-1 and Sunitinib,the expression of major proteins in EMT pathway and PI3K-AKT-VEGFR signaling pathway was determined by western experiments.7.In vivo experiments to verify the inhibition of tumor growth by HP-1 and combination drugs,and use PCR and western to analyze its internal biological mechanism.Results1.HP-1 reduces the viability of renal cancer cells.When sunitinib is used in combination with HP-1,it inhibits cell viability more than sunitinib alone,and both have time and concentration dependence of the drug.At the same time,the combined application of drugs with kidney cancer cells has a weaker ability to form colonies,and the morphological changes of the cells are obvious.2.HP-1 treatment significantly reduced the ability of the renal cancer cell line to exercise,and inhibited the ability of cell proliferation and invasion.After sunitinib combined with HP-1,sunitinib's tumor suppressive effect was more significant.3.G1 phase of cell block more than combined administration of single drug,and the expression of cyclin-D and cyclin-E decreased,while the expression of p21 Cipl and p27 Kip1 related to cycle inhibition increased.4.The expression of apoptosis-associated proteins caspase-3 and Bax increased,and flow cytometry showed that the combination of HP-1,HP-1 and sunitinib induced apoptosis,and the combination treatment was more significant than that of fresh fruit alone.5.Renal cancer cell viability weakened,proliferation and invasion were inhibited after drug treatment,which was the result of inhibition of CIP2A gene expression and decrease of CIP2A protein expression.6.The expression of N-cadherin,Vimentin,Snail,and Slug of EMT-related proteins decreased and the expression of E-cadherin increased after drug treatment.The phosphorylation activity of PI3K and AKT proteins decreased,and GSK-3? and VEGFR proteins also decreased after drug treatment.7.Compared with the control group,mouse xenograft tumors were significantly reduced after HP-1 treatment,and the sunitinib combined with HP-1 application group had the smallest tumor volume.PCR analysis showed that CIP2A gene expression was reduced in the tumors of the drug-treated group.Western experiments showed the expression of cell cycle,apoptosis,EMT process,PIEK-AKT-VEGFR signaling pathway related proteins.Conclusion1.HP-1 can inhibit the activity of renal cancer cells and weaken the ability of tumor cells to proliferate and invade.2.HP-1 can inhibit tumors by inducing apoptosis of renal cancer cells and blocking the progression of renal cancer cells.3.HP-1 attenuates tumor cell proliferation and invasion by regulating the expression of CIP2A gene and CIP2A protein.4.HP-1 can reverse the EMT process,induce apoptosis of renal cancer cells,and inhibit tumor angiogenesis by regulating EMT,PI3K-AKT-VEGFR/GSK-3? and other signal pathways.5.In vivo environment,HP-1 inhibits the growth of xenograft tumors in vivo by reducing the expression of CIP2A,reversing the EMT process,inducing apoptosis,blocking the cell cycle and reducing the activity of the PI3K-AKT-VEGFR pathway.6.When Sunitinib is combined with HP-1,the effect of Sunitinib is better than when used alone,indicating that HP-1 enhances the drug sensitivity of tumor cells to Sunitinib.