| ObjectiveProstate cancer is the most common malignant tumor in urology.According to the report of the American Cancer Society,there will be nearly 20000 new prostate cancer patients in 2020,accounting for nearly 20%of male new cancer patients.The clinical treatment of advanced prostate cancer,especially castration resistant prostate cancer(CRPC),is still a worldwide problem.In China,clinical experience has proved that the combination of traditional Chinese medicine can significantly improve the quality of life of patients with advanced prostate cancer.My team systematically summarized the TCM syndrome law of prostate cancer through the retrospective clinical investigation of large samples of TCM syndrome,and took the lead in putting forward the treatment strategy of"FuzhengYiliu method" based on syndrome differentiation and treatment by stages.The traditional Chinese medicine prescription "FuzhengYiliu Decoction" based on this strategy has been authorized by the national invention patent.However,the mechanism of FuzhengYiliu Decoction in the treatment of prostate cancer is not clear.This topic mainly focuses on the following two objectives:(1)To clarify the mechanism of FuzhengYiliu Decoction regulating PI3K-Akt signal pathway in the treatment of prostate cancer;(2)To explore the potential pharmacodynamic target of FuzhengYiliu Decoction in the effective treatment of prostate cancer,so as to provide experimental data reference for the promotion and transformation of FuzhengYiliu Decoction.Methods(1)Clinical retrospective cohort studyCollect the complete clinical data of prostate cancer cases who were treated in Guangdong traditional Chinese medicine hospital from January 2014 to December 2021,who were clearly diagnosed by histopathology and received endocrine therapy.According to whether to use FuzhengYiliu Decoction,the patients were divided into Fuzheng Yiliu Decoction group and routine treatment group.The time of progression to CRPC and the rate and amplitude of decline of prostate specific antigen(PSA)were compared between the two groups.(2)Network pharmacology analysisThe compounds and targets of Fuzheng Yiliu Decoction were collected by tcmsp data,the targets of prostate cancer related diseases were collected by gencards database,the protein-protein interaction(PPI)of FuzhengYiliu Decoction prostate cancer common target was analyzed by string 11.0 database,and the gene ontology(go)annotation and enrichment and genome Encyclopedia(KEGG)pathway enrichment of Fuzheng Yiliu Decoction prostate cancer common target were carried out by David 6.8 database,Using Cytoscape 3.8.0 to construct and analyze the "prescription drug compound target pathway"network of Fuzheng Yiliu Decoction in the treatment of prostate cancer.(3)To clarify the mechanism of Fuzheng Yiliu Decoction regulating PI3K-Akt signal pathway in the treatment of prostate cancer based on animal transplanted tumor modelThe mouse prostate cancer cell RM-1 was inoculated into the armpit of the right forelimb of male C57 mice to construct the mouse model of prostate cancer transplantation.The mice were given Fuzheng Yiliu Decoction by gavage(high dose:12.74 g/kg;medium dose:6.37 g/kg;low dose:3.19 g/kg),and the positive control group was given abiraterone(130 mg/kg)and prednisolone(1.3 mg/kg);During the administration,the body weight was recorded,the tumor radius was measured,and the body weight growth curve and tumor growth curve were drawn;After administration,the tumor tissue and organs such as heart,liver,spleen,lung and kidney were stripped and weighed,and the organ index and tumor inhibition rate were calculated;Blood was collected and serum was separated.The levels of prostate specific antigen(PSA)and testosterone(TTE)in serum were detected by enzyme-linked immunosorbent assay(ELISA);The total RNA was extracted by Trizol method,and the mRNA level of PI3K-Akt signal pathway related genes was detected by polymerase chain reaction(PCR);The tumor tissue was fixed with 4%paraformaldehyde,embedded in paraffin and sectioned,and the related proteins were detected by immunohistochemical(IHC)staining.Guided by the results of previous network pharmacology research,this paper discusses the pharmacodynamic effect of Fuzheng Yiliu Decoction on inhibiting the growth of prostate cancer and its regulation on PI3K-Akt signal pathway.Results(1)Clinical retrospective cohort studyA total of 124 cases of prostate cancer with complete clinical data and receiving endocrine therapy were collected in this clinical retrospective cohort study.They were divided into routine group and Fuzheng Yiliu Decoction group according to whether Fuzheng Yiliu Decoction was used or not.From the baseline analysis,there was significant difference in the prognosis stage of the American Joint Committee on cancer staging(AJCC)between the two groups(P<0.05),there was no significant difference in the Gleason score of puncture pathology between the two groups(P>0.05),and there was significant difference in PSA between the two groups before treatment(P<0.05).The results showed that there was significant difference in the time to reach the minimum value between the two groups(P<0.001),and there was no significant difference in the comparison between other project groups(P>0.05).The stratified analysis of the two groups showed that the time of progression to CRPC in FuzhengYiliu Decoction group was 9 months and 10 months longer than that in routine group,regardless of the age of patients<70 years old or 70-96 years old.The difference between the two groups was statistically significant(P<0.05).Patients with Gleason score of 7 and 8～10 in the Fuzheng Yiliu Decoction group were 12 months and 8 months longer than those in the conventional group,respectively.There was significant difference between the two groups(P<0.05).The results showed that the time of progression to CRPC in FuzhengYiliu Decoction group was 20 months and 9 months longer than that in routine group,respectively.There was significant difference between the two groups(P<0.05).According to the stratified analysis of body mass index(BMI),the time of progression to CRPC in FuzhengYiliu Decoction group was 20.5 months longer than that in routine group(P<0.001).The comparison of AJCC prognostic stage between the two groups suggests that the time of progression to CRPC in the Fuzheng Yiliu Decoction group is 9 months longer than that in the routine group.The difference between the two groups is statistically significant(P<0.01).(2)Network pharmacology analysisThrough searching and screening,a total of 158 compounds of FuzhengYiliu Decoction,255 potential targets of compounds,11719 genes of prostate cancer related diseases and 233 common targets of FuzhengYiliu Decoction and prostate cancer were obtained.The results of network analysis showed that quercetin,kaempferol and luteolin were the main active components of FuzhengYiliu Decoction.PTGS2,ncoa2 and ptgsl were the main targets of Fuzheng Yiliu Decoction in the treatment of prostate cancer.The common targets were enriched in multiple signal pathways such as PI3K-Akt and regulated biological processes such as cell proliferation,apoptosis and gene transcription.(3)Inhibitory effect of FuzhengYiliu Decoction on transplanted prostate cancer cells in RM-1 miceAfter 9 days of administration,the tumor volume of each dose group and positive drug group of FuzhengYiliu Decoction was significantly smaller than that of the model group.At the end of the experiment,the tumor weight in the model group was 10.53 g±1.54 g,and the average weight of transplanted tumor in the low,medium and high dose groups of FuzhengYiliu Decoction were 9.31 g ±0.79 g(P>0.05),8.70 g±1.36 g(P<0.05),8.46 g ±0.81 g(P<0.05),respectively.The tumor inhibition rates were about 11.61%±7.53%(P>0.05),17.41%±12.92%(P<0.05)and 19.70%±7.73%(P<0.05),showing a good dose dependence;In the positive drug group,the transplanted tumor weight was 9.19 g ± 1.29 g(P>0.05),and the tumor inhibition rate was about 12.76%±12.20%(P>0.05).The results of ELISA showed that the serum PSA level of untreated tumor bearing mice was 113.32 ± 6.49 ng/ml,which was not significantly different from that of normal mice(129.25 ± 24.57 ng/ml).After administration,the serum PSA level of FuzhengYiliu Decoction low,medium and high dose groups decreased to 45.53 ± 11.63 ng/ml(P<0.001),60.79±16.92 ng/ml(P<0.05)and 53.79 ± 8.36 ng/ml(P<0.01),respectively.The serum PSA level of positive drug group was 59.10±14.82 ng/ml(P<0.05).The serum TTE level of untreated tumor bearing mice was 32.72 ± 1.62 nmol/L,which was not significantly different from that of normal mice(36.23 ±6.05 nmol/L).After administration,the serum TTE level of FuzhengYiliu Decoction low,medium and high dose groups decreased to 20.64±4.23 nmol/L(P<0.05),12.91 ±2.04 nmol/L(P<0.001),23.69 ± 5.38 nmol/L(P>0.05),and the serum TTE level of positive drug group was 18.12 ±3.84 nmol/L(P<0.05).QPCR test results showed that FuzhengYiliu Decoction could significantly up regulate the mRNA levels of four genes,including IL6,IL4,hsp90aa1 and VEGFA(FIG.16),and significantly down regulate the mRNA levels of 19 genes,including bcl 211,KDR,CDK4,MCL1,EGF,CDK2,met,rafl,rxra,TP53,mapk1,EGFR,ITGB3,COL3A1,gsk3b,rela,CDKN1A,INSR and prkca(FIG.17),while on MDM2,PTEN,Chuk,SPP1,NOS3,AKT1,CDKN1A,CASP9,BCL2 There was no obvious regulation of the mRNA expression of 11 genes such as pik3cg and myc.IHC results showed that FuzhengYiliu Decoction and positive drug(abiraterone+prednisolone)could significantly reduce the level of PSA in transplanted tumor tissue,but the expression of androgen receptor(AR)protein did not change significantly.FuzhengYiliu Decoction can significantly reduce the phosphorylation level of Akt,the key protein of PI3K-Akt pathway,and significantly up regulate the protein expression of p53,the downstream protein of Akt.It has no significant effect on the expression and phosphorylation level of PI3K protein and Akt protein.In addition,FuzhengYiliu Decoction can significantly inhibit the expression of cell proliferation marker Ki67 and autophagy marker 1c3b,and significantly up regulate the protein expression of caspase 3,the main related protein of apoptosis,but has no significant effect on the expression level of p16,p21,cyclin D1 and other proteins.Conclusion(1)FuzhengYiliu Decoction group can prolong the time of prostate cancer patients progressing to CRPC compared with routine group.(2)Network pharmacological analysis suggests that quercetin,kaempferol,luteolin and other active components in FuzhengYiliu Decoction may inhibit the proliferation of prostate cancer cells,induce apoptosis and exert their pharmacological activity in the treatment of prostate cancer by inhibiting the abnormally activated PI3K Akt and other signal pathways in prostate cancer,regulating gene transcription and affecting the expression or activity of key targets such as PTGS2,ptgsl and ncoa2.(3)FuzhengYiliu Decoction can inhibit cell proliferation in prostate cancer transplanted tumor tissue,promote cell apoptosis,effectively inhibit the growth of prostate cancer transplanted tumor in mice,inhibit the phosphorylation and activation of Akt,a key protein in PI3K-Akt pathway,and regulate PI3K-Akt pathway,which is consistent with the results of network pharmacological analysis. |
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