The Anti-cervical Cancer Potential And Molecular Mechanisms Of Icariside ? In Epimedium Brevicornum Maxim

Epimedium brevicornum Maxim is one of the food materials with the same origin as medicine and food stipulated by National Health Commission of the People's Republic of China.Cervical cancer accounts for a huge mortality burden from female-related cancers.Using safe and low-toxic natural extracts as dietary supplements to prevent or treat cancer is widely accepted and is one of the global research hotspots.This dissertation firstly clarified the anti-cancer effect of Icariside ? on cervical cancer both in Hela cells and xenograft mice.Secondly,the integrated analysis of mi RNA-m RNA-protein omics was used to study the mechanisms of Icariside ? on Hela cells from the whole.Lastly,the inhibition effect of Icariside ? on Hela cell proliferation and migration was evaluated.The research in this paper will provide theoretical support for the development of functional food derived from E.brevicornum Maxim for medical purposes.The anti-cancer activities of Icariside ? and E.brevicornum Maxim water extraction on cervical cancer.Primarily,single factor experiment and response surface analysis were used to get the optimum extraction conditions of E.brevicornum Maxim water extract which rich in Icariside ? by macroporous resin elution.The process is 7.45 g/L E.brevicornum Maxim water extract flow through macroporous resin at rate of 3 BV/h.After absrption completely,3 BV and 60 %ethanol was used for elution.The best elution efficiency of Icariside ? was 89.38 %.The content of Icariside ? in E.brevicornum Maxim water extract reached 5.08 %.After given the final E.brevicornum Maxim water extract to Hela tumor-bearing mice by gavage at a dosage of 25 mg/kg body weight/2 day,the tumor volume decreased significantly.Then,four similar structure monomers(Icaritin,Icariin,Icariside ?,and Icariside ?)were experimented on MTT of Hela cells.And Icariside ? had the inhibitoriest effect on Hela cells comparing with other drugs.Finally,tumor-burdened mice were used,the tumor was markedly inhibited after Icariside ? treatment.In conclusion,the Icariside ? obtained from E.brevicornum Maxim had significant anti-cancer effects of cervical cancer.The effect of Icariside ? on the whole level of mi RNA in Hela cells.After 30 ?M Icariside ? treatment,expression of 63 mi RNA significantly changed(5 mi RNA were up-regulated and 58 mi RNAs were down-regulated)? Among them,hsa-mi R-4508,hsa-mi R-320a-5p,hsa-mi R-17-3p,hsa-mi R-182-5p,and hsa-mi R-345-5p presented the most obvious changes.Combining with GO analysis and KEGG analysis,these mi RNAs targeted genes were mainly enriched in cell cycle,apoptosis,antigen receptor signaling pathway(toll-like receptor and nod-like receptor signaling pathway),and related downstream signaling pathway(MAPK and NF-?B signaling pathway).The effect of Icariside ? on the whole level of m RNA in Hela cells.After treating Hela cells with 30 ?M Icariside ?,expression of 1502 genes significantly changed(680 m RNA were up-regulated and 822 m RNA were down-regulated).The significant m RNAs were mainly enriched in endoplasmic reticulum protein processing,cell proliferation,cell apoptosis,MAPK and Foxo signaling.Real-time PCR was used to verify the mi RNA-m RNA omics screened cell proliferation,apoptosis and,MAPK signaling pathway related genes,most of which were consistent with the projected results.The effect of Icariside ? on the whole level of protein in Hela cells.After treating Hela cells with 30 ?M Icariside ?,there were 2998 proteins with significant changes,among them 1591 proteins were up-regulated and 1407 proteins were down-regulated.Most of them were enriched in the nucleus,mitochondria,NF-?B,Wnt,p53,TNF,MAPK,endoplasmic reticulum protein processing,cell apoptosis,cell adhesion,autophagic,and AMPK signaling pathway.After the comprehensive analysis of all the mi RNA-m RNA-protein results,it was revealed that Icariside ? mainly regulated the following pathways in Hela cells: a)SEL1L,HSPA5 and HYOU1 in endoplasmic reticulum protein processing sigaling pathway;b)G1/S phase related key regulators in cell cycle;c)both intrinsic and extrinsic related parameters in apoptosis;d)JNK,ERK and P38 in MAPK signaling pathway and IKK?/?-I?B-P50/P65 in NF-?B signaling pathway.Icariside ? inhibited Hela cell proliferation and its mechanisms.Icariside ? had remarkable inhibitory effects on proliferation of Hela cells.On the one hand,the cell cycle of Hela cells was arrested by Icariside ? at G0/G1 phase through AKT/Cyclin E/CDK2.On the other hand,Flow Cytometry and Immunofluorescence showed that Icariside ? promoted apoptosis in a dose-dependent manner.And Icariside ? could activate the mitochondria dependent pathway Caspase 9/Caspase 3 much more significantly than death receptor pathway Caspase 8/Caspase 3.Finally,these results were further confirmed in transplanted models.Icariside ? inhibited Hela cell migration and its mechanisms.Fist of all,Immunohistochemistry and Western blot results showed Icariside ? inhibited the expression of MMP2/9 in cervcal cancer.Then,the cell migration assays revealed that Icarisdie ? diminished the ability of Hela cell crossing the transwell chambers and wound closure rate.Eventually,the key regulators in NF-?B and MAPK signaling pathway which can transcribed MMP2/9 were detected.JNK was screened from several important signaling molecules,which had the same expression trend with MMP2/9.And JNK activators Polyphyllin I and Anisomycin were used and concluded Icariside ? could inhibit cervical cancer cells migration through JNK-MMP2/9 signaling pathway.