The Mechanisms Of Oleanolic Acid In Preventing Hepatocellular Carcinoma Lung Metastasis Through Regulating Hypoxic Tumor-Derived Exosomes-Mediated Premetastatic Niche

BackgroundHepatocellular carcinoma(HCC)poses a significant health threat due to its high mortality rate,primarily resulting from treatment resistance and organ-specific metastasis.Despite therapeutic advancements,the 5-year survival rate for HCC patients remains low.Hypoxia,a common condition in tumors,is recognized as a risk factor for unfavorable outcomes.Transarterial chemoembolization(TACE),the primary treatment for intermediate-stage HCC,often induces extrahepatic metastasis due to severe local ischemia and hypoxia.Currently,the mechanism of liver cancer lung metastasis is unclear,lacking effective prevention and treatment methods,significantly impacting patient prognosis.Targeting pre-metastatic niche(PMN)development has been proposed as a potential therapeutic approach to prevent tumor metastasis.Traditional Chinese medicine(TCM)plays a crucial role in tumor treatment.Jiedu Recipe,an herbal formula composed of Cremastrae Pseudobulbus Pleiones Pseudobulbus,Actinidia valvata Dun,Salvia chinensis Benth,and Galli Gigerii Endothelium Corneum,is endorsed in the Chinese Guidelines for Diagnosis and Treatment of Primary Liver Cancer(2022 version)and is frequently used in clinical settings to impede the progression and metastasis of HCC.Oleanolic acid,a crucial component in Jiedu Recipe,exhibits a certain preventive effect on hepatocellular carcinoma lung metastasis.However,its therapeutic mechanism remains unclear.AimsThis study aimed to explore the mechanisms involved in the formation of PMN induced by hypoxic HCC tumor cells,as well as the preventive effect and mechanisms of oleanolic acid.MethodsPart 1: The regulatory effect of hypoxic tumor-derived exosomes on PMN formation1.Subcutaneous tumor resection models were generated to investigate the role of HIF-1α in tumor lung metastasis.2.TDEs derived from normoxic(N-TDEs)and hypoxic(H-TDEs)tumor cells were collected and were injected into mice by tail vein.Collagen deposition,fibroblast activating markers,and immunosuppressive M2 macrophage and MDSC were analyzed to investigate PMN development.3.H22-luciferase tumor cell were injected into PMN model mice to investigate the effect of TDEs on development of lung metastatic tumors.4.Plasma exosomes from HCC patients before and after TACE were collected to treat MRC5 lung fibroblast cells.The activation of inflammatory fibroblasts was determined by WB,qPCR and transwell.Part 2: The molecular mechanisms involved in the effect of H-TDEs activating lung fibroblasts1.Differentially expressed miRNAs in H-TDEs were determined through miRNA sequencing.2.miRNA extracted from plasma exosomes in HCC patients before and after TACE,normoxia and hypoxia-cultured tumor cells and TDEs were determined by qPCR.The WB assay followed by MRC5 cells transfected with miRNA mimic were performed to investigate the effect of miRNA on fibroblast activation.3.Utilizing miRNA target prediction and functional experiments to screen for direct targets of the identified key miRNAs that regulate the activation of lung fibroblasts.4.WB,immunofluorescence,and reporter gene experiment were used to investigate the effects of miRNA regulation on downstream targets and the major signaling pathways involved in the activation of lung fibroblasts.5.miR-4508-enriched TDEs were collected from the supernatant of lentivirus-infected tumor cells,and were intravenously injected into mice.The PMN formation rate was detected through immunofluorescence.Subsequently,tumor cells were intravenously injected and the development level of metastatic tumors were analyzed using in vivo fluorescence imaging.6.Pathway inhibitors were used to investigate the role of MAPK-NF-κB in the activating effect of H-TDEs on lung fibroblasts.Part 3: The suppressive effect of oleanolic acid on fibroblast activation and PMN development1.We treated H-TDE-activated fibroblast cells with individual compounds from Jiedu Recipe to screen for the component that inhibits inflammatory fibroblast activation.2.Jiedu Recipe and oleanolic acid were administered to PMN model mice at appropriate doses to investigate the preventive effect of drugs on PMN formation and metastatic tumor development.3.Oleanolic acid was used to treat H-TDEs-activated MRC5 fibroblasts to investigate its suppressive effect on inflammatory fibroblast activation.4.Conditioned medium collected from H-TDEs and oleanolic acid-treated MRC5 cells was used to treat HCC tumor cells,macrophages,splenocytes and endothelial cells to explore the educational effect of activated fibroblasts on PMN cells.Part 4: The molecular mechanisms involved in the effect of oleanolic acid suppressing inflammatory fibroblast activation1.Drug affinity responsive target stability proteomics assay combined with Swiss Target Prediction tool were used to investigate protein targets of oleanolic acid.WB,cellular thermal shift assay,and microscale thermophoresis assay were used to investigate the binding ability of protein target and oleanolic acid.2.Molecular docking were used to predict the binding site of protein target and oleanolic acid.3.Immunofluorescence was used to investigate nuclear translocation of key kinases in oleanolic acid-treated fibroblasts.4.The inhibitory effect of oleanolic acid on key signaling pathways underlying fibroblast activation was investigated by WB.ResultsPart 1: H-TDEs facilitated pulmonary PMN formation and promoted metastatic tumor development1.HIF-1α knockdown subcutaneous tumor developed less lung metastasis.2.H-TDEs promoted inflammatory activation of MRC5 cells.3.Plasma exosome collected from HCC patients after TACE promoted fibroblast activation.4.H-TDEs promoted pulmonary PMN development and metastatic tumor formation,while Jiedu recipe administration suppressed PMN formation.Part 2: H-TDEs activated lung fibroblast through miR-4508-RFX1-IL17A-p38 MAPK NF-κB signaling pathway1.miR-4508 was highly expressed in H-TDEs,which mediated fibroblast activation.2.miR-4508 was upregulated in plasma exosomes collected from HCC patients undergoing TACE and exosomes collected from hypoxic HCC tumor cells.3.miR-4508 enwrapped by TDEs can be ingested by fibroblasts and promoted inflammatory activation.4.miR-4508 inhibited RFX1 expression by binding to its m RNA 3’UTR.5.miR-4508 induced PMN formation in lung tissue of mice.6.Suppressed RFX1 activated IL-17A-p38 MAPK-NF-κB signaling pathway in lung fibroblasts.7.p38 MAPK inhibitor suppressed miR-4508-mediated metastatic tumor formation in PMN mice.8.p38 MAPK inhibitor suppressed lung fibroblasts activation.Part 3: Oleanolic acid inhibits H-TDEs-regulated fibroblast activation and PMN formation1.As one of the main monomeric components identified in Jiedu recipe,oleanolic acid significantly suppressed the activation of lung fibroblasts.2.Oleanolic acid significantly suppressed H-TDEs-facilitated expression of FN and α-SMA in premetastatic lung,which results in reduced development of lung metastasis.3.Oleanolic acid inhibited H-TDE-induced lung fibroblast activation,further blocking stem cell-like sphere formation in MRC5/97 H co-cultures,and reduced SOX2 and OCT4 expression.4.Oleanolic acid suppressed M2 marker expression(Arg-1,CD163,CD206)in RAW264.7/WML2 co-cultures,and inhibited CD11b+ Gr-1+ MDSC differentiation from WML2 co-cultured mouse splenocytes,reducing Arg-1,i NOS,and IL10 levels.Oleanolic acid also hampered tubule formation in EAhy926 endothelial cells induced by activated MRC5 cells.Part 4: Oleanolic acid inhibits H-TDEs-regulated fibroblast activation by targeting ERK MAPK-NF-κB signaling pathway1.The activation of ERK MAPK signaling pathway promoted fibroblast activation and PMN development.2.Oleanolic acid directly binding to ERK1/2 proteins and inhibited H-TDEs-mediated ERK MAPK signaling activation in MRC5 cells.3.Oleanolic acid suppressed the phosphorylation activation and nuclear translocation of ERK1/2 and NF-κB p65 kinase in MRC5 cells.4.Oleanolic acid inhibited H-TDEs-mediated fibroblast activation through suppressing ERK MAPK-NF-κB signaling pathway.Conclusions1.H-TDEs activate inflammatory fibroblasts and facilitate lung pre-metastatic niche formation in HCC through miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway.2.Oleanolic acid inhibits inflammatory fibroblast activation and suppresses H-TDEsinduced PMN formation in HCC by targeting ERK1/2-NFκB signaling.