The Study Of Phyllanthus Urinaria L Antineoplastic Decoction Inhibiting The Progress Of HBV Related HCC Via The Pathway Of HBx-microRNAs From Exosomes-VEGFR2

The etiology of hepatocellular carcinoma(HCC)is multifaceted,with hepatitis B virus(HBV)infection being the predominant cause of HCC in China.This is evidenced by epidemiological factors such as average age of onset,survival rate,and economic burden.HBx(Hepatitis B virus x gene)is an established oncogenic gene and an independent risk factor for HBV-HCC development.Exosomes play a crucial role in intercellular communication processes,impacting signaling pathways,tumor proliferation and metastasis,angiogenesis,and therapeutic response.Our previous research investigated the role of exosomal microRNA-21(miR-21)from HBx and HCC.Vascular endothelial growth factor(VEGF)is a critical factor in HCC angiogenesis,with VEGFR2 overexpression being associated with tumor angiogenesis and metastasis,possibly through the actions of HBx and exosomal miRs.However,the precise mechanism underlying such actions remain yet to be fully elucidated.According to TCM theory,hepatocellular carcinoma’s etiology and pathogenesis align with modern TCM fields.These theories hypothesize that the primary pathogenesis of HBV-related HCC is stasis,toxicity,and deficiency,with deficiency being the root cause,and stasis and toxicity serving as symptoms.Treatment should aim to resolve stasis and detoxify deficiency.In the first stage of our clinical anti-tumor effectiveness study,we initially focused on Phyllanthus urinaria L’s antioxidant and antiviral effects.However,we are now exploring the targets of HBx,vascular endothelial growth factor,and receptor to understand the proliferation and metastasis mechanism of HBV-related HCC,mainly affecting lymphatic metastasis.The antineoplastic decoction of Phyllanthus urinaria L.(PAD)is an optimal treatment formula for HBV-related HCC.It works through the method of resolving blood stasis,detoxifying toxins,and tonifying deficiency.This thesis aims to explore the microscopic immune mechanism of PAD’s inhibition of HBV-related HCC,identifying new targets and novel treatments for HBV-related HCC.The findings from this research aim to provide better theoretical support for the clinical treatment of HBV-related HCC.ObjectiveTo explore the mechanism by which Hepatitis B virus x gene-positive expressing cells promote tumor proliferation and metastasis through positive and negative regulation of the biological activity of secreted exosomes and specific derivatives miRs with the alteration of tumor microenvironment,affecting the expression of VEGFR2 and so on,and to verify the tumor-suppressive effect of Compound PAD on HBV-related HCC possibly through interfering with the exosomal miRs-VEGFR2 pathway.MethodsSection 1.Non-targeted metabolomics of serum containing PAD in different forms UPLC-Q/TOF-MS was utilized to analyze the metabolic profiles of serum non-targeted metabolomics in four herbal PAD forms,which included aqueous decoction,lyophilized powder,fluid infusion,and granules.Appropriate forms were then selected for in vitro and in vivo experiments.Section 2.Study on the effect of Phyllanthus urinaria L antineoplastic decoction on the proliferation phenotype of HBV-related HCC1.Various concentrations of Compound PAD(0,2.5,5,10,20,40,80,160,320,640μg/mL)were utilized to treat HepG2 and Huh-7 hepatocellular carcinoma cell lines,and CCK-8,cell counting,and clone formation assays were conducted to investigate the impact of PAD on proliferation and clone formation of hepatocellular carcinoma cells.2.Establish stable HepG2 cell lines that overexpress HBx and VEGFR2 genes,and perform Western blot experiments to confirm the success of the cell line construction.To evaluate the inhibitory effect of Compound PAD on the proliferation ability of HBV-related HCC cells,different concentrations(0,40,80,and 160ug/mL)of Compound PAD will be applied,and CCK-8 and clone formation assays will be conducted on both HepG2-HBx and HepG2-VEGFR2 cells.Section 3.Study on the effect of Phyllanthus urinaria L antineoplastic decoction on the metastatic ability of HBV-related HCC1.Scratch and Western blot assays were conducted to investigate the expression of proteins associated with the migratory and metastatic abilities of HepG2-HBx and HepG2-VEGFR2 cells.2.Different concentrations of Compound PAD(0,40,80,160 μg/mL)were utilized to treat HepG2-HBx and HepG2-VEGFR2 cells,while 160 μg/mL of Compound PAD was employed to interfere with HBV-related HCC cells for 0,24,48,and 72 hours.Scratch and Western blot experiments were performed to study the effect of PAD on the metastatic ability and markers of metastasis-related proteins in HBV-related HCC.Section 4.Related studies on the regulatory effects of exosomal miRs in Phyllanthus urinaria L antineoplastic decoction1.PCR experiments were conducted to determine the relative expression of miR-21 and miR-199a-3p in HepG2-HBx and HepG2-VEGFR2 cells.2.Experimental groups included HepG2,HepG2-HBx,and HepG2-VEGFR2 control groups,as well as HepG2-HBx and HepG2-VEGFR2 compound PAD co-culture groups(at a concentration of 160ug/ml).Exosomes were extracted and identified,and PCR experiments were performed to investigate the relative expression of exosomal miR-21 and miR-199a-3p in HBV-related HCC cells,as well as the regulatory effects of PAD on exosomal miRs.Section 5.Network pharmacology study of Phyllanthus urinaria L antineoplastic decoctionIn conjunction with the network pharmacology investigation,we constructed a network depicting the"active ingredients-disease targets-signaling pathways" of the antineoplastic decoction derived from Phyllanthus urinaria L.We analyzed the primary active components,essential targets,and signaling pathways influenced by the Phyllanthus urinaria L-derived antineoplastic decoction that functions on HBV-related HCC.Section 6.Study on the mechanism of PI3K-Akt inhibition of HBV-related HCC mediated by Phyllanthus urinaria L antineoplastic decoction1.Various concentrations of compound PAD(0,40,80,160 μg/ml)were applied to HepG2-HBx and HepG2-VEGFR2 cells.Meanwhile,HBV-related HCC cells were treated with 160 μg/ml of compound PAD for 0 h,24 h,48 h,and 72 h to assess the effects of PAD on PI3K-Akt and VEGFR2 signal transduction pathway-related protein expression levels using Western blot experiments.2.The selected compound PAD(40,160 μg/ml)was combined with Akt inhibitor LY294002(LY,25 μM)to intervene in HepG2-HBx and HepG2-VEGFR2 cells.Using Western blot experiments,the effects of PAD on PTEN,Akt,and p-Akt protein expression were evaluated.Section 7.Study on the inhibitory effect of Phyllanthus urinaria L antineoplastic decoction on transplanted tumors in nude mice with hepatocellular carcinoma1.HepG2-Luc and HepG2-HBx-Luc cell lines were selected to establish hepatocellular carcinoma transplanted tumor models in nude mice.Different concentrations of compound PAD(0,0.9,and 1.8 g/mL)were administered,and changes in tumor size were observed.The body weight,tumor volume/weight data of nude mice were recorded,and the tumor inhibition rate was calculated and analyzed.2.In vivo animal imaging experiments were conducted to determine the appropriate dose,duration of action,and reaction time of the substrate used.The successful construction of the transplanted tumor model was also verified.3.Animal sampling,HE,and IHC experiments were performed to study the liver and tumor pathology histology of nude mice.Section 8.Study on the mechanism of inhibitory effect of Phyllanthus urinaria L antineoplastic decoction on transplantation tumor in nude mice with liver cancer1.Conduct ELISA experiments to detect the serum of nude mice and investigate the impact of PAD on the level of serum VEGFR2 expression in nude mice.2.Carry out exosome extraction and identification as well as PCR experiments to examine the effects of PAD on exosomal miRs in different groups of nude mice.3.Perform Western blot experiments to analyze the influence of PAD on the expression levels of PI3K-Akt and VEGFR2 signal transduction pathway-related proteins in tumor tissues.ResultsSection 1.Non-targeted metabolomic of PAD-containing serum in different dosage formsThe secondary metabolites found in lyophilized powder and aqueous decoction forms of PAD-containing serum are similar in composition to the flavonoids present in the junmai leaf hypocretin.Lyophilized Compound PAD was chosen for in vitro experiments and aqueous decoction PAD for in vivo experiments.Section 2.Inhibitory ability of Compound PAD on proliferation of hepatocellular carcinoma cells and HBV-related HCC cells1.PAD demonstrated concentration-and time-dependent inhibition of the proliferation of HepG2 and Huh-7 hepatocellular carcinoma cells;however,no significant toxic effects of PAD were observed on human normal hepatocytes LO2.2.Stable strains of HepG2-HBx and HepG2-VEGFR2 cells were successfully developed,and HBV-related cells exhibited stronger proliferation ability than typical hepatocellular carcinoma cell lines(P<0.01).PAD displayed a concentration-dependent inhibitory effect on their proliferation ability.Section 3.Inhibition of HBV-related metastasis by Compound PAD1.The metastatic ability of HepG2-HBx and HepG2-VEGFR2 was significantly higher(P<0.01),and there was a significant alteration in the expression of EMT-related proteins.2.Compound PAD exhibited a concentration-and time-dependent inhibition of the wound healing ability of HBV-related HCC cells(P<0.01).Specifically,the content of N-cadherin and Vimentin proteins decreased,whereas the content of E-cadherin and PTEN proteins increased due to the effects of compound PAD.Section 4.Phyllanthus urinaria L antineoplastic decoction regulates the expression level of exosomal miRs1.Compared to HepG2 cells,the relative expression of miR-21 increased significantly(P<0.01)while the expression of miR-199a-3p decreased remarkably(P<0.01)in both HepG2-HBx and HepG2-VEGFR2 cells.2.The relative expression of exosomal miR-21 significantly increased(P<0.05)while the expression of exosomal miR-199a-3p significantly decreased(P<0.05)in HepG2-HBx and HepG2-VEGFR2 cells when compared to HepG2 cells.Notably,by regulating exosomal miR expression,compound PAD effectively reduced the relative expression of the proto-oncogene exosome miR-21(P<0.05)and increased the relative expression of the anti-oncogene exosome miR-199a-3p(P<0.05).Section 5.Pharmacological study on the network of Phyllanthus urinaria L antineoplastic decoctionThe analysis of the key nodes in the network of "the main active components of Phyllanthus urinaria L antineoplastic decoction-key disease targets-important signaling pathways"reveals that the main active components of the decoction are Luteolin,gallic acid,and kaempferol,among others.The identified key disease targets include PIM1,PTGS2,and KDR(VEGFR2),while the important signaling pathways comprise PI3K-Akt signaling pathway,metabolic pathway,and cancer pathway.These results shed light on the possible mechanisms underlying the multifunctional and multi-pathway effects of Phyllanthus urinaria L antineoplastic decoction for treating hepatocellular carcinoma(HCC).Section 6.Phyllanthus urinaria L antineoplastic decoction mediates PI3K-Akt and VEGFR2 signaling pathway against HCC1.PAD interferes with PI3K-Akt signaling pathway to inhibit HBV-related HCC proliferation;2.PAD regulates HBx-mediated VEGFR2 signaling pathway to inhibit HBV-related HCC metastasis.Section 7.Inhibition of transplantation tumor growth in nude mice with hepatocellular carcinoma by Phyllanthus urinaria L antineoplastic decoction1.PAD significantly suppressed the growth of transplanted hepatocellular carcinoma tumors in nude mice,and exhibited a dose-dependent effect.2.Evidence from live imaging experiments in animals indicated successful modeling;where a substrate concentration of 15mg/ml,a dose of 300ul,and an action time of 30 minutes were used.3.IHC assay showed that PAD could inhibit the expression of HBx,VEGFR2,SHC,Cyclin-D1,and Grb2 proteins in tumor cells.Section 8.Anti-HCC via exosomal miRs-VEGFR2 pathway with Phyllanthus urinaria L antineoplastic decoction1.PAD concentration-dependently reduces serum VEGFR2 expression levels in nude mice(P<0.01).2.PAD regulates serum-derived exosomal miRs expression,specifically decreasing proto-oncogene miR-21(P<0.01)and increasing anti-oncogene miR-199a-3p relative expression(P<0.01)in a concentration-dependent manner.3.PAD significantly decreases p-PI3K,p-Akt,VEGFR2,SHC,Cyclin-D1,Grb2,and HBx protein expression levels in tumor tissues.Conclusion1.Phyllanthus urinaria L antineoplastic decoction can effectively inhibit HBV-related HCC proliferation and metastasis both in vitro and in vivo.2.By integrating public database data and systematic analysis with the help of network pharmacology,VEGFR2 was identified as one of the core functional genes of HCC;pathway enrichment analysis suggested that PI3K-Akt signaling pathway was involved in the anti-cancer and anti-metastasis mechanism of PAD in the proliferation and metastasis of HBV-related HCC.3.Mechanistic exploration revealed that Phyllanthus urinaria L antineoplastic decoction inhibits HBV-related HCC proliferation and metastasis by reducing Hepatitis B virus x gene expression,interfering with the biological activities of exosomes and their specific derivatives miR-21 and miR-199a-3p,and regulating the expression of VEGFR2 and so on.