Role And Mechanism Of Ursodeoxycholic Acid In Inhibiting Hypoxia-induced Primary Liver Cancer Microenvironment

BackgroundUrsodeoxycholic acid(UDCA)is one of the active ingredients in bear bile powder.Studies have shown that UDCA has a protective effect on cell damage caused by hypoxia.At the same time,experiments suggest that UDCA can protect liver and gallbladder and promote hepatobiliary secretion.Transcatheter arterial chemoembolization(TACE)is currently the main method for treating malignant liver tumors that cannot be surgically removed or recurring liver cancer after surgery.However,due to incomplete tumor necrosis after TACE,increased secretion of angiogenic growth factors and promotion of angiogenesis,the long-term effects of TACE are not satisfactory.Hypoxia-inducible factor(HIF)-1α can act on downstream target genes,upregulate the expression of vascular endothelial growth factor(VEGF),and promote tumor angiogenesis.Therefore,these studies suggest that after TACE treatment of liver cancer,the high expression of HIF-1α in the microenvironment of absolute hypoxia inside tumor tissue can promote the expression of VEGF and angiogenesis,and then promote the recurrence,invasion and metastasis of liver cancer.ObjectiveTo determine whether UDCA can inhibit angiogenesis in the hypoxic microenvironment of liver cancer,and further explore the mechanism of UDCA’s inhibition of angiogenesis through signal pathways such as HIF-1α / VEGF.Methods1.MTT assay was used to detect the proliferation of human HCC cell line Huh 7 and human endothelial cell line EA.hy 926.Huh 7 cells or EA.hy 926 cells were treated with 25,50,100,200,and 400 μM UDCA for 24 or 48 h,respectively,and then subjected to MTT analysis to evaluate the antiproliferative effect of UDCA.EA.hy 926 cells were co-cultured with Huh 7 cells treated with 50 μM Co CL2 and 50 μM UDCA,respectively,and subjected to MTT analysis to evaluate the proliferation of EA.hy 926 cells.2.Tubular formation experiment to detect the effects of UDCA on the formation of tubules in Huh 7 cells under hypoxia.EA.hy 926 cells were blank or co-cultured with Huh 7 cells,treated with 50 μM Co CL2 and 25,50 μM UDCA,or cultured in a hypoxic incubator.Tubular formation experiments were performed using Image J to calculate the number of nodes,total segment length and Average grid size to quantify tube formation.3.RT-PCR was used to detect the m RNA expression of IL-8 and VEGF in Huh 7 cells treated with UDCA under hypoxia.Huh 7 cells were treated with 25,50 μM UDCA or 50 μM Co CL2,50 μM Co CL2 + 25 μM UDCA,50 μM Co CL2 + 50 μM UDCA,or cultured in a hypoxic incubator for 24 h.RT-PCR was used to detect IL-8 and VEGF.MRNA.4.Enzyme linked immunosorbent assay(ELISA)UDCA-treated Huh 7 cells VEGF and IL-8 concentration protein levels under hypoxic conditions.Huh 7 cells were treated with 25,50 μM UDCA or 50 μM Co CL2,50 μM Co CL2 + 25 μM UDCA,50 μM Co CL2 + 50 μM UDCA,or cultured in a hypoxic incubator for 24 h.VEGF and IL-8 protein concentrations were measured using ELISA Level.5.Tubular formation experiments to test the effects of UDCA on IL-8-induced tubule formation in vitro.EA.hy 926 cells were treated with 100 ng / m L IL-8 and different doses of UDCA(25 and 50 μM),and the number of tube formations and connections was calculated using Image J,total fragment length and mean grid size.6.Matrigel embolization in vivo angiogenesis.The role of UDCA in IL-8 induced tube formation in vivo was determined.Representative images of matrigel plugs in each group in vivo measurements,and determination of hemoglobin content of matrigel plugs.Representative images showing HE staining and IHC staining using CD31,VEGF and v WF antibodies from each group.7.RT-PCR detection of HIF-1α m RNA.Huh 7 cells were treated with 25,50 μM UDCA or 50 μM Co CL2,50 μM Co CL2 + 25 μM UDCA,50 μM Co CL2 + 50 μM UDCA or cultured in a hypoxic incubator for 24 h.RT-PCR was used to detect HIF-1α m RNA.8.Western Blot was used to determine the protein level of HIF-1α.Huh 7 cells were treated with 25,50 μM UDCA or 50 μM Co CL2,50 μM Co CL2 + 25 μM UDCA,50 μM Co CL2 + 50 μM UDCA or cultured in a hypoxia incubator for 24 h.Western Blot was used to determine the protein level of HIF-1α.9.Analysis of dual luciferase reporter gene.UDCA treatment of Huh 7 cells or Huh 7 cells transfected with HIF plasmid.Huh 7 cells or Huh 7 cells transfected with HIF plasmid were treated with UDCA,and the double luciferase reporter gene analysis was performed.The relative fluorescence intensity of each group was expressed as the ratio of firefly fluorescence intensity to Renilla fluorescence intensity(DF).10.RT-PCR analysis detected the m RNA of IL-8 and VEGF of Huh 7 cells or Huh 7 cells transfected with HIF plasmid after UDCA treatment.Huh 7 cells were transfected with HIF plasmid and treated with 50 μM UDCA for 24 h,and then the m RNA of IL-8 and VEGF was detected by RT-PCR analysis.11.ELISA was used to measure the levels of VEGF and IL-8 in Huh 7 cells under HIF overexpression and UDCA intervention.Determination of VEGF and IL-8 concentrations in Huh 7 cells overexpressed by HIF and UDCA.12.Tubular formation experiment to detect the effects of HIF plasmid Huh 7 cells transfected with UDCA on the formation of tubules under hypoxia.EA.hy 926 cells were blank or co-cultured with Huh 7 cells.They were transfected with HIF plasmid and treated with 25,50 μM UDCA in the presence of Co CL2 or cultured in a hypoxic incubator.Tubular formation experiments were performed and calculated using Image J Number of nodes,total segment length and average grid size to quantify tube formation.13.Western Blot method was used to detect the expression of p-P65,p-ERK,ERK,p-AKT and AKT.Huh 7 cells were treated with 25,50 μM UDCA or 50 μM Co CL2,50 μM Co CL2 + 25 μMUDCA,50 μM Co CL2 + 50 μMUDCA or cultured in a hypoxic incubator for 24 h,and then detected by Western Blot method for p-P65,p-ERK,ERK,pAKT and AKT expression.After treatment with IL-8 or IL-8 + UDCA for 2 h or 4 h,cytoplasmic P65(C-P65)and nuclear P65(N-P65)were detected.Treatment with IL-8 or IL-8 + UDCA for 15,30 or 60 minutes,detection of p-ERK,ERK-8.14.RT-PCR was used to detect the m RNA expression of HIF-1α in Huh 7 cells transfected with P65 plasmid and treated with UDCA under Co CL2 or hypoxia incubator conditions.Treated with Co CL2 or hypoxia incubator,transfected P65 plasmid into Huh 7 cells,treated with UDCA,and detected HIF-1α m RNA expression by RT-PCR.Results1.MTT showed that UDCA significantly inhibited the viability of Huh 7 and EA.hy 926 cells at high doses(> 100 μM).Treatment with UDCA(50 μM)with or without Co CL2(50 μM)for 24 h did not affect the viability of EA.hy 926 cells.Co-culture with Huh 7 cells can significantly increase the viability of EA.hy 926 cells.In Huh 7 cells,UDCA,Co CL2 and Co CL2 + UDCA did not affect the viability of EA.hy 926 cells.2.Tubular formation experiment: Under normal oxygen conditions,co-culture with Huh 7 cells could not promote tubule formation in EA.hy 926 cells(P> 0.05).Under Co CL2 treatment or in a hypoxia incubator,co-culture with Huh 7 cells can significantly increase tubule formation in EA.hy 926 cells(P <0.05).UDCA(25 and 50 μM)antagonizes the effects of Huh 7 cells on tube formation of EA.hy 926 cells under hypoxia.3.The m RNA expression of IL-8 and VEGF was detected by RT-PCR.Under normal oxygen conditions,there was no significant change in IL-8 and VEGF m RNA in Huh 7 cells between the control and UDCA groups.Co CL2 treatment significantly up-regulated the m RNA expression of IL-8 and VEGF.Both 25 and 50 μM UDCA inhibited Co CL2-induced upregulation of IL-8 and VEGF m RNA.Under hypoxic conditions,the expression of IL-8 and VEGF m RNA was also significantly increased in Huh 7 cells compared to normal oxygen conditions,which was also inhibited by UDCA.4.ELISA results showed that the protein levels of VEGF and IL-8 also increased significantly after Co CL2 or hypoxia treatment,and UDCA could partially reverse the protein levels of VEGF and IL-8.5.In the IL-8 group,compared with the control group,the number of nodes,total segment length and average mesh size increased significantly(P <0.05).UDCA treatment(25 and 50 μM)significantly inhibited IL-8-induced tubule formation in vitro.6.As determined by matrigel plug angiogenesis,in vivo,the hemoglobin content of the IL-8 group was higher than that of the control group.UDCA treatment significantly reduced hemoglobin content(P <0.05).In addition,HE staining showed that there were more newly formed blood vessels in the IL-8 group than in the control group,while UDCA reduced neovascularization.In addition,the expression of CD31,VEGF and v WF in the IL-8 group was up-regulated and suppressed by UDCA.7.By RT-PCR and Western Blot,both Co CL2 and hypoxia up-regulated the expression of HIF-1α m RNA and protein,while UDCA could inhibit the expression of HIF-1α m RNA and protein induced by hypoxia.In contrast,HIF-1α m RNA did not change when treated with 25 and 50 μMUDCA under normoxic conditions.8.Through dual luciferase reporter gene detection and analysis,the relative luciferase activity of Huh 7 cells remained low after UDCA treatment under normoxic conditions.Under Co CL2 and hypoxia conditions,the activity of hypoxia response elements(HRE)was significantly increased,while UDCA partially inhibited this effect.Under normoxic and hypoxic conditions,HRE-1α vector control transfection did not significantly change HRE activity.When treated with Co CL2 or incubated in a hypoxic incubator,UDCA inhibited the HRE activity of Huh 7 cells(compared to control or vehicle control,P <0.05).HIF-1α-expressing cells had an approximately 13-fold increase in luciferase activity under hypoxic conditions.After treatment with UDCA,HRE-driven luciferase activity was significantly inhibited(P <0.05).The results show that UDCA inhibits HRE activity by inhibiting HIF-1α expression.9.The results of RT-PCR and ELISA showed that under normoxic conditions,UDCA had little effect on the expression of IL-8 or VEGF.Both IL-8 and VEGF were significantly up-regulated under hypoxia(P <0.05).This effect was significantly enhanced after transfection with the HIF plasmid.UDCA significantly inhibits IL-8 and VEGF overexpression in Huh 7 cells under hypoxia.10.The results of tubule formation experiments show that HIF-1α transfection can significantly promote tube formation under normoxic or hypoxic conditions.Under normoxic or hypoxic conditions,25 and 50 μM UDCA can inhibit HIF-1α transfection.Tube formation.Inhibition of HIF-1α signal by UDCA can be achieved by down-regulating the expression of IL-8 and VEGF.11.Western blot analysis showed that both Co CL2 and hypoxic incubator significantly up-regulated the phosphorylation expression of P65,ERK and ATK,while the total protein of P65,ERK and ATK changed little.Under normal conditions,the changes of p-P65,p-ERK and p-AKT are small after UDCA processing.After IL-8 treatment for 2 h or 4 hours,IL-8 increased the expression of P65 in the nucleus,but reduced the level of P65 in the cytoplasm,and this effect was inhibited by UDCA,and IL-8 intervention was more obvious for 4 hours.IL-8 treatment for 15-60 minutes can promote the phosphorylation of ERK in a time-dependent manner.12.RT-PCR test confirmed that under hypoxic conditions,overexpression of P65 further up-regulates the expression of HIF-1α m RNA,which can be inhibited by UDCA.Conclusion1.UDCA inhibits angiogenesis induced by liver cancer cells under hypoxic conditions.2.UDCA inhibited the up-regulation of IL-8 and VEGF induced by hepatocellular carcinoma cells under hypoxia.3.UDCA inhibits IL-8-induced tube formation in vitro and in vivo.4.UDCA inhibits HIF signaling and inhibits HRE activity by inhibiting HIF-1α expression under hypoxic conditions.5.The inhibitory effect of UDCA on angiogenesis is mediated by blocking HIF / IL-8 and VEGF signaling pathways.6.UDCA inhibits NFκB and MAPK activation under hypoxic conditions and inhibits angiogenesis of liver cancer.