Effect Of Bear Bile Powder On Inflammatory Factors And Angiogenesis Of Primary Hepatocellular Carcinoma After TACE

BackgroundTranscatheter arterial chemoembolization?TACE?is one of the primary treatment methods for primary hepatocellular carcinoma.The embolization itself,emboliaztion agents and chemotherapeutic drugs,and so on,may cause the hepatic's ischemia and hypoxia,thus cause inflammatory reaction in ischemic liver through inducing kupffer cells,mononuclear cells and neutrophil cells,which secrete many kinds of inflammatory factors in the same time.Inflammatory factors are closely related to the development of liver cancer.Among them,IL-8 and TNF-?are closely related to the angiogenesis and metastasis of liver cancer,which are the significant events in the development of liver cancer.The anoxic microenvironment of liver after TACE may up-regulate the expression of hypoxia inducible factor?HIF?-1?,and then promote tumor angiogenesis by inducing the increased expression of vascular endothelial growth factor?VEGF?that finally promote the tumor recurrence and metastasis.VEGF and HIF-1?are the key factors which regulating the angiogenesis in anoxic microenvironment.The high expression level of VEGF promote tumor growth rate and metastatic tendency and inhibit the expression of VEGF may be an important strategy to prevent the metastasis rate of liver cancer and improve the survival rate of patients after TACE.In hypoxic microenvironment,the expression of HIF-1?may stimulates the expression of VEGF that may be relate to tumor angiogenesis.Numerous studies have revealed that Traditional Chinese Medicine?TCM?is an important therapeutic means in the treatment of various tumors.It has also been found that TCM combined with TACE may improve the curative effect compare to TACE alone.Thus combined therapy not only reduce embolism syndrome,but also play a role in reducing the recurrence and metastasis of liver cancer.Clearing heat and detoxification is one of the main treatment methods for primary liver cancer base on the basic theories of Chinese medicine.Chinese herbal compound for clearing heat and detoxification has certain curative effect in different stages of liver cancer.Recent studies indicated that,bear bile powder?BBP?,as a kind of traditional Chinese medicine,has anti-inflammatory and anti-tumor effect.It is found that BBP have effect in reducing inflammatory factor,regulating tumor microenvironment,inhibiting the activation of hepatic stellate cell,promoting T cell proliferation,inducing apoptosis of liver tumor cells,inhibiting the tumor proliferation and angiogenesis,etc.However,it is still lack evidence about whether BBP may decrease the secretion of inflammatory factors after TACE,whether the expression of VEGF can be down-regulated and whether the angiogenesis can be suppressed under hypoxia conditions.ObjectiveThe objective of the present study was to explore the effect of BBP on inflammatory factors and embolism syndrome after TACE.Furthermore,the effects of BBP on inflammatory factors and angiogenesis of hepatocellular carcinoma?HCC?was also investigated.We hope our study may provide experimental basis for improving the efficacy of TACE combine with BBP.Methods1.Effect of BBP on inflammatory factors and embolism syndrome in patients with hepatocellular carcinomaOne hundred and seven patients with HCC who met the inclusion criteria were finally enrolled in the present study and were randomly divided into two groups which included one of BBP group?taking BBP capsule?and another was control group?taking a placebo?.The drug was administered 1 day before TACE and continues for 6 days.The patients'inflammatory factor,liver levels and the clinical symptoms were detected and recorded in the 3rd and the 5th day after TACE.2.The effect of BBP on the expression of IL-8?HIF-1??VEGF in SMMC-7721 hepatocellular carcinoma cells induced by CoCl₂In the in vitro study,we firstly detected the effects of different concentrations of BBP on the proliferation of hepatocellular carcinoma cells through MTT assay.Then,CoCl₂ was used to establish anoxic model with hepatocellular carcinoma cells SMMC-7721.Concentrations of BBP with no obvious toxicity to SMMC-7721 cells was used to further detect its effects on the expression of IL-8,HIF-1?and VEGF in SMMC-7721 cells under hypoxia by RT-PCR method.At the same time,the level of IL-8in the culture medium of SMMC-7721 cells was detected by ELISA method.On the other hand,the expression of the protein expression of HIF-1??VEGF was detected by Western blot method.3.Effect of BBP on angiogenesis induced by IL-8We investigated the effect of BBP on angiogenesis induced by IL-8 both in vitro and in vivo assays.In vitro experiments,we used capillary tube formation assay to investigate the anti-angiogenic capacity of BBP.Cells mix with 100 ng/mL IL-8 and different concentrations of BBP was added to Matrigel matrix in 96 well plates.Number of junctions,length of master segments,and mesh size were observed under fluorescent microscope and photographed.Image J software was used for processing and statistics analysis of angiogenesis.The nude mice were divided into three groups,respectively for the blank group?matrix gel were injected into the nude mice?,IL-8 group?matrix gel and IL-8 mixture were injected into the nude mice?,IL-8+BBP group?matrix gel and IL-8and 0.4 mg/mL of BBP mixture were injected into the nude mice?.In vivo experiments,the matrix gel combined with EA.hy 926 cells,100 ng/mL IL-8 and different doses of BBP were injected into the nude mice.After 10 days,the subcutaneous matrix of the nude mice was dissected.The tissue was lysised and the protein concentration was detected by BCA method.Then all samples were adjusted to the same protein concentration,the hemoglobin concentration were then detected by the Bebo hemoglobin test kit.On the other hand,HE staining was used to observe the angiogenesis and immunohistochemical detection were used to observe the expression of CD31 and VEGF,which may relate to the angiogenesis of EA.hy 926 cells.Results1.Clinical studies have found that the expression of inflammatory cytokines such as IL-6,CRP,IL-8 in patients with HCC after TACE is significantly up-regulated?P<0.05?.In the 5th day after TACE,the level of IL-8,TNF-?in BBP's group was significantly lower than that in the control group?P<0.01,P<0.05?.It is suggested that BBP can inhibit the expression level of inflammatory cytokinesIL-8,TNF-?after TACE.2.It is also observed that TBIL,DBIL,ALT,AST in patients with primary hepatocellular carcinoma were elevated after TACE.The ratio of AST/ALT and AST level of BBP's group was significantly lower than that of the control group?P<0.01,P<0.05?.The duration of fever was significantly shorter than that in the control group?P<0.05?.It is suggested that BBP can improve the liver function injury after TACE,relieve the fever and relieve the syndrome after embolization.3.In the in vitro assays,we found that low doses BBP had not obvious cytotoxicity in SMMC-7721 cells.The expression of IL-8,HIF-1?,VEGF mRNA in hepatocellular carcinoma cells were detected,the results showed that CoCl₂ could significantly induce the expression of IL-8,HIF-1?,VEGF mRNA,the difference was statistically significant?P<0.01?,compared with the control group.In addition,the expression of IL-8,HIF-1?,VEGF mRNA in the SMMC-7721 hepatocellular carcinoma cells induced by CoCl₂ was significantly inhibited by BBP.In addition,ELISA was used to detect IL-8 level in the culture medium of SMMC-7721,and the results were consistent with the mRNA results.Protein expression was detected in hepatocellular carcinoma cells by Western blot method,which also showed that BBP could inhibit expression of HIF-1?and VEGF.It is suggested that BBP has the effect of inhibiting the expression of IL-8?HIF-1??VEGF in the hypoxia condition.4.Effects of BBP on angiogenesis induced by IL-8.The results showed that the number of vascular nodules and the size and area of vascular were significantly higher than that in the blank control group?P<0.05?,which showed that IL-8 can stimulate the formation of blood vessels.After the intervention of different concentrations of BBP,the vascular nodules,blood vessel length and area were lower than that of IL-8.The difference between the 0.2,0.4 mg/mL groups and IL-8 group were significant?P<0.05?.All of these indicated that BBP can effectively inhibit the effect of IL-8 in a dose dependence.5.The effect of BBP on blood vessel formation in vivo.It was observed that using HE staining,the angiogenesis was significantly increased under IL-8 stimulation,and BBP could effectively inhibit this effect.Immunohistochemistry was used to detect the expression of CD31 and VEGF in vivo.The expression of CD31 and VEGF in IL-8group were significantly higher than the control group,but the expression of CD31 and VEGF were significantly decreased in the group with the BBP than IL-8 group.The detection of hemoglobin concentration also showed that hemoglobin concentration in the IL-8 group was more abundant than those in the blank control group,and the BBP may inhibited it significantly?P<0.05?.All of these indicated that BBP could inhibit the angiogenesis induced by IL-8.Conclusion1.BBP can inhibit the expression level of IL-8?TNF-?after TACE.It may also improve the liver function and relief of embolization syndrome;2.BBP can inhibit the expression of VEGF,IL-8 in HCC cells induced by hypoxia.3.BBP can inhibit angiogenesis induced by IL-8;4.BBP may inhibit the hypoxia-induced angiogenesis of primary hepatocellular carcinoma after TACE,the mechanism may be associated with inhibition of HIF-1?/IL-8/VEGF.