Vascular Mechanism Of Spleen-invigorating And Blood Stasis-removing Recipe In Postoperative Tumor Dormancy Of Hepatocellular Carcinoma

Objective(1)To explore the academic origin and relevant theoretical basis of treating primary hepatocellular carcinoma based on the theory of spleen deficiency and blood stasis,and to grasp the basic core pathogenesis of spleen deficiency and blood stasis of hepatocellular carcinoma,so as to demonstrate the theoretical basis of spleen-invigorating and blood stasis-removing recipe in the treatment of postoperative recurrence and metastasis of hepatocellular carcinoma in clinical practice.(2)On the basis of previous research of spleen-invigorating and blood stasis-removing recipe,in vivo experiment in animals was carried out firstly to explore the dynamic temporal and spatial changes of angiogenesis in the process of postoperative dormancy/recurrence of hepatocellular carcinoma and the intervention effect of spleen-invigorating and blood stasis-removing recipe.Secondly,the effect of Jaggedl/Notch signaling pathway on angiogenesis in the dormancy of hepatocellular carcinoma and the intervention of spleen-invigorating and blood stasis-removing recipe were further studied by in vivo and in vitro double-level experiments.The present study was expected to provide theoretical basis for clinical application of spleen-invigorating and blood stasis-removing recipe in anti-angiogenesis therapy of tumor,and supply scientific basis for better clinical application of traditional Chinese medicine compound recipe in the future.MethodsPart ?:In accordance with ancient and modern Chinese medical books and literature on hepatocellular carcinoma,this paper explored the academic origin and theoretical basis of the treatment of hepatocellular carcinoma from spleen deficiency syndrome and blood stasis syndrome,respectively.Part ?:An animal model of hepatocellular carcinoma was established by subaxillary injection of human hepatocellular carcinoma SMMC7721 cells in nude mice according to previous animal modeling methods.An animal model of hepatocellular carcinoma dormancy was further constructed by tumor resection after tumor formation.To observe the growth of tumor after hepatocellular carcinoma recurrence in nude mice,four groups of recurrent group and spleen-invigorating and blood stasis-removing recipe groups(at high,middle and low dose of 24.96 g/kg,12.48 g/kg and 6.24 g/kg,respectively)were grouped with truncation trauma to break the postoperative dormancy of hepatocellular carcinoma.Subsequently,tumors of nude mice were removed on 1st,4th,8th and 15th days after administration,and the pathological conditions were observed by HE staining.Meanwhile,expression levels of HIF-1 a,VEGF,MMP-9,TIMP-1 and EMMPRIN were detected by qPCR,Western Blot and immunohistochemistry.In addition,by collecting peripheral blood of nude mice,the contents of circulating endothelial cells(CECs)and active circulating endothelial cells(vCECs)were measured by flow cytometry.Part ?:In vitro cell experiment:The spleen-invigorating and blood stasis-removing recipe medicated serum was prepared in SPF grade nude mice.Identification of traditional Chinese medicine fingerprint spectrum was achieved by HPLC in spleen-invigorating and blood stasis-removing recipe and medicated serum.Human hepatocellular carcinoma SMMC7721 cells were cultured in in vitro cultured complete medium and then divided into normal serum group,DAPT inhibitor control group and drug serum group according to different additives.Cell proliferation and angiogenesis were measured by CCK8 assay at 0,24,48 and 72h,respectively,followed by comparative observation of angiogenesis under inverted microscope.Subsequently,expression levels of Jaggedl,Notchl and VEGF were further detected by qPCR and Western blot.Co-IP assay was used to detect the binding of Jaggedl and Notchl.In vivo animal experiment:An animal model of hepatocellular carcinoma was established by subaxillary injection of human hepatocellular carcinoma SMMC7721 cells in nude mice according to previous animal modeling methods.The animal model of hepatocellular carcinoma dormancy was constructed by tumor resection after tumor formation.To observe the growth of tumors after the recurrence of hepatocellular carcinoma in nude mice,four groups of recurrent group and spleen-invigorating and blood stasis-removing recipe groups(at high,middle and low dose of 24.96 g/kg,12.48 g/kg and 6.24 g/kg,respectively)were divided with truncation trauma to break the postoperative dormancy of hepatocellular carcinoma.The tumor dormancy state of nude mice after operation was broke to observe the growth of tumors dynamically.Furthermore,the pathological condition was observed by HE staining in each group after the removal of tumors.The removed tumors were sectioned in the four groups and then labeled with CD43,followed by photograph under inverted microscope(×200).Expressions of Jaggedl,Notchl and VEGF were detected by qPCR and Western Blot.In addition,the binding of Jaggedl and Notchl in tumors of each group was detected by Co-IP test.ResultsPart ?:The basic core pathogenesis of hepatocellular carcinoma was spleen deficiency and blood stasis.Accordingly,the established method of invigorating spleen and removing blood stasis was the key therapeutic approach of hepatocellular carcinoma.Part ?:At the same time point,expressions of HIF-l?,VEGF,MMP-9,TIMP-1,EMMPRIN and proportions of CES and vCECs were significantly higher in the recurrent group than those in the spleen-invigorating and blood stasis-removing recipe groups.In addition,the high dose group of spleen-invigorating and blood stasis-removing recipe showed the lowest expression in comparison of above indexes correspondingly(P?0.01).Besides,the expression and quantity of these indicators showed certain decreasing trend in the low,middle and high dose group of spleen-invigorating and blood stasis-removing recipe.Part ?:In vitro cell experiment:At 24,48 and 72h,cell proliferation in the normal serum group was faster than that in the drug serum group,with significant difference(P?0.01).The expression levels of Jaggedl,Notchl and VEGF in the drug serum group were significantly lower than those in the normal serum group(P<0.01).Furthermore,the area of angiogenesis was smaller and blood vessels were more sparse in the drug serum group and the inhibitor control group than those in the normal serum group.Meanwhile,the electrophoretic gray level of Jaggedl,Notchl and VEGF in cells of the drug serum group and the inhibitor control group was significantly lower than that of the normal serum group(P<0.01).In addition,the binding level of Jagged-1 and Notch-1 was significantly lower in the drug serum group and the inhibitor control group than that in the normal serum group(P?0.01).In vivo animal experiment:(1)HE staining was used to observe pathological changes tumors in the four groups.Microscopic examination(×200)showed that the abnormal nuclear staining in the control group of hepatocellular carcinoma recurrence showed the highest density and the most disordered arrangement of tumor cells.By contrast,in the high dose group of spleen-invigorating and blood stasis-removing recipe,tumor cells with abnormal nuclear staining showed the lowest density and the arrangement was closest to normal.In the four groups,the rank(from high to low)of tumor cell density was the control group of hepatocellular carcinoma recurrence,followed by low dose group and middle dose group,and finally the high dose group of spleen-invigorating and blood stasis-removing recipe.According to the observation of tumor pathological sections in vivo,the pathological results of in vivo experiment were basically consistent with the proliferation ability of hepatocellular carcinoma cells cultured in vitro.(2)The angiogenesis ability of nude mice was further compared in the four groups.Experimental immunohistochemistry(CD43 labeling)of tumor microangiogenesis in tumors of the four groups revealed that the control group of hepatocellular carcinoma recurrence presented larger area of tumor angiogenesis and denser blood vessels of angiogenesis.By comparison,the high dose group of spleen-invigorating and blood stasis-removing recipe had smaller area of angiogenesis,less number of angiogenesis,and thinner density of blood vessels.The size and density of tumor microvessels in the four groups were ranked in sequence(from high to low):the control group of hepatocellular carcinoma recurrence>low dose group>middle dose group>high dose group of spleen-invigorating and blood stasis-removing recipe.Meanwhile,the angiogenesis ability of tumor cells in vivo was similar to that of cultured hepatocellular carcinoma cells in vitro.(3)The expression levels of Jagged1,Notch1 and VEGF in four groups of tumor cells were detected by qPCR.There were significant differences in expression levels of Jagged1,Notch1 and VEGF among the four groups(P?0.01).The expression levels of Jagged1,Notch1 and VEGF were significantly lower in the low,middle and high dose group of spleen-invigorating and blood stasis-removing recipe than those in the control group of hepatocellular carcinoma recurrence,among which the high dose group indicated a significant decreased trend.(4)Western blot was then used to detect the expression of Jaggedl,Notchl and VEGF in tumor cells of the high,middle and low dose groups of spleen-invigorating and blood stasis-removing recipe as well as the control group of hepatocellular carcinoma recurrence.Protein electrophoretic gray levels of Jaggedl,Notchl,and VEGF weakened successively in tumor cells of hepatocellular carcinoma recurrence model group as well as low,middle and high dose groups of spleen-invigorating and blood stasis-removing recipe.Significant inter-group differences were observed in the protein electrophoretic gray levels of Jaggedl,Notchl and VEGF among the four groups(P?0.01).Among them,the high dose group had the most obvious decrease in the gray level of Jaggedl,Notchl and VEGF by applying protein electrophoresis,accompanied by the weakest expression levels,indicating statistical significance between groups(P?0.01).Similar results were observed in in vitro cell culture experiments.(5)According to the binding of Jagged 1 and Notch 1 detected by Co-IP assay,the binding ability of Jagged 1 and Notch 1 decreased orderly in tumor cells of the control group of hepatocellular carcinoma recurrence and the low,middle and high dose groups of spleen-invigorating and blood stasis-removing recipe,with significant difference among the four groups(P?0.01).Besides,the binding ability of Jaggedl and Notchl was the weakest in the high dose group of spleen-invigorating and blood stasis-removing recipe,which was similar in in vitro cell culture experiments.Conclusion(1)By grasping the basic core pathogenesis of hepatocellular carcinoma and establishing a recipe to strengthen the spleen and remove blood stasis,it is hopeful to make a breakthrough in the prevention and treatment of postoperative recurrence and metastasis of hepatocellular carcinoma.(2)The present study achieves a successful establishment of nude mice model of human hepatocellular carcinoma SMMC7721 cells and postoperative tumor dormancy/recurrence model.(3)Spleen-invigorating and blood stasis-removing recipe can promote the dormancy state and dormancy time of tumor cells after hepatocellular carcinoma operation in a certain drug concentration-dependent manner.(4)The effect of spleen-invigorating and blood stasis-removing recipe on promoting tumor dormancy and preventing postoperative recurrence of hepatocellular carcinoma is intimately associated with the inhibition of tumor angiogenesis,and this effect also exhibits a certain drug concentration dependence.(5)The anti-angiogenesis mechanism of spleen-invigorating and blood stasis-removing recipe in inhibiting tumor angiogenesis may reflect significantly in its reduction of tumor micro-vessel density through multiple pathways and multiple targets,suppression of the expression of HIF-1 ?,VEGF,MMP-9,TIMP-1,EMMPRIN in tumor cells,as well as the decrease of CECs and vCECs contents.(6)Spleen-invigorating and blood stasis-removing recipe can inhibit the proliferation of hepatocellular carcinoma cells and tumor angiogenesis.The anti-tumor mechanism may lie in the inhibition of the expression of ligand Jaggedl in tumor cells and its binding to Notch signaling pathway.Consequently,it decreases the activity of VEGF and down-regulates the expression of VEGF,thus inhibiting tumor angiogenesis,and ultimately playing its role in preventing and treating the postoperative recurrence of hepatocellular carcinoma.