| Research background and purposeTumor growth and metastasis is a multi-path,multi-factor,tumor and tumor microenvironment interaction evolution process.The studies which related tumor tend to explore the mechanism of drug action on tumor cells,usually ignoring the effects of tumor heterogeneity and tumor microenvironment on their metabolism.The treatment by traditional Chinese medicine is characterized by multi-target and multi-gene comprehensive regulation.The comprehensive treatment of traditional Chinese medicine combined with chemotherapy is a common postoperative adjuvant treatment for the patients with gastric cancer,which mainly includes phlegm and dampness removing drugs,blood activating and stasis removing drugs,heat clearing and detoxification drugs.The theory of "invigorating the spleen and resolving phlegm"in traditional Chinese medicine is connected with the theory of tumor microenvironment,which is the method to intervene tumor recurrence and metastasis.The purpose of this experiment is to integrate the theory of "invigorating the spleen and resolving phlegm" of traditional Chinese medicine with the theory of tumor microenvironment from Western medicine.Based on the CXCL12-CXCR4-ERK pathway and the reverse"Warburg" effect of tumor microenvironment,through the experiments of nude mice in vivo and cell cultures in vitro,to explore the possible mechanism of Jianpi Huatan Formula in the prevention and treatment of gastric cancer metastasis,and a new possible research direction for multi-component and multi-target Chinese medicine in the prevention and treatment of tumor metastasis.Experimental methods1.By isolated and cultured the CAFs and NFs from the stomach cancer tissue,growth curve was drawn by MTT,the a-SMA and Vimentin was detected by Immunofluorescence,Western Blotting and qPCR.MGC-803 cell was co-cultured with CAFs and NFs in Transwell suspension mode.The migration and invasion ability of gastric cancer cells were detected by Transwell.The proliferation activity and AMD3100 on CAFs-gastric cancer co-culture system were compared by MTT.The acidic property,lactic acid and ROS contents of co-culture system were determined by pH meter,lactic acid and DCFH-DA method.2.The serum containing JPHTF was prepared from rats.The best concentration of serum containing JPHTF,JPHTF extract and 5-Fu were selected by MTT.After administration,the proliferation activity of MGC-803 and CAFs,the effect of AMD3100 and co-culture system on the proliferation of MGC-803 were detected by MTT.The migration and invasion of gastric cancer were detected by Transwell method.The cell cycle of MGC-803 was detected by flow cytometry.The expression of CXCR4 and ERK was detected by Western Blotting and qPCR.After administration,the mitochondrial membrane potential of CAFs was determined by JC-1 method.The contents of ATP,ROS,pH,LD,glucose uptake and MDA were measured by phosphomolybdic acid colorimetry,DCFH-DA,pH meter,lactate enzyme,glucose oxidase and TBA method.3.The nude mice were randomly divided into blank control group(sham operation group,saline i.g),model group(saline i.g),positive group(5-Fu 120mg/kg i.p),low dose JPHTF group(5-Fu 120mg/kg i.p,JPHTF 18.75g/kg i.g),high dose JPHTF group(5-Fu 120mg/kg i.p,JPHTF 37.50g/kg i.g),8 rats in each group,to establish the model of gastric cancer transplantation.Nude mice were killed 14 days later.Their Spleens,livers and kidneys were taken to observe the growth and metastasis of the tumor.And HE staining was carried out to further detect the above situation.The expression of a-SMA and MMP-2 in liver metastasis was detected by immunohistochemistry.The expression of CXCR4 and ERK in liver metastasis were detected by Western Blotting and qPCR.The content of ATP in liver metastasis was detected by phosphomolybdic acid colorimetry.The levels of LDH,MDA and SOD in spleen,liver and kidney were detected by colorimetry,TBA and hydroxylamine method.Experimental results1.The morphology of CAFs,NFs cells was long spindle or flat star.The proliferation ability and overlapping growth phenomenon of CAFs were higher than those in NFs.The expression of a-SMA and Vimentin cells was positive in CAFs,but low or negative in NFs.The activity of gastric cancer in low density co-culture group was stranger than in the medium density group,and more than in the high density group.The pH value of CAFs co-culture system was decreased.The content of lactic acid and ROS was increased.But only in CAFs low density co-culture group,the effect on promoting cancer was significant.2.10%serum containing JPHTF,40 mg/mL JPHTF extract and 4 ?g/mL 5-Fu were selected as the best concentration in vitro.The proliferation rate,migration and invasion of MGC-803 cells in the serum containing JPHTF and extract were lower than those in the negative control group,which was consistent with the effect of 5-Fu.The inhibition rate of JPHTF on MGC-803 in CAFs co-culture system was higher than that in the conventional culture group only containing MGC-803,and the inhibition decreased with the time of separation from the co-culture system.After administration,the cells in S phase proportion were increased.But in the G2 phase proportion,they were decreased.And the expression levels of CXCR4 and ERK were decreased,which was consistent with the results of AMD3100 experiment.By the way,the activity of CAFs cells was decreased.After administration,the contents of LD,ROS and MDA in MGC-803 and CAFs cells were decreased.But the levels of ATP and pH were increased.3.Compared with the blank control group,the weight of mice in the model group was continuously decreased after receiving the transplantation tumor in spleen.The tumor tissue invaded to the spleen,liver and kidney,which was mainly adhered to the liver and intestine.The metastasis rate in liver was higher than that in kidney.The expression of a-SMA,MMP-2,CXCR4 and ERK was high in liver metastasis tumor.And the levels of ATP and SOD were decrease.But the levels of LDH and MDA were increase.After administrated JPHTF,the effects in inhibiting weight loss,reducing the size of spleen,liver and kidney tumors,the expression of a-SMA,MMP-2,CXCR4 and ERK could be found significiently,and which was dose-dependent.The inhibition effects in JPHTF group were higher than that in the positive drug group.The level of ATP and total SOD activity in JPHTF group was increased.By the same time,the contents of LDH and MDA were reduced.Experimental conclusion1.The human CAFs cells were successfully isolated and purified.The method of CAFs-gastric cancer Transwell co-culture was established.The mutual affects in co-culture system between gastric cancer cells with CAFs or NFs were important in their proportion.Low density co-culture system could significantly improve the proliferation and metastasis ability of gastric cancer cells.But high density co-culture could inhibit the growth and metastasis of cancer cells,which would be related to the contents of lactic acid and ROS.2.After administrating JPHTF,the proliferation,migration and invasion of MGC-803 could be inhibited in dose-dependent manner.MGC-803 cells could be stagnated in S phase.The acidity of tumor microenvironment was reduced.And the aerobic glycolysis metabolism and oxidative stress chain reaction of MGC-803 and CAFs cells were inhibited.The effect mechnismes of JPHTF may play a role in inhibiting the activity of cancer cells through tumor microenvironment,which is related to CXCR4-ERK signal pathway and the reverse "Warburg"effect.3.Through successful established the model of transplantation tumor in the spleen of nude mice,the effects of JPHTF could be effectively showed in reducing the growth of situ tumor in spleen,and the metastasis of liver and kidney,down regulating the expression of MMP-2 and a-SMA to directly inhibit or indirectly inhibit gastric cancer cells by tumor microenvironment.In the same time,it could also play synergistic roles by inhibiting CXCR4-ERK pathway and through the reverse "Warburg" effect to prevent the gastric cancer metastasis. |
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