| Research background and purposeIn present,the number of patients and deaths of liver cancer in China is high among all malignant tumors,which has become one of the malignant diseases that seriously affect the health of our people.The early stage of liver cancer is relatively hidden.Once patients were found to have obvious clinical symptoms,they usually developed to the middle or last stage of the course of disease.At the time,the patients had less benefit by the therapy of surgical resection of liver cancer,and had to face greater risk of recurrence and metastasis.Therefore,chemotherapy was the main treatment choice for patients with advanced liver cancer.However,long-term chemotherapy not only caused serious toxic and side effects to the main organs such as liver and kidney,but also leaded to the drug resistance.In recent years,with the continuous exploration,Chinese medicine combined with chemotherapy had shown certain advantages.According to the basic theory of Chinese medicine and the accumulation of more than 30 years’clinical treatment experience,the oncology department of Jiangsu Second Traditional Chinese Medicine Hospital had developed Jianpi Huoxue Recipe.While adopting chemotherapy,oral administration of Jianpi Huoxue Recipe can help patients improve the quality of life and reduce the risk of recurrence and metastasis in the meanwhile.Jianpi Huoxue Recipe had been used in clinic for more than 20 years,and its curative effect was certain.However,the studies of chemical basis and pharmacological mechanism on Jianpi Huoxue Recipe were not deep enough.This research paper intends to detect and analyze the main chemical components that may be contained in decoction by modern analytical chemistry,and try to explain the potential mechanism of Jianpi Huoxue Recipe combined with chemotherapy exerting curative effect by molecular biology skills on the basis of establishing orthotopic transplanted tumor model of liver cancer.Experimental methods1.UPLC-QE-Orbitrap-MS combined with Chinese medicine chemical database was used to analyze the chemical constituents of Jianpi Huoxue recipe.UPLC BEH C18(2.1 × 100 mm,1.7μm)column was used,0.1%formic acid aqueous solution acetonitrile was used as mobile phase for gradient elution,the flow rate was 400 μL·min-1.Mass spectrometry was used to analyze the extract of the drug in positive and negative ion mode respectively,and the mass spectrometry information was collected and analyzed to explore the main chemical basis.The compounds with anti-hepatoma activity and their potential protein targets were screened through literature review.The binding activity of compounds with corresponding protein targets was predicted by molecular docking technology.2.The nude mice were randomly divided into sham operation group,model group,chemotherapy group(5-FU+oxaliplatin)and chemotherapy combined with Chinese medicine group.The subcutaneous tumor was made by subcutaneous injection of HCCLM3 cell suspension into nude mice,and the subcutaneous tumor was cut into small pieces and implanted into the liver of nude mice in each experimental group.In the sham operation group,no tumor was implanted and the other operation steps were the same as the model group.In the chemotherapy group(5-FU+oxaliplatin),oxaliplatin(10 mg·kg-1)and 5-FU(10 mg·kg-1)were injected intraperitoneally once a time,and the chemotherapy was given once on the 7th and 14th day after the operation.The dose of Chinese medicine by oral administration was 36.9 g raw drug·kg-1(calculated by raw drug quantity,the same below)once.On the basis of the same chemotherapy treatment plan,the chemotherapy combined with Chinese medicine group continued to give the Chinese medicine decoction by oral administration for 14 days from the 7th day after the model making operation,once a day.The sham operation group and model group were given the same amount of distilled water by oral administration at the same time point.During the experiment,the survival state of nude mice was observed carefully.All nude mice were killed on the 35th day after the model making.The ortho topic liver tumor and lung tissue were collected.If other organs were observed,the organs with suspected tumor metastasis should also be collected.HE staining was performed on the collected tissues and the invasion of cancer cells was observed under optical microscope.3.The protein expressions of MMP-2,Ki67,VEGFA and p-ERK in orthotopic liver tumor of nude mice were detected by immunohistochemstry,and the expression of MMP-2 in orthotopic liver tumor of nude mice was detected by Western blotting.4.BALB/c mice were randomly divided into sham operation group,model group,5-FU group and 5-FU combined with low-dose and high-dose Chinese medicine groups.5-FU group was given intraperitoneal injection of 20 mg·kg-1 every other day.5-FU combined with low-dose and high-dose Chinese medicine group was given intragastric administration of Chinese medicine once a day on the basis of 5-FU injection,with the dose of 36.9 g·kg-1 and 73.8 g·kg-1 respectively.The mice in sham operation group and model group were given the same amount of distilled water by oral administration once a day.The administration started from the third day after modeling and lasted for 14 days.During the experiment,the survival status of nude mice was observed carefully.On the 18th day after operation,all mice were killed,and the orthotopic liver tumor,peritoneal tissue,kidney and spleen tissue were collected.The presence of tumor metastasis and obvious ascites were observed by naked eyes.The average tumor weight,tumor inhibition rate,peritoneal metastasis rate of tumor and incidence of ascites in each group were counted.HE staining was used to detect the possible pathological changes caused by invasion of tumor cells in orthotopic liver tumor,peritoneum,kidney and spleen.5.The protein expressions of MMP-2,MMP-9,E-cadherin,Ki67,HIF-1α,VEGF,ERK and p-ERK in orthotopic liver tumor of BALB/c mice were detected by Western blotting.The expression of E-cadherin in orthotopic liver tumor of BALB/c mice was detected by immunohistochemistry.The expression of VEGF mRNA in orthotopic liver tumor of BALB/c mice was detected by qPCR.Experimental results1.Based on UPLC-QE-Orbitrap-MS,74 compounds were identified,including 12 amino acids,2 phenylpropanoids,14 phenolic acids,6 alkaloids,10 terpenoids,25 flavonoids,1 carbohydrate,2 fatty acids,1 aromatic acid and 1 vitamin.The potential anti-hepatoma active components selected by searching literature include Rosmarinicacid,Curcumol,Paeoniflorin,Baicalin,Baicalein,Quercetin,Isoquercetin and Apigenin.The results of molecular docking suggested that the above active components had better binding activity with the protein targets in the literature.2.The results in nude mice experiment were showed,compared with the model group,the weight of orthotopic liver tumor in the chemotherapy group and the chemotherapy combined with Chinese medicine group were significantly reduced.HE staining results showed that the pathological deterioration of liver tumor in situ in chemotherapy combined with Chinese medicine group was better than that in chemotherapy group,and there was no cancer metastasis in lung tissue.3.The results of immunohistochemistry showed that the expressions of MMP-2,Ki67,VEGFA and p-ERK in orthotopic liver tumor of nude mice were down-regulated significantly in the chemotherapy combined with Chinese medicine group compared with the model group and the chemotherapy group.The results of Western blotting showed that the expression of MMP-2 in orthotopic liver tumor of nude mice was down-regulated significantly in the chemotherapy combined with Chinese medicine group compared with the model group and the chemotherapy group.4.The results in BALB/c mice experiment were showed,compared with the model group and 5-FU group,the weight of liver tumor in situ of BALB/c mice in the 5-FU combined with Chinese medicine low-dose and high-dose groups was significantly reduced.The results of HE staining showed that the pathological deterioration of liver tumor in situ in 5-FU combined with low-dose and high-dose Chinese medicine groups was better than that in model group and 5-FU group.The peritoneal metastasis rate of tumor in model group was the highest,followed by 5-FU group,and the peritoneal metastasis rate of tumor in 5-FU combined with low-dose and high-dose Chinese medicine groups had a downward trend compared with 5-FU group.The incidence of ascites in the model group was the highest,followed by the 5-FU group.The incidence of ascites in the 5-FU combined with low-dose and high-dose Chinese medicine groups had a downward trend compared with 5-FU group.5.The results of Western blotting showed that 5-FU combined with high-dose Chinese medicine group could down-regulate significantly the expressions of MMP-2,MMP-9,Ki67,VEGF and HIF-1α in orthotopic liver tumor of BALB/c mice compared with model group and 5-FU group.Compared with model group and 5-FU group,5-FU combined with high-dose Chinese medicine group can lower significantly ERK phosphorylation level and up-regulate significantly the expression of E-cadherin in orthotopic liver tumor of BALB/c mice.Compared with the model group and 5-FU group,5-FU combined with low-dose and high-dose Chinese medicine groups can up-regulate significantly the expression of E-cadherin in orthotopic liver tumor of BALB/c mice through immunohistochemistry.The results of qPCR showed that 5-FU combined with high-dose Chinese medicine group could down-regulate significantly the expression of VEGF mRNA in orthotopic liver tumor of BALB/c mice compared with model group and 5-FU group.Experimental conclusion1.Through the preliminary analysis of the chemical components in Jianpi Huoxue Recipe,the basic research on chemical substances of Chinese medicine was enriched,which provided data support for further research on molecular biological mechanism in the future.2.Jianpi Huoxue recipe combined with 5-FU and oxaliplatin could inhibit the growth of orthotopic liver tumor in nude mice to some extent,and its mechanisms would be related to down-regulation of MMP-2,Ki67,VEGFA and p-ERK expression.3.Jianpi Huoxue recipe combined with 5-FU could inhibit the growth of orthotopic liver tumor in BALB/c mice,in order to reduce the risk of tumor metastasis in peritoneum and reduce the incidence of ascites.The mechanisms were possibly related to down-regulating the expression of MMP-2,MMP-9,Ki67,HIF-la and VEGF,decreasing the phosphorylation level of ERK and up-regulating the expression of E-cadherin. |
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