| Background:Lymphatic System is a major route of metastasis in gastric carcinoma, and lymph node metastasis is also one of the major prognostic factors. Studies have found that in the event of lymph node metastasis of tumors, lymphatic proliferation and expansion often can be observed.This indicates that lymphangiogenesis plays an important role in tumor metastasis.In his long-term medical practice, Professor Wei-Pinkang has established his theory of "Phlegm theory" by integrating modern medicine and Traditional Chinese Medicine treatment of gastric carcinoma, he consider that "Phlegm" is material foundation of generation and metastasis in tumor. He believes that "dispersing phlegm" is a basic treatment for gastric carcinoma, and creates Xiaotan Sanjie Recipe, which is applied to gastric carcinoma treatment and achieves satisfactory results. In prior studies, we have found that Xiaotan Sanjie Recipe can inhibit tumor growth and metastasis.But the mechanism of inhibiting lymph node metastasis in gastric carcinoma is not clear,so the purpose of this study is to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis on gastric carcinoma orthotropic transplantation model.Objective:Through the establishment of MKN-45 human gastric carcinoma orthotopic transplantation tumor model in nude mice,to discover the mechanism of Xiaotan Sanjie Recipe in inhibiting gastric carcinoma lymphatic metastasis by observing the expression of VEGF-C,VEGF-D and VEGFR-3 on gastric carcinoma orthotropic implantation model.All the factors were detected by immunohistochemical staining, RT-PCR, and other molecular biology methods.Methods:1. The MKN-45 cancer cell was orthotopic implanted on stomach near the greater curvature side in BALB/C nude mice,nude mice transplantation tumor model of human gastric carcinoma was set up, the growth of tumor and the life status of mice were observed regularly. The animals were sacrificed and the local growth of transplanted tumor was observed.2.40 mice were divided into 4 groups:saline group,5-Fu group, Elemene group, and Xiaotan Sanjie Recipe group randomly after the orthotopic transplantation model was built. The administrations began from the 48h after the surgery, which were as follows: the mice in saline group were given isotonic saline 0.4ml ig. once a day×6w; those in 5-Fu group were given 5-Fu dilution(60mg.kg-1.w-1) 0.2ml ip. once a week×3w; those in Elemene group were given Elemene 0.4ml (2.5mg/ml) ig. once a day×6w; and those in Xiaotan Sanjie Recipe group were given Xiaotan Sanjie Recipe 0.4ml ig. once a day×6w. The life status, weight change and abdominal signs were observed regularly3.The experiment was terminated on the 6th week. The mice were anesthetized, sacrificed and dissected, the tumor metastasis to the surrounding tissue was measured, the tumors were weighted, observed pathologic diagnosis,inhibition rate was calculated.4. The expressions of VEGF-C,VEGF-D and VEGFR-3 were detected and LMVD was counted and analyzed semi-quantitatively by means of Envision immunohistochemical staining in tumor.5. The mRNA was extracted from tumors for the cDNA synthesizing. Detect the mRNA expression of VEGF-C,VEGF-D and VEGFR-3 by means of RT-PCR.Results:1. We built an orthotopic implantation model of human gastric carcinoma MKN-45 in nude mice successfully.10 days after surgery, the lump could be touched in the upper abdominal about 5mm. On the 6rd week, the lump appeared and looked noduous through the skins. Gradually nude appeared deprivation and weight loss, slow action, such as failure symptoms. The abdomen opened, the tumors were gray-white and fish-shaped tumor tissues were the substantive, which were round or oval. The stomachs of mice were surrounded by the tumor tissues, and the some tissues adhered to the adjacent organs such as liver, gut, lungs and lymphaden. The formation rate was 100%. The HE staining illustrated the tumors was low-differentiated adencarcinoma.2. Animals in each group could be observed in enlarged abdominal lymph nodes, lymph node metastasis rates of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group were 60%,40%,40%, significantly lower than saline group,which was 90%.3. The tumor inhibition rate was 66.5%in 5-Fu group,51.0%in Elemene group and 53.25%in Xiaotan Sanjie Recipe group. Compared with saline group, average tumor weight of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was lower significantly (P< 0.01); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference(P>0.05); the difference between 5-Fu group and Elemene group was significant (P<0.05);4. The results of Xiaotan Sanjie Recipe affecting VEGF-C on nude mice tumor tissue:(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-C protein showed:Compared with saline group, the VEGF-C expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); the difference between Xiaotan Sanjie Recipe group and Elemene group had a statistical significance (P<0.05); Compared with 5-Fu group, Xiaotan Sanjie Recipe group and Elemene group had no significant difference (P> 0.05)(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-C mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).5. The results of Xiaotan Sanjie Recipe affecting VEGF-D on nude mice tumor tissue:(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGF-D protein showed:Compared with saline group, the VEGF-D expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.01); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P>0.05).(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGF-D mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).6. The results of Xiaotan Sanjie Recipe affecting VEGFR-3 on nude mice tumor tissue:(1) The results of Xiaotan Sanjie Recipe affecting the expression of VEGFR-3 protein showed:Compared with saline group, the VEGFR-3 expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);(2) The results of Xiaotan Sanjie Recipe affecting the expression of endostatin mRNA showed:Compared with saline group, the VEGFR-3 mRNA expression in 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group compared with each other had no statistical significance (P >0.05).7. The results of Xiaotan Sanjie Recipe affecting lymphatic microvessel density: LMVD count was as follows:saline group was 17.43±1.79,5-Fu group was 13.33±2.71, Elemene group was 10.20±3.64, and Xiaotan Sanjie Recipe group was 12.23±3.21. Compared with saline group, LMVD of 5-Fu group, Elemene group and Xiaotan Sanjie Recipe group was significantly decreased, the difference had a statistical significance (P<0.05); Compared with Xiaotan Sanjie Recipe group,5-Fu group and Elemene group had no significant difference (P>0.05);Conclusion: 1. Xiaotan Sanjie Recipe can obviously inhibite tumor growth and lymphatic metastasis of the orthotopic transplantation model of human gastric carcinoma MKN-45 in nude mice.2. Xiaotan Sanjie Recipe can decrease lymphatic microvessel density in gastric carcinoma, this may be the pathways that Xiaotan Sanjie Recipe inhibites lymph node metastasis of gastric carcinoma.3. Xiaotan Sanjie Recipe can down-regulate the expressions of VEGF-C,VEGF-D and VEGFR-3 proteins and mRNAs,which may be one of the mechanisms inhibiting lymphatic metastasis.
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