Experimental Study On The Tumor-inhibition Effects And Anti-angiogenesis Of Swelling-Dispersing Decoction In Mice Bearing S180

BackgroundMalignant tumor has become one of the most frequent diseases which are harmful to humans in 21 century. Moreover, the morbility of malignant tumor has sharply increased all over the world in the recent years.There are 10,000,000 persons suffering from malignant tumor every year according to WHO's reports. And the age of the patients tends to be young. It has been a key point in the medical field how to effectively prevent and treat tumor.Anti-cancer drug development has recently shifted in part to development of more innovative anti-cancer agents. The mechanisms of the drugs on the anti-tumor include:1. induce the tumor cells apoptosis; 2. cytotoxic action; 3. inhibit tumor cell proliferation; 4.inhibit tumor cell invasion and transfusion.The theory of tumor growth depends on the growth of new vascular which has been introuded by Folkman since 1971, blocking up the growth of new vascular and cutting off nutrition pathway of tumor tissue are becoming new antineoplaston target, and becoming the hotspot in the studies on malignant.At present angiogenesis regulating factors which have been confirmed include angiogenesis factors and anti-angiogenesis factors. And vascular endothelial growth factor as one which induced angiopoietic vascular growth factor play a core part in angiogenesis.In the developmet of malignant tumor, tumor cells to mionectic adaptation and angiogensis are committed steps. hypoxia inducible factor-la is a transcription factor, primary oxygen regulatory subunit and function subunit which generally resided in mammal and endosomatic,and play a center effectiveness in the process of oxygen reaction. VEGF is a most important angiogenesis factors which can increase vascular permeability, promote endothelial cell migration, hyperplasy and the growth of new vascular, promote growth, invasion and metastasis of malignant tumor. It has been proved in many studies all over the world that VEGF through the mechanisms of paracrine and autocrine thus promote angiogenesis and promote growth, invasion and metastasis of malignant tumor. With the gross tumor volum sharply multiplication, malignant tumor fit into oxygen deficiency condition thus make a great quantity HIF-lacumulation,which can combin together with target genes and make expression enhancement. VEGF is one of target genes. Therefore, it is very important to the pathway of "HIF-VEGF-Angiogenesis".Carcinosis is a generic name of many malignant tumors in the tradtion chinese medicine. The onset of carinosis accociated with disorder of entrails,yin-yang, qi and blood,invasion of six exogenous factors which cause diseases and cumulation of qi, phlegm, moist, heat and blood stasis. the traditional Chinese medicine have many different therapeutical principle in treating cancer. and these medicine which have different functions such as moving qi and resolving phlegm,activating blood and resolving stasis,heat-clearing and detoxicating medicinal,harmonizing qi and blood,promoting urination and draining dampness all have anti-tumor effect in different degree. With the development of molecular biology and traditional Chinese medicine, the simple herb and complex prescription in empirical study get great progression. Such as Curcuma, Hemlock parsley,Scorpion,Wall lizard,Biejiajian pills,Shenmai injection and Angelica Blood-Tonifying Decoction all have different function on inhibiting angiogenesis. Swelling-Dispersing Decoction is the outcome of supervisor Pro.Chen Dali's years of clinical practice. The recipe consists of Fritillaria,Pinellia,Centipede,Selfheal,Oldenlandia diffusa,and so on. It was proved that this Swelling-Dispersing Decoction has an exact in anti-tumor effect in vivo and inhibit tumor cell proliferation in vitro. The is likely to induce the tumor cells apoptosis. The mechanism of this decoction inducing apoptosis may be that it could reduce the expression of the bcl-2 gene, which is considered to repress apoptosis. This project researched on the tumor-inhibition effects and anti-angiogenesis of Swelling-Dispersing Decoction in mice bearing S180.In the experimental study on the tumor-inhibition effects of tradtion chinese medicine in vivo, a series of tumor animal models are set up,such as spontaneous tumor model,provocation tumor model,transplantation tumor model. We select the transplantation tumor model which is frequently used as an experimental model to study. because preparing this model is simple,easy,low cost and needn't long time. Further more,it is very useful to research on onset,treat,infiltration and transferment.Objective To observe the anti-tumor activities, immune regulation and the expression of VEGF and HIF-1α. of Swelling-Dispersing Decoction in mice bearing S180, and analyze the mechanisms on the anti-tumor effect of the decoction.MethodsEstablish mice bearing neoplasm (S180 cell line) as the experimental tumor models:In vitro experiment:culture the S180 cell line, choose the fourth or sixth generation as the experimental object. Use the already prepared stroke-physiological saline solution contain 2-3×106/ml cell density interfere in 10 healthy mice. then we use 0.4ml give per mice intraperitoneal injection; one week later, these 10 mice's abdomen increase in bulk very obviously, we draw-off abdominal dropsy which is ivory which after putting to death. At this time we use the already prepared stroke-physiological saline solution contain 1-2×107/ml cell density interfere in 40 healthy mice's right upper extremity in axillary fossa. And we control cumulative time within one hour.40 mice bearing neoplasm induced by S180 cell inoculation were randomly divide into 4 groups:tumor control group, cyclophosphamide(CTX) group, Swelling-Dispersing Decoction group,Swelling-Dispersing Decoction combined with CTX group.In tumor control group the mice were fed with 0.5ml distilled water and injected into 0.2ml distilled water to the mice's abdominal cavity. In CTX group the mice were fed with 0.5ml distilled water and injected into 20mg/kg cytoxan to the mice's abdominal cavity. Swelling-Dispersing Decoction group the mice were fed with corresponding dose Swelling-Dispersing Decoction and injected into 0.2ml distilled water to the mice's abdominal cavity. Swelling-Dispersing Decoction combined with CTX group the mice were fed with corresponding dose Swelling-Dispersing Decoction and injected into 20mg/kg cytoxan to the mice's abdominal cavity. The tumor-bearing mice received corresponding treatments for 15 days, then stripping and weighing thymuses and spleens of all the mice bearing neoplasm, calculate the indices of thymus and spleen, inhibitory rate of S180 tumor.Then the tumor tissues were fixed with 10% formaldehyde. After the treatment of H&E staining, observe the pathological changes of tissue through light microscope. Immunohistochemistry with SP staining method were used to detect the expression of VEGF and HIF-1αin planted tomor.Results1. Tumor control group the mice's tumor grow quickly, and the mice's drinking and eating decrease obviously. and hairs are confused and disorderly, then piloerection phenomenon is obvious. Other groups the mice's tumor grow slow relativly, Swelling-Dispersing Decoction group and the combined treatment the mice are lively than others groups'mice. CTX group the mice's condition is worst.2. Compared with tumor control group, the weights of the other groups'mice were significantly decreased(F-52.692, P=0.000);and the combined group the weights of tumor were significantly lighter than the Swelling-Dispersing Decoction group and CTX group(P＜0.05).Tumor inhibitory rate of the single-agent Swelling-Dispersing Decoction and CTX respectively are 30.26% and 51.55%,and the combined group the tumor inhibitory rate is 71.14%. at the same condition.q value is 1.08, which indicated that Swelling-Dispersing Decoction combined with CTX can significantly inhibit the growth of the neoplasm of the mice bearing neoplasm.3. Compared with tumor control group and CTX group, the index of thymus and spleen of Swelling-Dispersing Decoction group were significantly increased(P＜0.05); compared with CTX group, the index of thymus and spleen of the combined group were significantly increased(P＜0.05).4. After the treatment of H&E staining, the tumor cells of tumor control group are intensive.and the small vascular of tumor tissues is very plentiful. Then other different groups'can observe lamellar zone of necrosis in different degree and the blood capillary is significantly reduce,and the combined group is best.5. Immunohistochemistry study showed the expression of VEGF and HIF-1αin Swelling-Dispersing Decoction group, CTX group and combined group were lower than that in control group (F=54.982,P=0.000; F=88.759, P=0.000).Compared with Swelling-Dispersing Decoction group, the expression of VEGF and HIF-1αin CTX group is lower(P＜0.05).6. There is some positive correlation trend between VEGF and HIF-1α. (r=0.806, P=0.000).ConclusionsSwelling-Dispersing Decoction can significantly improve the mice's survival state and inhibit the growth of the neoplasm of the mice bearing neoplasm, then Decoction combine CTX have synergistic effect. Swelling-Dispersing Decoction can also increase immune function of the mice, and decrease the tumor's dysfunction and inhibition to the mice's immune organs. Swelling-Dispersing Decoction can significantly inhibit the expression of of the neoplasm block of the all mice bearing neoplasm. There is some positive correlation trend between VEGF and HIF-1α. Swelling-Dispersing Decoction has good effect in inhibiting the growth of S180 Tumor-bearing mice by regulating VEGF expression and inhibiting the protein synthesis of HIF-1α.