Study On Inhibition Effect And Mechanism Of Different Therapies Of Chinese Patent Medicine On Murine Hepatoma H₂₂ In Vivo

BackgroundPrimary hepatic carcinoma(PHC) is a high occured rate disease all over the world,IARC estimated that there are 564,000 people suffer from PHC and 549,000 are died;In China,there are 306,000 patients,and 300,000 are died,which is a half of total caicinomatous and death number,so PHC is one of the main diseases that threatens human's life in our country.There are 2,500,000 cancer patients in China,and one fifth is died from it,the medical care expense on cancer amounts to overï¿¥30 billions annually,the indirect loss is higher than hundreds of billion yuan.Now the main therapies are opration,radiotherapy and chemo,but lots of patient don't suit for opration at advanced stage when they go to hospital as the hide,malignant and growth nature of PHC,most chemotherapy drugs can restrain excess of proliferation,but side effects are so severe that patients can't afford by their weak bodys.However,Chinese patent medicine are extensive absorbed by advantages of comprehensive therapy,enhance the quality and prolong life and little side effect in clinic.Therefor,it is overwhelmingly significant in social benefit,economic benefit and especially academical values to exert the advantage of Chinese patent medicine,seek for some kind of high-effective and low-cytotoxic Chinese patent medicine,study the mechanism of it,to improve survival probability,reduce therapy expence and widen the range of Chinese patent medicine's application.PHC is a diease which is under mutual effect of intra and external factors,can lead to imbalance of body's Yin and Yang,disfunction of viscera,choken meridian,abnormal operation of Qi and blood,stagnation of Qi and blood,accumulation of wetness and phlegm, reduction evil fire,finally to fall ill combined with Qi,blood,wetness,heat,Liver Qi depression and pathogenic fire and toxin,we always choose promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy to treat the disease in clinic.But PHC is a thorny problem in clinic work since complicated pathogenesis,critical symptoms and contemporaneous syndrome types. The modern medicine shows that the mechanism of Chinese patent medicine cured PHC are various,embodied by:kill the cancer cells directly,restrain to form of cancer's blood vessel, restrain to proliferation of cancer cell,inducement apoptosis of cancer cell,regulate the function of body's immunity and influence gene's expression,but we haven't found the mechanism and relative study of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy yet.ObjectiveTo study the therapy mechanism of PHC by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy,compare the function targets,provide experimental evidence on the treatment of PHC with Chinese patent medicine.Methods1.Exsuct asepsis H₂₂ tumor cell which concentrated to 1×10⁷mL^-1,hypodermic inject 0.2mL at right oxter of mice after antisepsis.2.Repectively fed H₂₂ tumor-bearing mice by the water-solubility liquid of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy,whose best concentration of Chinese patent medicine have been confirmed by fore-experiment,observed their common situations(included by fur's colour,appetite situation,activity limit and so on).3.Weighed tumor up,accounted rate of anti-tumor,analysed inhibiting of growth of tumor cell treated with Chinese patent medicine.4.Observed shape's change of tumor cells by microscope.5.Weighed spleen up,accounted rate of its exponential,analysed immunity effect of tumor-bearing mice treated with Chinese patent medicine.6.TUNEL was applied to test the situation of apoptosis.7.Immunohistochemistry was applied to analyze the expression level of cell cycle and apoptosis cell of CyclinD1,PCNA,Bcl-2 and Bax proteins in the cell,to research the relationship between the mechanism of proliferating,apoptosis,the regulation and control of gene expression.Results1.H₂₂ tumor-bearing mice model was established successfully,,visible hypodermal tumor after 4 days,the rate of successful model is 100%.2.Rate of anti-tumor of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy groups are 43.64%,50.91%and 45.45%.3.The effect of splenic exponential was not obviously treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy(P＞0.05).4.The effect of proliferating was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy(P＜0.05).5.The effect of apoptosis was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy(P＜0.05).6.The data of immunohistochemistry indicated:compared with the model group,the expression of PCNA,CyclinD1 and Bcl-2 were reduced,Bax was increased remarkably after treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy (p＜0.05).Conclusion1.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,especial for heat clearing and wetness removing therapy.2.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can reduce cell's expression of PCNA,resist compose progress of DNA,then resist proliferating of carcinoma cell.3.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,which mechanism may reduced expression of CyclinD1,blocked the way of CyclinD1-CDK4-Rb.4.The apoptosis-associated proteins Bcl-2 and Bax were probably significant regulation factors of apoptosis-induced by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy.5.The result of experiment indicated that promoting blood circulation and removing blood stasis therapy can resist the growth of PHC mostly by resisted proliferating of carcinoma cell.6.The result of experiment indicated that heat clearing and wetness removing therapy and nourishing Yin of liver and kidney therapy can resist the growth of PHC mostly by abducted apoptosis of carcinoma cell.