| Aim:This research intends to build a mouse model of lung cancer through vaccinating lung cancer tumor strain to C57BL/6mice’s right axillaries.Then we intervene the model by using Yi Fei Qing Hua Granular and measure the levels of vascula the Lewis lung serum VEGF,ES generate impact factor by Elisa method.We determine the VEGF、ES protein levels of Lewis lung tumor tissue in mice by the method of Immunity histochemical.We determine the VEGF、ES mRNA levels of Lewis lung tumor tissue in mice by the method of RT-PCR.Therefore,we can probe these following questions:①The impact of each group drugs on the tumor weight and tumor inhibition rate of Lewis lung cancer.②The VEGF,ES expression level of serum in the control group, treatment group.③The relationship between the different dose Yi Fei Qing Hua Granular on the expression levels of VEGF,ES and its dose.④effect relationship of Yi Fei Qing Hua Granular combined with gifitinib.Through this,we can preliminary explore the mechanism of action of Yi Fei Qing Hua Granular treatment to non-small cell lung cancer angiogenesis and provide experimental evidence for the"supplementing qi and nourishing yin,heat-clearing and detoxifying"treatment of non-small cell lung cancer, which will provide better clinical treatment.Methods:C57BL/6J mice of clean grade,male,70,weight18-20g,lung cancer model established through the right armpit vaccination lung tumor strain.The mice were randomly divided into7groups:Model group,the Yi Fei Qing Hua Granular low dose group,the Yi Fei Qing Hua Granular middle dose group,the Yi Fei Qing Hua Granular high dose group,Gefitinib group, Gefitinib+Yi Fei Qing Hua Granular in dose group,and CTX group.Give drug intervention after inoculation the next day:Control group:0.4ml sterile water gavage,once a day.the Yi Fei Qing Hua Granular low dose group:5g/Kg,0.4ml per mice,gavage,once a day.The Yi Fei Qing Hua Granular middle dose group:10g/Kg,0.4ml per mice,gavage,once a day.The Yi Fei Qing Hua Granular high dose group:10g/Kg,0.4ml per mice,gavage,tiwce a day.Gefitinib group:100g/Kg,0.2ml per mice, gavage,once a day.Gefitinib+Yi Fei Qing Hua Granular in middle dose group:0.2ml gefitinib in the morning and0.4ml Yi Fei Qing Hua Granular in the afternoon.CTX group:60mg/kg,Intraperitoneal injection0.2ml on the third and seventh day after vaccination,14day continuous medication.Specimen after14days:specimens from mice eyeball blood bring mice to death and peel tumor block.2000rpm centrifugal for15minutes,separate the mice’s eye blood and specimen serum in the-70℃frozen environment for reservation.Detect the expression level of VEGF、ES in mice serum by the method of Elisa.Specimen2pieces of tumor tissue after peel out the tumor mass, put them in to imported EP tubes (without RNA enzyme) and dip tubes into nitrogen liquid immediately.Detect the expression level of VEGF、ES mRNA in mice tumor tissue by the method of RT-PCR.put the rest tumor tissues in to jars with formalin solution.Detect the expression level of VEGF ES mRNA in mice tumor tissue by the method of immunohistochemistry.Results:1The impact of each group drugs on the tumor weight of Lewis lung cancerThere is no explicit difference between the Yi Fei Qing Hua Granular low dose group and the model group(P>0.05).Tumor weight in the the Yi Fei Qing Hua Granular middle dose group is lower than the model group, which is significant to statistics (P<0.05).There is divergence on tumor weight between the Yi Fei Qing Hua Granular high dose group and the model group,which is significant to statistics (P<0.01).The tumor weight of the Gefitinib group,Gefitinib+Yi Fei Qing Hua Granular middle dose group and CTX group are lower than the model group,which is significant to statistics (P<0.001).2The effects of each group drugs on the tumor inhibition rate of Lewis lung cancer mice.Tumor inhibition rate=[1-(the tumor mass weight of the experimental group/tumor mass weight of the control group)] x100%).There is no explicit difference between the Yi Fei Qing Hua Granular low dose group and the model group(P>0.05). Inhibition rate in the the Yi Fei Qing Hua Granular middle dose group is higher than the model group,which is significant to statistics (P<0.05). There is divergence on Inhibition rate between the Yi Fei Qing Hua Granular high dose group and the model group,which is significant to statistics (P<0.01).The Inhibition rates of the Gefitinib group,Gefitinib+Yi Fei Qing Hua Granular middle dose group and CTX group are higher than the model group,which is significant to statistics(P<0.001).The top three tumor inhibition rates of groups are:CTX group,Gefitinib+Yi Fei Qing Hua Granular middle dose group and the Gefitinib group.3ELISA research on Groups of drugs to the Lewis lung serum VEGF&ES3.1VEGFEach group of drugs has significantly inhibited serum VEGF expression in mice with Lewis lung.There is difference between Yi Fei Qing Hua Granular low dose group and the Control group(P<0.05).There is also significant difference between Yi Fei Qing Hua Granular middle dose group and the Control group(P<0.01).Besides,the difference among Yi Fei Qing Hua Granular high dose group,Gefitinib group,Gefitinib+Yi Fei Qing Hua Granular middle dose group,and CTX group is very significant(P<0.001).3.2ESThe effects of each group drug to the serum Endostatin expression of Lewis lung mice are:there is no statistic meaning of Yi Fei Qing Hua Granular low dose groups. Compared with Control Group,the Yi Fei Qing Hua Granular middle and high dose groups have statistic meaning(P<0.05).Compared with Control Group,the Gefitinib+Yi Fei Qing Hua Granular group have clear statistic meaning(P<0.01).Compared with model group,Gefitinib and CTX Group have clear statistic meaning(P<0.001). 4Immunohistochemistry (IHC) research on the effects of each group drugs to the Lewis lung cancer mouse xenograft tumor tissue VEGFand ES4.1VEGFEach group of drugs has significantly inhibited serum VEGF expression in mice withLewis lung. Compared with the Model Group, the other Groups have more significan statistic meaning(P<0.001).4.2ESThe effects of each group drug to the serum Endostatin expression of Lewis lung mice are:Compared with the Model Group,the ES expression of Yi Fei Qing Hua Granular low dose group Significantly up-regulate,which has apparent statistic meaning(P<0.01). Compared with the Model Group,the ES expressions of Yi Fei Qing Hua Granular middle,high dose groups, Gifitinib Group,Gefitinib+Yi Fei Qing Hua Granular group,and CTX group significantly up-regulate,which has apparent statistic meaning(P<0.001).5real-time PCR research on the effects of each group drugs to the Lewis lung cancer mouse xenograft tumor tissue about VEGFand ES.5.1the results of VEGF real-time quantitative PCRThe effects of each group drug to of VEGF mRNA in mice with Lewis lung tumor tissue are:Compared with the Model Group,Yi Fei Qing Hua Granular low dose group decreases,with no significant difference (P>0.05). Compared with the Model Group,Yi Fei Qing Hua Granular middle dose group has statistic meaning(P<0.05). Compared with the Gifitinib Group,Yi Fei Qing Hua Granular high dose group,Gefitinib Nepal+Yi Fei Qing Hua Granular middle dose group and CTX group decrease significantly,which have significant staticric difference(P<0.001).5.2ES statistical results of the real-time PCRThe effects of each group drug to of Endostatin mRNA in mice with Lewis lung tumor tissue are:Compared with the Model Group,Yi Fei Qing Hua Granular low dose group increases,with no significant difference(P>0.01).Compared with the Model Group,Yi Fei Qing Hua Granular middle dose group and Gefitinib Nepal increase,with significant statistic meaning(P<0.01). Compared with the Gifitinib Group,Yi Fei Qing Hua Granular high dose group,Gefitinib Nepal+Yi Fei Qing Hua Granular middle dose group and CXT group increase significantly,whichave more significant staticric difference(P<0.001).Conclusions:1. Yi Fei Qing Hua Granular can reduce the tumor weight of Lewis lung cancer mouse.2. Yi Fei Qing Hua Granular have significant tumor inhibition rate.3. Yi Fei Qing Hua Granular can inhibit the expression of VEGF which is a promotion angiogenesis factor,And its effects in a dose-dependent relationship.4. Yi Fei Qing Hua Granular can promote the expression of ES which is a inhibition angiogenesis factor,And its effects in a dose-dependent relationship. 5. Gefitinib+Yi Fei Qing Hua Granular middle dose group is better than single Yifei the Yi Fei Qing Hua Granular in dose medication.But there is no synergistic action compared with single Gefitinib group.6. The mechanism to treat non-small cell lung cancer with Yi Fei Qing Hua Granular is related to the inhibition of tumor angiogenesis. |
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