Sijunzi Tang Improves The Efficacy Of Gefitinib Based On Pre-metastatic Niche

Objective:1.Lewis lung cancer(LLC)model was used to detect the efficacy of gefitinib combined with SJZ Tang.2.To examine the impact of gefitinib and SJZ on pre-metastatic niche and its underlying mechanism,so as to provide a scientific basis for the clinical treatment of lung cancer.Methods:To generate spontaneous lung metastatic models,1×106 luciferase-labeled LLC cells were injected subcutaneously in the shaved right flank of mice.Then the mice were randomly divided into model,gefitinib treatment,SJZ treatment and co-treatment groups with intragastrical administration next day.The study is composed of the following two parts:1.Research on the therapeutic effect of gefitnib and SJZ on tumor bearing mice:To observe the daily activities and body weight changes for evaluating the basic situation of tumor bearing mice in each group.After the experiment,the fluorescence intensity of tumor was detected by small animal imaging technology.For evaluating the effect of gefitinib and SJZ,the tumors were weighed and the its volume was measured with vernier caliper.The weight of heart,liver,spleen,lung,kidney and other organs were measured and the mass index of each organ was calculated.TUNEL staining and HE staining were used to investigate the apoptosis of tumor cells and inflammatory infiltration and metastasis in the lung.Western blot and immunohistochemistry were used to investigate the expression of EGFR/PI3K/Akt pathway related proteins in tumor tissues.2.Research on the effect of gefitinib and SJZ on pre-metastatic niche:This part attempts to elucidate the immunological mechanism of SJZ in improving the efficacy of gefitinib.According to the literature,LLC metastasis model was used and we choose 14days as the observation point.Flow cytometry was used to detect the number of immune cells and the related receptors on their surface in peripheral blood,spleen and lung tissues mice;Luminex xMAP was used to detect the expression level of tumor-derived factors in tumor tissues of miceResults:1.The therapeutic effect of gefitnib and SJZ on tumor bearing mice:1)During the experiment,we found that with the tumor size increasing,the mice in the model group showed slow activity and had bad mental state.The mice in the gefitinib or SJZ groups could still maintain their daily drinking and eating activities.The mice in the co-treatment group were in a good mental state,and their acted more agile.The weight of mice in each group increased slowly,and there was no significant difference between the groups.After21 days of continuous administration,the tumor inhibition rates of gefitinib group,SJZ group and co-treatment group were 45.7%,38.4%and 84.8%,respectively.After 45 days of administration,the survival rate of model group,gefitinib group,SJZ group and co-treatment group was 0%40%,60%and 60%,respectively.Compared with the model group,there was a significant difference in the organ coefficient of spleen but not in heart,liver,lung and kidney in co-treatment group,indicating that co-treatment can boost immunity and had no significant toxic and side effects on organs of tumor bearing mice.2)Tunel staining and HE staining results showed that Gefitinib and SJZ can promote the cell apoptosis of tumor tissues and inhibit the lung metastasis and the effect is more obvious in co-treatment group(P<0.01 or P<0.05).3)Western Blot results revealed that compared with the model group,the expression of p-EGFR,p-Akt,and p-PI3K in gefitinib or SJZ group showed a downward trend,and the trend was more obvious in co-treatment group.However,there were no significant changes in the expression of Akt and PI3K proteins.The gefitinib and co-treatment group could effectively down-regulate the expression of EGFR protein,while SJZ had no significant effect on the expression of EGFR protein.The results of immunohistochemistry experiments showed that compared with the model group,the expression of p-PI3K,p-AKT,EGFR and p-EGFR in the gefitinib group was significantly down-regulated and p-PI3K,p-AKT and p-EGFR in the SJZ group was significantly down-regulated.Compared with the gefitinib group or SJZ group,the expressions of p-PI3K,p-AKT,EGFR,and p-EGFR in the co-treatment were down-regulated.The above results suggest that the EGFR/PI3K/AKT pathway may play a central role in the mechanism of SJZ improving the efficacy of gefitinib.2.Research on the effect of gefitinib and SJZ on pre-metastatic niche:1)Flow cytometry results showed that compared with the model group,the percentage of neutrophils in the lung tissue were significantly decreased(P<0.05 or P<0.01)in the gefitinib group,SJZ group and co-treatment group.But there was no significant difference among the three groups(P>0.05).In the peripheral blood and lung tissue,compared with the model group,the expression levels of CXCR1,CCR2 and c-Kit on the surface of neutrophils and monocytes in SJZ group and co-treatment group decreased or decreased significantly.However,there was a significant increase in the expression level of c-Kit on the surface of monocytes.In the mouse spleen tissue,the expression levels of CXCR1,CCR2 and c-Kit in the gefitinib group increased significantly,while decreased significantly in SJZ or co-treatment group(P<0.05).4)The results of Luminex xMAP showed that in the tumor tissues,the content of TNF-α in gefitinib group decreased significantly and IL-23,RANTES increased compared with the model group(P<0.01 or P<0.05).And the content of GRO-α,IP-10 in SJZ group decreased significantly(P<0.05).Besides,the content of IL-12p70,IL-15 in co-treatment group decreased and IL-1α,IL-1β,IL-18 and GRO-α increased significantly(P<0.05).Furthermore,compared with the gefitinib group,the content of IL-1α,IL-6 in the cotreatment group and IL-6,IL-18 in SJZ group decreased significantly(P<0.05).The above results indicate that gefitinib increases the inflammatory level of the pre-metastatic niche by acting on immune cells and cytokines,while SJZ can improve the efficacy of gefitinib by down-regulating the inflammatory level.Conclusion:SJZ can improve the efficacy of gefitinib by regulating the inflammatory level of pre-metastatic niche.