Study On The Mechanism Of Qilian Mixture Regulating Intestinal Flora And Improving The Curative Effect Of EGFR-TKI In Lung Adenocarcinoma Based On The Theory Of "Spleen As The Guard"

Objective:The aim of this work is to explain the mechanism of Traditional Chinese Medicine（TCM）compound to improve the efficacy of targeted drugs by regulating intestinal flora,based on the observation of the clinical efficacy of Qilian mixture combined with targeted drug gefitinib on lung adenocarcinoma and its impact on intestinal flora,the analyses of the changes of intestinal flora in the process of targeted treatment of lung adenocarcinoma and the differences of antitumor efficacy of targeted treatment under different intestinal flora background,as well as the discussion of the relationship between synergistic effect of TCM in targeted treatment and intestinal flora.Methods:A randomized controlled design was used in the clinical study.46 cases were randomly divided into control group（targeted drug gefitinib）and experimental group（gefitinib combined with TCM compound Qilian mixture）,with 23 cases in each group.The objective efficacy,TCM syndrome score,tumor markers,Kamofsky score and adverse reactions were observed before and after treatment for 12 weeks to evaluate the clinical efficacy and safety.Furthermore,16SrDNA sequencing was used to analyze the structural differences of intestinal flora in lung adenocarcinoma patients and the effect of Qilian mixture combined with gefitinib on intestinal flora before and after treatment.Experimental study:The subcutaneous transplanted tumor animal model of BALB/c-nu nude mice with lung cancer was constructed,and the mice with intervention for 28 days were divided into blank control group（control）,transplanted tumor model group（model）,gefitinib group（targeting）,Qilian mixture group（medicine_targeting）,Qilian mixture combined with gefitinib group（medicine_targeting）,gefitinib+antibiotics group（targeting_antibiotics）,as well as gefitinib+probiotics group.The weight,tumor volume,tumor histopathology and serum tumor markers of tumor bearing mice were observed to evaluate the antitumor efficacy of different intervention groups.The correlation between the changes of intestinal flora and the efficacy indexes of tumor bearing mice was analyzed to find the different species affecting the efficacy of targeted therapy in tumor bearing mice,and further compare the anti-tumor efficacy of different combination targeted drugs such as TCM,antibiotics and probiotics under the background of different intestinal flora.Results:Clinical research:The objective remission rate of Qilian mixture combined with targeted therapy in patients with lung adenocarcinoma was 52.2%,which was higher than that for the single targeted therapy group（30.4%）,but there was no significant difference（P=0.134）.In terms of clinical symptom score,Qilian mixture combined with gefitinib group had more significant effect on improving the symptom score of mental fatigue,loss of appetite,dry mouth and pharynx,nausea and vomiting,as well as diarrhea（P<0.05 or P<0.01）,and the combination of TCM and Western medicine group had more advantages in improving KPS score than the simple targeted treatment group（T3,P<0.05）.For the reduction of blood tumor markers,Qilian mixture combined with targeted therapy could effectively reduce the level of serum CEA（P<0.01）.Moreover,Qilian mixture combined with targeted drugs could effectively reduce the incidence of EGFR-TKI related adverse reaction diarrhea（P<0.05）.In the intestinal flora study,the targeted treatment with gefitinib after 12 weeks caused the disorder of flora,for instance,the contents of p_Verrucomicrobiotag_uncultured_bacterium,g_Phascolarctobacterium,g_Eubacterium_xylanophilum_group,and g_Eubacterium_siraeum_group decreased,while the contents of g_Epulopiscium,g_Adlercreutzia,g_Turicibacter,and o_Erysipelotrichales increased.The combination of TCM and gefitinib could regulate the abundance of species of g_Phascolarctobacterium,o_Acidaminococcales,and f_Acidaminococcaceae,and had a certain recovery trend for the flora disorder caused by targeted therapy.Experiment study:The weight of mice in the model group decreased significantly（P<0.01 or P<0.001）,and the weight of mice in the targeting group also showed a downward trend.The weight of mice in the TCM compound combined with targeted treatment group was significantly higher than that in the model group（P<0.01 or P<0.001）,and had more advantages in stabilizing weight compared to the targeted treatment group alone（P<0.05 or P<0.01）.In terms of controlling tumor growth,each intervention group had the effect of inhibiting tumor growth in tumor bearing mice,and the effect of combined TCM and Western medicine group was more significant compared to the targeted treatment group after intervention for 3 weeks（P<0.05 or P<0.001）.By HE staining,it was found that Qilian mixture combined with gefitinib group had more obvious tumor necrosis and inflammatory reaction,reflecting a strong therapeutic effect.For the reduction of serum tumor markers,the targeted treatment group and integrated TCM and Western medicine group were lower than the model group,and the effect of integrated TCM and Western medicine group was more obvious in reducing CYP-19 level（P<0.05）.In the changes of intestinal flora in different intervention groups,the richness and evenness of intestinal flora in tumor bearing mice decreased with the growth of tumor.Compared with the model group,the mice in the targeted treatment group had increased relative abundance of S24_S24_7Group and Por_Parabacteroides,while the contents of Lac_Lachnospiraceae_Group,Rum_Ruminococcus,Lac_Blautia Rum_Oscillospira,Rik_Alistipes and Rik_Alistipes decreased.There were significant differences in the structure of intestinal flora between Qilian mixture,antibiotics and probiotics.TCM or probiotics combined with targeted drugs could restore the disorder of intestinal flora to a certain extent.In different intestinal flora environment,the weight of antibiotic group decreased significantly,and the inhibition of tumor growth was significantly lower than that of TCM or probiotics combined targeted treatment group.The combination of targeted therapy and TCM had the same effect as probiotics in stabilizing the body weight of tumor bearing mice,and had a more obvious effect in inhibiting tumor growth.By correlating the different strains in each group with the anti-tumor efficacy,it was found that 10 different strains of Alc_Sutterella,Bac_Bacteroide,Lac_Blauti,Lac Dore,Lac Ruminococcus,Por Parabacteroides,Rum Oscillospir,Rum Ruminiclostridium,Rum_Ruminococcaceae_Group,and Rum_Ruminococcus were correlated with antitumor efficacy.Among them,the abundance of Rum Ruminococcus decreased significantly（P<0.05）for tumor bearing mice during tumor formation（T3 and T4 time points）,and also decreased significantly in targeted therapy（T2 to T4 time point,P<0.01 or P<0.05）,while the combination of TCM and Western medicine group had the effect of increasing its abundance trend（T2 to T4 time point）,which was similar to the effect of probiotics.Conclusion:In the targeted therapy of lung adenocarcinoma,both the diversity of intestinal flora and the content of beneficial bacteria（such as Rum Ruminococcu）decreased.Qilian mixture developed based on the theory of "spleen is the guard" in TCM could improve the efficacy of EGFR-TKI targeted therapy in patients with lung adenocarcinoma and reduced adverse reactions such as diarrhea.The regulation of intestinal flora during targeted therapy is one of the mechanisms of the synergistic effect of TCM and Western medicine.