| Objective: to establish a mouse model of lung qi deficiency syndrome of non-small cell lung cancer,to extract the characteristic factors of the animal model of lung qi deficiency syndrome of non-small cell lung cancer,and to find the characteristic biomarkers of the model.Preliminary construction of animal model evaluation system of lung qi deficiency syndrome of non-small cell lung cancer based on intestinal flora-host metabolic mechanism.To evaluate the effects of Radix Astragali and Scutellaria barbata on plasma metabonomics,intestinal flora and inflammatory cytokines in the mouse model of the disease and syndrome,and to explain the intervention effect and mechanism of Radix Astragali and Scutellaria barbata on this model.to provide new ideas and targets for the diagnosis and treatment of non-small cell lung cancer.Methods:1.Study on the Mouse Model of combination of Disease and Syndrome of non-small Cell Lung Cancer.The mouse model of lung qi deficiency syndrome of non-small cell lung cancer was established by using the method of traditional Chinese medicine and modern medical etiology and pathology,and the mouse model of lung qi deficiency syndrome of non-small cell lung cancer was established by fumigation method and tumor inoculation method.the evaluation methods were the combination of macroscopic index and microscopic index,the combination of differential markers and whole metabolic pathway,and the combination of animal behavior index and biochemical index.Comprehensive application of animal behavior,biochemical indicators,immunohistochemistry,metabonomics,16s-r DNA sequencing and other technical means to analyze the prepared animal model,and construct the evaluation system of the disease-syndrome combination model from multiple angles.2.Study on the safety of Radix Astragali and Scutellaria barbata.Radix Astragali and Scutellaria barbata were treated with Radix Astragali and Scutellaria barbata.After 14 days of intragastric administration,the blood routine,liver and kidney function and other indexes were tested,and the pathological observation of liver and kidney tissue HE staining was carried out to evaluate the safety of Radix Astragali and Scutellaria barbata.3.Study on the microflora-metabolic mechanism of Radix Astragali and Scutellaria barbata.After intervention with Radix Astragali and Scutellaria barbata on the disease-syndrome combination model of mice,the samples of plasma,feces,bronchoalveolar lavage fluid and tumor tissue of the disease-syndrome combination model mice were collected.Using LC-MS/MS system combined quadrupole Orbitrap mass spectrometer to study the metabonomics of plasma samples of animal model,to find the differential metabolic markers and metabolic pathways of lung qi deficiency syndrome combined with non-small cell lung cancer after intervention of astragalus and Scutellaria barbata by traditional Chinese medicine,and to study the changes of intestinal flora composition and abundance in model mice by using 16 SNova Seq PE250 high-throughput sequencing platform and Shengxin analysis platform.At the same time,combined with the expression of IL-6,IL-10 in bronchoalveolar lavage fluid and IL-8,IL-10 in tumor tissue,to explore the mechanism of Radix Astragali and Scutellaria barbata on the intervention of lung qi deficiency syndrome of non-small cell lung cancer in flora-metabonomics level.Results:1.Study on the Mouse Model of combination of Disease and Syndrome of non-small Cell Lung Cancer.(1)Macro-characterization: there were significant differences in behavior,fur appearance,activity and mental state between the qi deficiency group,the tumor-bearing group and the control group,especially in the tumor-bearing group with qi deficiency.The amount of food and water consumption of mice in the qi deficiency tumor group were significantly lower than those in other groups at the 4th week.(2)body mass: during the fumigation period(within 20 days),the body mass of mice in qi deficiency group and qi deficiency tumor bearing group increased more slowly than that in control group and tumor bearing group,and decreased after a brief increase in body weight in tumor bearing group and qi deficiency tumor bearing group(about 20-30 days).(3)tumor mass: the tumor quality in qi deficiency tumor bearing group was significantly higher than that in qideficiency tumor bearing group(P > 0.05).(4)spleen index: spleen index in Qi deficiency group was lower than that in Qi deficiency group and tumor bearing group(P <0 05).There was no significant difference between Qi deficiency group and tumor bearing group(P > 0 05);(5)HE staining and immunohistochemistry: tumor cell density decreased and necrotic area increased in Qi deficiency tumor bearing group.The expression level of IL-8 and IL-10 in tumor tissue in Qi deficiency tumor bearing group was higher than that in tumor bearing group.(6)the levels of IL6 and IL10 in bronchoalveolar lavage fluid: the levels of IL6 and IL10 in bronchoalveolar lavage fluid in Qi deficiency group,tumor bearing group and Qi deficiency tumor bearing group were all increased,and those in Qi deficiency tumor bearing group were significantly higher than those in Qi deficiency tumor bearing group.(7)the results of plasma metabonomics: a total of 53 differential metabolites and 9 related metabolic pathways were selected from the comparison between the control group and tumor bearing group.28 differential metabolites and 9 related metabolic pathways were screened between the control group and qi deficiency group,49 differential metabolites and 5 related metabolic pathways were screened between tumor bearing group and qi deficiency tumor bearing group,59 differential metabolites and 7related metabolic pathways were screened between qi deficiency group and qi deficiency tumor bearing group.(8)Analysis of intestinal flora characteristics of mice: there was significant difference in flora structure among the four groups(P < 0.05),but there was no significant change in flora diversity(P > 0.05).The ratio of thick-walled bacteria to Bacteroides in the tumor-bearing group increased,and the abundance of Lactobacillus decreased in the Qi-deficiency tumor-bearing model group.It was found that the bacteria related to qi deficiency were Clostridia＿UCG＿014 and Allobaculum,the expression of Dubosiella,Allobaculum,Prevotellaceae＿UCG＿001,Prevotellaceae was up-regulated,while the expression of Ileibacterium,Ileibacterium＿valens was down-regulated;Prevotellaceae,Prevotellaceae＿UCG＿001,Erysipelotrichales,Erysipelotrichaceae,Allobaculum,Dubosiella were up-regulated in qi-deficiency tumor-bearing group,Prevotellaceae,Prevotellaceae＿UCG＿001,Dubosiella were up-regulated in qi-deficiency tumor-bearing group,and Ileibacterium and Ileibacterium＿valens were down-regulated in qi-deficiency tumor-bearing group compared with qi deficiency tumor-bearing group.2.Safety Evaluation of Radix Astragali and Scutellaria barbata.Mice were treated with Radix Astragali and Scutellaria barbata for 14 days.Liver function(ALT,AST,AST/ALT),renal function(UREA,CREA),blood routine,liver andkidney tissue HE staining pathology were observed.There was no significant difference between the normal group and the intervention group.3.Study on the microflora-metabolic mechanism of Radix Astragali and Scutellaria barbata.(1)body mass: during the fumigation period(within 21 days),the body mass of mice in qi deficiency group,qi deficiency drug pair group,qi deficiency tumor bearing group and qi deficiency tumor bearing group increased more slowly than that in control group,tumor bearing group and tumor bearing drug group.After fumigation and during the period of tumor inoculation(about 21-30 days),the body weight of mice in tumor-bearing group,qi-deficiency tumor-bearing group,tumor-bearing drug-bearing group and qi-deficiency tumor-bearing group decreased(excluding estimated tumor mass).The body weight of the tumor-bearing group with qi deficiency decreased significantly.During the end of fumigation(30-40 days),the body weight of qi deficiency group and qi deficiency medicine group increased slightly,but the body weight of tumor bearing group,tumor bearing group,qi deficiency tumor bearing group and qi deficiency tumor bearing group continued to decrease.(2)tumor quality: there were significant differences between tumor-bearing group and qi-deficiency tumor-bearing group,tumor-bearing group and qi-deficiency tumor-bearing group,qi-deficiency tumor-bearing group and tumor-bearing drug group,but there was no significant difference among other groups(P > 0.05);(3)spleen index in qi deficiency tumor-bearing group was lower than that in qi deficiency group and tumor-bearing group.After intervention,spleen index in qi deficiency group was higher than that in model group.(4)HE staining and immunohistochemistry: the density of tumor cells decreased and the necrotic area increased in the tumor-bearing group,while the density distribution was uniform in the tumor-bearing group,tumor-bearing drug pair and qi-deficiency tumor-bearing drug group,and there was no necrosis in the tumor-bearing group.The expression levels of IL-8 and IL-10 in tumor tissue were higher in Qi deficiency tumor bearing group,and the expression levels of IL-8 and IL-10 in drug pair intervention group were lower than those in model group.(5)the levels of IL-6 and IL-10 in bronchoalveolar lavage fluid: the levels of IL-6 and IL-10 in bronchoalveolar lavage fluid of mice in qi deficiency group,tumor bearing group and qi deficiency tumor bearing group were all increased,while the expression levels of IL-6 and IL-10 in drug pair intervention group were lower than those in model group.(6)the results of plasma metabonomics study: in the comparison between the control group and the qi deficiencydrug pair group,we screened 39 differential metabolites and 4 related metabolic pathways,79 differential metabolites and 7 related metabolic pathways were screened between the control group and the tumor-bearing drug group.90 differential metabolites and 9 related metabolic pathways were screened out in the control group and qi deficiency drug pair group,and 39 differential metabolites were screened between qi deficiency group and qi deficiency drug pair.There were 3 related metabolic pathways,76 differential metabolites were screened between tumor-bearing group and tumor-bearing drug group,7metabolic pathways related to differential metabolic biomarkers,65 differential metabolites were screened between qi deficiency tumor-bearing group and qi deficiency tumor-bearing drug group,and 6 related metabolic pathways were selected.(7)Analysis of the characteristics of intestinal microflora in mice: there was significant difference in the structure of intestinal flora among the seven groups(P < 0.05),but there was no significant difference between the control group and qi deficiency drug group(P > 0.05).There were significant differences in microflora diversity between control group and qi deficiency drug pair group,tumor-bearing drug pair group and qi deficiency drug pair group,qi deficiency drug pair group and qi deficiency drug pair group,qi deficiency tumor group and qi deficiency drug pair group.It was found that Klebsiella＿v and Klebsiella were specifically expressed in qi deficiency drug pair,Gammaproteobacteria and Proteobacteria were up-regulated in qi deficiency group,Clostridia＿UCG＿014 was up-regulated in qi deficiency group,Enterobacterales and Enterobacteriaceae were down-regulated in qi deficiency group,Clostridia,Lachnospirales and Lachnospiraceae were down-regulated in qi deficiency group,and further down-regulated in qi deficiency group.The expression of Allobaculum in qi deficiency drug pair group was higher than that in qi deficiency group.Prevotellaceae＿UCG＿001 and Allobaculum were up-regulated in Qi-deficiency tumor-bearing group,and the expression of Ileibacterium＿valens and Ileibacterium was decreased after intervention,while the expression of Qi-deficiency tumor-bearing drug in Qi-deficiency tumor-bearing drug was increased after intervention.The expression of Erysipelotrichaceae,Ileibacterium,Ileibacterium＿valens,Proteobacteria and Gammaproteobacteria decreased in the tumor-bearing group,but increased after the intervention of tumor-bearing drugs.Conclusion:1.The traditional etiology of traditional Chinese medicine and the pathological model of modern medical etiology(smoking + seeding tumor)were used to establish the model,which accorded with the pathological state of lung qi deficiency syndrome of non-small cell lung cancer.the characteristic macroscopic characterization,microscopic indexes,metabolic markers and intestinal flora of the syndrome which can reflect the mouse model of lung qi deficiency of non-small cell lung cancer were screened out.A preliminary evaluation system of animal model of lung qi deficiency syndrome of non-small cell lung cancer based on intestinal flora-host metabolic mechanism was established.2.Using Radix Astragali and Scutellaria barbata as drugs to interfere with the mouse model has no blood routine and hepatorenal toxicity,and the drug application is safe.3.Radix Astragali and Scutellaria barbata could improve and regulate the body weight,spleen index,tumor pathomorphology and inflammatory cytokines(tumor tissue IL-8,IL-10,bronchoalveolar lavage fluid IL-6,IL-10)in non-small cell lung cancer mice with lung qi deficiency syndrome,but had no significant effect on tumor quality.At the same time,based on the research methods of metabonomics and intestinal flora,it was found that the expression of related differential metabolic biomarkers and intestinal flora after the intervention of Radix Astragali and Scutellaria barbata may be the mechanism of Radix Astragali and Scutellaria barbata in the treatment of non-small cell lung cancer. |
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