Mechanisms Of Qiyusanlong Decoction Enhancing Th1 Immune Response To Inhibit Lung Cancer Through PD-1 Signaling Pathway And Its Metabonomic Study

Objective:Based on Qiyu Sanlong Decoction,an anti-lung cancer prescription with the function of "Fuzheng and Xiaoji" and take PD-1 signaling pathway as the research target,we observed the effect of Qiyu Sanlong Decoction on Th1 immune response and elaborated the molecular mechanism of Qiyu Sanlong Decoction enhancing Th1 immune response and inhibiting lung cancer through PD-1signaling pathway.At the same time,the role and intervention mechanism of Qiyu Sanlong Decoction in the treatment of lung cancer were explored from the level of endogenous metabolites in the body with the holistic thinking of Traditional Medicine.Potential biomarkers related to the therapeutic effect of Qiyu Sanlong Decoction were screened out,thus providing a scientific basis for optimizing Qiyu Sanlong Decoction from the perspective of modern biology.Methods:2.1 Effect of Qiyu Sanlong Decoction on Proliferation and Apoptosis of A549 CellsMTT method was used to evaluate the viability of A549 cells before and after treatment with Qiyu Sanlong Decoction.The 50% inhibitory concentration(IC50)of Qiyu Sanlong Decoction was screened out.DAPI method was used to observe the apoptosis and morphological changes of A549 cells before and after treatment with Qiyu Sanlong Decoction.2.2 Effect of Qiyu Sanlong Decoction on the weight and volume of tumor in mice bearing lung cancerUsing LLC cell culture and subcutaneous transplantation method,48 model mice were randomly divided into four groups: model group,low dose group of Qiyu Sanlong Decoction(L-QYSLD): 20g/kg(equivalent to the daily dose of clinical adults)Chinese herbal medicine liquid,medium dose group of Qiyu Sanlong Decoction(M-QYSLD):40g/kg(twice the daily dose of clinical adults)Chinese herbal medicine liquid.High dose group of Qiyu Sanlong Decoction(H-QYSLD): 80 g/kg(equivalent to 4 times the daily dose of clinical adults)Chinese herbal medicine liquid.Chinese herbal medicine solution was given by gastric lavage,the remaining 12 healthy mice were taken as control group.The model group and the control group were given 0.9% saline0.1m L/10 g per day.The general conditions of mice were observed daily.The growth of tumor was monitored every 3 days for 10 consecutive days.The vertical diameter of tumor was measured with vernier caliper to evaluate the therapeutic effect of Qiyu Sanlong Decoction on lung cancer transplanted mice.The best dose was selected as the treatment group of Qiyu Sanlong Decoction for further in vivo experiment and metabolomics study.2.3 Qiyu Sanlong Decoction Enhances Th1 Immune Response and Inhibits Lung Cancer through PD-1 Signaling PathwayThe subcutaneous transplantation model of lung cancer mice was established by LLC cell culture and subcutaneous transplantation.72 model mice were randomly divided into four groups: control group(0.1m L/10 g saline per day),chemotherapy group(DDP):0.2ml DDP solution was intraperitoneally injected on 1,3 and 5 days,Chinese Medicine group(QYSLD): 40g/kg(2 times the daily dose of clinical adults)and combined group(DDP+QYSLD).Every day,40g/kg of Chinese Medicine solution was given orally,and 0.2ml of DDP solution was injected intraperitoneally on 1,3 and 5 days.The general conditions of mice were observed daily.Six mice were sacrificed 7,14 and 21 days after treatment.Splenic lymphocytes were isolated from spleen.Tumor killing effect of CD8+T cells and immune response of CD4+T cells were detected by ELISA and flow cytometry at different time points.The number of CD4+CD25+Foxp3+regulatory T cells was changed.The proportion of PD-1 positive cells in T cells and apoptosis of memory T cells were analyzed.Cell proportion.For 21-day-old mice,the lungs were removed completely after execution and the metastatic nodules on the lung surface were counted under an anatomical microscope.2.4 Effect of Qiyu Sanlong Decoction on Plasma Metabolites in Tumor-bearing Mice with Lung CancerIn the experiment,blood samples were collected from the intraorbital reflux area of mice in model group,control group and middle dose group of Qiyu Sanlong Decoction before execution.The functional metabolites of tumor-bearing mice with lung cancerwere identified and quantified by non-targeted metabonomics based on UPLCQTOF/MS.The intervention effect of Qiyu Sanlong Decoction was also studied.Finally,MVDA was used to evaluate the changes of plasma metabolites in LLC lung cancer transplanted mice,and potential biomarkers related to the treatment of Qiyu Sanlong Decoction were screened.3.Result3.1 Effect of Qiyu Sanlong Decoction on Proliferation and Apoptosis of A549 CellsMTT staining showed that Qiyu Sanlong Decoction inhibited the growth of A549 cells in a concentration-related manner.The 50% inhibitory concentration of Qiyu Sanlong decoction was estimated to be 18.19(±0.51mg/ml)at 24 hours.After DAPI staining,the results of fluorescence microscopy showed that Qiyu Sanlong Decoction could induce A549 cell apoptosis in a concentration-dependent manner,and Qiyu Sanlong Decoction could effectively induce morphological changes of A549 cells,including mitosis and nuclear pyknosis.3.2 Effect of Qiyu Sanlong Decoction on the Weight and Volume of tumor in Lung Cancer-bearing MiceQiyu Sanlong Decoction can effectively reduce the weight and volume of tumor in lung cancer-bearing mice.The volume and weight of tumor in lung cancer-bearing mice treated with Qiyu Sanlong Decoction for 10 days were significantly lower than those in normal saline group.The volume and weight of tumor in M-QYSLD group were significantly lower than those in L-QYSLD group(P < 0.01),which was similar to that in H-QYSLD group.Therefore,we chose Qiyu Sanlong Decoction medium dose group as Qiyu Sanlong Decoction treatment group for further in vivo and metabolomics research.3.3 Qiyu Sanlong Decoction Enhances Th1 Immune Response and Inhibits Lung Cancer through PD-1 Signaling PathwayThe proliferation of T cells was the strongest in the traditional Chinese medicine group,and the secretion levels of IFN-gamma and TNF-alpha were the highest in the combined group,followed by the other groups(P < 0.01).The number of CD4 + CD25 + Foxp3 +regulatory T cells in combination group decreased significantly with the prolongation of treatment time.The number of CD4 + CD25 + Foxp3 + regulatory T cells in traditional Chinese medicine group and chemotherapy group also decreased,but not in Qiyu Sanlong Decoction group and combination group.The specific killing effect of CD8 +T cells in Chinese medicine group and combination group was significantly higher than that in other groups(P < 0.05).The proportion of PD-1 positive cells in combination group and traditional Chinese medicine group was significantly lower than that in other groups(P < 0.001),and the apoptotic rate of CD44 high CD4 + T cells in combination group was also significantly lower than that in other groups(P < 0.001).After 21 days of treatment,the mice were executed and their lungs were removed completely.It was found that the combination group was small.The pulmonary metastasis nodules of rats were the least,and the effect was better than that of chemotherapy group(P < 0.001).Compared with model group,Chinese medicine group also had obvious therapeutic effect(P < 0.001).3.4 Effect of Qiyu Sanlong Decoction on Plasma Metabolites in Tumor-bearing Mice with Lung CancerBy MVDA statistical analysis,PCA score chart showed that M-QYSLD group showed a regression trend compared with Model group,indicating that Qiyu Sanlong Decoction had the ability to intervene in metabolic disorders related to lung cancer.On this basis,21 potential biomarkers related to lung cancer and the intervention of Qiyu Sanlong Decoction were screened out.Potential biomarkers mainly involve glycerol phospholipid metabolism,sphingolipid metabolism,steroid hormone biosynthesis,fatty acid degradation and arachidonic acid metabolism.4.Conclusion4.1 Qiyu Sanlong Decoction inhibits the growth of A549 cells and induces apoptosis in a concentration-related manner.4.2 Qiyu Sanlong Decoction can effectively reduce the weight and volume of tumor in lung cancer-bearing mice.4.3 Qiyu Sanlong Decoction can enhance Th1 immune response by down-regulating PD-1 signal pathway,inhibit memory T cell apoptosis,improve the killing effect of CD8+T and CD4+T cell immune response in mice,and improve the cellular immunity of mice,so as to achieve the role of assistant treatment of lung cancer.4.4 The mechanisms of Qiyu Sanlong Decoction in the intervention of lung cancer mainly involve glycerol phospholipid metabolism,sphingolipid metabolism,steroid hormone biosynthesis,fatty acid degradation and arachidonic acid metabolism.The metabolic pathway of arachidonic acid and sphingolipid is significantly related to immunosuppression,cytotoxic T cells and Th1-mediated anti-tumor immunity.Metabolic pathway analysis showed that the activities of LCAT,PI synthase,phosphatidyltransferase and PS decarboxylase were up-regulated,while the downregulated activities of choline kinase,PC synthase,CL synthase,ASM,s PLA2 and c PLA2 alpha were involved in the intervention of Qiyu Sanlong Decoction.