| Objective:To sort out and explore the pathogenesis and transmission of lung cancer meridian disease,to evaluate the clinical efficacy and safety of the Xiaoliu Pingyi recipe combined with pemetrexed maintenance therapy for advanced lung adenocarcinoma based on the meridian disease theory through clinical research,and to observe the anti-tumor effect of the Xiaoliu Pingyi recipe in vitro and in vivo through experimental research,explore its impact on tumor angiogenesis and related regulatory mechanisms,further enrich the modern biological connotation of lung cancer meridian disease theory,and provide new scientific basis for the clinical application of the Xiaoliu Pingyi recipe.Methods:1.Clinical study:In this study,70 patients with advanced lung adenocarcinoma were selected according to the nadir criteria in a prospective randomized controlled study and randomly divided into an observation group（Xiaoliu Pingyi recipe combined with pemetrexed treatment）and a control group（pemetrexed treatment alone）.The patients were treated until disease progression and the differences in progression-free survival（PFS）,TCM syndrome score,quality of life,hematological toxicity,liver and kidney function abnormalities and gastrointestinal reactions between the two groups were observed to evaluate the clinical efficacy and safety of hematological toxicity.2.Experimental study:In the in vivo part of the study,lung cancer transplantation tumor models were established by subcutaneous injection of Lewis lung cancer cells into C57BL/6J mice,and each group of mice was treated with dd H2O gavage,pemetrexed intraperitoneal injection,Xiaoliu Pingyi recipe gavage and pemetrexed intraperitoneal injection combined with Xiaoliu Pingyi recipe gavage for 14 d.The general condition of each group of mice was observed and the tumor volumes were compared.Immunohistochemical fluorescence staining was used to detect the effects of tumor elimination panning formula on the structure and function of blood vessels in the tumor.CD31 andα-SMA fluorescence labeling were used to observe the density of microvessels and pericyte coverage in the tissues,and CD31and FITC-Dextran fluorescence labeling were used to observe the vascular permeability in the tissues.The expression levels of HIF-1α,VEGFA,Ang-2,and PI3K/Akt m RNA and protein in the tumor tissues of each group of mice were assessed using q RT-PCR and Western Blot techniques.In an in vitro study,the drug-containing serum was prepared by gavage of Xiaoliu Pingyi recipe in SD rats.The CCK-8 method was used to observe the proliferation trend of the drug-containing serum on human normal lung epithelial BEAS-2B cells and human lung adenocarcinoma A549 cells,and to screen the optimal intervention concentration of the drug-containing serum.Transwell and scratch assays were used to determine the effect of drug-containing serum on the invasion and migration ability of lung cancer A549 cells;ELISA was used to observe the effect of drug-containing serum on the secretion level of VEGF in the supernatant of lung cancer A549 cells.The expression levels of HIF-1α,VEGFA,Ang-2,and PI3K/Akt m RNA and protein were assessed using q RT-PCR and Western Blot techniques in each group of cells.Finally,the differences in their expression levels were determined.Results:1.Clinical study:70 patients with advanced lung adenocarcinoma who met the inclusion criteria were enrolled in the study.9 cases,including 4 from the observation group and 5from the control group,were excluded during the study.61 cases were finally evaluated for efficacy,31 cases in the observation group and 30 cases in the control group,with an overall dropout rate of 12.9%,which was estimated by statistical sample size and the number of cases met the requirements for efficacy evaluation.No statistical differences were found between the two groups in terms of baseline data such as age,gender,TNM stage,pre-treatment TCM syndrome score,weight,KPS score and the efficacy of previous first-line treatment.When comparing the PFS between the two groups,the observation group had a median PFS of 5.2 months（95%CI:4.949-5.451）,while the control group had a median PFS of 4.6 months（95%CI:3.735-5.465）.Despite the improvement in PFS in the observation group,no statistically significant difference was found between the two groups as determined by the Log-rank test（P>0.05）.Subgroup analysis revealed no significant differences in PFS between the two groups based on age and gender.Furthermore,no significant differences were observed between the two groups for TNM stage III patients.However,for stage IV patients,the observation group had a median PFS of 5.3 months,while the control group had a median PFS of 4.0 months,indicating a statistically significant difference between the two groups（P<0.05）.In terms of comparison of TCM syndrome score,a total of 23 patients in the observation group improved their TCM clinical symptoms,with a total effective rate of 74.19%,while a total of 16 patients in the control group improved their TCM clinical symptoms,with a total effective rate of 53.33%,with statistical differences between the two groups（P<0.05）.Regarding the comparison of quality of life,15 patients in the observation group showed improvement in KPS scores,12 patients showed stable scores,and 4 patients showed a decline.Additionally,80.65%of patients in the observation group had either gained weight or maintained a stable weight.On the other hand,9 patients in the control group showed improvement in KPS scores,10 patients showed stable scores,and 11 patients showed a decline.The proportion of weight gain and stability was 50%in the control group.The statistical analysis indicated a significant difference between the two groups（P<0.05）.In terms of safety evaluation,4 patients in the observation group developed bone marrow suppression,accounting for 12.9%of all patients,while a total of 11 patients in the control group developed bone marrow suppression,accounting for 36.67%of all patients,and the results of the two groups were statistically different after testing（P<0.05）.A total of 4 patients in the observation group developed gastrointestinal adverse reactions,accounting for 12.9%of all patients.In contrast,10patients in the control group,accounting for 33.33%of all patients,experienced gastrointestinal adverse reactions.There were no statistically significant differences between the two groups regarding abnormal liver and kidney functions.This suggests that the Xiaoliu Pingyi recipe did not increase adverse reactions related to treatment and had a favorable safety profile.2.Experimental study:In the in vivo part of the study,the drug intervention was started after the successful establishment of the tumor-bearing mouse model.No significant differences were observed in the general condition of the mice in each group during the intervention period.Concerning tumor volume,the combined and pemetrexed groups exhibited significantly smaller tumor volumes than the model group starting from day 6.The Xiaoliu Pingyi recipe group exhibited a significant difference from day 9.With the prolongation of drug intervention,the tumor suppression effect of each drug group gradually became more apparent.In terms of tumor inhibition rate,the tumor inhibition rate of pemetrexed group was 65.47%,the tumor inhibition rate of Xiaoliu Pingyi recipe group was 59.36%,and the highest tumor inhibition rate of 86.02%was achieved in the combination group.In terms of vascular structure of tumor tissues,immunohistochemical staining results showed that there was a significant decrease in microvessel density in all intervention groups,while the pericyte coverage was significantly increased in all intervention groups.Concerning vascular function,the tumor tissues of mice in the model group exhibited significantly higher FITC-Dextran leakage than those in the pemetrexed,Xiaoliu Pingyi recipe,and combination groups.Moreover,the FITC-Dextran leakage in the combination group was significantly lower than that in the pemetrexed group.According to the q RT-PCR results,the pemetrexed,Xiaoliu Pingyi recipe,and combination groups exhibited significant downregulation of HIF-1α,VEGFA,and Ang-2 m RNA expression levels.However,there were no significant differences in PI3K and Akt m RNA expression levels.The Western Blot results indicated significant downregulation of HIF-1α,VEGFA,and Ang-2 protein expression levels in each intervention group.Though total protein expression levels of PI3K and Akt were not significantly different,p-PI3K and p-Akt expression levels were significantly downregulated.In the in vitro part of the experiment,after the successful collection of the drug-containing serum of the Xiaoliu Pingyi recipe.Initially,the CCK-8 method was employed to compare the effects of different concentrations of blank serum with the corresponding drug-containing serum on human normal lung epithelial cells BEAS-2B.The results of the study showed no significant effect on the cell proliferation rate at 2.5%,5%and 10%serum concentrations（P>0.05）,but a decrease in cell proliferation rate at 20%serum concentration（P<0.05）.The results indicated that the inhibitory effect of Xiaoliu Pingyi recipe on lung cancer A549 cell proliferation gradually increased with increasing drug concentration and intervention duration,implying a time-concentration dependence of the drug’s inhibitory effect.However,at the same intervention duration,the relative cell proliferation rate did not significantly decrease when the concentration of the drug-containing serum was greater than 10%.The Xiaoliu Pingyi recipe serum was more sensitive to the inhibitory effect on A549 cells than BEAS-2B cells,therefore,combining the results of the serum-containing studies in both types of cells,10%was chosen as the optimal concentration of Xiaoliu Pingyi recipe containing serum for subsequent experiments.The results of the cell scratch and Transwell assays indicated that the pemetrexed,drug-containing serum,and combination groups demonstrated a significant decrease in cell migration and the number of cells penetrating the membrane,as compared to the control group（P<0.05）.Moreover,the combination group exhibited a more significant reduction in cell migration and the number of penetrating cells as compared to the pemetrexed group（P<0.05）.The ELISA results showed a statistically significant decrease in VEGF expression in the cell supernatant of the pemetrexed group,Xiaoliu Pingyi recipe containing serum group,and the combined group（P<0.05）.The q RT-PCR results demonstrated significant downregulation of the expression levels of HIF-1α,VEGFA,and Ang-2 in the pemetrexed,drug-containing serum,and combination groups（P<0.05）,and the differences were statistically significant compared with the control group,but the expression levels of PI3K and Akt m RNA did not show significant differences.The results of Western Blot showed that the expression levels of HIF-1α,VEGFA and Ang-2 were significantly downregulated in the pemetrexed,drug-containing serum and combination groups,and the difference was statistically significant compared with the control group（P<0.05）,which was generally consistent with the findings of in vivo experiments.Conclusion:1.Xiaoliu Pingyi recipe in combination with pemetrexed maintenance therapy has shown significant advantages over pemetrexed chemotherapy alone in terms of improving clinical symptoms and quality of life,reducing bone marrow suppression and gastrointestinal reactions,and has a good safety profile.2.Xiaoliu Pingyi recipe demonstrated a significant inhibition of the growth of Lewis lung cancer transplants in C57BL/6J mice.It also improved the structure and function of the microvascular system,thereby interfering with tumor angiogenesis and inducing normalization of tumor vessels.In addition,it inhibited the proliferation,migration,and invasion of lung cancer A549 cells and reduced the concentration of VEGF in the cell supernatant.The regulatory mechanism of this effect may be related to the inhibition of PI3K/Akt protein phosphorylation and down-regulation of the expression of HIF-1α,VEGF,and Ang-2 angiogenesis-related factors. |
