| Objective: Lung cancer is one of the most common malignant tumors with the highest morbidity and mortality in the world.In addition to the limited treatment and poor prognosis of advanced lung cancer,the recurrence and metastasis of early lung cancer after surgery is also an urgent medical problem to be solved.Metabolic reprogramming is one of the important characteristics of tumor and its microenvironment.Abnormal lipid metabolism affects the survival of tumor cells.Traditional Chinese Medicine plays an important role in preventing recurrence and metastasis of early lung cancer after surgery,and can correct metabolic abnormalities in patients with lung cancer.In this study,based on the serum metabolomics analysis of patients with early clinical lung cancer after surgery,the lung cancer treatment mechanism of Fu-Zheng-Qu-Xie Decoction through regulation of lipid metabolism is discussed.Methods:Part I Clinical Study1.This part adopts dual-center,retrospective,cohort research methods.Enrolled from January 2010 to December 2015,761 patients were enrolled in Shanghai Hospital of Traditional Chinese Medicine as the treatment group,and 373 patients enrolled in Shanghai Pulmonary Hospital as the control group.Treatment group: Take Fuzheng Quxie Decoction daily for at least 6 months or more,and regular outpatient follow-up.Control group: No treatment(including postoperative adjuvant treatment,Chinese herbal medicine and Chinese patent medicine,etc.),regular outpatient follow-up.With PFS as the end point,survival analysis and Cox regression analysis were performed.Then,the correlation analysis between the blood lipid level before treatment and the PFS of early lung cancer patients after surgery was carried out to explore the relationship between the improvement of the survival and prognosis of lung cancer patients and the change of blood lipids.2.This part adopts a two-center,prospective,cohort research method.Enrolled from January 2017 to April 2021,281 patients were enrolled in Shanghai Hospital of Traditional Chinese Medicine as the treatment group,and 287 patients enrolled in Shanghai Pulmonary Hospital as the control group.Treatment group: Take Fuzheng Quxie Decoction daily for at least 6 months or more,and regular outpatient follow-up.Control group: No treatment(including postoperative adjuvant treatment,Chinese herbal medicine and Chinese patent medicine,etc.),regular outpatient follow-up.Using DFS as the primary endpoint,the 1-year,2-year,3-year,and 4-year disease-free survival rates of the two groups of patients were observed;the changes in blood lipids and KPS scores of the two groups of patients were used as secondary endpoints,before and after treatment and between groups Comparison to further verify the clinical efficacy of Fuzheng Quxie Decoction in preventing the recurrence and metastasis of patients with early lung adenocarcinoma after surgery.3.A collection of 15 patients with stage Ia lung adenocarcinoma after radical resection in the Department of Oncology,Shanghai Hospital of Traditional Chinese Medicine from January 2019 to December 2019,started to take Fu-Zheng-Qu-Xie Decoction within 3 months after the operation,and took Chinese medicine continuously for more than half a year,serum was collected from each patient before and after taking the medicine for 3 months.Randomly recruit 10 healthy people to ensure that the age stratification of the two groups is not statistically significant,and serum is collected.Liquid chromatography mass spectrometry(LC-MS)was used to detect the serum of patients with early lung cancer before and 3 months after oral administration of the Traditional Chinese compound medicine Fu-Zheng-Qu-Xie Decoction,and the serum of healthy people was used as the control group to screen the different metabolites between patients with early lung cancer after operation and healthy people before and after oral administration of Fu-Zheng-Qu-Xie Decoction.Part II Experimental Research1.Western Blot was used to test the differential protein of lung cancer para-carcinoma tissues and cancer tissues,lung carcinoma cell as well as bronchial epithelial cells collected clinically,and it was found that the SREBP(Sterol Regulatory Element-binding Proteins)were highly expressed in lung cancer tissues compared with that in para-carcinoma tissues and in the meantime,it is highly expressed in lung cancer cell line.SREBP gene knockdown was performed on lung cancer cells A549 and H1299 using toxic infection technology,and the knockdown effect was verified by Western Blot,q PCR and Immunofluorescence to establish stable cell strains of A549-Sh-SREBP and H1299-Sh-SREBP.The proliferation,metastasis and invasion of lung cancer cells after knockdown SREBP were observed by plate cloning,soft agar and scratch,and Transwell validation respectively.CCK8 method was used to determine the IC50 concentration of Fu-Zheng-Qu-Xie Decoction freeze-dried powder on A549 cells and H1299 cells so as to verify the effect of Fu-Zheng-Qu-Xie Decoction on SREBP in A549 and H1299 cells.2.Using C57 mice subcutaneous transplantation tumor model,supporting with Fu-Zheng-Qu-Xie Decoction and SREBP inhibitors Fastostatin HBr respectively to get intervention in mice,we observed the effects on the tumor and the effects on a tumor-burdened mice immune microenvironment;using flow cytometry to detect the ratio effects on M2 type of tumor-associated macrophages(TAMs),T-cell,natural killer cells(NK),myeloid-derived suppressor cells(MDSCs)both in the spleen and tumors in mice;the contents of fatty acids,triglycerides and cholesterol in the serum of mice were determined by colorimetric method of Microplate Reader,and the relationship among Chinese compound medicine,lipid metabolism and tumor immune microenvironment was speculated.3.A549,H1299,A549-Sh-SREBP and H1299-Sh-SREBP were co-cultured with M0-TAM induced by PMA for 24 hours respectively to observe the direction of THP-1 differentiation.Compared with THP-1 tumor-associated macrophages induced by PMA and IL-4,Western Blot was used to detect the changes of cell pathway proteins after co-combination.Results:Part I Clinical Study1.Retrospective clinical studies have found that Fuzheng Quxie Decoction can prolong the progression-free survival of lung cancer patients and the overall survival of early lung cancer patients,and is an independent protective factor affecting the survival and prognosis of lung cancer patients.After propensity matching scores,Fuzheng Quxie Fang has a more significant impact on the progression-free survival of patients with lung cancer and the overall survival of patients with early lung cancer.Univariate analysis showed that gender(P<0.001),age(P<0.001),smoking history(P<0.001),family history(P<0.001),surgery(P=0.031),chemotherapy(P<0.001),traditional Chinese medicine Intervention(P<0.001)is a related factor affecting PFS in patients with lung cancer,gender(P=0.001),age(P=0.001),smoking history(P<0.001),family history(P=0.014),Chinese medicine intervention(P= 0.004)are related factors affecting the OS of lung cancer patients;multivariate analysis shows that young age(P<0.001)and traditional Chinese medicine(P<0.001)are independent protective factors affecting lung cancer PFS,and smoking(P=0.006)is an independent factor affecting lung cancer PFS Risk factors.Young age(P=0.001)and traditional Chinese medicine(P=0.04)are independent protective factors affecting lung cancer OS,and smoking(P=0.009)is an independent risk factor affecting lung cancer OS.By analyzing the correlation between blood lipids and the prognosis of lung cancer patients with progression-free survival,it was found that lung cancer patients with high serum cholesterol and high triglycerides have longer progression-free survival.2.A prospective clinical study found that the disease-free survival rates of the control group at 1,2,3,and 4 years were 97.5%,92.4%,90.1%,and 87.1%,respectively.The 4-year disease-free survival rates were 99.2%,97%,95.6%,and95.6% respectively,that is,Fuzheng Quxie Decoction can improve the disease-free survival of patients after radical resection of early lung adenocarcinoma,and improve the survival of patients after radical resection of early lung adenocarcinoma.The1-year,2-year,3-year,4-year disease-free survival rate of patients can effectively prevent the recurrence and metastasis of early lung cancer after surgery.At the same time,Fuzheng Quxie Decoction can also adjust the blood lipid level of patients after early lung cancer surgery.3.Statistics of blood lipids between patients with stage Ia lung adenocarcinoma and healthy people after surgery found that the cholesterol and triglycerides of patients with early lung cancer after surgery were higher than those of healthy people,and there were statistical differences,indicating that most patients with lung cancer have abnormal lipid metabolism;Statistics of blood lipids in patients with stage Ia lung adenocarcinoma after oral administration of Fu-Zheng-Qu-Xie Decoction found that after oral Fu-Zheng-Qu-Xie Decoction in patients with early lung cancer surgery,cholesterol and triglycerides decreased,and there was a statistical difference,indicating that Fu-Zheng-Qu-Xie Decoction not only plays an anti-tumor effect,and can also restore dyslipidemia in patients with lung cancer.Based on metabonomics preliminary screening,it was concluded that lipid metabolites were one of the most importantly different metabolites between patients after early lung cancer surgery and healthy people,as well as those who get and before and after oral Fu-Zheng-Qu-Xie Decoction after early lung cancer surgery.Among them,there are differences in lipid metabolites between healthy people and patients after early lung cancer surgery,mainly of them were pre-putrebactin,Lyso PC(18:1(11Z)),Lyso PC(18:0),1-heptadecanoyl-sn-glycero-3-phosphocholine and other metabolites;after the treatment of early lung cancer patients with the Traditional Chinese Medicine Fu-Zheng-Qu-Xie Decoction,their lipid metabolites changed,mainly were Lyso PC(18:1(11Z)),6’’,6’’-Dimethylpyraono[ 2``,3’’:7,8]kaempferol 4’-methyl ether3-rhamnoside,Thysanone,Patuletin and other metabolites.Part II Experimental Research1.After the SREBP gene was knocked out,functional experiment showed that the proliferation,migration and invasion ability of lung cancer cells A549 and H1299 were significantly reduced.CCK8 method was used to determine the IC50 concentration of Fu-Zheng-Qu-Xie Decoction freeze-dried power on A549 cells and H1299 cells at 8.02mg/m L and 10.85mg/ml,respectively.The further experiments showed that Fu-Zheng-Qu-Xie Decoction could reduce the SREBP expression in lung cancer cells and was concentration dependent.2.After Fu-Zheng-Qu-Xie Decoction and Fastostatin HBr exerting effects on tumor-bearing C57 mice,the tumor volume and weight showed that both of them had anti-tumor effects,but their effects would not superpose to each other.Flow cytometry results showed that the number of tumor-associated macrophage in the spleen and tumor of mice in the TCM group was lower than that in the inhibitor group,and the Fu-Zheng-Qu-Xie Decoction could reduce the content of fatty acids in the serum of mice.3.A549,H1299,A549-Sh-SREBP and H1299-Sh-SREBP were co-cultured with THP-1 cells induced by PMA for 24 hours.Flow cytometers showed that the knock-down of lung cancer cells of SREBP could reduce the differentiation of M0 macrophages into M2-type TAMs,and lower the pathway proteins PPAR γ and STAT6 activated protein.Conclusion:Clinical Research1.A retrospective study found that Fuzheng Quxie Decoction can prolong the progression-free survival of lung cancer patients and the overall survival of early lung cancer patients.After propensity matching scores,Fuzheng Quxie Decoction has a more significant clinical effect in improving the progression-free survival of lung cancer patients and the overall survival of early lung cancer patients;Cox regression analysis shows that young age and traditional Chinese medicine are independent protective factors affecting lung cancer PFS;young age,Traditional Chinese medicine is an independent protective factor affecting the OS of lung cancer;Fuzheng Quxie Decoction can effectively prevent the recurrence and metastasis of lung cancer patients,and improve the survival prognosis of lung cancer patients;through the correlation analysis between the blood lipids before treatment and the PFS prognosis of I lung cancer patients,it is found that the high TC group Of lung cancer patients have longer PFS.2.Prospective studies have shown that Fuzheng Quxie Decoction can prolong the disease-free survival of patients after radical resection of early lung adenocarcinoma,and increase the disease-free survival rate of patients with early radical resection of lung adenocarcinoma at 1,2,3,and 4 years.Effectively prevent recurrence and metastasis after radical resection of early lung adenocarcinoma.Fuzheng Quxie Decoction can adjust the blood lipid level of patients with early lung adenocarcinoma after surgery.We speculate that Fuzheng Quxie Decoction may prevent recurrence and metastasis after radical resection of early lung adenocarcinoma by regulating the blood lipid level of patients.3.Lipid metabolites were one of the most importantly different metabolites between patients after early lung cancer surgery and healthy people,as well as those who get oral Fu-Zheng-Qu-Xie Decoction after early lung cancer surgery;Fu-Zheng-Qu-Xie Decoction can regulate the lipid metabolism of patients with early lung cancer after surgery。Experimental Study1.SREBP gene is not only a key gene of lipid metabolism,but also a key gene that affects the malignant behavior of lung cancer cells,and can be used as an oncogene and therapeutic target for lung cancer.2.Fu-Zheng-Qu-Xie Decoction,via suppressing SREBP,can regulate lipid metabolism,thus inhibiting the invasion and metastasis of lung cancer cells.3.Fu-Zheng-Qu-Xie Decoction can affect the proportion of TAMs in the immune microenvironment of lung cancer by regulating lipid metabolism,remodeling tumor immune microenvironment so as to achieve the anti-lung cancer effect. |
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