The Association Between Fiber Intake,gut Microbiota,Short Chain Fatty Acid And Therapeutic Effect Of Lung Cancer And The Mechanism

Objective:1 A large number of studies have shown that there is a certain correlation between prediagnostic dietary intakes and the risk of lung cancer,but there are few reports on the correlation between dietary factors and lung cancer survival.Therefore,we analyzed the association between pre-diagnostic dietary factors（including food group and nutrition）and lung cancer survival.2 Previous studies have shown that the amount of fiber intake have influence on the distribution of human intestinal microbiota and the concentration of serum SCFA（Short Chain Fatty Acid）.We deeply analyzed the relationship between pre-diagnostic dietary fiber intake,gut microbiota distribution and the concentration of serum SCFA in two groups of patients with different therapeutic effects.3 Recently,some studies have shown that SCFA can inhibit the proliferation,migration and invasion of malignant cells,including lung cancer,but there is no evidence indicated the influence of isobutyric acid on the biological characteristics of lung cancer cells.Therefore,we studied the influence of isobutyric acid on the malignant biological behavior of lung cancer cells and its molecular mechanism.Methods:1 The participants of this study aged 30-75 years with lung cancer who were admitted and treated in the oncology center of Shengjing Hospital of China Medical University from 2019 to 2021.Baseline information on dietary intake over the past year before lung cancer diagnosed and other information were collected using a questionnaire with good reliability and validity that was collected by a trained interviewer.In this study,variance analysis and chi-square test were used to compare the differences in baseline characteristics among lung cancer patients with different dietary fiber intake.Using Cox proportional risk regression model respectively explore the food groups or nutrient intake and lung cancer survival.Subgroup analysis and interaction analysis were performed according to age at diagnosis,BMI,histological type,and smoking status.2 Patients were divided into responders and non-responders according to whether there was disease progression event or death within the first 6 months after treating with anti-tumor drugs.16 S r DNA sequencing was used to analyze the differences of gut microbiota between the two groups.The concentration differences of serum SCFA between the two groups was analyzed by gas chromatography-mass spectrometry.Through Spearman correlation analysis,the correlation between different gut microbiota and serum SCFA was analyzed.3 The proliferation of lung cancer cells treated with different concentrations of isobutyric acid was detected by CCK8 and plate clone formation assay.Transwell experiment was used to analyze the difference of migration and invasion ability in lung cancer cells between isobutyric acid treated group and control group.The differences of apoptosis rates and cell cycle distribution between isobutyric acid treated group and control groups were analyzed by flow cytometry.The difference of GPCR（G Protein-Coupled Receptor）m RNA levels between isobutyric acid treated group and control group was analyzed by real-time PCR.Western blot was used to quantitatively analyze the differences of GPR41,GPR43,GPRC5 A,PAR1,Acetyl-Histone H3 and H4 expression in lung cancer cells between isobutyric acid treatment group and the control groups.The expression differences of GPR41,GPR43,GPRC5 A,PAR1,Acetyl-Histone H3 and H4 in lung cancer cells between isobutyric acid treated group and the control group were further analyzed by immunocytochemistry.We also analyzed the expression location of GPCR and acetyl histones with immunofluorescence.Results:1 The association between food group and nutrition and lung cancer survival1.1 Baseline characteristics of patientsA total of 389 lung cancer patients were involved in this study.Among them 119 patients died during follow-up.According to the tertile of dietary fiber intake,compared with the lowest tertile,participants with higher dietary fiber intake included more men and were more likely to have higher educational level,higher proportion of smoking,higher proportion of drinking tea,less physical activity and higher total energy intake（all p < 0.05）.1.2 The association between food group and lung cancer survivalAfter adjusting for multiple model factors,the relationship between the intake of 12 food groups and the survival of lung cancer patients was analyzed.Finally,we observed that compared with patients in the lowest tertile,the overall survival（OS）of patients with the highest total intake of fruits was better,and the HR（Hazard Ratio）was 0.60（95% CI（Confidence Interval）: 0.38-0.94,p trend < 0.05）,and the result was statistically significant.1.3 The association between nutrition and lung cancer survivalAfter adjusting for multiple model factors,the relationship between the intake of 21 nutrients and the survival of patients with lung cancer was analyzed.Finally,we observed that patients in the highest tertile of insoluble dietary fiber intake had better survival than patients in the lowest tertile,HR = 0.60（95% CI = 0.38-0.96,p trend < 0.05）.Similarly,we also observed that patients in the highest tertile of carotene intake had better survival than those in the lowest tertile,HR = 0.57（95% CI = 0.36-0.91,p trend < 0.05）.2 The association between gut microbiota,serum SCFA and therapeutic effects of lung cancer2.1 Baseline characteristics and pre-diagnostic dietary fiber intake in the two groupsCompared the responders with the non-responders,the average intake of total dietary fiber（p < 0.01）,soluble dietary fiber（p < 0.01）and insoluble dietary fiber（p < 0.01）in the former were higher than those in the latter.In 60 patients whose stool and serum samples were detected,no significant difference was observed between the responders（n = 30）and the non-responders（n = 30）in age（p = 0.684）,gender（p = 0.077）,disease stage（p = 0.377）,smoking history（p = 0.067）,pathological type（p = 0.814）and ECOG score（p = 0.351）.2.2 Difference of gut microbiota distribution in two groups2.2.1 Difference of gut microbiota diversity between the two groupsThe α-diversity of gut microbiota was higher in non-responders,while the β-diversity was higher in responders.2.2.2 Difference of gut microbiota composition between the two groupsAt the phylum level,Proteobacteria is the most abundant bacterial phyla,followed by bacteroidota,Firmicutes and Actinobacteria.Further analysis showed that there were significant differences in 11 bacterial phyla between the two groups（p < 0.01）.In addition,at the genus level,the gut microbiota with high relative abundance are Ralstonia,Bacteroides,Prevotella₉,Pseudomonas,Ruminococcus,Dialister and Parabacteroides.The results showed that there were differences in 736 genera between the two groups.Among them,there were significant differences in 35 genera（p < 0.01）.2.3 Difference of SCFA concentration between the two groupsFrom the log2 abundant of SCFA,we observed that compared with responders,the levels of seven kinds of SCFA in non-responders were decreased.Among them,the concentrations of acetic acid（p < 0.01）and isobutyric acid（p < 0.01）in non-responders decreased significantly,and the results were statistically significant.2.4 Correlation analysis between different gut microbiota and SCFA concentration between the two groupsThe results of Spearman’s correlation analysis showed that there was a positive correlation between Deferribacteres and acetic acid.However,we also observed that Spirochaetota,unidentified_Archaea,Gemmatimonadota,Enterobacteriaceae,WPS-2 and Armatimonadota were negatively correlated with butyric acid.Similarly,negative correlations were also observed between Fibrobacterota,unidentified_Archaea and WPS-2,and valeric acid.3 Isobutyric acid inhibited the biological activity of lung cancer cells and the molecular mechanism3.1 Isobutyric acid inhibited the proliferation,migration and invasion of lung cancer cellsAfter treating A549 cell line with different concentrations of isobutyric acid for 48 hours,the proliferation of A549 cells decreased,indicating that isobutyric acid can inhibit the proliferation of lung cancer cells,and the stronger the inhibition of cell proliferation with the increase of concentration.The migration and invasion ability of A549 cells treated with isobutyric acid significantly decreased.3.2 Isobutyric acid promotes cell apoptosis and induces cell cycle arrestCompared with the control group,the number of early and late apoptosis of A549 cells treated with isobutyric acid significantly increased.After treated with isobutyric acid,the number of cells in G1 phase increase,while in S phase decreased,indicating G1/S phase arrest.3.3 Isobutyric acid regulates the expression of GPCR and the level of Acetyl-HistoneCompared with the control group,the results of PCR showed that the m RNA levels of GPR41,GPR43 and GPRC5 A increased significantly,while PAR1 decreased in the isobutyric acid treatment group.The results of western blot showed that the expression of GPR41,GPR43,GPRC5 A and Acetyl-Histone H3 and H4 increased significantly in isobutyric acid treated group,while PAR1 decreased.The results of immunocytochemistry showed that the expression of GPR41,GPR43,GPRC5 A and the levels of Acetyl-Histones H3 and H4 increased significantly in isobutyric acid treated group,while PAR1 decreased.Conclusion:1 We found that higher total intake of fruits was correlation with a better OS of patients diagnosed as lung cancer.Moreover,the increase of pre-diagnostic insoluble dietary fiber and carotene intake was positively correlated with the lung cancer survival.2 Compared responders with non-responders,the total dietary fiber intake,soluble dietary fiber intake and insoluble dietary fiber intake of the former were higher than those of the latter.Further analysis found that the α-diversity of gut microbiota in non-responders was higher than in responders.The serum SCFA concentration in the responders were higher than those in the non-responders.The composition of gut microbiota in the two groups was different,and there was a certain correlation between different gut microbiota and SCFA of the two groups.3 Isobutyric acid can inhibit the proliferation,migration and invasion of lung cancer cells.At molecular level,isobutyric acid may inhibit the biological activity of lung cancer cells by regulating the expression of GPCR and acetylated histone level.