| BackgroundThoracic tumors,such as lung cancer,breast cancer and esophageal cancer,are common malignant tumors in clinical practice.RT is very important to control tumor development and metastasis.RILI is one of the common side effects after radiotherapy for thoracic tumors,with the main symptoms including chest pain,cough,fever,fatigue,etc.With the increase of radiotherapy dose and frequency,RILI may progress from RP to RIPF,which is a major limiting factor for the application of RT,which is not good for the prognosis and quality of life of tumor patients.Traditional Chinese medicine believes that radiation belongs to the "fiery poison",and the lung is delicate,fiery poison attack is easy to damage lung Yin,with the prolongation of the course of disease can cause "lung asthenia",lung Yin long-term damage leads to the quality of life of patients decline,serious cases can lead to death of patients.At present,the main treatment means of modern medicine for RILI are hormone,antibiotic,oxygen and other supportive treatment,but because of the long course of RILI disease,long-term use of hormones can lead to a variety of side effects such as immune dysfunction.There is an urgent need to seek effective treatment for RILI.In order to inherit the experience of famous and veteran traditional Chinese medicine,our research group has long been committed to exploring the clinical efficacy of the effective treatment of RILI,"nourishing Yin and clearing lung and promoting blood circulation " method.It is found that this method has a significant clinical effect on the prevention and treatment of RILI,but there is still a lack of reliable mechanism research,which limits its further application.Based on this,this study first used UPLC-MS/MS technology to explore the overall m6A methylation level of YYQF in RILI model animals guided by "nourishing Yin and clearing lung",and combined with transcriptional sequencing technology(RNA-seq)to explore the differentially expressed genes of YYQF in RILI model animals.GO and KEGG pathways were enriched to explore their related pathways.Secondly,the therapeutic effect of YYQF on RILI was verified through in vivo experiments,and the influence of Mettl3 on Wnt/β-catenin pathway was explored.Finally,in vitro experiments were conducted to further verify the relevant mechanism of YYQF treatment for RILI based on Mettl3 mediated Wnt/β-catenin pathway.This study provides a new theoretical basis for exploring the pathogenic mechanism of RILI and the therapeutic mechanism of Chinese medicine "nourishing Yin and clearing lung and promoting blood circulation" on RILI,which is of great significance for the research and development of new drugs and the clinical promotion of traditional Chinese medicine.ObjectiveEarly treatment of RILI can clearly improve the prognosis and provide a guarantee for the application of RT.By studying the specific protective mechanism of YYQF on RILI model animals and cells,this study further clarified the pathway of action and the optimal administration time of traditional Chinese medicine in the treatment of RILI,and provided theoretical basis for the clinical application of YYQF.Methods1 The possible mechanism of YYQF treatment on RILI model animals was explored based on the combined analysis of UPLC-MS/MS technology and transcriptional sequencing technology1.1 Study on the overall m6A methylation level of lung tissue treated by YYQF in RILI model animalsThe 6-week-old male SD rats with a body weight of 200±20g were randomly divided into 3 groups:blank group(K),model group(M)and traditional Chinese medicine group(Z).Blank group,model group and traditional Chinese medicine were exposed to 16Gy radiation.After 24h of irradiation,lung tissues were taken by gavage for 8 weeks and frozen at-80℃.RNA was extracted and quantified,and after purification,an appropriate amount of mRNA was used for mass spectrometry.Multiple response monitoring(MRM)mode was used for UPLC-MS/MS analysis.Samples were tested by UPLC-MS/MS to calculate m6A levels(percentage of m6A/A).1.2 Analysis of differential gene expression in RILI model rats treated by YYQF based on transcriptional sequencingThe 6-week-old male SD rats with a body weight of 200±20g were randomly divided into 3 groups:blank group(K),model group(M)and traditional Chinese medicine group(ZY).Blank group,model group and traditional Chinese medicine were exposed to 16Gy radiation.After 24h of irradiation,lung tissues were taken by gavage for 8 weeks and frozen at-80℃.Total RNA was extracted and identified for purity and quantification.After assessing RNA integrity,a transcriptome Library was constructed using the VAHTS Universal V6 RNA-Seq Library Prep kit.Illumina Novaseq 6000 sequencing platform was used for sequencing,HISAT2 software was used for reference genome comparison,DESeq2 software was used for differential expression gene analysis,and hypergeometric distribution algorithm was used for GO and KEGG Pathway enrichment analysis of differential expression genes.It is used for screening and analysis of enrichment function items.2 The therapeutic effect and possible mechanism of YYQF on RILI mice were verified by in vivo experiments2.1 Establishment of an animal model of C57BL/6J mouse RILIC57BL/6J male mice aged 6 weeks with SPF grade and body weight of 20±2g were randomly divided into 4 groups:blank group,12Gy group,16Gy group and 20Gy group.The samples were taken 4 weeks after radiotherapy.Body weight,lung and spleen weights were recorded,lung and spleen coefficients were calculated,lung tissues of mice in each group were stained with HE,inflammatory response was observed,and the optimal radiotherapy dose of RILI mouse model was explored.2.2 Study on the optimal dose of YYQF for RILI miceC57BL/6J male mice aged 6 weeks with SPF grade and body weight of 20±2g were randomly divided into 5 groups:blank group(K),model group(M),low-dose group(D),medium-dose group(Z)and high-dose group(G).The blank group was exposed to fake radiation,and the model group and the low-dose,medium-dose and high-dose groups were exposed to radiation of 20Gy.After 24h of irradiation,intragastric administration was performed,and samples were collected 4 weeks later.Body weight,lung and spleen weights were recorded,lung and spleen coefficients were calculated,and serum levels of IL-17,IL-12,IL-10 and IFN-y of RILI mice were detected by ELISA.2.3 Effect and mechanism of YYQF on RILI mice6-week-old C57BL/6J male mice with SPF grade and body weight of 20±2g were randomly divided into 3 groups:blank group(K),model group(M)and traditional Chinese medicine group(Z).Blank group was exposed to fake radiation,model group and traditional Chinese medicine group were exposed to radiation 20Gy.After 24h of irradiation,6 specimens were taken by gavage at 4 weeks and 8 weeks.The body weight,lung and spleen weights were recorded,and the lung and spleen coefficients were calculated.The lung inflammation was evaluated by HE staining at the 4th and 8th week,and the degree of pulmonary fibrosis was evaluated by Masson staining and αSMA and Vimentin immunohistochemistry at the 4th and 8th week.Serum levels of IL17,IL-12,IL-10 and IFN-y of RILI mice were detected by ELISA,and the expressions of Mettl3,Wnt9b,β-Catenin,P-β-Catenin and α-SMA in lung tissue of mice were detected by Western Blot.mRNA levels of Mettl3 and Wnt9b in mouse lung tissue were detected by RT-PCR.3 The effect of Mett13 on the mechanism related to YYQF treatment on RILI mice was studied in vitro3.1 Study on the optimal dosage of YYQF on RILI cells in vitroSPF 6-week-old male SD rats weighing 200±20g were randomly divided into blank group and medication group,with 20 rats in each group.The blank group was given normal saline,and the medication group was given YYQF suspension.3 days after intragastric administration,blank serum and drug-containing serum were taken to intervene with mouse lung epithelial cell line MLE-12,respectively.CCK-8 method was used to explore the optimal dosage of blank serum and YYQF drug-containing serum for in vitro intervention.3.2 YYQF interfered with Mettl3 and Wnt/β-catenin pathway related proteins and mRNA expression in MLE-12 cells in vitroMLE-12 cells cultured to 70%in good condition were digested by pancreatic enzymes and counted.The cells were divided into blank group(K),model group(M)and traditional Chinese medicine group(Z),with 3 multiple Wells in each group,according to 4×105 cells/well.The blank group was given false irradiation,the model group and the Chinese medicine group were given 10Gy irradiation,the blank rat serum was used to intervene the blank group and the model group 24h after radiotherapy,and the medicated rat serum was used to intervene the Chinese medicine group 24h later cells and slides were collected.The content and distribution of β-Catenin in MLE-12 cells were quantitatively analyzed by immunofluorescence.The protein expressions of Mettl3,Wnt9b,β-catenin and p-β-catenin in MLE-12 cells of each group were detected by WB,and the mRNA relative expressions of Mettl3 and Wnt9b in MLE-12 cells of each group were detected by RT-PCR.3.3 The effect of Mettl3 plasmid transfection on Wnt/β-catenin pathway was investigated in vitroMLE-12 cells were transfected with overexpressed Mettl3 plasmid,and cell culture medium was changed 4-6h after transfection,and MLE-12 cells and slides were collected after 24h culture.The protein expressions of Mettl3,Wnt9b,β-catenin and pβ-catenin in MLE-12 cells of each group were detected by WB,and the mRNA relative expressions of Mettl3 and Wnt9b in MLE-12 cells of each group were detected by RTPCR.Results1 The possible mechanism of YYQF treatment on RILI model animals was explored based on the combined analysis of UPLC-MS/MS technology and transcriptional sequencing technology1.1 Study on the overall m6A methylation level of lung tissue treated by YYQF in RILI model animalsCompared with the blank group,the overall m6A methylation level of lung tissue of rats in RILI model group was relatively decreased,while the overall m6A methylation level of lung tissue of rats in medication group was relatively increased compared with the model group,but the difference was not statistically significant.1.2 Analysis of differential gene expression in RILI model rats treated by YYQF based on transcriptional sequencingAccording to the selection criteria for M v K hike or cut,ZY vs M |log2FC |≥1.2 and P values<0.05.Venn diagram was used to analyze the intersection of differentially expressed genes obtained by transcriptome sequencing.The results showed that when K,M and ZY groups were compared,there were 1703 genes upregulated by M vs K,410 genes down-regulated by ZY vs M,126 genes up-regulated by M vs K and down-regulated by ZY vs M.Through GO enrichment analysis,a total of 568 items were significantly enriched,and 3 significantly enriched clusters were formed through clustering,including 437 biological processes,86 molecular functions,and 45 cell components.KEGG enrichment analysis showed that the differential genes were enriched to 167 pathways.The top 10 pathways involved in environmental information processing categories include cell adhesion molecules,calcium signaling pathway,Apelin signaling pathway,TNF signaling pathway,Wnt signaling pathway,viral protein interaction with cytokines and cytokine receptors,ECM receptor interaction,TGF-β signaling pathway,and NF-kappa B signal path,HIF-1 signal path.2 The therapeutic effect and possible mechanism of YYQF on RILI mice were verified by in vivo experiments2.1 Establishment of an animal model of C57 mouse RILIAfter 4 weeks of radiation irradiation on the chest of mice,compared with the blank group,the mice in the 20Gy group had decreased appetite,lost hair,and significantly decreased body weight(P<0.05),and the lung and spleen coefficients were significantly increased(P<0.05).HE staining showed that the inflammatory cells in the lung tissue were clustered significantly.In subsequent experiments,20Gy can be used as the modeling dose of RILI to establish RILI animal model.2.2 Study on the optimal dose of YYQF for RILI mice2.2.1 At week 0,there was no statistical difference in body weight between model group and blank group(P>0.05),and the baseline was consistent.From week 2 onwards,the weight increase of the model group was less than that of the blank group(P<0.01).There was no significant difference in body weight increase between experimental groups and model group(P>0.05).2.2.2 At the 4th week,compared with the blank group,the lung coefficient of mice in the model group was significantly increased(P<0.001).Compared with the model group,the lung coefficient of mice at low and medium doses of Chinese medicine was not significantly different(P>0.05),while the lung coefficient at high doses of Chinese medicine was significantly decreased(P<0.05).In spleen coefficient,at the 4th week,compared with blank group,spleen coefficient of mice increased significantly(P<0.05);compared with model group,lung coefficient of mice at low dose of Chinese medicine had no significant difference(P>0.05),while lung coefficient at medium and high dose of Chinese medicine significantly decreased(P<0.05).2.2.3 The results showed that at the 4th week,compared with the blank group,the serum levels of IL-17,IL-10 and IL-12 in the model group were significantly increased(P<0.05),while the serum levels of IFN-y were significantly decreased(P<0.05).Compared with model group,there were no significant differences in serum levels of IL-17,IL-10,IL-12 and IFN-y in low-dose group(P>0.05).The serum IL-17 level was significantly decreased and IFN-γ level was significantly increased in the medium-dose group,with statistical significance(P<0.05),but there were no significant differences in IL-10 and IL-12 levels(P>0.05).The serum levels of IL-17 and IL-12 were significantly decreased(P<0.05),and the serum levels of IFN-y were significantly increased(P<0.01).IL-10 level was decreased without significant difference(P>0.05).2.3 Effect and mechanism of YYQF on RILI mice2.3.1 At week 0,there was no significant difference in body weight between model group,TCM group and blank group(P>0.05).At the 4th week,the weight increase of the model group was less than that of the blank group(P<0.05).There was no significant difference in body weight increase between TCM group and model group(P>0.05).At the 8th week,the weight increase in the model group was less than that in the blank group(P<0.05).The body weight of mice in TCM group was higher than that in model group,but the difference was not statistically significant(P>0.05).2.3.2 In terms of lung coefficient,at the 4th week,compared with the blank group,the lung coefficient of mice in the model group was significantly increased(P<0.001),while compared with the model group,the lung coefficient of mice in the Chinese medicine group was significantly decreased(P<0.05).At the 8th week,there was no significant difference in lung coefficient between model group and blank group(P>0.05),and there was no significant difference in lung coefficient between TCM group and model group(P>0.05).In spleen coefficient,at the 4th week,compared with blank group,spleen coefficient of mice in model group was significantly increased(P<0.01),while spleen coefficient of mice in traditional Chinese medicine group was significantly decreased(P<0.05).At the 8th week,compared with blank group,spleen coefficient of mice in model group was significantly increased(P<0.05),while spleen coefficient of mice in Chinese medicine group was significantly decreased(P<0.05)compared with model group.2.3.3 HE staining results showed that at the 4th and 8th week,compared with the blank group,the lung tissue inflammatory cell infiltration was more significant in the model group,the lung tissue morphology was incomplete,the alveolar wall was more broken,and the alveolar matrix was significantly thickened.Compared with the model group,the Chinese medicine group reduced the infiltration of inflammatory cells in lung tissue,the lung tissue morphology was more complete,and the alveolar matrix thickening degree was relatively reduced.2.3.4 Masson staining showed that at the 4th week,the average optical density of lung tissue in the model group was slightly increased,while that in the Chinese medicine group was slightly decreased(P>0.05).At the 8th week,the deposition of blue collagen fibers in the lung tissue of mice in the model group was significantly increased(P<0.001),while the average optical density in the Chinese medicine group was significantly decreased(P<0.01).Immunohistochemical results showed that at the 4th week,α-SMA protein expression was significantly increased(P<0.05),and vimentin protein expression was slightly increased(P>0.05)in lung tissue of model group.The protein expressions of α-SMA and Vimentin in TCM group were slightly decreased(P>0.05).At the 8th week,the expressions of α-SMA and Vimentin in lung tissue of mice in model group were significantly increased(P<0.05),while the expressions of α-SMA and Vimentin in TCM group were significantly decreased(P<0.05).2.3.5 ELISA results showed that at the 4th week,compared with blank group,serum levels of IL-17,IL-10 and IL-12 in model group were significantly increased(P<0.05),while serum levels of IFN-γ were decreased,but the difference was not statistically significant(P>0.05).Compared with model group,serum IL-17 level of mice in TCM group was significantly decreased,and serum IFN-γ level was significantly increased,with statistical significance(P<0.05),while serum IL-10 and IL-12 levels of mice in TCM group were not significantly different(P>0.05).At the 8th week,compared with blank group,serum levels of IL-17,IL-10,IL-12 and IFN-γ in model group were significantly increased(P<0.05).Compared with model group,serum levels of IL-17,IL-10 and IFN-γ in TCM group were significantly decreased,with statistical significance(P<0.05),while serum levels of IL-12 in TCM group were decreased,with no statistical significance(P>0.05).2.3.5 Western Blot results showed that at the 4th week,compared with blank group,Mettlβ protein expression was significantly decreased in model group(P<0.05),Wnt9b,p-β-catenin and α-SMA protein levels were slightly increased,while β-catenin protein expression was slightly decreased.The difference was not statistically significant(P>0.05).Compared with model group,Mettl3 protein expression in lung tissue of mice in TCM group was significantly increased(P<0.05),Wnt9b,p-β-catenin and α-SMA protein levels were slightly decreased,and β-catenin protein expression was slightly increased,with no statistical significance(p>0.05).At the 8th week,compared with blank group,Mettl3 protein expression in lung tissue of model group was significantly decreased(P<0.05),Wnt9b,β-catenin and α-SMA protein levels were significantly increased(P<0.01),and p-β-catenin protein expression was significantly decreased(P<0.01).Compared with model group,Mettl3 protein expression in lung tissue of mice in TCM group was slightly increased(P>0.05),Wnt9b,β-catenin and α-SMA protein levels were significantly decreased(P<0.05),and p-β-catenin protein expression was significantly increased(p<0.01).2.3.6 RT-PCR results showed that at the 4th week,compared with blank group,Mettl3 mRNA level in lung tissue of mice in model group was significantly decreased(P<0.001),while Wnt9b mRNA level was slightly increased,with no statistical significance(P>0.05).Compared with model group,mRNA level of Mettl3 was significantly increased in TCM group(P<0.05),while mRNA level of Wnt9b was slightly decreased,with no statistical significance(P>0.05).At the 8th week,compared with blank group,Mettl3 mRNA level in lung tissue of mice in model group was slightly decreased,with no statistical significance(P>0.05),while Wnt9b mRNA level was significantly increased(P<0.05).Compared with model group,the mRNA level of Mettl3 in lung tissue of mice in TCM group was slightly increased without statistical significance(P>0.05),while the mRNA level of Wnt9b was significantly decreased(P<0.05).3 The effect of Mett13 on the mechanism related to YYQF treatment on RILI mice was studied in vitro3.1 Study on the optimal dosage of YYQF on RILI cells in vitroAfter 24 days of intervention,compared with Control,the cell viability of K5%,K10%and K15%was significantly increased(P<0.01),while the cell viability of K2.5%was slightly increased(P>0.05).The cell viability of Y 10%was significantly increased(P<0.01),and the cell viability of Y2.5%,Y 5%and Y 15%was slightly increased(P>0.05).After 48h of intervention,compared with Control,K5%,K10%and K15%cell viability was significantly increased(P<0.0001),and K2.5%cell viability was slightly increased(P>0.05).The cell viability of Y 10%and Y 15%was significantly increased(P<0.01),and that of Y2.5%and Y 5%was slightly increased(P>0.05).Therefore,the optimal concentrations were K10%and Y10%,and the intervention time was at least 24h.。3.2 YYQF interfered with Mettl3 and Wnt/β-catenin pathway related proteins and mRNA expression in MLE-12 cells in vitro3.2.1 Quantitative immunofluorescence analysis showed that the β-catenin protein expression was significantly increased in model group compared with blank group 24h after drug intervention(P<0.001).Compared with model group,β-catenin protein expression in MLE-12 cells in Chinese medicine group was significantly decreased(P<0.001).3.2.2 Western Blot results showed that after 24h of intervention,compared with blank group,Mettl3 protein expression in model group was slightly decreased,with no statistical significance(P>0.05),while Wnt9b and β-catenin protein levels were slightly increased,with no statistical significance(P>0.05).The protein expression of P-β-catenin was significantly increased(P<0.001).Compared with model group,Mettl3 protein expression was slightly increased and Wnt9b and β-catenin protein levels were slightly decreased in MLE-12 cells in TCM group,with no statistical significance(P>0.05),while P-β-catenin protein level was significantly decreased(P<0.05).3.2.3 RT-PCR results showed that after 24h of drug intervention,compared with blank group,Mettl3 mRNA level of mouse MLE-12 cells in model group were significantly decreased(P<0.001),and Wnt9b mRNA level was significantly increased(P<0.01).Compared with model group,mRNA level of Mettl3 in MLE-12 cells in TCM group was significantly increased(P<0.05),while mRNA level of Wnt9b was significantly decreased(P<0.01).3.3 The effect of Mettl3 plasmid transfection on Wnt/β-catenin pathway was investigated in vitro3.3.1 Western Blot results showed that after 24h of intervention,compared with NC group,Mettl3 protein expression in MLE-12 cells with Mettl3 overexpression was significantly increased(P<0.01).The protein levels of Wnt9b,β-catenin and P-βcatenin were significantly decreased(P<0.05).3.3.2 RT-PCR results showed that after 24h of intervention,compared with NC group,Mettl3 overexpression of MLE-12 cells significantly increased Mettl3 mRNA level(P<0.05),and Wnt9b mRNA level was significantly decreased(P<0.01).ConclusionsThe early application of YYQF can regulate the expression of RILI-related inflammatory factors IL-1 7,IL-10,IL-12 and IFN-γ,increase the expression of Mettl3 and P-β-catenin proteins,and reduce the expression of Wnt9b,β-catenin and α-SMA proteins.Finally,the lung inflammation and initial pulmonary fibrosis of RILI model mice were alleviated.The mechanism of YYQF in the treatment of RILI may be related to the regulation of Mettl3 to affect the methylation level of Wnt9b,thereby inhibiting the activation of Wnt/β-catenin pathway.Therefore,the traditional Chinese medicine"nourishing Yin and clearing lung method" has a clear theoretical basis for the treatment of RILI,and it can be further used in clinical practice. |
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