| Part ? The model modification for radiation-induced intestinal fibrosis in rats and multi-photon imagingObjective: We still lack a simple and stable model of radiation-induced colorectal fibrosis and a timely and precise evaluation method.Modified radiation regimen included the Trendelenberg position with a 15 degree incline,the precise localization of CT-simulator,and down-moving of the superior border radiation field.This study aimed to explore the successful modeling rate of the radiation-induced rectal fibrosis with this regimen in rats and the feasibility of the multi-photon imaging to evaluate the radiation injuries and fibrosis of the rectum in rats.Materials and methods: Sixty Sprague-Dawley(SD)rats were randomly divided into control group,conventional radiation group,and modified radiation group at 1: 3: 2.A single dose of 20 Gy was prescribed for pelvic irradiation.In the conventional radiation group,rats were taken in a supine position.The upper boundary of the irradiation field is 1cm above the pelvis.The inferior border was 1cm below the anus,and the lateral edges were 1cm lateral to the ipsilateral sides of the body.In the modified radiation group,a 15-degree Trendelenberg position was taken.The upper boundary of the irradiation field was the sacral promontory,and the lower border was the anal verge,and the lateral edges were the inner wall of the pelvis.CT simulator and three-dimensional planning system were used.We calculated the survival rate and radiation injury score of rats.We used the multi-photon imaging on the rectum of the control group and the modified radiation group to evaluate the pathological characteristics,collagen density,and collagen flatness.Results: Ten weeks after radiation,the survival rates of the conventional radiation group and the modified radiation group were 70.0% and 95.0%,respectively(P =0.030),and the difference was statistically significant.The survival rate of the modified radiation group and the control group was not statistically significant(95.0 vs.100%,P= 0.480).Compared with the control group,the Langberg's scores of the conventional radiation group were significantly higher than those in the modified radiation group(P<0.001).The difference between the conventional control group and the modified radiation group was statistically significant(5.3 ± 3.7 vs.8.4 ± 2.2,P <0.001).Multi-photon imaging in the modified radiation group showed the pathological changes of radiation injuries and fibrosis,including mucosal ulcers,submucosa,muscular and serosal fibrosis,microvascular stenosis,occlusion,and enteritis cystica profunda,which were consistent with the Masson's trichrome staining.In multi-photon imaging,the collagen density of the modified radiation group was 0.214 ± 0.039,which was significantly higher than that in the control group 0.127 ± 0.032(P = 0.002);the collagen flatness was also markedly higher than that of the control group(0.926 ±0.003 vs.0.917 ± 0.002,P<0.001).Conclusion: The modified radiation regimen by adjusting the Trendelenberg position and radiation field with a CT simulator can reduce the mortality of radiation-induced rectal fibrosis in rats.The radiation injury scores are higher than the conventional radiation group,which is conducive to the establishment of a stable model.Multi-photon imaging can identify and evaluate the radiation injuries of rectum and realize quantitative analysis for intestinal fibrosis.Part ? The proteomics of radiation-induced intestinal fibrosis in ratsObjective: The detailed mechanism of radiation-induced intestinal fibrosis has not been fully elucidated,and its proteomics research is still rarely reported.This study aimed to use Tandem mass tags(TMT)quantitative proteomics to explore differentially abundant proteins and enrichment pathways related to radiation-induced rectal fibrosis of rats,and to validate altered candidate proteins through the parallel reactions monitoring.Materials and methods: Six SD rats were randomly divided into a control group and a radiation group at 1: 1.The control group did not receive radiation,and the radiation group was radiated by X-ray 20 Gy according to the modified radiation regimen.After ten weeks,the rectum was obtained from the sacral promontory to the dentate line for pathological examination and TMT quantitative proteomics analysis.After digestion and labeling,TMT,high-performance liquid chromatography,Liquid chromatography-tandem mass spectrometry,and database search were performed.Proteins with a P value less than 0.05 and a fold change greater than 1.5 or less than 0.67 are defined as differentially abundant proteins,gene ontology(GO)and Kyoto Gene and Genome Encyclopedia(KEGG)pathway analysis,protein interaction network analysis,and altered proteins were validated by the parallel reaction monitoring.Results: Compared with the control group,the rats in the radiation group had significantly thickened rectal walls,mucosal ulcers,inflammation,collagen deposition in the submucosa,and microvessel wall hyalinosis,stenosis,and even occlusion.The radiation injury scores(Langberg's scores)of the radiation group and the control group were 12.7 ± 1.5 vs.0.3 ± 0.6(P <0.001).A total of 6,692 proteins were identified,and 5,756 of them were quantified by the TMT proteomics.Finally,we detected 227 were up-regulated and 93 down-regulated proteins in the radiation group.The biological processes of altered proteins are mainly enriched in the negative regulation of the cellular metabolic process,the negative regulation of protein metabolic process,and the regulation of proteolysis.KEGG analysis suggested that the mainly enriched pathway was the complement and coagulation cascade pathway.After validation of the parallel reaction monitoring,we confirmed that FGG,THBS1,AGT,F2,C3,ITGAM,ITGB2,and CYBB were significantly up-regulated in rats with radiation-induced intestinal fibrosis.Conclusion: This study provides the quantitative proteomic profile of radiation-induced rectal fibrosis in rats.Through the parallel reaction monitoring validation,we confirmed that THBS1,AGT,F2,C3,ITGAM,ITGB2,and CYBB proteins are up-regulated.Part ? The transcriptomics of radiation-induced intestinal fibrosis in rats.Objective: The transcriptomic mechanism of radiation-induced intestinal fibrosis was rarely reported.This study aimed to explore the differences in transcriptome m RNA expression of radiation-induced rectal fibrosis in rats,and to select the key modules and hub genes.Combined with the validation of proteomics,we aimed to explore the mechanism and candidate altered proteins of radiation-induced intestinal fibrosis.Materials and methods: There were nine SD rats in the modified radiation group and three in the control group.The radiation group received modified pelvic radiation with a 20 Gy X-ray.After ten weeks,the rectum was obtained for pathological examination and m RNA transcriptomics analysis.The differentially expressed genes corresponding to m RNA were defined as P <0.01.The bioinformatics analyses such as GO,KEGG analyses and protein interaction network analysis were performed.According to weighted gene co-expression network analysis,key module genes related to radiation-induced intestinal fibrosis were identified.We defined the hub genes in the key module,which met the following two conditions.First,module connectivity,that is,the absolute value of the Pearson correlation coefficient of the gene,and the module was more than 0.8.Second,the absolute value of the Pearson correlation coefficient between the gene and radiation-induced intestinal fibrosis was more than 0.2.Combined with proteomic results,we detected the hub genes that expressed differentially.Results: According to m RNA transcriptomics,there were 2023 differentially expressed genes,of which 634 were up-regulated,and 1389 were down-regulated.The mainly enriched biological processes were calcium ion transport,cellular calcium homeostasis,and potassium ion transport.KEGG analysis showed the mainly enriched pathways were the calcium signaling pathway,c GMP-PKG signaling pathway,focal adhesion,regulation of actin cytoskeleton.Seventeen hub genes,including PPP1R12 C,FLNA,JPH2,STUM,SPEG,ITGA1,ARHEGEF25,CAVIN1,and SYNM,in the key modules,were highly related to the trait of radiation-induced intestinal fibrosis.Taking the intersection,three hub genes,including CAVIN1,SYNM,and PPP1R12 C,were significantly down-regulated in rats with radiation-induced intestinal fibrosis.Conclusion: According to the transcriptomic study of radiation-induced intestinal fibrosis in rats,the differentially expressed genes were enriched in the calcium signaling pathway,c GMP-PKG signaling pathway,focal adhesion,and regulation of actin cytoskeleton.Among the 17 hub genes closely related to radiation-induced intestinal fibrosis,PPP1R12 C,CAVIN1,and SYNM were validated in proteomics.Part ? Prevention and treatment exploration of bone marrow-derived mesenchymal stem cells and Pirfenidone for radiation-induced intestinal fibrosis in ratsObjective: There is no effective method of prevention and treatment for radiation-induced fibrosis.The the efficacy of bone marrow mesenchymal stem cells(BMSC)on radiation-induced intestinal fibrosis remains controversial.This study aimed to explore the efficacy of intravenous injection of allogeneic BMSC and the combined intervention with Pirfenidone(PFD)on radiation-induced rectal fibrosis in rats.Materials and methods: Forty SD rats were divided into control group,radiation group,BMSC group,PFD group,and combined intervention group with eight rats in each group.The control group did not receive radiation,and the remaining four groups received 20 Gy X-ray pelvic radiation.The modified protocol included a 15-degree Trendelenberg position,CT simulator,and a three-dimensional planning system to calculate the dose distribution of the irradiation field.The upper boundary of the irradiation field was the sacral promontory,and the lower boundary was the anal verge,and the lateral boundaries were the inner wall of the pelvis.PFD group and the combined intervention group were given intragastric administration of PFD 300 mg / kg once a day,and the BMSC group and the combined intervention group received BMSC injection slowly through the femoral vein on the 21 st,34th and 41 st days after radiation.Ten weeks after irradiation,the rectum from the sacral promontory to the dentate line was obtained for pathological examination to evaluate the Langberg's scores and submucosal thickness of radiation injury.Results: Compared with the control group,the pathological examination in the radiation group revealed obvious ulcers,thickening of the intestinal wall accompanied by submucosal and serosal fibrosis,and severe rats showed full-thickness fibrosis.The BMSC group still had thickened intestinal wall with submucosal fibrosis,but the ulcers were not obvious compared with the radiation group.The thickening of the submucosa of the PFD group was alleviated compared with the radiation group,but the mucosal ulcers were still more obvious.The mucosal ulcers in the combined intervention group not obvious,the thickness of the submucosa was reduced compared with the previous,and the mucosal hyperplasia was obvious.Langberg's scores in the control group were significantly different from other groups.Compared with the radiation group,the Langberg's scores in the BMSC group,PFD group,and combined intervention group were all decreased(P = 0.025,P = 0.001 and P = 0.009,respectively),and the differences were statistically significant.There was no significant difference in the thickness of the submucosa between the BMSC group and the radiation group(P = 0.356).No significant difference in the thickness of submucosa was detected between the PFD group and the combined intervention group,but both groups had significantly decreased thickness of submucosa compared with the radiation group(P <0.001)Conclusion: Intravenous multiple injections of allogeneic BMSC could alleviate the radiation intestinal injury in rats;however,the effect of BMSC alone on radiation-induced intestinal fibrosis was not significant.When combined with PFD,BMSC did not show a significant synergistic protective effect on radiation-induced intestinal fibrosis in rats.Further studies are required to explore a more efficient combination treatment in the future. |
PDF Full Text Request