Experimental Study Of Optical Diagnosis With Multiphoton Imaging And Intervention With Pirfenidone For Radiation-induced Pelvic Fibrosis

Part ⅠThe study of colonic radiation-induced fibrosis after neoadjuvant chemoradiotherapy for rectal cancer by multiphoton imagingObjective: To study the colonic radiation-induced fibrosis after neoadjuvant chemoradiotherapy for rectal cancer by the technique of multiphoton imaging.Materials and Methods: 12 cases of patients with radiation-induced pelvic fibrosis with severe bowel stenosis or bowel obstruction(fibrosis group) and 12 cases of patients without pelvic fibrosis after neoadjuvant chemoradiotherapy for rectal cancer(non-fibrosis group) were retrospectively included, and the proximal surgical resection margins of colonic specimens embedded in paraffin wax were included to represent the status of the colon above the anastomosis. The fresh specimens of proximal surgical resection colonic margins of 10 patients after neoadjuvant chemoradiotherapy for rectal cancer(chemoradiotherapy group) and 10 patients without neoadjuvant chemoradiotherapy(non-chemoradiotherapy group) were included. The collagen density and the collagen orientation index(COI) were measured by multiphoton imaging, and the HE staining and the VG staining were applied for comparative study.Result: Multiphoton imaging technique can clearly detect the collagen and the elastic fiber in the specimen. Radiation therapy can lead to the increased collagen density of submucosal layer and muscularis propria or the destruction of collagen fiber with abundant new and immature collagen fiber, and also cause the disordered arrangement of submucosal collagen fiber and the rupture of elastic fiber. In the specimens embedded in paraffin wax, the submucosal collagen density of two group of was higher than the collagen density of muscularis propria(p<0.0001), and the submucosal and muscularis propria collagen density of fibrosis group was higher than the non-fibrosis group(p=0.0016 and p<0.0001, respectively). The COI of fibrosis group was also higher(p<0.0001). In the fresh specimens, the submucosal collagen density of two group of was higher than the collagen density of muscularis propria(p<0.0001), and the submucosal and muscularis propria collagen density of chemoradiotherapy group was higher than the non-chemoradiotherapy group(p=0.0002 and p=0.0014, respectively). The COI of chemoradiotherapy group was also higher(p=0.0068)Conclusion: Multiphoton imaging can detect the early-staged colonic fibrosis after radiation.Part Ⅱ The interventional study of pirfenidone on the rat radiation-induced pelvic fibrosis modelObjective: To study the interventional function of pirfenidone on the radiation-induced fibrosis of irradiated bowel and surrounding muscles of rat model.Materials and Methods: Male Sprague-Dawley rats were used to construct the rat radiation-induced pelvic fibrosis model, and pirfenidone was applied for interventional study. The rats were randomly assigned to blank control group(group A), pirfenidone intervention group(group B) and the radiation control group(group C). 10 weeks after radiation, the rat tissues of rectum and the muscles surrounding rectum were harvested for experiment, 5 rats in each group. Multiphoton imaging, HE staining and VG staining were applied to analyze the collagen density and collagen orientation index of the specimen, and RT-PCR and western blot were applied to detect the expression level of TGF-β1, Smad3 and CTGF.Result: Rat radiation-induced pelvic fibrosis model was successfully constructed. The submucosal collagen density of group A and B was higher than the muscularis propria(p<0.0001 both), and no significant difference between group A and B(p=0.20). But in group C, the submucosal collagen density was significantly lower than the muscularis propria(p=0.0004), and the collagen density of muscularis propria increased significantly(p=0.0008). One-way ANOVA analysis show that the COIs of the groups were significantly different. The result of fluorescence quantitative PCR showed that there were significantly different expression of TGF-β1 in the rectal tissue and muscles surrounding rectum of three groups(p=0.04 and p=0.004, respectively), and the expression of TGF-β1 was significantly decreased in group B. There was no significant difference of the m RNA expression of Smad3 in three groups. There was no significant difference of the expression CTGF by RT-PCR in three groups with the analysis of one-way ANOVA, but the expression in group C was inclined to be higher than the group B. The results of western blot showed that the protein expression of TGF-β1 of group C rectal tissue increased significantly(p=0.036), but there were no significantly different in the muscle tissue surrounding rectum. The protein expression of Smad3 of three groups rectum and muscle tissue also was no significantly different. The expression of CTGF protein level of rectum and muscle tissue decreased significantly in group B.Conclusion: Pirfenidone can attenuate the fibrosis of bowel and the muscles surrounding rectum in the rat radiation-induced pelvic fibrosis model. It may function as the preventive factor for radiation-induced fibrosis by inhibiting the protein expression of TGF-β1 and CTGF. Multiphoton imaging can be applied to effectively diagnose the fibrosis status in submucosal layer and the muscularis propria in the rat radiation-induced pelvic fibrosis modelPart Ⅲ The analysis of gene expression profile of rat radiation-induced pelvic fibrosis model after the intervention of pirfenidoneObjective: To analyze the effect of the pirfenidone on the gene expression profile of rat radiation-induced pelvic fibrosis model.Materials and Methods: The rectal tissues of the each rat in blank control group(group A), pirfenidone intervention group(group B) and the radiation control group(group C) were analyzed by using the Affymetrix Gene Chip Rat Genome 230 2.0 Array to detect the changes of gene expression profile.Result: By comparing the gene expression profile of group A and group B, the functional annotation of the differentially expressed genes were mainly with respect to categories such as response to lipid, response to organic cyclic compound and response to glucocorticoid. By comparing the gene expression profile of group C and group A, the significantly enriched GO terms were mainly associated with the extracellular matrix, which indicated that the extracellular matrix of group C was apparently affected. By comparing the group B and group C, the enriched GO terms of differentially expressed genes were with respect to the extracellular space, extracellular matrix and cell migration. After the intervention of pirfenidone, the enriched pathways of group B were significantly different from group C.Conclusion: The analyses of the gene expression profile of the rat rectal specimens form 3 groups showed that pirfenidone could attenuate the radiation effect of extracellular matrix of rectal tissue, and indicated that pirfenidone was able to prevent the radiation-induced fibrosis.