Research On The Regulatory Mechanisim Of TGF-BETA/smad Signaling Pathway In The Critical Irreversible Point Of Radiationinduced Lung Injury

Radiotherapy is increasingly applied in thoracic tumor,e.g.,lung and esophageal carcinoma.In the process of thoracic radiation,lung injury is inevitable to a certain degree.Thus,radiation-induced lung injury has been a riddle and a hot spot in the field of radiation oncology.The pathogenesis of radiation-induced lung injury is complex,which is obvious dose-dependent.When irradiated by low-dose,lung injury is mild and reversible,and can be restored completely in late period.With the increase of dose,lung injury aggravates as well.When dose reaches the threshold,irreversible injury may occur.Obviously,whether irreversible mainly depends on dose-volume parameters of exceeding a threshold,i.e.point of no return（PNR）or not.This study focuses on exploring the critical irreversible point of radiation-induced lung injury in rat models,and preliminarily investigates physical parameters associated with irreversible pulmonary fibrosis by clinical analysis,simutaneously.Purpose:In this study,whole right lung of rats was respectively irradiated with the sequential different biological effective dose,inspected and assessed lung pathological damage at each observed time point arfter irradiation,and then explored the critical irreversible point of radiation-induced lung injury.Meanwhile,molecular regulatory mechanism was further researched by the TGF-beta/Smad signaling pathways.Methods:144 healthy male SD rats were randomly divided into six groups:normal control group（A）,8Gy irradiation group（B）,10Gy irradiation Group（C）,12Gy irradiation group（D）,14Gy irradiation group（E）,16Gy irradiation group（F）.Each group had 24 rats.After intraperitoneal anesthesia,whole right lung of all experimental group（irradiation group）rats was respectively irradiated with ⁶⁰Coγ-ray at doses of 8Gy,10Gy,12Gy,14Gy,16Gy.The control group is free of irradiation.At the time of 2,5,12 and 24 weeks after irradiation,five rats were randomly picked up from each group.They were drawn blood from the heart and exteriorized right lungs before sacrificed.The removed lungs were divided into two parts,one was fixed in 10%formaldehyde solution,another kept at-80℃liquid nitrogen refrigerator after packaging.After fixation and dehydration,the lung tissues were embedded in paraffin and cut in the coronal plane.The sections were processed for routine haematoxylin-eosin and Masson staining to observe the deposition of collagen fibers and pathological change of the lung tissue.The lung tissue obtained above were processed immunohistochemically to detect the presence of TGF-beta1.The protein levels of Smad2,Smad3 and Smad7 were detected by Western blot.The classification standard of Szapiel was used to evaluate acute radiation-induced pneumonia,chronic fibrosis of lung tissue,and judged the critical irreversible point of radiation-induced lung injury.Finally,determined the threshold dose of radiation-induced irreversible damage of lung tissue.Results:（1）Two weeks after radiation,mild cute inflammatory changes were found in rats lung tissue of all irradiated groups by HE dyeing,such as hyperemia,edema and a variety of inflammatory cells infiltration,without obvious fibrosis.Five weeks later,The irradiated lung tissue still had a series of appearance as hyperemia,edema,alveolar septa thickening and mixed inflammatory cells infiltration,was severer than at the 2th weekend,and brought out slight diminishing of alveolar cavity,mild thickening of alveolar walls and mild to moderate pulmonary interstitial fibrosis.With hyperemia and edema of pulmonary tissues alleviated,alveolar septa got wide,alveolar cavity got smaller,alveolar walls were moderately thickened and lymphocytes were the predominant cells infiltrating in the alveolar spaces in rats of all irradiated groups at the 12th weekend.Meanwhile,lung tissue collagen fibers were obviously absorbed in low dose irradiated groups（group B,C,D）,accompanied by mild degree of fibrosis.While in high-dose groups（group E,F）,collagen fibers were hyperplastic so as to result in moderate pulmonary fibrosis（gradeⅡ）.At the 24th weekend after radiation,the obvious differentiation presented in these irradiated groups.In the low dose groups,congestion and edema had been mainly faded away,collagen fiber had been basically absorbed,and accompanied few inflammatory cells in alveolar interstitium.While in the high dose groups,alveolar septa got obviously wide,alveolar cavity got stenosis,alveolar walls were obviously thickened and not complete in part of it,which more lymphocytes infiltrating and more hyperplastic fibrous tissue in alveolar interstitium resulted in pulmonary fibrosis of no less than moderate（≥magnitudeⅡ）.（2）In Masson staining,there was little expression of the pulmonary matrix collagen in all irradiated groups at the 2nd weekend after irradiation,and pulmonary interstitial collagen fibers turned positive expression at the 5th weekend after irradiated,and increased obviously after 12 weeks.After 24 weeks,pulmonary interstitial collagen fiber decreased obviously in low-dose groups,instead it showed a sharp increase in high-dose groups,which was significantly higher than the low dose irradiated groups（P<0.05）.With the irradiation dose increasing content of collagen fibers increased at each time point.（3）In immunohistochemical detecting,TGF-beta1 of rats lung tissue in the control groups were wholly negative.TGF-beta1 of rats Lung tissue in the low dose groups began to appear weakly positive expression at the 2th weekend after irradiation,and positive or moderate positive expression at the 5th and 12th weekends after irradiation,however,became weakly positive expression in turn after 24 weeks irradiation.In the high dose irradiated groups,TGF-beta1 of rats lung tissue had become positive even in partial to moderate positive expression at the 2th weekend after irradiation,further strengthened after 5 weeks,wholly reached wholly moderate positive expression,and became moderate positive expression even strongly positive expression after 12 weeks irradiation,and kept the high expression level up to after 24 weeks.（4）Western Blot test shows:（1）Smad2 protein expression in the lung tissue of rats in all irradiation groups were characterized by the process of increacing in the beginning stage and falling in the later period.Abnormal increasing of Smad2protein occurred at the 5th weekend after irradiation which were signally higher than the second weekend,P<0.01),especially in medium-low dose groups（group B,C,D,E）.Subsequentl,Smad2 protein expression in the lower dose irradiated groups droped rapidly with time,which were all signally lower at the 12th weekend than the 5th weekend（P<0.01）,and continually decreased obviously in group B and C at the 24th weekends（P<0.05）.In high dose group（F）,Smad2 protein expression in lung tissue of rats increased very fast,and rose rapidly at the 2th weekend after irradiated,being significantly higher than the control group（P<0.05）,and attained maximum at the 5th weekend,and then continually maintained at a high level ever since,without dropping significantly（P>0.05）.Smad2 protein expression in lung tissue of F group was obviously higher than that of all other groups at the 12th and24th weekends after radiation（P<0.05）.（2）Smad3 protein expression of the lung tissue rats in irradiation groups were similarly characterized by the process of increacing in the beginning stage and falling in the later period after irradiation,especially obvious in the low dose groups（group B,C,D,E）.Smad3 protein in these four groups reached its peak at 5th weekend after radiation,and then gradually droped over time.However,group F which received high dose irradiation increased more slowly,was still at a lower level at the 2th weekend,and increased significantly just at 5^thweekend（P<0.05）,and then reached the highest content at 12th weekend,and fell to some extent at the 24th weekend,which was also significantly higher than other groups（P<0.05）.（3）Smad7 protein expression of rat lung tissue in irradiation groups of increased significantly（P<0.05）at 2th and5th weekends after irradiation,and then gradually declined.Evidently,Smad7protein expression of rat lung tissue in the group B,C,D and E dropped sharply（P<0.05）at the 12^thweekend,only E and F groups did not decrease significantly（P>0.05）.Conclusion:（1）Whole right pulmonary irradiation to rats,the critical irreversible point of radiation-induced lung injury should probably be 12 Gy—14 Gy.（2）As the change of irradiation dose and the progress of the radiation-induced pulmonary injury（RPI）,TGF-beta1 and protein expression of Smad2,Smad3Smad7 presents dynamic change.It confirms that TGF-beta/Smads signal pathway play an important role in regulating the process of radiation-induced lung injury progress.（3）TGF-beta/Smads signal pathway regulate radiation-induced lung injury by its downstream target genes,proteins,etc.As TGF-beta1 and Smad3 of lung tissue significantly increase and without desent any more,the irreversible radiation-induced damage should be come about under regulating of these proteins.（4）The signal pathway not only participate in the regulation of early acute inflammation,also act on the entire process of injurying repair,hyperplasia and chronic fibrosis.We preliminary speculated that the early acute inflammation is mainly regulated by Smad2,and the chronic inflammation and fibrosis of lung tissue in the late period is principally regulated by Smad3 in the signal pathway.Objective: By analysing the relationship between the dosimetric parameters and the probability of radiation-induced lung fibrosis in the thoracic-tumor patients who received intensity-modulated radiatherapy（IMRT）,explored the optimum physical parameters of evaluating radiation-induced lung injury in those patients whose chest radiation.And preliminarily studied the critical irreversible point of radiation-induced pulmonary injury.Methods: Collected 81 hospitalized patients of the thoracic-tumor in our hospital from December 2012 to May 2015,which were received intensitymodulated radiation therapy（IMRT）.Follow-up at regular intervals after radiotherapy,and according to the NCI-Common Terminology Criteria for Adverse Events V3.0（CTCAE V3.0）for radiation-induced pulmonary fibrosis of 0-5 grade standard,evaluated the situation of chronic radiation-induced lung injury.Several clinical and dosimemc factors were analyzed retrospectively by univariate factor and multivariate factors,clinical factors including sex,age,smoking history,weight loss,chemotherapy,complication,and so on,dosimemc factors including Volume of irradiated certain dose（V—V40）,Dose of irradiated certain volume（D10—80）,mean lung dose（MLD）,prescription dose and fractionated dose.And explored the independent influence factors of radiation-induced lung injury.Results: In 81 patients,7（8.6%）had chronic pulmonary fibrosis≥grade 2.Univariate factor analysis showed that D50,D60,D70,D80,V5,V10,V15 and MLD were associated significantly with radiation-induced pulmonary fibrosis ≥grade 2.Multivariate factors regression analysis showed that the D50 was independent predictive factor for radiation-induced pulmonary fibrosis.According to ROC analysis,the area under the curve（AUC）was 0.794,P value was 0.01,and it was speculated that yueden index achieved maximum value 0.492 when the D50 attained 13.25,in other words,when D50 was more than 13.25 Gy the incidence of RPF≥grade 2 increased dramatically.