Protective Effect Of PMID On Radiation-induced Pulmonary Fibrosis

The lung is an important target organ damaged by nuclear radiation.While wartime nuclear radiation,radioactive nuclear accidents,bone marrow transplantation pretreatment,and chest tumor radiotherapy occur,normal lung tissue is damaged by radiation exposure,which is clinically called radiation-induced pulmonary injury(RPI).Early RPI from 1 to 6 months after irradiation refers to acute radiation pneumonitis(ARP).Late RP occurs from months to years after irradiation,and the clinical manifestation is radiation-induced lung fibrosis(radiation-induced lung fibrosis,RILF).Once RILF occurs,it is difficult to reverse,seriously affecting the patient's quality of life.In terms of treatment,two drugs have been approved by FDA including Nitinedanib(BF)and Pirfenidone(PFD),both of which have limited therapeutic effects and obvious sideeffects such as gastrointestinal reactions.Therefore,the development of new drugs has important clinical significance.2-indolinone derivative [3-(3-pyridylmethylidene)2-indolinone,PMID] is a small molecular compound,which is screened based on Nrf2 / Keap1(Nuclear Factor Erythroid 2-Related Factor 2 / Kelch-like erythroid cell-derived protein CNC homology-associated protein 1)pathway,is an amorphous crystalline yellow powder,and has antioxidant effect.Our previous study finds that PMID can promote the dissociation of Nrf2 and Keap1,enhance the combination of Nrf2 and anti-oxidant-response element(ARE),and upregulate the expression of the downstream antioxidant-related genes such as superoxide dismutase(SOD),Heme oxygenase-1(HO-1)and NADPH quinone oxidoreductase 1(NQO-1).PMID shows certain rules of absorption,elimination and distribution in the body,with stable metabolism and great bioavailability.Previous research also suggest that PMID has a great protective effect on bleomycin(BLM)-induced lung fibrosis in mice and rats,and silica(Si O2)-induced lung fibrosis in mice.Its synthetic method and its application to the treatment of pulmonary fibrosis have been patented(patent application number: 201610794652.6).However,the effect of PMID in radiation-induced pulmonary fibrosis has not been evaluated.In this study,we evaluated the protective effect of PMID on radiation-induced pulmonary fibrosis.We used a single whole chest radiation model of mice.Frequent long-term follow-up observations were performed to systematically evaluate the protective effect of PMID on radiation-induced lung fibrosis in mice.Co60 was used as irradiation source of model mice,and the appropriate radiation dose was selected through long-term observation of different radiation doses.The two administration modes,prophylactic and therapeutic,were evaluated separately.The mice were randomly divided into 6 experimental groups: normal control group(NC group),the model control group(MC group),the low,medium and high dose of PMID group and positive drug group(pirfenib,PFD).Long-term observations of changes of body weight and survival rates were performed,besides,lung respiratory function of mice was performed at the end stage.After that,lung samples were prepared for H&E staining,and Ashcroft scores were used to examine the degree of lung tissue pathological damage.MASSON staining and collagen area analysis were performed to evaluate the accumulation of collagen.Quantitative analysis of collagen formation in lung was performed by measuring the content of hydroxyproline.The expression of Fibronectin(FN),Type I Collagen(COL1A2),fibrosis core factor transcription growth factor-?(TGF-?)and Myeloperoxidase(MPO)in lung were measured by Immunohistochemical staining.The expression of inflammatory factors and pulmonary fibrosis-related factors in lung were analyzed by Real-Time PCR.In addition,we also verified the inhibitory effect of PMID on TGF-? stimulation in vitro.Human fibroblasts were pretreated with different concentrations of PMID and given TGF-? for 48 hours.Cells were collected to obtain protein samples for Western Blot.The expressions of fibrosis-related proteins such as FN,COL1A2 and smooth muscle protein-?(?-SMA)were analyzed.The results showed that PMID significantly prevent mice from radiation-induced lung fibrosis under 15 Gy or 17.5Gy irradiation exposure with prolonged survival time,alleviated decreased forced vital capacity and lung dynamic compliance of mice,reduced lung pathological damage and collagen deposition,decreased neutrophil infiltration in the lungs,and down-regulation of TGF-? and COL1A2 expression levels.Also,the protective activity of PMID with preventive administration is better than PFD.Meanwhile,PMID also has a good therapeutic effect of radiation-induced lung fibrosis in mice..However,under the same dose of radiation,the protective effect of treatment administration on pulmonary fibrosis was less effective than the prevention administration.The results of in vitro experiments showed that PMID inhibited TGF-?-induced expression of fibrosis-related proteins such as FN,COL1A2 and ?-SMA in a dosedependent manner,suggesting that PMID blocked the transformation of human fibroblasts to myofibroblasts.In summary,the present study show that PMID has a significant protective effect on radiation-induced lung fibrosis.