The Mitigating Effect Of Heptamethine Cyanine Dye IR-780 On Radiation-induced Lung Injury

Objective:To study the effect and mechanism of IR-780 in mitigating radiation-induced lung injury,and explore the potential role of glycolysis in radiation-induced pulmonary fibrosis.Methods:1.The establishment of radiation-induced lung injury model.The C57/BL6 mice were anesthetized and whole chests were exposed to a single dose of 15?Gy X-rays with lead shield on other body parts.Then collected the samples at week 6 and week 16.The injury was evaluated by HE and Masson staining.2.The accumulation level of IR-780 in irradiated lung tissue.To detect the accumulation of IR-780 in vivo,Mice were randomized into 3groups: control(ctrl),IR-780 and Radiation with IR-780(IR+IR-780),near-infrared imaging was performed 16 weeks after radiation.For live-imaging,the mice were imaged 24 hours after intraperitoneal injection under anesthesia using a Near-infrared imaging system.Then the mice were euthanized,and the organs including heart,liver,spleen,lung,kidney,intestine,muscle,were imaged to evaluate the accumulation level of IR-780 in different organs.3.The radioprotection of IR-780 on radiation-induced lung injury in vivo.Mice were randomized into 3 groups: control,IR and IR+IR-780,the mice of IR+IR-780 were treated with IR-780 for three weeks(twice a week)after radiation.To evaluate the radiation-induced acute lung injury in mice,the morphology of the irradiated lung,the inflammatory cell infiltration,and the m RNA level of pro-inflammatory factors including IL-Iβ,IL-6 and TNF-α were detected at week 6after radiation.And 16 weeks after radiation,lung tissue density was detected by micro-CT,histopathological studies including HE and Masson staining,and the expression of fibrosis-related factors(Collagen I,α-SMA and Fibronectin)detected by q-PCR and Western blot were performed to evaluate radiation-induced lung fibrosis.4.The radioprotection of IR-780 on radiation-induced lung injury in vitro.Mouse alveolar macrophages(MH-S)and human fetal lung fibroblasts(HFL1)were cultured,and cells were exposed to 8 Gy radiation to establish radiation-induced cell damage model in vitro.To evaluate the effect of IR-780 on inhibiting fibroblast activation,after 24 hours,the m RNA expression of pro-inflammatory factors(IL-1β,IL-6,TNF-α)and pro-fibrosis factors(YM-1,TGF-β1,IL-10,Arg-1)were evaluated by q-PCR.After 48 hours,the m RNA expression and protein expression of fibrosis-related factors(Collagen Ⅰ,α-SMA,Fibronectin)of HFL1 cells were detected by q-PCR and Western blot.To study the effect of IR-780 on the pro-fibrotic effects of macrophages,a co-culture model was constructed using a Transwell chamber,they were divided into Control,IR and IR+IR-780 group.Briefly,MH-S cells were pre-treated with IR-780 for 15 minutes,and exposed to 8 Gy radiation,then co-cultured with primary fibroblasts or murine lung epithelial-12(MLE-12)for 48 hours.Finally,the m RNA expression and protein expression of fibrosis-related factors(Collagen Ⅰ,α-SMA,Fibronectin)in fibroblasts or MLE-12 were detected by q-PCR and Western blot.5.The radioprotective mechanisms of the IR-780 on radiation-induced lung injury.In order to clarify the effect of IR-780 on glycolysis,we applied q-PCR and Western blot to detect the effect of IR-780 on the expression level of glycolysis-related kinases(HK1,HK2,PFK1,PFK2,GLUT1,GLUT3)in vivo and in vitro.Further,Seahorse XFp analysis was used to detect the extracellular acidification rate(ECAR).we also used a lactic acid detection kit to detect the level of extracellular lactic acid release.In order to evaluate the role of glycolysis in the radiation damage of fibroblasts and macrophages,fibroblasts and macrophages were treated with glycolysis inhibitor2-Deoxy-D-glucose(2-DG),then exposed to 8 Gy radiation,the m RNA expression level of fibrosis-related protein(Fibronectin)in fibroblasts was detected by q-PCR;and the m RNA expression of pro-fibrosis factors(YM-1,TGF-β1,IL-10,Arg-1)of macrophage was detected by q-PCR;the secretion levels of IL-10 and TGF-β1 in macrophages were detected by Enzyme-linked immunosorbent assay(ELISA).To study the effect of 2-DG on the pro-fibrotic effects of macrophages,a co-culture model was constructed using a Transwell chamber.Briefly,the macrophages were treated with 2-DG,and exposed to 8 Gy radiation,then co-cultured with primary fibroblasts or epithelial cells for 48 hours.Finally,the m RNA expression and protein expression of Collagen Ⅰ,α-SMA and Fibronectin in fibroblasts or epithelial cells were detected by q-PCR and Western blot.Results:1.Radiation-induced lung injury model was successfully established.6 weeks after radiation,HE staining revealed increased inflammatory cell infiltration in the irradiated lungs.Similarly,16 weeks after radiation,HE staining showed an upregulation in the proportion of inflammatory cells in the irradiated group;Masson staining showed that the irradiated lungs had more collagen deposition compared with the unirradiated mice.2.The results of near-infrared imaging showed that IR-780 could accumulate in the lung tissues,and we observed stronger fluorescence density in the irradiated lungs,comparing with the lungs from unirradiated mice.Besides,in the irradiated mice,IR-780 accumulated much more in the lung than other organs including heart,liver,spleen,kidney,intestine,muscle.These results suggested that IR-780 preferred to accumulate in the irradiated lung tissues.3.IR-780 protected mice from radiation-induced lung injury.6 weeks after radiation,we found that IR-780 reduced the production of lung tissue exudate.The results of q-PCR showed that IR-780 reduced the m RNA expression of IL-1β,IL-6,and TNF-α in lung tissue,and the results of HE staining also showed that IR-780 reduced the infiltration of inflammatory cells.16 weeks after radiation,the result of CT showed that IR-780 reduced the parenchymal opacity and lung density of the lung tissue after radiation.Masson staining and immunohistochemical results showed that IR-780 reduced the deposition of collagen in the lung.The results of q-PCR and Western blot showed that IR-780 reduced the m RNA expression and protein expression of Collagen Ⅰ,α-SMA and Fibronectin.The above results suggested that IR-780 could mitigate radiation-induced lung injury.4.IR-780 mitigated radiation-induced lung fibrosis in vitro.In vitro,the results of q-PCR showed that IR-780 reduced pro-inflammatory factors(IL-1β,IL-6,TNF-α)and pro-fibrotic factors(YM-1,TGF-β1,IL-10,Arg-1)m RNA expression level;IR-780 reduced the m RNA and protein expression of fibrosis-related proteins(Collagen Ⅰ,α-SMA,Fibronectin)in irradiated fibroblasts.The results of co-culture showed that IR-780 pretreatment reduced the effect of irradiated macrophages on promoting fibroblast activation and epithelial-mesenchymal transition(EMT).The above results indicated that IR-780 could inhibit the activation of fibroblasts after radiation,and could also reduce the pro-fibrosis effect of alveolar macrophages after radiation.5.The radioprotective effect of IR-780 on radiation-induced lung injury may be related to the regulation of glycolysis.Our results showed that IR-780 reduced the m RNA expression and protein expression of glycolysis-related kinases(HK1,HK2,PFK1,PFK2,GLUT1 and GLUT3)in vitro and in vivo after radiation.The results of Seahorse XFp analysis and lactate test showed that IR-780 reduced the ECAR and lactic acid release levels of macrophages after radiation exposure.These results suggested that IR-780 could reduce glycolysis.Furthermore,the glycolysis inhibitor2-DG could reduce m RNA expression of fibrosis-related protein(Fibronectin)in HFL1.Similarly,2-DG could reduce the m RNA expression of pro-fibrotic factors and the secretion of IL-10 and TGF-β1 in macrophages after radiation.In the co-culture model,we found that 2-DG could reduce the pro-fibrosis effect of alveolar macrophages after radiation.These results suggested that the effect of IR-780 on radiation lung injury may be related to glycolysis,but the detailed mechanism needs to be further explored.Conclusion:1.IR-780 can accumulate in the irradiated lung.2.IR-780 can mitigate radiation-induced lung injury.3.IR-780 may mitigate radiation-induced lung injury by regulating glycolysis.