The Biological Effects Of CBD-bFGF On Radiation-induced Lung Injury In Mice And Its Mechanisms

Background:Radiation- induced lung injury(RILI) is a complication of chest radiotherapy. RILI has attracted extensive attention, because of its unclear pathogenesis and ineffective clinical treatment.Basic Fibroblast growth factor(b FGF) has biological functions as revascularization, wound healing, neurotrophy and nerve regeneration. At present, recombination b FGF has been widely used in clinic, such as wound healing, diabetic ulcers, repair of spinal cord injury, Parkinson’s and Alzheimer’s disease. b FGF can contribute to the repair of radiation damage[1][2],and may also have protective effects and repair the damage in radiation- induced lung injury [3]. In this study,we adopted collagen binding domain(CBD) conjugated with b FGF to explore its biological effect on radiation- induced lung injury in mice.Part One: The biological activity and release of the CBD-b FGF in vitroObjective:To compare the differences between CBD-b FGF and commercialized b FGF in the aspects of biological activities, release rate from collangens.Methods:CCK 8 method was adopted to detect effects of C BD-b FGF and commercialized b FGF on promoting the proliferation of human skin fibroblasts(HS-865). The same concentration of CBD-b FGF solution and b FGF solution was loaded with collagen scaffold of similar size. BCA kit(Total protein quantification test kit) was used to test concentration of cytokine in solution and calculate the combination efficiency. The release curve of CBD-b FGF and b FGF from collagen was calculated at different time points in vitro.Results:In v itro, CBD-b FGF stimulated HS-865 proliferation stronglier than commercialized b FGF and its combination rate with collagen was higher than that of b FGF. The release curves showed that commercialized b FGF released faster than that of C BD-b FGF.Conclusion:CBD-b FGF, having same biological function as commercialized b FGF, can combine with collagen efficiently. Part Two: The Biological effects of CBD-b FGF on radiation-induced lung injury in miceObjective:To explore the early intervention function of C BD-b FGF to radiation- induced lung injury in mice and its possible mechanism.Methods:C57BL/6J female mice were randomly divided into four groups: control group without radiation(group A, n = 30), radiation-administrated group(group B, n = 50), CBD-b FGF-treated on the 1st day of radiation(group C, 2ug/mouse, n = 60) and CBD-b FGF- treated on the 7th day of radiation(D group, 2ug/mouse, n = 60). Mice were accepted X-ray linear accelerator radiation on their whole chest, single, 14 Gy.Observe and record the following indicators to all the mice respectively at 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks and 20 weeks after radiation:(1) weight change of mice;(2) appearance change of mice;(3) lung biopsy HE and Masson staining;(4) the expression of α-smooth muscle action(α-SMA), Collagen-I(Col-1), Collagen-3(Col-3) and Transforming growth factor-β1,(TGF-β1) in lung tissue of the mices by using immunohistochemical staining method.(4) O n the 20 t h week after radiation, 2 mice from group A, B, C and D for lung CT scan and 3D reconstruction.Results:Mice of group A can move freely with smooth breathing, normal drinking and diet and the bright luster hair all over the body. Radiatied mice of group B, C and D began to appear listlessness, unresponsiveness, respiratory frequency rising and less eating and drinking 3 days after radiation. In the 2nd week, there has a small white hair in the radiation field. With time goes on, the hair in radiation field turned white and fell off gradually, but there is no obvious skin ulceration hemorrhage in the radiation field.Weight of mice in group A continued to increase. Mice of group B, C and D are losing weight after radiation. 8 weeks later, their weight lost slowly. But their weight lost abruptly after week 16. The weight loss of mice in group C and D was more obvious than that in group B, from week 16(P < 0.05).Lung tissue HE and Masson staining showed that,, mice of group B, C and D exhibited early inflammatory cell infiltra tion, tissue exudation increasing, interstitial inflammatory change and thick septa after radiation. With the passage of time, the damage of lung tissue structure, the deposit of pulmonary interstitial collagen fibers, and pulmonary alveoli atrophy or disappearance were appeared. Compared with group B, group C and D were observed that acute radiation pneumonitis did not reduced but, the pulmonary fibrosis increased after the 16 t h of radiation(P < 0.05).Immunohistochemical detection of lung tissue in mice showed that, compared with mices in group A, the expressiones of α-SMA, Col-1, Col-3 and TGF-β1 in mices lung tissue were significantly higher(P<0.001). At the 20 th week, the expressiones of α-SMA, Col-1, Col-3 in group C and D were obviously higher than that of group B(P < 0.05). lung CT scan images and 3D reconstruction images of the mice at 20 th week showed that pulmonary diffuse interstitial took place in all radiated mice.Conclusion:14Gy single dose radiation on chest of C57BL/6J can successfully develop radiation- induced lung injury animal models. Early CBD-b FGF intervention on mice with radiation- induced lung injury, there are no effect on alleviating the lung injury repair, but instead of promoting the occurrence and development of radioactive pulmonary fibrosis.