Lung Endothelial Cell-targeted Peptide-modified BFGF Promotes The Repair After Radiation Induced Lung Injury

Background and objective:Radiation-induced lung injury(RILI)is the main complication of survivors in nuclear accidents,affecting the quality of life of the long-term survivors.It is also the main limiting factor of radiation dose for thoracic tumors,affecting the control effect of tumors.The mechanism of RILI is very complex,involving a series of cell and molecular interactions.The main pathological manifestations are migration and infiltration of inflammatory cells,proliferation and differentiation of fibroblasts,secretion and accumulation of collagen,thickening and occlusion of blood vessel walls,destruction and remodeling of normal alveolar structure,etc.Recent studies have found that apoptosis of vascular endothelial cells within a few hours after radiation injury may be the initiation mechanism leading to the above pathological manifestations,and blocking apoptosis of vascular endothelial cells can alleviate the degree of radiation injury.Basic fibroblast growth factor(bFGF)is widely involved in embryonic development,tissue regeneration,angiogenesis and other biological processes.It also plays a role in promoting repair in the damage of skin,bone,urethra,uterus,nerve and other tissues.Studies have found that bFGF also plays a protective role in radiation injury,and the main mechanism is to inhibit the apoptosis of vascular endothelial cells.In addition,the sensitivity of microvascular endothelial cells to radiation was significantly higher than that of large vessels,which may be related to the low or even lack of bFGF distribution on the basement membrane of microvessels.The lung has a very rich microvascular system,and the lack of bFGF may be one of the reasons why the lung is very sensitive to radiation.So,the supplementation of exogenous bFGF may be an effective method to alleviate and repair RILI.However,the application of bFGF is faced with a serials of problems,such as low targeting,poor permeability and short half-life.In order to achieve therapeutic effects,the dosage of bFGF should be increased,but high doses of bFGF may cause potential risks to other normal tissues.Targeted drug delivery has been a research focus in recent years.It can increase drug concentration in lesions and reduce drug distribution in normal tissues.So it can enhance therapeutic effect and reduce side effects.Among them,targeted polypeptide is a delivery ligand with good application prospects.It has a serials of advantages,such as good permeability,convenient synthesis,low immunogenicity,low cost,easy to modify.CGSPGWVRC is a lung endothelial cell-targeted peptide(LET)screened by phage display technology.Its good targeting ability of lung endothelial cells has been proved in vitro and in vivo experiments.To sum up,we intend to modify bFGF with LET,which can improve the lung endothelial cell-targeted ability of bFGF and enhance the repair function of bFGF on RILI.Methods:1.Preparation of LET-bFGF:Based on the pET28a-bFGF plasmid,a Linker sequence(15 amino acids,45 nucleotides)and a LET sequence(9 amino acids,27 nucleotides)were ligated to the carboxy terminus of bFGF.pET28a-LET-bFGF plasmid was constructed by PCR,restriction enzyme digestion,ligation,transformation,screening,etc.The LET-bFGF recombinant protein was then expressed by E.coli expression system,purified by nickel column affinity chromatography,and finally tested for biological activity by MTT assay.2.Lung-targeted validation of LET-bFGF:Twenty-seven SD rats were randomly divided into three groups: control group,bFGF group and LET-bFGF group.Rats in the bFGF group and the LET-bFGF group were injected with bFGF and LET-bFGF(25 nmol/kg)through tail vein,respectively,and the control group was treated with saline.At 10 min,1 h and 6 h after injection,Elisa was used to detect the levels of bFGF in lung tissue and serum.Twenty-four SD rats were randomly divided into 4 groups: normal+bFGF group,radiation+bFGF group,normal+LET-bFGF group and radiation+LET-bFGF group.Normal rats were used in the normal group and RILI rats were used in the radiation group.In the bFGF group and the LET-bFGF group,Cy7-conjugated bFGF(bFGF-Cy7)and Cy7-conjugated LET-bFGF(LET-bFGF-Cy7)(500 ?L,30 ?mol/L)were injected into rats through tail vein,respectively.At 1 h after injection,the rats were imaged under IVIS imaging system.Then,various organs were dissected and imaged by the IVIS imaging system.Twenty-four SD rats were randomly divided into 4 groups: normal+bFGF group,radiation+bFGF group,normal+LET-bFGF group and radiation+LET-bFGF group.Normal rats were used in the normal group,and RILI rats were used in the radiation group.In the bFGF group and the LET-bFGF group,bFGF and LET-bFGF(100 nmol/kg)were injected into the rats through tail vein after modeling,respectively.At 1 h after injection,immunofluorescence staining was used to detect the distribution of exogenous bFGF in lung tissues.3 The repair effect of LET-bFGF on RILI rat model:Sixty SD rats were randomly divided into 4 groups: control group,radiation group,radiation+bFGF group and radiation+LET-bFGF group.Normal rats were used in the control group,and RILI rats were used in the other three groups.In the radiation+bFGF group and the radiation+LET-bFGF group,bFGF and LET-bFGF(25 nmol/kg)were injected into the rats through tail vein after modeling,respectively.Rats in the control group and the radiation group were treated with saline.In the early stage after modeling(4 h),lung tissues were taken for double staining using TUNEL and CD31.Therefore,the protective effect of LET-bFGF on apoptosis of pulmonary vascular endothelial cells was examined.In the long-term stage(2 months),the lungs were scanned by CT.Then,HE staining,Masson staining and immunohistochemical staining of CD45 were performed.Therefore,the therapeutic effect of LET-bFGF on pneumonia and pulmonary fibrosis was examined.Results:1.On the basis of pET28a-bFGF plasmid,a 45-nucleotide Linker and a 27-nucleotide LET were successfully connected to the carboxyl terminal of bFGF sequence.Finally,pET28a-LET-bFGF plasmid was obtained.The sequence was verified by sequencing to be completely correct.2.Purified bFGF and LET-bFGF were obtained by escherichia coli expression system and nickel column affinity chromatography.SDS-PAGE confirmed that both bFGF and LET-bFGF were in line with theoretical molecular weights(19.42 kd and 21.50 kd,respectively).WB confirmed that both bFGF and LET-bFGF could specifically bind to anti-bFGF antibodies.3.Both bFGF and LET-bFGF had good biological activity.The fusion of LET and Linker did not affect the biological activity of bFGF.4.After injected into rats via tail vein,LET-bFGF targeted to the lung rapidly.However,it was also qucikly metabolized in vivo.The amounts in the lung dropped to the background level after 6 h after injection,possibly mainly through renal excretion.5.Radiation injury had a certain chemotaxis effect on LET-bFGF,which could enhance the lung-targeted ability of LET-bFGF.6.At 4 h after irradiation,apoptosis mainly occurred in pulmonary vascular endothelial cells.At 2 months after irradiation,apoptosis was observed in various types of cells,including vascular endothelial cells,alveolar epithelial cells and interstitial cells.LET-bFGF could inhibit early and long-term lung vascular endothelial apoptosis induced by radiation.The inhibitory effect of LET-bFGF on apoptosis was better than that of bFGF.7.At 2 months after irradiation,a serials of pathological manifestations were observed,such as severely damaged alveolar structures,markedly thickened alveolar septa,infiltrating inflammatory cells,thickened vessel walls,and dilated and congested pulmonary capillaries.LET-bFGF could inhibit the proliferation of pulmonary blood vessel wall and alleviate the disorder of structures.The protective effect of LET-bFGF was better than that of bFGF.8.At 2 months after irradiation,inflammatory cells were widely distributed in the lung interstitium and alveoli.LET-bFGF could reduce radiation-induced lung inflammation,while the inhibitory effect of inflammatory was not observed in bFGF.9.At 2 months after irradiation,fibrosis could be obviously observed in the lungs.CT images showed a large number of high-density shadows were widely distributed in the lungs.The average lung density was significantly increased.LET-bFGF could reduce the degree of radiation-induced pulmonary fibrosis,while the inhibitory effect of fibrosis was not observed in bFGF.Conclusion:bFGF modified with LET peptide had a good lung-targeted ability and could play a good role in repairing RILI.