| BackgroundIn the accident of disabled sunk nuclear submarine,the survivors may be exposed to radiation before carrying out submarine escape.They will suffer conjoint exposure to radiation and decompression injuries.Decompression injury is prevalent in hyperbaric exposure,especially severe in submarine escape.The preposed exposure of radiation may increase the probability and severity of decompression sickness(DCS)of following decompression,which will deteriorate the survivors’ illness status after surface and during waiting rescue.However,the poor understanding of conjoint effects of radiation exposure following with decompression exposure restricts the development of emergent treatment strategies.It is necessary to improve the cognition of this pathological process of combined injury to support more timely and effective treatment.DCS is a series of pathological results induced by systemic bubble production during inappropriate dropping of environmental pressure in hyperbaric exposure.Previous studies suggested that lung is the main target organs of decompression injury,as well as one of the sensitive tissues to radiation.According to the previous reports,the recruitment and activation of neutrophils to the injury tissues played an important role in the phase of acute lung injury(ALI)induced by either decompression or radiation.Interleukin-17(IL-17)is a characteristic chemokine of activated T helper cell 17(Th17)cells.Its primary biological effects are stimulating target cells to produce a variety of cytokines and inducing recruitment of neutrophils to affected tissues.It plays an important role in the process of inflammatory diseases and immune diseases.Recent studies suggested that IL-17 is also a critical factors in the inflammatory process of ALI.This research is to observe the individual or combined effects of this two types of injuries on the severity of lung damage,to define the role of IL-17 and various cytokines in the potential mechanism,which will improve the theoretical knowledge for developing the onsite emergence life support and hospital treatment strategies of the nuclear submarine accident survivors.MethodFirstly,the radiation dose of the combined injury model was determined by observing the effects of different doses of radiation exposure on the survival and myelogram of rats.By observing the effects of a certain dose of radiation combined with different decompression loads on the mortality of rats with decompression sickness and lung wet-dry weight ratio,the decompression exposure protocol of the combined injury model was determined.After that,sixty 7-week-old male SD rats weighing 220-250 g were selected to establish the radiation combined with decompression injury model,and they were divided into the control group,radiation group,decompression group and combined group.A single dose of 4 Gy whole-body irradiation and a constant rate of decompression 35 s after 45 min exposure to 57 m pressure in air were used,respectively.After decompression,the incidence of DCS was observed,the lung wet-dry weight ratio was measured and pathological lung tissue damage was observed by HE staining.The extent of neutrophil infiltration into lung tissue was demonstrated by immunohistochemistry.Meanwhile,the levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor α(TNF-α),glutathione peroxidase(GSH-Px),malondialdehyde(MDA)in bronchoalveolar lavage fluid(BALF)and the levels of intercellular adhesion molecule 1(ICAM-1),nitric oxide(NO)in peripheral serum were measured by enzyme-linked immunosorbent assay(ELISA)or biochemical method,in order to explore whether radiation exposure will aggravate the severity of lung injury caused by decompression.On this basis,single-cell transcriptome sequencing was used to identify the cell types that played a key role in the combined injury,and KEGG pathway enrichment analysis was used to screen out the pathways related to lung injury induced by radiation combined with decompression.The proportion of Th17 cells in lung tissue was further detected by flow cytometry.The Th17 cell differentiation related molecule IL-23 and retinoic acid receptorrelated orphan receptor γt(RORγt),IL-17 and its receptor,IL-17 pathway related molecules and its downstream chemokines and inflammatory factors were detected by RT-q PCR and Western Blot,so as to determine whether IL-17 was involved in the process of lung injury induced by radiation combined with decompression.In order to further verify the role of IL-17,sixty-four 7-week-old male SD rats weighing220-250 g were selected and divided into control group,combind group,isotype group and neutralizing antibody group.In the neutralizing antibody group,IL-17 neutralizing antibody(100 μg/kg)was intraperitoneally injected.Rats in the isotype group were intraperitoneally injected with Ig G1 isotype antibody(100 μg/kg),and rats in the other groups were intraperitoneally injected with an equal volume of normal saline.By observing the incidence of DCS,weighing the lung wet-dry weight ratio,observing the pathological injury of lung tissue,detecting the degree of neutrophil infiltration in lung tissue,the levels of inflammatory factors in BALF,the expression changes of IL-17 pathway related molecules and their downstream chemokines and inflammatory factors,this study verified whether blocking IL-17 could ameliorate lung injury induced by radiation combined with decompression.ResultIn the radiation dose screening experiment,the myelogram of the animals showed significant changes after 3 days of exposure to 4 Gy dose,and the dose did not reach the lethal dose,which could be used as a radiation dose in the study of the biological effect of combined injury.In the experiment of radiation combined with different decompression loads,the animals exposed to 4 Gy radiation combined with 45 min high pressure exposure had obvious damage changes and the mortality rate could be controlled below 50%,which was suitable for the subsequent study on the biological effects and mechanism of combined injury.Therefore,in the subsequent experiments,we selected a radiation and decompression protocol of 4 Gy and 45 min exposure to construct a combined injury animal model.In the combined injury effect experiment,the incidence of DCS in the combined group was significantly higher than that in the decompression group(93.33% vs.60.00%,P<0.05).Compared with the radiation group and the decompression group,the lung wet-dry weight ratio of the combined group(4.68 ± 0.19 vs.4.07 ± 0.32,P<0.01;4.68 ± 0.19 vs.4.23 ± 0.11,P<0.05)was significantly increased,and the degree of histopathological damage and neutrophil infiltration in lung tissue were increased.The results of ELISA showed that compared with the radiation group and decompression group,the levels of IL-1β(80.58 ±10.15 vs.53.78 ± 5.88 ng/L,P<0.01;80.58 ± 10.15 vs.64.68 ± 6.22 ng/L,P<0.01),IL-6(229.36 ± 10.64 vs.172.34 ± 12.30 ng/L,P<0.01;229.36 ± 10.64 vs.169.61 ± 16.64 ng/L,P<0.01),TNF-α(453.04 ± 40.71 vs.354.46 ± 25.97 ng/L,P<0.01;453.04 ± 40.71 vs.338.52± 14.86 ng/L,P<0.01)in BALF and ICAM-1(74.39 ± 11.83 vs.58.70 ± 4.19 ng/L,P<0.01;74.39 ± 11.83 vs.47.29 ± 5.45 ng/L,P<0.01)in serum were all significantly increased in the combined group.The results of biochemical tests showed that compared with the radiation group and decompression group,the levels of GSH-Px(106.64 ± 19.46 vs.145.95 ± 18.36 μmol/L,P<0.01;106.64 ± 19.46 vs.163.18 ± 20.60 μmol/L,P<0.01)in BALF of the combined group was significantly decreased,and the contents of MDA(18.63± 2.02 vs.13.99 ± 1.87 nmol/ml,P<0.01;18.63 ± 2.02 vs.12.03 ± 2.10 nmol/ml,P<0.01)in BALF and NO(67.25 ± 14.75 vs.50.86 ± 14.07 μmol/L,P<0.01;67.25 ± 14.75 vs.49.76 ±12.30 μmol/L,P<0.01)in serum were significantly increased.The results of single-cell transcriptome sequencing showed that the changes of neutrophils were the most significant in the combined injury,and the results of KEGG pathway enrichment analysis suggested that IL-17 signaling pathway may play an important role in the development of lung injury induced by radiation combined with decompression.The results of flow cytometry showed that the percentage of Th17 cells in the lung tissue of the combined group(2.59 ± 0.43 vs.2.16 ± 0.27,P<0.05;2.59 ± 0.43 vs.1.91 ±0.17,P<0.01)was significantly higher than that of the radiation group and decompression group.The results of RT-q PCR and Western Blot showed that the m RNA and protein expression levels of the Th17 cell differentiation related molecules IL-23 and RORγt,IL-17 and its receptor in the combined group were significantly higher(P<0.05)than those of the radiation group and decompression group.In addition,the expression levels of IL-17 pathway related molecules and their downstream chemokines and inflammatory factors were increased in different degrees(P<0.05)in the combined group.After blocking the binding of IL-17 and its receptor with IL-17 neutralizing antibody,the DCS incidence of rats in the neutralizing antibody group was significantly lower than that in the combined group(37.50% vs.75.00%,P<0.05).Compared with the combined group and the isotype group,the lung wet-dry weight ratio of the neutralizing antibody group(3.10 ± 0.29 vs.3.71 ± 0.35,P<0.01;3.10 ± 0.29 vs.3.69 ± 0.40,P<0.01)was significantly decreased,and the degree of lung pathological injury and neutrophil infiltration in the neutralizing antibody group were also decreased.The results of the detection of inflammatory factors showed that compared with the combined group and isotyp group,the levels of IL-1β(30.33 ± 1.15 vs.35.88 ± 2.68 ng/L,P<0.01;30.33 ± 1.15 vs.35.40 ± 1.58ng/L,P<0.05),IL-6(79.14 ± 5.40 vs.100.83 ± 9.26 ng/L,P<0.01;79.14 ± 5.40 vs.101.5 ±12.77 ng/L,P<0.01),TNF-α(271.12 ± 25.80 vs.305.21 ± 11.93 ng/L,P<0.05;271.12 ±25.80 vs.305.38 ± 23.89 ng/L,P<0.05)in BALF of the neutralizing antibody group were significantly decreased.At the same time,RT-q PCR and Western blot results showed that the expression levels of IL-17 pathway related molecules and its downstream chemokines and inflammatory factors were significantly decreased after blocking IL-17(P<0.05).ConclusionExposure to radiation can increase the incidence of DCS and aggravate the severity of lung injury caused by decompression.The injurious inflammatory response of lung tissue is strongly connected with the process of lung injury induced by radiation combined with decompression,which may be the result of upstream regulation of multiple inflammatory factors,rather than the role of a single inflammatory factor.As a pro-inflammatory factor,IL-17 mediates the recruitment of neutrophils in lung tissue and further leads to the expansion of inflammatory response,which is involved in the occurrence of lung injury induced by radiation combined with decompression.After blocking IL-17 with neutralizing antibody,the lung injury induced by radiation combined with decompression can be significantly alleviated.In this study,we observed that radiation exposure aggravated the severity of lung injury induced by decompression,and found that pre-radiation exposure aggravated the inflammatory response of decompression injury by activating proinflammatory factors,which in turn led to the aggravation of lung injury induced by decompression,confirming the key role of the pro-inflammatory factor IL-17.This study suggests that the medical assistance of escaped crew members in nuclear submarine accidents requires timely intervention in radiation injury combined with decompression injury to block the progression of the disease and improve the final probability of rescue. |
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