To Investigate The Effect Of LCN2 On Macrophage Polarization In Severe Hypothermia-induced Acute Lung Injury By RNA-sequencing

Objective:Acute lung injury（ALI）is a serious complication of severe hypothermia and an important research content of naval military medical service.In this study,a rat model of severe hypothermia-induced ALI was successfully established by low temperature seawater immersion,and the supernatant of Bronchoalveolar lavage fluid（BALF）was extracted and stimulated on alveolar macrophage line NR8383 cells.The gene with the highest differential expression was lipocalin-2（LCN2）by transcriptome sequencing.Then we constructed a NR8383 macrophage cell line with stable low expression of LCN2,and used adeno-associated virus vectors that interfere with LCN2 expression in ALI model rats to clarify the effect of LCN2 on macrophage polarization in vitro and in vivo animal levels.Methods:1.Establish a rat model of ALI induced by seawater immersion hypothermia and detect the polarization state of alveolar macrophagesNine-week-old healthy male Sprague-Dawley（SD）rats were randomly divided into three groups:seawater immersion 3h group（n=8）,seawater immersion 5h group（n=8）and seawater immersion 7h group（n=8）.After the rats were immersed in artificial seawater at 15℃for a corresponding period of time,the survival rate,blood gas indexes,lung injury pathology and wet-to-dry ratio of each group were tested,so as to judge whether the model was successfully established.Besides,the changes of main inflammation（IL-6,IL-10,TNF-α）,oxidation indexes（SOD,MDA,GSH-PX）,blood coagulation function,and liver and kidney function in rats were detected,to clarify the change rules of the above indexes in severe hypothermia-induced ALI.After successful modeling,the BALF supernatant of rats in the best modeling time,namely seawater immersion 5h group,was taken as the subsequent intervention factor.At the same time,the polarization state of alveolar macrophages in rats was detected by flow cytometry and q RT-PCR.2.Screen The differential genes of NR8383 macrophages stimulated by BALF supernatant on RNA-SeqNR8383 macrophages were divided into the Control group（n=3）and the Hypothermia group（n=3）.They were stimulated with PBS and BALF supernatant for24h,respectively.The cells were collected and subjected to transcriptome sequencing.After sequencing,the differential genes and pathways were screened out using DESeq2 software under the condition of Padj<0.05 and|log2Fold Change|≥1.Meanwhile,the differential genes were functionally classified based on GO and KEGG databases,and the screened differential genes were verified in another 10 SD rats by q RT-PCR（Quantitative real-time-PCR,q RT-PCR）.Finally,gene LCN2 with the highest differential expression multiple was used as the subsequent research object.3.Detect the polarization morphology of NR8383 macrophages stimulated by BALF supernatant after interfering with LCN2 expression at the cellular level in vitroThree lentivirus sh RNA sequences of LCN2 and one NC sequence were constructed and connected to a lentivirus vector,and the packed lentivirus was used to infect NR8383 target cells.After successful infection,the interference effect of the interference vector on the target gene LCN2 was evaluated by using a q RT-PCR,and a NR8383 cell line with low-stable expression of LCN2 was obtained by screening with puromycin.The constructed NR8383 cell line with stable and low expression of LCN2was used as the research object.The cells were divided into the Control group,Hypothermia group,Hypothermia+empty vector group and Hypothermia+si-LCN2group,and five holes were plated in each group.Cells in all groups were stimulated with BALF supernatant for 24h,except for the Control group which was treated with PBS.The expressions of intracellular proteins i NOS and Arg-1 were detected by Western blotting（WB）,and the polarization state of macrophages in all groups was detected by flow cytometry.4.Detect the polarization of alveolar macrophages and the recovery of lung injury after interfering with the expression of LCN2 in the severe hypothermia-induced ALI rat modelAn adeno-associated virus vector（AAV6-LCN2-RNAi）that interfered with the expression of LCN2 was constructed based on the screened sh RNA sequence with the best interference effect for subsequent animal experiments.The animal experiments were also divided into the Control group,Hypothermia group,Hypothermia+empty vector group and Hypothermia+si-LCN2 group,with five rats in each group.In the first two weeks of the experiment,40ul(virus titer:1.0×10¹³vg/ml)of negative control virus and AAV6-LCN2-RNAi adeno-associated virus were injected into nasal cavity of rats in the Hypothermia+empty vector group and Hypothermia+si-LCN2 group respectively,While the Control group and Hypothermia group did not receive any treatment.After rats were immersed in seawater at 15℃for 5h,WB and q RT-PCR were used to detect the interference effect of LCN2,and Immunofluorescence（IF）was used to detect the polarization state of macrophages in the lung of rats.Eventually,the effect of interfering LCN2 expression on the severity of lung injury was confirmed by pathological damage assessment and ELISA detection of inflammatory factor concentration.Results:1.Successfully established the animal model of ALI induced by seawater immersion hypothermia,and confirmed the imbalance of macrophage polarization in the lung of ALI model rats,mainly M1 macrophagesThe survival rate after seawater immersion for different times was 87.5%（3h group）.75%（5h group）;50%（7h group）.After 2h immersion in 15℃seawater,the core body temperature of the rats dropped to the ambient temperature.After 3h of immersion in seawater,the decreased respiratory rate,the destruction of alveolar structure and the infiltration of inflammatory cells in the lung were observed in the rats,indicating that ALI could be observed after 3h of immersion in 15℃seawater.With the prolongation of the immersion time,the wet-to-dry ratio（W/D）and the pathological score of lung injury reached the highest at 5h（P<0.05）,suggesting that5h of immersion in 15℃seawater was the optimal time for modeling.Studies on a variety of inflammatory and oxidative indicators showed that the short-term seawater immersion in the lung was dominated by the increase of inflammatory indicators（IL-6、TNF-α）,which reached the peak at 5h（P<0.05）.With the prolongation of the immersion time,the MDA increased gradually（P<0.05）.Compared with the control group,prothrombin time,partial thromboplastin time,ALT,total protein,creatinine and urea nitrogen in seawater immersion groups increased with the prolongation of immersion time,and some differences had statistical significance（P<0.05）.After successful modeling,M1 and M2 macrophages were labeled with i NOS and Arg-1respectively.The alveolar macrophages of Control group and Hypothermia group（seawater immersion 5h group）were detected by flow cytometry and verified by q RT-PCR.The results showed that compared with the Control group,the proportion of M1 cells in the Hypothermia group was significantly increased,and the difference was statistically significant（P<0.05）.2.The differentially expressed genes in NR8383 macrophages stimulated by BALF supernatant were screened by RNA-Seq,among which LCN2 had the highest fold changeThrough the difference comparison between the Control group and the Hypothermia group（NR8383 macrophages stimulated by BALF supernatant）,a total of 236 differential genes were screened out,including 147 up-regulated genes and 89down-regulated genes.The functions of these differential genes are mainly enriched in the processes of sterol,lipid biosynthesis/metabolism,IFN-γsynthesis,inflammatory response,ROS biosynthesis/metabolism and so on.By combining the Padj value,FPKM value and|log2Fold Change|value of the differential genes,we screened out 10 core genes:Ldlr,Dhcr24,Cyp51,LCN2,Ccl2,Fabp4,Clec7a,Acsl1,Cybb and Agt,and verified them in a rat model.Finally,we screened out the gene LCN2 with the highest differential expression multiple from it for subsequent research.3.After interfering with LCN2 expression in vitro,NR8383 macrophages stimulated by BALF supernatant polarized to M2The three constructed lentivirus interference vectors were sequenced,and after identified as error-free,the NR8383 cells were infected with lentivirus,and when the cells proliferated to about 2×10⁵cells/ml,puromycin with a final concentration of1ug/ml was added for screening.According to the verification of q RT-PCR,it displayed that compared with the negative control sequence,all three sh RNA fragments had a certainsilencingeffect.Amongthem,LCN2-sh RNA-3（CCGG-GCATCTGACCTTATTTG-CTCGAG-CAAATAAGGGGATGATGC-TTTT）demonstrated the most significant inhibition on LCN2 m RNA.The LCN2-sh RNA-3group was the finally constructed NR8383 cell line with low expression of LCN2.The cells in each group were stimulated by PBS and BALF supernatant respectively,WB results of cellular experiments indicated that compared with the Control group,the i NOS expression in NR8383 cells of the Hypothermia group and Hypothermia+empty vector group increased,while the Arg-1 expression decreased.In the Hypothermia+si-LCN2 group,the expression of i NOS decreased,and the expression of Arg-1 increased（P<0.05）.M1 and M2 macrophages were labeled with i NOS and Arg-1,separately,for flow cytometry detection.Compared with the Control group,the results showed that the proportion of M1 cells in the Hypothermia group and Hypothermia+empty vector group significantly grew,while the proportion of M2 cells remarkably shrank,and the proportion of M1/M2 dramatically rose.In the Hypothermia+si-LCN2 group,the percentage of M1 cells substantially fell,while that of M2 cells considerably expanded,and the proportion of M1/M2 was decreased（P<0.05）.4.In the rat model of severe hypothermia-induced ALI,after interfering with the expression of LCN2,macrophages in the lung polarized to M2,and the severity of lung injury was reducedThe adeno-associated virus vector interfering with LCN2 expression was successfully constructed and infected rats,which were immersed in low temperature seawater at 15°C,the immunofluorescence results of animal experiments showed that compared with the Control group,the proportion of M1 macrophage positive cells in the Hypothermia group and Hypothermia+empty vector group increased,while the proportion of M2 macrophage positive cells decreased.In the Hypothermia+si-LCN2 group,M1 macrophages descended,and M2 macrophages ascended（P<0.05）.Compared with the Control group,the pathological damage scores and wet-to-dry ratio in the Hypothermia group and Hypothermia+empty vector group grew,while the pathological damage score and wet-to-dry ratio in the Hypothermia+si-LCN2 group were declined significantly.The release of inflammatory factors IL-6 and TNF-αin serum and alveolar tissue was decreased（P<0.05）.Conclusion:1.A stable and easily replicated model of acute lung injury induced by immersion hypothermia of simulate personnel falling overboard was established,and the optimal modeling time was to immerse in seawater at 15℃for 5h.Otherwise,the imbalance of macrophage polarization in the lungs of severe hypothermia-induced ALI rats was confirmed,mainly M1 macrophages.2.10 key genes were screened from alveolar macrophages of severe hypothermia-induced ALI rats,and the differential gene LCN2 with the highest differential multiple expression was screened out at the same time.3.The lentiviral vector interfering with LCN2 expression was successfully constructed by RNA interference technology.After the lentivirus was infected into the target cells,the NR8383 macrophage strain with stable low expression of LCN2 was cultured and screened.After stimulation with BALF supernatant of severe hypothermia-induced ALI rats,it was found that interfering with LCN2 expression could promote macrophage polarization to M2 phenotype.4.The use of adeno-associated virus vector to interfere with LCN2 expression in severe hypothermia-induced ALI rats can change the polarization state of alveolar macrophages,increase the proportion of M2 macrophages,reduce the release of inflammatory factors,and reduce the degree of lung injury.