Influence of reactive oxygen species responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats
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摘要:
目的 探究负载焦亡抑制剂的活性氧响应性自组装纳米胶束对糖尿病大鼠全层皮肤缺损的影响。 方法 采用实验研究方法。用纳米胶束聚乙二醇-嵌段-聚丙烯硫醚（PEG-b-PPS）包封核苷酸结合寡聚化结构域（NOD）1/2抑制剂（NOD-IN-1），将所得产物称为PEPS@NOD-IN-1。利用透射电子显微镜和粒度分析仪分别观测PEG-b-PPS与PEPS@NOD-IN-1的形貌和水合粒径，用酶标仪测量并计算PEPS@NOD-IN-1对NOD-IN-1的包封率和载药率以及PEPS@NOD-IN-1在单纯磷酸盐缓冲液（PBS）和含过氧化氢的PBS中40 h内对NOD-IN-1的累积释放率，样本数均为3。取24只6~7周龄雄性SD大鼠，通过注射链脲佐菌素的方法诱导1型糖尿病，在每只大鼠背部制作6个全层皮肤缺损创面，按随机数字表法将致伤大鼠分为进行相应处理的PBS组、NOD-IN-1组、PEG-b-PPS组、PEPS@NOD-IN-1组，每组6只。伤后3、7、12 d观察创面愈合情况并计算创面愈合率；伤后3 d，采用免疫荧光法检测创面组织中活性氧水平；伤后7 d，利用苏木精-伊红染色评估创面肉芽组织厚度，采用实时荧光定量反转录PCR法检测创面组织中NOD1、NOD2的mRNA表达，采用蛋白质印迹法检测创面组织中NOD1、NOD2、GSDMD-N端的蛋白表达。前述指标均各取各组不同鼠的共6个创面检测。另取PBS组和PEPS@NOD-IN-1组大鼠伤后7 d创面组织（各3个样本），利用高通量测序技术平台进行转录组测序，筛选出PEPS@NOD-IN-1组相较于PBS组显著下调的差异表达基因（DEG），进行京都基因与基因组百科全书（KEGG）富集分析；制作焦亡相关通路NOD样受体通路DEG热图；通过STRING数据库对热图中的DEG进行蛋白质-蛋白质相互作用（PPI）分析，筛选PEPS@NOD-IN-1调控NOD样受体通路的关键基因。对数据行重复测量方差分析、单因素方差分析、Tukey检验。 结果 PEG-b-PPS与PEPS@NOD-IN-1均为大小较为均一的球形结构，水合粒径分别为（134.2±3.3）、（143.1±2.3）nm。PEPS@NOD-IN-1对NOD-IN-1的包封率为（60±5）%、载药率为（15±3）%。PEPS@NOD-IN-1在单纯PBS中对NOD-IN-1的释放较缓慢，40 h累积释放率仅为（12.4±2.3）%；PEPS@NOD-IN-1在含过氧化氢的PBS中10 h内对NOD-IN-1的释放十分迅速，10 h累积释放率已达（90.1±3.6）%。伤后3、7 d，4组大鼠创面均逐渐愈合，PEPS@NOD-IN-1组愈合情况优于其余3组；伤后12 d，PBS组创面结痂面积较大，NOD-IN-1组、PEG-b-PPS组创面上皮化明显，PEPS@NOD-IN-1组创面接近完全上皮化。与PBS组、NOD-IN-1组及PEG-b-PPS组比较，PEPS@NOD-IN-1组大鼠伤后7、12 d创面愈合率均显著增高（P<0.05），伤后3 d创面组织中活性氧水平显著下降（P<0.05），伤后7 d创面肉芽组织厚度显著增厚（P<0.05），伤后7 d创面组织中NOD1、NOD2的mRNA表达以及NOD1、NOD2、GSDMD-N端的蛋白表达均显著下降（P<0.05）。KEGG通路分析显示，PEPS@NOD-IN-1组相较于PBS组显著下调的DEG在NOD样受体、缺氧诱导因子、丝裂原活化蛋白激酶和肿瘤坏死因子（TNF）通路方面显著富集。在NOD样受体通路的DEG热图中，可见调控细胞焦亡的基因主要涉及NOD1、NOD2、NOD样受体热蛋白结构域相关蛋白3、Jun、信号转导及转录激活因子1（STAT1）、TNF-α诱导蛋白3。PPI结果显示，NOD1、NOD2、STAT1为PEPS@NOD-IN-1调控NOD样受体通路的关键基因。 结论 PEPS@NOD-IN-1能下调创面局部活性氧水平及细胞焦亡关键调节因子NOD1、NOD2、GSDMD-N端的表达，进而促进糖尿病大鼠全层皮肤缺损创面修复；PEPS@NOD-IN-1还可显著下调创面的焦亡、炎症及缺氧相关通路，通过下调关键基因NOD1、NOD2、STAT1调控NOD样受体通路。 Abstract:Objective To investigate the influence of reactive oxygen species (ROS) responsive self-assembled nanomicelle loaded with pyroptosis inhibitor on full-thickness skin defects in diabetic rats. Methods Experimental research methods were employed. A nucleotide-binding oligomerization domain (NOD) 1/2 inhibitor (NOD-IN-1) was encapsulated with nanomicelle polyethylene glycol-block-polypropylene sulfide (PEG-b-PPS), and the resulting product was called PEPS@NOD-IN-1. The morphology and hydration particle size of PEG-b-PPS and PEPS@NOD-IN-1 were observed by transmission electron microscope and particle size analyzer, respectively, and the encapsulation rate and drug loading rate of PEPS@NOD-IN-1 to NOD-IN-1 and the cumulative release rate of NOD-IN-1 by PEPS@NOD-IN-1 in phosphate buffer solution (PBS) alone and hydrogen peroxide-containing PBS within 40 h were measured and calculated by microplate reader, and the sample number was 3. Twenty-four male Sprague-Dawley rats aged 6-7 weeks were injected with streptozotocin to induce type 1 diabetes mellitus. Six full-thickness skin defect wounds were made on the back of each rat. The injured rats were divided into PBS group, NOD-IN-1 group, PEG-b-PPS group, and PEPS@NOD-IN-1 group with corresponding treatment according to the random number table, with 6 rats in each group. The wound healing was observed on post injury day (PID) 3, 7, and 12, and the wound healing rate was calculated. The ROS levels in wound tissue were detected by immunofluorescence method on PID 3. On PID 7, the granulation tissue thickness in wound was assessed by hematoxylin-eosin staining, the mRNA expressions of NOD1 and NOD2 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction, and the protein expressions of NOD1, NOD2, and GSDMD-N terminals were detected by Western blotting. Six wounds from different rats in each group were taken for detection of the above indicators. Wound tissue (3 samples per group) was taken from rats in PBS group and PEPS@NOD-IN-1 group on PID 7, and transcriptome sequencing was performed using high-throughput sequencing technology platform. Differentially expressed genes (DEGs) significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were screened, and the enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. The DEG heatmap of the NOD-like receptor pathway, a pyroptosis-related pathway, was made. Protein-protein interaction (PPI) analysis of DEGs in heatmap was performed through the STRING database to screen key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey test. Results PEG-b-PPS and PEPS@NOD-IN-1 were in spherical structures of uniform size, with hydration particle sizes of (134.2±3.3) and (143.1±2.3) nm, respectively. The encapsulation rate of PEPS@NOD-IN-1 to NOD-IN-1 was (60±5)%, and the drug loading rate was (15±3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in PBS alone was slow, and the cumulative release rate at 40 h was only (12.4±2.3)%. The release of NOD-IN-1 from PEPS@NOD-IN-1 in hydrogen peroxide-containing PBS within 10 h was very rapid, and the cumulative release rate at 10 h reached (90.1±3.6)%. On PID 3 and 7, the wounds of rats in the four groups were gradually healed, and the healing in PEPS@NOD-IN-1 group was better than that in the other three groups. On PID 12, the wound scab area in PBS group was large, the wound epithelialization in NOD-IN-1 group and PEG-b-PPS group was obvious, and the wound in PEPS@NOD-IN-1 group was close to complete epithelialization. Compared with those in PBS group, NOD-IN-1 group, and PEG-b-PPS group, the wound healing rates on PID 7 and 12 in PEPS@NOD-IN-1 group were significantly increased (P<0.05), the level of ROS in wound tissue on PID 3 was significantly decreased (P<0.05), the thickness of granulation tissue in wound on PID 7 was significantly thickened (P<0.05), and the mRNA expressions of NOD1 and NOD2 and the protein expressions of NOD1, NOD2, and GSDMD-N terminals in wound tissue on PID 7 were significantly decreased (P<0.05). KEGG pathway analysis showed that DEGs significantly down-regulated in PEPS@NOD-IN-1 group as compared with PBS group were significantly enriched in NOD-like receptors, hypoxia-inducible factors, mitogen-activated protein kinases, and tumor necrosis factor (TNF) pathways. In the DEG heatmap of NOD-like receptor pathway, the genes regulating pyroptosis mainly involved NOD1, NOD2, NOD-like receptor thermoprotein domain-related protein 3, Jun, signal transduction and transcriptional activator 1 (STAT1), TNF-α-induced protein 3. The PPI results showed that NOD1, NOD2, and STAT1 were the key genes of PEPS@NOD-IN-1 regulating the NOD-like receptor pathway. Conclusions PEPS@NOD-IN-1 can down-regulate the level of local ROS in wounds and the expression of NOD1, NOD2, and GSDMD-N terminals, the key regulators of pyroptosis, thereby promoting the repair of full-thickness skin defect wounds in diabetic rats. PEPS@NOD-IN-1 can also significantly down-regulate the pyroptosis, inflammation, and hypoxia-related pathways of wounds, and regulate NOD-like receptor pathways by down-regulating key genes NOD1, NOD2, and STAT1. -
参考文献
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1 PEG-b-PPS和PEPS@NOD-IN-1的表征。1A、1B.分别为PEG-b-PPS、PEPS@NOD-IN-1形貌，均为大小较为均一的球形结构 透射电子显微镜×20 000；1C.PEG-b-PPS和PEPS@NOD-IN-1的水合粒径；1D.PEPS@NOD-IN-1在单纯PBS和含过氧化氢的PBS中40 h内对NOD-IN-1的释放曲线(样本数为3，
注：PEG-b-PPS为聚乙二醇-嵌段-聚丙烯硫醚，NOD-IN-1为核苷酸结合寡聚化结构域1/2抑制剂，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得，PBS为磷酸盐缓冲液；图1C为横坐标经过lg处理的数据形成的描记图
2 4组糖尿病全层皮肤缺损大鼠伤后各时间点创面大体愈合情况。2A、2B、2C.分别为PBS组伤后3、7、12 d创面，愈合较慢；2D、2E、2F和2G、2H、2I.分别为NOD-IN-1组和PEG-b-PPS组伤后3、7、12 d创面，图2D、2G创面较图2A干燥，图2E、2H创面开始结痂且创面面积稍小于图2B，图2F、2I创面结痂面积均小于图2C；2J、2K、2L.分别为PEPS@NOD-IN-1组伤后3、7、12 d创面，图2J创面较图2A干燥，图2K创面面积明显小于图2B、2E和2H，图2L创面上皮化接近完全且基本无痂皮
注：创面上方白色硅胶环为参照物，直径为10 mm；PBS为磷酸盐缓冲液，NOD-IN-1为核苷酸结合寡聚化结构域1/2抑制剂，PEG-b-PPS为聚乙二醇-嵌段-聚丙烯硫醚，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得
3 4组糖尿病全层皮肤缺损大鼠伤后7 d创面肉芽组织厚度及伤后3 d创面活性氧水平。3A、3B、3C、3D.分别为PBS组、NOD-IN-1组、PEG-b-PPS组和PEPS@NOD-IN-1组伤后7 d肉芽组织厚度（黑色双箭头的长度）情况，图3D肉芽组织厚度较图3A、3B和3C明显增厚 苏木精-伊红×20；3E、3F、3G、3H.分别为PBS组、NOD-IN-1组、PEG-b-PPS组和PEPS@NOD-IN-1组伤后3 d活性氧（红色）水平，图3H活性氧水平显著低于图3E、3F、3G 二氢乙锭+4'，6-二脒基-2-苯基吲哚×120
注：PBS为磷酸盐缓冲液，NOD-IN-1为核苷酸结合寡聚化结构域1/2抑制剂，PEG-b-PPS为聚乙二醇-嵌段-聚丙烯硫醚，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得
4 蛋白质印迹法检测4组糖尿病全层皮肤缺损大鼠伤后7 d创面组织中焦亡相关指标蛋白表达
注：条带上方1、2、3、4分别指示磷酸盐缓冲液组、核苷酸结合寡聚化结构域（NOD）1/2抑制剂（NOD-IN-1）组、聚乙二醇-嵌段-聚丙烯硫醚（PEG-b-PPS）组和PEPS@NOD-IN-1组，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得
5 PEPS@NOD-IN-1组糖尿病全层皮肤缺损大鼠伤后7 d创面相较于PBS组显著下调的DEG的KEGG富集分析与NOD样受体通路DEG热图。5A.KEGG富集分析中排名前20的通路；5B.NOD样受体通路DEG热图
注：DEG为差异表达基因，PBS为磷酸盐缓冲液，PEPS@NOD-IN-1为用聚乙二醇-嵌段-聚丙烯硫醚包封核苷酸结合寡聚化结构域（NOD）1/2抑制剂（NOD-IN-1）所得，KEGG为京都基因与基因组百科全书；图5B中左侧3列为PBS组样本，右侧3列为PEPS@NOD-IN-1组样本；MAPK为丝裂原活化蛋白激酶，TNF为肿瘤坏死因子，OAS为寡聚腺苷酸合成酶，NAIP2为神经元凋亡抑制蛋白2，NLRP3为NOD样受体热蛋白结构域相关蛋白3，GBP为鸟苷酸结合蛋白，IRF为干扰素调节因子，IFI为干扰素激活基因，STAT为信号转导及转录激活因子，CCL为趋化因子配体，GABARAPL1为γ氨基丁酸受体相关蛋白样1，TRAF为TNF受体相关因子，RIPK2为受体相互作用丝氨酸/苏氨酸激酶2，ITPR2为肌醇1,4,5-三磷酸受体2型，IKBKG为K轻链多肽抑制基因，TLR4为Toll样受体4，NAMPT为烟酰胺磷酸核糖转移酶，TNFAIP3为TNF-α诱导蛋白3
6 PEPS@NOD-IN-1组糖尿病全层皮肤缺损大鼠伤后7 d创面相较于PBS组显著下调的NOD样受体通路差异表达基因的蛋白质-蛋白质相互作用分析中存在相互作用者
注：PEPS@NOD-IN-1为用聚乙二醇-嵌段-聚丙烯硫醚包封核苷酸结合寡聚化结构域（NOD）1/2抑制剂（NOD-IN-1）所得，PBS为磷酸盐缓冲液；一个圆圈代表一个基因，圆圈之间的连线代表相互作用，连线越多说明相互作用越强
表1 4组糖尿病全层皮肤缺损大鼠伤后各时间点创面愈合率比较（%，
组别 样本数 3 d 7 d 12 d PBS组 6 11.5±3.4 33.8±4.1a 43.7±5.2a NOD-IN-1组 6 14.8±1.9 39.7±2.8a 49.2±3.4a PEG-b-PPS组 6 14.3±2.5 42.8±3.7a 51.3±4.6a PEPS@NOD-IN-1组 6 15.7±4.2 63.2±3.8 82.4±4.9 F值 1.09 37.15 43.99 P值 0.068 <0.001 <0.001 注：PBS为磷酸盐缓冲液，NOD-IN-1为核苷酸结合寡聚化结构域1/2抑制剂，PEG-b-PPS为聚乙二醇-嵌段-聚丙烯硫醚，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得；处理因素主效应，F=68.20，P<0.001；时间因素主效应，F=396.80，P<0.001；两者交互作用，F=14.33，P<0.001；与PEPS@NOD-IN-1组比较，aP<0.05 
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表2 4组糖尿病全层皮肤缺损大鼠伤后7 d创面组织中焦亡相关指标mRNA和蛋白表达比较（
组别 样本数 mRNA 蛋白 NOD1 NOD2 NOD1 NOD2 GSDMD-N端 PBS组 6 1.000±0.045a 1.000±0.053a 1.00±0.04a 1.00±0.03a 1.00±0.04a NOD-IN-1组 6 0.863±0.052a 0.783±0.067a 0.72±0.04a 0.71±0.06a 0.76±0.07a PEG-b-PPS组 6 0.834±0.025a 0.741±0.076a 0.76±0.05a 0.68±0.03a 0.69±0.13a PEPS@NOD-IN-1组 6 0.132±0.031 0.153±0.024 0.15±0.03 0.16±0.04 0.21±0.05 F值 1 235.23 698.73 897.79 750.64 219.73 P值 <0.001 <0.001 <0.001 <0.001 <0.001 注：PBS为磷酸盐缓冲液，NOD-IN-1为核苷酸结合寡聚化结构域（NOD）1/2抑制剂，PEG-b-PPS为聚乙二醇-嵌段-聚丙烯硫醚，PEPS@NOD-IN-1为用PEG-b-PPS包封NOD-IN-1所得；与PEPS@NOD-IN-1组比较，aP<0.05
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