PGRN缺陷抑制IMQ诱导小鼠皮肤炎症组织的自噬和凋亡

PGRN deficiency inhibits autophagy and apoptosis of skin tissues in mice with IMQ-induced dermatitis

目的:探讨颗粒蛋白前体(progranulin,PGRN)缺陷型小鼠与野生(wild type,WT)型小鼠在咪喹莫特(Imiquimod,IMQ)诱导皮肤炎症模型中炎症及自噬的差异性。方法:运用IMQ涂抹WT(44只)和PGRN-/-(16只)小鼠背部皮肤构建银屑样皮肤炎症模型,并通过蛋白质印迹(Western blot)、定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)、苏木精-伊红(hematoxylin and eosin,HE)染色等方法检测小鼠组织结构、炎性因子[白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、一氧化氮合酶2(nitric oxide synthase 2,NOS2)、环氧化酶2(cyclo-oxygen-ase 2,COX2)、白细胞介素-1β(interleukin-1β,IL-1β)]、凋亡相关蛋白[半胱氨酸天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase 3,Caspase 3)]以及自噬相关蛋白[微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、泛素结合蛋白(ubiquitin binding protein,P62)、自噬相关蛋白5(autophagy related protein 5,ATG5)、自噬相关蛋白7(autophagy related protein 7,ATG7)、自噬相关蛋白5和12的复合物(complex of autophagy related protein 5 and 12,ATG5-ATG12)]的变化。结果:成功建立了小鼠皮肤炎症模型,在WT小鼠模型中观察到上皮组织明显增厚,炎性细胞浸润增多,血管生成增加,炎性因子IL-6、IL-10、TNF-α、NOS2、COX2在第6天表达最高(P<0.05),自噬随着时间的增加而增加;此外,第6天脾脏比重明显增加(P<0.05),炎性因子IL-6、IL-10、TNF-α、NOS2、COX2、IL-1β表达最高(P<0.05)。与WT模型小鼠相比,PGRN-/-模型小鼠背部皮肤加厚更加明显,炎性因子IL-6、NOS2表达更高(P<0.05),组织自噬与凋亡水平明显降低(P<0.05)。结论:WT小鼠和PGRN-/-小鼠都能够在第6天成功建立小鼠皮肤炎症模型;与WT小鼠皮肤炎症模型相比,PGRN-/-小鼠加重了皮肤炎症模型症状,这一过程可能与PGRN缺陷阻碍了自噬的产生有关。

Objective:To investigate the differences in inflammation and autophagy in imiquimod(IMQ)-induced dermatitis model between progranulin-deficient(PGRN-/-)mice and wild-type(WT)mice.Methods:The psoriasis-like dermatitis model was established by smearing the back skin of mice(44 WT mice and 16 PGRN-/-mice)with IMQ.Western blot,quantitative polymerase chain reaction(qPCR),hematoxylin and eosin(HE)staining were used to detect the changes in the tissue structure,inflammatory factors[(interleukin-6,IL-6),(interleukin-10,IL-10),(tumor necrosis factor-α,TNF-α),(nitric oxide synthase 2,NOS2),(cyclo-oxygen-ase2,COX2),(interleukin-1β,IL-1β)],apoptosis-related protein(cysteinyl aspartate specific proteinase 3,Caspase3)and autophagyrelated proteins[(microtubule-associated protein 1 light chain 3,LC3),(ubiquitin binding protein,P62),(autophagy related protein5,ATG5),(autophagy related protein 7,ATG7),(complex of autophagy related protein 5 and 12,ATG5-ATG12)]of the mice.Results:The mouse model of psoriasis-like dermatitis was successfully established.Significant thickening of epithelial tissues,increased infiltration of inflammatory cells,and more angiogenesis were observed in the WT mouse model,with the highest expression levels of IL-6,IL-10,TNF-α,NOS2,and COX2 observed on the sixth day(P<0.05),and autophagy increased with time.In addition,the proportion of spleen increased significantly on the sixth day(P<0.05),and the expression levels of inflammatory factors IL-6,IL-10,TNF-α,NOS2,COX2,and IL-1βwere highest(P<0.05).Compared with the WT mouse model,the PGRN-/-mouse model showed more significant thickening of the back skin,higher expression levels of inflammatory factors IL-6 and NOS2(P<0.05),and significantly reduced levels of autophagy and apoptosis(P<0.05).Conclusion:The dermatitis model is successfully established both in the WT mice and the PGRN-/-mice on the sixth day.The symptoms are more severe in the PGRN-/-mouse model than in the WT mouse model,which may be related to PGRN deficiency due to its role in impeding autophagy.