伪狂犬病病毒对小鼠脾脏细胞焦亡相关因子表达的影响

Effect of Pseudorabies virus on the expression of pyroptosis-related factors in mouse spleen

为了研究感染猪伪狂犬病病毒(Pseudorabies virus,PRV)对小鼠脾脏细胞焦亡相关因子的影响,试验将40只Balb/c小鼠随机分为对照组和试验组(包括48小时组、72小时组、96小时组),试验组小鼠皮下接种1×10TCID/100μL PRV 200μL,对照组小鼠注射等量生理盐水。采集小鼠脾脏,采用酶联免疫吸附试验检测脾脏组织中白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-18(interleukin-18,IL-18)水平;分别采用实时荧光定量PCR和Western-blot方法,检测细胞焦亡相关因子核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、天冬氨酸蛋白水解酶1(Caspase-1)、Gasdermins家族蛋白D(GSDMD)、IL-1β和IL-18基因和蛋白表达水平;采用免疫组织化学方法检测脾脏组织中Caspase-1和IL-β蛋白表达量。结果表明:与对照组相比,48,72,96小时组脾脏组织中IL-1β极显著升高(P<0.01);48小时组IL-18显著升高(P<0.05),72,96小时组极显著升高(P<0.01)。感染PRV后48小时组NLRP3基因相对表达量极显著升高(P<0.01),72,96小时组显著升高(P<0.05);48,72小时组Caspase-1基因相对表达量显著升高(P<0.05),96小时组极显著升高(P<0.01);96小时组GSDMD基因相对表达量极显著升高(P<0.01);72,96小时组IL-1β基因相对表达量极显著升高(P<0.01);48,72小时组IL-18基因相对表达量显著升高(P<0.05),96小时组极显著升高(P<0.01)。与对照组比较,48,72,96小时组小鼠脾脏细胞中NLRP3、Caspase-1和IL-18蛋白相对表达量均呈现上升趋势,而IL-1β蛋白在72,96小时,GSDMD蛋白在96小时时呈现明显上升趋势。免疫组织化学法进一步证实,Caspase-1和IL-β蛋白在小鼠脾脏中呈上调表达。说明PRV感染小鼠后,可通过上调表达细胞焦亡相关因子促进炎性反应的发生。

In order to study the effect of porcine Pseudorabies virus(PRV) infection on pyroptosis-related factors in mouse spleen, in the experiment, 40 Balb/c mice were randomly divided into control group and test group(including 48-hour group, 72-hour group, and 96-hour group). The mice in the test group were subcutaneously inoculated with 1×10~3 TCID/100 μL PRV 200 μL, and the mice in the control group were injected with the same volume of normal saline. The spleens of mice were collected;and the levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) in spleen tissue were detected by enzyme-linked immunosorbent assay;the gene transcription and protein expression levels of pyroptosis-related factors NLRP3, Caspase-1, GSDMD, IL-1β and IL-18 were detected by real-time quantitative PCR and Western-blot, respectively;the expression of Caspase-1 and IL-β protein in spleen tissue was detected by immunohistochemistry method. The results showed that compared with the control group, IL-1β in the spleen tissue of the 48, 72, and 96-hour groups was very significantly increased(P<0.01);IL-18 was significantly increased(P<0.05) in the 48-hour group, and was very significantly increased(P<0.01) in the 72, and 96-hour groups. In terms of transcription level, the NLRP3 gene was very significantly increased(P<0.01) after PRV infection in the 48-hour group, and significantly increased(P<0.05) in the 72, and 96-hour groups. The Caspase-1 gene was significantly increased(P<0.05) in the 48, and 72-hour groups, and was very significantly increased(P<0.01) in the 96-hour group. The GSDMD was very significantly increased(P<0.01) in the 96-hour group. The IL-1β gene was very significantly increased(P<0.01) in the 72, and 96-hour groups. The IL-18 gene was significantly increased(P<0.05) in the 48, and 72-hour groups, and was very significantly increased(P<0.01) in the 96-hour group. Compared with the control group, the expression of NLRP3, Caspase-1 and IL-18 proteins in the spleen cells of mice in the 48, 72, and 96-hour groups showed an upward trend, while the IL-1β protein at 72, 96 h, and the GSDMD protein at 96 h, showed a clear upward trend. Immunohistochemistry method further confirmed that Caspase-1 and IL-β proteins were up-regulated in mouse spleen. The results suggested that the PRV infection in mice could promote the inflammatory response by up-regulating the expression of pyroptosis-related factors.