核孔蛋白160基因对肾病综合征小鼠肾脏辅助性T细胞17/调节性T细胞免疫平衡的影响

Effect of nucleoporin 160 gene on immune balance of renal helper T cell 17/regulatory T cell in mice with nephrotic syndrome

目的探讨核孔蛋白160(NUP160)基因对肾病综合征小鼠肾脏辅助性T细胞17(Th17)/调节性T细胞(Treg)免疫平衡的影响。方法将30只小鼠随机分为空白组(n=10)和造模组(n=20)。造模组小鼠经尾静脉注射阿霉素溶液(7.5 mg·kg-1)制备原发性肾病综合征(PNS)模型,空白组小鼠经尾静脉注射等容积的生理盐水。造模成功后将造模组小鼠随机分为PNS组和NUP160组,每组10只。NUP160组小鼠经尾静脉注射NUP160(10 mg·kg-1),每日1次;PNS组和空白组小鼠经尾静脉注射等量生理盐水,每日1次;3组小鼠均连续干预4周。给药0、2、4周时,应用生化自动检测仪检测小鼠24 h尿蛋白定量(24 h HUPQ);给药结束后,采用酶联免疫吸附试验法检测各组小鼠血清白细胞介素(IL)-17和IL-10水平;苏木精-伊红(HE)染色、过碘酸希夫(PAS)染色、Masson染色法观察各组小鼠肾组织病理学变化;流式细胞术检测各组小鼠肾组织中Th17、Treg细胞比例。结果给药0、2、4周时,PNS组和NUP160组小鼠24 h HUPQ显著高于空白组(P<0.05)。给药0周时,PNS组与NUP160组小鼠24 h HUPQ比较差异无统计学意义(P>0.05);给药2、4周时,NUP160组小鼠24 h HUPQ显著低于PNS组(P<0.05)。与空白组比较,PNS组和NUP160组小鼠血清IL-17水平显著升高,IL-10水平显著降低(P<0.05);与PNS组比较,NUP160组小鼠血清IL-17水平显著降低,IL-10水平显著升高(P<0.05)。空白组小鼠肾组织无明显病理学变化。与空白组相比,PNS组小鼠HE染色发现肾小球细胞排列紊乱,肾小球轻度萎缩,少许炎症细胞浸润,球囊壁轻度增厚,小球囊腔轻度扩张;PAS染色发现系膜出现增生,肾间质可见炎症细胞浸润;Masson染色发现小鼠肾小球基底膜和间质蓝色胶原沉积明显增多,组织纤维化程度显著升高。与PNS组相比,NUP160组小鼠肾组织以上症状均得到明显改善。与空白组比较,PNS组和NUP160组小鼠肾组织中Th17占比、Th17/Treg比值显著增加,Treg占比显著降低(P<0.05);与PNS组比较,NUP160组小鼠肾组织中Th17占比、Th17/Treg比值显著降低,Treg占比显著增加(P<0.05)。结论NUP160可调节PNS小鼠的Th17/Treg免疫平衡,进而发挥改善PNS的作用。

Objective To investigate the effect of the nucleoporin 160(NUP160)gene on the immune balance of renal helper T cell 17(Th17)/regulatory T cell(Treg)in mice with nephrotic syndrome.Methods Thirty mice were randomly divided into blank group(n=10)and modeling group(n=20).Mice in the modeling group were injected with doxorubicin solution(7.5 mg·kg-1)via the tail vein to prepare primary nephrotic syndrome(PNS)models,while mice in the blank group were injected with an equal volume of normal saline via the tail vein.After successful modeling,the modeled mice were randomly divided into PNS group and NUP160 group,with 10 mice in each group.Mice in the NUP160 group were injected with NUP160(10 mg·kg-1)via the tail vein,once a day;mice in the PNS group and blank group were injected with an equal volume of normal saline via the tail vein,respectively,once a day;all mice in the 3 groups were intervened continuously for 4 weeks.At 0,2,and 4 weeks of administration,the 24-hour urine protein quantification(24 h HUPQ)of mice was detected by using a biochemical automatic detector;after the end of administration,the levels of serum interleukin(IL)-17 and IL-10 of mice in each group were detected by using the enzyme-linked immunosorbent assay;hematoxylin-eosin(HE)staining,periodic acid-Schiff(PAS)staining,and Masson staining were used to monitor the pathological changes in renal tissues;and the proportion of Th17 and Treg cells in renal tissues of mice in each group were detected by using flow cytometry.Results At 0,2,and 4 weeks of administration,24 h HUPQ of mice in the PNS group and NUP160 group was significantly higher than that in the blank group(P<0.05).There was no statistically significant difference in 24 h HUPQ of mice between the PNS group and the NUP160 group at 0 week of administration(P>0.05);at 2 and 4 weeks of administration,24 h HUPQ of mice in the NUP160 group was significantly lower than that in the PNS group(P<0.05).Compared with the blank group,the serum IL-17 levels of mice in the PNS group and NUP160 group significantly increased,while the IL-10 levels significantly decreased(P<0.05).Compared with the PNS group,the serum IL-17 level of mice in the NUP160 group significantly decreased,while the IL-10 level significantly increased(P<0.05).The renal tissues of mice in the blank group showed no significant pathological changes.Compared with the blank group,HE staining of mice in the PNS group revealed a disordered arrangement of glomerular cells,mild atrophy of glomeruli,infiltration of a few inflammatory cells,mild thickening of the balloon wall,and mild dilation of the glomerular lumen;PAS staining revealed mesangial proliferation and inflammatory cell infiltration in the renal interstitium;Masson staining revealed a significant increase in the deposition of blue collagen in the glomerular basement membrane and interstitium,and also a significant increase in the degree of tissue fibrosis.Compared with the PNS group,mice in the NUP160 group showed a significant improvement in the above symptoms of renal tissues.Compared with the blank group,the proportion of Th17 cells and the Th17/Treg ratio in renal tissues of mice in the PNS group and NUP160 group significantly increased,while the proportion of Treg cells significantly decreased(P<0.05);compared with the PNS group,the proportion of Th17 cells and the Th17/Treg ratio in renal tissues of mice in the NUP160 group significantly decreased,while the proportion of Treg cells significantly increased(P<0.05).Conclusion NUP160 can regulate the Th17/Treg immune balance in PNS mice and thus improve PNS.