微小RNA-19b-3p靶向调控信号传导与转录激活因子1信号通路对膜性肾病小鼠辅助性T17/调节性T免疫平衡的影响

宋晓薇; 全正莉; 臧华龙; 郭成坤

doi:10.3969/j.issn.1671-2390.2025.04.009

微小RNA-19b-3p靶向调控信号传导与转录激活因子1信号通路对膜性肾病小鼠辅助性T17/调节性T免疫平衡的影响

Effect of miR-19b-3p on Th17/Treg immune balance in membranous nephropathy mice by targeting the STAT1 signaling pathway

摘要

摘要: 目的探究微小RNA-19b-3p（microRNA-19b-3p，miR-19b-3p）靶向调控信号传导与转录激活因子1（signal transducer and activator of transcription 1，STAT1）信号通路对膜性肾病（membranous nephropathy，MN）小鼠辅助性T（helper T，Th）17/调节性T（regulatory T，Treg）免疫平衡的影响。方法将小鼠按照随机数字表法随机分为对照组、MN组、MN+激动剂（agonist，agomir）-阴性对照（negative control，NC）组、MN+miR-19b-3p agomir组，每组10只，采用阳离子化牛血清白蛋白诱导MN模型。检测各组小鼠24 h尿蛋白定量（24 hour urinary protein，24 hUTP）、血清总蛋白（total protein，TP）、白蛋白（albumin，Alb）、血肌酐（serum creatinine，Scr）、血尿素氮（blood urea nitrogen，BUN）水平；实时荧光定量聚合酶链反应检测肾组织miR-19b-3p、STAT1 mRNA表达，HE、Masson、PAS染色观察肾脏组织病理改变；免疫荧光染色观察免疫球蛋白G（immunoglobulin G，IgG）沉积；酶联免疫吸附试验检测血清白细胞介素（interleukin，IL）17、IL-22、IL-10、转化生长因子β（transforming growth factor-β，TGF-β）水平；流式细胞术检测Th17、Treg细胞比例；蛋白质印迹法检测STAT1、IL-17、叉头框转录因子P3（forkhead box transcription factor P3，Foxp3）蛋白表达；双荧光素酶检测miR-19b-3p与STAT1的靶向关系。结果与对照组相比，MN组、MN+agomir-NC组小鼠肾小球损伤严重，间质胶原纤维沉积明显，基底膜厚度增加，IgG在肾小球毛细血管襻颗粒状沉积，24 hUTP［（103.62 ±12.47）μg、（108.93 ±14.15）μg比（11.26 ±2.14）μg］、Scr［（68.42 ±7.01）μmol/L、（69.57 ±7.13）μmol/L比（37.82 ±3.96）μmol/L］、BUN［（13.59 ±1.46）mmol/L、（13.83 ±1.39）mmol/L比（6.15 ±0.74）mmol/L］、IL-17［（126.43 ±13.95）ng/L、（118.57 ±13.83）ng/L比（54.72 ±5.83）ng/L］、IL-22［（288.63 ±29.71）ng/L、（293.47 ±30.62）ng/L比（136.82 ±15.07）ng/L］、Th17细胞比例［（3.39 ±0.42）%、（3.41 ±0.38）%比（1.02 ±0.11）%］、Th17/Treg比值［（2.46 ±0.27）、（2.44 ±0.29）比（0.24 ±0.03）］、IL-17蛋白［（0.95 ±0.10）、（0.97 ±0.11）比（0.30 ±0.04）］、STAT1蛋白［（1.28 ±0.14）、（1.25 ±0.12）比（0.41 ±0.05）］及mRNA［（1.72 ±0.19）、（1.75 ±0.18）比（1.01 ±0.11）］水平明显上升（P<0.05）；TP［（46.28 ±4.93）g/L、（45.72 ±4.76）g/L比（57.24 ±5.83）g/L］、Alb［（28.36 ±2.91）g/L、（27.55 ±2.83）g/L比（35.94 ±3.85）g/L］、miR-19b-3p［（0.38 ±0.06）、（0.35 ±0.05）比（1.00 ±0.10）］、IL-10［（71.92 ±7.36）ng/L、（75.83 ±7.79）ng/L比（182.05 ±19.36）ng/L］、TGF-β［（115.92 ±13.62）ng/L、（107.84 ±12.68）ng/L比（235.17 ±26.39）ng/L］、Treg细胞比例［（1.38 ±0.17）%、（1.40 ±0.16）%比（4.21 ±0.48）%］、Foxp3蛋白［（0.42 ±0.05）、（0.41 ±0.06）比（0.86 ±0.09）］表达明显下降（P<0.05）。与MN组、MN+agomir-NC组相比，MN+miR-19b-3p agomir组小鼠肾组织损伤明显减轻，间质胶原纤维沉积和基底膜厚度减少，IgG沉积减少，24 hUTP［（51.64 ±6.83）μg比（103.62 ±12.47）μg、（108.93 ±14.15）μg］、Scr［（42.04 ±4.83）μmol/L比（68.42 ±7.01）μmol/L、（69.57 ±7.13）μmol/L］、BUN［（6.15 ±0.74）mmol/L比（13.59 ±1.46）mmol/L、（13.83 ±1.39）mmol/L］、IL-17［（68.51 ±7.26）ng/L比（126.43 ±13.95）ng/L、（118.57 ±13.83）ng/L］、IL-22［（151.86 ±17.42）ng/L比（288.63 ±29.71）ng/L、（293.47 ±30.62）ng/L］、Th17细胞比例［（1.81 ±0.21）%比（3.39 ±0.42）%、（3.41 ±0.38）%］、Th17/Treg比值［（0.56 ±0.07）比（2.46 ±0.27）、（2.44 ±0.29）］、IL-17蛋白［（0.46 ±0.06）比（0.95 ±0.10）、（0.97 ±0.11）］、STAT1蛋白［（0.57 ±0.06）比（1.28 ±0.14）、（1.25 ±0.12）］及mRNA［（1.23 ±0.14）比（1.72 ±0.19）、（1.75 ±0.18）］水平明显下降（P<0.05）；TP［（55.94 ±5.73）g/L比（46.28 ±4.93）g/L、（45.72 ±4.76）g/L］、Alb［（34.06 ±3.72）g/L比（28.36 ±2.91）g/L、（27.55 ±2.83）g/L］、miR-19b-3p［（0.83 ±0.09）比（0.38 ±0.06）、（0.35 ±0.05）］、IL-10［（164.82 ±17.39）ng/L比（71.92 ±7.36）ng/L、（75.83 ±7.79）ng/L］、TGF-β［（213.82 ±22.71）ng/L比（115.92 ±13.62）ng/L、（107.84 ±12.68）ng/L］、Treg细胞比例［（3.26 ±0.37）%比（1.38 ±0.17）%、（1.40 ±0.16）%］、Foxp3蛋白［（0.71 ±0.08）比（0.42 ±0.05）、（0.41 ±0.06）］表达明显上升（P<0.05）。结论 MiR-19b-3p可能通过靶向调控STAT1，恢复MN小鼠Th17/Treg免疫平衡，改善MN小鼠肾损伤。

Abstract: Objective To investigate the effect of microRNA-19b-3p （miR-19b-3p） on T helper 17 （Th17）/regulatory T （Treg） immune balance in membranous nephropathy （MN） mice by targeting the regulation of signal transducer and activator of transcription 1 （STAT1） signaling pathway.Methods The mice were randomly grouped into the control group， MN group， MN+agonist （agomir）-negative control （NC） group， and MN+miR-19b-3p agomir group according to random number table method， with 10 mice in each group. Cationic bovine serum albumin was used to induce the MN model. The 24 hour urinary total protein （24 hUTP）， serum total protein （TP）， albumin （Alb）， creatinine （Scr）， and blood urea nitrogen （BUN） were measured. Real-time fluorescence quantitative polymerase chain reaction （RT-qPCR） was applied to detect the mRNA expressions of miR-19b-3p and STAT1 in renal tissue. The hematoxylin and eosin （H&E）， Masson， and periodic acid-Schiff （PAS） staining were used to observe pathological changes in renal tissue. Immunoglobulin G （IgG） deposition was observed by immunofluorescence staining. Enzyme-linked immunosorbent assay （ELISA） was applied to detect serum levels of interleukin （IL）-17， IL-22， IL-10， and transforming growth factor-β （TGF-β）. Flow cytometry was applied to detect the proportions of Th17 and Treg cells. Western blot was applied to detect the protein expressions of STAT1， IL-17， and forkhead box transcription factor P3 （Foxp3）. Dual luciferase reporter assay was applied to detect the targeting relationship between miR-19b-3p and STAT1.Results Compared with the control group， mice in the MN group and MN+agomir-NC group showed severe glomerular injury， obvious deposition of interstitial collagen fibers， increased basement membrane thickness， granular deposition of IgG in glomerular capillary loops， significantly higher 24 hUTP ［（103.62 ±12.47）μg、（108.93 ±14.15）μg vs （11.26 ±2.14）μg］， Scr ［（68.42 ±7.01）μmol/L、（69.57 ±7.13）μmol/L vs （37.82 ±3.96）μmol/L］， BUN ［（13.59 ±1.46）mmol/L、（13.83 ±1.39）mmol/L vs （6.15 ±0.74）mmol/L］， IL-17［（126.43 ±13.95）ng/L、（118.57 ±13.83）ng/L vs （54.72 ±5.83）ng/L］， IL-22［（288.63 ±29.71）ng/L、（293.47 ±30.62）ng/L vs （136.82 ±15.07）ng/L］， proportion of Th17 cells ［（3.39 ±0.42）%、（3.41 ±0.38）% vs （1.02 ±0.11）%］， Th17/Treg ［（2.46 ±0.27）、（2.44 ±0.29） vs （0.24 ±0.03）］， protein level of IL-17［（0.95 ±0.10）、（0.97 ±0.11） vs （0.30 ±0.04）］， protein level of STAT1 ［（1.28 ±0.14）、（1.25 ±0.12） vs （0.41 ±0.05）］ and mRNA level of STAT1 ［（1.72 ±0.19）、（1.75 ±0.18） vs （1.01 ±0.11）］， but significantly lower TP ［（46.28 ±4.93）g/L、（45.72 ±4.76）g/L vs （57.24 ±5.83）g/L］， Alb ［（28.36 ±2.91）g/L、（27.55 ±2.83）g/L vs （35.94 ±3.85）g/L］， miR-19b-3p ［（0.38 ±0.06）、（0.35 ±0.05） vs （1.00 ±0.10）］， IL-10 ［（71.92 ±7.36）ng/L、（75.83 ±7.79）ng/L vs （182.05 ±19.36）ng/L］， TGF-β［（115.92 ±13.62）ng/L、（107.84 ±12.68）ng/L vs （235.17 ±26.39）ng/L］， proportion of Tregs ［（1.38 ±0.17）%、（1.40 ±0.16）% vs （4.21 ±0.48）%］， and protein level of Foxp3 ［（0.42 ±0.05）、（0.41 ±0.06）比（0.86 ±0.09）］（P<0.05）. Compared with the MN group and MN+agomir-NC， mice in the MN+miR-19b-3p agomir group had significantly alleviated renal tissue damage， reduced interstitial collagen fiber deposition and basement membrane thickening， decreased IgG deposition， lower 24 hUTP ［（51.64 ±6.83）μg vs （103.62 ±12.47）μg、（108.93 ±14.15）μg］， Scr［（42.04 ±4.83）μmol/L vs （68.42 ±7.01）μmol/L、（69.57 ±7.13）μmol/L］， BUN ［（6.15 ±0.74）mmol/L vs （13.59 ±1.46）mmol/L、（13.83 ±1.39）mmol/L］， IL-17 ［（68.51 ±7.26）ng/L vs （126.43 ±13.95）ng/L、（118.57 ±13.83）ng/L］， IL-22 ［（151.86 ±17.42）ng/L vs （288.63 ±29.71）ng/L、（293.47 ±30.62）ng/L］， proportion of Th17 cells ［（1.81 ±0.21）% vs （3.39 ±0.42）%、（3.41 ±0.38）%］， Th17/Treg ［（0.56 ±0.07） vs （2.46 ±0.27）、（2.44 ±0.29）］， protein level of IL-17［（0.46 ±0.06） vs （0.95 ±0.10）、（0.97 ±0.11）］， protein level of STAT1［（0.57 ±0.06） vs （1.28 ±0.14）、（1.25 ±0.12）］ and mRNA level of STAT1［（1.23 ±0.14） vs （1.72 ±0.19）、（1.75 ±0.18）］， but higher TP［（55.94 ±5.73）g/L vs （46.28 ±4.93）g/L、（45.72 ±4.76）g/L］， Alb ［（34.06 ±3.72）g/L vs （28.36 ±2.91）g/L、（27.55 ±2.83）g/L］， miR-19b-3p ［（0.83 ±0.09） vs （0.38 ±0.06）、（0.35 ±0.05）］， IL-10［（164.82 ±17.39）ng/L vs （71.92 ±7.36）ng/L、（75.83 ±7.79）ng/L］， TGF-β［（213.82 ±22.71）ng/L vs （115.92 ±13.62）ng/L、（107.84 ±12.68）ng/L］， proportion of Tregs ［（3.26 ±0.37）% vs（1.38 ±0.17）%、（1.40 ±0.16）%］， and protein level of Foxp3［（0.71 ±0.08） vs （0.42 ±0.05）、（0.41 ±0.06）］（P<0.05）.Conclusion MiR-19b-3p may restore Th17/Treg immune balance in MN mice and improve renal injury by targeting the regulation STAT1.

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