维生素D治疗小鼠狼疮性肾炎的机制:基于上调miR145的表达进而抑制TGF-β1/Smad3通路

Inhibit TGF-β1/Smad3 pathway by upregulation of miR145 with active vitamin D in lupus nephritis

目的研究1,25(OH)_(2)D_(3)是否通过上调miR145从而抑制TGF-β1/Smad3通路,探讨其在狼疮性肾炎中的可能治疗机制。方法共42只8周龄雌性ApoE^(-/-)小鼠,分为7组,每组6只。空白对照组1、2每天予腹腔注射0.5 mL无菌0.9%生理盐水至实验结束;空白模型组1、2予腹腔注射降植烷0.5 mL/只1次后高脂饲料喂养3个月建立狼疮性肾炎小鼠模型;1,25(OH)_(2)D_(3)治疗组、miR145过表达组、miR145空载体组在空白模型组的基础上,分别予灌胃维生素D、载入miR145空载腺病毒、载入miR145过表达腺病毒。CBB法检测各组小鼠第8周尿蛋白含量,ELISA法分别检测各组小鼠外周血ANA抗体、抗dsDNA抗体,评估小鼠模型建立情况。通过RT-qPCR检测各组小鼠外周血PBMCs中miR145表达,观察1,25(OH)_(2)D_(3)处理后miR145表达情况。通过Western blot检测各组小鼠外周血PBMCs及肾脏中TGF-β1、Smad3、p-smad3表达,观察上调1,25(OH)_(2)D_(3)、miR145过表达对TGF-β1/Smad3通路的影响。对空白对照组1、空白模型组1、miR145过表达组、miR145空载体组小鼠肾脏进行病理检测,比较miR145过表达后肾脏病理变化。结果miR145对小鼠狼疮性肾炎的作用实验组:miR145过表达组中小鼠肾脏损害减轻;外周血PBMCs中miR145表达水平显著高于miR145空载体组(P<0.01),TGF-β1、Smad3、p-smad3表达均显著低于miR145空载体组(均P<0.01),肾脏中TGF-β1、Smad3、p-smad3表达均显著低于miR145空载体组(均P<0.01)。1,25(OH)_(2)D_(3)对小鼠狼疮性肾炎的治疗作用实验组:第24周,1,25(OH)_(2)D_(3)治疗组尿蛋白量明显低于空白模型组(P<0.01);1,25(OH)_(2)D_(3)治疗组小鼠血清1,25(OH)_(2)D_(3)浓度明显高于空白模型组(P<0.01),外周血PBMCs中miR145表达水平显著高于空白模型组(P<0.01),而TGF-β1、Smad3、p-smad3表达明显低于空白模型组(均P<0.01)。结论1,25(OH)_(2)D_(3)通过上调miR145表达,从而负性调节TGF-β1/Smad3通路,减轻小鼠狼疮性肾炎肾脏损害。miR145可能是治疗狼疮性肾炎的新靶点。

Objective To investigate whether 1,25(OH)_(2)D_(3)inhibits the TGF-β1/Smad3 pathway through upregulation of miR145 and its possible therapeutic mechanism in lupus nephritis.Methods A total of 428-week-old female ApoE^(-/-)mice were divided into 7 groups of 6 mice each.Blank control group 1 and group 2 were given 0.5 mL of sterile 0.9%saline intraperitoneally every day until the end of the experiment;blank model group 1 and group 2 were given 0.5 mL of hypocretin intraperitoneally once and fed with high-fat diet for 3 months to establish a mouse model of lupus nephritis.1,25(OH)_(2)D_(3)treatment group,miR145 overexpression group and miR145 empty vector group were treated with vitamin D by gavage,miR145 by gavage and miR145 by gavage,respectively.Mice were treated with vitamin D by gavage,loaded with miR145 empty vector adenovirus and loaded with miR145 overexpressing adenovirus,respectively.Urine protein level was measured by CBB at week 8 in each group,and peripheral blood ANA antibody and anti-dsDNA antibody were measured by ELISA in each group to assess the establishment of the mouse model.The expression of miR145 in peripheral blood PBMCs was measured by RT-qPCR and the expression of miR145 was observed after treatment with 1,25(OH)_(2)D_(3).1,25(OH)_(2)D_(3),miR145 overexpression on the TGF-β1/Smad3 pathway.The kidneys of normal control group 1,blank model group 1,miR145 overexpression group and miR145 empty vector group were examined pathologically to compare the renal pathological changes after miR145 overexpression.Results Kidney damage was reduced in the miR145 overexpression group.Expression levels of miR145 in peripheral blood PBMCs were significantly higher than those in the miR145 empty vector group(P<0.01),and the expression of TGF-β1,Smad3 and p-smad3 were significantly lower than those in the miR145 empty vector group(P<0.01).Expression of TGF-β1,Smad3 and p-smad3 in the kidney were significantly lower than those in the miR145 empty vector group(P<0.01).1,25(OH)_(2)D_(3)on the treatment of lupus nephritis in mice experimental group:the amount of urinary protein in the 1,25(OH)_(2)D_(3)treated group was significantly lower than that in the blank model group at week 24.The serum 1,25(OH)_(2)D_(3)concentration in the 1,25(OH)_(2)D_(3)treated group was significantly higher than that in the blank model group(P<0.01),and the expression level of miR145 in peripheral blood PBMCs was significantly higher than that in the blank model group(P<0.01),while expressions of TGF-β1,Smad3 and p-smad3 were significantly lower than those in the blank model group(all P<0.01).Conclusion 1,25(OH)_(2)D_(3)attenuated kidney damage of lupus nephritis in mice by upregulating miR145 expression and thus negatively regulating the TGF-β1/Smad3 pathway.miR145 may be a new target for the treatment of lupus nephritis.