miR-451调控早期糖尿病肾病小鼠肾小球肥大的机制

Involvement of miR-451 in the regulation of glomerular hypertrophy in db/db mouse with diabetic nephropathy at early stage

摘要:

目的：观察miR-451对早期糖尿病肾病（diabetic nephropathy，DN）db/db小鼠肾脏形态学影响，探讨miR-451在DN发病机制中的作用。方法：采用24只7周龄雄性db/db小鼠和8只正常对照db/m小鼠依次分为未处理组、空质粒组、miR-451组及对照组。腹腔注射法进行质粒注射处理2周后，光镜下观察肾小球形态学变化，实时荧光定量PCR（real-time PCR）检测肾脏组织miR-451的表达水平，Western blot和激光共聚焦检测肾脏组织中miR-451的靶基因酪氨酸-3单氧化酶/色氨酸单氧化酶活化蛋白（tyrosine3-monooxy-genase/tryptophan 5-monooxygenase activation protein，Ywhaz）及其相关的丝裂原激活蛋白激酶（mitogen-activated protein kinase，MAPK）途径中p38丝裂原激活的蛋白激酶（p38MAPK）及其上游激酶MKK3（MAPK kinase 3）的表达水平。结果：7周龄db/db小鼠未处理组相比对照组出现高血糖且尿白蛋白排泄率（urinary albumin excretion rate，UAE）增加，提示进入早期DN阶段，给予30 mg/（kg?d）质粒注射实验2周。光镜观察和肾小球直径测量发现，miR-451处理组肾小球形态减小（P=0.000）。Real-time PCR检测显示miR-451组小鼠肾脏组织中miR-451的表达水平增高1.79倍（P=0.002），West－ern blot和激光共聚焦结果显示，miR-451组较未处理组和空质粒组肾脏组织Ywhaz的表达受到下调，同时p38MAPK及上游激酶MKK3蛋白表达水平减少（P均=0.000）。结论：miR-451可能通过直接靶向Ywhaz基因，调控MAPK途径中关键蛋白p38MAPK、MKK3的表达，从而抑制肾小球肥大，在DN发病机制中可能起着重要的作用。

Abstract:

Objective：To observe the effect of miR-451 on glomerular morphological changes in diabetic nephmpathy（DN） of type 2 diabetic db/db mice and to explore the role of miR-451 in pathogenesis of early DN. Methods：Thirty-two 7-week-old db/db and normal db/m male mice were divided into miR-451 group，empty plasmid group，untreated group and normal control group. After in－traperitoneal injection of plasmid for two weeks，the glomerular morphological changes were observed under the light microscope. Also，the expression of miR-451 was tested by real-time PCR and the tyrosine3-monooxy-genase/tryptophan 5-monooxygenase activation protein（Ywhaz），p38MAPK and MAPK kinase 3（MKK3） protein levels were examined by Western blot and immunofluorescence. Results：Elevation of urinary albumin excretion rate（UAE） was shown in the untreated group of 7-week-old db/db mice，suggesting that the 7-week-old db/db mice had the features similar to the diabetic nephropathy at early stage. Then plasmid injection ex－periment was executed on mice for two weeks. Real-time RT-PCR results showed that miR-451 was 1.79 folds high in miR-451 group than in empty plasmid group（P=0.002）. Also，glo-meruli acreage was smaller in miR-451-treated group than in the empty plasmid and untreated groups（P=0.000）. Moreover，Western blot and immunofluorescence results demonstrated that Ywhaz，p38MAPK and MKK3 proteins were reduced in miR-451 group（P=0.000）. Conclusion：miR-451 can repress glomerular hypertrophy by targeting Ywhaz gene and affecting MAPK signal pathway. Therefore，miR-451 might play an important role in the pathogenesis of DN.