肾原位抑制 miR-146b-5p 表达改善 UUO 小鼠肾纤维化
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中西医结合研究中心,西南医科大学附属中医医院,四川 泸州 646000

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Renal inhibition of miR-146b-5p expression in situ improves renal fibrosis in UUO mice
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Research Center of Integrated Traditional Chinese and Western Medicine, Affiliated Chinese Medicine Hospital of Southwest Medical University, Luzhou 646000, China

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    摘要:

    目的 明确 miR-146b-5p 在小鼠肾纤维化模型中的表达情况,并探讨体内敲低 miR-146b-5p 对小鼠肾损伤及纤维化的影响。 方法 将 24 只 8 周龄 C57BL/ 6 雄性小鼠随机分为假手术组( sham),UUO 模型组(UUO),UUO+肾 miR-146b-5p 电转敲低组(UUO-KD),每组 8 只。 Sham 组仅切开皮肤,暴露且游离右侧肾输尿管,不做结扎或离断处理。 UUO 组,行单侧输尿管梗阻(UUO)动物模型。 UUO-KD 组通过先电转 CRISPR/ RfxCas13 d质粒于小鼠肾进行特异性 miR-146b-5p 敲低,24 h 后按模型组方法建立 UUO 小鼠模型,7 d 后处死小鼠收集肾标本。 HE 染色观察肾病理变化,Masson 检测肾间质纤维化程度,免疫组化检测纤维化相关蛋白(α-SMA、FN、Col-1)表达,Western Blot、Real-time PCR 检测 miR-146b-5p、α-SMA、FN、IL-1β、IL-6、TNF-α 等基因的变化。 结果 miR-146b-5p 在 UUO 模型中显著升高,电转敲低 miR-146b-5p 后该基因显著下降(P<0. 05),同时,IL-1、IL-6、TNF-α 等炎性因子表达出现显著下调(P<0. 05)经 HE、Masson 染色后观察到,UUO-KD 组较 UUO 组相比肾结构良好,肾小管变形轻微,肾损伤及纤维化程度均明显改善。 且免疫组化结果发现:α-SMA、FN、Col-1 等纤维化指标在 UUO-KD 组也显著降低(P<0. 0001)。 结论 抑制 UUO 中高表达的 miR-146b-5p 可明显改善肾纤维化,miR-146b-5p 可能是肾纤维化的一个潜在治疗靶标。

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    Objective To investigate miR-146b-5p expression in mice model of renal fibrosis induced by unilateral renal ureteral ligation, and to suppress miR-146b-5p expression to improve renal fibrosis induced by unilateral renal ureteral ligation in mice. Methods Twenty-four 8-week-old C57BL/ 6 male mice were randomly divided into sham operation group (sham), UUO model group (UUO), UUO+kidney miR-146b-5p knockdown group (UUO-KD), 8 mice in each group. In the sham group,the skin was only cut to expose and free the right kidney and ureter without ligation or disconnection. In the UUO group, the animal model of unilateral ureteral obstruction (UUO) was performed. In the UUOKD group, miR-146b-5p was specificly knocked down by electrotransferring the CRISPR/ RfxCas13 d plasmid in the mouse kidney. After 24 hours, the UUO mouse model was established according to the method of the model group, and the mice were sacrificed 7 days later to collect kidney samples. HE staining was used to observe renal pathological changes, Masson was used to detect the degree of renal interstitial fibrosis, immunohistochemistry was used to detect the expression of fibrosis-related proteins (α-SMA, FN, Col-1), and Western Blot and Real-time PCR were used to detect miR-146b-5p,α-SMA, FN, IL-1β, IL-6, TNF-α and other gene changes. Results Real-time PCR showed that miR-146b-5p was significantly increased in UUO model, and the gene was significantly decreased after electroporation knockdown of miR-146b-5p (P< 0. 05). Meanwhile,the expression of IL-1β, IL-6, TNF-α and other inflammatory factors was significantly down-regulated (P< 0. 05). It was observed after HE and Masson staining. Compared with the UUO group, the UUO-KD group had a better kidney structure, slightly deformed renal tubules, and less severe renal damage. The degree of fibrosis was significantly improved. And the results of immunohistochemistry showed that α-SMA, FN, Col-1 and other fibrosis indicators were also significantly reduced in the UUO-KD group ( P< 0. 0001). Conclusions Inhibition of highly expressed miR-146b-5p in UUO can significantly improve renal fibrosis, and miR-146b-5p may be a potential therapeutic target for renal fibrosis.

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谢柯欢,郭怀英,韩壤乐,王丽.肾原位抑制 miR-146b-5p 表达改善 UUO 小鼠肾纤维化[J].中国实验动物学报,2022,30(7):927~934.

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  • 收稿日期:2021-11-08
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  • 在线发布日期: 2023-03-10
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