肾原位抑制miR-146b-5p表达改善UUO小鼠肾纤维化

Renal inhibition of miR-146b-5p expression in situ improves renal fibrosis in UUO mice

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。

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 UUO-KD 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-αand 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-αand 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 thatα-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.