骨形成蛋白4在单侧输尿管梗阻小鼠肾间质纤维化中的作用

Role of bone morphogenetic protein 4 on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction

目的探讨骨形成蛋白4(BMP4)对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的影响。方法将雄性C57BL/6小鼠随机分为腹腔注射生理盐水假手术组(saline-Sham,n=8)、腹腔注射生理盐水UUO组(saline-UUO,n=8)、腹腔注射抗BMP4抗体(anti-BMP4)假手术组(anti-BMP4-Sham,n=8)、腹腔注射anti-BMP4 UUO组(anti-BMP4-UUO,n=8)。各组于手术日起每天给予anti-BMP4(200μl/g·体重)或saline腹腔注射。于术后7d处死取材。免疫荧光观察正常小鼠肾脏BMP4的表达与分布;Masson染色观察肾间质纤维化;免疫组织化学观察肾组织α-平滑肌肌动蛋白(α-SMA)、胶原-Ⅲ(COL-Ⅲ)及p-Smad3的表达;实时荧光定量聚合酶链反应检测肿瘤坏死因子-α(TNF-α)mRNA、单核细胞趋化蛋白-1(MCP-1)mRNA的表达。应用SPSS18.0统计软件分析。计量资料以均值±标准差(Mean±SD)表示,参数资料采用单因素方差分析(One-way ANOVA),组间比较采用t检验。结果正常小鼠肾组织表达BMP4,主要分布于肾小管。Masson染色显示,anti-BMP4-Sham组和saline-Sham组肾间质纤维化面积分别为(1.01±0.21)%比(0.81±0.19)%,两组间比较差异无统计学意义(t=0.983,P>0.05);anti-BMP4 UUO组和saline-UUO组纤维化面积分别为(15.08±2.43)%比(21.23±3.19)%,anti-BMP4-UUO组胶原纤维明显减少,纤维化程度明显减轻(t=3.651,P<0.01);免疫组织化学显示,anti-BMP4-Sham组和saline-Sham组α-SMA表达阳性面积分别为(1.51±0.35)%比(1.68±0.44)%、COL-Ⅲ分别为(0.84±0.22)%比(0.65±0.17)%、p-Smad3分别为(0.44±0.11)%比(0.71±0.28)%,各项指标在两组间比较差异无统计学意义(t=0.557,P>0.05;t=0.896,P>0.05;t=0.753,P>0.05);anti-BMP4-UUO组和saline-UUO组α-SMA表达阳性面积分别为(9.11±1.03)%比(16.18±2.15)%、COL-Ⅲ分别为(7.08±0.83)%比(12.55±1.88)%、p-Smad3分别为(6.14±0.76)%比(11.66±1.91)%,anti-BMP4-UUO组各项指标表达均明显减少(t=2.991,P>0.05;t=3.051,P<0.01;t=2.753,P<0.05);实时荧光定量聚合酶链反应显示,anti-BMP4-Sham组和saline-Sham组TNF-α mRNA和MCP-1 mRNA表达差异无统计学意义(t=0.661,P>0.05;t=0.715,P>0.05);与saline-UUO组比较,anti-BMP4-UUO组TNF-α mRNA、MCP-1mRNA表达均明显降低(t=3.117,P<0.01;t=3.762,P<0.01),差异有统计学意义。结论BMP4通过激活Smad3信号通路参与梗阻性肾病肾间质纤维化的进展,抑制BMP4可能作为抑制肾间质纤维化的治疗靶点。

Objective To observe the effects of bone morphogenetic protein 4 (BMP4) on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO). Methods C57BL/6 mice were randomly divided into four groups: injection intra-abdominally with saline-sham-operated group (saline-sham, n=8), injection intra-abdominally with saline-UUO-operated group (saline-UUO, n=8), injection intra-abdominally with anti-BMP4-sham-operated group (anti-BMP4-sham, n=8), and injection intra-abdominally with anti-BMP4-UUO-operated group (anti-BMP4-UUO, n=8). Either saline or anti-BMP4 (200 μl/g of body weight per day) were injection intra-abdominally for 7 days after surgery. Mice were sacrificed at 7 day to evaluate the fibrosis by Masson staining. The expression of BMP4 in renal tissue of normal mice was assayed by immunofluorescence. Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), collagen Ⅲ(COL-Ⅲ) and p-Smad3 in each group. Besides, the TNF-α mRNA and MCP-1 mRNA levels were also analyzed by fluorescent quantitative Real-time polymerase chain reaction in four groups. Results The expression of BMP4 was mainly distributed in the renal tubules of mice. The area of renal interstitial fibrosis in anti-BMP4-Sham group and saline-Sham group were (1.01±0.21)% and (0.81±0.19)%, respectively. There was no significant difference in the expression of collagen fibers in 2 Sham groups (t=0.983, P>0.05). A significantly reduced collagen deposition was observed in anti-BMP4-UUO group [(15.08±2.43)%] compared to the saline-UUO group [(21.23±3.19)%, t=3.651, P<0.01]. Similar to that, the expression of α-SMA, COL-Ⅲ and p-Smad3 in anti-BMP4-Sham group and saline-Sham group were (1.51±0.35)% vs.(1.68±0.44)%,(0.84±0.22)% vs.(0.65±0.17)%,(0.44±0.11)% vs.(0.71±0.28)%, respectively. There was no significant difference in 2 Sham groups (t=0.557, P>0.05;t=0.896, P>0.05;t=0.753, P>0.05). the expression of α-SMA, COL-Ⅲ and p-Smad3 were significantly decreased in anti-BMP4-UUO group than in saline-UUO group [α-SMA:(9.11±1.03)% vs.(16.18±2.15)%, COL-Ⅲ:(7.08±0.83)% vs.(12.55±1.88)%, p-Smad3:(6.14±0.76)% vs.(11.66±1.91)%, t=2.991, P<0.05;t=3.051, P<0.01;t=2.753, P<0.05]. Meanwhile, fluorescent quantitative Real-time polymerase chain reaction showed that there was no significant difference in expression of TNF-α mRNA and MCP-1 mRNA between 2 Sham groups (t=0.661, P>0.05;t=0.715, P>0.05). The expression of TNF-α mRNA and MCP-1 mRNA were obviously decreased in anti-BMP4-UUO group than in saline-UUO group (t=3.117, P<0.01;t=3.762, P<0.01). Conclusion BMP4 contributes to renal interstitial fibrosis in obstructive nephropathy through activation Smad3 pathway, and inhibition of BMP4 may be a choice for preventing interstitial fibrosis.