白藜芦醇抑制蛋白激酶B磷酸化缓解大鼠肌成纤维细胞形成及肾纤维化

Resveratrol ameliorates renal myofibroblastic phenotype and fibrosis by inhibiting protein kinase B phosphorylation

目的探讨白藜芦醇对大鼠肌成纤维细胞形成及肾纤维化的影响。方法制备大鼠单侧输尿管梗阻(UUO)模型,白藜芦醇干预组于手术当天ig给予白藜芦醇20 mg·kg^(-1),术后第7天取其梗阻侧肾脏进行检测。HE和Masson染色分别观察肾组织病理改变和纤维化程度;免疫组织化学染色检测肌成纤维细胞标志物α平滑肌肌动蛋白(α-SMA)的表达。体外分别培养肾小管上皮细胞(NRK-52E)和成纤维细胞(NRK-49F),各分为3组:细胞对照组,转化生长因子β_1(TGF-β_1)5μg·L^(-1)诱导组和TGF-β_15μg·L^(-1)+白藜芦醇10或100 mmol·L^(-1)干预组。细胞免疫荧光染色检测α-SMA、细胞外基质成分(Ⅲ型胶原)和上皮细胞标志物E-钙黏着蛋白的表达;Western蛋白印迹法检测细胞或组织中蛋白激酶B(AKT)磷酸化。结果与假手术组相比,HE染色显示UUO模型组肾间充质面积扩大(P<0.01),Masson染色显示UUO模型组胶原累积明显(P<0.05);白藜芦醇干预组上述改变均被抑制(P<0.05),α-SMA阳性肌成纤维细胞的形成亦被抑制(P<0.05)。体外应用TGF-β_1刺激后,与细胞对照组相比,TGF-β_15μg·L^(-1)诱导组α-SMA表达增加(P<0.01),提示细胞表型转化为肌成纤维细胞数量明显增加,同时伴随着Ⅲ型胶原过度累积(P<0.01);白藜芦醇干预组上述改变被抑制(P<0.05)。进一步研究发现,在肌成纤维细胞产生过程中,AKT磷酸化水平增加(P<0.05);白藜芦醇干预后,体内外AKT磷酸化升高均被抑制(P<0.01)。结论白藜芦醇可能通过拮抗AKT的磷酸化,抑制TGF-β_1所诱导的NRK-52E和NRK-49F表型转化为肌成纤维细胞,进而减少基质累积,缓解肾纤维化。

OBJECTIVE To investigate the effect of resveratrol on renal myofibroblastic phenotype and fibrosis.METHODS A rat model of unilateral ureteral obstruction(UUO)was established.From the day of surgery,resveratrol 20 mg·kg-1was given daily and for 7 d.The pathological changes and fibrosis of renal tissues were observed by HE and Masson staining.The expression of myofibroblastic markerα-smooth muscle actin(α-SMA)in renal tissues was detected by immunohistochemical staining Renal tubular epithelial cells(NRK-52E)and fibroblasts(NRK-49F)were cultured in vitro,and divided into four groups:normal cell control group,TGF-β15μg·L-1group,TGF-β15μg·L-1+resveratrol 10 or100 mmol·L-1group.In vitro,the expressions ofα-SMA,E-cadherin and extracellular matrix component(collagenⅢ)were detected by immunofluorescence staining.The expressions of protein kinase B(AKT)and phospholated AKT(p-AKT)in cells or renal tissues were determined by Western blotting.RESULTS Compared with normal control group,HE staining showed an enlarged interstitial area in the UUO model group(P<0.01),Masson staining showed significant accumulation of collagen in the UUO model group(P<0.05),but these changes were inhibited in the resveratrol intervention group(P<0.05).At the same time,the formation ofα-SMA positive myofibroblasts was also inhibited(P<0.05)After TGF-β1stimulation in vitro,α-SMA was significantly increased(P<0.05).All this suggested that the number of myofibroblasts transformed from renal cells was significantly increased,accompanied by excessive accumulation of collagen(P<0.01),but these changes were inhibited in the resveratrol intervention group.Western boltting results showed that AKT phosphorylation was increased during myofibroblast production(P<0.01).After intervention with resveratrol,AKT phosphorylation was inhibited not only in UUO model group(P<0.05)but also in vitro(P<0.05).CONCLUSION Resveratrol may inhibit myofibroblastic phenotype by antagonizing the activation of AKT,thereby reducing the accumulation of extracellular matrix and alleviating renal fibrosis.