Yuan Ji-Li, Tao Yan-Yan, Wang Qing-Lan, Shen Li, Liu Cheng-Hai
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Shanghai Key Laboratory of Traditional, Chinese Clinical Medicine, Shanghai, 201203, China.
Chin J Integr Med. 2017 Aug;23(8):598-604. doi: 10.1007/s11655-016-2540-z. Epub 2017 Feb 15.
To investigate the mechanism of action of Fuzheng Huayu Formula (, FZHY) against renal interstitial fibrosis (RIF) relating to oxidative injury and nuclear factor-kappa B (NF-κB) activity.
Thirty-two Sprague-Dawley rats were randomly divided into 3 groups: normal group, model group and FZHY treatment group. The RIF model was induced by oral administration of HgCl at a dose of 8 mg/kg body weight once a day for 9 weeks. Meanwhile, rats in FZHY treatment group orally took FZHY at a dose of 4.0 g/kg rat weight for 9 weeks. The content of hydroxyproline (Hyp) and collagen deposition in kidney were observed. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the content of glutathione (GSH) and malondialdehyde (MDA) of kidney were tested. The expressions of inhibitor-κappa B (IκB), phospho-IκB (p-IκB), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. α-SMA expression was also observed by immunofluorescent staining. MMP-2 activity was measured by gelatin zymography. NF-κB activation was determined by electrophoretic mobility shift assay.
Renal interstitial fibrosis was induced by HgCl, demonstrated by remarkably increased Hyp contents and excessive collagen deposition in kidney (P<0.01). FZHY significantly inhibited renal interstitial collagen deposition and reduced Hyp content of the HgCl-treated rats (P<0.01). GSH content decreased obviously, and MDA content increased signifificantly in HgCl-treated rats compared with that of normal rats (P<0.01). FZHY significantly increased GSH content and decreased MDA content in the model rats (P<0.01). The expression α-SMA was increased in model rats compared with that of normal rats, FZHY signifificantly decreased its expression (P<0.01). The expressions of p-IκB and TNF-α and MMP-2, MMP-2 activity, and NF-κB activation were increased in model group compared with that in normal group (P<0.01), FZHY signifificantly decreased NF-κB activation, MMP-2 activity and p-IκB and TNF-α expressions (P<0.01).
FZHY could protect kidney from oxidative injury intoxicated by HgCl, and antagonized oxidative stress-stimulated NF-κB activity through inhibition of IκB phosphorylation in the interstitial fibrotic kidney, these effects importantly contributed to FZHY action mechanism against renal interstitial fifibrosis.
探讨扶正化瘀方（FZHY）抗肾间质纤维化（RIF）与氧化损伤及核因子-κB（NF-κB）活性相关的作用机制。
将32只Sprague-Dawley大鼠随机分为3组：正常组、模型组和FZHY治疗组。通过每日一次口服8mg/kg体重的HgCl2诱导RIF模型，持续9周。同时，FZHY治疗组大鼠以4.0g/kg大鼠体重的剂量口服FZHY，持续9周。观察肾脏中羟脯氨酸（Hyp）含量及胶原沉积情况。检测肾脏中超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性、谷胱甘肽（GSH）和丙二醛（MDA）含量。采用蛋白质免疫印迹法分析抑制蛋白-κB（IκB）、磷酸化IκB（p-IκB）、肿瘤坏死因子-α（TNF-α）、基质金属蛋白酶-2（MMP-2）和α-平滑肌肌动蛋白（α-SMA）的表达。通过免疫荧光染色观察α-SMA表达。采用明胶酶谱法测定MMP-2活性。通过电泳迁移率变动分析确定NF-κB激活情况。
HgCl2诱导了肾间质纤维化，表现为肾脏中Hyp含量显著增加和胶原过度沉积（P<0.01）。FZHY显著抑制了HgCl2处理大鼠的肾间质胶原沉积并降低了Hyp含量（P<0.01）。与正常大鼠相比，HgCl2处理大鼠的GSH含量明显降低，MDA含量显著增加（P<0.01）。FZHY显著增加了模型大鼠的GSH含量并降低了MDA含量（P<0.01）。与正常大鼠相比，模型大鼠中α-SMA表达增加，FZHY显著降低了其表达（P<0.01）。与正常组相比，模型组中p-IκB、TNF-α和MMP-2的表达、MMP-2活性及NF-κB激活增加（P<0.01），FZHY显著降低了NF-κB激活、MMP-2活性及p-IκB和TNF-α表达（P<0.01）。
FZHY可保护肾脏免受HgCl2所致的氧化损伤，并通过抑制间质纤维化肾脏中IκB磷酸化来拮抗氧化应激刺激的NF-κB活性，这些作用对FZHY抗肾间质纤维化的作用机制具有重要贡献。
