马兜铃酸肾病慢性缺氧损伤的机制与温阳活血方的干预作用

Injury mechanism of chronic renal hypoxia and interventional effect of Wenyang Huoxue Recipe on rats with aristolochic acid nephropathy

目的研究温阳活血方(WYHXF)对慢性马兜铃酸肾病(CAAN)的慢性缺氧损伤及间质纤维化的干预作用及机制。方法按随机数表法将大鼠分为5组:正常组给以0.9%NaCl 10 mL·kg^(-1)·d^(-1);模型组给予木通水煎液10m L·kg^(-1)·d^(-1)(相当于关木通40 g·kg^(-1)·d^(-1),马兜铃酸2.6mg·kg^(-1)·d^(-1));中药组在模型组基础上,同时予温阳活血方30 g·kg^(-1)·d^(-1)灌胃,每组7只;西药组在模型组基础上,同时予氯沙坦33.3 mg·kg^(-1)·d^(-1)灌胃;联用组在模型组基础上,同时再予温阳活血方+氯沙坦灌胃,连续给药12周。12周末,腹主动脉取血,用于测定血清肌酸酐(SCr)、超氧化物岐化酶(SOD)和丙二醛(MDA)含量;留取肾组织,免疫组化方法检测肾组织缺氧诱导因子-1α(HIF-1α)、结缔组织生长因子(CTGF)、基质金属蛋白-9(MMP-9)和组织基质金属蛋白酶抑制剂-1(TIMP-1)的表达。结果正常组、模型组、中药组、西药组和联用组的SCr分别为(81.13±14.25),(136.85±2.72),(113.86±21.69),(110.20±25.04),(90.45±15.78)μmoL·L^(-1);这5组的MDA分别为(5.03±1.27),(7.20±0.54),(5.99±0.70),(6.00±0.97),(5.88±0.55)nmoL·mL^(-1);这5组的SOD分别为(154.03±11.81),(128.79±1.91),(146.46±17.21),(150.77±19.78),(145.48±8.85)U·mL^(-1),模型组与正常组比较,大鼠血MDA明显升高而SOD降低,差异均有统计学意义(均P<0.05);而3个给药组与模型组比较,差异均有统计学意义(均P<0.05)。给予温阳活血方治疗后,大鼠CTGF和TIMP-1表达明显下降,MMP-9表达上调,说明温阳血方有改善缺血缺氧,减轻肾间质纤维化的作用。结论温阳活血方可以通过抑制氧化应激和抑制HIF-1α,调节MMP-9与TIMP-1平衡,改善CAAN慢性缺氧损伤,降低尿蛋白,延缓肾功能进展。

Objective To study the effects and its mechanism of Wenyang Huoxue Recipe（ WYHXF） on chronic renal hypoxia and interstitial fibrosis in rats with chronic aristolochic acid nephropathy（ CAAN）.Methods The male SD rats were randomly divided into 5 groups with 7 rats in each group. Normal group were treated with normal saline（ 10 mL·kg^-1·d^-1）; model group were treated with Caulis Aristolochia Manshuriensis decoction 10 mL·kg^-1·d^-1（ Aristolochia Manshuriensis40 g·kg^-1·d^-1,Aristolochic acid A 2. 6 mg·kg^-1·d^-1）; Traditional Chinese medicine（ TCM） group were treated with WYHXF 30 g·kg^-1·d^-1 after the rats being modeled with the above-mentionedmethod; western medicine group were treated with losartan potassium tablets 33. 3 mg·kg^-1·d^-1 after the rats being modeled; the combined group were treated with WYHXF and losartan potassium tablets after the rats being modeled. The abdominal aorta blood samples were taken for the determination of serum creatinine（ SCr）,superoxide dismutase（ SOD）,and malondialdehyde（ MDA）. And immunohistochemical method was used to measure the protein of hypoxia inducible factor-1 alpha（ HIF-1α）,connective tissue growth factor（ CTGF）,matrix protein-9（ MMP-9） and metal tissue inhibitor of matrix metalloproteinase-1（ TIMP-1）. Results SCr in normal control group,model group,TCM group,western medicine group and combine group were（ 81.13 ± 14.25）,（ 136.85 ± 2.72）,（ 113.86 ± 21.69）,（ 110.20 ± 25.04）,（ 90.45 ± 15.78） μmo L·L^-1 respectively. Serum MDA in the 5 groups were（ 5.03 ± 1.27）,（ 7.20 ± 0.54）,（ 5.99 ± 0.70）,（ 6.00 ± 0.97）,（ 5.88 ± 0.55） nmo L·m L^-1,respectively; serum SOD in the 5 groups were（ 154.03 ± 11.81）,（ 128.79 ± 1.91）,（ 146.46 ± 17.21）,（ 150.77 ± 19.78）,（ 145.48 ± 8.85）U·m L^-1,respectively. Comparison between model group and normal group,the difference of the factors were significant（ all P〈0.05）; comparison between TCM group,western medicine group and combine group with model group,the difference of the factors were significant（ all P〈0.05）. Expression of HIF-1α,CTGF and TIMP-1 were decreased while expression of MMP-9 was increased after administration WYHXF in each group of rats. Conclusion WTHXF recipe can reduce the urinary protein and postpone the progression of renal hypoxia and renal function in CAAN by inhibiting oxidative stress and HIF-1 alpha expression and regulating the balance of MMP-9 and TIMP-1.