基于Rho相关卷曲螺旋激酶(ROCK)/IκB激酶(IKK)/核因子-κB(NF-κB)通路,探讨真武汤对脾肾阳虚型糖尿病肾病(DN)小鼠的干预作用及其机制。适应性地饲喂95只7周龄db/db雄性小鼠和25只7周龄db/m雄性小鼠一周。大黄汤联合氢化可的松灌胃法制备脾肾阳虚型DN模型,然后对模型进行了评估。建模后,他们被随机分为一个模型组,高剂量,中等剂量,和低剂量真武汤组(33.8、16.9和8.45g·kg〜(-1)·d〜(-1)),和厄贝沙坦组(25mg·kg〜(-1)·d〜(-1)),每组至少15只动物。干预持续了八周。干预之后,测量体重和食物摄入量。血清肌酐(Scr),血尿素氮(BUN),空腹血糖(FBG),尿白蛋白(uALb),测定尿肌酐(Ucr)。计算uALb/Ucr比值(ACR)和24h尿蛋白(UTP)。HE染色和Masson染色评价肾脏病理形态。Westernblot检测ROCK/IKK/NF-κB通路中关键分子蛋白的水平。采用酶联免疫吸附试验(ELISA)检测白细胞介素-1β(IL-1β),白细胞介素-6(IL-6),白细胞介素-8(IL-8),白细胞介素-10(IL-10),和肿瘤坏死因子-α(TNF-α)。与空白组相比,模型组BUN含量增加,UALb,SCr,24hUTP和ACR值增加,Ucr含量降低(P<0.05),增大的肾小球,增厚的基底膜,系膜基质增殖,炎性细胞浸润,和胶原纤维沉积。ROCK1、ROCK2、IKK、NF-κB,磷酸化IKK(p-IKK),磷酸化NF-κB(p-NF-κB),磷酸化NF-κB抑制剂(p-IκB)增加(P&lt;0.05),而NF-κB抑制剂(IκB)的蛋白表达降低(P&lt;0.05)。炎症因子IL-1β水平,IL-6,IL-8和TNF-α升高(P&lt;0.05),IL-10水平降低(P<0.05)。与模型组相比,药物治疗组显示BUN水平下降,UALb,SCr,24hUTP,ACR,Ucr水平增加(P<0.05),肾脏病理状态有不同程度的改善。真武汤高、中剂量组及厄贝沙坦组ROCK1、ROCK2、IKK蛋白表达降低,NF-κB,p-IKK,p-NF-κB,和肾脏中的p-IκB(P&lt;0.05),IκB蛋白表达增加(P&lt;0.05),血清炎症因子IL-1β水平降低,IL-6、IL-8和TNF-α(P<0.05),IL-10水平升高(P<0.05)。真武汤能明显改善脾肾阳虚型DN小鼠肾功能及肾脏病理损害,其具体机制可能与下调肾脏ROCK/IKK/NF-κB通路关键分子的表达抑制炎症反应有关。
To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.