多因素诱导高尿酸肾病大鼠模型的建立与芪苓颗粒的干预作用研究

Establishment of a multi-factor-induced hyperuricemic nephropathy rat model to study the intervention effect of Qiling granules

目的采用“氧嗪酸钾联合腺嘌呤加酵母饲料饲喂”的多因素诱导法建立高尿酸肾病(HN)大鼠模型,并观察芪苓颗粒(QLG)的干预作用。方法取58只SPF级雄性SD大鼠,随机取10只大鼠作正常对照(normal control,NC)组,其余大鼠采用多因素诱导法建立HN大鼠模型,造模2周后颌下取血检测血清UA、CREA、BUN、TG和TC,筛选出血清UA和体重接近均值的HN大鼠40只,采用分层随机法分为模型对照(Model,M)组、QLG低剂量(Qiling granules low dose,QLG-L)组、QLG高剂量(Qiling granules high dose,QLG-H)组和阳性对照(positive control,PC)组,每组10只。各组每天给予相应药物灌胃,连续给药4周后颌下采血检测血清UA、CREA、BUN、TG和TC,行安死术,取肝组织检测XOD、ADA活性,取肾组织进行HE、Gomori六胺银染色,并用免疫组化和Western blot法观察肾GLUT9、OAT1、VCAM-1和TGF-β的蛋白表达。结果与NC组比,M组血清UA、CREA、BUN、TC、TG水平和肝XOD、ADA活性均显著升高(P<0.01),肾组织病变明显,肾小管尿酸盐含量和肾GLUT9、VCAM-1、TGF-β蛋白表达均显著升高(P<0.01,P<0.05)、OAT1表达显著降低(P<0.01)。与M组比,各给药组大鼠的血清UA水平和肝XOD、ADA活性以及肾VCAM-1蛋白表达均显著降低(P<0.01,P<0.05),QLG-L组大鼠的血清CREA、BUN水平和肾TGF-β蛋白表达亦显著降低(P<0.05,P<0.01),QLG-H组大鼠的血清CREA、BUN水平和肾GLUT9蛋白表达亦显著降低(P<0.01,P<0.05),各给药组大鼠的尿酸盐沉积及其引发的肾损伤均有不同程度的减轻但无显著差异(P>0.05)。结论给予大鼠酵母饲料饲喂的同时给予氧嗪酸钾与腺嘌呤联合灌胃可诱导建立稳定的HN大鼠模型;QLG可通过改善HN模型大鼠UA代谢紊乱、减轻肾炎症和尿酸盐沉积及其引发的肾损伤来有效治疗HN,其作用机制与降低血清UA、CREA、BUN、TG水平和肝XOD、ADA活性以及肾GLUT9、OAT1、VCAM-1、TGF-β蛋白的表达有关。

Objective To establish a rat model of hyperuricemic nephropathy(HN)using a multifactorial induction method of potassium oxazinate combined with adenine and yeast feed to observe the intervention effect of Qiling granules(QLG).Methods Fifty-eight SPF-grade male SD rats were selected,and 10 rats were randomly allocated to the normal control(NC)group.The remaining rats were induced by multiple factors to establish HN rat models.After 2 weeks of modeling,submandibular blood samples were taken to detect serum UA,CREA,BUN,TG,and TC.Forty HN rats with bleeding clearance UA and body weight close to the mean were selected.They were randomly divided into a model(M)group,QLG low dose(QLG-L)groups,QLG high dose(QLG-H)group,and a positive control(PC)group,with 10 rats in each group,using a stratified randomization method.Each group was given corresponding drugs by gavage daily,and after continuous administration for 4 weeks,submandibular blood samples were taken to detect serum UA,CREA,BUN,TG,and TC.After euthanasia of the rats,liver tissue was taken to detect XOD and ADA activity.Renal tissue was taken for HE and Gomori hexamine silver staining,and the protein expression of GLUT9,OAT1,VCAM-1,and TGF-βin the kidneys was observed using immunohistochemistry and Western blot method.Results Compared with the NC group,the M group’s serum levels of UA,CREA,BUN,TC,and TG,as well as liver XOD and ADA activities,were significantly increased(P<0.01).The renal tissue of the model rats showed significant pathological changes.The area of renal tubules positive for urate and the expression of GLUT9,VCAM-1,and TGF-βproteins in the kidneys were significantly increased(P<0.01,P<0.05),while the expression of OAT1 was significantly reduced(P<0.01).Compared with the M group,each treatment group showed significantly reduced serum UA levels,liver XOD,ADA activity,and renal VCAM-1 protein expression(P<0.01,P<0.05).The serum CREA and BUN levels and renal TGF-βprotein expression of rats in the QLG-L group were significantly reduced(P<0.05,P<0.01).The serum CREA and BUN levels and renal GLUT9 protein expression of rats in the QLG-H group were also significantly reduced(P<0.01,P<0.05).The urate deposition and renal injury caused by each treatment were reduced to varying degrees,but there were no significant differences among groups(P>0.05).Conclusions A stable HN rat model can be induced by gavage of potassium oxyzinate and adenine in combination with yeast feed.QLG can effectively treat HN by improving UA metabolic disorders,reducing the renal inflammation and urate deposition that cause renal damage in HN model rats.Its mechanism of action is related to a reduction in serum UA,CREA,BUN,and TG levels;liver XOD and ADA activities;and the expression of GLUT9,OAT1,VCAM-1,and TGF-βproteins in the kidneys.