养阴祛瘀方对高尿酸性肾病大鼠保护作用及对肾脏TGF-β_(1)/Smad3信号通路的影响

Protective effects of decoction for nourishing yin and dispeling stasis on rats with hyperuricemic nephropathy and its effects on renal TGF-β_(1)/Smad3 signaling pathway

目的观察养阴祛瘀方对高尿酸性肾病大鼠肾保护作用及对炎症因子和转化生长因子-β_(1)(TGF-β_(1))/Smad3通路的影响,探讨其作用机制。方法取56只雄性SD大鼠,随机选取13只作为空白组,其余大鼠采用氧嗪酸钾联合腺嘌呤灌胃法建立高尿酸性肾病模型。将造模成功的40只大鼠随机分为模型组及养阴祛瘀高、中、低剂量组,每组10只。养阴祛瘀高、中、低剂量组分别给予养阴祛瘀方37.2 g/(kg·d)、18.6 g/(kg·d)、9.3 g/(kg·d)灌胃,空白组及模型组给予等量生理盐水灌胃,均每天1次,连续灌胃3周。收集尿液检测24 h尿蛋白定量;取下腔静脉血,检测血肌酐(SCr)、尿素氮(BUN)、尿酸(UA)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;留取肾组织,HE染色观察肾组织病理形态,荧光显微镜下计算阳性细胞百分比;RT-PCR法检测肾组织中TGF-β_(1)、Smad3 mRNA相对表达量。结果模型组24 h尿蛋白定量及SCr、BUN、UA、IL-6、TNF-α水平均明显高于空白组(P均<0.05);肾小管萎缩,管内见尿酸盐晶体沉积,间质中可见较多炎性细胞浸润;肾组织中阳性细胞百分比及TGF-β_(1)、Smad3 mRNA相对表达量均明显高于空白组(P均<0.05)。养阴祛瘀各组24 h尿蛋白定量及血清IL-6、TNF-α水平和养阴祛瘀高、中剂量组SCr、BUN、UA水平均明显低于模型组(P均<0.05),且养阴祛瘀高剂量组血清UA、IL-6、TNF-α水平均明显低于养阴祛瘀中、低剂量组(P均<0.05);养阴祛瘀中、高剂量组尿酸盐沉积、炎性细胞浸润、肾小管损伤较前减轻,而养阴祛瘀低剂量组改善不明显;养阴祛瘀各组肾组织中阳性细胞百分比及TGF-β_(1)、Smad3 mRNA相对表达量均明显低于模型组(P均<0.05),且养阴祛瘀高剂量组各指标均明显低于养阴祛瘀低剂量组(P均<0.05)。结论养阴祛瘀方可保护高尿酸性肾病大鼠肾脏,减少蛋白尿,机制与下调炎症因子IL-6、TNF-α表达,抑制肾组织中TGF-β_(1)/Smad3通路激活有关。

Objective It is to observe the protective effects of decoction for nourishing yin and dispeling stasis on rats with hyperuricemic nephropathy and its effect on inflammatory factors and transforming growth factor-β_(1)(TGF-β_(1))/Smad3 pathway,and to explore its mechanism.Methods Fifty-six male SD rats were taken,in which 13 ones were randomly selected as the blank group,and the rest rats were given potassium oxyzincate combined with adenine by gavage to establish the models of hyperuricemic nephropathy.The 40 successfully modeled rats were randomly divided into model group and the high,medium and low dose groups of decoction for nourishing yin and dispeling stasis,with 10 rats in each group.The high,medium and low dose groups of decoction for nourishing yin and dispeling stasis were given decoction for nourishing yin and dispeling stasis 37.2 g/(kg·d),18.6 g/(kg·d),9.3 g/(kg·d)by gavage,respectively,the blank group and model group were given equal amount of normal saline by gavage,all once daily,continuously treated for 3 weeks.The urine of the rats was collected to determine 24 h urine protein quantification;their blood was collected from the inferior vena cava to detect the levels of blood creatinine(SCr),urea nitrogen(BUN),uric acid(UA),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α);their kidney tissues were gotten to observe the pathological morphology of kidney tissues by HE staining,and the percentage of positive cells was calculated under fluorescence microscope;the relative expression of TGF-β_(1) and Smad3 mRNA in kidney tissues were detected by RT-PCR.Results The 24 h urine protein quantification and the levels of SCr,BUN,UA,IL-6,and TNF-αwere significantly higher in the model group than those in the blank group(all P<0.05);renal tubular was atrophic with urate crystal deposits,and more inflammatory cell infiltration was found in the interstitium;the percentage of positive cells and the relative expression of TGF-β_(1) and Smad3 mRNA in the renal tissue were significantly higher than those in the blank group(all P<0.05).The 24 h urine protein quantification and the levels of serum IL-6 and TNF-αin each group of decoction for nourishing yin and dispeling stasis and the levels of SCr,BUN and UA in the high and medium dose groups of decoction for nourishing yin and dispeling stasis were significantly lower than those in the model group(all P<0.05),and the levels of serum UA,IL-6 and TNF-αin the high dose group were significantly lower than those in the medium and low dose groups(all P<0.05);the uric acid deposition,inflammatory cell infiltration,renal tubular injury were improved in the high and medium dose groups of decoction for nourishing yin and dispeling stasis,but the improvement was not significant in the low dose group;the percentage of positive cells and the relative expression of TGF-β_(1) and Smad3 mRNA in the kidney tissues of each groups of decoction for nourishing yin and dispeling stasis were significantly lower than those of the model group(all P<0.05),and the indexes of the high-dose group were significantly lower than those of the low-dose group(all P<0.05).Conclusion Decoction for nourishing yin and dispeling stasis can protect the kidney and reduce proteinuria of rats with hyperuricemic nephropathy,the mechanism may be related to down-regulating the expression of inflammatory factors IL-6 and TNF-α,and inhibiting the activation of TGF-β_(1)/Smad3 pathway in renal tissues.