世界中医药

作者	单位
吕森浩1，李怀玉1，2，涂欣欣1，欧阳毅3，魏泽宏1，李玮婷4，杨军平4	1 江西中医药大学，南昌，330004； 2 广州中医药大学第一附属医院，广州，510405； 3 瑞金市人民医院，瑞金，342500； 4 江西中医药大学附属医院，南昌，330006

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中文摘要:

目的：观察肾衰泄浊汤对腺嘌呤灌胃致慢性肾衰竭（CRF）大鼠和转化生长因子β1(TGF-β1)干预人肾皮质近曲小管上皮细胞(HK-2细胞)纤维化的影响，并从let-7b-5p微小RNA（let-7b-5p）/转化生长因子-β受体Ⅰ(TGF-βR1)轴探究其可能的作用机制。方法：将32只斯泼累格·多雷(SD)大鼠采用完全随机法随机分为正常组、模型组、中药（18.75 g/kg、37.5 g/kg）组，每组组8只。采用腺嘌呤灌胃21 d构建CRF大鼠，中药组给予相应浓度肾衰泄浊汤灌胃，其余组给予等量生理盐水，给药28 d。HK-2细胞分为正常组、模型组、10%含药血清组以及20%含药血清组，TGF-β1处理HK-2细胞24 h以构建纤维化模型，含药血清组中加入相应浓度含药血清。检测大鼠血清血肌酐(Scr)、尿素氮(BUN)水平，苏木精-伊红染色法（HE染色）评估肾组织的病理改变，免疫组织化学检测肾组织中TGF-βR1、Ⅰ型胶原蛋白(Collagen Ⅰ)、α肌动蛋白(α-SMA)及纤连蛋白(FN)表达，实时荧光定量逆转录PCR（qRT-PCR）法检测肾组织及HK-2细胞中let-7b-5p微小RNA（let-7b-5p）表达，蛋白质免疫印迹法检测HK-2细胞中TGF-βR1、Collagen Ⅰ、α-SMA、FN蛋白表达。结果：1）体内实验结果：与正常组比较，模型组大鼠血清Scr、BUN水平高（P<0.05）；与模型组比较，中药18.75 g/kg组、中药37.5 g/kg组血清Scr、BUN水平低（P<0.05）；与中药18.75 g/kg组比较，中药37.5 g/kg组血清Scr、BUN水平低（P<0.05）。与模型组比较，中药组肾组织炎症细胞浸润、纤维化病变及结构破坏改善，结晶沉淀减少。与正常组比较，模型组大鼠肾脏组织中TGF-βR1、Collagen Ⅰ、α-SMA、FN蛋白表达高（P<0.05）；与模型组比较，中药18.75 g/kg组、中药37.5 g/kg组大鼠肾脏组织中TGF-βR1、Collagen Ⅰ、α-SMA、FN蛋白表达低（P<0.05）；与中药18.75 g/kg组比较，中药37.5 g/kg组大鼠肾脏组织中Collagen Ⅰ、FN蛋白表达低（P<0.05）。与正常组比较，模型组大鼠肾脏组织中let-7b-5p表达低（P<0.05）；与模型组比较，中药18.75 g/kg组、中药37.5 g/kg组大鼠肾脏组织中let-7b-5p表达高（P<0.05）；与中药18.75 g/kg组比较，中药37.5 g/kg组大鼠肾脏组织中let-7b-5p表达高（P<0.05）。2）体外实验结果：含药血清干预HK-2细胞24、48 h的半数抑制浓度（IC50）分别为81.35%、54.53%。因此设定后续给药时间为24 h，含药血清浓度为10%、20%。与正常组比较，模型组HK-2细胞let-7b-5p表达低（P<0.05）；与模型组比较，10%、20%含药血清组HK-2细胞let-7b-5p表达高（P<0.05）；与10%含药血清组比较，20%含药血清组HK-2细胞let-7b-5p表达高（P<0.05）。与正常组比较，模型组HK-2细胞中TGF-βR1、Collagen Ⅰ、α-SMA、FN蛋白高（P<0.05）；与模型组比较，10%、20%含药血清组HK-2细胞中中TGF-βR1、Collagen Ⅰ、α-SMA、FN蛋白表达低（P<0.05）；与10%含药血清组比较，20%含药血清组HK-2细胞中TGF-βR1、Collagen Ⅰ、FN蛋白表达低（P<0.05）。结论：肾衰泄浊汤能减轻体内外模型的肾纤维化，其作用机制可能与let-7b-5p/TGF-βR1轴有关。

English Summary:

To observe the effects of Shenshuai Xiezhuo Decoction(SSXD) on chronic renal failure(CRF) rats induced by adenine and on fibrosis in human renal proximal tubular epithelial cells(HK-2 cells) intervened by transforming growth factor-β1(TGF-β1)，and to explore its potential mechanism via the let-7b-5p microRNA/TGF-β receptor I(TGF-βR1) axis.Methods:Thirty-two Sprague-Dawley(SD) rats were randomly divided into four groups:normal，model，and SSXD(18.75 g/kg，37.5 g/kg) groups，with 8 rats in each group.CRF was induced by oral administration of adenine for 21 days，and SSXD was administered to the corresponding groups for 28 days.The normal and model groups were given an equal volume of saline.HK-2 cells were divided into normal，model，10% drug-containing serum，and 20% drug-containing serum groups，with TGF-β1 treatment for 24 hours to construct a fibrosis model.Drug-containing serum groups were treated with the corresponding concentrations of drug-containing serum.Serum creatinine(Scr) and blood urea nitrogen(BUN) levels in rats were measured.Hematoxylin-eosin(HE) staining was used to evaluate kidney tissue pathology.Immunohistochemical analysis was performed to detect the expression of TGF-βR1，collagen I(Collagen I)，α-smooth muscle actin(α-SMA)，and fibronectin(FN) in kidney tissues.Quantitative real-time PCR(qRT-PCR) was used to measure the expression of let-7b-5p microRNA in kidney tissues and HK-2 cells.Western blot was used to detect the protein expression of TGF-βR1，Collagen I，α-SMA，and FN in HK-2 cells.Results:1) In vivo results:Compared with the normal group，the model group showed higher serum levels of Scr and BUN(P<0.05).Compared with the model group，the SSXD 18.75 g/kg and 37.5 g/kg groups had lower serum Scr and BUN levels(P<0.05).The SSXD 37.5 g/kg group showed significantly lower Scr and BUN levels than the 18.75 g/kg group(P<0.05).Compared with the model group，kidney tissue inflammation，fibrosis，and structural damage were improved in the SSXD groups，and crystal precipitation was reduced.In the model group，TGF-βR1，Collagen I，α-SMA，and FN protein expression in kidney tissue was higher than that in the normal group(P<0.05).Compared with the model group，the SSXD 18.75 g/kg and 37.5 g/kg groups had lower expression of TGF-βR1，Collagen I，α-SMA，and FN proteins in kidney tissue(P<0.05).The SSXD 37.5 g/kg group had significantly lower expression of Collagen I and FN than the 18.75 g/kg group(P<0.05).Compared with the normal group，let-7b-5p expression in the model group was lower(P<0.05)，while it was higher in the SSXD 18.75 g/kg and 37.5 g/kg groups compared to the model group(P<0.05).The SSXD 37.5 g/kg group had significantly higher let-7b-5p expression compared to the 18.75 g/kg group(P<0.05). 2) In vitro results:The half-maximal inhibitory concentrations(IC50) for 24-hour and 48-hour drug-containing serum intervention on HK-2 cells were 81.35% and 54.53%，respectively，and the subsequent treatment time was set at 24 hours with 10% and 20% drug-containing serum concentrations.Compared with the normal group，let-7b-5p expression in HK-2 cells of the model group was lower(P<0.05)，while the 10% and 20% drug-containing serum groups showed significantly higher expression of let-7b-5p compared to the model group(P<0.05).The 20% drug-containing serum group had significantly higher expression of let-7b-5p than the 10% group(P<0.05).Compared with the normal group，the model group showed higher expression of TGF-βR1，Collagen I，α-SMA，and FN proteins in HK-2 cells(P<0.05).The 10% and 20% drug-containing serum groups showed lower expression of TGF-βR1，Collagen I，α-SMA，and FN proteins compared to the model group(P<0.05).The 20% drug-containing serum group showed significantly lower expression of TGF-βR1，Collagen I，and FN proteins than the 10% drug-containing serum group(P<0.05).Conclusion:SSXD can alleviate kidney fibrosis in both in vivo and in vitro models.Its mechanism of action may be related to the let-7b-5p/TGF-βR1 axis.

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