大黄不同炮制品和煎煮时间对TGF-β1诱导人肾皮质近曲小管上皮细胞中E-cadherin和α-SMA浓度的影响

Effects of Difference Processed Products and Decoction Time of Dahuang(Radix et Rhizoma Rhei) on TGF-β1 Induced E-cadherin and α-SMA Concentrations in Human Renal Cortex Proximal Tubule Epithelial Cells

目的探讨不同大黄炮制品干预人肾皮质近曲小管上皮细胞(HK-2细胞)的作用差异及筛选最佳的煎煮时间点。方法取生大黄、熟大黄、酒大黄、大黄炭饮片各350 g,均分为7份。每份药物分别煎煮5、7、9、10、15、20、30 min,最终制备浓度约1000 g/L药液。将145只大鼠随机分为生大黄组、熟大黄组、酒大黄组、大黄炭组,各组又分为5、7、9、10、15、20、30 min时间点的亚组,每个亚组5只,另设对照组5只。将各药物组大鼠分别给予相应的大黄煎剂以1.35 g/(kg·d)的剂量灌胃,对照组大鼠给予0.9%氯化钠2 ml灌胃,每日2次,第6日灌胃后制备含药血清。用大黄不同炮制品不同煎煮时间的含药血清干预经转化生长因子β1(TGF-β1)诱导的HK-2细胞24 h,检测各组HK-2细胞中上皮细胞钙黏蛋白(E-cadherin)和α平滑肌肌动蛋白(α-SMA)的浓度。结果α-SMA浓度最低的分别为生大黄10 min亚组(52.086 ng/ml)、熟大黄10 min亚组(44.596 ng/ml)、酒大黄9 min亚组(49.768 ng/ml)、大黄炭9 min亚组(58.470 ng/ml)。E-cadherin浓度最高的分别为生大黄10 min亚组(1.017 ng/ml)、熟大黄10 min亚组(1.325 ng/ml)、酒大黄9 min亚组(1.163 ng/ml)、大黄炭9 min亚组(0.787 ng/ml)。与大黄炭组比较,其余3组α-SMA浓度降低,E-cadherin浓度升高(P<0.05或P<0.01)。与熟大黄组比较,生大黄组和酒大黄组α-SMA浓度升高,E-cadherin浓度降低(P<0.05或P<0.01)。酒大黄组与生大黄组比较,α-SMA和E-cadherin浓度差异亦有统计学意义(P<0.05)。结论大黄的四种炮制品均能有效抑制经TGF-β1诱导的HK-2细胞上皮间充质转化,其作用效果由强到弱依次均为熟大黄、酒大黄、生大黄、大黄炭,生大黄和熟大黄最佳煎煮时间为10 min,酒大黄和大黄炭最佳煎煮时间为9 min。

Objective To investigate the effects of different processed products of Dahuang(Radix et Rhizoma Rhei)on human renal cortex proximal tubule epithelial cells(HK-2 cells)and to screen the best decoction time.Methods Decoction pieces of raw Radix et Rhizoma Rhei,prepared Radix et Rhizoma Rhei,wine processed Radix et Rhizoma Rhei,and charred Radix et Rhizoma Rhei were collected 350 g each and divided into 7 equal parts.Each medicine was respetively decocting for 5,7,9,10,15,20,30 minutes,and finally a concentration of about 1000 g/L was prepared.Totally 145 rats were randomly divided into a raw Radix et Rhizoma Rhei group,a prepared Radix et Rhizoma Rhei group,a wine processed Radix et Rhizoma Rhei group,and a charred Radix et Rhizoma Rhei group,and each group was further divided into subgroups at decocting time 5,7,9,10,15,20,and 30 minutes,with 5 rats in each subgroup.Additional 5 rats were set as control group.Rats in each drug group were given the corresponding Radix et Rhizoma Rhei decoction by gavage at a dose of 1.35 g/(kg·d),while the control group was given 0.9%Sodium chloride twice a day,and drug-containing serum was prepared after gavage on the 6 th day.The drug containing serum with different decocting time and different processed products of Radix et Rhizoma Rhei was used to interfere with the induction of HK-2 cells by transforming growth factorβ1(TGF-β1)for 24 hours.The epithelia-cadherin(E-cadherin)and alpha smooth muscle actin(α-SMA)concentration in HK-2 cells of each group were measured.Results The lowestα-SMA concentrations were in the raw Radix et Rhizoma Rhei 10 min subgroup(52.086 ng/ml),the prepared Radix et Rhizoma Rhei 10 min subgroup(44.596 ng/ml),the wine processed Radix et Rhizoma Rhei 9 min subgroup(49.768 ng/ml),and the charred Radix et Rhizoma Rhei 9 min subgroup(58.470 ng/ml).The highest concentrations of E-cadherin were the raw Radix et Rhizoma Rhei 10 min subgroup(1.017 ng/ml),the prepared Radix et Rhizoma Rhei 10 min subgroup(1.325 ng/ml),the wine processed Radix et Rhizoma Rhei 9 min subgroup(1.163 ng/ml),and the charred Radix et Rhizoma Rhei 9 min subgroup(0.787 ng/ml).Compared with the charred Radix et Rhizoma Rhei group,the concentration ofα-SMA decreased and the concentration of E-cadherin increased in the other 3 groups(P<0.05 or P<0.01).Compared with the prepared Radix et Rhizoma Rhei group,theα-SMA concentration increased and the E-cadherin concentration decreased in the raw Radix et Rhizoma Rhei group and the wine processed Radix et Rhizoma Rhei group(P<0.05 or P<0.01).The differences betweenα-SMA and E-cadherin concentrations in the wine processed Radix et Rhizoma Rhei group and the raw Radix et Rhizoma Rhei group were also statistically significant(P<0.05).Conclusion The 4 processed products of Radix et Rhizoma Rhei can effectively inhibit the transformation of epithelial mesenchyme of HK-2 cells induced by TGF-β1,and their effects are in order from strong to weak:prepared Radix et Rhizoma Rhei,wine processed Radix et Rhizoma Rhei,raw Radix et Rhizoma Rhei,and charred Radix et Rhizoma Rhei.The best decocting time for raw Radix et Rhizoma Rhei and prepared Radix et Rhizoma Rhei is 10 min,and 9 min for wine processed Radix et Rhizoma Rhei and charred Radix et Rhizoma Rhei.