目的 研究益母草碱(LEO)调节Ras同源基因家庭成员A(RhoA)/Rho相关卷曲螺旋形成的蛋白激酶(ROCK)信号通路对原发性肾病综合征(PNS)大鼠的改善作用.方法 培养大鼠肾小球系膜细胞HBZY-1,随机分为对照组、增生组[100 ng·mL-1脂多糖(LPS)]、低浓度实验组(100 ng·mL-1 LPS+5 μmol·L-1 LEO)、高浓度实验组(100 ng·mL-1 LPS+10 μmol·L-1 LEO)和联合处理组(100 ng·mL-1 LPS+10 μmol·L 1 LEO+10 nmol·L 1 RhoA 激活药U46619).以细胞计数试剂盒法检测细胞增殖活性,用流式细胞仪检测细胞凋亡情况.将60只SPF级Wistar大鼠随机分为正常组、模型组、低剂量实验组(10 mg·kg-1 LEO)、高剂量实验组(20 mg·kg-1 LEO)和联合组(20 mg·kg-1 LEO+10 mmol·L-1 U46619),每组12只.用考马斯亮蓝法检测大鼠24 h尿蛋白含量,用酶联免疫吸附测定法检测大鼠肾组织中炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-4水平,用蛋白质印迹法检测大鼠肾组织RhoA和ROCK1蛋白的表达水平.结果 在细胞实验中,对照组和增生组的HBZY-1细胞增殖活性(光密度值)分别为0.32±0.03和0.70±0.07,细胞凋亡率分别为(9.23±1.04)％和(1.64±0.22)％,在统计学上差异均有统计学意义(均P＜0.05).在动物实验中,正常组、模型组、低剂量实验组、高剂量实验组和联合组的24 h尿蛋白含量分别为(21.45±2.28)、(127.38±14.70)、(120.85±13.34)、(43.15±6.68)和(96.20±10.63)mg,TNF-α 水平分别为(0.27±0.05)、(1.58±0.16)、(1.56±0.16)、(0.44±0.05)和(1.03±0.10)ng·mL-1,IL-4 水平分别为(0.17±0.02)、(1.24±0.12)、(1.20±0.12)、(0.29±0.03)和(0.87±0.09)ng·mL-1,RhoA蛋白相对表达水平分别为0.27±0.03、0.78±0.08、0.76±0.07、0.34±0.03 和0.72±0.07,ROCK1 蛋白相对表达水平分别为 0.22±0.02、0.85±0.09、0.83±0.08、0.41±0.04 和0.75±0.08,联合组的上述指标与高剂量实验组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 LEO可能通过下调RhoA/ROCK信号通路对大鼠PNS起到改善作用.
Objective To explore the improvement effect of leonurine(LEO)on primary nephrotic syndrome(PNS)rats by regulating Ras homolog gene family member A(RhoA)/Rho associated coiled-coil containing protein kinase(ROCK)signaling pathway.Methods Rat glomerular mesangial cells HBZY-1 were cultured and randomly grouped into control group,proliferation group(100 ng·mL-1 LPS),low concentration experimental group(100 ng·mL-1 LPS+5 μmol·L-1 LEO),high concentration experimental group[100 ng·mL-1 lipopolysaccharide(LPS)+10 μmol·L-1LEO],and combined treatment group(100 ng·mL-1LPS+10 μmol·L-1 LEO+10 nmol·L-1 RhoA activator U46619).Cell counting kit-8 assay was applied to detect cell proliferation activity;flow cytometry was applied to detect apoptosis.Sixty SPF Wistar rats were randomly grouped into normal group,model group,low-dose experimental group(10 mg·kg-1 LEO),high-dose experimental group(20 mg·kg-1 LEO),and combination group(20 mg·kg-1 LEO+10 mmol·L-1 U46619),with 12 rats in each group.Except the normal group,the other groups were injected with adriamycin hydrochloride via tail vein to establish PNS rat model;coomassie brilliant blue method was applied to detect 24-hour urinary protein content in rats.Enzyme linked immunosorbent assay was used to detect the levels of inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-4 in renal tissue.Western blot was used to detect RhoA and Rock1 protein expression in rat kidney.Results In the cell experiment,the proliferation activities(optical density value)of HBZY-1 cells in control group and hyperplasia group was 0.32±0.03 and 0.70±0.07;the apoptosis rate were(9.23±1.04)％and(1.64±0.22)％,with statistical significance(all P＜0.05).In animal experiments,24 h urinary protein content of normal group,model group,low-dose experimental group,high-dose experimental group and combination group were(21.45±2.28),(127.38±14.70),(120.85±13.34),(43.15±6.68)and(96.20±10.63)mg;TNF-α levels were(0.27±0.05),(1.58±0.16),(1.56±0.16),(0.44±0.05)and(1.03±0.10)ng·mL-1;IL-4 levels were(0.17±0.02),(1.24±0.12),(1.20±0.12),(0.29±0.03)and(0.87±0.09)ng·mL-1;RhoA protein expression levels were 0.27±0.03,0.78±0.08,0.76±0.07,0.34±0.03 and 0.72±0.07;the expression levels of ROCK1 protein were 0.22±0.02,0.85±0.09,0.83±0.08,0.41±0.04 and 0.75±0.08.The differences of above indexes were statistically significant between the high-dose experimental group and the combination group(all P＜0.05).Conclusion LEO may improve PNS in rats by down-regulating RhoA/ROCK signaling pathway.
宫璞;王晴;赵振
天津市第五中心医院急诊内科,天津 300450天津市第一中心医院急诊科,天津 300190
中医学
益母草碱;Ras同源基因家庭成员A/Rho相关卷曲螺旋形成的蛋白激酶信号通路;原发性肾病综合征
leonurine;Ras homolog gene family member A/Rho associated coiled-coil containing protein kinase signaling pathway;primary nephrotic syndrome
《中国临床药理学杂志》 2024 (011)
1603-1607 / 5
天津市滨海新区卫生健康委科技基金资助项目(2022BWKQ002)
10.13699/j.cnki.1001-6821.2024.11.013
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