四君子汤对慢性肾脏病-蛋白质能量消耗模型小鼠的改善作用及机制研究(1)
摘 要 目的：研究四君子湯对慢性肾脏病-蛋白质能量消耗(CKD-PEW)模型小鼠骨骼肌萎缩的改善作用，并探讨其可能的作用机制。方法：将80只小鼠随机分为假手术组(n=10)和造模组(n=70)。造模组小鼠采用切除5/6肾联合低蛋白(4%酪蛋白)饮食法建立CKD-PEW模型。将造模成功的50只小鼠随机分为模型组，四君子汤低、中、高剂量组[2.34、4.68、9.36 g/(kg·d)，以生药量计]和复方α-酮酸片组[阳性对照，1 g/(kg·d)]，每组10只。各给药组小鼠灌胃相应药物，假手术组和模型组小鼠灌胃等体积生理盐水，每天1次，连续灌胃14 d。末次给药后，称定小鼠体质量、左侧胫骨前肌(TA)湿质量，并测定TA横截面积;检测小鼠TA蛋白合成与分解代谢情况;采用实时荧光定量-聚合酶链式反应法检测小鼠TA中B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)及胱天蛋白酶3(Caspase-3) mRNA表达水平;采用Western blotting法检测小鼠TA中肌萎缩蛋白Fbox-1(Atrogin-1)、肌环指蛋白1(MuRF-1)、Rho相关蛋白激酶1(ROCK1)、磷酸化肿瘤抑制基因(p-PTEN)、磷脂酰肌醇3 激酶(PI3K)及磷酸化蛋白激酶B(p-Akt)蛋白的表达水平。结果：与假手术组比较，模型组小鼠的体质量、TA湿质量、TA蛋白合成代谢能力以及PI3K、p-Akt蛋白表达水平显著降低(P<0.05)，TA横截面积显著减小(P<0.05)，TA蛋白分解代谢能力、Bax/Bcl-2比值、Caspase-3 mRNA表达水平和Atrogin-1、MuRF-1、ROCK1、p-PTEN蛋白表达水平显著升高(P<0.05)。与模型组比较，四君子汤中、高剂量组和复方α-酮酸片组小鼠上述指标均显著改善(P<0.05)。结论：四君子汤能够增加CKD-PEW模型小鼠体质量，抑制其骨骼肌萎缩;其机制可能与调控ROCK1/PTEN/Akt信号通路，抑制Atrogin-1和MuRF-1表达有关。
关键词 四君子汤;慢性肾脏病;蛋白质能量消耗;Rho相关蛋白激酶1;磷酸化肿瘤抑制基因;磷脂酰肌醇-3-激酶;蛋白激酶B;小鼠
ABSTRACT OBJECTIVE： To study the improvement effects of Sijunzi decoction on skeletal muscle atrophy in chronic kidney disease-protein energy wasting (CKD-PEW) model mice， and to explore its potential mechanism. METHODS： A total of 80 mice were randomly divided into sham operation group (n=10) and modeling group (n=70). CKD-PEW model was established by removing 5/6 kidneys and giving a low-protein diet (4% casein) for mice in modeling group. Totally 50 modeled mice were randomly divided into model group， Sijunzi decoction low-dose， medium-dose and high-dose groups [2.34， 4.68， 9.36 g/(kg·d)，by crude drug]， Compound α-ketoacid tablets group [positive control， 1 g/(kg·d)]， with 10 mice in each group. Administration groups were given relevant medicine intragastrically; sham operation group and model group were given constant volume of normal saline intragastrically， once a day， for consecutive 14 d. After last medication， body weight of mice and wet mass of left tibialis anterior muscle (TA) were weighed; TA cross-sectional area was determined; protein synthesis and decomposition metabolism ability of TA were detected; mRNA expressions of Bcl-2， Bax and Caspase-3 in TA were detected by Real-time PCR; protein expressions of muscular dystrophin Fbox-1 (Atrogin-1)， myofloin-1 (MuRF-1)， Rho-related protein kinase 1 (ROCK1)， phosphorylated PTEN (p-PTEN)， phosphatidylinositol-3-kinase (PI3K) and phosphorylated Akt (p-Akt) in TA were detected by Western blotting. RESULTS： Compared with the sham operation group， the body weight， TA wet weight， protein synthesis metabolism ability of TA as well as protein expressions of PI3K and p-Akt were decreased significantly in model group (P<0.05); the cross-sectional area of TA decreased significantly (P<0.05); protein decomposition metabolism ability of TA， Bax/Bcl-2 ratio， Caspase-3 mRNA expression， protein expressions of Atrogin-1， MuRF-1， ROCK1 and p-PTEN were increased significantly (P<0.05). Compared with model group， above indexes of mice were all improved significantly in Sijunzi decoction medium-dose， high-dose groups and Compound α-ketoacid tablets group (P<0.05). CONCLUSIONS： Sijunzi decoction can increase the body weight of CKD-PEW model mice and alleviate the skeletal atrophy; the mechanism may be related to regulating ROCK1/PTEN/Akt signaling pathway activity， inhibiting the expression of Atrogin-1 and MuRF-1., http://www.100md.com(许烨 李志明 远方)
关键词 四君子汤;慢性肾脏病;蛋白质能量消耗;Rho相关蛋白激酶1;磷酸化肿瘤抑制基因;磷脂酰肌醇-3-激酶;蛋白激酶B;小鼠
ABSTRACT OBJECTIVE： To study the improvement effects of Sijunzi decoction on skeletal muscle atrophy in chronic kidney disease-protein energy wasting (CKD-PEW) model mice， and to explore its potential mechanism. METHODS： A total of 80 mice were randomly divided into sham operation group (n=10) and modeling group (n=70). CKD-PEW model was established by removing 5/6 kidneys and giving a low-protein diet (4% casein) for mice in modeling group. Totally 50 modeled mice were randomly divided into model group， Sijunzi decoction low-dose， medium-dose and high-dose groups [2.34， 4.68， 9.36 g/(kg·d)，by crude drug]， Compound α-ketoacid tablets group [positive control， 1 g/(kg·d)]， with 10 mice in each group. Administration groups were given relevant medicine intragastrically; sham operation group and model group were given constant volume of normal saline intragastrically， once a day， for consecutive 14 d. After last medication， body weight of mice and wet mass of left tibialis anterior muscle (TA) were weighed; TA cross-sectional area was determined; protein synthesis and decomposition metabolism ability of TA were detected; mRNA expressions of Bcl-2， Bax and Caspase-3 in TA were detected by Real-time PCR; protein expressions of muscular dystrophin Fbox-1 (Atrogin-1)， myofloin-1 (MuRF-1)， Rho-related protein kinase 1 (ROCK1)， phosphorylated PTEN (p-PTEN)， phosphatidylinositol-3-kinase (PI3K) and phosphorylated Akt (p-Akt) in TA were detected by Western blotting. RESULTS： Compared with the sham operation group， the body weight， TA wet weight， protein synthesis metabolism ability of TA as well as protein expressions of PI3K and p-Akt were decreased significantly in model group (P<0.05); the cross-sectional area of TA decreased significantly (P<0.05); protein decomposition metabolism ability of TA， Bax/Bcl-2 ratio， Caspase-3 mRNA expression， protein expressions of Atrogin-1， MuRF-1， ROCK1 and p-PTEN were increased significantly (P<0.05). Compared with model group， above indexes of mice were all improved significantly in Sijunzi decoction medium-dose， high-dose groups and Compound α-ketoacid tablets group (P<0.05). CONCLUSIONS： Sijunzi decoction can increase the body weight of CKD-PEW model mice and alleviate the skeletal atrophy; the mechanism may be related to regulating ROCK1/PTEN/Akt signaling pathway activity， inhibiting the expression of Atrogin-1 and MuRF-1., http://www.100md.com(许烨 李志明 远方)