济川煎对CaMKⅡγ沉默及过表达结肠平滑肌细胞的影响及机制研究  

Effect and Mechanism of Jichuan Decoction(济川煎)on CaMKIIγ Silencing or Over-expression in Colonic Smooth Muscle Cells

作　　者：刘聪[1] 张悦 张润涛 荆然 郭丁丁[1] 倪艳[1] 康永[1] LIU Cong;ZHANG Yue;ZHANG Runtao;JING Ran;GUO Dingding;NI Yan;KANG Yong(Key Laboratory of Critical Technology Development of TCM New Products,Institute of Chinese Medicine Prescriptions,Shanxi Provincial Academy of Traditional Chinese Medicine,Taiyuan 030045)

机构地区：[1]山西省中医药研究院中药方剂研究所,中药新产品关键技术开发重点实验室,太原030045

出　　处：《中药药理与临床》2023年第10期2-7,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：山西省青年科技研究基金项目(编号:201801D221385);中药新产品关键技术开发省级重点培育实验室[山西省“四个一批”科技兴医创新计划项目](编号:2020SYS09)。

摘　　要：目的:观察济川煎对钙调蛋白依赖性激酶Ⅱγ(Calmodulin-dependent protein kinases Ⅱγ,CaMKⅡγ)沉默及过表达大鼠结肠平滑肌细胞的影响及机制。方法:基于慢病毒介导构建原代大鼠结肠平滑肌细胞CaMKⅡγRNA干扰载体,建立CaMKⅡγ沉默及过表达结肠平滑肌细胞模型。将模型成功并计数的细胞悬液,随机分为空白对照组、阴性载体对照组、CaMKⅡγ沉默及过表达组、莫沙必利+CaMKⅡγ沉默及过表达1.88 mg/kg组、济川煎+CaMKⅡγ沉默及过表达4.31 g/kg组。每组细胞给药后培养24 h,采用流式细胞仪检测胞内钙离子(Ca^(2+))浓度变化;采用实时荧光定量聚合酶链反应(RT-PCR)及蛋白免疫印迹法(Western blot)法检测胞内钙调蛋白(CaM)、CaMKⅡγ、L型钙离子通道Cav1.2、SR钙离子依赖性ATP酶1(SERCA1)、兰尼碱受体(RYR2)的表达情况。结果:与空白对照组比较,模型对照CaMKⅡγ沉默组CamkⅡγ mRNA表达下调,Ca^(2+)浓度显著降低(P<0.01),CaM、CaMKⅡγ、SERCA1、RYR2蛋白表达明显下调,CaV1.2蛋白表达明显上调(P<0.05或P<0.01);与模型对照CaMKⅡγ沉默组比较,Ca^(2+)浓度降低,CaMKⅡγ蛋白表达下调(P<0.05或P<0.01);与空白对照组比较,模型对照CaMKⅡγ过表达组CamkⅡγ mRNA表达上调,Ca^(2+)浓度显著升高(P<0.01),CaM、CaMKⅡγ、SERCA1、RYR2蛋白表达明显上调(P<0.05或P<0.01),CaV1.2蛋白表达明显下调(P<0.05);与模型对照CaMKⅡγ过表达组比较,济川煎4.31 g/kg组CamkⅡγ mRNA表达下调,Ca^(2+)浓度显著降低(P<0.01),CaM、CaMKⅡγ、SERCA1、RyR2蛋白表达明显下调(P<0.05或P<0.01)。结论:济川煎可能通过抑制CaMKⅡγ的表达调控结肠平滑肌细胞功能,达到治疗阳虚便秘目的。Objective:To explore the effect and mechanism of Jichuan Decoction(济川煎)on silencing or overexpression of calcium/calmodulindependent protein kinase II(CaMKIIγ)in colonic smooth muscle cells of rats.Methods:Primary rat colonic smooth muscle cells were established with CaMKIIy RNA interference vectors mediated by lentivirus to achieve silencing or overexpression of CaMKIIγ.The model counted cell suspension was randomLy divided into eight groups:a blank control group,a negative vector control group,CaMKIIγ silencing or overexpression groups,mosapride+CaMKIIγ silencing or overexpression groups(1.88 mg/kg),and Jichuan Decoction+CaMKIIγ silencing or overexpression groups(4.31 g/kg).After drug administration and 24 hours of incubation,intracellular calcium ion(Ca^(2+))concentration was measured using flow cytometry.Real-time fluorescence-based quantitative polymerase chain reaction(RT-PCR)and Western blot were performed to assess the expression of intracellular calcium modulator(CaM),CaMKIIγ,L-type calcium channel Cav1.2,sarcoplasmic reticulum calcium ATPase 1(SERCA1),and ryanodine receptor 2(RYR2).Results:Compared with the blank control group,the model CaMKIIγ silencing group showed downregulated CaMKIIγ mRNA expression,decreased intracllular Ca^(2+)concentration(P<0.01),decreased protein expression of CaM,CaMKIIγ,SERCA1,and RYR2,and upregulated expression of CaV1.2(P<0.05 or P<0.01).The intracellular Ca^(2+) concentration was reduced and the expression of CaMKIIγ protein was downregulated(P<0.05 or P<0.01)as compared with those in the model CaMKIIγ silencing group.Compared with the blank control group,the model CaMKIIγ overexpression group showed upregulated CaMKIIγ mRNA expression,increased intraceular Ca^(2+) concentration(P<0.01),upregulated protein expression of CaM,CaMKIIγ,SERCA1,and RYR2(P<0.05 or P<0.01),and downregulated expression of CaV1.2(P<0.05).Compared with the model CaMKIIγ overexpression group,the Jichuan Decoction group(4.31 g/kg)showed downregulated CaMKIIγ mRNA expression,reduc

关 键 词：济川煎 钙调蛋白依赖性激酶Ⅱγ 结肠平滑肌细胞 沉默 过表达 兰尼碱受体 

分 类 号：R285[医药卫生—中药学]