基于Ca^(2+)-CaMKⅡ信号通路的济川煎治疗阳虚便秘的作用及分子机制  

Study on molecular mechanism of Jichuanjian improving Yang deficiency constipation based on Ca^(2+)-CaMKⅡsignaling pathway

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 Run-tao;JING Ran;GUO Ding-ding;NI Yan;KANG Yong(Shanxi Provincial Academy of Traditional Chinese Medicine,Taiyuan 030045,China)

机构地区：[1]山西省中医药研究院中药方剂研究所,山西太原030045

出　　处：《中国药理学通报》2023年第2期366-372,共7页Chinese Pharmacological Bulletin

基　　金：山西省青年科技研究基金项目(No 201801D221385);山西省“四个一批”科技兴医创新计划项目(No 2020SYS09)。

摘　　要：目的基于Ca^(2+)-CaMKⅡ通路观察济川煎对阳虚便秘大鼠的治疗作用及分子机制。方法采用白醋+活性炭冰水联合复方地芬诺酯片复制阳虚便秘大鼠模型。将模型成功大鼠随机分为5组,即模型组、莫沙必利组(1.88 mg·kg^(-1))、济川煎高(8.26 g·kg^(-1))、中(4.31 g·kg^(-1))、低(2.16 g·kg^(-1))剂量组,灌胃给药每日1次,连续7 d。末次给药,记录大鼠状态并评分,进行排便功能测定;分离模型大鼠结肠平滑肌细胞并鉴定,FCM检测结肠平滑肌细胞内Ca^(2+)浓度变化;采用ELISA法检测大鼠血清中cAMP、cGMP含量及其比值;采用RT-PCR及Western blot法检测大鼠结肠平滑肌细胞CaM、CaMKⅡ亚型的表达情况。结果济川煎可明显缓解模型大鼠腹胀,摄食、饮水量、排便减少及体质量降低的症状(P<0.05),评分明显升高(P<0.01);能明显缩短首粒排便时间(P<0.01),增加排便粒数(P<0.05);济川煎还可明显增加模型大鼠血清中cAMP含量(P<0.05),减少cGMP含量(P<0.01),cAMP/cGMP比值明显增加(P<0.01);明显降低大鼠结肠平滑肌细胞内Ca^(2+)浓度(P<0.01);PCR及Western blot检测结果显示,济川煎可明显降低CaM及CaMKⅡβ、γ、δ亚型蛋白表达(P<0.01)。结论济川煎对阳虚便秘具有明显的治疗作用,可能与其调节Ca^(2+)-CaMKⅡ信号通路有关。Aim To investigate the effect of JiChuanjian(JCJ)on the Yang deficiency constipation based on Ca^(2+)-CaMKⅡsignaling pathway and the molecular mechanisms.Methods Yang deficiency constipation model of SPF SD rats(half male and half female)was set up by white vinegar and activated carbon ice water combined with compound diphenoxylate.The rats of Yang deficiency constipation model were randomly divided into five groups:control group,model group,mosapride citrate group(1.88 mg·kg^(-1)),JCJ high-does group(8.26 g·kg^(-1)),JCJ middle-does group(4.31 g·kg^(-1))and JCJ low-does group(2.16 g·kg^(-1)).Gavage was administered once a day for seven consecutive days.The general state was observed and scored at the end of the last administration of rats.The time of first defecation and dejection amount were observed by defecation function was measured of rats.Colonic smooth muscle cells were isolated and identified of model rats,and calcium concentration was detected with flow cytometry inside the colonic smooth muscle cells.The contents of cAMP and cGMP in serum and ratio of rats were detected by Elisa.The protein expression of CaM,CaMKⅡ and its isoforms in colonic smooth muscle cells of rats were detected by RT-PCR and Western blot.Results JCJ could significantly promote the abdominal distention,reduce intake,water intake,defecation,weight loss(P<0.05),and higher scores in rats(P<0.01).It could significantly shorten first dejection time(P<0.01)and increase dejection amounts(P<0.05).JCJ also significantly increased the content of cAMP in serum(P<0.05),decreased the cGMP(P<0.01),and increased the cAMP/cGMP ratio(P<0.01).It could significantly decreased calcium ion concentration in colonic smooth muscle cells of rats(P<0.01).The results were shown in PCR and Western blot that JCJ could significantly reduce the protein expression of CaM andβ,γ,δsubtype of CaMKII(P<0.01).Conclusions JCJ may show effects on Yang deficiency constipation in rats remarkably,which may be related to signaling pathway of the Ca^(2+)-CaMKII.

关 键 词：济川煎 阳虚便秘 Ca^(2+)-CaMKⅡ 治疗作用 信号通路 分子机制 

分 类 号：R-332[医药卫生] R256.35R289.5R348.1R442.2R574.62