Sun Lan, Liu Lina, Zong Shaobo, Wang Zhengzhong, Zhou Jun, Xu Zhiliang, Ding Gang, Xiao Wei, Kou Junping
Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, Jiangsu 211198, PR China; State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
J Ethnopharmacol. 2016 Sep 15;191:273-279. doi: 10.1016/j.jep.2016.06.042. Epub 2016 Jun 20.
Guizhi Fuling formula, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, is composed of Cinnamomum cassia (L.) J.Presl (Cassia bark), Poria cocos (Schw.) Wolf (Poria), Paeonia suffruticosa andrews (Moutan Cortex), Paeonia lactiflora Pall (Herbaceous peony), and Amygdalus persica L.(Persicae Semen). It has clinical efficacy of activating blood circulation to dissipate blood stasis and is commonly used for the treatment of primary dysmenorrhea. However, its therapeutic mechanism has not been clearly elucidated. The aim of this study is to reveal molecular mechanisms of action using in vivo and in vitro experimental models.
The ICR mouse uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment. Mice were given GZFLC (0.54, 1.08g/kg) by gavage. The levels of NO, PGF2α and Ca(2+) in uterine tissue were determined according to instructions. Cyclooxygenase-2 (COX-2) and oxytocin receptor (OTR) proteins in uterine tissue were assessed by Western Blot. Mouse isolated uterus strips were mounted in tissue organ baths containing Locke's solution. The contractile responses were recorded with Power Lab recording system. The effect of GZFLC on spontaneous uterine contraction, and uterine contraction induced by oxytocin, PGF2α was observed. Myometrial cells were exposed to oxytocin (5U/L) to induce calcium release, and the effect of GZFLC and its components (PL, PGG, CA) on intracellular Ca(2+) was analyzed with fluorometry imaging.
In vivo study demonstrated that GZFLC significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 55%. It also decreased the levels of NO, PGF2α and Ca(2+) in oxytocin-induced mice uterine tissue. Moreover, Western blot analysis showed that COX-2 and OTR expressions in uterine tissue of dysmenorrhea mice were significantly reduced. GZFLC inhibited spontaneous uterus contractions in a dose-dependent manner, and the IC50 value was 0.99mg/ml. The IC50 values of GZFLC on PGF2α, oxytocin-induced contractions were 1.45mg/ml, 3.53mg/ml, respectively. Further in vitro studies indicated that GZFLC and its components (PL, PGG, CA) could restrain intracellular calcium levels in favour of uteri relaxation.
Both in vivo and in vitro results indicated that GZFLC possessed a significant spasmolytic effect on uterine tetanic contraction. The present study provides in vivo and in vitro experimental evidence to support the use of GZFLC for the clinical treatment of primary dysmenorrheal (PD).
桂枝茯苓方是东汉时期记载的著名中药方剂，由肉桂（桂皮）、茯苓、牡丹皮、白芍、桃仁组成。它具有活血化瘀的临床疗效，常用于治疗原发性痛经。然而，其治疗机制尚未明确阐明。本研究的目的是使用体内和体外实验模型揭示其分子作用机制。
经苯甲酸雌二醇预处理后，用催产素诱导ICR小鼠子宫收缩。小鼠灌胃给予桂枝茯苓方（0.54、1.08g/kg）。按照说明书测定子宫组织中一氧化氮（NO）、前列腺素F2α（PGF2α）和钙离子（Ca²⁺）的水平。通过蛋白质免疫印迹法评估子宫组织中环氧合酶-2（COX-2）和催产素受体（OTR）蛋白。将小鼠离体子宫条安装在含有洛克溶液的组织器官浴槽中。用Power Lab记录系统记录收缩反应。观察桂枝茯苓方对子宫自发收缩以及催产素、PGF2α诱导的子宫收缩的影响。将子宫肌层细胞暴露于催产素（5U/L）以诱导钙释放，并用荧光成像分析桂枝茯苓方及其成分（芍药苷、茯苓聚糖、桂皮醛）对细胞内Ca²⁺的影响。
体内研究表明，桂枝茯苓方显著降低催产素诱导的扭体反应，最大抑制率为55%。它还降低了催产素诱导的小鼠子宫组织中NO、PGF2α和Ca²⁺的水平。此外，蛋白质免疫印迹分析表明，痛经小鼠子宫组织中COX-2和OTR的表达显著降低。桂枝茯苓方以剂量依赖方式抑制子宫自发收缩，IC50值为0.99mg/ml。桂枝茯苓方对PGF2α、催产素诱导的收缩的IC50值分别为1.45mg/ml、3.53mg/ml。进一步的体外研究表明，桂枝茯苓方及其成分（芍药苷、茯苓聚糖、桂皮醛）可抑制细胞内钙水平，有利于子宫松弛。
体内和体外结果均表明，桂枝茯苓方对子宫强直性收缩具有显著的解痉作用。本研究提供了体内和体外实验证据，支持桂枝茯苓方用于原发性痛经（PD）的临床治疗。
