The effect of Wujiadan Recipe on the role of PLC-β/IP3/CaM signaling pathway of hypothalamus in ovariectomized rats

Objective:To investigate the effect of Wujiadan Recipe on PLC-β1,IP3,IP3R,Cam,PLC-β1,IP3R1 mRNA and GnRH,Cam protein from hypothalamus tissue of ovariectomized female rats,and to further clarify whether Wujiadan Recipe plays a role through PLC-β/IP3/CaM signal pathway.Methods:We randomly divided 64 SD adult female rats into NG group(normal group),MG group(the model group),TMC group(the control group with traditional Chinese medicine),WMC group(the control group with western medicine)with 10 rats in each,and Wujiadan Recipe high(WH),Wujiadan Recipe medium(WM)and Wujiadan Recipe low dose group(WL)with 8 rats in each.Except the NG group,we removed the ovarian tissues of other six groups to establish the ovariectomized model.When the model is successfully replicated,we give the medicine to the rats in each group once a day.The TMC and WMC group were given the corresponding dose of Gengnian'an tablet suspension and Bujiale suspension by gavage respectively.The NG and MG group were given equal measure of normal saline.After 4 weeks,the rats were killed.We use ELISA to detecte the levels of PLC-β1,IP3,IP3R and Cam;and we use PCR to detecte the mRNA expression levels of PLC-βand IP3R1;and use WesternBlot to detecte the protein expression levels of Cam and GnRH in rat hypothalamus tissue.Results:The test results of PLC-β1,IP3,IP3R and Cam in the hypothalamus homogenate of the model group were significantly increased than the NG group(P<0.01).The results of PLC-β1 and IP3R1 and GnRHmRNA were significantly increased(P<0.01),and the expressions of GnRH and Cam protein were higher than NG(P<0.01).Wujiadan Recipe could reduce the results of PLC-β1,IP3,IP3R and Cam in hypothalamus of ovariectomized rats,and reduce the expression of PLC-β1 and IP3R1 and GnRH mRNA.Conclusion:Wujiadan Recipe down-regulates GnRH expression,which may be caused by acting on PLC-β1,IP3,IP3R,CaM in hypothalamus of ovariectomized rats,so as to coordinate the function of reproductive axis and improve the reproductive endocrine level of patients with menopausal syndrome.

The effect of Wujiadan Recipe on serum sex hormone and expression of Kiss-1, GPR54 and GnRH in arcuate nucleus of hypothalamus in ovariectomized rats

Objective:To explore the effect of wujiadan Recipe on the expression of FSH,E2 in serum and KiSS-1,GPR54,GnRH mRNA in hypothalamic arcuate nucleus tissue homogenate of ovariectomized rats,and to further explore the mechanism of wujiadan Recipe on menopausal syndrome.Methods:Sixty-four adult female rats were randomly divided into three groups:blank group,model group,Chinese medicine control group,western medicine control group,and wujiadan high,medium and low dose groups.Except for the blank group,all the other groups were ovariectomized.The rats in the normal group and the model group were given the same dose of saline once a day.One rat in the blank group died during the experiment.After 4 weeks of continuous administration,blood was collected from abdominal aorta and brain stereotaxic localization was performed to preserve the arcuate nucleus of hypothalamus.The levels of FSH and E2 in serum were detected by enzyme-linked immunosorbent assay(ELISA),and the expression of Kiss-1,GPR54 and GnRH mRNA in arcuate nucleus of hypothalamus were detected by PCR.Results:1.Compared with the blank group,FSH level in the model group was significantly higher(P<0.01),and E2 level was significantly lower(P<0.01).Compared with the model group,the level of serum E2 in the western medicine control group increased significantly(P.The serum FSH content in each group decreased,especially in the high dose group and the western medicine control group(P<0.01),and in the Chinese medicine control group,the middle dose group and the low dose group(P<0.05).Compared with the blank group,the expression of KiSS-1 mRNA,GPR54 mRNA and GnRH mRNA in the model group increased significantly(P<0.01);compared with the model group,the expression of KiSS-1 mRNA and GPR54 mRNA in the high dose group,middle dose group and Western medicine control group of wujiadan prescription decreased significantly(P<0.05).Compared with the model group,the expression of GnRH mRNA in the high dose group and the western medicine control group decreased significantly(P<0.01);the middle dose group of wujiadan decreased significantly(P<0.05).Conclusion:Wujiadan recipe can improve the serum sex hormone level of castrated rats,and reduce the over expression of KiSS-1,GPR54 and GnRH mRNA in hypothalamic arcuate nucleus of castrated rats,which may be the reproductive endocrine mechanism of wujiadan recipe in the treatment of menopausal syndrome.

五加丹方对绝经综合征模型大鼠下丘脑PLC-β/IP3/CaM通路作用的研究

目的:探讨中药方剂五加丹方对去卵巢雌性大鼠下丘脑组织匀浆中PLC-β1、IP3、IP3R、CaM及PLC-β1、IP3R1、GnRHmRNA和GnRH、CaM蛋白的影响,试图阐释五加丹方对PLC-β/IP3/CaM通路及绝经综合征的影响。方法:取雌性SD大鼠64只,随机分为空白组(NG)、模型组(MG)、中药对照组(TMC)、西药对照组(WMC)各10只,五加丹方高(WH)、中(WM)、低剂量组(WL)各8只。除NG组外,其余6组均手术摘除大鼠双侧卵巢复制去势模型。模型复制成功后,各组采用灌胃给药1次/日,TMC组及WMC组分别给予相应剂量更年安片混悬液及补佳乐混悬液灌胃,NG组、MG组予等剂量生理盐水灌胃。4周后,处死大鼠,检测脑组织中PLC-β1、IP3、IP3R、CaM的含量;PCR法检测PLC-β、IP3R1、GnRH mRNA表达;Western Blot法检测各组脑组织CaM和GnRH蛋白的表达。结果:与NG组比较,大鼠去势后下丘脑组织PLC-β1、IP3、IP3R、CaM含量明显升高(P<0.01),PLC-β1、IP3R1、GnRHmRNA的表达显著升高(P<0.01),GnRH、CaM蛋白表达较空白组均升高(P<0.01),五加丹方可降低去势模型大鼠下丘脑组织中PLC-β1、IP3、IP3R、CaM水平,并下调PLC-β1、IP3R1、GnRHmRNA的表达。结论:五加丹方下调GnRH的表达,可能是通过作用于GnRH上游调节因子PLC-β1、IP3、IP3R、CaM等而产生,从而起到协调生殖轴功能,改善绝经综合征患者生殖内分泌水平。

五加丹方对去势模型大鼠血清性激素及下丘脑弓状核Kiss-1、GPR54、GnRH基因表达的影响

目的:探讨五加丹方对去卵巢大鼠血清中FSH、E2及下丘脑弓状核组织匀浆Kiss-1、GPR54、GnRH·RNA表达的影响,进一步探讨五加丹方对绝经综合征的作用机制。方法:将64只成年期雌性大鼠随机分为7组,空白组、模型组、中药对照组、西药对照组各10只,五加丹方高、中、低剂量组各8只。除空白组外,其余各组均手术去势。各组灌胃给药每日1次,正常组及模型组给予等剂量生理盐水灌胃每日1次,实验过程中空白组大鼠灌胃死亡1只。连续给药4周后,腹主动脉取血并处死大鼠,脑立体定位留存下丘脑弓状核组织。采用酶联免疫吸附测定法(ELISA)检测大鼠血清促卵泡激素(FSH)、雌二醇(E2)含量,PCR技术检测各组大鼠下丘脑弓状核Kiss-1、GPR54、GnRH mRNA表达水平。结果:(1)与空白组比较,模型组FSH水平明显升高(P<0.01),E2水平明显下降(P<0.01)。与模型组比较,西药对照组血清E2水平明显升高(P<0.01),五加丹方高剂量组、中剂量组、中药对照组血清E2水平也明显升高(P<0.05)。各给药组血清FSH含量均下降,五加丹方高剂量组与西药对照组下降明显(P<0.01),中药对照组、五加丹方中剂量组、低剂量组也明显降低(P<0.05)。(2)与空白组比较,模型组Kiss-1 mRNA、GPR54 mRNA、GnRH mRNA表达均明显上升(P<0.01);与模型组比较,五加丹方高剂量组、中剂量组、西药对照组的Kiss-1 mRNA、GPR54 mRNA表达明显降低(P<0.05)。与模型组比较,五加丹方高剂量组、西药对照组GnRH mRNA表达明显下降(P<0.01);五加丹方中剂量组明显降低(P<0.05)。结论:五加丹方可以改善去势模型大鼠的血清性激素水平,可以降低去势模型大鼠下丘脑弓状核Kiss-1、GPR54、GnRH mRNA的过高表达,这可能是五加丹方临床治疗绝经综合征的生殖内分泌机制。

王孝莹教授治疗绝经综合征经验介绍

本文通过整理王孝莹教授有关著作、书稿、临床医案及临床教学讲稿,总结王孝莹教授治疗绝经综合征学术思想。王孝莹教授认为肝肾亏损是本病的发病基础,阴阳失调是本病的发病关键;同时认为烘热汗出是本病的首发症状,又是主症,绝经综合征应属妇人血劳范畴。因此治疗以滋补肝肾、调和阴阳、养血安神为主,采用辨病与辨证相结合的方式。王孝莹教授运用由古方油煎散为基础方化裁的自拟五加丹方,加减治疗绝经综合征。