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.