Compatibility Mechanism Of Qing'E Fomula Based On Pharmacodynamic Interaction

Objective:Based on the estrogen-like effect of the traditional kidney-tonifying prescription-Qing'E formula?QEF?,a comparative study on the pharmacodynamics of Qing'E Formula before and after its compatibility with other ingredients was carried out,and the compatibility mechanism of QEF was discussed from the point of pharmacodynamic interaction in vivo and in vitro.Method:1.The estrogenic effect of QEF and its individual ingredient was compared using the ovariectomized rat.ELISA kit was used to detect the levels of serum hormone,blood lipids,neurotransmitters,aromatase,SOD and MDA in ovariectomized rats.To investigate the characteristic of the morphology of target organs and the expression of estrogen receptors in ovariectomized rats,HE staining and immunohistochemistry were used.Western blotting assay was used to analyze the activation of the estrogen pathway-related proteins in ovariectomized rats.2.MCF-7 cell proliferation assay and fluorescence expression assay of HEK293 cells stably transfected with ER?-ERE and ER?-ERE luciferase reporter genes were used to study the estrogen-like effect of QEF and its compatible compositions.CCK-8 assay was used to detect the effect of QEF and its combination on the viability of MCF-7cells.Luciferase reporter gene assay was used to compare the selectivity of QEF and its compatibility to ER receptor subtypes.The expression of estrogen pathway-related proteins ER?,ER?and G protein-coupled receptor?GPR30?in MCF-7 cells was detected by Western blotting assay;and the expression of estrogen receptor ER-related genes ER?,ER?and PS2 was detected by real-time quantitative PCR.Result:1.The comparative pharmacodynamic study of estrogen-like effects of QEF and its single herbs showed that QEF,Eucommia and Psoralea could resist the decline of uterine index caused by ovariectomy?OVX?,which was significantly different from that of model group;QEF,Eucommia and Psoralea could reduce the levels of luteinizing hormone LH and follicle-stimulating hormone FSH and increase the level of estradiol E2 in serum of OVX rats.and moreover,QEF and Psoralea could restore the level of serum estradiol to normal level.The body weight of ovariectomized rats was significantly higher than that sham-operated rats,but there was no significant difference between QEF group and sham group,suggesting that QEF can counteract the weight gain of ovariectomized rats.QEF and Psoralea group could significantly reduce the total cholesterol T-CHO content in rat serum.Each group can significantly reduce the content of triglyceride TG.Only QEF and Psoralea group could significantly increase the HDL-C content and return it to the normal level.QEF,Eucommia,Psoralea and garlic group can significantly reduce the LDL-C content.Both QEF and its combination group could improve the disorder of lipid metabolism in OVX rats caused by estrogen deficiency in varying degrees,but the effects of Qing'e Fomula and Psoralea were the best.QEF and garlic group could significantly increase the level of aromatase in serum of OVX rats.Only QEF could significantly increase the level of SOD and decrease the level of MDA in serum of OVX rats.QEF and its single drug could significantly reduce the content of monoamine neurotransmitters 5-hydroxytryptamine?5-HT?,except Eucommia,all groups could significantly reduce the content of 5-hydroxyindole acetic acid?5-HIAA?,especially the trend of QEF is the best.HE staining morphological study showed that QEF,Eucommia and Psoralea could antagonize uterine atrophy induced by OVX.QEF,Eucommia and Psoralea could improve the vacuole of liver fat caused by OVX.Immunohistochemical method showed that QEF,Eucommia and Psoralea could significantly increase the expression of ERs in uterus of OVX rats,and QEF could raise the level of ERs to the same level as sham-operated group.Western Blotting assay showed that QEF could significantly up-regulate the expression of ER?and ER?in uterus of OVX rats.In the expression of ER?,EC has a trend of up-regulation,but it is not statistically significant,but it can still return to the level of blank group.At the same time,E2,QEF and EC can up-regulate the receptor expression of ER?in uterus,and PF has an upward trend but no Statistically significant,but can be restored to the equivalent of the sham group.The food intake rate of Psoralea group was significantly lower than that in sham-operated group,and no similar side effects were observed in Qing'e Fomula.3.The estrogen-like effects of QEF and its compatible compositions in vitro showed that:?1?After the decomposition of QEF,Eucommia,Psoralea and Juglandis could promote the proliferation of MCF-7 cells except garlic,but Psoralea had the strongest activity.The concentration of Psoralea at 1 mg/ml,0.5 mg/ml and 0.1 mg/ml could promote the proliferation of MCF-7 cells,followed by the activity of Eucommia at 1 mg/m.The activity of Juglandis was the weakest,and only at 1 mg of crude drug/ml the proliferation of MCF-7 cells could be promoted.The combination of Eucommia and Psoralea showed the better effect.When they were compatible with Juglandis and garlic respectively,the proliferation rate of the compatible Juglandis group was higher than the compatible garlic group,which accorded with their assistant status.However,QEF extract,which was composed of four herbs,had the strongest proliferation effect.It can promote the proliferation of MCF-7 cells at the concentration of 1 mg/ml,0.5mg/ml,0.1 mg/ml,and 0.05 mg/ml.?2?The expression of luciferase in HEK293 cells transfected with ER?-ERE reporter gene was significantly promoted by QEF at 0.05-1 m g/ml,Psoralea at 0.01-1 mg/ml and Eucommia at 1 mg/ml,while the expression of luciferase in HEK293 cells transfected with ER?-ERE reporter gene was significantly enhanced by QEF at 0.1-1mg/ml,Psoralea at 0.01-1 mg/ml and Eucommia at 1 mg/ml.Garlic and Juglandis had no significant effect on luciferase expression in HEK293 cells transfected with ER?-ERE and ER?-ERE reporter genes.The results suggested that estrogen-like effects of QEF,Psoralea and Eucommia could be mediated through estrogen receptor pathway and through binding with ERE binding sites.?3?It was found that at 0.1 and 0.5 mg crude drug/ml,QEF could significantly up-regulate the expression of ER?,ER?,PS2 protein and gene in MCF-7 cells;at 0.5 mg crude drug/ml,Eucommia could up-regulate the expression of PS₂ protein and gene;at0.5 mg/ml,Psoralen could up-regulate the expression of ER?protein;at 0.1 mg/ml,both monarch and Minister drugs had no significant up-regulation on all proteins.And in addition,Eucommia can also up-regulate the expression of estrogen membrane receptor GPR30.QEF,Eucommia,Psoralea could up-regulate the expression of ER?,ER?and PS₂ genes,but QEF has a greater effect than single drug,and could play a role at a lower concentration.The experimental results show that QEF still has a good activity at a lower concentration.Conclusion:1.In vivo pharmacodynamics results showed that QEF and its monarch drug Eucommia and Minister drug Psoralea could increase uterine coefficient and serum hormone levels in ovariectomized rats,and had estrogen-like effect.QEF and its decomposed compatibility groups could improve the disorders of lipid metabolism and neurotransmitter caused by estrogen deficiency in OVX rats to varying degrees,and enhance the serum antioxidant capacity of OVX rats.At the same time,Qing'e Fomula could also resist the weight gain of ovariectomized rats.HE staining results showed that QEF,Eucommia and Psoralea could antagonize uterine atrophy and improve hepatic fat vacuolation of OVX rats,and had mild stimulating effect on adrenal gland.Immunohistochemical and Western Blotting analysis showed that QEF,Eucommia and Psoralea could all up-regulate the ERs receptor level of uterus in OVX rat.To sum up,Eucommia and Psoralea in QEF had estrogen-like effect,which accords with their status as Monarch drug and Minister drug.However,the estrogen-like effect of QEF is the strongest,which is the result of synergistic effect of four herbs.Qing'e Formula could also antagonize the anorexia of OVX rats caused by Psoralea.The compatibility mechanism of QEF is explained from the perspective of pharmacodynamics in vivo.2.The results of in vitro experiments showed that QEF,Eucommia and Psoralea could promote the proliferation of MCF-7 cells.When used in combination,the results of drug compatibility analysis showed that the two drugs had synergistic effect,but the QEF had the strongest proliferation promoting effect.QEF,Eucommia and Psoralea mainly played a role through estrogen receptor pathway,while QEF still had a relatively lower concentration.The compatibility of QEF is accurate and scientific from the point of view of in vitro experiment.