Study on mechanism of Pulsatilla saponin B4 in treatment of uterine fibroids in rats
Objective To investigate the therapeutic mechanism of Pulsatilla saponin B4 on leiomyomas in rats based on in vivo experiments and network pharmacology.Methods Rats were randomly divided into six groups according to body weight:control group,model group,mifepristone(positive drug,1.25 mg·kg-1)group,and Pulsatilla saponin B4 low,medium,and high dose(5,10,20 mg·kg-1)groups,with eight rats in each group.The leiomyoma model was established by hormone loading,and ip injection was given after the model was established.The control group and model group were injected with an equal volume of 0.9％sodium chloride solution daily.The general behavior of rats was observed,the uterine tissue was observed under bright field,and the pathological HE staining sections were observed.The expression levels of estradiol,progesterone,and estrogen receptor in serum and uterine tissue were detected by ELISA kits,to determine whether Pulsatilla saponin B4 had a therapeutic effect on rat leiomyomas.The interactive targets of Pulsatilla saponin B4 and leiomyoma were collected by network pharmacology,and the protein interaction network information was constructed and gene function enrichment analysis was performed.The targets and drugs were subjected to AutoDock Vina molecular docking analysis to determine the action targets.Finally,the protein levels of related targets Bcl-2,Bax,and Caspase-3 in the tissue were detected by Western blotting,and validated.Results The in vivo experiment results showed that compared with the model group,the uterine morphology of rats treated with Pulsatilla saponin B4 improved significantly,and the uterine coefficient was significantly lower(P＜0.001);HE staining showed that rat uterine smooth cells were arranged in a regular pattern,with normal morphology,uniform color,and reduced inflammatory cell infiltration in the muscle layer.Serum and uterine tissue levels of estradiol,progesterone,and estrogen receptors were significantly lower(P＜0.05,0.01,0.001).Using the Pubchem database,23 targets related to Pulsatilla saponin B4 were screened,8 of which were common targets with leiomyoma.Sixty-nine KEGG signaling pathways were enriched,including apoptosis,lipid and atherosclerosis,IL-17,NF-κB,small cell lung cancer,and MAPK related signaling pathways.The AutoDock Vina results showed that Pulsatilla saponin B4 bound well to key targets BCL2,BAX,and Caspase3.Compared with the model group,the middle and high dose groups of Pulsatilla saponin B4 showed significantly increased levels of Bax and Caspase-3 protein(P＜0.001),and significantly decreased levels of Bcl-2 protein(P＜0.001).Conclusion Pulsatilla saponin B4 may induce apoptosis through key targets such as BCL2,BAX,and Caspase3,while reducing the expression of estrogen,progesterone,and estrogen receptors,thereby treating leiomyoma.
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