Study On The Effect And Mechanism Of The Treatment Of Hyperplasia Of Mammary Gland By The Traditional Chinese Medicine Compound Li-Ru-Kang

Aim and significance: Li-Ru-Kang(LRK),a formula of eight traditional Chinese medicines(TCM),has been used to treat hyperplasia of mammary gland(HMG)in TCM in clinics.The clinical effect of LRK on HMG is remarkable.However,how LRK works in HMG is unclear.In this study,we combined the methods of network pharmacology,molecular biology and metabolomics to explore the therapy effect and mechanism of LRK on HMG from various angles and multi-levels,and provide theoretical basis for clinical promotion.Methods:(1)To investigate the treatment effect of LRK on HMG,estrogen combined with progesterone was used to induce HMG rats model.The parameters,including rat breast height,breast diameter,uterine index and pathological tissue observation were detected to evaluate the therapeutic effects of LRK on HMG.(2)Network pharmacology method was used to screen and predict the active components and targets of the treatment of HMG by LRK.(3)The expression levels of p-p38,pJNK and p-ERK proteins in mammary gland tissues were detected by western blotting,and the expression levels of NF-κB,IL-1β,TNF-α,COX-2,iNOS,NT,and 8-OHdG in mammary gland tissues were detected by immunohistochemistry to investigate the mechanism of action of LRK on HMG.(4)Metabolomics technology and network pharmacology method were combined to clarify the metabolic markers change law and related metabolic pathways of LRK on HMG.Finally,immunohistochemical method was performed to validate the screened metabolic markers.Results:(1)The therapeutic effect of LRK on HMGThe therapeutic effect of LRK on HMG was investigated through the observation of breast height,breast diameter,uterine index and pathological tissue in rats.The results showed that estrogen and progesterone combined induction could lead to significantly increased breast height,breast diameter and uterine index in rats.The TCM compound LRK can improve the breast height and breast diameter of HMG rats,reduce the increased uterine index of rats,and improve the pathological changes of mammary tissue at the same time.(2)Network pharmacology research of LRK on HMG.The "drug-target-disease" interactive network for the treatment of HMG by LRK was constructed based on the chemical composition of each component of TCM and the action targets of HMG,and 19 active components and 13 direct action targets were predicted.The KEGG pathway analysis predicted that there might be 5 action pathways related to the treatment of HMG by LRK,including MAPK signaling pathway,Toll-like receptor signaling pathway,TNF signaling pathway,NOD-like receptor signaling pathway and chemokine signaling pathway.These pathways are directly or indirectly related to inflammation,suggesting that LRK may play an anti-hyperplasia role by acting on these inflammatory pathways.(3)Mechanism study of LRK on HMGThe results showed that the expression of ERα and PR in rat mammary tissue was not significantly affected by the traditional Chinese medicine compound LRK.LRK can alleviate the inflammatory reaction in the HMG rats,significantly reduce the expression levels of the inflammatory factors IL-1,TNF-α,COX-2,iNOS in the mammary gland tissues of rats,significantly reduce the levels of 8-OHdG and NT,and alleviate the oxidative stress reaction.The phosphorylation levels of JNK,ERK and p38 and the expression levels of NF-κB p65 were decreased.The results indicated that LRK could inhibit inflammatory reaction and oxidative stress by regulating the MAPKNF-κB signaling pathway,thus achieving the effect of treating HMG.(4)Metabolomics study on LRK treatment HMGThe metabolomics results showed that there were 10 characteristic metabolic markers related to the treatment of mammary gland hyperplasia in rats with rirukang,including sulfate glycine,leukotriene-β4,3-hydroxytryptamine,coenzyme Q9,gammaglutamine l-alanine,phosphoesterase choline,ceramide,prostaglandin,5-amino-4-imidazolate and phosphatidymide.The results of metabolic pathway analysis showed that: arachidonic acid metabolism,glyceride metabolism,linoleic acid metabolism,sphingomyelin metabolism,-linolenic acid metabolism,glycine,serine and triacid metabolism,arginine and proline metabolism,purine metabolism and many other metabolic pathways were related to the treatment of HMG by LRK.The results of network pharmacology and molecular biology analysis showed that PKC protein played an important role in the treatment of HMG by LRK.Conclusion: The results showed that the LRK could significantly improve the HMG on rats induced by estrogen and progesterone.MAPK signal pathway as the breakthrough point,using molecular biology experiment method is verified with the result of network pharmacology,results show that the molecular mechanism of LRK treatment of hyperplasia of mammary glands may be through the effect on MAPK-NF-κB pathway predominate,reduce the secretion of inflammatory factors associated inflammation,relieve oxidative stress at the same time,realize its effect in treatment of hyperplasia of mammary glands,confirmed the prediction results of network pharmacology.Metabolomics LRK hints of traditional Chinese medicine compound interest by arachidonic acid metabolism,alpha linolenic acid metabolism,arginine and proline metabolism and purine metabolic multiple metabolic pathways,such as play to the role of the treatment of mammary gland hyperplasia,joint network pharmacology,and molecular biology research results suggest PKCα protein in the valley of LRK treatment of hyperplasia of mammary glands play an important role in the process of LRK is elaborated from metabolism mechanism for the treatment of HMG.This study preliminarily clarified the scientific connotation and potential mechanism of the treatment of mammary gland hyperplasia by the traditional Chinese medicine compound LRK,providing a theoretical basis for the clinical application of LRK.