Academician Wang Qi's Clinical Prescription For The Treatment Of Semen Liquefaction And Its Mechanism Of Action Researc

Semen non-liquefaction refers to the phenomenon that semen expelled from the body after 3-5 days of abstinence at room temperature in adult men does not liquefy within 60 min.The pathogenic factors of semen non-liquefaction are complex,such as chronic prostatitis,varicocele and other diseases can induce the production of semen non-liquefaction.Therefore,it is necessary to develop and find new drugs for the treatment of seminal fluid indigestion.Professor Wang Qi is a master of Chinese medicine and academician of the Chinese Academy of Engineering,and has been clinically treating male semen opacification for many years.Therefore,this study explores the core formula and mechanism of action of academician Wang Qi’s clinical treatment of seminal fluid non-liquefaction based on the ancillary platform of TCM inheritance and network pharmacology,and validates and explores with animal experiments to provide ideas for the treatment of seminal fluid non-liquefaction.Objective:Based on the software of "TCM Inheritance Support System(TCMISS)(V2.5)",the core prescription HXZF of academician Wang Qi in the clinical treatment of patients with semen non-liquefaction were explored,and the main active ingredients,action targets and signaling pathways were predicted by network pharmacology based on the core prescription HXZF,and the preliminary validation of the active ingredients and action targets was carried out by molecular docking technology.Finally,the experimental autoimmune prostatitis(EAP)model was used to induce a decrease in the secretion of PSA,PAP and other major proteolytic enzymes in the prostate of rats to prepare a rat semen non-liquefaction model to further validate the predicted results of network pharmacology.The mechanism of action was deeply explored to provide data support for the clinical application of the core prescription HXZF for the treatment of semen non-liquefaction.Methods:1.Data mining:The case data of patients with non-liquefaction of semen admitted to academician Wang’s outpatient clinic were collected and standardized,data mining and visual analysis of the cases were conducted through frequency analysis,association rules,cluster analysis and other methods,the core prescription HXZF was clarified and potential new prescriptions was discovered and summarize the rules of medicine.2.Network pharmacology:Firstly,we used TCMSP and Uniprot database to collect the components and targets of the core prescription HXZF of academician Wang,and then collected the disease targets from GeneCards database and OMIM,and took the "drug-disease"intersection with the drug targets to obtain the core targets,and used String database to map the protein-protein interaction(PPI)network.Using R language platform.GO and KEGG signaling pathway enrichment analysis was performed to construct the core prescription HXZF"component-target-disease" pathway map,and the key components and core targets were molecularly docked and visualized.3.Animal experiment:Sixty Sprague-Dawley(SD)male rats were divided into control group,model group,Ningmitai group(0.3 g/kg),HXZF-low dose group(5 g/kg),HXZF-middle dose group(10 g/kg)and HXZF-high dose group(20 g/kg).EAP rat models were established in all groups except the control group.And rat semen non-liquefaction models were prepared by inducing the decrease of secretion of major proteolytic enzymes such as PSA and PAP in prostate.After 28 days of drug intervention,the organ index of prostate tissue of rats in each group was counted,and the pathological morphology of prostate tissue of rats in each group was observed by Hematoxylin-eosin staining(HE).Testosterone(T),estradiol(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH),prostate specific antigen(PSA)and prostatic acidic phosphatase(PAP)in serum of rats in each group were detected by enzyme linked immunosorbent assay(ELISA).Tumor necrosis factors(TNF),interleukin-6(IL-6),serine protease inhibitor family member 1(SERPINE1),interleukin-1β(IL-1β)and interleukin1β(IL-1β)in prostate tissue were detected by ELISA.The contents of PSA and PAP in prostatic fluid were also detected by ELISA.The contents of citric acid(CA)and Zn2+ in prostate tissue of rats in each group were detected by colorimetry.Immunohistochemical method was used to detect the expression of PSA and PAP protein in prostate tissue of rats in each group.The protein expressions of interleukin-17(IL-17),interleukin-17 receptor A(IL-17RA)and nuclear factor kappa B activator 1(Act1)of IL-17/IL-17RA/Act1 in prostate tissues of rats in each group were detected by Western blot.Beside,the relative expressions of IL-17,IL-17RA and Act1 mRNA in prostate tissue of rats in each group were detected by real-time polymerase chain reaction(RT-PCR).Results:1.Data mining:In this study,28 valid medical records were included,including 51 prescriptions involving 77 kinds of traditional Chinese medicines,and 15 kinds of highfrequency drugs were obtained.The top 5 drugs were Angelica sinensis,Radix Astragali,Radix Morindae Officinalis,Semen Cuscutae,Fructus Lycii.The first frequency of five kinds of flavour is sweet taste,followed by pungent taste,bitter taste,sour taste and salty taste,and the last is astringent taste;The frequency of four nature of drugs is warm at first,then flat,cold and cool,and finally is hot.Drugs mainly enter liver,spleen,kidney,lung and stomach meridians.According to the results of Academician Wang’s clinical medication habits and association rules,the core prescription for Academician Wang Qi’s treatment of non-liquefaction of semen was obtained as follows:10 g of Angelica sinensis,10 g of Radix Astragali,10 g of Morinda officinalis,10 g of Semen Cuscutae and 10 g of Fructus Lycii.Finally,three potential new prescriptions were obtained by unsupervised entropy hierarchical clustering,and it was found that most of the drugs used in the new prescriptions were drugs for nourishing liver and kidney,promoting qi circulation and promoting digestion,clearing away heat and eliminating dampness,and their simultaneous therapeutic effects also met the common accompanying symptoms in the course of semen non-liquefaction.2.Network pharmacology:29 active components in the core prescription HXZF were predicted,and the top five active ingredients in the core prescription HXZF were quercetin,kaempferol,β-sitosterol,isorhamnetin and daidzein according to the Degree value.There are 194 component targets in the core prescription HXZF,and there are 24 common targets between component targets and disease targets.The top 5 core targets in terms of Degree value are TNF,IL6,SERPINE1,IL1B,VEGFA respectively.GO enrichment found that the biological processes involved in the core prescription HXZF were significantly enriched in the processes related to vascular growth and inflammation and immunity.KEGG enrichment analysis found that the core prescription HXZF mainly focused on the ways of AGE-RAGE signaling pathway,IL-17 signaling pathway.After molecular docking of key components with core targets,it was found that quercetin,β-sitosterol and isorhamnetin formed stable conformations with five core target protein residues through hydrogen bonds.3.Animal experiment:(1)EAP model can significantly reduce the contents of PSA and PAP in rat prostate tissue and prostatic fluid,and affect the positive expression of PSA and PAP protein in rat prostate tissue,which can affect the normal liquefaction process of rat semen.(2)At first,the results of HE staining of prostate tissue showed that the interstitial tissue of prostate in the model group was congested and edema,showing loose reticulation,disordered arrangement of glandular epithelial cells,proliferation of a large number of fibrous tissues,partial atresia of glandular cavities,and decreased secretion in glandular cavities.The damage degree of prostate gland epithelium in each dose group of the HXZF was improved to varying degrees,and the glands were enlarged to varying degrees,and the secretion of gland cavity was also increased to some extent.Secondly,the results of prostate index and ELISA of TNF-α,IL6 and IL-1β in prostate tissue showed that compared with the model group,all dosage groups of the HXZF could significantly reduce the prostate index of rats(P<0.01)and the contents of TNF-α,IL-6 and IL-1β in prostate tissue(P<0.05).Finally,according to the ELISA results of PSA and PAP in serum,compared with the model group,the contents of PSA and PAP in serum of rats in each dose group of the HXZF decreased significantly(P<0.01).(3)The ELISA results of serum T,E2,FSH and LH showed that compared with the model group,the serum T content in the middle and high dose group of the HXZF increased significantly(P<0.01),while the serum E2,LH and FSH content decreased significantly(P<0.01).(4)The detection results of Zn2+ and CA in prostate tissue showed that the contents of Zn2+ and CA in prostate tissue of rats in each dose group of the HXZF increased significantly(P<0.05).(5)The ELISA results of PSA,PAP,SERPINE1 in prostate tissue and PSA and PAP in prostatic fluid showed that the contents of PSA and PAP in prostate tissue and prostatic fluid of rats in the middle and high dose group increased significantly(P<0.01),while the content of SERPINE1 which inhibited serine protease activity decreased significantly(P<0.01).(6)Immunohistochemical staining of prostate tissue showed that compared with the model group,the HXZF-high dose group could significantly improve the positive expression of PSA and PAP in rat prostate tissue(P<0.01).(7)Western blot results showed that the levels of IL-17/β-actin,IL-17RA/β-actin and Act1/β-actin in each dose group of the HXZF were significantly lower than those in the model group(P<0.01).(8)RT-PCR results showed that compared with the model group,the mRNA expression levels of IL-17,IL-17RA and Act1 in prostate tissue in the middle and high dose groups of the HXZF were significantly decreased(P<0.05).Conclusion:1.Academician Wang’s medication experience in treating semen non-liquefaction:(1)Academician Wang’s treatment of semen non-liquefaction starts with the liver,kidney and spleen,and focuses on the etiology of "imbalance of kidney yin and yang and abnormal Qi",emphasizing the medication principle of "strengthening the vital to eliminate pathogenic factor and restoring the function of Qi".The medication has the characteristics of supplementing Qi and blood and regulating Yin and Yang simultaneously.(2)Academician Wang’s core prescription HXZF for clinical treatment of semen non-liquefaction consists of five tonic drugs:Angelica sinensis,Radix Astragali,Morinda officinalis,Semen Cuscutae and Fructus Lycii.2.Network pharmacology research revealed that the core prescription HXZF has a potential therapeutic effect on semen non-liquefaction,and regulates the biological processes related to prostate vascular growth and inflammation and immunity through the characteristics of "multi-component,multi-target,multi-pathway".Quercetin,β-sitosterol and other key components in the core prescription HXZF may regulate multiple pathways by acting on TNF,SERPINE1 and other targets,and may treat semen liquefaction by inhibiting inflammatory reaction,antioxidation and regulating fibrin.3.Overall animal experiments showed that,firstly,it is found that EAP model can affect the secretion of PSA and PAP in rat prostate,so it is predicted that this EAP model can make rat semen not liquefied,and this model is used as an experimental method to induce rat semen not to liquefy.It is further found that the core prescription HXZF has a good therapeutic effect on rats with semen non-liquefied,and its preliminary pharmacological mechanism is related to alleviating the inflammatory injury of prostate,restoring the secretion function of prostate,regulating the disorder of serum sex hormones,promoting the secretion of proteolytic enzymes by prostate tissue and inhibiting the IL-17/IL-17RA/Actl signaling pathway.