UPLC-MS/MS Combined With Network Pharmacology To Investigate The Treatment Of Spermatogenic Dysfunction In Rats With Kidney Yang Deficiency By Penis Et Testis Cervi

Penis et Testis Cervi is the penis and testis of the Cervusnippon Temminck or Cervuselaphus Linnaeus.After slaughter,the penis and testicles are cut,the residual meat and grease are removed,and fixed on the board for drying.It can also be blanched with boiling water and then dried in an oven at 80℃.It has the effect of tonifying kidney essence,strengthening kidney yang and strengthening waist and knee.Objective: In order to explore the potential active ingredients in Penis et Testis Cervi;To study the biological mechanism of Penis et Testis Cervi in treating spermatogenic dysfunction in rats with kidney yang deficiency;To explore the effect and curative effect of Penis et Testis Cervi on the dysfunction of spermatogenesis due to deficiency of kidneyyang;It provides a theoretical basis for the promotion and application of Penis et Testis Cervi in the treatment of kidney yang deficiency and spermatogenesis dysfunction.Methods: 1.Firstly,BCA protein concentration kit was used to determine the protein concentration of the water extract of Penis et Testis Cervi;Then,the molecular weight distribution of the water extract protein was detected by gel permeation chromatography;Finally,the amino acids,sterols,long-chain fatty acids,short-chain fatty acids,nucleotides and biogenic amines in the water extract of Penis et Testis Cervi were analyzed by liquid chromatography,ultra-high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.The main targets of the disease are obtained through the Genecards database,the active components and action targets of the Penis et Testis Cervi are collected using the BAT-MAN database,and the target genes are enriched with GO and KEGG using the STRING database,and the "drug-component-target-pathway" network is also constructed.The network is visually analyzed by using Cytoscape.Screening out the pathway and target of Penis et Testis Cervi to treat the potential spermatogenic dysfunction of kidney-yang deficiency;Molecular docking was used for validation analysis.Kidney-yang deficiency model induced by hydrocortisone;The pathological changes of testis,kidney and adrenal gland were observed by H&E staining;Serum testosterone(T),follicle-stimulating hormone(FSH),luteinizing hormone(LH),adrenocorticotropic hormone(ACTH)and cortisol(CORT)were measured by ELISA;The expression level of reproductive related genes was detected by q RT-PCR;The expression of ARG1 in testis was detected by immunohistochemistry;The concentration and viability of rat sperm were measured using the wet sheet method..Results: 1.The protein content of the water extract of Penis et Testis Cervi is 50.7%.The protein of 70-130 k Da accounted for 10.71%,and the protein of less than 70 k Da accounted for 82.7%;There are 16 kinds of amino acids in the water extract of Penis et Testis Cervi,the top five are glycine,proline,glutamic acid,alanine,arginine,15 kinds of hormones in the water extract of Penis et Testis Cervi,the top five are 17-hydroxypregnenolone,estriol,17-hydroxyprogesterone,21-hydroxyprogesterone,estradiol;Among the 24 long-chain fatty acids in the water extract of Penis et Testis Cervi,the top 5are palmitic acid,stearic acid,oleic acid,docosahexaenoic acid methyl ester and arachidonic acid;The water extract of Penis et Testis Cervi contains 8 short chain fatty acids,including acetic acid,caproic acid,n-butyric acid,propionic acid,isovaleric acid,isobutyric acid and n-valeric acid;The water extract of Penis et Testis Cervi contains 5 kinds of nucleotides,including guanine nucleotides,adenine nucleotides,uracil nucleotides,hypoxanthine nucleotides and cytosine nucleotides;The water extract of Penis et Testis Cervi contains four biological amines,including tyramine,β-Phenylethylamine,tryptamine,histamine.2.The results of network pharmacology analysis showed that there were 303 cross targets between the potential compounds of Penis et Testis Cervi and diseases;Penis et Testis Cervi can regulate 2802 GO entries and 206 KEGG entries to play a role in the treatment of spermatogenic dysfunction;Arginine biosynthesis pathway is a potential pathway to treat the dysfunction of spermatogenesis due to deficiency of kidney-yang;Molecular docking results showed that NOS1,NOS2,NOS3,ARG1,CPS1 and GLUL in the biosynthetic pathway of arginine had strong binding energy with estradiol.3.The water extract of Penis et Testis Cervi can significantly restore the levels of testosterone,luteinizing hormone,follicle-stimulating hormone,adrenocorticotropic hormone and cortisol in the serum of rats with kidney yang deficiency.At the same time,Penis et Testis Cervi can activate the biosynthetic pathway of arginine and improve the pathological state of testicles,kidneys and adrenal glands in rats with kidney yang deficiency;The expression level of reproductive markers in testis was significantly restored,and the expression level of ARG1 protein in testis was inhibited;The water extract of Penis et Testis Cervi can significantly restore sperm concentration and sperm vitality of rats with kidney yang deficiency.Conclusion: The content of protein in the water extract of Penis et Testis Cervi is as high as 50.7%,and it is rich in 16 amino acids,15 hormones,24 long-chain fatty acids,8medium-short-chain fatty acids,5 nucleotides and 4 biological amines.It is a highly nutritious dietary supplement,providing a material basis for the treatment of kidney yang deficiency and spermatogenesis dysfunction.There are 303 cross targets of potential compounds of Penis et Testis Cervi and diseases,and 2802 GO entries and 206 KEGG entries in deer whip can play a role in the treatment of spermatogenic dysfunction.There are many potential compounds of Penis et Testis Cervi,and the biological mechanism of regulating spermatogenic dysfunction in rats with kidney yang deficiency is complex,which provides theoretical basis for the treatment of spermatogenic dysfunction of kidney yang deficiency.The water extract of Penis et Testis Cervi can improve the pathological changes of testicles,kidneys and adrenal glands of rats with kidney-yang deficiency;Restore the levels of testosterone,luteinizing hormone,follicle-stimulating hormone,adrenocorticotropin,cortisol hormones in rats with kidney yang deficiency,and restore the expression level of reproductive related markers in the testis.To provide data support for the treatment of kidney yang deficiency and spermatogenesis dysfunction.The aqueous extract of Penis et Testis Cervi may regulate the spermatogenesis dysfunction of rats with kidney yang deficiency and restore the sperm vitality and concentration of rats through the arginine biosynthesis pathway.