| Research objectives:Bos grunniens mainly live in the Qinghai-Tibet Plateau and its adjacent alpine and sub-alpine alpine alpine regions.They are highly adaptable to the harsh environment such as high pressure,low oxygen and extreme cold.With the development of animal husbandry,yaks have been gradually large-scale and intensive breeding.Artificial semen collection and insemination have become one of the necessary measures to improve its fertility.Frequent semen collection and mating can result in listlessness and semen quality poor in male breeder stocks,thus inducing the occurrence of kidney-yang deficiency syndromes such as oligospermatism and asthenospermia.In the previous study,according to the physiology and kidney-yang deficiency characteristics of breeding bulls,our research group added the new formula made of Cnidium cotoniformis and Morbidiaceae as feed additives on the basis of Yi-Jing Decoction(YJD),which had achieved the purpose of filling lean pulp,strengthening Yang and tonifying kidney.However,the multiple action sites and targets of the 11 ingredients of this formula also increase the difficulty in studying the mechanism of action.This study is expected to explore the effect of the modified Yi-Jing Decoction(YJD)"Drug-Targets-Disease" by using network pharmacology and molecular docking technology.By establishing a drug model of "kidney-yang deficiency" rats,and combining metabonomics techniques,the effects of modified Yi-Jing Decoction(YJD)on serum metabolome of rats are explored.By adding the modified Yi-Jing Decoction(YJD)to the feed of yaks with deficiency of kidney Yang,the effect of it on semen quality is studied.According to the research,the addition of Yi-Jing Decoction(YJD)can prevent the low conception rate caused by low semen quality and the impotence caused by long-term frequent mating,greatly increase the number of healthy sperm per unit volume in semen,thus prolongating the service life of male breeding yaks and improving their reproductive performance;It can also replace hormones to provide theoretical and experimental basis for improving the immunity and reproductive performance of yaks,and escort the development of modern and large-scale breeding of male yaks.Research methods:1.Network pharmacological analysis: Through TCMSP,TCMIP,Herb and other databases,the targets of Yi-Jing Decoction(YJD)drug-disease interaction were screened,and the core targets were screened;Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were used to predict the mechanism of action of Chinese medicine in the treatment of oligasthenospermia.The large molecules and small molecules with top topological parameters were molecule-docking,and the results were imported into Pymol for visualization of docking results.2.Animal experiments: The rat model of kidney-yang deficiency was established as follows: 60 male SD rats(200g±20g)were randomly divided into 6 groups.The normal control group(NC)was injected with normal saline,and the other groups were injected with hydrocortisone at the dose of 2.5mg/100 g.The modeling lasted for 10 days.After intramuscular injection,the high-dosage YJD(YJD-H),medium-dosage YJD(YJD-M),and low-dosage YJD(YJD-L)groups were given by intragastric administration every day.The doses were 2.04g/100 g in YJD-H,1.02g/100 g in YJD-M and 0.51g/100 g in YJD-L,twice a day,once in the morning and once in the evening.The positive control group(PC)was given L-carnitine at a dose of10mg/100 g,once every morning,the model control group(MC)and NC group were given normal saline,and the NC,MC and PC groups were given normal saline at night,and the administration days were 15 days in total.2.1 Metabolomics analysis: Metabolomics technology was used to conduct high-throughput sequencing of metabolites in serum samples of SD rats.2.2 HPLC-MS was used for qualitative and quantitative detection of the effective ingredients of YJD: The relevant verification in animal experiments was as follows:HE staining was used to explore the morphological changes of rat testis,ELISA was used to detect the concentration of cAMP/cGMP and T/E2 in serum of kidney-yang deficiency rats,western blot and qRT-PCR were used to detect the protein and mRNA of Bcl-2,Bax,Cyt-C and Caspase 3.3.Clinical verification: 8 yaks(3-6 years old)with kidney-yang deficiency syndrome were randomly divided into two groups,namely the YJD group and the negative control group,and 4 normal yaks were selected as normal control.The YJD was mixed into coarse powder and mixed into yak feed,once a day for each yak,5days as a course of treatment,and the yak was fed the second course of treatment after20 days and the third course of treatment after 30 days,a total of 3 courses of treatment for 65 days.The other two groups were fed normally,and the semen quality was investigated.The concentrations of cAMP/cGMP and T/E2 in serum of kidney-yang deficiency yak were detected by ELISA,and the proteins and mRNA of Bcl-2,Bax,Cyt-C and Caspase 3 in semen were detected by western blot and qRT-PCR.Research results:1.Through screening,there were 81 effective ingredients and 491 effective targets in the YJD,and 4436 effective targets in oligasthenospermia,with 251 targets in total.The key compounds were quercetin,kaempferol,icariin,luteolin and catechin.There were 10 core targets of AKT1,INS,TP53,IL6,TNF and Bcl-2 in the treatment of oligasthenospermia.The biological processes of GO mainly include drug response,serine/threonine protein kinase complex,and nuclear receptor activity.The pathways of KEGG were TNF,HIF-1 and PI3K-Akt.Catechins,kaempferol,quercetin,luteolin and icariin in molecular docking showed lower binding energy with Bcl-2,but better binding activity.2.The serum metabolomics of rats with kidney-yang deficiency induced by hydrocortisone showed that the expression level of Sphingsine 1-phosphate(S1P)was significantly different among all groups.3.HPLC-MS results showed that the contents of catechin,quercetin,kaempferol,luteolin and icariin were 21700ng/mL,1050ng/mL,5280ng/mL,1180ng/mL and38800ng/mL,respectively.ELISA results showed that compared with NC group,the concentrations of cAMP and T in serum of all experimental groups were decreased,but the concentrations in JYD-H group were the highest(P<0.01),while cGMP and E2 were opposite to cAMP and T;The ratio of cAMP/cGMP and T/E2 were down-regulated in all experimental groups,but the ratio was the largest in YJD-H group(P<0.01);HE results showed that in the NC and YJD-H group,testicular structure were complete,with closely and clearly arranged spermatocytes and Sertoli cells at all levels and regular hierarchy.Western-blot and qRT-PCR results showed that Bcl-2 in each YJD group was significantly higher than that in the other groups(P<0.01),YJD-H group was significantly up-regulated(P<0.01);Bax,Cyt-C and Caspase 3 were down-regulated in each YJD group compared with the NC group and lcarnitine group,and the down-regulation was most significant in YJD-H group(P<0.01);4.The clinical experiment showed that YJD as a feed additive greatly improved the semen quality of yaks,and compared with the control group,the concentrations of serum cAMP and T were up-regulated(P<0.01),the concentrations of cGMP and E2 was decreased(p<0.01),and the ratio of cAMP/cGMP to T/E2 increased(P<0.01);The expression of Bcl-2 in semen was up-regulated(P<0.01),and the expression of Bax,Cyt-C and Caspase 3 was down-regulated(P<0.01).Conclusion:1.The Yi-Jing Decoction(YJD)can promote the secretion of T and other reproductive hormones.2.The Yi-Jing Decoction(YJD)can effectively improve the spermatogenesis environment of rats with kidney-yang deficiency caused by hydrocortisone and promote spermatogenesis through mitochondrial apoptosis pathway.3.The Yi-Jing Decoction(YJD)can improve semen quality and promote sperm vitality and density of yaks. |
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