Data Mining Of The Treatment Of Oligoasthenospermia And Its Mechanism Of Regulating Spermatogenic Cells By Invigorating The Kidney And Producing Sperm

Oligoasthenospermia has become a research hotspot in andrology.Western medicine treatment has certain limitations,and clinical treatment cannot rely on evidence-based medicine evidence drug recommendation and most empirical treatment as well.As a common treatment of oligoasthenospermia,kidney-tonifying and spermatogenerating method has unique therapeutic advantages and it can effectively make up for the deficiency of western medicine in treating oligoasthenospermia.Guangci yulin decoction refers to a prescription for treating oligoaspermia in the Male Department of Dongzhimen Hospital,and it has achieved better clinical effects.This prescription is an empirical prescription for tonifying kidney and raising sperm,with dodder seed and wolfberry as the king medicine.Objective1 To explore the origin and development of tonifying kidney and generating essence.2 To explore the experience and principles of treating oligoasthenospermia by Professor Li Haisong and other Traditional Chinese Medicine(TCM)scholars in the national patent database.3 To explore the clinical efficacy of Guangci Yulin Decoction in the treatment of oligoasthenospermia.4 To explore the experimental mechanism of dodder and wolfberry in the treatment of oligoasthenospermia.MethodsLiterature research:Based on TCM literature and database,the literatures on the treatment of male infertility through tonifying kidney and generating essence and the theory of storing essence by kidney in ancient works were sorted out.On the other hand,the development context and origin of the method of tonifying kidney and generating essence were sorted out as well.Data mining:Based on the TCM inheritance auxiliary platform,438 prescriptions for the treatment of oligoasthenospermia by Professor Li Haisong and 70 prescriptions of TCM for the treatment of oligoasthenospermia from the national patent database were respectively enrolled as the mining objects for the exploration of drug application rules and treatment principles of Professor Li Haisong and other TCM scholars.Clinical study:According to the data mining results of Professor Li Haisong’s prescription for the treatment of oligoasthenospermia,namely "Guangsi Yulin Decoction",the clinical efficacy of this prescription was evaluated.By adopting the method of case-control study,120 patients suffering from oligoasthenospermia were included in the 12-week treatment,and the evaluation indexes included semen quality and TCM syndrome score.Experimental study:Based on the data mining results of the representative drug pair of kidney-tonifying and sperm-generating method ’Cusder and Lycium wolfberry’(SC-FL),the experimental mechanism was verified,and the specific mechanism of the representative drug pair of kidney-tonifying and sperm-generating was explored through the combination of cell experiment and animal experiment.The spermatogonial cells(GC-1SPG)were divided into 4 groups:normal group(NC group),model group(GTW group),levocarnitine treatment group(GTW+LEV group),and Cuscuta-Lycii treatment group(GTW+SC-FL group).The spermatogenic dysfunction cell model was prepared by in vitro intervention of Tripterygium wilfordii polyglycosides that contain drug serum.The treatment group was respectively treated with levocarnitine(LEV)and SC-FL intervention.The animal experiment was also divided into 4 groups,with 6 rats in each group.The rat model of oligoasthenospermia was prepared by GTW gavage for 4 weeks.The treatment group was treated with LEV and SC-FL gavage for 4 weeks,respectively.The general status and serum hormone levels of rats in each group were observed.The cell cycle,mitochondrial membrane potential,proliferation and apoptosis of GC-1SPG in each group were observed by flow cytometry.The organelles and ultrastructures of GC-1SPG cells and testis were observed by transmission electron microscopy.Immunofluorescence staining,WB and RT-qPCR were applied to detect the distribution and content of PI3K/Akt pathway-related proteins and mRNA in GC-1SPG cells and rat testis tissues.Results1 Literature research:The method of tonifying kidney and generating essence is developed from the theory of kidney storage in TCM.As early as pre-qin period,kidney essence law basis was born.During the period of Wei,Jin,and Sui,literatures on kidney in raw liquor method had certain inheritance and innovation.After experiencing Tang,Song and Yuan dynasties,kidney essence theory was perfected and systematic.In Ming and Qing dynasties,ancient records of kidney literature theory has been booming,and ultimately formed a complete system.2 On the basis of the tutor’s prescriptions and experience,the tutor discovered that the etiology of oliospermia is usually kidney sperm deficiency,involving liver,kidney and spleen.The treatment is based on fine-tuning Yin and Yang,and the aim is to nourish kidney and generate sperm,with holistic treatment.The most frequently used drugs were the two herbs of dodder and wolfberry,followed by raspberry,schizandrae,rehmannia rehmannii,astragalus,plantago seed,oyster and angelica.The above nine herbs,along with yam,constituted"Guangsi Yulin Decoction".Dodder and wolfberry were the medicines of Guangsi Yulin Decoction.By digging the prescription of national patent database,it was obvious that the most frequent prescriptions were also dodder and wolfberry,which indicated that this drug had certain curative effects in clinical practices,and its mechanism still needs to be verified by experiment.3 Through clinical studies,it was clear that,after treatment with Guangsi Yulin Decoction,the semen concentration and activity rate of patients were significantly increased,and the main and secondary syndromes of Chinese medicine were also remarkably improved(P<0.05 or P<0.01).The clinical effective rate of Guangsi Yulin Decoction in the treatment of oligoasthenospermia was 92.7%,without adverse reactions during the trial.4 The results of cell experiment revealed that,in terms of cell cycle,mitochondrial membrane potential and apoptosis,the percentage of G0/G1 and G2/M phase of GC-1SPG cells in GTW group was obviously decreased and the percentage of S phase was greatly increased compared with NC group(P<0.01).Compared with GTW group,the percentage of spermatogenic cells in the G0/G1 and G2/M phases of GC-1SPG cells in GTW+LEV group and the GTW+SC-FL group was increased,while the percentage of spermatogenic cells in the S phase was decreased(P<0.05,P<0.01).Compared with GTW+LEV group,the percentage of GC-1SPG cells in GTW+SC-FL group was extremely increased in G0/G1 and G2/M phase,while the percentage of S phase was decreased(P<0.01).As for cell ultrastructure:Compared with NC group,GC-1SPG in GTW group had a large area of cell membrane rupture,organelle disassociation,a few pseudopods and processes around the membrane,and a large area of cell matrix density reduction,vacuolar degeneration and overall disintegration.The shape of the nucleus was seriously deformed,with local depression,multiple nucleoli and clear nuclear membrane.The number of mitochondria was abundant:Most of them were severely swollen and deformed,and the cristae were broken and disappeared.In some severe cases,the membrane was disintegrated and the matrix disappeared.The rough endoplasmic reticulum was obviously dilated.The number of autophagy lysosomes was more,Golgi apparatus was hypertrophy,and Golgi apparatus pool was obviously expanded.Fat droplets were present in small amounts.Compared with GTW group,the ultrastructure and morphology of each organelle of GC-1SPG in GTW+LEV and GTW+SC-FL groups were significantly restored,while compared with NC group,the structure of GC-1SPG was not completely restored.For protein and mRNA expression,compared with NC group,the protein and mRNA expressions of PI3K,p-Akt and Bcl-2 in GC-1SPG cells in GTW group were remarkably decreased(P<0.01 or P<0.05).Compared with GTW group,the protein and mRNA expressions of PI3K,p-Akt,Bcl-2 in GC-1SPG cells in GTW+LEV group and GTW+SC-FL group were obviously increased(P<0.01 or P<0.05).Compared with GTW+LEV group,the protein and mRNA expressions of PI3K and Bcl-2 in GC-1SPG cells in GTW+SC-FL group were greatly increased(P<0.01 or P<0.05).There were no significant differences in p-Akt protein and mRNA expression in GC-1SPG cells in GTW+SC-FL group(P>0.05).Compared with NC group,the protein and mRNA expressions of Bad and Bax in GC-1SPG cells in GTW group were obviously increased(P<0.01 or P<0.05).Compared with GTW group,the protein and mRNA expressions of Bad and Bax in GC-1SPG cells in GTW+LEV group and GTW+SC-FL group were extremely decreased(P<0.01 or P<0.05).Compared with GTW+LEV group,the protein and mRNA expressions of Bad and Bax in GC-1SPG cells in GTW+SC-FL group were greatly decreased(P<0.01 or P<0.05).The results of animal experiments proved that the general condition of rats after treatment was improved compared with model group.Routine semen:Compared with NC group,sperm concentration and PR+NP in GTW group were decreased(P<0.01).Compared with GTW group,sperm concentration and PR+NP in GTW+SC-FL and GTW+LEV groups were increased(P<0.01).Compared with GTW+LEV group,sperm concentration in GTW+SC-FL group was increased(P<0.01),while PR+NP in GTW+SC-FL group had no significant differences(P>0.05).Serum sex hormone levels:Compared with GTW group,serum FSH,LH and T in GTW+LEV and GTW+SC-FL groups were significantly increased(P<0.01 or P<0.05).Compared with GTW+LEV group,serum FSH,LH and T in GTW+SC-FL group were obviously increased(P<0.01).In terms of tissue structure and ultrastructure,the morphology and structure rules of spermatocytes in NC group were observed.The nuclei(N)were plump and round with normal structure and abundant euchromatin.The nucleolus morphology and structure were obvious(NU).Cytoplasmic endoplasmic reticulum(ER)was common,and lysosomes(AAS)and autophagosomes(AP)were more or less distinct.The morphology and structure of spermatocytes in GTW group were irregular.The cell membrane was damaged,and the structure and morphology of N were changed to be round or oval,with abundant euchromatin and a small amount of heterochromatin agglutinating.Intracytoplasmic ER dilatation was also observed with various amounts of AAS and AP.After SC-FL treatment,the damaged organelles of GTW recovered somewhat.In terms of protein and mRNA expression,compared with GTW group,the protein and mRNA expressions of PI3K,AKT and Bcl-2 in GTW+LEV group and GTW+SC-FL group were remarkably increased(P<0.01 or P<0.05).Compared with GTW+LEV group,the protein and mRNA expressions of PI3K,Akt and Bcl-2 in GTW+SC-FL group were obviously increased(P<0.01 or P<0.05).Conclusion1 Data mining results displayed that Cuscuta and Lycii are the representative drug pairs for the treatment of oligoasthenospermia by tonifying kidney and generating sperm;Professor Li Haisong treats oligoasthenospermia with the principle of fine-tuning Yin and Yang and the aim of tonifying kidney and generating sperm.2 The experimental results illustrated that SC-FL could effectively improve the general state of spermatogenic dysfunction model rats,improve semen quality,participate in the regulation of hypothalamic-pituitary-gonadal axis sex hormones,and repair the ultrastructure of GC-1SPG and testicular tissue.Meanwhile,SC-FL can promote the proliferation and inhibit the apoptosis of spermatogenic cells through PI3K/Akt signaling pathway.3 Clinical study results demonstrated that Guangsi Yulin Decoction can effectively improve the semen quality of oligoasthenospermia patients and reduce the TCM symptom scores of patients suffering from kidney essence deficiency syndrome.In conclusion,kidney tonifying and spermatogenic therapy can effectively treat oligoasthenospermia with renal sperm deficiency,and its regulation of PI3K/Akt signaling pathway protein and mRNA is one of the mechanisms of action.