基于蛋白质组学技术探究龟鹿二仙口服液改善肾阳虚型少弱精子症大鼠的作用机制OA

Objective:To investigate the mechanisms of Guilu Erxian Oral Liquid(GLEX)in the treatment of oligoasthenospermia(OAS)of kidney-yang deficiency type.Methods:Fifty-six Sprague-Dawley(SD)rats were randomly divided into a control(C)group,a model(M)group,GLEX low-(GLEX-L),medium-(GLEX-M),and high-(GLEX-H)dose groups,a Wuzi Yanzong Pills(WZYZP)group,and a Levocarnitine Oral Solution(LOS)group,with 8 rats in each group.A kidney-yang deficiency type OAS model was established by intragastric administration of adenine(200 mg·kg-1·d-1)for 30 days.The treatment groups were administered GLEX-L,GLEX-M,and GLEX-H(2.7,5.4,and 10.8 mL·kg-1·d-1),WZYZP(1.269 mL·kg-1·d-1),and LOS(2.0 mL·kg-1·d-1),respectively.General conditions of rats were observed,and testicular and epididymal organ indices were calculated.Semen quality was assessed using a sperm analyzer.Serum hormone levels of the hypothalamic-pituitary-target gland axis were measured by enzyme-linked immunosorbent assay(ELISA).Testicular histomorphology was observed by hematoxylin-eosin(HE)staining,and Johnsen scores were calculated.Mitochondrial membrane potential(MMP)was detected by JC-1 flow cytometry.Data-independent acquisition(DIA)quantitative proteomics was used to screen key pathways involved in the therapeutic effects of GLEX on kidney-yang deficiency type OAS.Western blot(WB)was performed to validate the expression of key proteins,including Toll-like receptor 4(TLR4),inhibitor of nuclear factor kappa B(IκB),and nuclear factor kappa B p65(NF-κB p65),in rat testicular tissues.Results:Compared with the C group,rats in the M group exhibited lethargy,sluggishness,hair loss with huddling,loose stools,cold back,and slow weight gain.The testicular index was significantly increased(P<0.01).Sperm motility and rapid progressive motility were markedly decreased,whereas the sperm deformity rate was significantly increased(P<0.05,P<0.01).Serum levels of thyroid-stimulating hormone(TSH),follicle-stimulating hormone(FSH),luteinizing hormone(LH),and estradiol(E2)were significantly elevated(P<0.01),while no statistically significant differences were observed in corticotropin-releasing hormone(CRH),cortisol(Cor),or testosterone(T).Histological examination showed seminiferous tubule atrophy,exfoliation of spermatogenic cells,and reduced sperm count,with a marked reduction in Johnsen score(P<0.01).The MMP polymer/monomer ratio was significantly reduced(P<0.01).After treatment,the above indicators were improved to varying degrees.Proteomic analysis indicated that GLEX exerts its effects in tonifying kidney-yang and treating OAS mainly through the Toll-like receptor signaling pathway,autophagy-animal pathway,and NOD-like receptor signaling pathway.Validation of the TLR4/IκB/NF-κB pathway showed that,compared with the C group,the M group had TLR4 upregulation and enhanced phosphorylation of IκB-α and NF-κB p65 in testicular tissues(P<0.05,P<0.01).After GLEX intervention,the excessive expression of these proteins was significantly suppressed(P<0.05).Conclusion:GLEX can improve sperm quality,testicular tissue damage,mitochondrial function,and hormone levels in rats with kidney-yang deficiency type OAS.Its mechanism may be associated with the TLR4/IκB/NF-κB signaling pathway.