Effect Of Jinkui Shenqi Pills On Mitophagy In Preventing Testicular Endocrine Dysfunction In Subacute Aging Rats

Objective To investigate the molecular mechanism of Jinkui Shenqi Pills（JP）regulating mitophagy to prevent D-galactose-induced testicular endocrine dysfunction in subacute aging rats and to provide an experimental basis for clinical Jinkui Shenqi Pills to prevent testicular aging.Methods Sixty SD rats were randomly divided into a control group,a model group,JP low,medium,and high experimental groups,and a metformin group.The control group was given 0.9%normal saline subcutaneously,the aging group,JP experimental group,and metformin group were given 125mg/kg/d D-galactose subcutaneously for 8 weeks,and the rats were given normal saline,Jinkui Shenqi Pills Solution（1.25g/kg/d,2.5g/kg/d,5g/kg/d）,and Metformin Hydrochloride Solution 300mg/kg intragastrically from the 5th week at the same time of subcutaneous injection for 4 weeks.Observe the general condition of rats,and record the body weight and body temperature of rats.After 8 weeks,blood was collected and sex hormone content in rat testicular tissue was measured by ELISA;rat testicular tissue was taken and HE staining was used to observe the effect of Jinkui Shenqi Pills on rat testicular morphology;immunoblotting was used to detect the expression of P16^INK4A,PINK1,Parkin,LC3,Beclin-1,P62,St AR,CYP11A1,CYP17A1 and HSD17β3 protein in rat testicular tissue;immunohistochemical staining was used to detect the expression of aging-related proteinβ-galactosidase,P16^INK4A,P21^Waf1/Cip1,autophagy-related protein PINK1,Parkin,LC3,Beclin-1,P62,and testosterone synthesis key enzymes St AR,CYP11A1,CYP17A1,and HSD17β3 protein.Transmission electron microscopy was used to observe the mitochondrial ultrastructure of testicular cells at all levels.Results1.Effect of JP on general condition,body temperature,and body weight in subacute aging ratsRats in the model group showed obvious signs of kidney deficiency and aging such as reduced diet,lack of energy,arched back curling,thin body shape,slow movement,and reduced spontaneous activity.Compared with the aging group,the rats in the JP experimental group and the metformin group were significantly better than those in the model group in terms of spirit,diet,and activity.Compared with the control group,the body temperature and body weight of rats in the model group decreased as a whole,and the body temperature and body weight of rats in the JP experimental group increased significantly at weeks 6 and 8compared with the model group.2.Effect of JP on testes weight and testes coefficient in subacute aging ratsCompared with the control group,the aging group could significantly down-regulate the testis weight and testis coefficient（P<0.05）;Compared with the aging group,JP could significantly increase testis weight and testis coefficient（P<0.01）.3.Effect of JP on Serum Hormones in Subacute Aging RatsCompared with the control group,JP could significantly down-regulate the levels of T,FSH,and E₂ in serum and increase the content of LH;compared with the aging group,JP could increase the levels of T,FSH,and E₂ in serum to different extents and decrease the level of LH,especially in the medium dose group.4.Evaluation of the aging level of testis in Subacute aging rat model by JPAfter D-galactose administration,the expression level of aging-related protein P16^INK4Awas significantly up-regulated compared with the control group（P<0.01）.Microscopically,immunohistochemical observation showed that P16^INK4A,P21^Waf1/Cip1,and the aging markerβ-galactosidase in the testis were increased in cytoplasmic expression in the aging model group compared with the control group（P<0.05）;the expression of P16^INK4A,P21^Waf1/Cip1,andβ-galactosidase in the testis of JP-treated rats was decreased compared with the aging model group,with the medium-dose group being the most significant（P<0.05）.5.Effect of JP on Histomorphology of Testis in Subacute Aging RatsThe results of HE staining showed that compared with the control group,the testicular tissue structure of the model group lost its normal morphology,the space of adjacent seminiferous tubules became larger,the spermatogenic cells were disorganized,and the number of Sertoli cells and germ cells at all levels was significantly reduced.After JP intervention,the seminiferous tubule wall of testicular tissue was intact,the adjacent seminiferous tubules were closely connected,and the spermatogenic cells were arranged relatively neatly,especially in the medium-dose group.6.Protective effect of mitophagy regulated by JP on testicular function in subacute aging ratsThe results showed that the expression levels of autophagy-related proteins Parkin,PINK1,Beclin-1,LC3,and p62 were significantly decreased in the aging model group compared with the control group;however,compared with the aging group,JP intervention up-regulated the expression of autophagic proteins and down-regulated P62 protein expression（P<0.05）;in addition,immunohistochemical staining results suggested that the expression of autophagic proteins in testicular tissues was consistent with Western blot results,and compared with the model group,JP treatment showed that the positive expression of cells at all levels in the cytoplasm of the testis increased after WB treatment.It was observed by immunofluorescence that the expression of Beclin-1 and LC3,key proteins of autophagy,was attenuated and the expression of P62 was enhanced in Leydig cells of the aging group compared with the blank control group;while Beclin-1 and LC3 were strongly stained in Leydig cells of the testicular tissue and the expression of P62 was attenuated in Leydig cells after JP treatment.7.Protective effect of JP on testicular endocrine dysfunction in subacute aging ratsThe immunohistochemical results showed that the key enzymes of testosterone synthesis,St AR,CYP11A1,CYP17A1,and HSD17β3,were brownish-yellow positive in testicular cells in the control group,and the staining in the aging model group was weakened compared with the control group;the expression increased after JP treatment,most significantly in the medium-dose group（P<0.05）.WB showed that the protein expression of St AR,CYP11A1,CYP17A1,and HSD17β3,which are key enzymes of testosterone synthesis,was significantly down-regulated in the aging model group compared with the control group;however,the protein expression was increased to varying degrees after JP treatment,most significantly in the medium-dose group（P<0.05 for all）.8.Effect of JP on the ultrastructure of mitochondria in the testis of subacute aging ratsTransmission electron microscopy was used to observe the mitochondrial morphology in testicular cells.In the control group,the mitochondria were arranged neatly,the morphology was normal,the mitochondrial cristae structure was intact,and a small amount of autophagy occurred;in the model group,different degrees of mitochondrial cristae disorder and dissolution occurred,the mitochondria were swollen and vacuolated significantly,the number of mitochondria decreased,the volume became smaller,and no typical mitophagosomes were observed;after JP treatment,the mitochondrial partial edema and vacuole-like changes were significantly improved compared with the aging model group,the number of mitochondria increased,and typical mitophagosomes with bilayer membrane structure could be observed.ConclusionJP plays a protective role in preventing testicular endocrine dysfunction in subacute aging male rats by protecting mitochondrial ultrastructure,activating the mitophagy process,regulating testicular autophagy levels,and increasing the content of key enzymes for testosterone synthesis.