Mechanism Of Apoptosis In Spermatogenic Cells Of Varicocele Rats Regulated By The Prescription Of "Tongjingling" Mediated By Mitochondrial Signaling Pathway

The first part:Effect of Tongjingling Prescription on oxidative stress in the Testis of Rat with experimental VaricoceleObjective To explore the protective effect of"Tongjingling"on the level of oxidative stress of experimental varicocele rats.Methods 72 Wistar male rats are divided into six groups:sham operation group,model group,"Tongjingling"Prescription high dose group,middle dose group,low dose group and L-carnitine group.12 mice of each group.Experimental varicocele is created in the rats by partial ligation of left renal vein.4 weeks after establishing the model,medicine is given for 8weeks.Observe the general situation of rats,The changes in the testicular tissue structure are examined using HE staining.Detection of epididymal sperm DFI by SCSA.The content of hydroperoxide（H₂O₂）and the activities of catalase（CAT）,superoxide dismutase（SOD）in the testes are detected by colorimetry,and the ultrastructural change of spermatogenic cell by the transmission electron microscope is observed.Results 1.The results of HE staining show that compare with the sham group,the testicular tissue of the model group has pathological changed.The spermatogenic cells in the testis are significantly reduced,the arrangement is irregular,the supporting cells are detached,the stromal cells are swelling,and there are no spermatozoa or spermatozoa scarcity.Compare with the model group,the pathological changes of each treatment group are lighter;TEM results show that:the model group spermatogenic cell cytoplasm seen hyperplastic,partially broken nuclei,nuclear membrane shrinkage,mitochondrial swelling,cristae fuzzy endoplasmic reticulum cords changed;There are ultrastructural changes in spermatogenic cells in different groups and the L-carnitine group,but the degree of change is lighter than that in the model group.2.Compare with the sham group,DFI in the model group increase（P<0.01）.Compare with the model group,DFI is reduced in each treatment group after intervention（P<0.01）.There is no significant difference between the groups.3.Compare with the sham group,the H₂O₂ content in the model group increase（P<0.01）;Compare with the model group,the H₂O₂content in the high dose group,the middle dose group and the L-carnitine group decrease,the difference is statistically significant（P>0.05）,there is no significant difference among the treatment groups（P>0.05）.4.Compare with the sham group,the CAT activity in the model group decrease（P<0.01）.Compare with the model group,the high dose group,the middle dose group and the L-carnitine group are able to increase the activity of CAT,the difference is statistically significant（P<0.01）.The CAT activity in the low dose group does not significantly increase（P>0.05）.5.Compare with the sham group,SOD activity in the model group decrease,and the difference is statistically significant（P<0.01）.Compare with the model group,the SOD activity in each treatment group increase,and the difference is statistically significant（P<0.01）.Conclusion VC can lead to high level of oxidative stress in rat testis and the pathological damage of ultrastructure of testis and spermatogenic cells.Sperm DNA integrity is also compromised.“Tong Jingling”can reduce the level of oxidative stress in testis of VC rats,as well the pathological damage of tissue structure to protect sperm DNA integrity.The second part:Effect of“Tongjingling”Prescription on apoptosis of spermatogenic cells of experimental varicocele rats in mitochondrial apoptosis wayObjective To explore the effects of"Tongjingling"on apoptosis of spermatogenic cells of experimental varicocele rats in mitochondrial apoptosis way.Methods 72 Wistar male rats are divided into six groups:sham group,model group,"Tongjingling"prescription high dose group,middle dose group,low dose group and L-carnitine group.12 mice of each group.Experimental varicocele is created in the rats by partial ligation of left renal vein.4 weeks after establishing the model,medicine is given for 8weeks.Observe the general situation of rats,apoptosis、ROS、MMP and Ca²⁺are examined by flow cytometric analysis and the expression of Cyt-c、caspase-3 and caspase-9 are measured by WB analysis.The mitochondrial ultrastructure of spermatogenic cells is examined with transmission electron microscope.Results 1.Compare with the sham group,the ROS content in the model group increase,the difference is statistically significant（P<0.01）;Compare with the model group,the ROS content in the high,middle dose group and the L-carnitine group decrease,the difference is statistically significant.（P<0.01）.The decrease of ROS content in the low dose group is not significant,and the difference is not statistically significant（P>0.05）.2.Compare with the sham group,the MMP in the model group is decreased（P<0.05）.Compare with the model group,each treatment group is able to increase MMP,the difference is statistically significant（P<0.01）.3.Compare with the sham group,the Ca²⁺content of the spermatogenic cells in the model group increase（P<0.01）.Compare with the model group,the Ca²⁺content in the high dose group and the L-carnitine group decrease（P<0.05）.4.Compare with the sham group,the apoptotic rate of spermatogenic cells in the model group increase（P<0.05）.Compare with the model group,the apoptotic rate of the spermatogenic cells decrease in the high dose group,the middle dose group,and the L-carnitine group.The difference is statistically significant（P<0.01,P<0.05）.5.Compare with the sham group,the expression of Cyt-c protein in the model group is significantly higher（P<0.01）.Compare with the model group,the expression of Cyt-c protein in each treatment group is significantly decrease,and the difference is statistically significant（P<0.01）.6.Compare with the sham group,the expression of caspase-3 and caspase-9 protein in the model group significantly increase,and the difference is statistically significant（P<0.01）.Compare with the model group,the expression of caspase-3 and caspase-9 protein in each treatment group is decreased,and the difference is statistically significant（P<0.05,P<0.01）.7.The number of mitochondria in the spermatogenic cells of the model group decrease,the ultrastructure changed,and the mitochondria are damaged.The ultrastructure of mitochondria in each treatment group had different degrees of change,but there are fewer changes Compare with the model group.Conclusion In experimental varicocele rats,the ultrastructure of mitochondria in spermatogenic cells is damaged obviously.Changes in mitochondrial membrane structure and function lead to Cyt-c being released into cytoplasm from mitochondrial membrane gap,activating caspase apoptosis cascade,which lead to the excessive apoptosis of spermatogenic cells in mitochondrial apoptosis way.By protecting mitochondrial structure in spermatogenic cells,"Tongjingling"reduce the releasing level of Cyt-c in the mitochondria,inhibite the high expression of Caspase-9 upstream of cascade reaction and reduce the expression of Caspase-3downstream of cascade reaction.So we infer that"Tongjingling"could reduce apoptosis of spermatogenic cells of experimental varicocele by regulating the mitochondrial apoptosis way.The third part:Effect of“Tongjingling”Prescription on ERK1/2、PI3K/Akt pathway of spermatogenic cells of experimental varicocele ratsObjective To explore the Effect of“Tongjingling”Prescription on ERK1/2、PI3K/Akt pathway of spermatogenic cells of experimental varicocele rats.Methods 72 Wistar male rats are divided into six groups:sham group,model group,"Tongjingling"Prescription high dose group,middle dose group,low dose group and L-carnitine group.12 mice of each group.Experimental varicocele is created in the rats by partial ligation of left renal vein.4 weeks after establishing the model,medicine is given for 8weeks.Observe the general situation of rats,the expression of Bad and Bcl-2 proten is measured by Immunohistochemical analysis;Western Blot are used to test the proten expression of ERK1、ERK2、PI3K and Akt.Results 1.Compare with the sham group,the expression of Bad protein in the model group increase（P<0.01）.Compare with the model group,Bad protein expression is significantly decreased in the high dose group and the L-carnitine group（P<0.01）.The expression of Bad protein in the low dose group and the middle dose group is not significantly decreased（P>0.05）.2.Compare with the sham group,Bcl-2 protein expression is decreased in the model group（P<0.01）.Compare with the model group,Bcl-2 protein expression significantly increase in the medium dose group,high dose group,and L-carnitine group（P<0.01）.3.Compare with sham group,the expression of p-ERK1/2、p-Akt proten is significantly reduced in model group（P<0.01）.Compare with model group,the expression of p-ERK1/2、p-Akt proten significantly increase in"Tongjingling"groups（P<0.01）.Conclusion ERK1/2 and PI3K/Akt pathways play an important role in the process of apoptosis of spermatogenic cells in VC rats.Varicocele inhibit the anti-apoptotic function of ERK1/2 and PI3K/Akt pathways,resulting the activation of mitochondrial apoptosis pathway,the increase of pro-apoptotic protein Bad,the decrease of anti-apoptotic protein Bcl-2,eventually lead to excessive apoptosis of spermatogenic cells.So we infer that"Tongjingling"may activate ERK1/2 and Akt proteins through affecting the ERK1/2 and PI3K/Akt pathways,and regulate the mitochondrial pathway to decrease excessive apoptosis of spermatogenic cells in VC rats.