| ObjectiveThe secondary Varicocele(VC)rat model was established to study the expression level of heatshockprotein70(HSP70)in the testis,to detect the changes of spermatogenic function and oxidative stress indexes in the testis of rats,and to explore the relationship between the expression level of HSP70 and spermatogenic function in the testis of VC rats.Further,in vitro cell experiments were conducted to investigate whether HSP70 played the role of anti-oxidative stress and apoptosis through mitogen-activated protein kinase(MAPK)and Bcl-2-associated X protein(Bax)signaling pathways,and to explore the relationship between HSP70,c-Jun N-terminal kinase(JNK)and Bax in the VC rat model.Methods1.The secondary VC rat model was established by partial ligation of the left renal vein,and the sham operation group was set up.The general condition of the rats was observed,and histopathological examination was performed after HE staining to evaluate the effectiveness of the model construction.2.The rats were divided into operation group and sham operation group.VC model was established in the operation group,and the sham operation group was not treated.At the same time,VC rats were divided into alanyl-glutamine(Ala-Gln)group and control group.The Ala-Gln group was treated with Ala-Gln to induce high expression of HSP70 in VC rats,and the control group was not treated.The expression of HSP70 in the testis of VC rats was detected by quantitative PCR and Western blot.The apoptosis of spermatogenic cells was detected by Tunel method,and the spermatogenic function of VC rats was evaluated by Makler score.The activities of superoxide dismutase(SOD)and catalase(CAT)and the content of 8-hydroxydeoxyguanosine(8-OHDG)in testicular tissue were detected by ELISA.The relationship between HSP70 level and testicular spermatogenic function of VC rats was analyzed.3.Mouse spermatogonial cell line GC-1 spg cells were cultured in vitro.The HSP70 overexpression plasmid and siRNA were used to up-regulate and down-regulate the expression of HSP70,respectively.The oxidative stress response was induced by hydrogen peroxide stimulation.The activities of SOD and CAT and the content of 8-OHDG were detected.Co-ip was used to detect whether HSP70 binds directly to Bax.Gc-1 spg cells were pretreated with JNK inhibitor SP600125 and Bax inhibitor BAI1 to knockdown HSP70 and stimulated with hydrogen peroxide.The activities of SOD and CAT,8-OHdG content,phosphorylation levels of JNK and Bax,and Bax nuclear translocation were detected.Finally,the levels of HSP70,JNK and Bax in the testis tissue of the Ala-Gln rat model and the control group were detected and the correlation among them was explored.Results1.The secondary VC rat model was successfully induced by partial ligation of the left renal vein after 4 weeks.The left testicular mass of VC rats was significantly reduced compared with the contralateral one(P<0.05);The injury rate of the left seminiferous tubules was significantly higher than that of the right side(P<0.05).2.Compared with the sham operation group,the HSP70 mRNA level in the left testis of the operation group was significantly decreased(P<0.001),and the HSP70 protein level was significantly decreased(P<0.05).Compared with the control group,the Ala-Gln group had a significant increase in the mRNA level of HSP70 in the left testis(P<0.001)and the protein level of HSP70(P<0.01).These results indicate that the expression level of HSP70 in the testis of VC rats is lower than that of the sham-operated group,while Ala-Gln can induce the increase of HSP70 expression in VC rats.3.Compared with the sham operation group,the apoptosis rate of spermatogenic cells in the operation group was significantly increased(P<0.001),and the Makler score was significantly decreased(P<0.05),indicating that the spermatogenic function of VC rats was more damaged than that of the sham operation group.Compared with the control group,the VC rats in the Ala-Gln group had a significant reduction in the apoptosis rate of spermatogenic cells(P<0.01)and a significant increase in the Makler score(P<0.05),indicating that the spermatogenic function of VC rats in the Ala-Gln group was less damaged than that in the control group.4.Compared with the sham operation group,the activity of SOD and CAT in the operation group was significantly decreased(P<0.01),and the content of 8-OHDG was significantly increased(P<0.01),indicating that the oxidative stress injury of VC rats was more serious than that of the sham operation group.Compared with the control group,the Ala-Gln group had a significant increase in SOD activity(P<0.001).CAT activity was significantly increased(P<0.001).The content of 8-OHDG was significantly decreased(P<0.001),indicating that the Ala-Gln group had reduced oxidative stress damage compared with the control group.5.Under oxidative stress conditions,the apoptosis of GC-1 spg cells was significantly increased after HSP70 siRNA knockdown(P<0.001),while the apoptosis of GC-1 spg cells was significantly reduced when HSP70 was overexpressed(P<0.01).These results indicate that HSP70 has an inhibitory effect on the apoptosis of GC-1 spg cells under oxidative stress.6.After the GC-1 spg cells were treated with hydrogen peroxide,the SOD activity(P<0.001)and CAT activity(P<0.01)in the HSP70 overexpression group were increased compared with the control group,and the 8-OHdG content was decreased(P<0.01),while the SOD activity in the HSP70 knockdown group was decreased compared with the control group(P<0.01).CAT activity was decreased(P<0.001),and 8-OHdG content was increased(P<0.001).These results indicate that HSP70 can inhibit the oxidative stress response of GC-1 spg cells.7.After the GC-1 SPG cells were treated with hydrogen peroxide,the total protein and phosphorylated protein levels of ERK and p38 in the HSP70 overexpression group did not change significantly(P>0.05),while the phosphorylation level of JNK was significantly decreased(P<0.01),indicating that the activation of JNK was inhibited.The total and phosphorylated protein levels of ERK and p38 in the HSP70 knockdown group also did not change significantly(P>0.05),while the phosphorylation level of JNK was significantly increased(P<0.05),indicating that JNK activation was increased.The transfer of Bax to mitochondria was decreased in the HSP70 overexpression group(P<0.01),while the transfer of Bax to mitochondria was increased in the HSP70 knockdown group(P<0.05),indicating that HSP70 had a regulatory effect on JNK signaling pathway and Bax activity.8.The Co-IP method was used to detect the binding of HSP70 to Bax after overexpression,and the results showed that HSP70 did not bind to Bax after overexpression.Before HSP70 knockdown,GC-1 SPG cells were intervened with JNK inhibitor SP600125,and then transfected with HSP70 siRNA and treated with hydrogen peroxide again.The transfer of Bax to mitochondria was detected.Compared with the control group,the HSP70 knockdown group had no significant increase in Bax activation transfer to mitochondria,indicating that the activity of Bax is regulated by JNK.9.The spermatogenic cells were pretreated with JNK inhibitor SP600125 and Bax inhibitor BAI1,followed by HSP70 siRNA to knockdown HSP70 expression and hydrogen peroxide treatment again.Compared with the HSP70 knockdown group,the JNK inhibitor intervention group significantly inhibited cell apoptosis(P<0.001),and the Bax inhibitor intervention group significantly inhibited cell apoptosis compared with the HSP70 knockdown group(P<0.001).These results indicate that JNK/Bax signaling pathway mediates the effect of HSP70 on reducing apoptosis.The SOD activity and CAT activity of JNK inhibitor group were significantly higher than those of HSP70 siRNA group(P<0.01),while the 8-OHdG content was significantly lower than that of HSP70 siRNA group(P<0.01).However,there was no significant difference in SOD activity,CAT activity and 8-OHdG content between the Bax inhibitor group and the HSP70 siRNA treatment group(P>0.05).These results suggest that JNK signaling pathway mediates the regulation of HSP70 on the expression of SOD,CAT and 8-OHdG.10.Compared with the control group,the expression of HSP70 in the testicular tissue of VC rats in the Ala-Gln group was increased(P<0.01),and the level of phosphorylated JNK was decreased(P<0.01),The distribution of Bax in mitochondria of VC rats in Ala-Gln group was lower than that in control group(P<0.01).It was verified that high expression of HSP70 could inhibit oxidative stress and apoptosis by inhibiting JNK/Bax signaling pathway in VC rat model.Conclusion1.Secondary VC model in rats was successfully established by partial ligation of the left renal vein.2.The expression of HSP70 in the testicular tissue of VC rats was decreased,the apoptosis of spermatogenic cells was increased,the Makler score was decreased,the activities of SOD and CAT were decreased,the content of 8-OHdG was increased,and the testicular tissue damage was aggravated.In the testicular tissue of VC rats with high expression of HSP70,the apoptosis of spermatogenic cells was reduced,the Makler score was increased,the activities of SOD and CAT were increased,the level of 8-OHdG was decreased,and the testicular spermatogenic function was improved.It is suggested that HSP70 can protect the spermatogenic function of VC rats by improving the anti-oxidative stress state of testicular tissue and reducing the damage of OS.3.In GC-1 SPG cells cultured in vitro,the activation of JNK and Bax in GC-1 SPG cells was inhibited after HSP70 overexpression,and the level of cell apoptosis and oxidative stress was reduced.However,the activation of JNK and Bax increased after HSP70 knockdown,and the level of apoptosis and oxidative stress increased,and the activation of Bax was mediated by JNK.These results indicate that HSP70 in GC-1 SPG can mediate the regulation of HSP70 against oxidative stress through JNK signaling pathway,and reduce cell apoptosis by inhibiting JNK/Bax signaling pathway.Finally,it was verified that HSP70 high expression could inhibit oxidative stress and apoptosis by inhibiting JNK/Bax signaling pathway in VC rat model. |
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