| Objective:In this study,we aimed to establish a rabbit model of complete acute testicular torsion/reduction and investigate the feasibility of applying ultrasound technology to predict the long-term spermatogenic function of the testis after the reduction of testicular torsion.The possible mechanisms of the protection of spermatogenic function during testicular torsion and reduction were explored based on network pharmacology and molecular docking.Methods:1.Sixty healthy adult male New Zealand rabbits were randomly divided into four groups:sham-operated group,2h-reversal group,6h-reversal group and 12h-reversal group.A rabbit model of complete acute testicular torsion/reduction was constructed,and conventional ultrasound(grayscale and Doppler ultrasound),shear wave elastography(SWE)and contrast-enhanced ultrasound(CEUS)were performed at different time points before,after the torsion and after the torsion/reduction of the testis.The pathology of hematoxylin-eosin(HE)staining was performed at 14 d after the reduction,and the pathological results were used as the gold standard to validated ultrasound indices for predicting long-term spermatogenic function after reduction.2.Forty-five healthy adult male New Zealand rabbits were randomly divided into 3groups: sham-operated group,6h-reversal group and torsion reduction dosing group.A rabbit model of complete acute testicular torsion/reduction(6h of torsion)was constructed,in which the torsion reduction dosing group was given Shengmai yin by gavage after the reduction.Routine ultrasound,SWE and CEUS examinations were performed before,2h,6h and 14 d after the reduction.The testicular spermatogenic cell apoptosis assay was performed in each group at 14 d after the reduction to observe the protective effect of Shengmai yin on spermatogenic function after the reduction of testicular torsion.3.The TCMSP database was used to screen the active ingredients and related targets in Shengmai yin.The Gene Cards and OMIM databases were used to retrieve the targets related to testicular torsion.The Cytoscape 3.8.2 and String databases were used to construct the "ingredient-target" network and protein interaction network,and the DAVID database was used to perform gene function annotation and pathway enrichment analysis.Suitable targets were selected for molecular docking studies.Theoretical derivation of a possible mechanism for the protection of spermatogenic function by Shengmai yin during testicular torsion/reduction.Results:1.Immediately after testicular torsion/reduction,each group had different ultrasonographic changes: In the 6h-reversal group,SWE showed a green or yellowgreen color(hardening)in the peripheral region of the testis on the operated side;CEUS showed that the peak of the time-intensity curve of the testis on the operated side was reduced and shifted backward,showing a "slow-in,slow-out" phenomenon.In the 12h-reversal group,the SWE images of the testicular parenchyma on the operated side were mixed in color(red and yellow-green predominant,hard);CEUS showed a significant decrease in the peak of the time-intensity curve of the operated testis.Pathological HE staining showed Johnsen scores of(9.93 ± 0.26),(8.20 ± 1.15),(6.07 ± 1.98),and(1.80 ± 1.08)in the sham-operated group,2h-reversal group,6hreversal group and 12h-reversal group,respectively,with statistically significant differences between groups(all P < 0.05).The above ultrasonographic findings,combined with the pathological findings,indicated that the hardening of the peripheral region of the testis and the "slow-in,slow-out" phenomenon of blood perfusion after testicular torsional repositioning may suggest that the distant spermatogenic function of the testis is impaired after repositioning;while the mixed color of the testicular parenchymal SWE images and the significant decrease of blood perfusion may suggest that the distant spermatogenic function of the testis is significantly impaired after repositioning.The apparently impaired spermatogenic function of the testis after repositioning may suggest that the affected testis should be considered for preservation.2.There was a moderate negative correlation between shear wave velocity(SWV)and spermatogenic function in the central region of the testis on the side of immediate resetting(r =-0.483)and a very strong negative correlation between SWV values in the peripheral region and postoperative spermatogenic function in the testis(r =-0.881).The AUCs of SWV values in the central and peripheral regions on the surgical side of testis of immediately after testicular torsion/reduction to predict whether the distal spermatogenic function of the testis is impaired after resuscitation were 0.677 and 0.952,respectively,with sensitivities of 0.536 and 0.929 and specificities of 1.000 and 0.969,respectively.3.The germ cell apoptosis index was(47.00 ± 10.38)% and(25.59 ± 6.54)% in the 6hreversal and torsion reduction dosing group,respectively,with statistically significant differences(P < 0.05).The ultrasound results showed that the 6h-reversal group and the torsion reduction dosing group had different sonographic manifestations at 6 h and14 d after resetting: at 6 h after resetting,some rabbits in the 6h-reversal group had slightly more syringomyelia than before,and the SWE of the testis on the operated side showed a "hard ring sign",and the CEUS showed a "slow-in,slow-out" phenomenon on the operated testis.In the torsion reduction dosing group,the sphincter fluid decreased or subsided,and the color of the peripheral area of the testis on the operated side changed(softer),and CEUS showed that the testis on the operated side was "advancing and receding".At 14 d of resetting,the SWE maps of testes both in the 6h-reversal and torsion reduction dosing group were predominantly green,and the quantitative analysis showed that the SWV values in the peripheral area of the torsion reduction dosing group were significantly lower than those of the 6hreversal group,with statistically significant differences(2.51 ± 0.37 vs.2.90 ± 0.43,P< 0.05).Thus,ultrasound manifestation and germ cell apoptosis assay indicated the protective effect of Shengmai yin on testicular function in testicular torsion/reduction.4.The network pharmacological analysis and molecular docking results showed that the key active ingredients and core targets of Shengmai yin were able to combine and act well.Further gene function annotation and pathway enrichment analysis showed that the protective effects of Shengmai yin on testicular function involved multiple targets and pathways,including increasing energy metabolism,reducing oxidative stress and inflammatory response,and inhibiting apoptosis.Conclusion:Different patterns of stiffness changes in the central and peripheral regions after torsion/reduction of the testis in rabbits with different degrees of ischemia.The hardness of the peripheral region can better reflect the degree of damage to the distant spermatogenic function of the testis,and is expected to be an objective indicator for clinical prediction of testicular spermatogenic function after testicular torsion/reduction.Various ultrasound techniques and apoptosis assays can confirm the protective effect of Shengmai yin on testicular spermatogenic function in testicular torsion/reduction,and its mechanism of action mainly involves increasing energy metabolism,reducing oxidative stress and inflammatory response,and inhibiting apoptosis,etc.Shengmai yin is expected to be a therapeutic candidate for protecting testicular function after testicular torsion/reduction. |
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