| Objective:Fertility rates are declining around the world,and infertility caused by reduced or impaired male fertility is one of the key factors influencing this decline.Although some progress has been made in preclinical research and the clinical application of current methods to improve male fertility,limitations related to long treatment cycles,high costs,the existence of genetic risks,and public opinion about ethics have created obstacles to greater technical breakthroughs.Therefore,seeking a novel safe and effective technology to promote male fertility is still a long-term clinical goal.Low energy shock wave(LESW),a type of micro-energy mechanical force therapy,is defined as a therapeutic intervention that aims to reduce and reverse tissue damage or promote homeostasis of healthy tissues by mechanical means at the molecular,cellular or tissue level.LESWs are mainly used to positively affect cell function,improve the local microenvironment,and improve tissue repair capacity.To date,the effects of LESW therapy on cells of different tissues have been extensively and thoroughly studied by scholars and some promising results have been obtained.However,there are few studies on the effects of LESWs on testicular tissue function,with limited related studies on testicular injury induced by the treatment of peripheral testicular tissue and organ diseases have been reported.However,the direct effect of LESWs on testicular tissue has not been reported.Therefore,it is of great significance to explore the effect of LESW therapy,a new treatment technology,on testicular spermatogenic epithelium function when LESW therapy is directly applied to testicular tissue.In this study,the safety of LESWs directly applied to the testes of rats is first discussed,to determine optimal shock parameters;this is of great significance for the later exploration of the potential effects of LESWs on testicular spermatogenic epithelium and the underlying mechanism.After the initial experiment that successfully determined relatively suitable shock parameters for direct use of LESWs in rat testis,we discuss whether these shock parameters for direct use in the rat testis improve protection of testicular sperm production or exert a steady and continuous positive biological effect via regulation of the testicular microenvironment.Finally,the possible mechanism by which LESWs affect testicular function in rats,mechanotransduction,is preliminarily discussed.Methods:1.The scrotal area of sexually mature male Sprague-Dawley rats was directly exposed to LESWs at three different energy densities(i.e.,0.02m J/mm~2,0.04m J/mm~2and 0.06m J/mm~2)and three different numbers of pulse(i.e.,500,1000 and 1500 pulses).According to mutual composition intervention of LESW,shock cycles lasting 2 weeks and 8 weeks were received,and the shock frequency was set to one shock every two days.After each shock,the rats were weighed,blood samples were taken to measure the concentrations of reproductive endocrine hormones,and scrotal ultrasound was used to observe the morphology and hemodynamics of the testis.Then,the caudal epididymis and testicular tissues were collected to analyze semen quality,the oxidative stress level of the testicular tissue was measured,and histopathological biopsy and transmission electron microscopy(TEM)were used to observe ultrastructural changes of the testicular tissue in order to determine suitable shock parameters for LESWs applied to rat testicular tissue.2.After LESW shock parameters(i.e.,500 pulses at0.02m J/mm~2and 500 pulses at 0.04m J/mm~2)that were relatively suitable for direct use in rat testis were successfully determined in the previous experiment,two groups were exposed to these LESW shock parameters;then TUNEL staining was performed to detect the expression levels of proteins related to the mitochondrial apoptosis pathway(Bax,Cytochrome C,Caspase-3,Bcl-2)and proteins involved in cell proliferation and repair(VEGF and e NOs)were further determined.The effects of the LESW pulses parameters in the two groups were compared to determine whether they promoted anti-apoptotic processes and cell proliferation of testicular spermatogenesis.The morphology of testicular spermatogenic tubules and testicular interstitium was quantitatively analyzed by stereology.In this manner this study was able to identify LESW shock parameters that could be directly and safely applied to rat testicular tissue and effectively regulate testicular spermatogenic function.3.On the basis of results from the above experiments,we continued to explore LESWs as a type of micro-energy mechanical force therapy,and how its main mechanism of action,namely,mechanotransduction,affects testicular function in rats.First,according to the results and standards determined in the previous experiments,we established a testicular tissue injury model using shock parameters that were harmful to rat testis,and then used the safe and effective shock parameters verified in the experiment(i.e.,500 pulses at 0.02m J/mm~2)as the treatment parameters for intervention.The protein expression levels ofβ1-Integrin and Laminin,the changes in phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway and mitogen-activated signal-regulated kinases(MEK/ERK1/2)signaling pathway-related proteins,Cyclin D1 and mitochondrial apoptosis pathway-related protein(Bax,Cytochrome C,Caspase-3,and Bcl-2)expression were measured to preliminarily explore the possible mechanism by which LESWs exert their biological effects on testicular function in rats through mechanotransduction.Results:1.The adverse effects of LESWs delivered at 500 pulses at0.02m J/mm~2and 500 pulses at 0.04m J/mm~2on testicular morphology,oxidative stress function,sperm quality and the general physiological state of rats were relatively small.Specifically,there was no significant difference in testicular morphology,structure or function between the experimental group receiving 500 pulses at 0.02m J/mm~2regardless of short-term(2 weeks)or long-term(8 weeks)treatment,and no adverse reaction occurred in the general physiological state of the whole body.However,LESW shock with energy densities and pulse numbers higher than the two parameters mentioned above damaged the tissue structure and morphology of rat testes to varying degrees or induced harmful biochemical reactions,leading to reduced or even obstructed sperm function.The longer the cycle of continuous shocks,the worse the impact.2.LESWs delivered at 500 pulses at 0.02m J/mm~2had no adverse effects on testicular spermatogenesis in rats,and could balance and maintain cell apoptosis and cell proliferation effectively,which was helpful to promote constructive regulatory effects of histocytes.3.When LESW therapy delivered at 500 pulses at 0.02m J/mm~2was used on testes damaged by LESWs delivered at 1500 pulses at 0.04m J/mm~2,the therapeutic dose of LESWs mainly mediated adhesion between cells and the extracellular matrix by enhancing the binding ofβ1-integrin to its ligand laminin.In addition,PI3K/AKT and MEK/ERK1/2 signals were transmitted into the cell,which promoted the upregulation of the expression of downstream factor Cyclin D1,effectively promoting cell cycle progression and promoting cell proliferation.In addition,the expression of anti-apoptotic protein Bcl-2 was upregulated,and the expression of pro-apoptotic proteins Bax,Caspase-3 and Cytochrome C was downregulated,effectively regulating initiation of the mitochondrial apoptosis program.Therefore,this treatment could effectively regulate the internal balance between apoptosis and proliferation of testicular tissue cells and ultimately play an important role in improving the ability of testicular tissue to repair itself.When used in testes damaged by LESWs delivered at 1500 pulses at 0.04m J/mm~2,similar to LESWs delivered at 1500 pulses at 0.04m J/mm~2and 1500 pulses at 0.06m J/mm~2without therapeutic intervention,levels of phosphorylated PI3K,AKT,MEK,and ERK1/2and protein expression of Cyclin D1 were downregulated,and expression levels of pro-apoptotic proteins and anti-apoptotic proteins were significantly upregulated.Conclusions:1.LESWs affect testicular tissue in rats in a dose-dependent manner.2.LESWs delivered at shock parameters of 500pulses at 0.02m J/mm~2is safer for testicular shock in rats,has better protective effects on testicular spermatogenic function,and has effective balancing and sustaining biological effects on testicular spermatogenic function.3.LESW therapy delivered at shock parameters of 500 pulses at0.02m J/mm~2activates the PI3K/AKT and MEK/ERK1/2 signaling pathways by upregulating the protein expression ofβ1-integrin and laminin.Furthermore,this treatment inhibits the initiation of mitochondrial apoptosis and promotes the expression of Cyclin D1,thus effectively regulating the internal balance between apoptosis and proliferation of testicular cells,and ultimately plays an important role in improving the reparative ability of testicular tissue. |
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