Sulforaphane Reduction Of Testicular Apoptotic Cell Death In Diabetic Mice Is Associated With The Upregulation Of Nrf2Expression And Function

DIABETES MELLITUS (DM) is a complicated metabolic disorderthat impactsmultiple systems, leading to the development ofvarious complications. Infertility indiabetic men is well knownto be among these but heretofore has not beenclarifiedmechanistically in its entirety. In diabetic humans，increased sperm apoptosishas been noted. However, this has not been demonstrated in basic science studies inanimals with type2diabetes (T2DM).In fact, testicular apoptotic cell death occurs during normalspermatogenesis andappears to be accelerated in certainchronic disease conditions, such as diabetes. Abodyof evidence has indicated the induction of testicular apoptotic celldeath,predominantly via mitochondrial and endoplasmic reticulum(ER) stress-mediated celldeath pathways, in the type1diabetic (T1DM) model. Testicular cell deathhas beenstudied extensively under other pathological conditions, among which inflammation seems to play animportant role in inducing cell death inthe testis.Hyperglycemia, hyperlipidemia, and insulin resistance, as hallmarksof T2DM, aremajor risk factors for the development ofdiabetic complications. All of these factorsinduce intracellularenergy metabolic imbalance, resulting in extra generation ofreactive oxygen (ROS) and/or nitrogen species (RNS) as oxidativestress, tissuechronic hypoxia, and inflammation such as NF-κB activation in the vasculature ofvarious organs. In thetestis, abnormal vasculature changes often leads to testicularhypoxia due to the reduction of blood supply, resulting in structuralremodeling. In fact,sperm apoptotic cell deathwas found to be associated with increased oxidative stressanddamage, suggesting the pivotal role of oxidative stress and damage in promotingtesticular cell death in diabetic individuals.Oxidative stress occurs when extra generation of ROS orRNS in cells or tissuesoverwhelms endogenous antioxidantdefense. Evidence that defective signaling of the transcriptionfactor nuclear factor (erythroid-derived2)-like2(Nrf2) leadsto defectivespermatogenesis exists. It is known that Nrf2regulates basal and inducibletranscription of genes encodingprotective molecules against various oxidative stresses.In response to a range of oxidative and electrophilicstimuli, including ROS and RNS,heavy metals, and certaindisease processes, Nrf2is activated and mediates theinductionof a spectrum of cytoprotective proteins; these include phase IIenzymes suchas catalase and NAD(P)H-quinone oxidoreductase(NQO-1) and antioxidant proteinssuch as heme oxygenase-1(HO-1) through the antioxidant responseelement-dependentpathway. Nrf2is broadly expressed in tissues and hasbeenrecognized to play a critical role in the testis.Furthermore, deletion of the Nrf2genehas been shown tocause age-dependent testicular and epididymal oxidative stress，which disrupts spermatogenesis. All of these pieces ofinformation suggest a criticalrole for Nrf2in preventingoxidative disruption of spermatogenesis.Sulforaphane (SFN) is an Nrf2activator that has beenreported to preventoxidative damage as well as cardiovasculardisease. Therefore, SFN may beadvantageous inameliorating the problem of diabetic male infertility. Although SFNhas been used extensively within in vitro cell culture andanimal studies for itsexpansive and potent antioxidative propertiesvia Nrf2, there is currently no studydemonstratingwhether SFN protects from testicular oxidative damage,particularlyfrom diabetes-induced testicular toxicity.Therefore, the objectives of the present study were to determine1) whether SFNcan induce testicular Nrf2expression,2) whetherT2DM also induces testicular celldeath and what is the cell deathpathway, if any, and3) whether SFN is able to protectfromT2DM-induced testicular cell death.Diabetes-induced testicular celldeath is due predominantly to oxidative stress.Nuclear factor (erythroid-derived2)-like2(Nrf2) is an important transcription factorincontrolling the antioxidative system and is inducible by sulforaphane (SFN). To testwhether SFN prevents diabetes-induced testicular celldeath, an insulin-defective stageof type2diabetes (T2DM) wasinduced in mice. This was accomplished by feedingthem a high-fatdiet (HFD) for3mos to induce insulin resistance and then givingoneintraperitoneal injection of streptozotocin to induce hyperglycemiawhileage-matched control mice were fed a normal diet (ND). T2DM and ND-fed controlmice were then further subdivided intothose with or without4-mos SFN treatment. T2DM inducedsignificant increases in testicular cell death presumably throughreceptorand mitochondrial pathways, shown by increased ratio ofBax/Bcl2expressionand cleavage of caspase-3and caspase-8withoutsignificant change of endoplasmicreticulum stress. Diabetes alsosignificantly increased testicular oxidative damage andinflammation.All of these diabetic effects were significantly prevented bySFNtreatment with upregulated Nrf2expression. These results suggest thatT2DMinduces testicular cell death presumably throughcaspase-8activation andmitochondria-mediated cell death pathwaysand also by significantly downregulatingtesticular Nrf2expressionand function. SFN upregulates testicular Nrf2expressionand its targetantioxidant expression, which was associated with significantprotectionof the testis from T2DM-induced germ cell death.