Diabetes mellitus (DM) has been reported to affect the male gonad negatively, with a decrease in fertility rate [1
]. Mounting evidence from both clinical and experimental studies have demonstrated decreased fertility rates, and have linked the same with testicular oxidative stress, inflammation and apoptosis [3
]. In fact, in our previous studies, we found that not only did DM increase testicular oxidative stress [6
], but it also increased oxidative stress in the cauda epididymis [2
], thus exposing the stored spermatozoa to further risk of oxidative stress. Particularly, down-regulation of nuclear factor erythroid 2-related factor 2/antioxidant response elements (Nrf2/ARE) and up-regulation of nuclear factor kappa B (NF-κB)-mediated inflammation have been reported in the testes of diabetic rats [3
Oxidative stress and inflammation are common features of DM, and are reportedly responsible for the multi-organ complications associated with the disease. Both conditions reportedly share the same activation stimulus, reactive oxygen species (ROS), which are produced in large concentrations as a result of the increase in glucose auto-oxidation in DM [9
]. Furthermore, DM-mediated oxidative stress and inflammation can trigger apoptotic signals, leading to cellular/tissue damage and resultant organ failure. Therefore, the use of antioxidants as complementary medicines to DM treatment becomes pertinent, in other to curb its ROS-associated complications.
Propolis is a gluey mixture of resinous substances which are deployed by honey bees to repair broken hives. Propolis contains sap, exudates from tree and leaf buds, and wax [10
]. Generally, propolis is reported to contain 50% resin, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% other organic substances [11
]. To date, over 300 chemical components have been identified in propolis, and studies have concluded that the chemical composition of propolis may differ depending on its geographical region and bee species [10
Propolis from many regions have been chemically characterised. The main components common to propolis from Turkey, Greece, the United States of America, Malaysia, Thailand, Australia, China, Brazil, Bulgaria, Algeria, Egypt, Cameroon and Congo, are flavonoids, aromatic acids, phenolic compounds, and esters of caffeic and ferulic acids [14
]. Some biological activities have been ascribed to propolis, and they include: anti-hyperglycaemic, antioxidant, anti-inflammatory, anti-apoptotic and anti-microbial effects [13
]. Others include renoprotective [21
], cardioprotective [23
] and hepatoprotective [25
Clinical trials of propolis on diabetic patients have yielded conflicting results [27
]. However, most studies have demonstrated significant decreases in blood glucose, oxidative stress and inflammatory biomarkers [27
], which are consistent with animal studies [20
]. There is a need, therefore, to study the complementary effects of propolis with established anti-hyperglycaemic agents as a step towards its use as a complementary therapy in the event that existing medications fail to achieve normoglycaemia, which however, is often the case.
We previously demonstrated significant α-glucosidase inhibition and antioxidant activities of Malaysian propolis (MP) in vitro [20
]. We also demonstrated a significant decrease in fasting blood glucose and an increase in insulin levels in rats treated with MP + metformin (Met). Worthy of noting is that the combined therapy presented better results relative to the monotherapy-treated (MP or Met) rats. Herein, we attempt to probe MP’s effect on DM-induced testicular oxidative stress, inflammation and apoptosis, in a bid to find answers regarding its ability to protect the male reproductive system from oxidative damage, inflammation and apoptosis in a diabetic state. Further, its possible complementary effects with the anti-hyperglycaemic medication, Met, was assessed with a view to recommend its use should Met alone fail to contain the negative effects of DM on the male reproductive system.
Several biological activities have been ascribed to propolis. In the present study, we explored its potential to abrogate oxidative stress, inflammation and apoptosis in the gonads of STZ-induced diabetic male rats. Further, we examined the possibility of MP serving as a complementary therapy with Met in the event that the monotherapies do not sufficiently counteract the negative effects of the disease. Consistent with our previous reports, MP demonstrated a significant anti-hyperglycaemic effect, which we previously attributed to pancreatic β-cell regeneration, increased insulin secretion, decreased hepatic gluconeogenesis and inhibition of α-glucosidase activity [20
]. Worthy of noting is that the combined therapy (MP+Met) showed the best anti-hyperglycaemic effects, as demonstrated by a 4.87-fold decrease in final blood glucose level relative to DC group, while MP and Met only decreased the final blood glucose level by 2.32 and 2.98 fold, respectively. Further, the absolute and relative weights of the testes, epididymis, prostate and seminal vesicle decreased in the DC group in the present study, consistent with previous investigations [42
]. Tissue wasting, organ scaring and loss of organ function are common in type 1 DM [45
]. This may be attributable to a chronic lack of insulin, which triggers proteolysis in several tissues and organs, thus depleting structural proteins [46
]. However, significant improvements were observed in the treatment groups. MP increased reproductive organs’ absolute and relative weights, while the combined treatment group showed the best improvements, with values comparable to NC group.
Seminiferous tubular atrophy and the depletion of germ cells were described as morphological indicators of spermatogenesis failure [48
]. Earlier studies reported increased seminiferous tubule thickness, germ cell depletion and Sertoli cells’ vacuolization in diabetic rats, and in diabetic human testicular biopsies [49
]. The histopathological findings in the untreated diabetic rats’ testes in the present study suggests a decline in spermatogenesis, and were consistent with the epididymal histopathology, which revealed large areas that were either devoid of spermatozoa or had decreased spermatozoan density. Furthermore, the epithelial height of the tubules in the epididymis increased in DC group, consistent with previous reports [51
]. MP attenuated those negative changes in the testes and epididymis, comparably to metformin. Although there are no reports on the effects of propolis on testicular histology in a diabetic state, studies using Indian, Turkish and Egyptian propolis have demonstrated significant increases in testicular weight and seminiferous tubular diameter in rats after exposure to a heavy metal (cadmium) or chemotherapy (methotrexate, doxorubicin and mitomycin C) [53
]. In those studies, counteracting oxidative stress was suggested as the possible mechanism of action of propolis when improving testes’ histopathological findings, as also observed in the present study.
Oxidative stress is generally considered as a major pathway for the development of diabetic complications [57
]. It occurs when there is an imbalance between pro and anti-oxidants in favour of pro-oxidants. In DM, an increase in mitochondrial glucose oxidation, which results from hyperglycaemia, releases a large amount of ROS into the cytoplasm of cells, leading to an imbalance between pro and antioxidants in favour of pro-oxidants [9
]. It has been reported that testicular germ cells are more susceptible to oxidative damage than somatic cells, because their plasma membrane contains more polyunsaturated fatty acids which are prone to oxidation by free radicals [59
]. Therefore, excessive generation of ROS at a rate that outweighs the antioxidant defence system, as observed in DM, and the resulting oxidative stress, triggers germ cell death [6
], thus impacting negatively on spermatogenesis and fertility potential [2
]. Beyond the testes, oxidative stress may impact negatively on mature spermatozoa in the epididymis during storage. It is on that premise that markers of oxidative stress and antioxidant enzymes were assessed in both testes and epididymis in the present study.
The down-regulation of Nrf2 and some members of the antioxidant response element group (SOD, CAT and GPx) were seen in the testes of DC group, which are consistent with previous investigations [60
]. Previous studies reported down-regulation of the other members of the antioxidant response elements (haem oxygenase-1 (HO-1) and NAD(P)H dehydrogenase(quinone)1 (NQO1)) controlled by Nrf2, in the testes of diabetic rats [60
HO-1 plays a role in haem degradation, yielding bilirubin (which has antioxidant property) in the process, while NQO1 plays a role in reduction of ubiquinone and vitamin E derivatives to their antioxidant forms [64
]. Nrf2 plays a critical role in the control of the expression and function of oxidative stress response genes, which is the reason it is referred to as the master regulator of redox status [65
]. In fact, a previous study showed testicular oxidative stress and poor spermatogenesis in Nrf2 knockout mice, thus emphasising the crucial role of Nrf2 in maintaining redox status and spermatogenesis [66
]. In the present study, by down-regulating Nrf2, the activities of antioxidant enzymes, particularly SOD, CAT, GPx, GST and GR, and the GSH level, decreased in the DC group, which is consistent with previous studies [44
]. Similarly, epididymal antioxidant enzymes’ activities and GSH’s level decreased in DC group. From these results, we hypothesise that surviving spermatozoa from the testes may be prone to oxidative damage during storage in the epididymis.
The master regulator of redox status, Nrf2, is reported to be controlled by Kelch-like ECH-associated protein 1 (Keap1). Under normal conditions, Nrf2 is sequestered by Keap1 which results in its rapid degradation [68
]. Inhibition of Keap1, therefore, prevents Nrf2 degradation, allowing the latter to be translocated into the nucleus, where it binds to the antioxidant response elements’ gene sites and up-regulates targeted gene expression (HO-1, NQO1, CAT and SOD) [68
]. Though not assessed in the present study, previous studies have reported significant increases in Keap1 and cytosolic Nrf2 protein levels, and a decrease in the nuclear Nrf2 protein level in the testes of diabetic rats, clearly showing the inhibition of Nrf2 translocation to the nucleus by Keap1 [63
]. Interestingly, polyphenols have been reported to up-regulate Nrf2 levels as part of their mechanisms of combating oxidative stress [63
]. MP whole extract that was used in the present study was previously reported to contain high flavonoid and phenol contents [20
]. Hence, the up-regulation of Nrf2 in the present study may be attributed to MP’s polyphenolic components.
Antioxidant enzymes play a major role in keeping the body free from oxidants. SOD is reported to catalyse the dismutation of O2−
, which is then converted to H2
O by CAT and/or GPx, without which the accumulated H2
will destroy lipid membranes and release large amounts of malondialdehyde (MDA) [71
]. The action of GPx is mediated through reduced glutathione, and forms glutathione disulfide in the process. The latter is reduced to the sulfhydryl form GSH by GR [71
]. The present study, therefore, shows that the SOD-CAT-GSH antioxidant defence system was significantly impaired in the testes and epididymis in the DC group, which may have resulted in the increased H2
level as previously reported [72
Consistent with previous investigations [43
], intra-testicular and epididymal TAC decreased significantly in the DC group of the present study. TAC is a measure of the synergistic interactions of the endogenous enzymatic and non-enzymatic antioxidant systems [73
]. Decreased TAC, as seen in the DC group, is indicative of a significant decrease in the activity of the antioxidant defence system. This may have been orchestrated by increased intra-cellular ROS production and increased lipid peroxidation, as a previous study has reported increased intra-testicular total oxidative status in diabetic state [62
]. Treatment with MP significantly increased antioxidant enzymes’ activities in both the testes and epididymis in the present study. These effects were comparable or better when compared with Met, with the best outcome following the combined treatment. Improvement in intra-testicular and epididymal antioxidant status may have played a significant role in improving testicular and epididymal cytoarchitecture, as observed in the treated diabetic groups, since oxidative stress inflicts structural damage. This assertion is supported by the fact that intra-testicular and epididymal TBARS, which is a product of lipid peroxidation, was significantly decreased following treatment with the various regimens.
Interestingly, MP demonstrated strong anti-oxidant activities in vitro (DPPH, FRAP and H2
scavenging activities), in addition to having high phenolic and flavonoid contents [20
]. By scavenging H2
, MP may have decreased its peroxidation effects on lipid membranes, thus decreasing TBARS’ level. The observed significant antioxidant activities may be attributable to the presence of gallic acid derivatives, coumaric acid derivatives, caffeic acid derivatives, ellagic acid and resveratrol in MP, which were previously identified by our group [31
]. These findings are similar to those from previous studies, where propolis from Egypt, Turkey and India, were reported to decrease testicular MDA level and increase TAC after chemotherapy [53
] or exposure to cadmium [54
]. Furthermore, studies using MP obtained from H. itama
demonstrated significant decreases in placental MDA and protein carbonyl levels, and an increase in TAC in diabetic dams [74
Considering the above-discussed effects, it is plausible to hypothesise that MP may have improved testicular antioxidant status through three possible mechanisms: (i) a direct mechanism involving a synergy between MP and endogenous antioxidants, leading to scavenging of more ROS than only the endogenous antioxidants would do, thus sparing endogenous antioxidants and increasing their overall levels; (ii) up-regulation of Nrf2 and its downstream antioxidant response elements genes, thus up-regulating the mRNA levels and the activities of antioxidant enzymes; and (iii) an indirect mechanism involving a decrease in hyperglycaemia, thus decreasing the overall production of ROS, since the primary source of increased ROS production in DM is glucose auto-oxidation associated with hyperglycaemia [9
]. The first two propositions are drawn from the fact that studies have demonstrated that the administration of antioxidants (quercetin, curcumin and resveratrol) significantly increases intra-testicular antioxidant activity, decreases lipid peroxidation and improves spermatogenesis without substantially decreasing blood glucose levels [57
]. In fact, the fold changes in FBG level following treatment with quercetin and curcumin when compared to untreated diabetic rats were reported to be 1.09 and 1.13, respectively [57
]. Since MP whole extract used in the present study demonstrated anti-hyperglycaemic effects, it is conceivable that the synergy of its phytoconstituents may have caused the significant decrease in FBG level, and improvement in antioxidant status, thus validating the third hypothesis above, which is centred on MP’s anti-hyperglycaemic effect.
The occurrence of oxidative damage in tissues is an indication of a likely occurrence of inflammation, since the duo are closely related, and to some extent, share common activation stimuli (ROS). The NF-κB-mediated inflammatory pathway is reportedly one of the targeted pathways of ROS [76
]. Studies have reported that NO, which is released by vascular endothelial smooth muscle cells, plays a substantial role in the occurrence of both oxidative stress and inflammation [71
]. In DM, NO is reported to be produced in high concentrations as a result of the up-regulation of iNOS [77
]. The high circulating NO then interacts with other nitrogen and/or oxygen species, triggering nitrosative and/or oxidative stress [71
]. NF-κB, a transcription factor, serves as a critical link between oxidative stress, inflammation and apoptosis. Upon activation by oxidative stress, NF-κB up-regulates iNOS’s level, leading to an increase in NO production. On the other hand, a high NO level can also trigger NF-κB up-regulation, thus initiating an inflammatory signalling cascade that, in turn, triggers the release of numerous inflammatory cytokines [71
Consistent with our present study, previous studies have reported testicular inflammation in diabetic rats. Specifically, the activation of NF-κB, with increases in TNF-α, iNOS and IL-6 levels, have been reported in diabetic rats’ testes [6
]. In the present study, the intra-testicular NO level increased notably in the DC group, in addition to increases in the mRNA and protein levels of NF-κB, iNOS, TNF-α and IL-1β, and a decrease in IL-10. Significant improvements were seen in the treated diabetic groups. Particularly, D+MP+Met group was comparable to NC group, while MP treatment yielded better results when compared to Met. The combined therapy, thus, appears to offer better protection from DM-induced inflammation. This effect may not be unconnected to the near-normal blood glucose level recorded in this group, since inflammation in the gonad is orchestrated by persistent hyperglycaemia and oxidative stress. The anti-inflammatory effect of MP in the testes is consistent with previous report from our group, where MP significantly decreased NF-κB (p65), TNF-α and IL-1β protein expressions, and increased IL-10 protein expression in the pancreases and livers of diabetic rats [20
Studies have reported increased testicular germ cell apoptosis in DM, which occurs as a result of the interaction between oxidative stress and inflammation. In the present study, the up-regulation of pro-apoptotic p53, Bax/Bcl-2 ratio, caspase-8 and caspase-9, are indicative of the contributions from both intrinsic and extrinsic apoptotic signalling, both of which lead to the activation of caspase-3, as previously reported [8
]. Following a build-up of oxidants, p53 up-regulates pro-apoptotic Bax, releasing cytochrome-c which drives the intrinsic apoptotic pathway, and subsequent activation of the executioner, caspase-3 [80
]. Although a TUNEL assay was not carried out on the testes in the present study, up-regulation of both mRNA and protein levels of caspase-3 in this study may suggest increased testicular germ cell apoptosis. Further, there were less PCNA-positive germ cells in the DC group, which implies poor cell proliferation, and supports the idea that most of the germ cells could have undergone apoptosis. Previous studies have reported an increase in TUNEL-positive germ cells in the testes of diabetic rats [57
], thus corroborating our hypothesised increase in apoptosis in diabetic rats’ testes in the present study. Interestingly, MP treatment decreased apoptosis in the testes, as seen with the significant decreases in the mRNA and protein levels of caspase-3. The result of the present study is consistent with previous studies using Egyptian and Turkish propolis, which reported decreased testicular germ cell apoptosis after exposure to chemotherapy [54
]. The inhibitory effects of MP on apoptosis in the present study may be associated with decreases in oxidative stress and inflammation.
Overall, MP demonstrated comparable effects with Met, and better effects in some regard. Whether or not MP could substitute Met or any other anti-hyperglycaemic medication, cannot be inferred in the present study. However, treatment with MP+Met offered a better protection against DM-induced oxidative stress, inflammation and apoptosis in the gonad, relative to MP and Met monotherapies. This maybe attributable to the fact that Met decreases hepatic gluconeogenesis, improves the sensitivity of cells to circulating insulin and improves glucose uptake [82
], while MP acts on the pancreas to increase insulin level, and decreases hepatic gluconeogenesis, in addition to having inherent antioxidant potential [20
]. The combination proved more beneficial, as seen in the present study, and therefore, is a promising therapy that requires further mechanistic investigations.