The Possible Ameliorating effects of Rooibos, Honeybush and Sutherlandia on diabetes-induced reproductive impairment in adult male Wistar rats

Omolaoye, Temidayo Siyanbola (2020-03)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: The global prevalence of infertility is on the rise, and so is male factor infertility. Out of the approximately 72.4 million infertile couples worldwide, male factor is responsible for 50%. It has been reported that sperm quality is on the decline, even in healthy men. Several aetiologies have been identified, and this includes disease-related male infertility, such as diabetes mellitus (DM). DM is a chronic non-communicable disease, marked by increased hyperglycaemia which occurs as a result of lack in insulin secretion or due to the insensitivity of the target tissue to the metabolic effect of insulin. It has been reported that 422 million people are affected with DM globally, of which ≤10% is type 1 diabetic and ≤90% is type 2. Studies have shown that although most autoimmune diseases usually affect a higher percentage of women, type 1 diabetes is more prevalent in boys and men. The consequential detrimental effects of DM on male fertility have been reported in both experimental animals and diabetic men. This includes, decreased sperm motility, reduced normal sperm morphology, increased fragmented spermatozoa and many more defects. In the course of unravelling the pathways through which DM affect male fertility, studies have elucidated the role of endocrine dysfunction, formation of reactive oxygen species (ROS) and the subsequent development of oxidative stress (OS). However, other possible related pathways such as the involvement of glycated proteins, are yet to be explored. Since there are evidences that DM is detrimental to male fertility, it is important to firstly understand the underlying pathophysiology and then develop and explore strategies to combat this disease and its associated male reproductive complications. Many natural and artificial agents have been proposed to have antioxidant properties due to their potential in reducing oxidation. Antioxidants work either by scavenging ROS or by preventing its formation. Rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) and sutherlandia (Lessertia frutescens) are plants endemic to Southern Africa. Herbal teas are derived from the leaves and stems of these plants. These infusions have been shown to be caffeine-free. Studies have also reported that the infusions from these plants contain bioactive chemical compounds with therapeutic benefits. Additionally, studies have shown their beneficial role in preventing the development of diseases and some have shown their ameliorative effects. However, their role in male reproduction is little understood as this represents the first study to report the effects of honeybush and sutherlandia on male reproductive function. Based on the expressed gaps in knowledge, the study was divided into two main parts, with each having several objectives. For the first part, thirty adult male rats were randomly divided into three groups of ten, which included a vehicle and two streptozotocin (STZ) groups receiving either 30mg/kg (STZ30) or 60mg/kg (STZ60) intraperitoneally. For the second part, ninety animals were divided into nine groups of ten without bias. The groups included a control (water only), vehicle (STZ control, citrate buffer + water), rooibos (2% fermented rooibos), honeybush (4% fermented honeybush), sutherlandia (0.2% unfermented sutherlandia), diabetic control (STZ45mg/kg + water), diabetic + rooibos (STZ45mg/kg + 2% rooibos), diabetic + honeybush (STZ45mg/kg + 4% honeybush), and diabetic + sutherlandia (STZ45mg/kg + 0.2% sutherlandia) group. Animals in the diabetic infusion treated groups were pre-treated with the respective infusion one week before DM induction. Animals were monitored closely throughout the treatment period and were sacrificed seven weeks after DM induction. Blood, testes and epididymides were collected for further analysis.The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal morphology. Paradoxically, they presented with increased sperm concentrations. Hence, rooibos, honeybush and sutherlandia may enhance sperm concentration, which represents the sperm quantity. However, they may impair sperm quality (sperm motility and morphology), when consumed by healthy animals. Subsection two of the second part of the study investigated the testicular oxidative stress and apoptosis status in diabetic rats following treatment with rooibos, honeybush and sutherlandia infusions. This section highlighted the negative impact of DM on sperm functional parameters through increased lipid peroxidation and reduced antioxidant activity. The infusion treatment groups displayed increased antioxidant enzyme activity, which may be partly responsible for the observed improvement in sperm motility and morphology of diabetic animals receiving rooibos (DRF) and diabetic animals receiving sutherlandia (DSL). Additionally, the current study have shown the increased expression of apoptotic biomarkers in the diabetic control (DC) animals, which were not alleviated by the infusions. This suggests that these infusions play a role in alleviating diabetes-induced sperm function impairment through suppression of OS, but their role in apoptosis is still unclear. Subsection three of the second part of the study investigated the role of rooibos, honeybush and sutherlandia on insulin signalling in the testes of diabetic rats. All diabetic groups presented with a significant increase in blood glucose levels after diabetes induction. However, the diabetic animals treated with the infusions showed only a mild decrease in fasting blood glucose. The diabetic control animals showed a decrease in testis protein expression of IRS-1, PkB/Akt and GLUT4. Diabetic animals treated with rooibos (DRF) and honeybush (DHB) displayed an upregulation in IRS-1, while diabetic animals treated with sutherlandia (DSL) showed improvement indecrease in PkB/Akt, DHB and DSL animals displayed upregulation compared to control. All diabetic animals showed increased phosphorylated ERK1/2 and reduced total ERK1/2 when compared to control and vehicle. Additionally, diabetic control animals presented with a non significant decrease in plasma testosterone concentration compared to control, while DRF and DSL showed a significant decrease and DHB showed upregulation. This suggests that (i) the activation of IRS, PkB/Akt, ERK and GLUT translocation is important in testicular insulin signalling (ii) rooibos, honeybush and sutherlandia may play a role in testicular insulin signalling, however, through different pathways (iii) honeybush may mildly enhance testosterone production in diabetes. Lastly, the fourth subsection of the second part of the study investigated the descriptive histological evaluation of the testis and cauda epididymis after treating with rooibos, honeybush and sutherlandia in both healthy and diabetic rats. The infusion control groups (RF, HB and SL) showed normal seminiferous tubule cellular association, presence of spermatozoa in the epididymal lumen and normal overall architecture. Both the testicular and epididymal morphology were altered in DM, but these disruptions were mildly alleviated by rooibos, honeybush and sutherlandia. It appears, from appreciating the results of the entire study, that diabetes does have detrimental effects on male reproduction, both at the tissue and cellular levels. The cellular impact may be exerted through protein glycation, development of OS and apoptosis induction. The use of rooibos, honeybush and sutherlandia, when healthy, should be taken moderately and with caution, as too much may be detrimental and may impair sperm functional parameters. However, in disease conditions such as diabetes, these infusions may be beneficial, as: (i) Rooibos and sutherlandia may mildly improve sperm quality (motility and morphology), (ii) Rooibos, honeybush and sutherlandia may mildly reduce hyperglycaemia by enhancing insulin signalling, (iii) Honeybush may enhance testosterone production, (iv) Rooibos, honeybush and sutherlandia may mildly improve the histomorphological architecture of the seminiferous tubule and cauda epididymal tubule.

AFRIKAANSE OPSOMMING: Die wêreldwye voorkoms van onvrugbaarheid, en in spesifiek manlike-faktor onvrugbaarheid, is besig om toe te neem. Die manlike faktor is verantwoordelik vir 50% van alle gevalle van onvrugbaarheid in die ongeveer 72,4 miljoen onvrugbare paartjies wêreldwyd. Gepaardgaande hiermee word daar berig dat spermkwaliteit aan die afneem is, selfs in gesonde mans. Verskeie etiologieë is geïdentifiseer, en dit sluit in siekteverwante manlike onvrugbaarheid soos diabetes mellitus (DM). DM is ʼn chroniese nie-oordraagbare siekte, gekenmerk deur hiperglukemie, wat voorkom as gevolg van ʼn gebrek aan insulienafskeiding of weens die ongevoeligheid van die teikenweefsel vir die metaboliese effek van insulien. Daar is berig dat 422 miljoen mense wêreldwyd deur DM geaffekteer word, waarvan ≤10% tipe 1-diabete en ≤90% tipe 2-diabete is. Studies het getoon dat hoewel die meeste outo-immuun siektes gewoonlik ʼn hoër persentasie vroue aantas, tipe 1-diabetes meer algemeen voorkom in seuns en mans. Die gevolglike nadelige gevolge van DM op manlike vrugbaarheid is in sowel eksperimentele diere as in diabetiese mans aangemeld. Dit sluit in verlaagde spermmotiliteit, verminderde normale spermorfologie, verhoging in spermatosoa met DNS-fragmentasie en nog vele meer defekte. In die proses om die roetes waardeur DM ʼn invloed op manlike vrugbaarheid het te ontrafel, het studies die rol van endokriene disfunksie, die vorming van reaktiewe suurstofspesies (RSS) en die daaropvolgende ontwikkeling van oksidatiewe spanning (OS) toegelig. Ander moontlike verwante roetes, soos die betrokkenheid van geglikeerde proteïene, moet egter nog ondersoek word. Aangesien daar bewyse is dat DM nadelig is vir manlike vrugbaarheid, is dit belangrik om eers die onderliggende patofisiologie te verstaan en dan strategieë te ontwikkel en te ondersoek om hierdie siekte en die gepaardgaande komplikasies vir manlike voortplanting te bestry. Daar word voorgehou dat baie natuurlike en kunsmatige middels antioksidant-eienskappe het as gevolg van hul potensiaal om oksidasie te verminder. Antioksidante werk óf deur RSS op te vang óf deur die vorming daarvan te voorkom. Rooibos (Aspalathus linearis), heuningbos (Cyclopia intermedia) en sutherlandia (Lessertia frutescens) is plante wat inheems is aan Suider-Afrika. Kruietee word gemaak van die blare en stamme van hierdie plante. Daar is bewys dat hierdie infusies kafeïenvry is. Studies berig ook dat die infusies van hierdie plante bioaktiewe chemiese verbindings met terapeutiese voordele bevat. Daarbenewens het studies getoon dat hulle ʼn voordelige rol speel in die voorkoming van die ontwikkeling van siektes, en sommige het verbeterende effekte getoon. Hulle rol in manlike voortplanting word egter nog nie goed verstaan nie, en hierdie is die eerste studie wat die gevolge van heuningbos en sutherlandia op manlike voortplantingsfunksie aantoon. Op grond van die gemelde kennisleemtes is die studie in twee hoofafdelings verdeel, elk met verskeie doelstellings. Vir die eerste deel is dertig volwasse manlike rotte lukraak in drie groepe van tien elk verdeel, wat ʼn draer en twee streptosotosien (STS) groepe insluit wat óf 30 mg/kg (STS30) óf 60 mg/kg (STS60) intraperitoneaal ontvang het. Vir die tweede deel is negentig diere sonder vooroordeel in nege groepe van tien elk verdeel. Die groepe het ʼn kontrole- (slegs water), draer- (STS-kontrole, sitraatbuffer + water), rooibos- (2% gefermenteerde rooibos), heuningbos- (4% gefermenteerde heuningbos), sutherlandia- (0,2% ongefermenteerde sutherlandia), diabeteskontrole- (STS45 mg/kg + water), diabetes + rooibos- (STS45 mg/kg + 2% rooibos), diabetes + heuningbos- (STS45 mg/kg + 4% heuningbos), en diabetes + sutherlandia- (STS45 mg/kg + 0,2% sutherlandia) groep ingesluit. Diere in groepe wat behandel is met ʼn diabetiese infusie is ʼn week voor die DM-induksie met die onderskeie infusies behandel. Diere is noukeurig gemonitorgedurende die behandelingsperiode en is sewe weke ná DM-induksie geoffer. Bloed, testes en epididimisse is ingewin vir verdere ontleding. Die uitslae van die eerste deel van hierdie studie het die negatiewe gevolge van DM op manlike voortplantingsfunksie getoon, aangesien daar ʼn afname in spermatosoa met normale morfologie en ʼn verhoogde aantal spermatosoa met ʼn hoër deformiteitsindeks in diere met diabetes voorgekom het. Histologiese en histomorfometriese analise van die testis het veranderinge in die sellulêre assosiasie van die seminale tubules in diabetiese diere getoon. Daarbenewens lig die eerste afdeling die betrokkenheid/uitdrukking van gevorderde glikasie eindprodukte (AGE), sy reseptor (RAGE), mitogeen-geaktiveerde proteïenkinases (MGPK) en die aktivering van apoptose in diabetes, wat kan bydra tot die afwykings gesien in manlike vrugbaarheid, toe. Kortliks, in DM is daar ʼn veranderde antioksidant-ensiemaktiwiteit (↓ Katalase) wat kan lei tot OS. Die ontwikkeling van OS kan enersyds die afbreek van poli onversadigde vetsure (POVS) aanhelp, wat gemeet kan word aan die vlakke van malondialdehied (MDA), en andersyds ook die vorming van AGE indirek verhoog. Die peroksidasie van POVS lok meer RSS-vorming, wat apoptotiese induksie kan aktiveer, wat lei tot beskadigde spermfunksie. AGE's werk deur te bind aan hul reseptor (RAGE). Die AGE RAGE-kompleks inisieer die aktivering van die MGPK’s, wat apoptose kan veroorsaak deur die splitsing van PARP te verhoog, wat gevolglik lei tot nDNA-skade. Al hierdie effekte lei gesamentlik tot ʼn verlaagde vrugbaarheid in mans. Om die rol van die infusies (rooibos, heuningbos en sutherlandia) op manlike voortplanting van beide gesonde en diabetiese diere behoorlik te verstaan, is die tweede deel van die studie in vier substudies verdeel. Onderafdeling een van die tweede deel van die studie het ondersoek of behandeling met rooibos, heuningbos en sutherlandia sperm se funksionele parameters in gesonde rotte positief of andersins sal beïnvloed. Diere wat met die onderskeie infusies behandel is, het met ʼn persentasie toename in superoksieddismutase-aktiwiteit gepresenteer, maar het ʼn verlaging in spermmotiliteit en normale morfologie getoon. Paradoksaal genoeg het hulle verhoogde spermkonsentrasies gehad. Rooibos, heuningbos en sutherlandia kan dus moontlik spermkonsentrasie, wat die hoeveelheid sperma verteenwoordig, verhoog. Die gebruik hiervan kan egter moontlik die spermkwaliteit (spermmotiliteit en morfologie) van gesonde diere benadeel. Onderafdeling twee van die tweede deel van die studie het die testikulêre OS en apoptose status in diabetiese rotte ondersoek na behandeling met rooibos, heuningbos en sutherlandia-infusies. Hierdie afdeling het die negatiewe impak van DM, as gevolg van verhoogde lipiedperoksidasie en verlaagde antioksidant aktiwiteit, op die funksionele parameters van sperms beklemtoon. Die infusiebehandelingsgroepe het ʼn verhoogde antioksidant-ensiemaktiwiteit getoon, wat moontlik deels verantwoordelik kan wees vir die waargenome verbetering in spermbeweeglikheid en -morfologie van diabetiese diere wat rooibos (DRF) ontvang, en diabetiese diere wat sutherlandia (DSL) ontvang. Daarbenewens het die huidige studie die toenemende uitdrukking van apoptotiese biomerkers in die diabeteskontrole(DK)-diere getoon, wat nie deur die infusies verlig is nie. Dit dui daarop dat hierdie infusies ʼn rol speel in die verligting van die verswakking van diabetes-geïnduseerde spermfunksies deur onderdrukking van OS, maar hulle rol in apoptose is nog onduidelik. Onderafdeling drie van die tweede deel van die studie het die rol van rooibos, heuningbos en sutherlandia op insulienseingewing in die testes van diabetiese rotte ondersoek. Al die diabetesgroepe het ʼn beduidende toename in bloedglukosevlakke gehad ná die induksie van diabetes. Die diabetesdiere wat met die infusies behandel is, het egter slegs ʼn effense afname in die vastende bloedglukose getoon. Die DK diere het ʼn afname in testisproteïenuitdrukking van IRS-1, PkB/Akt en GLUT4 getoon. Diabetiese diere wat met rooibos (DRF) en heuningbos (DHB) behandel is, het ʼn opregulering in IRS-1 getoon, terwyl diabetiese diere wat met sutherlandia (DSL) behandel is, verbetering in IRS-2 getoon het. Alhoewel DRF-diere ʼn afname in PkB/Akt getoon het, het DHB- en DSL-diere opregulering getoon in vergelyking met kontrole. Alle diabetiese diere het verhoogde gefosforileerde ERK1/2 en verlaagde totale ERK1/2 getoon in vergelyking met die kontrole- en draerdiere. Daarbenewens het DK-diere ʼn nie-noemenswaardige afname in plasma-testosteroonkonsentrasie in vergeleke met kontrole getoon, terwyl DRF en DSL ʼn beduidende afname en DHB opregulering getoon het. Dit dui daarop dat (i) die aktivering van IRS, PkB/Akt, ERK en GLUT translokasie belangrik is in testikulêre insulienseingewing (ii) rooibos, heuningbos en sutherlandia moontlik ʼn rol kan speel in testikulêre insulienseingewing deur verskillende roetes en (iii) heuningbos moontlik testosteroonproduksie in diabetes effens kan verhoog. Laastens het die vierde onderafdeling van die tweede deel van die studie die beskrywende histologiese evaluering van die testis en cauda epididimis ondersoek na behandeling met rooibos, heuningbos en sutherlandia in beide gesonde en diabetiese rotte. Die infusie kontrolegroepe (RF, HB en SL) het normale seminale tubulêre sellulêre assosiasie, teenwoordigheid van spermatosoa in die epididimale lumen en normale algehele argitektuur getoon. Beide die testikulêre en epididimale morfologie was veranderd in DM, maar hierdie ontwrigtings is effens verlig deur rooibos, heuningbos en sutherlandia. Met inagname van die resultate van die hele studie blyk dit dat diabetes wel ʼn nadelige uitwerking op manlike voortplanting het, beide op die weefsel- en sellulêre vlak. Die sellulêre impak kan uitgeoefen word deur proteïenglikasie, ontwikkeling van OS en induksie van apoptose. Rooibos, heuningbos en sutherlandia moet matig en met omsigtigheid deur gesonde persone geneem word, aangesien te veel daarvan nadelig kan wees en die funksionele parameters van spermatosoa kan benadeel. In siektetoestande soos diabetes kan hierdie infusies egter voordelig wees, aangesien: (i) Rooibos en sutherlandia die spermkwaliteit (beweeglikheid en morfologie) effens kan verbeter, (ii) Rooibos, heuningbos en sutherlandia hiperglukemie effens verlaag deur insulien seingewing te verbeter, (iii) Heuningbos testosteroonproduksie moontlik kan verhoog, (iv) Rooibos, heuningbos en sutherlandia moontlik die histomorfologiese argitektuur van die seminale buisie en die cauda epididimale buisie effens kan verbeter.

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