The role of the intestinal immune system in the development and treatment of type 2 diabetes
Date
2022-11
Authors
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Background: Type 2 diabetes (T2D) is largely correlated with obesity. Low-grade systemic
inflammation and altered immune homeostasis in the gut are implicated in the pathogenesis of
T2D. The intestinal mucosal immune response and tissue repair mechanisms are reduced while
a state of dysbiosis is induced in T2D. Rooibos has been associated with hypoglycaemic and
anti-inflammatory effects. Therefore, rooibos potentially improves the intestinal immune status
in a type 2 diabetic state. This study aims to investigate the role of the intestinal immune
homeostasis and tissue repair in animal models of early and late stages of T2D development,
and to elucidate the effects of rooibos treatment on the intestinal immune status and tissue
repair.
Method: Male Wistar rats (n=60) were fed either a control, high-fat diet (HFD) or high-sugar
diet (HSD) for 10 weeks and thereafter, green rooibos extract (GRT) (60 mg/kg) was
supplemented (n=10 per group) for 7 weeks. Jejunum and ileum, and stool were harvested to
assess immunoglobulin A (IgA) content by immunohistochemistry staining and ELISA
respectively to measure intestinal mucosal immune responses.
Diabetic, db/db and non-diabetic, db/+ mice (n=64) were treated with vehicle control,
pioglitazone as a positive control (15 mg/kg), GRT low dose (74 mg/kg), or GRT high dose
(740 mg/kg) for 16 weeks. Small intestinal tissue were stained to quantify IgA-expressing cells,
Foxp3 to measure regulatory T-cells (Tregs), and Ki-67 to measure proliferating cells. Colonic
IgA was assessed as well as Cox-2 as a measure of tissue repair using Western blot. IL-10 and
TGF-β were assessed in the colon using ELISA for differentiation of Tregs. Alterations of the
gut microbiota was assessed from cecum DNA by sequencing hypervariable regions via an
Ion-torrent platform. Statistical significance was determined by 2-way ANOVA and Tukey
Kramer post-hoc test.
Results: HSD increased IgA-expressing cells in the jejunum of rats compared to the HFD-
(p=0,00001) and control-fed rats (p=0,02056), while GRT was able to increase IgA-expressing
cells in the jejunum compared to the non-treated rats (p=0,01044). No significant changes in
IgA expression was observed in the ileum or stool of HSD- or HFD-fed rats.
AFRIKAANS OPSOMMING: Agtergrond: Tipe 2-diabetes (T2D) word met vetsug geassosieer. Laegraadse sistemiese inflammasie en veranderde immuunhomeostase van die dermkanaal is betrokke by die patogenese van T2D. Die intestinale mukosale immuunrespons en weefsel herstel meganismes word onderdruk, terwyl 'n toestand van disbiose in T2D geïnduseer word. Aangesien rooibos met hipoglisemiese en anti-inflammatoriese effekte geassosieer word, mag rooibos moontlik ook die intestinale immuunstatus in 'n tipe 2-diabetiese toestand verbeter. Hierdie studie het ten doel om die rol van die intestinale immuunhomeostase en weefselherstel in diermodelle van vroeë en laat stadiums van T2D-ontwikkeling te ondersoek, en om die uitwerking van rooibosbehandeling op die intestinale immuunstatus en weefselherstel te bepaal. Metodes: Manlike Wistar-rotte (n=60) is óf 'n kontrole-, hoëvet-dieet (HFD) óf hoë-suiker dieet (HSD) vir 10 weke gevoer en daarna met groenrooibos ekstrak (GRT) (60 mg/kg) daagliks (n=10 per groep) vir 7 weke behandel. Jejunum- en ileumweefsels monsters is gebruik om immunoglobulien A (IgA) inhoud te bepaal. Immunohistochemiese kleuring en ELISA is onderskeidelik gebruik om die intestinale mukosale immuunresponse te meet. Diabetiese, db/db en nie-diabetiese, db/+ muise (n=64) was as middelkontrole, pioglitazoon as 'n positiewe kontrole (15 mg/kg), GRT lae dosis (74 mg/kg), of GRT hoë dosis (740 mg/kg) vir 16 weke behandel. Dundermweefsel is gekleur om IgA-positiewe selle, Foxp3 om regulatoriese T-selle (Tregs), en Ki-67 om prolifererende selle, onderskeidelik te kwantifiseer. IgA asook Cox-2, wat saam kan dien as 'n maatstaaf van weefselherstel, was met behulp van “Western blot” bepaal. IL-10 en TGF-β wat met die differensiasie van Tregs geassosieer word, is deur ELISA geassesseer. Veranderinge van die dermmikrobiota is geassesseer vanaf sekum -DNS deur die sekwensie van die hiperveranderlike streke met 'n “Ion-torrent platform” te bepaal. Statistiese betekenis is deur 2-rigting ANOVA en Tukey Kramer post-hoc toets bepaal. Resultate: HSD het die voorkoms van IgA-positiewe selle in die jejunum verhoog vergeleke met die HFD- (p=0,00001) en kontrole-dieet rotte (p=0,02056), terwyl GRT in staat was om die voorkoms on die IgA-positiewe selle in die jejunum te verhoog vergeleke met die onbehandelde rotte (p=0,01044). Geen betekenisvolle veranderinge van die IgA-vlakke was in die ileum of stoelgang van HSD- of HFD rotte waargeneem nie.
AFRIKAANS OPSOMMING: Agtergrond: Tipe 2-diabetes (T2D) word met vetsug geassosieer. Laegraadse sistemiese inflammasie en veranderde immuunhomeostase van die dermkanaal is betrokke by die patogenese van T2D. Die intestinale mukosale immuunrespons en weefsel herstel meganismes word onderdruk, terwyl 'n toestand van disbiose in T2D geïnduseer word. Aangesien rooibos met hipoglisemiese en anti-inflammatoriese effekte geassosieer word, mag rooibos moontlik ook die intestinale immuunstatus in 'n tipe 2-diabetiese toestand verbeter. Hierdie studie het ten doel om die rol van die intestinale immuunhomeostase en weefselherstel in diermodelle van vroeë en laat stadiums van T2D-ontwikkeling te ondersoek, en om die uitwerking van rooibosbehandeling op die intestinale immuunstatus en weefselherstel te bepaal. Metodes: Manlike Wistar-rotte (n=60) is óf 'n kontrole-, hoëvet-dieet (HFD) óf hoë-suiker dieet (HSD) vir 10 weke gevoer en daarna met groenrooibos ekstrak (GRT) (60 mg/kg) daagliks (n=10 per groep) vir 7 weke behandel. Jejunum- en ileumweefsels monsters is gebruik om immunoglobulien A (IgA) inhoud te bepaal. Immunohistochemiese kleuring en ELISA is onderskeidelik gebruik om die intestinale mukosale immuunresponse te meet. Diabetiese, db/db en nie-diabetiese, db/+ muise (n=64) was as middelkontrole, pioglitazoon as 'n positiewe kontrole (15 mg/kg), GRT lae dosis (74 mg/kg), of GRT hoë dosis (740 mg/kg) vir 16 weke behandel. Dundermweefsel is gekleur om IgA-positiewe selle, Foxp3 om regulatoriese T-selle (Tregs), en Ki-67 om prolifererende selle, onderskeidelik te kwantifiseer. IgA asook Cox-2, wat saam kan dien as 'n maatstaaf van weefselherstel, was met behulp van “Western blot” bepaal. IL-10 en TGF-β wat met die differensiasie van Tregs geassosieer word, is deur ELISA geassesseer. Veranderinge van die dermmikrobiota is geassesseer vanaf sekum -DNS deur die sekwensie van die hiperveranderlike streke met 'n “Ion-torrent platform” te bepaal. Statistiese betekenis is deur 2-rigting ANOVA en Tukey Kramer post-hoc toets bepaal. Resultate: HSD het die voorkoms van IgA-positiewe selle in die jejunum verhoog vergeleke met die HFD- (p=0,00001) en kontrole-dieet rotte (p=0,02056), terwyl GRT in staat was om die voorkoms on die IgA-positiewe selle in die jejunum te verhoog vergeleke met die onbehandelde rotte (p=0,01044). Geen betekenisvolle veranderinge van die IgA-vlakke was in die ileum of stoelgang van HSD- of HFD rotte waargeneem nie.
Description
Thesis (MSc) -- Stellenbosch University, 2022.
Keywords
Medicinal plants -- Therapeutic use, Type 2 diabetes, Immune response, Intestinal mucosa, Rooibos tea