| Dietary pattern’s shift to high-fat and calorie-rich diet is the main cause of obesityprevalence. Obesity is regarded as low grade chronic inflammation，which is closelyassociated with the occurrence and development of metabolic syndrome. Regulatory T cell(Treg) had a efficient control of inflammation, however, there was a marked decrease of Tregin obesity mice and human model without eluciating the mechanism clearly. Gamma-aminobutyric acid (GABA) was found to be a potential regulator of high fat diet (HFD)induced immune responses and insulin resistance through up-regulating splenic Tregfrequency. The objective of this study was to investigate the effect of HFD and GABA on theimmune competence and the modulation effect of splenic Tregs.(1) Male C57BL/6mice (18.5±0.54g) were fed with normal diet (3.85Kcal/g) andhigh fat food (4.73Kcal/g) provided with plain water or water containing0.06%and0.2%of GABA for20weeks. Spleens were dissected out immediately, weighted and divided intothree parts. A portion of the spleen was store at-80℃for mRNA extraction, and the othertwo were used for CD4+, CD8+and CD4+CD25+CD127-/lowTreg FACS cell sort and analysisof biomarkers of oxidative stress, respectively. ROS (Reactive oxygen species) andmitochondrial membrane potential (ΔΨm) of splenic Tregs were analysed by flow cytometry.Genes that were crucial for Treg function, apoptosis, metabolism and spleen antioxidantmechanism were measured by RT-PCR.(2) To evaluate the direct protection of GABA onimmune function and its influence on lymphocyte secretion function, we put forward the invitro experiment. The experiment selected the high-fat induced C57/BL6obesity mice for sixweeks and control mice spleens. The experiment was divided into5groups: Control group(Control); High-fat group (HFD); High fat group with different concentration of GABAtreatment groups: HFD+0.1μM GABA, HFD+1μM GABA and HFD+10μM GABA. TheROS level, ΔΨm and the secretion of IL–35, IL-10, IL-6and IL-2were determined.The body weight in HFD mice were significantly (P<0.05) higher than those in controlmice, oral treatment with GABA for20weeks can significantly reduce the gain of bodyweights. HFD induced marked decrease (P<0.05) of thymus index, spleen index and GABAcould improve the situation of spleen and thymus atrophy. There was a significant (P<0.05)decrease of total antioxidant capacity (T-AOC), GSH/GSSG ratio and a significant increase ofROS and MDA level in of spleen in HFD group. However, oral treatment with differentconcentrations of GABA could both bring about an improvement in the antioxidant defense(P<0.05). Compared with control group, anti-oxidant genes mRNA expression in spleen PI3K,Akt1, Nrf2, GST and GCLC is decreased significantly (P<0.05) in HFD group, however,supplementation of GABA could increase these genes mRNA expression significantly (P<0.05). CD4+/CD8+ratio and regulatory T cells percentages in spleen was significantlydecreased (P<0.05), demonstrated the immune imbalance status in HFD group mice.CD4+/CD8+ratio and regulatory T cells percentages are significantly correlated with redoxstate in spleen. Genes that were crucial for Treg structure and function Foxp3, IL-35, IL-10,TGF-β, SOCS2,4-1BB and OX40were abnormal expressed in HFD group, indicating thatHFD induced the imbalance of splenic Treg function (P<0.05), oral treatment with GABA canimprove the situation obviously (P<0.05). AMPK mRNA expression was significantly(P<0.05) increased and mTORC1, S6K1and4E-BP1were significantly (P<0.05) decreased,indicated that AMPK inhibited the mTORC1mediated synthesis metabolism, oral treatmentwith GABA could significantly increase mTORC1mediated anabolism. Tregs’ ROS level wassignificant increased in HFD group when compared with the control group (P<0.05). GABAincreased splenic Tregs frequency and ameliorated oxidative stress in splenic Tregs (P<0.05).Splenic Tregs demonstrated significant loss (P<0.05) of ΔΨm and mRNA analysis revealedthe inactivation of anti-apoptotic Nrf2/Bcl-2pathway under HFD exposure. GABA treatmentimproved the loss ofΔΨm and activated the Nrf2/Bcl-2pathway in splenic Tregs.(2)Compared with HFD group, GABA could improve the ΔΨm loss of splenic mixedlymphocytes and decrease its ROS production significantly (P<0.05). IL-2, IL-10and IL-35was significantly decreased (P<0.05), while IL-6was significantly increased in HFD group(P<0.05), indicted the chronic inflammation state in spleen. Treatment with GABA canmodulate the cytokine production to contol level. ROS level and ΔΨm of splenic lymphocytesis significantly correlated with the production of cytokines.Our research demonstrates that long period of HFD induced oxidative stress give rise tothe decreased frequency and dysfunction of splenic Tregs. Oral treatment with GABA canprotect Treg via activate the intracellular anti-apoptosis mechanism and improve the redoxstate. Experiment in vitro indicates that GABA can protect the lymphocytes directly. Differentdoses of GABA could improve the cytokine production to contol level without significantdose-dependent effect. |
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