| Objective: Oxidative stress and obesity induced by high-fat diet (HFD) could result inabnormal bone metabolism and poor bone quality. Previous studies have shown thatγ-aminobutyric acid (GABA) could play a role in modulating blood pressure, inhibitinginflammation and alleviating oxidative stress in peripheral tissues. The anti-oxidative effect ofGABA may be related to the inhibiting effect of GABA-B receptor (GABAbR) activation onGSK3β activity. However, the relationship among GABA-ergic signaling, GABAantioxidation and bone performance is unclear. This rsearch aimed to investigate therelationship among oxidative stress markers, GABA-ergic signaling system and boneperformance in HFD-fed mice. We further confirmed GABA-ergic signaling effect inregulating Nrf2antioxidant system and bone performance by oral GABA admistration toHFD-fed mice.Methods: Six-week-old male C57/BL6mice were assigned randomly into five dietarygroups: control group (fed normal diet), HFD group (fed high-fat diet), and other three groupsfed with HFD plus three different doses of GABA in drinking water (0.06%,0.12%,0.20%).The mice were weighted weekly and HFD mice further divided into three groups: HFL (thelower1/4), HFM (the intermediate1/2), and HFH (the upper1/4). Urine of mice in eachgroup was collected for measuring urinary calcium and hydroxyproline content at19th. Micewere sacrificed under general anesthesia at20th, and femur, tibia and duodenum were resectedfor analyzing redox state and bone performance. RT-PCR was applied to test the mRNAexpression of femoral GABA-ergic related genes GAD, GABAbR, GSK3β/Nrf2, andosteoblast-, osteoclast-specific genes.Results: Compared with the control group, the femoral MDA in HFD mice was elevated,GSK3β and GABAbR1mRNA expression increased, and GAD65mRNA expression reducedaccompanied by significantly elevated (P＜0.05) urinary calcium and hydroxyproline contentas well as poor bone performance. The osteoblast-specific genes including Col1a1, BGLAP2,BMP2and Nrf2were down-regulated, while the ratio of RANKL/OPG genes expression andthe mRNA levels of CTSK and TRAP were enhanced in HFL and HFM group. In addition,Nrf2, osteoblast-and osteoclast-specific genes were up-regulated significantly (P＜0.05) inHFH group, suggesting a compensatory stage. Compared with HFD group, treatment with0.06%-0.20%GABA markedly decreased (P＜0.05) femoral MDA content, partiallynormalizing GABAbR1, GSK3β and Nrf2mRNA expression. Furthermore, GABA treatmentpartially or competedly restored osteoblast-and osteoclast-specific genes mRNA expression,decreased urinary calcium and hydroxyproline content and improved femur and tibiaperformance. Supplementing0.12%GABA showed a better intervention effect than0.06%and0.20%in improving bone performance. Correlation analysis showed a strong relationshipof GAD65, GABAbR to GSK3β, Nrf2, femorla MDA respectively, as well as strongcorrelation of femur performance to MDA, GAD65and GABAbR1. Additionally, duodenumoxidative stress, and CaT1, Cal-D9kmRNA expression disorders were observed in HFD.GABA supplementation alleviated oxidative stress, and normalized CaT1and Cal-D9kmRNAexpresson. Conclusion: The HFD induced femoral oxidative damage and markedly up-regulatedGSK3β and GABAbR mRNA expression, and thus maybe important mediators ofHFD-induced poor bone perofrmace. The HFL and HFM group showed inhibited osteoblastdifferentiation and enhanced osteoclast differentiation, while HFH showed both up-regulatedosteoblast-and osteoclast-genes expression, implying a high rate of bone tunover. GABAsupplementation could balance bone metabolism on the molecular level and improve boneperformance by significantly reducing femoral MDA content, partially or completelynormalizing GSK3β, Nrf2and GABAbR1mRNA expression. Hence, oxidative stress inducedby HFD may have caused GABA-ergic signaling system and Nrf2antioxidant system relatedgenes mRNA expression disorders which could mediate the occurrence and development ofpoor bone performance. |
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