Yanning Li

Microglial activation is a key event in neuroinflammation, which, in turn, is a central process in neurological disorders. In this study, we investigated the protective effects of D-beta-hydroxybutyrate (BHB) against microglial activation in lipopolysaccharide (LPS)-treated mice and BV-2 cells. The effects of BHB in mice were assessed using behavioral testing, morphological analysis and immunofluorescence labeling for the microglial marker ionizing calcium-binding adaptor molecule 1 (IBA-1) and the inflammatory cytokine interleukin-6 (IL-6) in the hippocampus. Moreover, we examined the levels of the inflammatory IL-6 and tumor necrosis factor-α (TNF-α), as well as those of the neuroprotective brain-derived neurotrophic factor (BDNF) and transforming growth factor-β (TGF-β) in the brain. In addition, we examined the effects of BHB on IL-6, TNF-α, BDNF, TGF-β, reactive oxygen species (ROS) level and cell viability in LPS-stimulated BV-2 cells. BHB treatments attenuated behavioral abnormalities, reduced the number of IBA-1-positive cells and the intensity of IL-6 fluorescence in the hippocampus, with amelioration of microglia morphological changes in the LPS-treated mice. Furthermore, BHB inhibited IL-6 and TNF-α generation, but promoted BDNF and TGF-β production in the brain of LPS-treated mice. In vitro, BHB inhibited IL-6 and TNF-α generation, increased BDNF and TGF-β production, reduced ROS level, ameliorated morphological changes and elevated cell viability of LPS-stimulated BV-2 cells. Together, our findings suggest that BHB exerts protective effects against microglial activation in vitro and in vivo, thereby reducing neuroinflammation.

Microglia activation is the key player in neuro-inflammation, which is the central process of neuro-disorders. The protective effects of D-beta-hydroxybutyrate (BHB) against microglia activation were evaluated in lipopolysaccharide (LPS)-treated mice and BV-2 cells. Behavioral test, morphological change and immunofluorescence of microglia marker ionizing calcium-binding adaptor molecule 1 (IBA-1) in the hippocampus, and the generation of inflammatory interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and protective brain-derived neurotrophic factor (BDNF) and transforming growth factor-β (TGF-β) in the brain, were measured in LPS and/or BHB treated mice. In addition, the effects of BHB on the generation of IL-6, TNF-α, BDNF and TGF-β, and reactive oxygen species (ROS) level were detected in LPS-stimulated BV-2 cells. We found that BHB treatments attenuated behavioral abnormality of LPS-treated mice and reduced the number of IBA-1-positive cells with attenuation of cell morphological changes in the hippocampus. Meanwhile, BHB inhibited IL-6 and TNF-α generations; but promoted BDNF and TGF-β generations in the brain in LPS-treated mice. Furthermore, BHB concentration-dependently inhibited IL-6 and TNF-α generations, promoted BDNF and TGF-β generations, and reduced the level of reactive oxygen species with attenuation of cellular changes in LPS-stimulated BV-2 cells. In conclusion, BHB exhibits protective effects against microglia activation in LPS-treated mice and BV-2 cells, antagonizing neuro-inflammation.

Aims/hypothesis Microvascular endothelial hyperpermeability, mainly caused by claudin-5 deficiency, is the initial pathological change that occurs in diabetes-associated cardiovascular disease. The ketone body β-hydroxybutyrate (BHB) exerts unique beneficial effects on the cardiovascular system, but the involvement of BHB in promoting the generation of claudin-5 to attenuate cardiac microvascular hyperpermeability in diabetes is poorly understood. Methods The effects of BHB on cardiac microvascular endothelial hyperpermeability and claudin-5 generation were evaluated in rats with streptozotocin-induced diabetes and in high glucose (HG)-stimulated human cardiac microvascular endothelial cells (HCMECs). To explore the underlying mechanisms, we also measured β-catenin nuclear translocation, binding of β-catenin, histone deacetylase (HDAC)1, HDAC3 and p300 to the Claudin-5 (also known as CLDN5) promoter, interaction between HDAC3 and β-catenin, and histone acetylation in the Claudin-5 promoter. Results We found that 10 weeks of BHB treatment promoted claudin-5 generation and antagonised cardiac microvascular endothelial hyperpermeability in rat models of diabetes. Meanwhile, BHB promoted claudin-5 generation and inhibited paracellular permeability in HG-stimulated HCMECs. Specifically, BHB (2 mmol/l) inhibited HG-induced HDAC3 from binding to the Claudin-5 promoter, although nuclear translocation or promoter binding of β-catenin did not change with BHB treatment. In addition, BHB prevented the binding and co-localisation of HDAC3 to β-catenin in HG-stimulated HCMECs. Furthermore, using mass spectrometry, acetylated H3K14 (H3K14ac) in the Claudin-5 promoter following BHB treatment was identified, regardless of whether cells were stimulated by HG or not. Although reduced levels of acetylated H3K9 in the Claudin-5 promoter were found following HG stimulation, increased H3K14ac was specifically associated with BHB treatment. Conclusions/interpretation BHB inhibited HDAC3 and caused acetylation of H3K14 in the Claudin-5 promoter, thereby promoting claudin-5 generation and antagonising diabetes-associated cardiac microvascular hyperpermeability. Graphical abstract

Aims Besides energy supply, β-hydroxybutyrate (BHB) acts as a bioactive molecule to play multiple protective roles, even in diabetes and its complications. The aim of this study was to investigate the antagonizing effects of BHB against diabetic glomerulosclerosis and the underlying mechanism. Methods Male Sprague–Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes and then treated with different concentrations of β-hydroxybutyrate. After 10 weeks, body weight, blood glucose, serum creatinine and 24-h urine protein were examined. Glomerular morphological changes and the contents of collagen type IV (COL IV) were evaluated. Then, transforming growth factor (TGF)-β/Smad3 contents and matrix metalloproteinase-2 (MMP-2) generation were detected. Moreover, the total contents of trans-activating histone H3K9 β-hydroxybutyrylation (H3K9bhb) and the contents of H3K9bhb in the Mmp-2 promoter were measured. Results It was firstly confirmed that BHB treatments reduced renal biochemical indicators and attenuated glomerular morphological changes of the diabetic rats, with COL IV content decreased in a concentration-dependent manner. Then, BHB treatments were found to up-regulate renal MMP-2 generation of the diabetic rats significantly, while not affecting the increased TGF-β/Smad3 contents. Furthermore, the contents of H3K9bhb in the Mmp-2 promoter were elevated significantly for the middle and high concentrations of BHB treatments, up-regulating MMP-2 generation. Conclusion BHB treatments could up-regulate MMP-2 generation via causing elevated H3K9bhb in its promoter to antagonize glomerulosclerosis in the diabetic rats.