Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.     

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Characterization of a quinol peroxidase mutant in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.     

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains

Non-serotypeable Actinobacillus actinomycetemcomitans strains may be derived from the serotypeable ones. In the present study, we compared the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of serotypeable and non-serotypeable A. actinomycetemcomitans strains (n=24) of the same genotype in the same subject (n=6) to find out if alterations on the cell-surface contribute to the non-serotypeability. Serotypeable and non-serotypeable A. actinomycetemcomitans strains showed great similarity in the OMP patterns both within and between subjects. Using serotype-specific antisera, clear immunoblotting LPS profiles in the O-antigenic region were seen in serotype b and c strains but not in non-serotypeable strains from the same subjects. The results suggest that changes in LPS lead to the altered antigenicity of non-serotypeable A. actinomycetemcomitans strains.     

Leukotoxin from Aggregatibacter actinomycetemcomitans causes shrinkage and P2X receptor‐dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat‐in‐toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α‐haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA‐mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA‐mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS‐exposure is preceded by increments in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Interestingly, LtxA‐mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA‐induced [Ca²⁺]_i increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2‐mediated auto‐ and paracrine signalling may prevent LtxA‐mediated cell damage.     

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1

Repeats‐in‐toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function‐associated antigen 1 (LFA‐1, integrin α_L/β₂)‐dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA‐1‐expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA‐1 with a cyan fluorescent protein‐tagged cytosolic α_L domain and a yellow fluorescent protein‐tagged β₂ domain. Phorbol 12‐myristate 13‐acetate activation of LFA‐1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both α_L and β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ subunits (K_d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α_M, α_X, and β₃ subunits (K_d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane‐proximal domain of α_L and intermediate domain of β₂.     

Cytolethal distending toxin‐induced release of interleukin‐1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro‐inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase‐1 (canonical inflammasome), Cdt treatment leads to caspase‐4 activation and involvement of the noncanonical inflammasome. Cdt‐treated cells exhibit pyroptosis characterised by cleavage of gasdermin‐D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase‐1) or noncanonical (caspase‐4) inflammasome blocks both Cdt‐induced release of IL‐1β and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt‐induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt‐induced activation of GSK3β. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt‐producing organisms such as A. actinomycetemcomitans.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation

Actinobacillus actinomycetemcomitans is an important pathogen in periodontitis. In the present study we localized the GroEL- and DnaK-like heat shock proteins (Hsp) in subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and compared their effects on periodontal epithelial cell proliferation and viability. In all strains, GroEL-like protein was found in the membrane, cytoplasm, and periplasm, whereas DnaK-like protein was present in the cytoplasm and periplasm. No correlation was observed between the Hsp expression and the serotype or origin of A. actinomycetemcomitans strains. The bacterial membrane fractions that expressed the GroEL-like protein moderately or strongly induced epithelial cell proliferation more strongly than strains that expressed the protein weakly. The results suggest that GroEL-like Hsp may play a role in the virulence of A. actinomycetemcomitans by increasing epithelial proliferation.     

NMR structure of rALF‐Pm3, an anti‐lipopolysaccharide factor from shrimp: Model of the possible lipid A‐binding site

The anti‐lipopolysaccharide factor ALF‐Pm3 is a 98‐residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter as a folded and ¹⁵N uniformly labeled rALF‐Pm3 protein. Its 3D structure was established by NMR and consists of three α‐helices packed against a four‐stranded β‐sheet. The C³⁴? C⁵⁵ disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF‐Pm3 binds to LPS, lipid A and to OM®‐174, a soluble analogue of lipid A. Biophysical studies of rALF‐Pm3/LPS and rALF‐Pm3/OM®‐174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF‐Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A‐binding site of ALF‐Pm3. Such a binding site, located on the ALF‐Pm3 β‐sheet and involving seven charged residues, is well conserved in ALF‐L from Limulus polyphemus and in ALF‐T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that β‐hairpin synthetic peptides corresponding to ALF‐L β‐sheet bind to LPS. Delineating lipid A‐binding site of ALFs will help go further in the de novo design of new antibacterial or LPS‐neutralizing drugs. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 207–220, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Scale‐dependent longitudinal patterns in mussel communities

1. Species richness and assemblage patterns of organisms are dictated by numerous factors, probably operating at multiple scales. Freshwater mussels (Unionidae) are an endangered, speciose faunal group, making them an interesting model system to study the influence of landscape features on organisms. In addition, landscape features that influence species distributions and the scale at which the factors have the greatest impact are important issues that need to be answered to conserve freshwater mussels. 2. In this study, we quantified freshwater mussel communities at 16 sites along three mid‐sized rivers in the south‐central United States. We addressed the following questions: (i) Are there predictable longitudinal changes in mussel community composition? (ii) What landscape variables best explain shifts in community composition? and (iii) At what scale do landscape variables best predict mussel community composition? 3. After controlling for the influence of longitudinal position along the stream, we compared mussel distributions to a suite of hypothesised explanatory landscape variables across multiple scales – catchment scale (entire drainage area), buffer scale (100‐m riparian buffer of the entire catchment) and reach scale (100‐m riparian buffer extending 1 km upstream from the sampling site). 4. We found a significant and consistent longitudinal shift in dominant mussel species across all three rivers, with community composition strongly related to distance from the headwaters, which is highly correlated with stream size. After accounting for stream size, variables at the buffer scale were the best predictors of mussel community composition. After accounting for catchment position, mean channel slope was the best explanatory variable of community composition and appeared in all top candidate models at the catchment and buffer scales. Coverage of wetland and urban area were also correlated with community composition at the catchment and buffer scales. 5. Our results suggest that landscape‐scale habitat factors influence mussel community composition. Landscape features at the buffer scale performed best at determining community composition after accounting for position in the catchment; thus, further protection of riparian buffers will help to conserve mussel communities.     

Branching patterns in phylogenies cannot distinguish diversity‐dependent diversification from time‐dependent diversification

One of the primary goals of macroevolutionary biology has been to explain general trends in long‐term diversity patterns, including whether such patterns correspond to an upscaling of processes occurring at lower scales. Reconstructed phylogenies often show decelerated lineage accumulation over time. This pattern has often been interpreted as the result of diversity‐dependent (DD) diversification, where the accumulation of species causes diversification to decrease through niche filling. However, other processes can also produce such a slowdown, including time dependence without diversity dependence. To test whether phylogenetic branching patterns can be used to distinguish these two mechanisms, we formulated a time‐dependent, but diversity‐independent model that matches the expected diversity through time of a DD model. We simulated phylogenies under each model and studied how well likelihood methods could recover the true diversification mode. Standard model selection criteria always recovered diversity dependence, even when it was not present. We correct for this bias by using a bootstrap method and find that neither model is decisively supported. This implies that the branching pattern of reconstructed trees contains insufficient information to detect the presence or absence of diversity dependence. We advocate that tests encompassing additional data, for example, traits or range distributions, are needed to evaluate how diversity drives macroevolutionary trends.     

Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation‐dependent mechanism

The O‐antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O‐antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation.     

Cyclic AMP signaling enhances lipopolysaccharide sensitivity and interleukin‐33 production in RAW264.7 macrophages

While it has been suggested that IL‐33 plays pathogenic roles in various disorders, the factors that stimulate IL‐33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL‐33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High‐dose LPS treatment induced IL‐33 and TNF protein production in RAW264.7 macrophages. In contrast, low‐dose LPS failed to induce IL‐33 production while significantly inducing TNF production. In the presence of the membrane‐permeable cAMP analog 8‐Br‐cAMP, low‐dose LPS induced vigorous IL‐33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP‐increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL‐33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8‐Br‐cAMP and adrenaline on IL‐33 production, suggesting that PKA is involved in IL‐33 induction. Taken together, cAMP‐mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL‐33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL‐33; this process may be associated with the pathogenesis of various disorders involving IL‐33.     

Gene expression profile of mouse white adipose tissue during inflammatory stress: age‐dependent upregulation of major procoagulant factors

Tolerance to physiological stress resulting from inflammatory disease decreases significantly with age. High mortality rates, increased cytokine production, and pronounced thrombosis are characteristic complications of aged mice with acute systemic inflammation induced by injection with lipopolysaccharide (LPS). As adipose tissue is now recognized as an important source of cytokines, we determined the effects of aging on visceral white adipose tissue gene expression during LPS‐induced inflammation in male C57BL/6 mice. Microarray analysis revealed that the expression of 6025 genes was significantly changed by LPS; of those, the expression of 667 showed an age‐associated difference. Age‐associated differences were found in many genes belonging to the inflammatory response and blood clotting pathways. Genes for several procoagulant factors were upregulated by LPS; among these, tissue factor, thrombospondin‐1, and plasminogen activator inhibitors‐1 and ‐2, exhibited age‐associated increases in expression which could potentially contribute to augmented thrombosis. Further analysis by qRT–PCR, histological examination, and cell fraction separation revealed that most inflammatory and coagulant‐related gene expression changes occur in resident stromal cells rather than adipocytes or infiltrating cells. In addition, basal expression levels of 303 genes were altered by aging, including increased expression of component of Sp100‐rs (Csprs). This study indicates that adipose tissue is a major organ expressing genes for multiple inflammatory and coagulant factors and that the expression of many of these is significantly altered by aging during acute inflammation. Data presented here provide a framework for future studies aimed at elucidating the impact of adipose tissue on age‐associated complications during sepsis and systemic inflammation.     

β‐Hydroxybutyrate exacerbates lipopolysaccharide/ d‐galactosamine‐induced inflammatory response and hepatocyte apoptosis in mice

β‐Hydroxybutyrate (BHB), one of ketone body, has been traditionally regarded as an alternative carrier of energy, but recent studies found that BHB plays versatile roles in inflammation. It has been previously reported that the level BHB declined in mice with lipopolysaccharide (LPS)/d ‐galactosamine (d ‐Gal)‐induced liver damage, but the pathological significance remains unclear. In the present study, the pathophysiological roles of BHB in LPS/d ‐Gal‐induced hepatic damage has been investigated. The results indicated pretreatment with BHB further enhanced LPS/d ‐Gal‐induced elevation of aspartate aminotransferase and alanine aminotransferase, exacerbated the histological abnormalities and increased the mortality. Pretreatment with BHB upregulated the level of tumor necrosis factor α and interleukin‐6 in plasma, promoted the activities of caspase‐3, caspase‐8, and caspase‐9 and increased the count of terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive cells. In addition, post‐insult supplement with BHB also potentiated LPS/d ‐Gal‐induced apoptotic liver damage. Therefore, BHB might be a detrimental factor in LPS/d ‐Gal‐induced liver injury via enhancing the inflammation and the apoptosis in the liver.