Amphiregulin promotes hair regeneration of skin‐derived precursors via the PI3K and MAPK pathways

ObjectivesThere are significant clinical challenges associated with alopecia treatment, including poor efficiency of related drugs and insufficient hair follicles (HFs) for transplantation. Skin‐derived precursors (SKPs) exhibit great potential as stem cell‐based therapies for hair regeneration; however, the proliferation and hair‐inducing capacity of SKPs gradually decrease during culturing.Materials and MethodsWe describe a 3D co‐culture system accompanied by kyoto encyclopaedia of genes and genomes and gene ontology enrichment analyses to determine the key factors and pathways that enhance SKP stemness and verified using alkaline phosphatase assays, Ki‐67 staining, HF reconstitution, Western blot and immunofluorescence staining. The upregulated genes were confirmed utilizing corresponding recombinant protein or small‐interfering RNA silencing in vitro, as well as the evaluation of telogen‐to‐anagen transition and HF reconstitution in vivo.ResultsThe 3D co‐culture system revealed that epidermal stem cells and adipose‐derived stem cells enhanced SKP proliferation and HF regeneration capacity by amphiregulin (AREG), with the promoted stemness allowing SKPs to gain an earlier telogen‐to‐anagen transition and high‐efficiency HF reconstitution. By contrast, inhibitors of the phosphoinositide 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways downstream of AREG signalling resulted in diametrically opposite activities.ConclusionsBy exploiting a 3D co‐culture model, we determined that AREG promoted SKP stemness by enhancing both proliferation and hair‐inducing capacity through the PI3K and MAPK pathways. These findings suggest AREG therapy as a potentially promising approach for treating alopecia.     

LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling

BackgroundAlopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear.Methods3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3′UTR.ResultsXIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development.ConclusionXIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.     

Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti‐Thy‐1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway

ObjectivesWe aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis.Materials and methodsWe established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression.ResultsIn anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.ConclusionsOur study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.     

L‐Se‐methylselenocysteine sensitizes lung carcinoma to chemotherapy

ObjectivesOrganic Selenium (Se) compounds such as L‐Se‐methylselenocysteine (L‐SeMC/SeMC) have been employed as a class of anti‐oxidant to protect normal tissues and organs from chemotherapy‐induced systemic toxicity. However, their comprehensive effects on cancer cell proliferation and tumour progression remain elusive.Materials and MethodsCCK‐8 assays were conducted to determine the viabilities of cancer cells after exposure to SeMC, chemotherapeutics or combined treatment. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation levels were assessed via fluorescence staining. The efficacy of free drugs or drug‐loaded hydrogel against tumour growth was evaluated in a xenograft mouse model.ResultsAmong tested cancer cells and normal cells, the A549 lung adenocarcinoma cells showed higher sensitivity to SeMC exposure. In addition, combined treatments with several types of chemotherapeutics induced synergistic lethality. SeMC promoted lipid peroxidation in A549 cells and thereby increased ROS generation. Significantly, the in vivo efficacy of combination therapy was largely potentiated by hydrogel‐mediate drug delivery.ConclusionsOur study reveals the selectivity of SeMC in the inhibition of cancer cell proliferation and develops an efficient strategy for local combination therapy.     

Sirtuin 1 ameliorates defenestration in hepatic sinusoidal endothelial cells during liver fibrosis via inhibiting stress‐induced premature senescence

ObjectivePremature senescence is related to progerin and involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the mechanisms of premature senescence in defenestration of hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects HSECs fenestrae remain elusive.MethodsWe employed the CCl₄‐induced liver fibrogenesis rat models and cultured primary HSECs in vitro, administered with the SIRT1‐adenovirus vector, the activator of SIRT1 and knockdown NOX2. We measured the activity of senescence‐associated β‐galactosidase (SA‐β‐gal) in HSECs. Meanwhile, the protein expression of SIRT1, NOX2, progerin, Lamin A/C, Ac p53 K381 and total p53 was detected by Western blot, co‐immunoprecipitation and immunofluorescence.ResultsIn vivo, premature senescence was triggered by oxidative stress during CCl₄‐induced HSECs defenestration and liver fibrogenesis, whereas overexpressing SIRT1 with adenovirus vector lessened premature senescence to relieve CCl₄‐induced HSECs defenestration and liver fibrosis. In vitro, HSECs fenestrae disappeared, with emerging progerin‐associated premature senescence; these effects were aggravated by H₂O₂. Nevertheless, knockdown of NOX2, activation of SIRT1 with resveratrol and SIRT1‐adenovirus vector inhibited progerin‐associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co‐localized with the abnormal accumulation of actin filament (F‐actin) in the nuclear envelope of H₂O₂‐treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1.ConclusionSIRT1‐mediated deacetylation maintains HSECs fenestrae and attenuates liver fibrogenesis through inhibiting oxidative stress‐induced premature senescence.     

G6PD overexpression protects from oxidative stress and age‐related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL⁺ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.     

Enhanced effect of recombinant adenoviruses co‐expression of ING4 and OSM on anti‐tumour activity of laryngeal cancer

The inhibitor of growth family member 4 (ING4) is one of the ING family genes, serves as a repressor of angiogenesis or tumour growth and suppresses loss of contact inhibition. Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin (IL)‐6 subfamily with several biological activities. However, the role of recombinant adenoviruses co‐expressing ING4 and OSM (Ad‐ING4‐OSM) in anti‐tumour activity of laryngeal cancer has not yet been identified. Recombinant Ad‐ING4‐OSM was used to evaluate their combined effect on enhanced anti‐tumour activity in Hep‐2 cells of laryngeal cancer in vivo. Moreover, in vitro function assays of co‐expression of Ad‐ING4‐OSM were performed to explore impact of co‐expression of Ad‐ING4‐OSM on biological phenotype of laryngeal cancer cell line, that is Hep‐2 cells. In vitro, Ad‐ING4‐OSM significantly inhibited the growth, enhanced apoptosis, altered cell cycle with G1 and G2/M phase arrest, and upregulated the expression of P21, P27, P53 and downregulated survivin in laryngeal cancer Hep‐2 cells. Furthermore, in vivo functional experiments of co‐expressing of Ad‐ING4‐OSM demonstrated that solid tumours in the nude mouse model were significantly suppressed, and the co‐expressing Ad‐ING4‐OSM showed a significant upregulation expression of P21, P53, Bax and Caspase‐3 and a downregulation of Cox‐2, Bcl‐2 and CD34. This study for the first time demonstrated the clinical value and the role of co‐expressing Ad‐ING4‐OSM in biological function of laryngeal cancer. This work suggested that co‐expressing Ad‐ING4‐OSM might serve as a potential therapeutic target for laryngeal cancer patients.     

20‐HETE synthesis inhibition attenuates traumatic brain injury–induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC‐1α pathway: A translational study

Objectives20‐hydroxyeicosatetraenoic acid (20‐HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20‐HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms.Materials and methodsNeurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post‐TBI effects of HET0016, an inhibitor of 20‐HETE synthesis, and their underlying mechanisms.ResultsThe level of 20‐HETE was found to be increased significantly after TBI in mice. 20‐HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post‐TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC‐1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC‐1α and the abnormal mitochondrial dynamics induced by 20‐HETE in vitro. Furthermore, plasma 20‐HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score.ConclusionsThe inhibition of 20‐HETE synthesis represents a novel strategy to mitigate TBI‐induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC‐1α pathway.     

Quantitative contributions of TNF receptor superfamily members to CD8+ T‐cell responses

T‐cell responses to infections and cancers are regulated by co‐signalling receptors grouped into the binary categories of co‐stimulation or co‐inhibition. The co‐stimulation TNF receptor superfamily (TNFRSF) members 4‐1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T‐cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8⁺ T‐cell cytokine production. Although both 4‐1BB and CD27 increased production, only 4‐1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4‐1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co‐stimulation increases 4‐1BB expression and subsequent 4‐1BB co‐stimulation. GITR and OX40 displayed only minor effects on their own but, like 4‐1BB, CD27 could enhance GITR expression and subsequent GITR co‐stimulation. Thus, different co‐stimulation receptors can have different quantitative effects allowing for synergy and fine‐tuning of T‐cell responses.     

Reduced ELANE and SLPI expression compromises dental pulp cell activity

BackgroundPatients with ELANE variants and severe congenital neutropenia (SCN) commonly develop oral complications. Whether they are caused only by low neutrophil count or the combination of neutropenia and aberrant dental cells is unknown.MethodsGenetic variant was identified with exome sequencing. Dental pulp cells isolated from the SCN patient with an ELANE mutation were investigated for gene expression, enzyme activity, proliferation, colony formation, wound healing, apoptosis, ROS, attachment, spreading and response to lipopolysaccharide.ResultsELANE cells had diminished expression of ELANE and SLPI and reduced neutrophil elastase activity. Moreover, ELANE cells exhibited impaired proliferation, colony forming, migration, attachment and spreading; and significantly increased ROS formation and apoptosis, corresponding with increased Cyclin D1 and MMP2 levels. The intrinsic levels of TGF‐β1 and TNF‐α were significantly increased; however, IL‐6, IL‐8 and NF‐kB1 were significantly decreased in ELANE cells compared with those in controls. After exposure to lipopolysaccharide, ELANE cells grew larger, progressed to more advanced cell spreading stages and showed significantly increased SLPI, TNF‐α and NF‐kB1 and tremendously increased IL‐6 and IL‐8 expression, compared with controls.ConclusionThis study, for the first time, suggests that in addition to neutropenia, the aberrant levels and functions of ELANE, SLPI and their downstream molecules in pulp cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation.     

Stromal cell‐derived factor‐1/Exendin‐4 cotherapy facilitates the proliferation,migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo

ObjectivesStromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo.MethodsCell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo.ResultsSDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4⁺ cells and CD90⁺/CD34^‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo.ConclusionsSDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.     

Ferulic acid (FA) protects human retinal pigment epithelial cells from H2O2‐induced oxidative injuries

The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H₂O₂)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H₂O₂‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H₂O₂‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H₂O₂‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.     

Insight into the antibacterial resistance of graphdiyne functionalized by silver nanoparticles

ObjectivesSilver nanoparticles (AgNPs) tend to aggregate spontaneously due to larger surface‐to‐volume ratio, which causes decreased antibacterial activity and even enhanced antimicrobial resistance (AMR). Here, we aim to improve the stability of AgNPs by employing a growth anchor graphdiyne (GDY) to overcome these shortcomings.Materials and Methods Bacillus subtilis and Escherichia coli were selected to represent gram‐positive and gram‐negative bacteria, respectively. Transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM)‐EDS mapping and inductively coupled plasma mass spectrometry (ICP‐MS) were carried out to characterize the physiochemical properties of materials. The antimicrobial property was determined by turbidimetry and plate colony‐counting methods. The physiology of bacteria was detected by SEM and confocal imaging, such as morphology, reactive oxygen species (ROS) and cell membrane.ResultsWe successfully synthesized a hybrid graphdiyne @ silver nanoparticles (GDY@Ag) by an environment‐friendly approach without any reductants. The hybrid showed high stability and excellent broad‐spectrum antibacterial activity towards both gram‐positive and gram‐negative bacteria. It killed bacteria through membrane destruction and ROS production. Additionally, GDY@Ag did not induce the development of the bacterial resistance after repeated exposure.ConclusionsGDY@Ag composite combats bacteria by synergetic action of GDY and AgNPs. Especially, GDY@Ag can preserve its bacterial susceptibility after repeated exposure compared to antibiotics. Our findings provide an avenue to design innovative antibacterial agents for effective sterilization.

Graphdiyne@silver nanoparticles (GDY@Ag) composite preserves its bacterial susceptibilities after repeated exposure compared to antibiotics.     

Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

TSP50 promotes hepatocyte proliferation and tumour formation by activating glucose‐6‐phosphate dehydrogenase (G6PD)

Background & AimsHepatocellular carcinoma (HCC) is a common malignant tumour with high morbidity and mortality. Metabolic regulation by oncogenes is necessary for tumour growth. Testes‐specific protease 50 (TSP50) has been found to promote cell proliferation in multiple tumour types. However, the mechanism that TSP50 promotes HCC progression are not known.MethodsHepatocyte proliferation was analysed by MTT and BrdU incorporation after TSP50 transfection. Furthermore, LC‐MS/MS, co‐immunoprecipitation and GST pull‐down assays were performed to analyse protein(s) binding to TSP50. Moreover, the site‐specific mutation of G6PD was used to reveal the key site critical for G6PD acetylation mediated by TSP50. Finally, the role of G6PD K171 acetylation regulated by TSP50 in cell proliferation and tumour formation was investigated.ResultsOur data suggest that the overexpression of TSP50 accelerates hepatocyte proliferation. In addition, G6PD is an important protein that binds to TSP50 in the cytoplasm. TSP50 activates G6PD activity by inhibiting the acetylation of G6PD at the K171 site. In addition, TSP50 promotes the binding of G6PD to SIRT2. Furthermore, the K171ac of G6PD regulated by TSP50 is required for TSP50‐induced cell proliferation in vitro and tumour formation in vivo. Additionally, according to The Cancer Genome Atlas (TCGA) programme, TSP50 and G6PD are negatively correlated with the survival of HCC patients.ConclusionsCollectively, our findings demonstrate that TSP50‐induced cell proliferation and tumour formation are mediated by G6PD K171 acetylation.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.