Repeat exposure to polyinosinic:polycytidylic acid induces TLR3 expression via JAK-STAT signaling and synergistically potentiates NFκB-RelA signaling in ARPE-19 cells

Dry age-related macular degeneration (AMD), accounting for approximately 90% of AMD cases, is characterized by photoreceptor death, retinal pigment epithelium (RPE) dysfunction and, ultimately, geographic atrophy – the localized death of RPE leading to loss of the center of the visual field. The pathological etiology of AMD is multifactorial, but innate immune signaling and inflammation are involved in early stages of the disease. Although numerous single-nucleotide polymorphisms in innate immune genes are associated with dry AMD, no single gene appears to cause dry AMD.Here, we hypothesized that activation of TLR3 potentiates expression of TLR3 itself and the NFκB-p65 (RelA) subunit as part of pro-inflammatory RPE signaling. Furthermore, we hypothesized that TLR3 activation can ‘prime’ cells to future RelA stimulation, leading to enhanced, persistent RelA expression and signaling following a second TLR3 activation. We used the human RPE-derived cell line ARPE-19 as a model system for RPE signaling and measured NFκB expression and activity in response to TLR3 stimulation with its ligand, polyinosinic:polycytidylic acid (pI:C).Activation of TLR3 with pI:C led to increased TLR3 and RelA expression that was sustained for at least 24 h. Cells exposed for a second time to pI:C after an initial pI:C exposure displayed elevated RelA expression and RelA nuclear translocation above the level generated by individual primary or secondary exposures alone. Such an elevated response could also not be generated by a single application of higher concentrations of the agonist pI:C. Additionally, we determined the mechanism for TLR3 mediated TLR3 and RelA expression by using inhibitors of canonical TLR3-TBK1-IKKε and JAK-STAT signaling pathways.These data suggest that initial exposure of ARPE-19 cells to pI:C upregulates TLR3 and RelA signaling, leading to potentiated and persistent RelA signaling potentially generated by a positive feedback loop that may cause exacerbated inflammation in AMD. Furthermore, inhibition of JAK-STAT signaling may be a possible therapeutic treatment to prevent induction of TLR3 expression subsequent to pI:C exposure. Our results identify possible therapeutic targets to reduce the TLR3 positive feedback loop and subsequent overproduction of pro-inflammatory cytokines in RPE cells.     

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 ^−/−) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.     

Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of mortality and morbidity in the premature infant. Premature infants have a delay in intestinal colonization by commensal bacteria and colonization with potentially pathogenic organisms. Lactobacillus reuteri is a probiotic that inhibits enteric infections, modulates the immune system, and may be beneficial to prevent NEC. In previous studies, L. reuteri strains DSM 17938 and ATCC PTA 4659 differentially modulated inflammation in vitro; however, the strains had equivalent anti-inflammatory responses in LPS feeding-induced ileitis in neonatal rats in vivo. The impact of these two strains in the prevention of NEC has not been previously investigated. NEC was induced in newborn rats by orogastric formula feeding and exposure to hypoxia. L. reuteri was added to the formula to prevent NEC. NEC score, Toll-like receptor (TLR)-signaling genes, phospho-IκB activity, and cytokine levels in the intestine were examined. Both strains significantly increased survival rate and decreased the incidence and severity of NEC, with optimal effects from DSM 17938. In response to probiotic, mRNA expression of IL-6, TNF-α, TLR4, and NF-κB was significantly downregulated, while mRNA levels of anti-inflammatory cytokine IL-10 were significantly upregulated. In parallel, L. reuteri treatment led to decrease intestinal protein levels of TLR4 and cytokine levels of TNF-α and IL-1β in newborn rats with NEC. Both strains significantly inhibited not only intestinal LPS-induced phospho-IκB activity in an ex vivo study but also decreased the levels of phospho-IκB in the intestines of NEC rat model. Cow milk formula feeding produced a similar but milder proinflammatory profile in the intestine that was also ameliorated by 17938. Our studies demonstrate that each of the two L. reuteri strains has potential therapeutic value in our NEC model and in enteritis associated with cow milk feeding. These results support the concept that L. reuteri may represent a valuable treatment to prevent NEC.     

Exhaustive exercise increases inflammatory response via Toll like receptor-4 and NF-κBp65 pathway in rat adipose tissue

Cytokines (IL-6, IL-10, and TNF-α) are increased after exhaustive exercise in the retroperitoneal adipose tissue (RPAT) and mesenteric adipose tissue (MEAT). An exhaustive acute exercise protocol induces inflammation in adipose tissue that lasts 6 h after the exercise has ended. It is well-established that this protocol increases circulating plasma levels of non-esterified fatty acids (NEFAs) and lipopolysaccharides (LPS), compounds that are important in stimulating signaling via toll like receptor-4 (TLR-4) in different type cells. In the present study, we investigated the regulation of TLR-4 and DNA-binding of nuclear factor-κBp65 (NF-κBp65) in different depots of adipose tissue in rats after exhaustive exercise. Rats were killed by decapitation immediately (E0 group, n=6), 2 (E2 group, n=6), and 6 h (E6 group, n=6) after the exhaustive exercise, which consisted of running on a treadmill (approximately 70% V(O2max) ) for 50 min and then running at an elevated rate that increased at 1 m/min, until exhaustion. The control group (C group, n=6) was not subjected to exercise. In RPAT, TLR-4, MYD-88, and IkBα increased in the E2 group after exercise. MYD-88 and TRAF6 remained increased in the E6 group in comparison with the control group. DNA-binding of NF-κBp65 was not altered. In MEAT, TLR-4, MYD-88, TRAF6, and DNA-binding of NF-κBp65 were increased only in the E6 group. In conclusion, we have shown that increases in pro-inflammatory cytokines in adipose tissue pads after exhaustive exercise may be mediated via TLR-4 signaling, leading to increases in NF-κBp65 binding to DNA in MEAT.     

NF-κB signaling participates in both RANKL- and IL-4-induced macrophage fusion: receptor cross-talk leads to alterations in NF-κB pathways

NF-κB activation is essential for receptor activator for NF-κB ligand (RANKL)-induced osteoclast formation. IL-4 is known to inhibit the RANKL-induced osteoclast differentiation while at the same time promoting macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 inhibition of osteoclastogenesis is mediated by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block proximal, canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in nfκb1(-/-) bone marrow-derived macrophages (BMM), the formation of both multinucleated osteoclast and MNG induced by RANKL or IL-4, respectively, was impaired. This suggests that NF-κB signaling also plays an important role in IL-4-induced macrophage fusion. Indeed, we found that the RANKL-induced and IL-4-induced macrophage fusion were both inhibited by the NF-κB inhibitors IκB kinase 2 inhibitor and NF-κB essential modulator inhibitory peptide. Furthermore, overexpression of p50, p65, p52, and RelB individually in nfκb1(-/-) or nfκb1(+/+) BMM enhanced both giant osteoclast and MNG formation. Interestingly, knockdown of nfκb2 in wild-type BMM dramatically enhanced both osteoclast and MNG formation. In addition, both RANKL- and IL-4-induced macrophage fusion were impaired in NF-κB-inducing kinase(-/-) BMM. These results suggest IL-4 influences NF-κB pathways by increasing p105/p50 and suppressing RANKL-induced p52 translocation and that NF-κB pathways participate in both RANKL- and IL-4-induced giant cell formation.     

κ-Opioid receptor stimulation modulates TLR4/NF-κB signaling in the rat heart subjected to ischemia–reperfusion

It is well documented that the Toll-like receptor 4 (TLR4)/NF-κB signaling mediates early inflammation during myocardial ischemia and reperfusion. Our previous study has demonstrated that κ-opioid receptor stimulation with U50,488H produces cardioprotective and anti-inflammatory effects. The aim of the present study was to investigate whether κ-opioid receptor stimulation could modulate the TLR4/NF-κB signaling and reduce neutrophil accumulation and TNF-α induction in an ischemia–reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), and MI/R + U50,488H in the absence or presence of Nor-BNI, a selective κ-opioid receptor antagonist. The results demonstrated that after MI/R, the expressions of myocardial TLR4 and NF-κB increased significantly both in ischemia area and risking area. Compared with MI/R, κ-opioid receptor stimulation with U50,488H significantly attenuated the expressions of TLR4 and NF-κB. At the mean time, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-α production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R, all the effects of U50,488H were abolished by Nor-BNI. These data provide evidence for the first time that κ-opioid receptor stimulation inhibits TLR4/NF-κB signaling in the rat heart subjected to MI/R.     

Enhanced Wnt/β-catenin signalling during tooth morphogenesis impedes cell differentiation and leads to alterations in the structure and mineralisation of the adult tooth

BACKGROUND INFORMATION: Previous studies have indicated that over-activation of the wingless interaction site (Wnt)/β-catenin signalling pathway has important implications for tooth development, at the level of cell differentiation and morphology, as well as for the production of supernumerary teeth. Here, we provide evidence for a crucial role of this signalling pathway during the stage of tooth morphogenesis. We have developed an in vitro model consisting of 14.5-day-old mouse embryo first molars, in which the Wnt pathway is overactivated by the glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3'-oxime (BIO; 20 μM). RESULTS: We found that over-activation of the Wnt/β-catenin pathway delayed the differentiation and growth of the inner dental epithelium. In addition, in contrast to controls in which Nestin protein expression was restricted to differentiated odontoblasts, in BIO-treated molars, Nestin expression spread through sub-odontoblastic cellular layers. This alteration appears to be related to: (i) the over-expression of Bmp4 in the same region, (ii) the delay in odontoblast precursor cell differentiation and (iii) increased proliferation of mesenchymal cells. Furthermore, treatments longer than 6 days induced the malformation of typical dental structures and led to a total lack of cell differentiation. Finally, over-activation of the Wnt route during odontogenesis resulted in adult teeth which presented altered size, morphology and mineralisation. CONCLUSIONS: Our results indicate that Wnt/β-catenin over-activation during tooth morphogenesis is sufficient to cause dramatic alterations in the adult tooth, by delaying cellular differentiation and stimulating proliferation of the dental mesenchyme of developing teeth.     

Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae

The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pieris brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13·0 per cent and in the fat body of 4-day pupae 6·3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). The label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-1-¹⁴C 39·0 per cent of label was in PTC, 52·8 per cent in PTE, and 2·0 per cent in sphingomyelin. In late fifth instar 45·0 per cent was in PTC, 45·5 per cent in PTE, and 6·5 per cent in sphingomyelin, while in 4-day female pupae 45·2 per cent was in PTC, 41·3 per cent in PTE, and 13·5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51·8 per cent being in PTC and 48·2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55·8 and 43·2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58·6 per cent), PTE (24·4 per cent), and sphingomyelin (17·0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the rôle of the investigated fatty acids in the biosynthesis of PL are discussed.     

Role of TLR4/NF-κB in Damage to Intestinal Mucosa Barrier Function and Bacterial Translocation in Rats Exposed to Hypoxia

The role of Toll-like receptor 4 (TLR4)/nuclear factor-kappa-B (NF-κB) in intestinal mucosal barrier damage and bacterial translocation under hypoxic exposure is unclear. Here, we investigated their role using an acute hypobaric hypoxia model. Adult Sprague-Dawley rats were divided into control (C), hypoxia (H), hypoxia+NF-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) (100 mg. kg) (HP), hypoxia+0.5 mg/kg lipopolysaccharide (HPL), and hypoxia+PDTC+LPS (HPL) group. Except control group, other four groups were placed in a hypobaric chamber set at 7000 m. Samples were collected at 72 h after pressure reduction. Damage in ultrastructure of the intestinal tract was examined by transmission electron microscopy and bacterial translocation was detected by cultivation. Kinetic turbidimetric assay was used to measure the serum LPS.ELISA was performed to detect TNF-α and IL-6 serum concentrations. Fluorescent quantitative RT-PCR was used to measure TLR4 mRNA levels was measured using quantitative RT-PCR and protein of NF-κB p65 was measured by western blotting. Different degrees of intestinal mucosa damage were observed in groups H and HL. The damage was significantly alleviated after blockage of the TLR4/NF-κB signaling pathway. PDTC- treatment also reversed hyoxia- and LPS-induced bacterial translocation rate and increased serum levels of LPS, TNF-α, and IL-6. TLR4 mRNA levels and NF-κB p65 expression were consistent with the serum factor results. This study suggested that TLR4 and NF-κB expression increased in rat intestinal tissues after acute hypoxia exposure. PDTC-treatment reversed TLR4 and NF-κB upregulation and alleviated damage to the intestinal tract and bacterial translocation. Thus, the TLR4/NF-κB signaling pathway may be critical to the mechanism underlying hypoxia-induced damage to intestinal barrier function and bacterial translocation.     

Exposure to a maternal n-3 fatty acid-deficient diet during brain development provokes excessive hypothalamic–pituitary–adrenal axis responses to stress and behavioral indices of depression and anxiety in male rat offspring later in life

Brain docosahexaenoic acid (DHA, 22:6n-3) accumulates rapidly during brain development and is essential for normal neurological function. The aim of this study was to evaluate whether brain development was the critical period in which DHA deficiency leads to dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis in response to stress later in life. Rats were exposed to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet either throughout the preweaning period from embryo to weaning at 3 weeks old or during the postweaning period from 3 to 10 weeks old. Exposure to the n-3 fatty acid-deficient diet during the preweaning period resulted, at weaning, in a significant decrease in hypothalamic DHA levels and a reduced male offspring body weight. DHA deficiency during the preweaning period significantly increased and prolonged restraint stress-induced changes in colonic temperature and serum corticosterone levels, caused a significant increase in GABA_A antagonist-induced heart rate changes and enhanced depressive-like behavior in the forced swimming test and anxiety-like behavior in the plus-maze test in later life. These effects were not seen in male rats fed the n-3 fatty acid-deficient diet during the postweaning period. These results suggest that brain development is the critical period in which DHA deficiency leads to excessive HPA responses to stress and elevated behavioral indices of depression and anxiety in adulthood. We propose that these effects of hypothalamic DHA deficiency during brain development may involve a GABA_A receptor-mediated mechanism.     

Galectin-3 deficiency protects lipopolysaccharide-induced chondrocytes injury via regulation of TLR4 and PPAR-γ-mediated NF-κB signaling pathway

The aim of the present study was to identify the functional role of galectin-3 (Gal-3) in lipopolysaccharide (LPS)-induced injury in ATDC5 cells and to explore the probable molecular mechanisms. Here, we identified that LPS is sufficient to enhance the expression of Gal-3 in ATDC5 cells. In addition, repression of Gal-3 obviously impeded LPS-stimulated inflammation damage as exemplified by a reduction in the release of inflammatory mediators interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, as well as the production of nitric oxide and prostaglandin E2 (PGE2) concomitant with the downregulation of matrix metalloproteinases (MMP)-13 and MMP-3 expression in ATDC5 cells after LPS administration. Moreover, ablation of Gal-3 dramatically augmented cell ability and attenuated cell apoptosis accompanied by an increase in the expression of antiapoptotic protein Bcl-2 and a decrease in the expression of proapoptotic protein Bax and caspase-3 in ATDC5 cells subjected with LPS. Importantly, we observed that forced expression of TLR4 or blocked PPAR-γ with the antagonist GW9662 effectively abolished Gal-3 inhibition–mediated anti-inflammatory and antiapoptosis effects triggered by LPS. Mechanistically, depletion of Gal-3 prevents the NF-κB signaling pathway. Taken together, these findings indicated that the absence of Gal-3 exerted chondroprotective properties dependent on TLR4 and PPAR-γ-mediated NF-κB signaling, indicating that Gal-3 functions as a protector in the development and progression of osteoarthritis.     

PPARγ agonist rosiglitazone ameliorates LPS-induced inflammation in vascular smooth muscle cells via the TLR4/TRIF/IRF3/IP-10 signaling pathway

The activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) can induce inflammation that are one of key etiological conditions for the development of many chronic inflammatory diseases including atherosclerosis and diabetes. Peroxisome proliferator-activated receptor γ (PPARγ) agonists play a crucial role in improving glucose and lipid homeostasis in the development of cardiovascular diseases. Evidence is growing that benefits of PPARγ agonists may also be derived from the anti-inflammatory and anti-atherosclerotic properties of these agents. However, the role of rosiglitazone in regulating LPS-induced vascular inflammation has yet to be fully elucidated. The current study demonstrated that rosiglitazone exerted a potent anti-inflammatory action via decreasing interleukin-18 (IL-18), tissue inhibitor of metalloproteinase-1 (TIMP-1), TLR4 and increasing PPARγ in LPS-induced VSMCs. Furthermore, treatment of VSMCs with the TLR4 blocker or TLR4 small-interfering RNA presented that the regulatory effects of rosiglitazone on LPS-mediated inflammation in VSMCs were dependent on TLR4. Interestingly, the results indicated that beneficial effects of rosiglitazone on LPS-induced inflammation in VSMCs were mediated via interference of TLR4 and its downstream signaling components including Toll-interleukin-1 (IL-1) receptor domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF3) and interferon-gamma inducible protein 10 (IP-10). In summary, PPARγ agonist rosiglitazone exerts anti-inflammatory property by antagonizing LPS-mediated inflammation in VSMCs. More importantly, the regulation of the TRIF-dependent TLR4 signaling pathway (TLR4/TRIF/ IRF3/IP-10) provides new insight to understand the mode of action of rosiglitazone for its anti-inflammatory effects.     

5-Hydroxy-4-methoxycanthin-6-one alleviates dextran sodium sulfate-induced colitis in rats via regulation of metabolic profiling and suppression of NF-κB/p65 signaling pathway

Background5-Hydroxy-4-methoxycanthin-6-one (PQ-A) is the main active compound in Ramulus et Folium Picrasmae, a Chinese herbal medicine commonly used in colitis treatment.PurposeTo clarify PQ-A's role and mechanism in colitis treatment based on a non-targeted metabolomics study.MethodsRats with ulcerative colitis (UC) established with 4% dextran sulfate sodium (DSS) were orally treated with PQ-A. Body weight, disease activity index (DAI), colon length, biochemical parameters (MDA and SOD), and histopathological score in colon tissue were measured. A UPLC-Q-TOF-MS/MS approach-based metabolomics analysis was conducted to explore the underlying mechanisms of PQ-A in colitis treatment. Inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) concentrations in serum and their protein levels in the colon were determined. CD3 and NF-κB/p65 immunohistochemistry in the colon was semi-quantified. The related protein or mRNA in IKK-NF-κB/p65 signaling pathway was measured by Western blotting or RT-PCR, respectively. Potential molecular interactions between PQ-A and NF-κB/p65 was predicted using DS 2.5 software.ResultsPQ-A significantly prevented body weight loss and colonic shortening in colitic rats, and reduced the DAI and histopathologic score as well. PQ-A decreased MDA levels in the UC rat serum and increased those of SOD. Metabolomics results revealed forty-nine differential metabolites as biomarkers of DSS-induced colitis, demonstrating that the path-mechanism of colitis involved the perturbation of eight metabolic pathways, including alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, retinol metabolism, bile acid metabolism, et al. Thirty-six biomarkers were especially reversed to normal-like levels by PQ-A via regulation of alpha-linolenic acid and linoleic acid metabolism, sphingolipid metabolism, and retinol metabolism, which effectively hinted the potential pharmacological mechanism of PQ-A related to NF-κB/p65 inflammatory signaling. Molecular docking results predicted high affinity interaction between PQ-A and NF-κB/p65, involving hydrogen-bond interactions at five amino acid residues, suggesting NF-κB/p65 as a target. PQ-A decreased TNF-α, IL-1β, and IL-6 concentrations in serum and their protein levels in colon tissue in colitic rats. CD3, MYD88, p-IκBα, NF-κB/p65, and p-NF-κB/p65 expression levels decreased, whereas those of IKKβ and IκBα increased in colitic tissue following PQ-A treatment. PQ-A strongly inhibited nuclear translocation of NF-κB/p65.ConclusionsWe provide an overview of PQ-A's possible mechanism of action in colitis treatment based on serum non-targeted metabolomics. PQ-A treatment can protect rats against DSS-induced colitis by suppressing the NF-κB/p65 signaling pathway.     

Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κB-p53/p21 signaling

Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The β-galactosidase (SA-β-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-β-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.