Inhibition of p38 MAPK Reduces Expression of Vascular Endothelial Growth Factor in Allergic Airway Disease

Background

The p38 mitogen-activated protein kinase (MAPK) appears to play an important role in various pathophysiological responses and has been suggested to be involved in many processes considered critical to the inflammatory response and tissue remodeling. Bronchial asthma is a chronic inflammatory disorder of the airway accompanied by increased vascular permeability. Vascular endothelial growth factor (VEGF) is a potent stimulator of bronchial inflammation, airway remodeling, and physiologic dysregulation that augments antigen sensitization and T-helper type 2 cell (Th2)-mediated inflammation in allergic airway diseases. However, there are little data on the relationship between p38 MAPK signaling and VEGF expression in allergic airway disease.

Objective

This study aimed to investigate the role of p38 MAPK on the pathogenesis of allergic airway disease, more specifically in VEGF expression.

Methods

Using ovalbumin (OVA)-inhaled mice and a selective p38 MAPK inhibitor, SB 239063, the involvement of p38 MAPK in allergen-induced VEGF expression in the airway was evaluated.

Results

The increases of phosphorylation of p38 MAPK, VEGF protein expression, and vascular permeability in the lung after OVA inhalation were decreased substantially by the administration of SB 239063. In addition, SB 239063 significantly reduced the increase of Th2 cytokines and OVA-specific IgE. The inhibition of p38 MAPK or VEGF signaling prevented and also decreased the increases in the number of inflammatory cells and airway hyperresponsiveness in OVA-induced allergic airway disease.

Conclusions

These results indicate that inhibition of p38 MAPK may attenuate allergen-induced airway inflammation and vascular leakage through modulation of VEGF expression in mice.     

Carrageenan-induced transient inflammation in a rabbit knee model: molecular changes consistent with an early osteoarthritis phenotype

Objective

Inflammation following a knee injury is one of the factors associated with initiation of cartilage degeneration leading to osteoarthritis (OA). The hypothesis tested was that inflammation results in elevated expression of proteinases implicated in OA.

Methods

Mature female rabbits received a single carrageenan injection to the right hind knee and the left knee served as the control. Five animals were killed at time points of 1, 2 and 4?weeks. The synovium and cartilage from both knees were collected and analysed for specific mRNA levels.

Results

Interleukin (IL)-1β and IL-6 mRNA levels peaked at 2?weeks and returned to normal levels in tissues by 4?weeks post-carrageenan treatment. Matrix metalloproteinase (MMP)-13, MMP-1, MMP-3 and cathepsin K followed the trend set by the inflammatory cytokines. Both synovium and cartilage tissues exhibited similar patterns of molecular expression, with cartilage from the tibial plateau responding more strongly than the femoral condyles.

Conclusions

The acute inflammatory milieu controls the transient expression of many degradative proteinases in the knee. However, a single acute exposure to inflammation in the rabbit knee is insufficient to create a chronic inflammatory environment and other complementary factors, such as persistent mechanical instability and/or injury, may contribute to the establishment of OA.     

The p38 mitogen-activated protein kinases modulate endothelial cell survival and tissue repair

Objective and design

This study is designed to investigate the role of p38 MAPK in modulating human pulmonary artery endothelial cells (HPAECs) survival and tissue repair functions.

Methods

HPAECs (passage 8?C12) were used for all experiments. Cells were treated with IL-1?? (0.5 or 2?ng/ml) or p38 inhibitor (SB203580 or SB220025, 5???M each). Cells were also transfected with 50?nM siRNAs. Cell length was measured using ImageJ software. Collagen gel contraction and wound close assay were performed to evaluate tissue repair functions.

Results

IL-1?? activated p38 MAPK and induced morphologic change of HPAECs. The p38 inhibitors further augmented IL-1??-induced cell morphologic change, prevented cell death, and augmented collagen gel contraction. Suppression of p38??, ??, or ??, but not p38?? resulted in cell morphologic alteration, and suppressing any one of p38 isoforms by siRNAs increased cell survival. Suppression of p38?? or ?? augmented gel contraction. While p38?? suppression stimulated cell migration, suppressing the rest of three isoforms inhibit cell migration. Nuclear factor p65-siRNA blocked IL-1??-induced cell morphologic change, but did not affect p38 inhibitor-induced change.

Conclusion

These findings suggest that p38 MAPK may negatively modulate tissue repair functions of endothelial cells via p65 independent pathway.     

CD133+CXCR4+ colon cancer cells exhibit metastatic potential and predict poor prognosis of patients

Background

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. It is caused by mutations/deletions of the survival motor neuron 1 (SMN1) gene and is typified by the loss of spinal cord motor neurons, muscular atrophy, and in severe cases, death. The SMN protein is ubiquitously expressed and various cellular- and tissue-specific functions have been investigated to explain the specific motor neuron loss in SMA. We have previously shown that the RhoA/Rho kinase (ROCK) pathway is misregulated in cellular and animal SMA models, and that inhibition of ROCK with the chemical Y-27632 significantly increased the lifespan of a mouse model of SMA. In the present study, we evaluated the therapeutic potential of the clinically approved ROCK inhibitor fasudil.

Methods

Fasudil was administered by oral gavage from post-natal day 3 to 21 at a concentration of 30 mg/kg twice daily. The effects of fasudil on lifespan and SMA pathological hallmarks of the SMA mice were assessed and compared to vehicle-treated mice. For the Kaplan-Meier survival analysis, the log-rank test was used and survival curves were considered significantly different at P < 0.05. For the remaining analyses, the Student's two-tail t test for paired variables and one-way analysis of variance (ANOVA) were used to test for differences between samples and data were considered significantly different at P < 0.05.

Results

Fasudil significantly improves survival of SMA mice. This dramatic phenotypic improvement is not mediated by an up-regulation of Smn protein or via preservation of motor neurons. However, fasudil administration results in a significant increase in muscle fiber and postsynaptic endplate size, and restores normal expression of markers of skeletal muscle development, suggesting that the beneficial effects of fasudil could be muscle-specific.

Conclusions

Our work underscores the importance of muscle as a therapeutic target in SMA and highlights the beneficial potential of ROCK inhibitors as a therapeutic strategy for SMA and for other degenerative diseases characterized by muscular atrophy and postsynaptic immaturity.     

The role of Rho/Rho-kinase pathway in the expression of ICAM-1 by linoleic acid in human aortic endothelial cells

Linoleic acid (LA), a dietary unsaturated fatty acid, has been known to increase the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) through the activation of nuclear factor-kappa B. Rho/Rho-kinase (ROCK) pathway mediates various cellular functions related to cardiovascular disease and affects the expression of ICAM-1. However, the exact mechanism underlying this action has not been fully elucidated. In this study, we aimed to find out the role of Rho/ROCK pathway in LA-induced ICAM-1 expression in human aortic endothelial cells (HAECs). We found that LA increased ICAM-1 expression and phosphorylation of ROCK and MYPT-1, a distal signal of ROCK. Y-27632, a ROCK inhibitor, suppressed ICAM-1 expression and phosphorylation of MYPT-1 induced by LA. The effect of LA on the increased phosphorylation of MYPT1 and expression of ICAM-1 was abolished by knocking down RhoA and ROCK2 protein level expression using small interfering RNA. LA increased NF-κB DNA-binding activity, which was inhibited with pretreatment with Y-27632. This study suggests that Rho/ROCK pathway plays a role in LA-induced ICAM-1 expression, which is possibly mediated by NF-κB in HAECs.     

Cyclic strain-induced HSP27 phosphorylation modulates actin filaments in airway smooth muscle cells

Mechanical stress (cyclic deformational strain) increases proteins of cytoskeletal and contractile domains in airway smooth muscle (ASM) cells in a manner that increases cell contractility. Here we studied the role of HSP27 in strain-induced microfilament formation and stability. Cultured ASM cells showed rapid phosphorylation of HSP27 upon cyclic strain within a few minutes that continued for 30 to 40 minutes. Such increases in HSP27 phosphorylation were abolished with SB 202190, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by PD 98059 (an inhibitor of extracellular regulated kinase), GF109203X (an inhibitor of protein kinase C), or Y27632 (an inhibitor of Rho kinase). Direct activation of RhoA by GTPgammaS did not alter the level of HSP27 phosphorylation. Confocal microscopy revealed that cells pre-incubated with SB 202190, and/or Y27632 resulted in disorganization of stress fibers upon strain, unlike PD 98059 and GF 1092030X, suggesting that both p38 MAPK and Rho kinase were necessary for strain-induced microfilament formation. To determine the relationship between HSP27 and RhoA in strain-induced microfilament formation, cells were transfected with various isoforms of HSP27 and RhoA before strain. Co-expression of inactive HSP27 (3A-HSP27) with constitutively active EGF-RhoA (RhoV14) caused diminution of microfilaments compared with constitutive active EGFP-RhoA (RhoV14) alone, suggesting that HSP27 is necessary for microfilament stability. Similarly, expression of phosphomimicking HSP27 (3D-HSP27) was sufficient for retaining microfilament formation even when co-expressed with the dominant-negative RhoA (EGFP-RhoN17). Thus, HSP27 activation is necessary for microfilament stability independently of RhoA activation.     

Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity

Objectives

Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions.

Methods

Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry.

Results

In the presence of a TNF-α inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-α, and this increased expression was also inhibited by simvastatin. TNF-α also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression.

Conclusion

Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.     

Sorbitol-modified hyaluronic acid reduces oxidative stress,apoptosis and mediators of inflammation and catabolism in human osteoarthritic chondrocytes

Objective and design

Our study was designed to elucidate the precise molecular mechanisms by which sorbitol-modified hyaluronic acid (HA/sorbitol) exerts beneficial effects in osteoarthritis (OA).

Methods

Human OA chondrocytes were treated with increasing doses of HA/sorbitol ± anti-CD44 antibody or with sorbitol alone and thereafter with or without interleukin-1beta (IL-1β) or hydrogen peroxide (H₂O₂). Signal transduction pathways and parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated.

Results

HA/sorbitol prevented IL-1β-induced oxidative stress, as measured by reactive oxygen species, p47-NADPH oxidase phosphorylation, 4-hydroxynonenal (HNE) production and HNE-metabolizing glutathione-S-transferase A4-4 expression. Moreover, HA/sorbitol stifled IL-1β-induced metalloproteinase-13, nitric oxide (NO) and prostaglandin E2 release as well as inducible NO synthase expression. Study of the apoptosis process revealed that this gel significantly attenuated cell death, caspase-3 activation and DNA fragmentation elicited by exposure to a cytotoxic H₂O₂ dose. Examination of signaling pathway components disclosed that HA/sorbitol prevented IL-1β-induced p38 mitogen-activated protein kinase and nuclear factor-kappa B activation, but not that of extracellular signal-regulated kinases 1 and 2. Interestingly, the antioxidant as well as the anti-inflammatory and anti-catabolic effects of HA/sorbitol were attributed to sorbitol and HA, respectively.

Conclusions

Altogether, our findings support a beneficial effect of HA/sorbitol in OA through the restoration of redox status and reduction of apoptosis, inflammation and catabolism involved in cartilage damage.     

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor ��1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation

Objective and design

To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs).

Material

Using human LX-2 HSC cells, we examined the effects of PPC on expression of ??-smooth muscle actin (??-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor ??1 (TGF??1).

Results

PPC suppressed ROS which are induced by TGF??1, phosphorylation of p38MAPK, and expression levels of ??-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels.

Conclusion

These results suggest that ROS and Nox4 induced by TGF??1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.     

Over-expression of caveolin-1 aggravate LPS-induced inflammatory response in AT-1 cells via up-regulation of cPLA2/p38 MAPK

Objective and design

The aim of this study was to study the effect of caveolin-1 on the cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB (NF-κB) in mouse lung alveolar type-1 cells' (AT-1 cells) inflammatory response induced by LPS.

Materials and methods

Gene clone technique was used to over-express caveolin-1 in AT-1 cells by lentivirus vector. The level of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), cPLA2, p38 MAPK and NF-κB was measured by ELISA, western blotting and EMSA.

Treatment

AT-1 cells were treated with LPS.

Results

Over-expression of caveolin-1 not only increased the production of pro-inflammatory cytokine TNF-α and IL-6, but also enhanced the expression of the cPLA2, p38 MAPK, and NF-κB.

Conclusions

Our data demonstrated that over-expression of caveolin-1 aggravates the AT-1 injury induced by LPS, involving in modulation of the cPLA2 mediated by the cPLA2/p38 MAPK pathway.     

M3 mAChR-mediated IL-8 expression through PKC/NF-κB signaling pathways

Objective

M3 muscarinic acetylcholine receptor (mAChR) plays an important role in the regulation of cytokine production in inflammatory diseases. In this study, we explored the precise role of M3 mAChR under stimulation with agonist in IL-8 expression and of the signaling pathway involved in this process.

Materials and methods

Recombinant U2OS cells stably expressing M3 mAChR as a model system were stimulated by carbachol to evaluate the role of M3 mAChR in the expression of IL-8.

Results

Activation of M3 mAChR with carbachol increased both IL-8 mRNA and protein expression in a concentration-dependent manner. Elevated IL-8 expression was completely antagonized by atropine, 4-DAMP and tiotropium. M3 mAChR-mediated IL-8 expression was almost completely inhibited by the NF-κB inhibitor BAY11-7082 and, to a lesser extent, by U0126, SB203580, and SP600125, which are inhibitors for ERK1/2, p38, and JNK, respectively. Furthermore, M3 mAChR-mediated NF-κB activation and IL-8 expression were simultaneously attenuated by the PKC inhibitor calphostin C, whereas PMA, a PKC activator, mimicked the effects of carbachol, inducing IL-8 expression.

Conclusions

Our findings offer insights into the specific and critical role of M3 mAChR in regulating inflammatory response and indicate M3 mAChR/PKC/NF-κB signaling axis driven by endogenous acetylcholine as a potential therapeutic targets for inflammatory diseases.     

Anti-arthritic effects of crocin in interleukin-1β-treated articular chondrocytes and cartilage in a rabbit osteoarthritic model

Objective

Interleukin-1β-mediated production of matrix metalloproteinases (MMPs) plays a pivotal role in the process of osteoarthritis. Crocin, a pharmacologically active component of Crocus sativus L. (saffron), has been used in Chinese traditional medicine. In this study, we aimed to investigate the effects of crocin on MMP-1, MMP-3 and MMP-13 expression in rabbit chondrocytes induced by interleukin-1β (IL-1β) and in an experimental rabbit model induced by anterior cruciate ligament transection.

Methods

Chondrocytes isolated from the articular cartilage of 4-week-old rabbits were cultured and passaged. Confluent chondrocytes were treated with various concentrations of crocin in the presence or absence of IL-1β (10 ng/ml) for 24 h. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting were used to investigate the expression of inducible MMP-1, MMP-3 and MMP-13. In addition, the in-vivo effects of crocin were assessed by morphological and histological analysis.

Results

IL-1β markedly upregulated the expression of MMP-1, -3 and -13 in chondrocytes, and this activation was inhibited by co-incubation with crocin in a dose-dependent manner, in contrast with the control group. Moreover, crocin inhibited IL-1β-induced activation of the nuclear factor kappa B pathway through suppressing degradation of inhibitory-kappa-B-α. In-vivo investigations showed that crocin ameliorated cartilage degeneration and that expression of the MMP-1, -3 and -13 genes in cartilage was significantly inhibited by crocin.

Conclusion

Taken together, our findings suggest that the anti-inflammatory activity of crocin may be of potential value in the prevention and treatment of osteoarthritis.     

p38/ATF-2通路参与C反应蛋白诱导的内皮细胞活化

目的:考察p38 MAPK/ATF-2通路在C反应蛋白(CRP)诱导的内皮细胞活化中的作用。方法:采用培养的人冠状动脉内皮细胞(HCAEC)第3~7代用于实验。CRP刺激诱导内皮细胞活化,给予p38抑制剂SB203580和SB202190干预。免疫印迹法检测p-e NOS、p-p38和p-ATF2的水平;ELISA法测定HCAEC分泌的黏附分子ICAM-1、VCAM-1和MCP-1的变化。结果:CRP呈浓度依赖性地抑制p-e NOS水平,CRP诱导HCAEC分泌ICAM-1、VCAM-1和MCP-1;CRP激活p38/ATF-2通路;SB203580和SB202190部分恢复p-e NOS水平和抑制CRP诱导的ICAM-1、VCAM-1和MCP-1分泌。结论:p38 MAPK/ATF-2通路参与CRP诱导的HCAEC活化。     

Urinary trypsin inhibitor attenuates lipopolysaccharide-induced acute lung injury by blocking the activation of p38 mitogen-activated protein kinase

Objective

To investigate the protective effect of urinary trypsin inhibitor (UTI) in a rat model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the underlying molecular mechanism.

Methods

Rats were randomly assigned into three groups: control group, LPS treatment group and LPS/UTI treatment group. The serum concentrations of tumor necrosis factor (TNF)-?? and interleukin (IL)-10 were measured by ELISA. The expression of p38 mitogen-activated protein kinase (MAPK) in lung tissues was determined by Western blot analysis.

Results

Administration of UTI reduced the lung wet/dry weight ratio and ameliorated the tissue damage. In the LPS/UTI treatment group, levels of TNF-?? were significantly lower than those in the LPS treatment group, while the levels of IL-10 were significantly higher than those in the LPS treatment group. Western blot analysis revealed that UTI inhibited the phosphorylation of p38 MAPK in lung tissues.

Conclusions

UTI attenuates LPS-induced ALI, probably by adjusting the balance between proinflammatory and anti-inflammatory cytokines. The mechanism responsible for the decreased TNF-?? expression may be related to the inhibitory effect of UTI on p38 MAPK activation.     

Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells

Objective and design

Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at ?1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC.

Results

IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC.

Conclusions

Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.     

Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

Background

WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes.

Methods

The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed.

Results

WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1β-stimulated chondrocytes.

Conclusions

WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it’s up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.

     

Chemokine CXCL13 activates p38 MAPK in the trigeminal ganglion after infraorbital nerve injury

Recent data demonstrated that chemokine CXCL13 mediates neuroinflammation and contributes to the maintenance of neuropathic pain after nerve injury in the spinal cord. Pro-nociceptive chemokines activate mitogen-activated protein kinases (MAPKs) which are potential signaling pathways contributing to the nociceptive behavior in inflammatory or neuropathic pain. However, whether activation of p38 and JNK MAPK signaling pathway in the trigeminal ganglion (TG) are involved in CXCL13 and its receptor CXCR5-mediated orofacial pain has not yet been clarified. Here, we show that the unilateral partial infraorbital nerve ligation (pIONL) induced a profound orofacial pain in wild-type (WT) mice. Western blot results showed that pIONL induced p38 but not JNK activation in the TG of WT mice. However, the orofacial pain induced by pIONL was alleviated in Cxcr5 ^?/? mice, and the activation of p38 was also abrogated in Cxcr5 ^?/? mice. Furthermore, intra-TG injection of CXCL13 evoked mechanical hypersensitivity and increased p-p38 expression in WT mice. But CXCL13 had no effect on pain behavior or p-p38 expression in Cxcr5 ^?/? mice. Finally, pretreatment with p38 inhibitor, SB203580, attenuated the pIONL-induced mechanical allodynia and decreased the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the TG. Taken together, our data suggest that CXCL13 acts on CXCR5 to increase p38 activation and further contributes to the pathogenesis of orofacial neuropathic pain.