Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Xuebijing alters tumor necrosis factor-alpha, interleukin-1beta and p38 mitogen activated protein kinase content in a rat model of cardiac arrest following cardiopulmonary resuscitation

We established a rat model of cardiac arrest by clamping the endotracheal tube of adult rats at expiration. Twenty-four hours after cardiopulmonary resuscitation, nerve cell injury and expression of tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase content were increased. Rats injected with Xuebijing, a Chinese herb compound preparation, exhibited normal cellular structure and morphology, dense neuronal cytoplasm, and decreased tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase expression at 24 hours following cardiopulmonary resuscitation. These data suggest that Xuebijing can attenuate neuronal injury induced by hypoxia and reperfusion during cardiopulmonary resuscitation.

Role of Activator Protein-1 in the Transcription of Interleukin-5 Gene Regulated by Protein Kinase C Signal in Asthmatic Human TLymphocytes

Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

“Three Methods and Three Points” regulates p38 mitogen-activated protein kinase in the dorsal horn of the spinal cord in a rat model of sciatic nerve injury

Tuina is a traditional Chinese treatment for sensory disturbances caused by peripheral nerve injury and related diseases. Our previous studies showed that tuina regulates relevant regions and indices of the spinal dorsal horn using the Dian, Bo, and Rou method in Yinmen（BL37）, Yanglingquan（GB34）, and Weizhong（BL40）. Treatment prevents muscle atrophy, protects spinal cord neurons, and promotes sciatic nerve repair. The mechanisms of action of tuina for treating peripheral nerve injury remain poorly understood. This study established rat models of sciatic nerve injury using the crushing method. Rats received Chinese tuina in accordance with the principle of ＂Three Methods and Three Points,＂ once daily for 20 days. Tuina intervention reduced paw withdrawal latency and improved wet weight of the gastrocnemius muscle, as well as promoting morphological recovery of sciatic nerve fibers, Schwann cells, and axons. The protein expression levels of phospho-p38 mitogen-activated protein kinase, tumor necrosis factor-α, and interleukin-1β also decreased. These findings indicate that ＂Three Methods and Three Points＂ promoted morphological recovery and improved behavior of rats with peripheral nerve injury.

Efficacy of Sishen Wan(四神丸) on dinitrobenzene sulfonic acid-induced ulcerative colitis and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB signal pathway

OBJECTIVE:To investigate the therapeutic effect of Sishen Wan(四神丸,SSW)on ulcerative colitis(UC)induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB(TLR2/IRAK4/NF-κB)signaling pathway in colonic tissue.METHODS:In this study,120 Sprague–Dawley rats were randomly divided into blank and model groups.The experimental UC model in rats was established by subcutaneous injection of hydrocortisone+senna gavage for 21 d+dinitrobenzene sulfonic acid(DNBS)/ethanol solution enema.The successful model rats were randomly divided into the model group;mesalazine(0.36 g/kg)group;and high-,medium-,and low-dose SSW(24,12,and 6 g/kg)groups.The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d.The general condition of the rats was observed,and the body mass,fecal properties,and occult blood were recorded for calculating the disease activity index(DAI)score.The colonic tissue of the rats was collected,and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index(CMDI)score.Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group,apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling staining,and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2,myeloid differentiation primary response gene 88(My D88),IRAK4,and NF-κB p65 mRNA in the colon tissue.The expressions of TLR2,My D88,IRAK4,and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay,and the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the colon tissue were determined using enzyme linked immunosorbent assay.RESULTS:Compared with the blank group,the general condition of the model group was relatively poor.The DAI and CMDI scores of the model group increased significantly(P<0.01),the glands and intestinal mucosa disappeared partially,and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group.Furthermore,the cell apoptosis and expression levels of TLR2,My D88,IRAK4,and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly(P<0.01).The levels of IL-1βand TNF-αincreased significantly in the colon tissue of rats in the model group(P<0.01).After treatment with SSW,compared with the model group,the general condition of the UC rats improved.Moreover,the DAI and CMDI scores of the UC rats decreased significantly(P<0.05),and the pathological changes in the colon tissue of the UC rats tended to be normal.The cell apoptosis and expression levels of TLR2,My D88,IRAK4,and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually(P<0.01),and the levels of IL-1βand TNF-αdecreased significantly(P<0.01).CONCLUSION:SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats.Additionally,SSW can inhibit the TLR2/IRAK4/NF-κB signaling pathway,but further studies are required to confirm it.

Functional polymorphism in exon 5 and variant haplotype of the interleukin-1 receptor-associated kinase 1 gene are associated with susceptibility to and severity of sepsis in the Chinese population

Background The interleukin-1 （IL-1） receptor-associated kinase 1 （IRAK1） is believed to play an important role in the pathogenesis of sepsis. Recent studies have suggested that the I RAK1 functional genetic variant could affect the severity of sepsis in Caucasians. In this report, we have investigated whether polymorphisms at the IRAK1 gene are associated with the susceptibility to and severity of sepsis among the Chinese population. Methods Haplotype-tagging single nucleotide polymorphisms （htSNPs） were selected from the HapMap database. They.were genotyped in 255 patients with sepsis and 260 control subjects by PCR/restriction fragment length polymorphism （RFLP） analysis. The association between the selected htSNPs and the susceptibility to and severity of sepsis were estimated by Logistic regression with adjustments for age, sex, smoking, drinking, chronic disease status, Acute Physiology and Chronic Health Evaluation （APACHE） II score and primary diseases. Results rs1059702 was selected to represent the six linked htSNPs for IRAKI. Genotype frequencies of the htSNPs were in Hardy-Weinberg equilibrium for females, as were allele frequencies for both sex groups. Associations were observed in females between the htSNPs C/C genotype and increased susceptibility to sepsis （odds ratio （OR）, 5.46; 95% confidence interval （C/）, 1.12-26.67; P=0.018）, and such associations were also observed between the IRAK1 variant haplotype （CC/C-allele） and increased susceptibility to sepsis （OR, 1.68; 95% C/, 1.05-2.70; P=0.031） when compared with the T/T ＋ T/C genotype and the wild-type haplotype （TC ＋ TT/T-allele）. In the multiple organ dysfunction syndrome （MODS） subgroup, the variant haplotype was also associated with increased severity of sepsis （OR, 2.37; 95% Cl, 1.13-4.94; P=-0.02） when compared with the wild haplotype. This association was not significant in male patients. Conclusions The functional polymorphism in exon 5 and the variant haplotype of IRAK1 gene mediate susceptibility to and severity of sepsis. IRAK1 might be a genetic risk factor for the occurrence and development of sepsis in the Chinese population.

Approach to loss of response to advanced therapies in inflammatory bowel disease

BACKGROUND Remarkable progress over the last decade has equipped clinicians with many options in the treatment of inflammatory bowel disease.Clinicians now have the unique opportunity to provide individualized treatment that can achieve and sustain remission in many patients.However,issues of primary non-response(PNR)and secondary loss of response(SLOR)to non-tumour necrosis factor inhibitor(TNFi)therapies remains a common problem.Specific issues include the choice of optimization of therapy,identifying when dose optimization will recapture response,establishing optimal dose for escalation and when to switch therapy.AIM To explores the issues of PNR and SLOR to non-TNFi therapies.METHODS This review explores the current evidence and literature to elucidate management options in cases of PNR/SLOR.It will also explore potential predictors for response following SLOR/PNR to therapies including the role of therapeutic drug monitoring(TDM).RESULTS In the setting of PNR and loss of response to alpha-beta7-integrin inhibitors and interleukin(IL)-12 and IL-23 inhibitors dose optimization is a reasonable option to capture response.For Janus kinase inhibitors dose optimization can be utilized to recapture response with loss of response.CONCLUSION The role of TDM in the setting of advanced non-TNFi therapies to identify patients who require dose optimization and as a predictor for clinical remission is not yet established and this remains an area that should be addressed in the future.

Inflammation-mediated age-dependent effects of casein kinase 2-interacting protein-1 on osteogenesis in mesenchymal stem cells

Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.

Adaptive and regulatory mechanisms in aged rats with postoperative cognitive dysfunction

Inflammation may play a role in postoperative cognitive dysfunction. 5＇ Adenosine monophos- phate-activated protein kinase, nuclear factor-kappa B, interleukin-1β, and tumor necrosis factor-a are involved in inflammation. Therefore, these inflammatory mediators may be involved in postoperative cognitive dysfunction. Western immunoblot analysis revealed 5＇ adenosine mo- nophosphate-activated protein kinase and nuclear factor-kappa B in the hippocampus of aged rats were increased 1-7 days after splenectomy. Moreover, interleukin-1β and tumor necrosis fac- tor-α were upregulated and gradually decreased. Therefore, these inflammatory mediators may participate in the splenectomy model of postoperative cognitive dysfunction in aged rats.

Regulation of CD137 expression through K-Ras signaling in pancreatic cancer cells

Background:The interaction between CD137 and its ligand(CD137L)plays a major role in the regulation of immune functions and affects cancer immunotherapy.CD137 is a cell surface protein mainly located on activated T cells,and its regulation and functions in immune cells are well established.However,the expression of CD137 and its regulation in cancer cells remain poorly understood.The main purposes of this study were to examine the expression of CD137 in pancreatic cancer cells and to investigate its underlying mechanisms.Methods:Cells containing inducible K-RasG12V expression vector or with different K-Ras mutational statuses were used as in vitro models to examine the regulation of CD137 expression by K-Ras.Various molecular assays were employed to explore the regulatory mechanisms.Tumor specimens from 15 pancreatic cancer patients and serum samples from 10 patients and 10 healthy donors were used to test if the expression of CD137 could be validated in clinical samples.Results:We found that the CD137 protein was expressed on the cell surface in pancreatic cancer tissues and cancer cell lines.Enzyme-linked immunosorbent assay revealed no difference in the levels of secreted CD137 in the sera of patients and healthy donors.By using the K-Ras inducible cell system,we further showed that oncogenic K-Ras up-regulated CD137 through the activation of MAPK(mitogen-activated protein kinases)and NF-κB(nuclear factor kappa-light-chain-enhancer of activated B cells)pathways,as evidenced by significantly reduced CD137 mRNA expression led by genetic silencing of MAPK1 and p65,the key proteins involved in the respective pathways.Further-more,we also found that the NF-κB pathway was mainly stimulated by the K-Ras-induced secretion of interleukin-1α(IL-1α)which promoted the transcription of the CD137 gene in pancreatic cancer cell lines.Analysis of the TCGA(the cancer genome atlas)database also revealed a significant correlation between IL-1αand CD137 expression(r=0.274)in tumor samples from pancreatic cancer patients(P<0.001).Conclusions:The present study has demonstrated that the CD137 protein was expressed on pancreatic cancer cell surface,and has identified a novel mechanism by which K-Ras regulates CD137 in pancreatic cancer cells through MAPK and NF-κB pathways stimulated by IL-1α.

Atorvastatin attenuates IL-6 production partly via activating heme oxygenase-1 in LPS-stimulated RAW264.7 macrophages

Background Statins are known as a lipid-lowering drug as well as anti-inflammatory effect, this article aimed to evaluate the effect of atorvastatin on LPS-induced interleukin-6 （IL-6） production and determine the related mechanisms in RAW264.7 macrophages. Methods The levels of IL-6 were determined by enzyme linked immunosorbent assay （ELISA）. The levels of mRNA and protein expression of IL-6 and heme oxygenase-1 （HO-1） were respectively determined by quantitative PCR and western-blot. Results LPS could significantly increase mRNA expression of IL-6 and its secretion in dose- and time-dependent manners, which could be significantly attenuated by atorvastatin. In addition, HO-1 expression could be significantly increased by atorvastatin treatment, and it could be remarkably attenuated by SB203580 and PD98059 but not SP600125, which suggests that extracellular signal-regulated kinase （ERK） and p38 mitogen-activated protein kinase （MAPK） pathways participate in regulating the above-mentioned effects of atorvastatin on HO-1 expression. In addition, SnPP, a kind of HO-1 activity inhibitor could significantly attenuate atorvastatin’s effects on IL-6 expression and secretion in LPS-stimulated RAW264.7 macrophages. Conclusions Atorvastatin can attenuate LPS-induced IL-6 expression and secretion by activating HO-1 via ERK and p38 MAPK pathways, which helps to explain atorvastatin has pleiotropic benefits for the treatment of diseases associated with inflammation.

Celastrol targets IRAKs to block Toll-like receptor 4-mediated nuclear factor-κB activation

OBJECTIVE： Celastrol has been established as a nuclear factor-κB（NF-κB） activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that int erleukin-1 receptor-associated kinases（IRA Ks） are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB（e.g., the inter leukin-1 receptor（IL-1R）/Toll- like receptor（TLR） superfamily）.METHODS： The human hepatocellular cell line（Hep G2） treated with palmitic acid（PA） was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions.RESULTS： The results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the Hep G 2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation.CONCLUSION： The results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.