Diacerein mitigates renal ischemia/reperfusion injury via inhibition of renal inflammation, dendritic cells maturation and apoptosis: the role of TLR4/NF-kB/NLRP3/IL-1beta signaling pathway / La diacereína mitiga la lesión por isquemia/reperfusión renal a través de la inhibición de la inflamación renal, la maduración de las células dendríticas y la apoptosis: el papel de la vía de señalización TLR4/NF-kB/NLRP3/IL-1beta

SUMMARY: One of the reasons for acute kidney damage is renal ischemia. Nevertheless, there are limited protective and therapeutic approaches for this problem. Diacerein is an anti-inflammatory drug characterized by numerous biological activities. We aimed to determine the ameliorative impact of diacerein on renal ischemia/reperfusion injury (I/R) condition, exploring the underlying mechanisms. Twenty-four male rats were allotted into four groups (n= 6): sham group; Diacerein (DIA) group; I/R group, in which a non-crushing clamp occluded the left renal pedicle for 45 min, and the right kidney was nephrectomized for 5 min before the reperfusion process; I/R + diacerein group, injected intraperitoneally with 50 mg diacerein/kg i.m 30 minutes prior to I/R operation. Ischemia/ reperfusion was found to affect renal function and induce histopathological alterations. The flow cytometry analysis demonstrated an elevated expression of innate and mature dendritic cells in I/R renal tissues. Moreover, upregulation in the expression of the inflammatory genes (TLR4, Myd88, and NLRP3), and overexpression of the pro-inflammatory cytokines (IL-1β), apoptotic (caspase-3) and pyroptotic (caspase-1) markers were observed in I/R-experienced animals. The aforementioned deteriorations were mitigated by pre-I/R diacerein treatment. Diacerein alleviated I/R-induced inflammation and apoptosis. Thus, it could be a promising protective agent against I/R.

La isquemia renal es una de los motivos del daño renal agudo. Sin embargo, los enfoques protectores y terapéuticos para este problema son limitados. La diacereína es un fármaco antiinflamatorio caracterizado por numerosas actividades biológicas. Nuestro objetivo fue determinar el impacto de mejora de la diacereína en la condición de lesión por isquemia/ reperfusión renal (I/R), explorando los mecanismos subyacentes. Veinticuatro ratas macho se distribuyeron en cuatro grupos (n= 6): grupo simulado; grupo de diacereína (DIA); grupo I/R, en el que una pinza no aplastante ocluyó el pedículo renal izquierdo durante 45 min, y el riñón derecho fue nefrectomizado durante 5 min antes del proceso de reperfusión; Grupo I/R + diacereína, inyectado por vía intraperitoneal con 50 mg de diacereína/kg i.m. 30 min antes de la operación I/R. Se encontró que la isquemia/ reperfusión afecta la función renal e induce alteraciones histopatológicas. El análisis de citometría de flujo demostró una expresión elevada de células dendríticas innatas y maduras en tejidos renales I/R. Además, se observó una regulación positiva en la expresión de los genes inflamatorios (TLR4, Myd88 y NLRP3) y una sobreexpresión de las citoquinas proinflamatorias (IL-1β), marcadores apoptóticos (caspasa-3) y piroptóticos (caspasa-1) en animales con experiencia en I/R. Los deterioros antes mencionados fueron mitigados por el tratamiento previo a la diacereína I/R. La diacereína alivió la inflamación y la apoptosis inducidas por I/R. Por lo tanto, podría ser un agente protector prometedor contra I/R.

Methylene blue reduces IL-1β levels by enhancing ERK1/2 and AKT phosphorylation to improve diabetic retinopathy in rats / 细胞与分子免疫学杂志

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.

"Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy / 中国中药杂志

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Prostate-derived IL-1β upregulates expression of NMDA receptor in the paraventricular nucleus and shortens ejaculation latency in rats with experimental autoimmune prostatitis / 亚洲男科学杂志(英文版)

Experimental autoimmune prostatitis (EAP)-induced persistent inflammatory immune response can significantly upregulate the expression of N-methyl-D-aspartic acid (NMDA) receptors in the paraventricular nucleus (PVN). However, the mechanism has not yet been elucidated. Herein, we screened out the target prostate-derived inflammation cytokines (PDICs) by comparing the inflammatory cytokine levels in peripheral blood and cerebrospinal fluid (CSF) between EAP rats and their controls. After identifying the target PDIC, qualified males in initial copulatory behavior testing (CBT) were subjected to implanting tubes onto bilateral PVN. Next, they were randomly divided into four subgroups (EAP-1, EAP-2, Control-1, and Control-2). After 1-week recovery, EAP-1 rats were microinjected with the target PDIC inhibitor, Control-1 rats were microinjected with the target PDIC, while the EAP-2 and Control-2 subgroups were only treated with the same amount of artificial CSF (aCSF). Results showed that only interleukin-1β(IL-1β) had significantly increased mRNA-expression in the prostate of EAP rats compared to the controls (P < 0.001) and significantly higher protein concentrations in both the serum (P = 0.001) and CSF (P < 0.001) of the EAP groups compared to the Control groups. Therefore, IL-1β was identified as the target PDIC which crosses the blood-brain barrier, thereby influencing the central nervous system. Moreover, the EAP-1 subgroup displayed a gradually prolonged ejaculation latency (EL) in the last three CBTs (all P < 0.01) and a significantly lower expression of NMDA NR1 subunit in the PVN (P = 0.043) compared to the respective control groups after a 10-day central administration of IL-1β inhibitors. However, the Control-1 subgroup showed a gradually shortened EL (P < 0.01) and a significantly higher NR1 expression (P = 0.004) after homochronous IL-1β administration. Therefore, we identified IL-1β as the primary PDIC which shortens EL in EAP rats. However, further studies should be conducted to elucidate the specific molecular mechanisms through which IL-1β upregulates NMDA expression.

Increased serum interleukin-9 and interleukin-1β are associated with depression in type 2 diabetes patients / Interleucina-9 e interleucina-1β séricas aumentadas estão associadas à depressão em pacientes com diabetes tipo 2

ABSTRACT Background: Co-morbid diabetes and depression are prevalent chronic conditions negatively affecting quality of life (QoL). Inflammation has been considered as an integral mechanism in patients with both diabetes and depression. Objective: The aim of the present study was to investigate depression and its association with interleukins (IL)-1β and IL-9 in type 2 diabetic patients (T2DM) and controls. The QoL in diabetic patient was also assessed. Methods: Eighty subjects were included, distributed among three groups: Group 1 - Healthy controls; Group 2 - T2DM patients without depression; Group 3 - T2DM patients with depression. Depression and QoL were assessed using Patient Health Questionnaire (PHQ-9) and The Audit of Diabetes-Dependent QoL (ADDQoL), respectively. IL-1β and IL-9 were measured in serum samples of all the patients using ELISA kit. Results: The PHQ score in the Group 3 was significantly higher as compared to Group 1. The ADDQoL scores in the Group 3 were significantly higher as compared to Group 2. Levels of IL-9 and IL-1β were elevated in Group 3, as compared to the other groups. Conclusion: This study showed positive association between depression and IL-1β, IL-9 in T2DM patients. Additionally, the diabetic patients have poorer quality of life, which is further worsened by the presence of depression. Thus, routine assessment for the presence of depression is suggested in T2DM patients.

RESUMO Introdução: O diabetes e a depressão comórbidas são condições crônicas prevalentes que afetam negativamente a qualidade de vida (QdV). A inflamação tem sido considerada como um mecanismo integral em pacientes com diabetes e depressão. Objetivo: Investigar a depressão e sua associação com interleucinas (IL)-1β e IL-9 em pacientes diabéticos tipo 2 (DM2) e controles. A QdV em diabéticos também foi avaliada. Métodos: Foram incluídos 80 indivíduos, divididos em três grupos: Grupo 1 - controles saudáveis; Grupo 2 - pacientes com DM2 sem depressão; Grupo 3 - pacientes com DM2 com depressão. A depressão e a QdV foram avaliadas usando o Questionário de Saúde do Paciente (Patient Health Questionnaire - PHQ-9) e a auditoria de QdV dependente de diabetes (Audit of Diabetes-Dependent Quality of Life - ADDQoL), respectivamente. IL-1β e IL-9 foram medidas em amostras de soro de todos os pacientes utilizando kit de ELISA. Resultados: O escore do PHQ no grupo 3 foi significativamente maior em comparação ao grupo 1. Os escores de ADDQoL no grupo 3 foram significativamente maiores em comparação ao grupo 2. Os níveis de IL-9 e IL-1β foram elevados no grupo 3, como em comparação com os outros grupos. Conclusão: Este estudo mostrou associação positiva entre depressão e IL-1β, IL-9 em pacientes com DM2. Além disso, os pacientes diabéticos têm pior QdV, o que é ainda piorado pela presença de depressão. Assim, a avaliação rotineira da presença de depressão é sugerida em pacientes com DM2.

Cytotoxicity, inflammation, biomineralization, and immunoexpression of IL-1β and TNF-α promoted by a new bioceramic cement

Abstract Aim To evaluate the cytotoxicity, biocompatibility and mineralization capacity of BIO-C PULPO, and MTA. Methodology L929 fibroblasts were cultured and MTT assay was used to determine the material cytotoxicity on 6, 24, and 48 h. A total of 30 male rats (Wistar) aged between 4 and 6 months, weighing between 250 and 300 g were used. Polyethylene tubes containing BIO-C PULPO, MTA, and empty tubes were implanted into dorsal connective tissue. After the experimental periods (7, 15, 30, 60, and 90 days) the tubes were histologically analyzed using hematoxylin-eosin (H&E), immunolabeling of IL-1β and TNF-α, and von Kossa staining, or without staining for polarized light analysis. The average number of inflammatory cells was quantified; the mineralization assessment was determined by the area marked in μm2 and semiquantitative immunolabeling analyses of IL-1β and TNF-α were performed. Then, data underwent statistical analysis with a 5% significance level. Results It was observed that BIO-C PULPO and MTA presented cytocompatibility at 6, 24, and 48 similar or higher than control for all evaluated period. On periods 7 and 15 days, BIO-C PULPO was the material with the highest number of inflammatory cells (p<0.05). On periods 30, 60, and 90 days, BIO-C PULPO and MTA presented similar inflammatory reactions (p>0.05). No statistical differences were found between Control, BIO-C PULPO, and MTA for immunolabeling of IL-1β and TNF-α in the different periods of analysis (p<0.05). Positive von Kossa staining and birefringent structures under polarized light were observed in all analyzed periods in contact with both materials, but larger mineralization area was found with BIO-C PULPO on day 90 (p<0.05). Conclusion BIO-C PULPO was biocompatible and induced mineralization similar to MTA.

Baicalin suppresses Propionibacterium acnes-induced skin inflammation by downregulating the NF-κB/MAPK signaling pathway and inhibiting activation of NLRP3 inflammasome

Acne is a kind of common, chronic skin condition caused by the inflammation of the sebaceous glands in hair follicles. Recent studies have demonstrated that baicalin (BA) possesses potential anti-inflammatory properties. In this study, we evaluated the anti-inflammatory activity of BA in vitro and in vivo. Heat-killed Propionibacterium acnes-induced THP-1 cells and live P. acnes-injected male Sprague Dawley rats were used for establishing the acne model. The rate of ear swelling was calculated, and the severity was determined by hematoxylin and eosin staining. The production of cytokines [interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF-α)] in the cell supernatant and ear tissue homogenates was measured by ELISA. Protein levels of JNK, ERK, P38, IκBα, P65, Nod-like receptor pyrin domain-containing 3 (NLRP3), pro-caspase-1, and IL-1β in THP-1 cells and ear tissues were detected by western blotting. NLRP3 and IL-1β were detected by immunohistochemistry, and the NLRP3, IL-1β and pro-caspase-1 mRNAs were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that BA decreased the expression of pro-inflammatory cytokines in vitro and in vivo. Moreover, BA down-regulated the phosphorylation of JNK, ERK1/2, and κBα and inhibited the nuclear translocation of p65. Furthermore, BA inhibited the activation of NLRP3 inflammasome, at both the gene and protein levels. Taken together, the results demonstrated that BA might exert its anti-inflammatory activity by inhibiting NF-κB/MAPK signaling pathways and consequently suppressing the activation of the NLRP3 inflammasome both in vivo and in vitro.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Involvement of TLR4 in the protective effect of intra-articular administration of curcumin on rat experimental osteoarthritis

Abstract Purpose In view of the principal role of Toll-like receptor 4 (TLR4) in mediating sterile inflammatory response contributing to osteoarthritis (OA) pathogenesis, we used lipopolysaccharide (LPS), a known TLR4 activator, to clarify whether modulation of TLR4 contributed to the protective actions of intra-articular administration of curcumin in a classical rat OA model surgically induced by anterior cruciate ligament transection (ACLT). Methods The rats underwent ACLT and received 50μl of curcumin at the concentration of 1 mg mL-1 and 10 μg LPS by intra-articular injection once a week for 8 weeks. Morphological changes of the cartilage and synovial tissues were observed. Apoptotic chondrocytes were detected using TUNEL assay. The concentrations of IL-1β and TNF-ɑ in synovial fluid were determined using ELISA kits. The mRNA and protein expression levels of TLR4 and NF-κB p65 were detected by real-time PCR and Western blotting, respectively. Results Intra-articular administration of curcumin significantly improved articular cartilage injury, suppressed synovial inflammation and down-regulated the overexpression of TLR4 and its downstream NF-κB caused by LPS-induced TLR4 activation in rat osteoarthritic knees. Conclusion The data suggested that the inhibition of TLR4 signal might be an important mechanism underlying a protective effect of local curcumin administration on OA.

Glycogen synthase kinase-3 beta inhibitors protectagainst the acute lung injuries resulting from acute necrotizing pancreatitis

Abstract Purpose The research is intended for clarification of the efficacy as well as the underlying mechanism of GSK-3β inhibitors on the advancement of acute lung injuries in acute necrotizing pancreatitis (ANP) in rats. Methods Seventy-two rats were randomly divided into 6 groups: (1)ANP-vehicle; (2)ANP-TDZD-8;(3)ANP-SB216763;(4)Sham-vehicle;(5)Sham-TDZD-8;(6)Sham-SB216763; Blood biochemical test, histopathological examination and immunohistochemical analysis of rats pancreas and lung tissues were performed. The protein expression of GSK-3β, phospho-GSK-3β (Ser9), iNOS, ICAM-1, TNF-α, and IL-10 were detected in lung tissues by Western-blot. Results The outcomes revealed that the intervention of GSK-3β inhibitors alleviated the pathological damage of pancreas and lung (P<0.01), reduced serum amylase, lipase, hydrothorax and lung Wet-to-Dry Ratio, attenuated serum concentrations of IL-1β and IL-6 (P<0.01), inhibited the activation of NF-κB, and abated expression of iNOS, ICAM-1 and TNF-α protein, but up-regulated IL-10 expression in lung of ANP rats (P<0.01). The inflammatory response and various indicators in ANP-TDZD-8 groups were lower than those in ANP-SB216763 groups. Conclusions Inhibition of GSK-3β weakens acute lung injury related to ANP via the inhibitory function of NF-κB signaling pathway. Different kinds of GSK-3β inhibitors have different effects to ANP acute lung injury.

Effects of hypothermia on lung inflammation in a rat model of meconium aspiration syndrome

Abstract Purpose: To evaluate the effects of hypothermia treatment on meconium-induced inflammation. Methods: Fifteen rats were instilled with human meconium (MEC, 1.5 mL/kg, 65 mg/mL) intratracheally and ventilated for 3 hours. Eight rats that were ventilated and not instilled with meconium served as a sham group. In MEC-hypothermia group, the body temperature was lowered to 33±0.5°C. Analysis of the blood gases, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage (BAL) fluid samples, and histological analyses of the lungs were performed. Results: The BAL fluid TNF-α, IL-1β, IL-6 and IL-8 concentrations were significantly higher in the MEC-hypothermia group than in the MEC-normothermia (p < 0.001, p < 0.001, p = 0.001, p < 0.001, respectively) and sham-controlled groups (p < 0.001, p < 0.001, p < 0.001, p < 0.001, respectively). Conclusion: Meconium-induced inflammatory cytokine production is affected by the body temperature control.

NLRP3 inflammasome signaling as an early molecular response is negatively controlled by miR-186 in CFA-induced prosopalgia mice

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the most frequently studied in the central nervous system and has been linked to neuropathic pain. In this study, a post-translational mechanism of microRNA (miR)-186 via regulating the expression of NLRP3 in the complete Freund's adjuvant (CFA)-treated mice was investigated. The injection of CFA was used to induce trigeminal neuropathic pain in mice. miRs microarray chip assay was performed in trigeminal ganglions (TGs). CFA treatment significantly increased the mRNA expression of NLRP3, interleukin (IL)-1β, and IL-18 in TGs compared to the control group. Moreover, 26 miRs were differentially expressed in TGs from trigeminal neuropathic pain mice, and the expression of miR-186 showed the lowest level of all the miRs. Further examination revealed that NLRP3 was a candidate target gene of miR-186. We delivered miR-186 mimics to CFA-treated mice. The head withdrawal thresholds of the CFA-treated mice were significantly increased by miR-186 mimics injection compared with CFA single treatment. The mRNA and protein expression of NLRP3, IL-1β, and IL-18 in TGs from trigeminal neuropathic pain mice were significantly inhibited by miR-186 mimics treatment compared to the CFA group. miR-186 was able to suppress the neuropathic pain via regulating the NLRP3 inflammasome signaling.

Exogenous neuregulin-1 attenuates STZ-induced diabetic peripheral neuropathic pain in rats

Abstract Purpose: To investigate whether modulating NRG1 could attenuate diabetic neuropathic pain and analyze the underlying mechanism. Methods: Male SD rats were randomly divided into control group, diabetic group, NRG1 intervention group. After STZ-induced 2 weeks, NRG1 intervention daily for consecutive 7 days. 4 weeks after NRG1 intervention, both the mechanical withdrawal threshold and the morphological changes of the dorsal root ganglion and sural nerve were observed. Meanwhile, the expression of NGF, IL-1β, TNF-α in spinal cord were determined. Results: Compared with the diabetic group, NRG1 treatment improved the mechanical withdrawal threshold in diabetic rats, pathological changes of dorsal root ganglion and sural nerve were alleviated by NRG1 treatment with electron microscopy imagine. Moreover, compared with the control group, the expression of NGF was significantly decreased and the production of IL-1β, TNF-α were markedly induced in diabetic group. Furthermore, NRG1 treatment could normalized the above effect as compared to diabetic group. Conclusion: NRG1 exerted positive effects on the behavioral and pathological changes of rats with STZ-induced diabetic neuropathic pain, the underlying mechanism might be related to the promotion of NGF excretion and the inhibition of inflammatory cytokines excretion.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

Different engagement of TLR2 and TLR4 in Porphyromonas gingivalis vs. ligature-induced periodontal bone loss

Abstract This study was conducted to investigate the roles of different Toll-like receptor (TLR) signaling in Porphyromonas gingivalis (P. gingivalis)-induced and ligature-induced experimental periodontal bone resorption in mice. Wild-type (WT), TLR2 knockout (KO), TLR4KO, and TLR2&4 KO mice with C57/BL6 background were divided into three groups: control, P. gingivalis infection, and ligation. Live P. gingivalis or silk ligatures were placed in the sulcus around maxillary second molars over a 2-week period. Images were captured by digital stereomicroscopy, and the bone resorption area was measured with ImageJ software. The protein expression level of gingival RANKL was measured by ELISA. The gingival mRNA levels of RANKL, IL-1β, TNF-α, and IL-10 were detected by RT-qPCR. The results showed that P. gingivalis induced significant periodontal bone resorption in WT mice and TLR2 KO mice but not in TLR4 KO mice or TLR2&4 KO mice. For all four types of mice, ligation induced significant bone loss compared with that in control groups, and this bone loss was significantly higher than that in the P. gingivalis infection group. RANKL protein expression was significantly increased in the ligation group compared with that in the control group for all four types of mice, and in the P. gingivalis infection group of WT, TLR2 KO, and TLR4 KO mice. Expression patterns of RANKL, IL-1β, TNF-α, and IL-10 mRNA were different in the P. gingivalis infection group and the ligation group in different types of mice. In summary, P. gingivalis-induced periodontal bone resorption is TLR4-dependent, whereas ligation-induced periodontal bone resorption is neither TLR2- nor TLR4-dependent.

Effect of fresh frozen plasma on the in vitro activation of U937 monocytes: a potential role for the age of blood donors and their underlying cytokine profile

BACKGROUND: Fresh frozen plasma (FFP) administration may increase the risk of nosocomial infections in parallel with the development of immune modulation. This could be driven by soluble mediators, possibly influencing the in vitro activation of human U937 monocyte cells, in a manner dependent on the age of the donors. METHODS: FFP donors were stratified into groups of 19-30 years, 31-40 years or 41-50 years, and U937 cells were cultured with FFP (alone or plus lipopolysaccharide-LPS) for 24 h. Both in FFP and supernatants, TNF, IL-1ß, IL-6, and IL-10 levels were measured by ELISA. Additionally, CD11B, TLR2, and CASP3 gene expression were measured by qtPCR in U937 cells. Total phagocytic activity was also assayed. RESULTS: Elevated IL-10, but low TNF and IL-1ß levels were measured in FFP from individuals aged 19-40 years, whereas in individuals aged 41-50 years FFP were characterized by equalized TNF and IL-10 levels. Elevated IL-6 levels were found in all FFP samples, especially in those from the oldest individuals. FFP stimulation was associated with striking modifications in cytokine production in an age-dependent way. Exposure to FFP attenuates the response to LPS. TLR2 and CD11B expression were enhanced regardless of the age of plasma donors, although CASP3 expression was increased only when FFP from individuals aged 19-40 years were tested. Phagocytosis decreased after exposure to FFP regardless of donor age. CONCLUSION: Our results suggest that soluble mediators in FFP may modulate the functioning of monocytes. Interestingly, this effect appears to be partially influenced by the age of donors.

LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

Analysis of the effects of topical renal hypothermia on lung tissue after kidney ischemia and reperfusion in rats

PURPOSE: To evaluate whether topical renal hypothermia (TRH) at different levels of temperature has protective effects on lung tissue after renal I/R, through an analysis of organ histology and inflammatory markers in lung tissue. METHODS: Twenty-eight male Wistar rats were randomly allocated across four groups and subjected to renal ischemia at different levels of topical renal temperature: normothermia (no cooling, 37°C), mild hypothermia (26°C), moderate hypothermia (15°C), and deep hypothermia (4°C). To induce I/R, the vessels supplying the left kidney of each animal were clamped for 40 minutes, followed by reperfusion. After four hours, another procedure was performed to harvest the tissues of interest. TNF-α, IL-1β and myeloperoxidase activity were measured in lung tissue. Histological analysis was performed in hematoxylin and eosin-stained lung specimens. RESULTS: Induction of renal I/R under deep topical hypothermia resulted in a significant decrease in lung concentrations of TNF-α compared with normothermic I/R (p<0.05). A trend toward significant correlation was found between lung IL-1β concentration and intensity of hypothermia (Spearman r=−0.37; p=0.055). No difference was found in myeloperoxidase activity or histologic injury between groups. CONCLUSION: Topical renal hypothermia reduces activation of the inflammatory cascade in the lung parenchyma. However, tissue-protective effects were not observed. .

Effects of Er-Miao-San extracts on TNF-alpha-indueed MMP-1 expression in human dermal fibroblasts

BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.

Electroacupuncture ameliorates experimental colitis induced by TNBS through activation of interleukin-10 and inhibition of iNOS in mice

PURPOSE: To study the anti-inflammatory actions of electroacupuncture (EAc) on an experimental colitis model in mice. METHODS: Thirty-eight male Swiss mice, divided in five groups, were subjected to induction of colitis by TNBS in 50% ethanol. Saline (SAL) and ethanol (ETNL) groups served as controls. TNBS+EAc and TNBS+ dexamethasone subgroups were treated with EAc 100Hz and dexamethasone (DEXA) 1 mg/Kg/day, respectively. After three days, a colon segment was obtained for quantification of myeloperoxidase (MPO) activity, immunohistochemistry for iNOS, malondialdehyde (MDA) and cytokines (IL-1β and IL-10). RESULTS: Neutrophilic activity, assayed as MPO activity, was significantly higher in the TNBS colitis group than that in the saline control group. TNBS+EAc group showed suppression of IL-10 in the colon. EAc treatment significantly reduced the concentration of MDA and the expression of iNOS, as compared to the other groups. CONCLUSION: Electroacupuncture 100Hz applied to acupoint ST-36 promotes an anti-inflammatory action on the TNBS-induced colitis, mediated by increase of IL-10 and decrease of iNOS expression. .