IL-6 Protein Expression Research Articles

Metal chelation attenuates oxidative stress, inflammation, and vertical burn progression in a porcine brass comb burn model

Oxidative stress and inflammation may mediate cellular damage and tissue destruction as the burn wound continues to progress after the abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of a metal chelator proprietary lotion (Livionex Formulation (LF) lotion), that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent progression or reduce burn wound severity in a porcine model.We have reported earlier that in a rat burn model, LF lotion reduces thermal injury progression. Here, we used the porcine brass comb burn model that closely mimics the human condition for contact burns and applied LF lotion every 8 h starting 15 min after the injury. We found that LF lotion reduces the depth of cell death as assessed by TUNEL staining and blood vessel blockage in the treated burn sites and interspaces. The protein expression of pro-inflammatory markers IL-6, TNF-a, and TNFα Converting Enzyme (TACE), and lipid aldehyde production (protein-HNE) was reduced with LF treatment. LF lotion reversed the burn-induced decrease in the aldehyde dehydrogenase (ALDH-1) expression in the burn sites and interspaces. These data show that a topically applied EDTA-containing lotion protects both vertical and horizontal burn progression when applied after thermal injury. Curbing burn wound conversion and halting the progression of second partial burn to third-degree full-thickness burn remains challenging when it comes to burn treatment strategies during the acute phase. Burn wound conversion can be reduced with targeted treatments to attenuate the oxidative and inflammatory response in the immediate aftermath of the injury. Our studies suggest that LF lotion could be such a targeted treatment.

Long‐term exposure to cigarette smoke influences characteristics in human gingival fibroblasts

Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria accumulated in the gingival sulcus and periodontal pocket. Cigarette smoking is a well-established risk factor for periodontal disease, and periodontal tissues in smokers are chronically exposed to cigarette smoke on a long-term basis. In this study, we investigated the effects of long-term exposure to nicotine or cigarette smoke condensate (CSC) on cellular functions of human gingival fibroblasts (HGFs). In vitro-maintained HGFs were divided into two groups. The HGFs of the short-term and the long-term culture groups were cultured for 4 and 25days, respectively, in the presence or absence of nicotine, which is one of the main components of cigarette smoke, or CSC. The cellular proliferation and migration capacities of HGFs exposed to nicotine or CSC were evaluated by WST-1 and wound healing assays. The effects of exposure to nicotine or CSC on the expression of various extracellular matrix (ECM) components, inflammatory cytokines, and senescence-related genes were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The cellular senescence of HGFs exposed to nicotine or CSC was detected by the senescence-associated β-galactosidase (SA-β-gal) assay. To explore the senescence-associated microRNA (miRNA), we extracted miRNA from the HGFs and the expression profiles were examined by miRNA array. In short-term culture, no significant changes were observed. Long-term exposure of HGFs to nicotine or CSC significantly suppressed their cellular proliferation and migration and upregulated type Ⅰ collagen, type Ⅲ collagen, interleukin (IL)-6, IL-8, p16, p21, and p53mRNA expression, and IL-6 and IL-8 protein expression. Furthermore, long-term nicotine or CSC exposure significantly increased the percentage of SA-β-gal-positive HGFs. In addition, long-term nicotine or CSC exposure reduced miR-29b and miR-199a expression to less than 50% of that in the unstimulated HGFs. These data suggest that long-term smoking habits may reduce wound healing ability, modulate ECM protein homeostasis, stimulate the inflammatory response, and accelerate cellular senescence in HGFs, and consequently accelerate the progression of periodontal diseases.

Allicin Could Potentially Alleviate Oral Cancer Pain by Inhibiting "Pain Mediators" TNF-alpha, IL-8, and Endothelin.

To evaluate the effects of allicin on mediators of pain secreted by oral cancer cells in vitro, single-cell suspensions were prepared by enzymatic method from oral squamous cell carcinoma (OSCC). Cancer stem cells were isolated by the CD133+ selection method with magnetic cell sorting. Stemness markers were checked in both cancer cells and cancer stem cells by RT-PCR. Comparative analysis of pain mediators TNF-alpha, IL-8, and endothelin at both RNA and protein levels for normal epithelial cells, cancer cells, and cancer stem cells was carried out with and without allicin treatment. CD133 and CD44 expression levels were checked in cancer cells and cancer stem cells flow cytometrically. Allicin inhibited both gene and protein expression of TNF-alpha, IL-8, and endothelin in both cancer cells and cancer stem cells. Allicin is more likely to be a promising treatment in alleviating the levels of pain and inflammation in OSCCs.

Systematic Pharmacology Reveals the Antioxidative Stress and Anti-Inflammatory Mechanisms of Resveratrol Intervention in Myocardial Ischemia-Reperfusion Injury.

Objective To explore the oxidative stress and inflammatory mechanisms of resveratrol intervention in myocardial ischemia-reperfusion injury (MIRI). Methods The potential targets of resveratrol were predicted by PharmMapper. The MIRI genes were collected by Online Mendelian Inheritance in Man (OMIM), GeneCards is used to collect related disease genes, and String is used for enrichment analysis. Animal experiments were then performed to verify the systematic pharmacological results. Hematoxylin-eosin (HE) staining was used to observe myocardial damage. The levels of serum interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in each experimental group were detected. The protein and mRNA expressions of Toll-like receptor 4 (TLR4), nuclear factor-kappa (NF-κB) p65, IL-1β, IL-6, and TNF-α in rat myocardial tissue were measured. Results The results of systematic pharmacology showed that insulin resistance, FoxO signaling pathway, adipocytokine signaling pathway, insulin signaling pathway, PI3K-Akt signaling pathway, ErbB signaling pathway, T-cell receptor signaling pathway, peroxisome proliferator-activated receptors (PPAR) signaling pathway, Ras signaling pathway, TNF signaling pathway, and so on were regulated to improve MIRI. The results of animal experiments showed that the myocardial cells of the sham operation group were arranged in fibrous form, and the myocardial ischemia-reperfusion injury group had obvious cell morphology disorder. Compared with the MIRI group, the resveratrol group had a certain degree of relief. Compared with the MIRI group, serum IL-1β, TNF-α, and IL-6 in the resveratrol group was significantly reduced (P < 0.05), and myocardial tissue TLR4, NF-κB p65, IL-1β, IL-6, and TNF-α mRNA and protein expressions were significantly reduced (P < 0.05). Conclusion Resveratrol can effectively improve MIRI, and its mechanism may be related to antioxidative stress and anti-inflammatory.

Haplopine Ameliorates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in Mice and TNF-α/IFN-γ-Induced Inflammation in Human Keratinocyte.

This study aimed to investigate the anti-inflammatory, antioxidant, and anti-atopic dermatitis (AD) effects of haplopine, which is one of the active components in D. dasycarpus. Haplopine (12.5 and 25 μM) inhibited the mRNA expressions of inflammatory cytokines IL-6, TSLP, GM-CSF, and G-CSF and the protein expressions of IL-6 and GM-CSF in TNF-α/INF-γ-stimulated HaCaT cells. In H2O2-induced Jukat T cells, haplopine (25 and 50 μM) suppressed the productions of proinflammatory cytokines (IL-4, IL-13, and COX-2) and increased the mRNA and protein expressions of oxidative stress defense enzymes (SOD, CAT, and HO-1) in a concentration-dependent manner. In vivo, haplopine significantly attenuated the development of AD symptoms in 2,4-dinitrochlorobenzene (DNCB)-stimulated Balb/c mice, as evidenced by reduced clinical dermatitis scores, skin thickness measurements, mast cell infiltration, and serum IgE concentrations. These findings demonstrate that haplopine should be considered a novel anti-atopic agent with the potential to treat AD.

New use of old medicine: Nifedipine acts on the TRP family and inflammatory proteins in the treatment of chilblain

As a calcium antagonist, the mechanism of nifedipine for treating chilblain has not been reported. In the present study, we established the chilblain model by using −20 ℃ 95% ethanol to freeze the right back foot of SD rats, and investigated the effects of this drug. Hematoxylin–eosin (HE) examination indicated most of pannus in the skin tissue of chilblain rats had disappeared, and the local inflammatory cells were also greatly reduced when given nifedipine at 15.0 mg/kg/d. The enzyme-linked immunosorbent assay (ELISA) revealed that nifedipine inhibited release of inflammatory factors TNF-α, IL-6, IL-1β and VEGF in serum. The RT-PCR analysis showed that nifedipine down regulated mRNA levels of TRPC-6 and VEGF in skin tissue. Furthermore, immunohistochemical examination showed nifedipine inhibited expression of IL-1β, IL-6, and TNF-α inflammatory protein and further inhibited expression of TRP (transient receptor potential) family proteins TRPM-7, TRPC-1, TRPC-3 and TRPC-6 and reduced expression of VEGF in skin and relieved erythema and oedema. This study demonstrated that nifedipine as an old medicine can be new use for the treatment of chilblain by acting on TRPs family and inflammatory proteins.

IL-18 Promotes Erythrophagocytosis and Erythrocyte Degradation by M1 Macrophages in a Calcific Microenvironment

BackgroundCalcific aortic-valve disease (CAVD) is the most common cause of aortic valve replacement in developed countries. Intraleaflet hemorrhage has been found to be positively correlated with the progression of CAVD. Although most research has focused on erythrocyte degradation products, which promote progression of CAVD, the process of erythrophagocytosis and erythrocyte degradation by macrophages in intraleaflet hemorrhage areas has remained unexplored. MethodsThe erythrocyte degradation products of aortic valve specimens were detected by Perls’ staining and quantified. The gene and protein expression levels of interleukin (IL)-18 in THP-1–polarized macrophages cultured in osteogenic medium were tested. We also quantified the iron and heme degraded by macrophages and analyzed the expression of ferroportin (FPN) and heme oxygenase-1 (HO-1) in the osteogenic medium. Furthermore, we tested the mRNA and protein levels of osteoblast markers in valve interstitial cells after co-culture with M1 macrophages treated with IL-18 and erythrocytes. ResultsOur experiments demonstrated that IL-18 activates HO-1 and FPN to promote erythrophagocytosis and erythrocyte degradation by macrophages in a calcific microenvironment via p38 and Erk1/2. We also found that the calcific microenvironment promotes IL-18 mRNA and protein expression in THP-1-polarized macrophages. ConclusionsIn conclusion, IL-18 promotes M1 macrophage-mediated erythrophagocytosis and erythrocyte degradation by regulating the activation of HO-1 and FPN via p38 and Erk1/2 in a calcific microenvironment.

Juglone regulates gut microbiota and Th17/Treg balance in DSS-induced ulcerative colitis

Juglone, mainly isolates from the green walnut husks of Juglans mandshurica, exhibits anti-cancer and anti-inflammaroty activities. But its protection on ulcerative colitis (UC) has never been explored. In this study, we first evaluated whether juglone ameliorated UC, and investigated its effects on gut microbiota and Th17/Treg balance in DSS-induced UC mice model. The model was established by administrating 2.7% DSS for seven days. Juglone was given daily by gavage for ten days, once a day. The disease activity index (DAI) decrease and pathological characteristics improvement demonstrated that the UC in mice was alleviated by juglone. Juglone treatment significantly inhibited the protein levels of IL-6, TNF-α and IL-1β, improved the protein expression of IL-10. In addition, juglone altered microbial diversity and gut microbiota composition, including the enhancement of the ratio of Firmicutes to Bacteroidota and the abundance of Actinobacteriota, and decrease of the abundance of Verrucomicrobiota. Juglone treatment also inhibited the protein expressions of IL-6, STAT3 and RORγt, meanwhile improved the protein level of FOXP3. Furthermore, juglone inhibited Th17 development and increased Treg generation, beneficial to Th17/Treg balance. Together, we herein provided the first evidence to support that juglone, especially the high dose, possibly protected mice against UC by modulating gut microbiota and restoring Th17/Treg homeostasis.

Porphyromonas gingivalis lipopolysaccharide promotes T-hel per17 cell differentiation by upregulating Delta-like ligand 4 expression on CD14+ monocytes.

BackgroudTo investigate the effect and mechanism of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on Th17 cell differentiation mediated by CD14+ monocytes. Methods P. gingivalis LPS-activated CD14+ monocytes were co-cultured with CD4+T cells in different cell ratios. An indirect co-culture system was also established using transwell chambers. Furthermore, anti- Delta-like ligand 4 (Dll-4) antibody was used to investigate the role of Dll-4 in Th17 cell response. The mRNA expression was analyzed using quantitative reverse transcription-polymerase chain reaction, and secreted cytokines in culture supernatant were detected using enzyme-linked immunosorbent assay. Flow cytometry was used to determine the frequencies of Th17 cells. IL-17 protein expression levels were determined using western blotting assay. ResultsP. gingivalis LPS increased the expressions of interleukin (IL)-1β, IL-6, IL-23 and transforming growth factor (TGF)-β in CD14+ monocytes. Th17 cell frequency upregulated, which is not solely cytokine-dependent but rather requires cell-cell contact with activated monocytes, particularly in the 1:10 cell ratio. Furthermore, P. gingivalis LPS increased t he expression of Dll-4 on CD14+ monocytes, whereas the anti- Dll-4 a ntibody decreased the response of Th17 cells. The results suggest that P. gingivalis LPS enhances Th17 cell response via Dll-4 upregulation on CD14+ monocytes.

Microglial IL-10 and β-endorphin expression mediates gabapentinoids antineuropathic pain

Gabapentinoids are recommended first-line treatments for neuropathic pain. They are neuronal voltage-dependent calcium channel α2δ-1 subunit ligands and have been suggested to attenuate neuropathic pain via interaction with neuronal α2δ-1 subunit. However, the current study revealed their microglial mechanisms underlying antineuropathic pain. Intrathecal injection of gabapentin, pregabalin and mirogabalin rapidly inhibited mechanical allodynia and thermal hyperalgesia, with projected ED50 values of 30.3, 6.2 and 1.5 µg (or 176.9, 38.9 and 7.2 nmol) and Emax values of 66%, 61% and 65% MPE respectively for mechanical allodynia. Intrathecal gabapentinoids stimulated spinal mRNA and protein expression of IL-10 and β-endorphin (but not dynorphin A) in neuropathic rats with the time point parallel to their inhibition of allodynia, which was observed in microglia but not astrocytes or neurons in spinal dorsal horns by using double immunofluorescence staining. Intrathecal gabapentin alleviated pain hypersensitivity in male/female neuropathic but not male sham rats, whereas it increased expression of spinal IL-10 and β-endorphin in male/female neuropathic and male sham rats. Treatment with gabapentin, pregabalin and mirogabalin specifically upregulated IL-10 and β-endorphin mRNA and protein expression in primary spinal microglial but not astrocytic or neuronal cells, with EC50 values of 41.3, 11.5 and 2.5 µM and 34.7, 13.3 and 2.8 µM respectively. Pretreatment with intrathecal microglial metabolic inhibitor minocycline, IL-10 antibody, β-endorphin antiserum or μ-opioid receptor antagonist CTAP (but not κ- or δ-opioid receptor antagonists) suppressed spinal gabapentinoids-inhibited mechanical allodynia. Immunofluorescence staining exhibited specific α2δ-1 expression in neurons but not microglia or astrocytes in the spinal dorsal horns or cultured primary spinal cells. Thus the results illustrate that gabapentinoids alleviate neuropathic pain through stimulating expression of spinal microglial IL-10 and consequent β-endorphin.

PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Borneol-modified tanshinone IIA liposome improves cerebral ischemia reperfusion injury by suppressing NF-κB and ICAM-1 expression

Objective To investigate the metabolism and brain tissue distribution of borneol-modified tanshinone IIA liposome (BO-TA-Lip) and its effect on NF-κB and ICAM-1 in cerebral ischemia reperfusion rats, thereby exploring the ameliorative mechanism of BO-TA-Lip on ischemic encephalopathy. Methods Particle size, entrapment efficiency, drug loading were measured to evaluate the preparation comprehensively. Metabolism and brain tissue distributions in vivo were measured by HPLC, and the pharmacokinetic parameters were calculated. In addition, 24 SD rats were randomly divided into sham, model, STS (sodium tanshinone IIA sulfonate, 30 mg/kg) and BO-TA-Lip groups (44 mg/kg). The middle cerebral artery occlusion (MCAO) rats were constructed with thread embolism method. Neurological deficits were scored using Zea Longa scoring standard. TTC and HE staining were used for the cerebral infarction and histopathological examination, respectively. The protein expression was examined by immunohistochemistry and Western blot. Results The average particle size, encapsulation efficiency and drug loading of BO-TA-Lip were (135.33 ± 7.25) nm, (85.95 ± 3.20)% and (4.06 ± 0.31)%, respectively. Both in the pharmacokinetic analysis of plasma and brain tissue, in BO-TA-Lip group, the peak concentration and the area under the curve increased, and the clearance rate decreased. The neurological deficit scores and infarct area of the BO-TA-Lip group were significantly lower than that of the model and STS groups. Besides, BO-TA-Lip reduced the protein expression of NF-κB, ICAM-1, IL-1β, TNF-α and IL-6 in the brain tissue. Conclusion BO-TA-Lip had higher bioavailability and better absorption in brain tissue, and could improve cerebral ischemia reperfusion injury, which might be related to the inhibitory effect of BO-TA-Lip in expression of NF-κB and ICAM-1.

Antiviral effect of fufang yinhua jiedu (FFYH) granules against influenza A virus through regulating the inflammatory responses by TLR7/MyD88 signaling pathway

Ethnopharmacological relevanceFufang-Yinhua-Jiedu Granules (FFYH) optimized from a Yin-Qiao-San, as traditional Chinese medicine (TCM), was used to treat influenza and upper respiratory tract infection and was recommended for the prevention and treatment of SARS in 2003 and current COVID-19 in Anhui Province in 2020. Aim of studyIn the clinical studies, FFYH was very effective for the treatment of influenza, but the mechanism of action against influenza A virus remains unclear. In the present study, we investigated the antiviral effect of FFYH against influenza A virus in vitro and vivo. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was investigated for the first time. Materials and methodsCPE inhibition assay and HA assay were used to evaluate the in vitro antiviral effects of FFYH against influenza A virus H1N1, H3N2, H5N1, H7N9 and H9N2. Mice were used to evaluate the antiviral effect of FFYH in vivo with ribavirin and lianhuaqingwen as positive controls. RT-PCR was used to quantify the mRNA transcription of TNF-α, IL-6, IFN-γ, IP10, and IL-1β mRNA. ELISA was used to examine the expression of inflammatory factors such as TNF-α, IL-6, IFN-γ, IP10, and IL-1β in sera. The blood parameters were analyzed with auto hematology analyzer. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was also investigated. ResultsFFYH showed a broad-spectrum of antiviral activity against H1N1, H3N2, H5N1, H7N9, and H9N2 influenza A viruses. Furthermore, FFYH dose-dependently increased the survival rate, significantly prolonged the median survival time of mice, and markedly reduced lung injury caused by influenza A virus. Also, FFYH significantly improve the sick signs, food taken, weight loss, blood parameters, lung index, and lung pathological changes. Moreover, FFYH could markedly inhibit the inflammatory cytokine expression of TNF-α, IL-6, IFN-γ, IP10, IL-10, and IL-1β mRNA or protein via inhibition of the TLR7/MyD88/NF-κB signaling pathway in vivo. ConclusionFFYH not only showed a broad-spectrum of anti-influenza virus activity in vitro, but also exhibited a significant protective effect against lethal influenza virus infection in vivo. Furthermore, our results indicated that the in vivo antiviral effect of FFYH against influenza virus may be attributed to suppressing the expression of inflammatory cytokines via regulating the TLR7/MyD88/NF-κB signaling pathway. These findings provide evidence for the clinical treatment of influenza A virus infection with FFYH.

Atorvastatin ameliorates early brain injury after subarachnoid hemorrhage via inhibition of pyroptosis and neuroinflammation.

Subarachnoid hemorrhage (SAH) is a subtype of stroke with high mortality and morbidity due to the lack of effective therapy. Atorvastatin has been reported to alleviate early brain injury (EBI) following subarachnoid hemorrhage (SAH) via reducing reactive oxygen species, antiapoptosis, regulated autophagy, and neuroinflammation. Which was the related to the pyroptosis? Pyroptosis can be defined as a highly specific inflammatory programmed cell death, distinct from classical apoptosis and necrosis. However, the precise role of pyroptosis in atorvastatin-mediated neuroprotection following SAH has not been confirmed. The present study aimed to investigate the neuroprotection and potential molecular mechanisms of atorvastatin in the SAH-induced EBI via regulating neural pyroptosis using the filament perforation model of SAH in male C57BL/6 mice, and the hemin-induced neuron damage model in HT-22. Atorvastatin or vehicle was administrated 2 h after SAH and hemin-induced neuron damage. The mortality, neurological score, brain water content, and neuronal death were evaluated. The results show that the atorvastatin treatment markedly increased survival rate, neurological score, greater survival of neurons, downregulated the protein expression of NLRP1, cleaved caspase-1, interleukin-1β (IL-1β), and IL-18, which indicated that atorvastatin-inhibited pyroptosis and neuroinflammation, ameliorated neuron death in vivo/vitro subjected to SAH. Taken together, this study demonstrates that atorvastatin improved the neurological outcome in rats and reduced the neuron death by against neural pyroptosis and neuroinflammation.

Astaxanthin inhibits inflammation of human periodontal ligament cells induced by lipopolysaccharide.

Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms. hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA. Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all P<0.05). Compared with the LPS group, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+AST (5, 10, 20, and 50 μmol/L) group were down-regulated (all P<0.05). Compared with the Con group, the levels of IKBα and p65 in cytoplasm of the LPS group were significantly downregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS group were significantly up-regulated (both P<0.05). Compared with the LPS group, the levels of IKBα and p65 in cytoplasm of the LPS+AST (20 μmol/L) group were significantly upregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS+AST (20 μmol/L) group were significantly downregulated (both P<0.05). The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+PDTC (10 μmol/L) group were lower than those in the LPS group (all P<0.05). AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.

Amentoflavone inhibits M1 polarization of THP-1-derived foam cells by activating PPAR-α/γ

To investigate the mechanism by which amentoflavone inhibits polarization of THP-1-derived foam cells to M1 phenotype. Human monocyte cell line THP-1 was stimulated to differentiate into M1-type macrophages using phorbol 12-myrislate13-acetate (PMA) combined with lipopolysaccharide (LPS) and recombinant human interferon-γ (rhlFN-γ). M1 polarization of THP-1-derived macrophages was confirmed by observing morphological changes of the cells and detecting the mRNA expression of L-6 and TNF-α with RT-qPCR. THP-1-derived foam cells treated with 5 or 10 μmol/L amentoflavone for 24 h were examined for cytokines using ELISA. The mRNA and protein expressions of IL-6, IL-10, TNF-α, TGF-β, PPAR-α/γ, Arg-1 and Fizz1 in the cells were detected using RT-qPCR and Western blotting. Amentoflavone prevented induced M1 polarization of THP-1 cells. Amentoflavone down-regulated the mRNA expressions of IL-6 and TNF-α, up-regulated mRNA expressions of IL-8 and TGF-β mRNA (P < 0.05), and increased the protein expressions of PPAR-α/γ, Arg-1 and Fizz1. Molecular docking simulation showed that amentoflavone could bind to the surface of PPARα/γ. Amentoflavone can inhibit the differentiation of macrophages into M1 type by activating PPAR-α/γ and restoring the expressions of the gene Arg-1 and Fizz1.

LJ529 attenuates mast cell-related inflammation via A3R-PKCε-ALDH2 pathway after subarachnoid hemorrhage in rats

Background and purposeMast cells (MCs) has been recognized as an effector of inflammation or a trigger of inflammatory factors during stroke. LJ529 was reported to attenuate inflammation through a Gi protein-coupled Adenosine A3 receptor (A3R) after ischemia. Here, we aim to study the protective effect and its mechanism of LJ529 in subarachnoid hemorrhage (SAH) rat model for mast cell-related inflammation. Methods155 Sprague–Dawley adult male rats were used in experiments. Endovascular perforation was used for SAH model. Intraperitoneal LJ529 was performed 1 h after SAH. Neurological scores were measured 24 h after SAH. Rotarod and morris water maze tests were evaluated for 21 days after SAH. Mast cell degranulation was assessed with Toluidine blue staining and Chymase/Typtase protein expressions. Mast cell-related inflammation was evaluated using IL-6, TNF-α and MCP-1 protein expressions. MRS1523, inhibitor of GPR18 and ε-V1-2, inhibitor of PKCε were respectively given intraperitoneally (i.p.) 1 h and 30 min before SAH for mechanism studies. Pathway related proteins were investigated with western blot and immunofluorescence staining. ResultsExpression of A3R, PKCε increased after SAH. LJ529 treatment attenuated mast cell degranulation and inflammation. Meanwhile, both short-term and long-term neurological functions were improved after LJ529 treatment. Administration of LJ529 resulted in increased expressions of A3R, PKCε, ALDH2 proteins and decreased expressions of Chymase, Typtase, IL-6, TNF-α and MCP-1 proteins. MRS1523 abolished the treatment effects of LJ529 on neurobehavior and protein levels. ε-V1-2 also reversed the outcomes of LJ529 administration through reduction in protein expressions downstream of PKCε. ConclusionsLJ529 attenuated mast cell-related inflammation through inhibiting degranulation via A3R-PKCε-ALDH2 pathway after SAH. LJ529 may serve as a potential treatment strategy to relieve post-SAH brain injury.

5-Aminoisoquinolinone, a PARP-1 Inhibitor, Ameliorates Immune Abnormalities through Upregulation of Anti-Inflammatory and Downregulation of Inflammatory Parameters in T Cells of BTBR Mouse Model of Autism.

Autism spectrum disorder (ASD) covers a range of neurodevelopmental disorders involving impairments in communication and repetitive and stereotyped patterns of behavior and reciprocal social interaction. 5-Aminoisoquinolinone (5-AIQ), a PARP-1 inhibitor, has neuroprotective and anti-inflammatory effects. We investigated the influence of 5-AIQ-treatment in BTBR T+ Itpr3tf/J (BTBR) mice as an autism model and used flow cytometry to assess the effect of 5-AIQ on FOXP3, Helios, GATA3, IL-9, IL-10 and IL-17A production by CXCR6+ and CD4+ T cells in the spleen. We also confirmed the effect of 5-AIQ treatment on expression of FOXP3, Helios, GATA3, IL-17A, IL-10, and IL-9 mRNA and protein expression levels in the brain tissue by quantitative PCR and western blotting. Our results demonstrated that 5-AIQ-treated BTBR mice had significantly increased numbers of CXCR6+FOXP3+, CXCR6+IL-10+, and CXCR6+Helios+ cells and decreased numbers of CD4+GATA3+, CD4+IL-9+, and CD4+IL-17A+ cells as compared with those in untreated BTBR mice. Our results further demonstrated that treatment with 5-AIQ in BTBR mice increased expression for FOXP3, IL-10, and Helios, and decreased expression for GATA3, IL-17A, and IL-9 mRNA. Our findings support the hypotheses that 5-AIQ has promising novel therapeutic effects on neuroimmune dysfunction in autism and is associated with modulation of Treg and Th17 cells.

Evaluation of the cutaneous expression of IL-17, IL-22, IL-31, and their receptors in canine atopic dermatitis

Interleukins (IL)-17, IL-22, and IL-31 play roles in human atopic dermatitis (AD), but scant information is available on canine AD. Histopathological assessment for interleukin expression is a challenge due to a lack of canine specific antibodies.To evaluate the mRNA and protein expression of IL-17 and IL-22, and mRNA expression of IL-31 and their receptors in the skin of healthy and atopic dogs, seventeen atopic (10 with and 7 without an active infection) and 13 healthy privately owned dogs were sampled. RNAscope® In situ hybridization (ISH) for IL-17, IL-22, IL-31, and their receptors was performed on archived canine skin samples. Simultaneously, indirect immunofluorescence (IIF) was performed for IL-17 and IL-22. RNAscope® ISH probes were validated by RT-PCR and RNAscope® ISH on cytospin preparations of peripheral blood mononuclear cells from atopic dogs.IL-17, IL-22, IL-31, and their receptors were successfully detected by RNAscope® ISH and by IIF (IL-17 and IL-22) in both atopic and healthy canine skin. There was no significant difference in the expression of interleukins and their receptors between healthy and atopic skin with or without active infection. Data from both methodologies were similar.The role and the relationship among those proteins in atopic skin is unclear from this study results. Data from IIF and ISH were overlapping and support each other. Fresh skin samples taken at different times during the development of atopic dermatitis might better assess the role that interleukins and their receptors play in AD.

Notch signaling inhibition protects against root resorption in experimental immature tooth movement in rats

This study aimed to build an experimental immature tooth movement model and verify less resorption of incompletely developed roots than those fully developed during the same orthodontic treatment, followed by investigating the cellular and molecular mechanism. The development of Wistar rat tooth was investigated using invivo microcomputed tomography and hematoxylin and eosin staining to decide the optimal ages of rats for immature tooth and mature tooth groups. The rats in the immature tooth and mature tooth groups were divided into experimental, sham control, and blank control groups. After orthodontic treatment for 3weeks, the mesial root volume, crown movement distance, neck movement distance, root inclination, and apical distance were measured by microcomputed tomography. The expressions of TRAP, Jagged1, Notch2, IL-6, and RANKL were analyzed by immunohistochemical staining and real-time polymerase chain reaction. The repair of root resorption was also investigated after removing orthodontic force for 3 and 6weeks. The root achieved the development stage around 10weeks, so 4-week-old rats and 10-week-old rats were used in the immature tooth group and mature tooth group, respectively. The volume of root resorption in the experimental immature tooth group was 0.0869±0.0244mm3, which was less than that in the mature tooth group (0.1218±0.0123mm3)(P<0.001). Immature tooth movement decreased TRAP-positive odontoclasts on the compression side while having no statistically significant effect on osteoclasts. The protein expression of Jagged1, Notch2, IL-6, and RANKL in the mature tooth group increased significantly compared with the immature tooth group, not only on the compression side but also on the tension sides. The mRNA expression of Jagged1, Notch2, and RANKL was significantly lower in the immature tooth group, whereas the expression of IL-6 had no significance but a strong tendency. The root volume after repairing for 3weeks was still less than that of blank control, whereas after repairing for 6weeks, the difference was not statistically significant. The experimental immature tooth movement model for the Wistar rat was achieved for the first time. The immature tooth will suffer less root resorption than the mature tooth, which may be due to odontoclastogenesis inhibition by decreased expression of Jagged1/Notch2/IL-6/RANKL signaling.