Protein Levels Of Inflammatory Cytokines Research Articles

Isorhamnetin ameliorates cisplatin-induced acute kidney injury in mice by activating SLPI-mediated anti-inflammatory effect in macrophage

Objective Isorhamnetin (IH) has been reported to have significant anti-inflammatory effects in various diseases, but its role and mechanism in AKI remain unclear. This study aimed to explore the potential role and mechanism of isorhamnetin in inhibiting macrophage related inflammation and improving AKI injury. Methods We established an AKI mouse model by intraperitoneal injection of cisplatin in vivo, and constructed an inflammatory cell model by stimulating RAW264.7 cells with LPS. Creatinine and urea nitrogen were measured to evaluate the changes of renal function in AKI mice. The changes of renal pathological structure were observed by H&E staining. The inflammatory factor-related proteins and RNA expression levels were detected by Western blot and real time PCR. Results Isorhamnetin protected the kidney from cisplatin induced AKI and significantly inhibited the mRNA and protein levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) both in AKI kidney and LPS-stimulated RAW264.7 cells. Interestingly, the data also demonstrated that isorhamnetin significantly upregulated the expression of secretory leukocyte peptidase inhibitor (SLPI), an anti-inflammatory factor, in AKI kidney and LPS-stimulated macrophages, as well as inhibited the M1 macrophage and activated M2 macrophage in vitro. Blocking of SLPI by siRNA activated Mincle-associated inflammatory signaling in macrophages, and the inhibitory effect of isorhamnetin on inflammation was significantly attenuated. Conclusion Isorhamnetin inhibits macrophage inflammation and protects kidney in AKI may be related to downregulating Mincle/Syk/NF-κB-maintained macrophage phenotype by activating SLPI.

Synergic effects of EP2 and FP receptors co-activation on Blood-Retinal Barrier and Microglia

Macular edema (ME) is caused with disruption of the blood-retinal barrier (BRB) followed by fluid accumulation in the subretinal space. Main components of the outer and inner BRB are retinal pigment epithelial (RPE) cells and retinal microvascular endothelial cells, respectively. In addition, glial cells also participate in the functional regulation of the BRB as the member of 'neurovascular unit'. Under various stresses, cells in neurovascular units secrete inflammatory cytokines. Neuroinflammation induced by these cytokines can cause BRB dysfunction by degrading barrier-related proteins and contribute to the pathophysiology of ME. Prostaglandins (PGs) are crucial lipid mediators involved in neuroinflammation. Among PGs, a novel EP2 agonist, omidenepag (OMD) acts on not only the uveoscleral pathway but also the conventional pathway, unlike F prostanoid (FP) receptor agonists. Moreover, the combination use of the EP and the FP agonist is not recommended because of the risk of inflammation. In this study, we investigated effects of OMD and latanoprost acid (LTA), a FP agonist, on BRB and microglia in vitro and in vivo.To investigate the function of outer/inner BRB and microglia, in vitro, ARPE-19 cells, human retinal microvascular endothelial cells (HRMECs), and MG5 cells were used. Cell viability, inflammatory cytokines mRNA and protein levels, barrier morphology/function, and microglial activation were evaluated using proliferation assays, qRT-PCR, ELISA, immunocytochemistry, trans-epithelial electrical resistance, and permeability assay. Moreover, after vitreous injection into the mouse, outer BRB morphology, glial activation, and cytokine expression were assessed. Each OMD and LTA alone did not affect the viability or cytokines expression of the three types of cells. In ARPE-19 cells, the co-stimulation of OMD and LTA increased the mRNA and protein levels of inflammatory cytokines (IL-6, TNF-α, and VEGF-A) and decreased the barrier function and the junction-related protein (ZO-1 and β-catenin). By contrast in HRMECs, the co-stimulation affected significant differences in the mRNA levels of some cytokine (IL-6 and TNF-α) but enhanced the barrier function. In MG5 cells, the cytokines mRNA and size of Iba1-expressed cell were increased. A non-steroidal anti-inflammatory inhibited the barrier dysfunction and the junction-related protein downregulation in ARPE-19 cells and activation of MG5 cells. Also in vivo, the co-stimulation induced outer BRB disruption, cytokine increase, and retinal glial activation. Therefore, the co-stimulation of EP2 and FP induced the inflammatory cytokine-mediated outer BRB disruption, the enhanced inner BRB function, and the microglial activation. The BRB imbalance and the intrinsic prostaglandin production may be involved in OMD-related inflammation.

Network pharmacology and in vivo and in vitro experiments to determine the mechanism behind the effects of Jiawei Yanghe decoction via TLR4/Myd88/NF-κB against mastitis

BackgroundIn the Qing dynasty, Yanghe decoction was as a therapeutic soup for effectively treating chronic inflammatory disorders. It was used as a therapeutic soup for effectively treating chronic inflammatory disorders. In the clinical use of Yanghe decoction, the adjustment of the medication for a variety of inflammatory diseases have therapeutic effect, including mastitis. Therefore, Jiawei Yanghe decoction (JWYHD) may be used to treat inflammatory breast diseases. MethodsFirst, LM- and JWYHD-related components were retrieved from the database and analysis platform. Next, protein–protein interaction networks were constructed to screen the key targets, and gene ontology and Kyoto encyclopedia of gene and genome enrichment analyses were performed to predict the potential biological functions and mechanisms of JWYHD. Simultaneously, the JWYHD samples were collected and analyzed by UPLC–HRMS. Finally, in vivo and in vitro experiments were conducted to construct animal and cellular inflammation models of mastitis with LPS. Pathological changes in the mammary tissues were detected. Enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blotting was performed to determine the mRNA and protein levels of inflammatory cytokines and toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor kappa B signaling pathway in the breast tissues to elucidate the potential underlying mechanisms of anti-mastitis effects of JWYHD from different aspects. ResultsIn total, 103 compounds were detected in JWYHD by UPLC–HRMS. 691 active ingredients of JWYHD were screened by network pharmacology, and 47 LM-related targets were identified. The PPI network analysis of the targets revealed the 5 core targets. The KEGG enrichment results established the NF-κB signaling pathways as the core. After JWYHD intervention, low inflammatory enrichment and mild inflammatory damage in breast tissues were observed. Furthermore, JWYHD treatment affected mammary gland inflammatory cytokines and the TLR4/Myd88/NF-κB signaling pathway by considerably reducing the respective protein levels and gene expression; thus, JWYHD alleviated LM symptoms. ConclusionsWe hypothesized and demonstrated the anti-inflammatory effects of JWYHD by cytokine regulation via the TLR4/Myd88/NF-κB signaling pathway. In conclusion, JWYHD showed its potential in LM treatment and in treating other acute and chronic inflammatory diseases.

The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy.

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE-/- mice were then assessed by performing hematoxylin-eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE-/- mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

Apigenin Alleviates Autoimmune Uveitis by Inhibiting Microglia M1 Pro-Inflammatory Polarization.

Apigenin is a natural small molecule compound widely present in various vegetables and fruits. Recently, Apigenin was reported to inhibit lipopolysaccharide (LPS)-simulated microglial proinflammatory activation. Considering the important role of microglia in retinal disorders, we wonder whether Apigenin could exert a therapeutic effect on experimental autoimmune uveitis (EAU) through reprogramming retinal microglia to a beneficial subtype. EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein (IRBP)651-670, followed by intraperitoneal administration of Apigenin. Disease severity was assessed based on clinical and pathological scores. In vivo, Western blotting was used to quantify protein levels of classical inflammatory factors, microglial M1/M2 markers and the tight junction protein of the blood-retinal-barrier (BRB). Immunofluorescence was used to determine the Apigenin's efficacy on microglial phenotype. In vitro, Apigenin was added in LPS and IFN-γ stimulated human microglial cell line. Western blotting and Transwell assays were used to analyze the phenotype of microglia. In vivo, we found that Apigenin significantly reduced the clinical and pathological scores of EAU. The protein levels of inflammatory cytokines were significantly decreased in retina, and BRB disruption was ameliorated after Apigenin treatment. Meanwhile, Apigenin inhibited microglia M1 transition in EAU mice retina. In vitro functional studies showed that Apigenin decreased LPS and IFN-γ-induced microglial inflammatory factor production and M1-activation via the TLR4/MyD88 pathway. Apigenin can ameliorate retinal inflammation in IRBP induced autoimmune uveitis through inhibiting microglia M1 pro-inflammatory polarization via TLR4/MyD88 pathway.

MicroRNA-338-3p suppresses lipopolysaccharide-induced inflammatory response in HK-2 cells

BackgroundInflammation is the most common cause of kidney damage, and inflammatory responses in a number of diseases are mediated by microRNA-338-3p (miR-338-3p). However, there are only a few reports which described the regulation of miR-338-3p in human proximal tubular cells. The goal of this study was to see how miR-338-3p affected lipopolysaccharide (LPS)-caused inflammatory response in HK-2 cells.MethodsLPS was used to construct an inflammatory model in HK-2 cells. miR-338-3p mimic was used to increase the levels of miR-338-3p in HK-2 cells. MTT, JC-1 staining, and apoptosis assays were used to detect cell viability, mitochondrial membrane potential (MMP), and apoptosis, respectively. The production of inflammatory factors and the levels of p38, p65, phospho-p65, phospho-p38, Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 were investigated using real-time polymerase chain reaction, western blotting, or enzyme-linked immunosorbent assay.ResultsThe levels of miR-338-3p were significantly lower in serum from patients with sepsis-induced kidney injury compared to the serum from healthy volunteers (P < 0.05). LPS reduced the level of miR-338-3p in HK-2 cells (P < 0.05). HK-2 cell viability, mitochondrial membrane potential, and Bcl-2 mRNA and protein levels were decreased by LPS (all P < 0.05). Apoptosis, the mRNA and protein levels of inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) and Bax, and the levels of cleaved caspase-9 and caspase-3 were increased by LPS (all P < 0.05). Raising the level of miR-338-3p mitigated these effects of LPS (all P < 0.05).ConclusionLPS-induced inflammation in HK-2 cells is reduced by miR-338-3p.

Suppressors of Cytokine Signaling Are Decreased in Major Depressive Disorder Patients.

There is strong evidence for an association between major depressive disorder (MDD) and inflammation. However, some studies have not observed an increase in inflammatory cytokines in MDD, and the mechanism behind this is unknown. In the present study, we evaluated MDD severity using the Montgomery–Åsberg Depression Rating Scale (MADRS) and quantified mRNA levels of the blood inflammatory cytokines interleukin (IL) 1β, IL-6 and tumor necrosis factor alpha (TNF-α), as well as negative regulators of cytokine signaling—comprising IL-10, IL-1RA, SOCS1, SOCS2 and SOCS3—in MDD patients (n = 36), with a focus on mild MDD, and normal controls (NC, n = 30). We also measured the serum levels of IL-1β and IL-6. Neither the blood mRNA nor the protein levels of inflammatory cytokines were significantly elevated in the MDD group compared with the NC group. However, we observed significant decreases in SOCS1, SOCS2 and SOCS3 mRNA in the MDD group compared to the NC group. A significant finding was a decrease in SOCS3 mRNA after remission from MDD, suggesting that SOCS3 is a trait marker in depressive symptoms. We consider that our findings would be useful in elucidating the pathophysiological mechanism of depression.

Albendazole ameliorates inflammatory response in a rat model of acute mesenteric ischemia reperfusion injury

BackgroundAcute mesenteric ischemia is known as a life threatening condition. Re-establishment of blood flow in this condition can lead to mesenteric ischemia reperfusion (MIR) injury which is accompanied by inflammatory response. Still, clear blueprint of inflammatory mechanism underlying MIR injury has not been provided. Interestingly, Albendazole has exhibited notable effects on inflammation and cytokine production. In this study, we aimed to evaluate outcomes of MIR injury following pretreatment with Albendazole with respect to assessment of mesenteric inflammation and ischemia threshold. MethodsMale rats were randomly divided into sham operated, vehicle treated, Albendazole 100 mg/kg and Albendazole 200 mg/kg groups. MIR injury was induced by occlusion of superior mesenteric artery for 30 min followed by 120 min of reperfusion. Samples were utilized for assessment of epithelial survival and villous height. Immunohistochemistry study revealed intestinal expression of TNF-α and HIF-1-α. Gene expression of NF-κB/TLR4/TNF-α/IL-6 was measured using RTPCR. Also protein levels of inflammatory cytokines in serum and intestine were assessed by ELISA method. ResultsHistopathological study demonstrated that pretreatment with Albendazole could ameliorate decline in villous height and epithelial survival following MIR injury. Also, systemic inflammation was suppressed after administration of Albendazole. Analysis of possible participating inflammatory pathway could demonstrate that intestinal expression of NF-κB/TLR4/TNF-α/IL-6 is significantly attenuated in treated groups. Eventually, IHC study illustrated concordant decline in mesenteric expression of HIF-1-α/TNF-α. ConclusionSingle dose pretreatment with Albendazole could ameliorate inflammatory response and enhance ischemia threshold following induction of MIR injury. More studies would clarify existing causality in this phenomenon.

Semaphorin 7A Accelerates the Inflammatory Osteolysis of Periapical Lesions

IntroductionSemaphorin 7A (SEMA7A), the only class VII semaphorin member, has been considered as a potent immunomodulatory regulator whose function in periapical lesions remains unclear. In our previous study, we found that SEMA7A was up-regulated in human periapical periodontitis and might be involved in the immune response and tissue destruction of periapical lesions. In this research, we aimed to further explore the specifical regulatory role of SEMA7A as well as its regulatory mechanisms in the inflammatory progression of periapical lesions. MethodsHuman periodontal ligament cells (hPDLCs) were collected from intact, caries-free, and healthy third molars and stimulated with recombinant human/mouse SEMA7A (rSEMA7A). Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, and enzyme-linked immunosorbent assay were used to detect the messenger RNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs) in hPDLCs. Twenty C57BL/6 mice were randomly divided into 4 groups: the healthy control group, pulp exposure group, pulp exposure and saline treatment group, and pulp exposure and rSEMA7A treatment group. Twenty microliters of sterile saline or 4 μg rSEMA7A were injected respectively into the buccal mucosa around the root apex at day 0, 7, and 14. Mandibular tissues were collected at day 21. Micro–computed tomographic and immunohistochemical staining were used to identify the bone destruction and inflammatory infiltration in periapical areas. Finally, an AKT inhibitor (LY294002) was used to pretreat hPDLCs before rSEMA7A stimulation to determine the role of AKT signaling activation in this process. ResultsAfter treatment with rSEMA7A, the messenger RNA and protein levels of interleukin (IL)-1β, IL-18, cyclooxygenase-2, MMP-1, and MMP-3 were remarkably up-regulated in hPDLCs. For in vivo experiments, compared with the other 3 groups, the treatment of rSEMA7A aggravated the osteolysis of alveolar bone and promoted the infiltration of immune cells into the apex area, along with increased expression levels of IL-1β, IL-18, MMP-1, and MMP-3. Furthermore, we found that the proinflammatory role of SEMA7A could be inhibited by the application of the AKT inhibitor (LY294002). ConclusionsSEMA7A likely aggravates the inflammatory reaction and bone destruction of existing periapical lesions. The proinflammatory role of SEMA7A in hPDLCs could partially be mediated through the AKT signaling transduction pathway.

Antioxidant glutathione inhibits inflammation in synovial fibroblasts via PTEN/PI3K/AKT pathway: An in vitro study.

Objectives In this study, we aimed to investigate whether glutathione (GSH) could decrease the secretion of reactive oxygen species (ROS), reduce inflammation, and modulate the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol 3-kinase/AKT (PTEN/PI3K/AKT) in synovial fibroblasts (SFs).Patients and methods A total of 30 DBA/1J female mice were used in this study. The release of ROS in MH7A cells was examined using a ROS assay kit. The effects of GSH on the messenger ribonucleic acid (mRNA) expression and protein levels of inflammatory cytokines were determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) in mouse SFs and MH7A cells, respectively. The PTEN/PI3K/AKT pathway was investigated via Western blotting. The effects of buthionine-sulfoximine (BSO), as an inhibitor of GSH, on these molecules were examined.Results The ROS were decreased after GSH treatment, and the mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)-1, MMP-3, were also significantly inhibited after GSH stimulation. However, the IL-10 levels were enhanced, and GSH increased the expression of PTEN. The GSH suppressed the activation of phosphorylated (p)-PI3K and p-AKT. The supplementation of the BSO restored the activation of PI3K/AKT pathway with a high production of ROS. The levels of TNF-α, IL-1β and IL-6 were also elevated, when the BSO was added.Conclusion These findings suggest that GSH can act as an inflammatory suppressor by downregulating the PTEN/PI3K/AKT pathway in MH7A cells. These data indicated a novel function of GSH for improving the inflammation of RA SFs and may help to alleviate the pathological process of RA.

Placenta-specific 8 (PLAC8) mediates inflammation and mobility of the hPDLCs via MEK/ERK signaling pathway

BackgroundPlacenta-specific 8 (PLAC8) is reported to regulate cellular functions in the progression of various diseases. However, its role in periodontitis is still unclear. MethodsHuman periodontal ligament cells (hPDLCs) were treated with lipopolysaccharide of Porphyromonas Gingivalis (LPS-PG) to mimic periodontitis in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression levels and western blot was for protein levels. Wound healing and transwell migration assays were performed to assess the cell mobility of hPDLCs. Both mRNA and protein levels of inflammatory cytokines including IFN-γ, IL-17, TNF-α, IL-4, IL-10 and IL-13 were accessed to evaluated process of periodontitis in vitro. Furthermore, the protein expressions of mitogen-activated protein kinase kinase (MEK), extracellular regulated protein kinase (ERK) and their phosphorylated products quantified by western blotting assay were determined to confirm the activation of the MEK/ERK signaling pathway. ResultsThe microarray analysis results showed that PLAC8 was most significantly downregulated in periodontium samples of patients with periodontitis, which participates in blood coagulation and integrin-mediated signaling pathway. PLAC8 was also markedly downregulated in the LPS-PG-treated hPDLCs. Moreover, overexpression of PLAC8 ameliorated inflammation and promoted cell mobility of LPS-PG-treated hPDLCs, while inhibition of PLAC8 exhibited the opposite effects. MEK/ERK was selected based on analyses of the protein-protein interaction (PPI) network as the potential signaling pathway interacted with PLAC8, and PLAC8 showed regulatory function on activation of the MEK/ERK pathway. Additionally, U0126, the inhibitor of MEK, abrogated the effects of PLAC8 on inflammation and cell mobility of LPS-PG-treated hPDLCs. ConclusionOverexpression of PLAC8 protected hPDLCs from dysfunction of inflammation and cell mobility via activating MEK/ERK pathway, indicating a novel therapeutic target for periodontitis.

Protein Kinase CK2 Maintains Reciprocal Balance Between Th17 and Treg Cells in the Pathogenesis of UC.

T helper 17 and regulatory T cells balance have crucial effects on the development of ulcerative colitis (UC). Currently, how to break this balance has not yet been found. Protein kinase CK2 is involved in the pathogenesis of immune-related disorders. However, its effects on the development of UC are obscure. The level of CK2 in the colonic tissues of UC patients was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and immune-histochemistry. Peripheral blood CD4+ T cells were treated with CK2 inhibitor CX4945 or transfected with Csnk2-interfering lentivirus; the mRNA expression and protein levels of inflammatory cytokines were detected by qRT-PCR, enzyme-linked immunosorbent assay, and flow cytometry. Moreover, CX4945 was administered to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice model for determining the function of CK2 on the regulation of intestinal inflammation. The CK2 level was markedly increased in inflamed mucosa of UC and highly expressed in CD4+ T cells. Blockade of CK2 by CX4945 inhibited Th17 but promoted regulatory T-cell (Treg) immune responses in CD4+ T cells from patients with UC. Moreover, CK2 blockade alleviated TNBS-induced colitis in mice. Inhibition of CK2 suppressed Th17 but promoted Treg differentiation by decreasing the phosphorylation level of signal transducer and activator of transcription (STAT) 3 and increasing the phosphorylation level of STAT5. The RNA-Seq and co-immunoprecipitation analysis further showed that CK2 could interact with Sirtuin 1 (SIRT1) and downregulate SIRT1 expression, which participated in Th17 inhibition but promoted Treg differentiation. Sirtuin 1 upregulation ameliorated TNBS-induced colitis, whereas SIRT1 blockade aggravated TNBS-induced colitis in mice. CK2 have crucial effects on the development of UC by maintaining reciprocal balance between Th17 and Treg cells. Protein kinase CK2 blockade might be considered as a new therapeutic approach for UC treatment.

Lymphatic Dysfunction Exacerbates Cutaneous Tumorigenesis and Psoriasis-Like Skin Inflammation through Accumulation of Inflammatory Cytokines

Lymphatic transport plays an important role in coordinating local immune responses. However, the biologic effects of impaired lymphatic flow invivo are not fully understood. In this study, we investigated the roles ofthe lymphatic system in skin carcinogenesis and psoriasis-like inflammation using k-cyclin transgenic (kCYC+/-) mice, which demonstrate severe lymphatic dysfunction. kCYC+/- mice showed augmented tumor growth in the two-stage skin carcinogenesis model and severe clinical scores in imiquimod-induced psoriasis-like skin inflammation compared with wild-type mice. Although mRNA levels of inflammatory cytokines in skin after topical application of 12-O-tetradecanoylphorbol-13-acetate or imiquimod were comparable between kCYC+/- and wild-type mice, protein levels of inflammatory cytokines, such as IL-17A, IL-22, and IL-23, were significantly upregulated in kCYC+/- mice in both models. Consistently, signal transducer and activator of transcription 3 pathway and NF-κB signaling were augmented in epidermal keratinocytes in kCYC+/- mice. These results suggest that lymphatic dysfunction in kCYC+/- mice caused accumulation of inflammatory cytokines, leading to the exacerbation of two-stage skin carcinogenesis and imiquimod-induced psoriasis-like skin inflammation. These findings add insight into the clinical problems of secondary malignancies and inflammatory dermatoses that may occur with extremity lymphedema.

Curcumol Alleviates the Inflammation of Nucleus Pulposus Cells via the PI3K/Akt/NF-κB Signaling Pathway and Delays Intervertebral Disk Degeneration

Intervertebral disk degeneration (IVDD) is closely associated with inflammatory environments. Curcumol has been shown to alleviate inflammation in various disease models, but its effects on IVDD remain unclear. In this study, we sought to determine the mechanism of curcumol in tumor necrosis factor (TNF)-α-induced nucleus pulposus cells and a mouse IVDD model. Nucleus pulposus cells were pretreated with curcumol and then exposed to TNF-α. Cell viability was analyzed using CCK-8, and the messenger ribonucleic acid and protein levels of inflammatory cytokines and PI3K/Akt/NF-κB-related signaling molecules were detected using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. The mouse IVDD model was established by puncturing the C6/7 level of the caudal spine, and then it was treated with curcumol after surgery. Alcian blue/orange G staining was performed to evaluate the severity of intervertebral disk damage, and immunohistochemistry was performed to detect the expression of TNF-α. Toxicologic effects of curcumol were measured by performing hematoxylin-eosin staining and enzyme-linked immunosorbent assay. Curcumol reduced IL-1β, IL-6, and TNF-α production in NPCs, and the phosphorylation of proteins in the PI3K/Akt/NF-κB signaling pathway was also decreased. The PI3K/Akt/NF-κB-related signaling molecules decreased when TNF-α-induced NPCs were treated with a PI3K inhibitor; however, curcumol did not reverse these effects. Invivo, curcumol ameliorated the progression of IVDD at the early stage and did not exert toxicologic effects. These results suggest a potential therapeutic use of curcumol to alleviate inflammation via the PI3K/Akt/NF-κB signaling pathway and delay the progression of IVDD.

Mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress

This study aims to investigate the beneficial effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on trabecular meshwork cells under oxidative stress and predict candidate genes associated with this process. Trabecular meshwork cells were pretreated with BMSC-derived exosomes for 24 h, and exposed to 0.1 mM H2O2 for 6 h. Survival rate of trabecular meshwork cells was measured with CCK-8 assay. Production of intracellular reactive oxygen species (iROS) was measured using a flow cytometer. RT-PCR and ELISA were used to detect mRNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs). Sequencing of RNA and miRNA for trabecular meshwork cells from Exo and control groups was performed on BGISEQ500 platform. Phenotypically, pretreatment of BMSC-derived exosomes improves survival rate of trabecular meshwork cells exposed to H2O2, reduces production of iROS, and inhibits expression of inflammatory cytokines, whereas increases expression of MMPs. There were 23 miRNAs, 307 lncRNAs, and 367 mRNAs differentially expressed between Exo and control groups. Exosomes derived from BMSCs may protect trabecular meshwork cells from oxidative stress. Candidate genes responsible for beneficial effects, such as DIO2 and HMOX1, were predicted.

The role of NLRP3 in lead-induced neuroinflammation and possible underlying mechanism

BackgroundNeuroinflammation induced by lead exposure (Pb) is a major cause of neurotoxicity of Pb in the central nervous system (CNS). The NLR family, domain of pyrin containing 3 (NLRP3) involves in various neurological diseases, while the question of whether NLRP3 plays a role in lead-induced neuroinflammation has not yet been reported. MethodsDevelopmental and knockout (KO) NLRP3 mice were used to establish two in vivo models, and BV2 cells were used to establish an in vitro model. Behavioral and electrophysiologic tests were used to assess the neurotoxicity of Pb, and immunofluorescence staining was used to assess neuroinflammation. Real-time PCR and western blot were performed to examine the mRNA and protein levels of inflammatory cytokines and NLRP3 inflammasomes. siRNA technology was used to block NLRP3 expression. ResultsPb exposure led to neural injure and microglial activation in the hippocampus region, while minocycline intervention attenuated Pb-induced neurotoxicity by inhibiting neuroinflammation. Pb increased the expression of NLRP3 and promoted cleavage of caspase-1 in mRNA and protein levels, and minocycline partially reversed the effects of Pb on NLRP3 inflammasomes. Blocking of NLRP3 by KO mice or siRNA attenuated neural alterations induced by Pb, weakened microglial activation in vivo and in vitro as well, without affecting the accumulation of Pb. Pb increased autophagic protein levels and phosphorylation of NF-κB, while suppressing autophagy or NF-κB inhibited Pb's effects on NLRP3. ConclusionsNLRP3 is involved in the regulation of Pb-induced neurotoxicity. These findings expand mechanism research of Pb neurotoxicity and may help establish new prevention strategies for Pb neurotoxicity.

HMGB1 Mediated Inflammation and Autophagy Contribute to Endometriosis.

AimHigh mobility group box (HMGB)-1 has been implicated in endometriosis due to the important regulatory roles of inflammation in endometriosis. The aim of the present study was to explore the roles of HMGB-1 in endometriosis and to elucidate the underlying mechanism.MethodsEndometrial specimens were collected from women with endometriosis and healthy volunteers. Immunohistochemistry staining was used to determine the expression patterns and localization of HMGB-1 in the normal, eutopic and ectopic endometrial tissues. Western blotting and qRT-PCR were used to determine the mRNA and protein levels of inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β], autophagy-related markers [beclin-1, autophagy-related (atg)13, microtubule-associated protein light chain (LC)3-I, LC-II and p62] and HMGB-1, respectively. Spearman’s rank correlation analysis was employed to investigate the correlation between HMGB-1 with inflammatory cytokines and beclin-1. Besides, human endometrial stromal cells (HESCs) were isolated from ectopic endometrium and subsequently transfected with shRNA against HMGB-1. After the transfected cells were subjected to hypoxia, ELISA was used to determine the levels of HMGB-1 and inflammatory cytokines in the cell supernatant. Western blotting was used to determine the expression levels of autophagy-related markers in the cells.ResultsPositive correlations were observed between HMGB-1 and the inflammatory cytokines. In addition, a positive correlation was also identified between HMGB-1 and beclin-1 in the ectopic endometrium. Further results demonstrated that autophagy-related markers beclin-1, atg13 and p62 were significantly upregulated in the ectopic endometrium. In addition, HMGB-1 knockdown suppressed the levels of inflammatory cytokines IL-6, TNF-α and IL-1β and autophagy-related markers beclin-1 and atg13, while upregulated p62 in HESCs under hypoxic condition.ConclusionKnockdown of HMGB-1 under hypoxic condition regulated inflammatory cytokines and autophagy-related markers. HMGB-1 might contribute to the development of endometriosis in part through regulating inflammatory response and autophagy.

Steroid Receptor Coactivator-3 Is Required for Inhibition of the Intestinal Muscularis Inflammatory Response of Postoperative Ileus in Mice.

Inflammation theory has suggested that the pathogenesis of postoperative ileus (POI) involves the steroid receptor coactivator-3 (SRC-3). Therefore, we investigated the role of SRC-3 in the muscles of the small intestine using a mouse POI model. Here, we reported that intestinal manipulation (IM) significantly reduced the extent of phenol red migration in the entire gastrointestinal tract, and the calculated geometric center (GC) value in wild-type (WT) mice at 24 h after surgery was higher than that in the knockout (KO) mice and in the sham-operated control group. The expression of SRC-3 was upregulated in the mouse intestinal muscularis at 24 h after surgical manipulation, and the mRNA and protein levels of inflammatory cytokines were upregulated compared with those in the control group. At 24 h after IM, the number of neutrophils in the experimental group was significantly higher than that in the control group; in the IM group, the number of neutrophils in the SRC-3-/- mice was markedly higher than that in the WT mice. At 24 h after IM, the myeloperoxidase (MPO) activity in the experimental group was significantly higher than that in the control group. In the IM group, the MPO activity of the SRC-3-/- mice was markedly higher than that of the WT mice. In summary, proinflammatory cytokines, the number of neutrophils, and the MPO activity were significantly increased in the muscularis of the jejunum and ileum of KO mice after IM compared with those of the WT mice, indicating that SRC-3 might play a protective role in POI.

Ursolic acid alleviates heat stress-induced lung injury by regulating endoplasmic reticulum stress signaling in mice

Acute lung injury has been reported to be associated with heat stress in various animals. Ursolic acid is a natural pentacyclic triterpenoid compound with multiple bioactivities. However, it remains unknown whether ursolic acid supplementation alleviates heat stress-induced lung injury. In the present study, male Institute of Cancer Research mice were left untreated under a normal temperature condition (23±1°C), receiving orally administrated with vehicle (phosphate buffered saline) or ursolic acid (40 mg/kg BW−1·d−1 for 2 d), and then were subjected to high temperature (41±1°C) for 2 h. Histological alterations, activities of antioxidative enzymes, apoptosis, generation of reactive oxygen species, abundance of inflammatory cytokines, and endoplasmic reticulum stress-related proteins were analyzed. Compared with the controls, heat stress treatment led to enhanced apoptosis, increased H2O2 production, and upregulated protein levels of inflammatory cytokines in the serum, including tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta. Activities of malondialdehyde, lactate dehydrogenase, and myeloperoxidase were increased, while the activities for superoxide dismutase and catalase were reduced in lung tissues of mice. All these alterations were significantly prevented by ursolic acid administration. Further study showed that heat stress led to activation of protein kinase-like ER kinase eukaryotic initiation factor 2 alpha -the transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) signaling, which was attenuated by ursolic acid supplementation. These findings indicated that ursolic acid pretreatment protected lung tissues against heat stress-induced injury by regulating inflammatory cytokines and unfolded protein response in mice. Ursolic acid supplementation might be a therapeutic strategy to alleviate high temperature-induced lung injury in humans and animals.

The Protective Effects of VVN001 on LPS-Induced Inflammatory Responses in Human RPE Cells and in a Mouse Model of EIU.

To investigate protective effects of VVN001 on lipopolysaccharide (LPS)-induced inflammatory response in human retinal pigment epithelial (RPE) cells and in a mouse model of endotoxin-induced uveitis (EIU), and to explore the underlying mechanisms. Human primary RPE (hRPE) and ARPE-19 cells were pretreated with or without VVN001 for 1h followed by 10μg/mL LPS stimulation for 24h. mRNA, and protein levels of inflammatory cytokines were analyzed with real-time PCR, western blotting, and ELISA. EIU was induced by intravitreal injection of 125ng LPS in female BALB/c mice. VVN001 eye drops (1%) were locally administrated every 4h for 24h after LPS injection. Clinical scores were assessed with a slit lamp. mRNA and protein levels of inflammatory cytokines were investigated simultaneously. Compared with the LPS group, VVN001 pretreatment significantly reduced mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), IL-6, IL-8, TNF-α, IL-1β, IL-18, caspase-1 in hRPE, and ARPE-19 cells. Protein overproduction of ICAM-1, TNF-α, IL-1β, NLRP3, caspase-1 P20, and p-IκBα/IκBα stimulated by LPS was suppressed by VVN001 pretreatment. In vivo, VVN001 significantly reduced the average clinical score from 5.0 to 1.3 in EIU mice. Furthermore, overproduction of ICAM-1, IL-1β, NLRP3, caspase-1 P20, and p-IκBα/IκBα at mRNA and protein levels were remarkably suppressed by VVN001. VVN001 alleviated the inflammatory response induced by LPS both in vitro and in vivo. The effect of anti-inflammation is associated with inhibiting the overproduction of ICAM-1 and blocking the activation of NLRP3 inflammasome and the NF-κB signaling pathway.