M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Interleukin-18 in chronic pain: Focus on pathogenic mechanisms and potential therapeutic targets.

Chronic pain has been proven to be an independent disease, other than an accompanying symptom of certain diseases. Interleukin-18 (IL-18), a pro-inflammatory cytokine with pleiotropic biological effects, participates in immune modulation, inflammatory response, tumor growth, as well as the process of chronic pain. Compelling evidence suggests that IL-18 is upregulated in the occurrence of chronic pain. Antagonism or inhibition of IL-18 expression can alleviate the occurrence and development of chronic pain. And IL-18 is located in microglia, while IL-18R is mostly located in astrocytes in the spinal cord. This indicates that the interaction between microglia and astrocytes mediated by the IL-18/IL-18R axis is involved in the occurrence of chronic pain. In this review, we described the role and mechanism of IL-18 in different types of chronic pain. This review provides strong evidence that IL-18 is a potential therapeutic target in pain management.

Quercetin alleviates DEHP exposure-induced pyroptosis and cytokine expression changes in grass carp L8824 cell line by inhibiting ROS/MAPK/NF-κB pathway.

Bis(2-ethylhexyl) phthalate (DEHP) is not only a widely used plasticizer but also a common endocrine disruptor that frequently lingers in water, posing a threat to the health of aquatic organisms. Quercetin (Que) is a common flavonol found in the plant kingdom known for its antioxidant, anti-inflammatory, and immunomodulatory effects. However, it is still unclear whether DEHP can cause pyroptosis and affect the expression of cytokines of grass carp L8824 cells and whether Que has antagonistic effect in this process. In our study, grass carp L8824 cells were treated into four groups after 24 h, namely NC group, DEHP group (1000 µM DEHP), Que group (5 µM Que), and DEHP + Que group (1000 µM DEHP + 5 µM Que). Our results indicate a significant increase in the level of ROS in L8824 cells after exposure to DEHP. DEHP upregulated oxidative stress markers (H2O2 and MDA) and downregulated antioxidant markers (CAT, GSH, SOD, and T-AOC). DEHP also upregulated MAPK and NF-κB signal pathway-related proteins and mRNA expressions (p-p38, p-JNK, p-EPK, and p65). As for cell pyroptosis and its related pathways, DEHP upregulated pyroptosis-related protein and mRNA expressions (GSDMD, IL-1ß, NLRP3, Caspase-1, LDH, pro-IL-18, IL-18, and ASC). Finally, DEHP can up-regulated cytokines (IL-6 and TNF-α) expression, down-regulated cytokines (IL-2 and IFN-γ) expression, and antimicrobial peptides (ß-defensin, LEAP2, and HEPC). The co-treatment of L8824 cells with DEHP and Que inhibited the activation of the ROS/MAPK/NF-κB axis, alleviated pyroptosis, and restored expression of immune-related indicators. Finally, NAC was applied to reverse intervention of oxidative stress. In summary, Que inhibited DEHP-induced pyroptosis and the influence on cytokine and antimicrobial peptide expression in L8824 cells by regulating the ROS/MAPK/NF-κB pathway. Our results demonstrate the threat to fish health from DEHP exposure and confirmed the harm of DEHP to the aquatic ecological environment and the detoxification effect of Que to DEHP, which provides a theoretical basis for environmental toxicology.

Mechanism of METTL3-Mediated m6A Modification in Cardiomyocyte Pyroptosis and Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: Myocardial ischemia/reperfusion (MI/R) injury is a complicated pathophysiological process associated with cardiomyocyte pyroptosis. Methyltransferase-like protein 3 (METTL3) catalyzes the formation of N6-methyl-adenosine (m6A) and participates in various biological processes. This study probed into the mechanism of METTL3 in cardiomyocyte pyroptosis in MI/R injury. METHODS: A rat model of MI/R was established. Rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment for the establishment of a cell model in vitro. METTL3 expression in myocardial tissues of MI/R rats and OGD/R-treated cardiomyocytes was determined using RT-qPCR and Western blot. The pathological changes of rat myocardial tissues were observed using hematoxylin and eosin staining. The positive expression of NLRP3 in myocardial tissues or cardiomyocytes was observed through immunohistochemistry or immunofluorescence. The activity of caspase-1 was measured using the colorimetric method. The expressions of GSDMD and cleaved caspase-1, as well as the levels of IL-1ß and IL-18 in rat myocardial tissues or cardiomyocytes were determined. m6A modification level was quantified. The binding relationship between pri-miR-143-3p and DGCR8 and the enrichment of m6A on pri-miR-143-3p were detected. The binding relationship between miR-143-3p and protein kinase C epsilon (PRKCE) was verified. RESULTS: METTL3 expression was elevated in MI/R rats and OGD/R cardiomyocytes. METTL3 silencing alleviated myocardial injury, reduced the number of NLRP3-positive cardiomyocytes, suppressed caspase-1 activity, decreased the protein levels of GSDMD-N and cleaved caspase-1, and decreased IL-1ß and IL-18 levels. METTL3 increased the total m6A level in MI/R rats and injured cardiomyocytes, promoted DGCR8 binding to pri-miR-143-3p, and enhanced miR-143-3p expression. miR-143-3p suppressed PRKCE transcription, and miR-143-3p overexpression reversed the inhibitory effect of METTL3 silencing on cardiomyocyte pyroptosis. CONCLUSION: METTL3 promoted DGCR8 binding to pri-miR-143-3p through m6A modification, thus enhancing miR-143-3p expression to inhibit PRKCE transcription and further aggravating cardiomyocyte pyroptosis and MI/R injury.

The effect of a-Lipoic acid (ALA) on oxidative stress, inflammation, and apoptosis in high glucose-induced human corneal epithelial cells.

PURPOSE: Oxidative stress and inflammation had been proved to play important role in the progression of diabetic keratopathy (DK). The excessive accumulation of AGEs and their bond to AGE receptor (RAGE) in corneas that cause the formation of oxygen radicals and the release of inflammatory cytokines, induce cell apoptosis. Our current study was aimed to evaluate the effect of ALA on AGEs accumulation as well as to study the molecular mechanism of ALA against AGE-RAGE axis mediated oxidative stress, apoptosis, and inflammation in HG-induced HCECs, so as to provide cytological basis for the treatment of DK. METHODS: HCECs were cultured in a variety concentration of glucose medium (5.5, 10, 25, 30, 40, and 50 mM) for 48 h. The cell proliferation was evaluated by CCK-8 assay. Apoptosis was investigated with the Annexin V- fluorescein isothiocyanate (V-FITC)/PI kit, while, the apoptotic cells were determined by flow cytometer and TUNEL cells apoptosis Kit. According to the results of cell proliferation and cell apoptosis, 25 mM glucose medium was used in the following HG experiment. The effect of ALA on HG-induced HCECs was evaluated. The HCECs were treated with 5.5 mM glucose (normal glucose group, NG group), 5.5 mM glucose + 22.5 mM mannitol (osmotic pressure control group, OP group), 25 mM glucose (high glucose group, HG group) and 25 mM glucose + ALA (HG + ALA group) for 24 and 48 h. The accumulation of intracellular AGEs was detected by ELISA kit. The RAGE, catalase (CAT), superoxide dismutase 2 (SOD2), cleaved cysteine-aspartic acid protease-3 (Cleaved caspase-3), Toll-like receptors 4 (TLR4), Nod-like receptor protein 3 (NLRP3) inflammasome, interleukin 1 beta (IL-1 ß), and interleukin 18 (IL-18) were quantified by RT-PCR, Western blotting, and Immunofluorescence, respectively. Reactive oxygen species (ROS) production was evaluated by fluorescence microscope and fluorescence microplate reader. RESULTS: When the glucose medium was higher than 25 mM, cell proliferation was significantly inhibited and apoptosis ratio was increased (P < 0.001). In HG environment, ALA treatment alleviated the inhibition of HCECs in a dose-dependent manner, 25 µM ALA was the minimum effective dose. ALA could significantly reduce the intracellular accumulation of AGEs (P < 0.001), activate protein and genes expression of CAT and SOD2 (P < 0.001), and therefore inhibited ROS-induced oxidative stress and cells apoptosis. Besides, ALA could effectively down-regulate the protein and gene level of RAGE, TLR4, NLRP3, IL-1B, IL-18 (P < 0.05), and therefore alleviated AGEs-RAGE-TLR4-NLRP3 pathway-induced inflammation in HG-induced HCECs. CONCLUSION: Our study indicated that ALA could be a desired treatment for DK due to its potential capacity of reducing accumulation of advanced glycation end products (AGEs) and down-regulating AGE-RAGE axis-mediated oxidative stress, cell apoptosis, and inflammation in high glucose (HG)-induced human corneal epithelial cells (HCECs), which may provide cytological basis for therapeutic targets that are ultimately of clinical benefit.

Wuzi Yanzong Pill relieves MPTP-induced motor dysfunction and neuron loss by inhibiting NLRP3 inflammasome-mediated neuroinflammation.

Parkinson disease (PD) is an age-related neurodegenerative disease, which is associated with the loss of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc), and neuroinflammation may lead to the occurrence of PD. Wuzi Yanzong Pill (WYP) has demonstrated neuroprotective and anti-inflammatory properties, but its molecular mechanism of action is still unclear. In this study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and LPS-mediated BV2 microglia to explore WYP intervention, anti-inflammatory effect and molecular mechanism in vivo and in vitro. The results showed that oral administration of WYP in MPTP-induced PD mice for 2 weeks ameliorated abnormal motor dysfunction, attenuated the loss of TH + neurons in SNpc, protected dopaminergic neurons, and inhibited the activation of microglia in MPTP-induced PD mice and LPS-stimulated BV2 cell. Meanwhile, WYP intervention inhibited the expression of IL-6, TNF-α, Pro-IL-1ß, IL-1ß, Pro-IL-18, IL-18 and enhanced the expression of IL-10 in the SNpc of PD mice. Simultaneously, WYP intervention inhibited the expression of NLRP3 inflammasome, accompanied by the decrease of the TLR4/MyD88/NF-κB pathway. However, the exact target and interaction of WYP on NLRP3 inflammasome and TLR4/MyD88/NF-κB pathway still needs to be further investigated.

Celastrol attenuates Guillain-Barré syndrome by inhibiting TLR4/NF-κB/STAT3 pathway-mediated Th1/Th17 cell differentiation.

Guillain-Barré syndrome (GBS) is an acute immune-mediated paralytic neuropathy with variable disease course and outcome. In this study, we aimed to investigate the therapeutic effects of celastrol on GBS and uncover its underlying mechanisms. Experimental autoimmune neuritis (EAN) is a typical animal model for GBS, and thus an EAN rat model was established with the injection of celastrol or/and LPS. We assessed the body weights and EAN clinical scores of rats. HE staining, flow cytometry, RT-qPCR, and Western blotting were respectively employed to measure pathological damage, proportions of cells (Th1, Th17, and Treg), Th1/Th17 cell differentiation-related mRNAs (IFN-γ, TBX21, IL-18, RORγT, IL-17, and IL-23) and TLR4/NF-κB/STAT3 pathway-related proteins (TLR4, NF-κB, p-NF-κB, STAT3, and p-STAT3). We found that celastrol attenuated clinical symptoms and pathological damage of GBS in EAN rats. Moreover, celastrol down-regulated Th1 and Th17 cell proportions, and the levels of IFN-γ, TBX21, IL-18, RORγT, IL-17, and IL-23 in EAN rats. Meanwhile, the levels of TLR4, p-NF-κB, and p-STAT3 were decreased by celastrol. Taken together, celastrol could restrain Th1/Th17 cell differentiation through inhibition of the TLR4/NF-κB/STAT3 pathway in EAN rats. Our findings suggest that celastrol may exert therapeutic effects on GBS by suppressing TLR4/NF-κB/STAT3 pathway-mediated Th1/Th17 cell differentiation.

Bone marrow mesenchymal stem cell-derived exosomal miR-30e-5p ameliorates high-glucose induced renal proximal tubular cell pyroptosis by inhibiting ELAVL1.

BACKGROUND: The rapid increase in the prevalence of diabetes has resulted in more cases of diabetic kidney disease (DKD). Treatment with bone marrow mesenchymal stem cells (BMSCs) may represent an alternative strategy to manage DKD. METHODS: HK-2 cells were treated with 30 mM high glucose (HG). Bone marrow MSC-derived exosomes (BMSC-exos) were isolated and internalized into HK-2 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays were used to measure viability and cytotoxicity. The secretion of IL-1ß and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. Quantitative RT-PCR was used to measure the levels of miR-30e-5p, ELAV like RNA binding protein 1 (ELAVL1), IL-1ß, and IL-18. The expression of ELAVL1 and pyroptosis-associated cytokine proteins was determined by western blot analysis. A dual-luciferase reporter gene assay was conducted to confirm the relationship between miR-30e-5p and ELAVL1. RESULTS: BMSC-exos decreased LDH, IL-1ß, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1ß, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion derived from BMSC-exos promoted HK-2 cell pyroptosis. Besides, miR-30e-5p over-expression or ELVAL1 knockdown could directly inhibit pyroptosis. ELAVL1 was a target of miR-30e-5p and knocking down ELAVL1 reversed the effect of miR-30e-5p inhibition in BMSC-exos-treated HK-2 cells. CONCLUSIONS: BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis by targeting ELAVL1 in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.

Transauricular Vagal Nerve Stimulation at 40 Hz Inhibits Hippocampal P2X7R/NLRP3/Caspase-1 Signaling and Improves Spatial Learning and Memory in 6-Month-Old APP/PS1 Mice.

OBJECTIVES: Transauricular vagal nerve stimulation (taVNS) at 40 Hz attenuates hippocampal amyloid load in 6-month-old amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice, but it is unclear whether 40-Hz taVNS can improve cognition in these mice. Moreover, the underlying mechanisms are still unclear. MATERIALS AND METHODS: 6-month-old C57BL/6 (wild type [WT]) and APP/PS1 mice were subjected to 40-Hz taVNS. Novel Object Recognition and the Morris Water Maze were used to evaluate cognition. Hippocampal amyloid-ß (Aß)1-40, Aß1-42, pro-interleukin (IL)-1ß, and pro-IL-18 were measured using enzyme-linked immunosorbent assays. Hippocampal Aß42, purinergic 2X7 receptor (P2X7R), nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), Caspase-1, IL-1ß, and IL-18 expression were evaluated by western blotting. Histologic assessments including immunofluorescence, immunohistochemistry, Nissl staining, and Congo red staining were used to assess microglial phagocytosis, neuroprotective effects, and Aß plaque load. RESULTS: 40-Hz taVNS improved spatial memory and learning in 6-month-old APP/PS1 mice but did not affect recognition memory. There were no effects on the cognitive behaviors of 6-month-old WT mice. taVNS at 40 Hz modulated microglia; significantly decreased levels of Aß1-40, Aß1-42, pro-IL-1ß, and pro-IL-18; inhibited Aß42, P2X7R, NLRP3, Caspase-1, IL-1ß, and IL-18 expression; reduced Aß deposits; and had neuroprotective effects in the hippocampus of 6-month-old APP/PS1 mice. These changes were not observed in 6-month-old WT mice. CONCLUSION: Our results show that 40-Hz taVNS inhibits the hippocampal P2X7R/NLRP3/Caspase-1 signaling and improves spatial learning and memory in 6-month-old APP/PS1 mice.

Mechanism for effect of tanshinone IIA on alleviating cardiomyocyte injury induced by oxygen glucose deprivation. / ä¸¹å‚é ®IIA减轻氧糖剥夺诱导心肌细胞损伤的机制.

OBJECTIVES: Tanshinone IIA has a wide range of myocardial protective effects. AK003290 is a long noncoding RNA (lncRNA) that is highly expressed in myocardial tissue, and its expression is down-regulated when myocardial injury occurs. This study aims to explore the mechanism for tanshinone IIA in alleviating myocardial cell damage induced by oxygen glucose deprivation (OGD). METHODS: OGD model was established in rat H9C2 cardiomyocytes. siRNA was transfected to reduce AK003290 expression. H9C2 cells were divided into 6 groups: A control group, a tanshinone IIA (TAN) group, an OGD group, a tanshinone IIA+OGD (TAN+OGD) group, a scrambled siRNA transfection+tanshinone IIA+OGD (scrambled siRNA+TAN+OGD) group, and a AK003290 siRNA transfection+tanshinone IIA+OGD (AK003290 siRNA+TAN+OGD) group. H9C2 cells in the TAN group were treated with 40 µmol/L tanshinone IIA for 12 h. The TAN+OGD group was treated with 40 µmol/L tanshinone IIA for 12 h, followed by OGD treatment for 12 h. The scrambled siRNA+TAN+OGD group and AK003290 siRNA+TAN+OGD group were transfected with the scrambled siRNA or AK003290 siRNA. Twenty-four hours later, the cells were treated with tanshinone IIA and OGD. Real-time RT-PCR was used to detect the expression of AK003290. Spectrophotometry was used to detect the content of lactate dehydrogenase (LDH) in cell culture medium to reflect LDH leakage rate, and enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). Western blotting was used to detect the protein expression of phospho-nuclear factor- κB (p-NF-κB). RESULTS: Compared with the control group, the leakage rate of LDH, the content of IL-1ß and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the OGD group (P<0.01 or P<0.001). Compared with the OGD group, the leakage rate of LDH, the content of IL-1ß and IL-18 in culture medium, and the protein expression level of p-NF-κB were decreased in the TAN+OGD group (P<0.05 or P<0.01). Compared with the control group, the AK003290 expression was increased in the TAN group (P<0.01) and it was decreased in the OGD group (P<0.05). Compared with the OGD group, the AK003290 expression was increased in the TAN+OGD group (P<0.05). Compared with the scrambled siRNA+TAN+OGD group, the leakage rate of LDH, the content of IL-1ß and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the AK003290 siRNA+TAN+OGD group (P<0.05 or P<0.01). CONCLUSIONS: Tanshinone IIA inhibits NF-κB activity and attenuates OGD-induced inflammatory injury of cardiomyocytes through up-regulating AK003290.

Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18.

NK cell-based therapies have shown promise for hematological cancer forms, but their use against solid tumors is hampered by their poor ability to infiltrate the tumor. NK cells release extracellular vesicles (EVs) containing cytolytic proteins, indicating that NK-cell derived EVs may have therapeutic potential. In this study, we compared the tumor-targeting potential of EVs derived from either primary NK cells or the NK cell lines NK-92 and KHYG-1 cultured in IL-15 alone or in combination with IL-12 and IL-18. Primary NK cells were also stimulated through the activating receptor CD16. Tumor cell apoptosis was measured using a panel of human colon, melanoma, glioblastoma, prostate, breast, and ovarian tumor cell line spheroids. NK cells or NK-92 cells stimulated with IL-12, IL-15, and IL-18 generated EVs with higher efficiency than EVs from resting cells, although similar amounts of EVs were produced under both conditions. Proteomic analysis indicated similar distribution of cytolytic proteins in EVs from primary NK cells and NK-92, but lower levels in KHYG-1 EVs that translated into poor capacity for KHYG-1 EVs at targeting tumor cell lines. Further, we show that CD16-stimulated NK cells release low amounts of EVs devoid of cytolytic proteins. Importantly, EVs from cytokine-stimulated NK cells penetrate into the spheroid core, and tumor spheroid susceptibility to NK-cell derived EVs was linked to differential expression of the NKG2D ligands MICA/B, which was blocked with an anti-NKG2D antibody. We conclude that EVs from activated primary NK cells or NK-92 cells has the best potential to infiltrate and target solid tumors.

Mechanism of LSD1 in oxygen-glucose deprivation/reoxygenation-induced pyroptosis of retinal ganglion cells via the miR-21-5p/NLRP12 axis.

BACKGROUND: Retinal ganglion cells (RGCs) are important retinal neurons that connect visual receptors to the brain, and lysine-specific demethylase 1 (LSD1) is implicated in the development of RGCs. This study expounded the mechanism of LSD1 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced pyroptosis of RGCs. METHODS: Mouse RGCs underwent OGD/R exposure, and then RGC viability was examined using the cell counting kit-8 method. The mRNA levels of Caspase 1, the protein levels of NOD-like receptor family pyrin domain-containing 3 (NLRP3), N-terminal fragment of gasdermin D (GSDMD-N), and cleaved-Caspase1, and the concentrations of interleukin (IL)-1ß and IL-18 were respectively examined. Subsequently, LSD1 expression was intervened to explore the underlying effect of LSD1 on OGD/R-induced pyroptosis of RGCs. Afterwards, the enrichments of LSD1 and histone H3 lysine 4 methylation (H3K4me) 1/2 on the microRNA (miR)-21-5p promoter were determined using chromatin-immunoprecipitation assay. And the binding interaction between miR-21-5p and NLRP12 was detected using dual-luciferase and RNA pull-down assays. Finally, the effects of miR-21-5p/NLRP12 on LSD1-mediated pyroptosis of RGCs were verified through functional rescue experiments. RESULTS: OGD/R treatment increased pyroptosis of RGCs and LSD1 expression. Silencing LSD1 declined levels of Caspase 1 mRNA, NLRP3, GSDMD-N, cleaved-Caspase1, IL-1ß, and IL-18 and limited pyroptosis of OGD/R-treated RGCs. Mechanically, LSD1 suppressed miR-21-5p expression via demethylation of H3K4me2 on the miR-21-5p promoter to hamper the binding of miR-21-5p to NLRP12, and thereby increased NLRP12 expression. Silencing miR-21-5p or overexpressing NLRP12 facilitated OGD/R-induced pyroptosis of RGCs. CONCLUSION: LSD1-mediated demethylation of H3K4me2 decreased miR-21-5p expression to increase NLRP12 expression, promoting pyroptosis of OGD/R-treated RGCs.

Paeonol regulates NLRP3 inflammasomes and pyroptosis to alleviate spinal cord injury of rat.

BACKGROUND: Spinal cord injury (SCI) is a life-threatening traumatic disorder. Paeonol has been confirmed to be involved in a variety of diseases. The purpose of this study is to investigate the role of paeonol on SCI progression. METHODS: Sprague Dawley (SD) rat was used for the establishment of SCI model to explore the anti-inflammation, anti-oxidation, and neuroprotective effects of paeonol (60 mg/kg) on SCI in vivo. For in vitro study, mouse primary microglial cells (BV-2) were induced by lipopolysaccharide (LPS)/adenosine triphosphate (ATP) treatment. The effect of paeonol on the polarization of LPS/ATP-induced BV-2 cells was determined by detection the expression inducible nitric oxide synthase (iNOS), tumour necrosis factor alpha (TNF-α), arginase-1 (Arg-1), and interleukin (IL)-10 using qRT-PCR. ELISA was used to assess the levels of IL-1ß, IL-18, TNF-α, malondialdehyde (MDA), and glutathione (GSH). Western blotting was conducted to determine the levels of NLRP3 inflammasomes and TLR4/MyD88/NF-κB (p65) pathway proteins. RESULTS: Paeonol promoted the recovery of locomotion function and spinal cord structure, and decreased spinal cord water content in rats following SCI. Meanwhile, paeonol reduced the levels of apoptosis-associated speck-like protein (ASC), NLRP3, active caspase 1 and N-gasdermin D (N-GSDMD), repressed the contents of IL-1ß, IL-18, TNF-α and MDA, and elevated GSH level. In vitro, paeonol exerted similarly inhibiting effects on pyroptosis and inflammation. Meanwhile, paeonol promoted BV-2 cells M2 polarization. In addition, paeonol also inactivated the expression of TLR4/MyD88/NF-κB (p65) pathway. CONCLUSION: Paeonol may regulate NLRP3 inflammasomes and pyroptosis to alleviate SCI, pointing out the potential for treating SCI in clinic.

Cytokine-mediated programmed proliferation of virus-specific CD8(+) memory T cells.

During infection, CD8(+) T cells not only respond to antigenic signals through their T cell receptor (TCR) but also incorporate inflammatory signals from cytokines produced in the local infected microenvironment. Transient TCR-mediated stimulation will result in programmed proliferation that continues despite removal of the antigenic stimulus, but it remains unclear whether brief exposure to specific cytokines will elicit similar effects. Here, we have demonstrated that brief stimulation of memory T cells with interleukin-12 (IL-12) and interleukin-18 (IL-18) results in tightly regulated programmed proliferation, in addition to acquisition of enhanced virus-specific cytokine production and cytolytic activity. CD8(+) T cells briefly exposed to IL-12 and IL-18 in vitro showed improved antiviral activity in vivo, as demonstrated by increased proliferation and reduced viremia. These results indicate that even transitory exposure to inflammatory cytokines can provide a selective advantage to infiltrating CD8(+) T cells by triggering a developmental program that is initiated prior to direct contact with virus-infected cells.

Electroacupuncture Prevents the Depression-Like Behavior by Inhibiting the NF-κB/NLRP3 Inflammatory Pathway in Hippocampus of Mice Subjected to Chronic Mild Stress.

BACKGROUND: The precise physiological mechanisms of acupuncture in the treatment of depression are still unknown. This study aimed to observe the effects of electroacupuncture (EA) on depression-like behavior of mouse in chronic mild stress (CMS) model and explore the underlying mechanism. METHODS: The depression model was established by using CMS method for 6 weeks. After the third week of the CMS paradigm, EA treatment was performed daily for 15 min over a period of 3 weeks. The antidepressant-like effects of EA were evaluated using the sucrose preference test and the forced swimming test (FST). The protein levels of the nuclear factor-kappa B (NF-κB), p-NF-κB, inhibitor of NF-κB, p-IκBα, NOD-like receptor protein 3, interleukin (IL)-6, IL-1ß, IL-18, and tumor necrosis factor-α (TNF-α) in hippocampus of mice were detected. RESULTS: Sucrose preference was decreased after 6 weeks of CMS and the effects of CMS was reversed by EA. CMS increased immobility time and decreased latency to the first immobility in the FST test, but these effects were reversed by EA. CMS-induced nuclear entry of NF-κB (nuclear/cytoplasmic ratio of NF-κB) with an increase in protein levels of p-NF-κB and p-IκBα in the hippocampus. The CMS also increased NLRP3 levels in the hippocampus. However, these effects were reversed by EA. In addition, the levels of IL-6, IL-1ß, IL-18, and TNF-α in the hippocampus were increased by CMS, and these effects of stress were reversed by EA. CONCLUSION: EA prevented CMS-induced depressive-like behaviors by inhibiting NF-κB/NLRP3 inflammatory pathway.

Vitamin C alleviates LPS-induced myocardial injury by inhibiting pyroptosis via the ROS-AKT/mTOR signalling pathway.

BACKGROUND: The efficacy of vitamin C in sepsis remains controversial. Whether vitamin C can alleviate lipopolysaccharide (LPS)-induced myocardial injury by inhibiting pyroptosis has not been studied. This study aimed to evaluate the effects of vitamin C on LPS-induced myocardial injury in vitro. METHODS: H9C2 cells were treated with indicated concentrations of LPS, and the cell viability was then assessed by CCK-8 assay. The levels of lactate dehydrogenase (LDH), CK-MB, IL-18 and IL-1ß were examined by enzyme-linked immunosorbent assay (ELISA). The levels of intracellular reactive oxygen species (ROS) were measured using the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA). Western blot assays were conducted to determine the levels of the ROS-associated protein nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) and pyroptosis-associated proteins, such as NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3), caspase-1 and gasdermin D (GSDMD). The AKT inhibitor MK-2206 was then applied to explore the signalling pathway. Finally, H9C2 cells were divided into the control group, LPS group, vitamin C + LPS group, and N-acetyl-L-cysteine (NAC) + LPS group. The intracellular ROS, levels of associated proteins, cell viability, and release of LDH, CK-MB, IL-18 and IL-1ß were examined. RESULTS: LPS decreased cell viability and induced ROS and pyroptosis in H9C2 cells in a dose-dependent manner. Moreover, LPS activated the AKT/mTOR pathway in H9C2 cells. The AKT inhibitor MK-2206 protected H9C2 cells from LPS-induced death by suppressing pyroptosis, without changing intracellular ROS level. Vitamin C significantly inhibited intracellular ROS and cell pyroptosis in LPS-treated H9C2 cells. Moreover, vitamin C suppressed the activation of the AKT/mTOR pathway. CONCLUSIONS: Our data suggest that vitamin C alleviates LPS-induced myocardial injury by inhibiting pyroptosis via the ROS-AKT/mTOR signalling pathway and thus provide novel insights into the prevention of sepsis-induced myocardial dysfunction.

MicroRNA-22 suppresses NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokine production by targeting the HIF-1α and NLRP3 in human dental pulp fibroblasts.

AIM: To investigate the synergetic regulatory effect of miR-22 on HIF-1α and NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1ß and IL-18 in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. METHODOLOGY: Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to determine the localization of miR-22-3p, NLRP3 and HIF-1α in human dental pulp tissues (HDPTs). The miR-22 mimics and inhibitor or plasmid of NLRP3 or HIF-1α were used to upregulate or downregulate miR-22 or NLRP3 or HIF-1α in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay were conducted to confirm target association. The mRNA and protein expression of HIF-1α, NLRP3, caspase-1, IL-1ß and IL-18 were determined by qRT-PCR and western blotting, respectively. The release of IL-1ß and IL-18 was analysed by ELISA. The significance of the differences between the experimental and control groups was determined by one-way analysis of variance, p < .05 indicated statistical significance. RESULTS: A decrease in miR-22 and an increase in HIF-1α and NLRP3 in HDPTs occurred during the transformation of reversible pulpitis into irreversible pulpitis compared with that in the healthy pulp tissues (p < .05). In the normal HDPTs, miR-22-3p was extensively expressed in dental pulp cells. HIF-1α and NLRP3 were mainly expressed in the odontoblasts and vascular endothelial cells. Whereas in the inflamed HDPTs, the odontoblast layers were disrupted. HDPFs were positive for miR-22-3p, HIF-1α and NLRP3. Computational prediction via TargetScan 5.1 and luciferase reporter assays confirmed that both NLRP3 and HIF-1α were direct targets of miR-22 in HDPFs. The miR-22 inhibitor further promoted the activation of NLRP3/CASP1 inflammasome pathway induced by ATP plus LPS and hypoxia (p < .05). In contrast, the miR-22 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS and hypoxia (p < .05). CONCLUSION: MiR-22, as a synergetic negative regulator, is involved in controlling the secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3 and HIF-1α. These results provide a novel function and mechanism of miR-22-HIF-1α-NLRP3 signalling in the control of proinflammatory cytokine secretion, thus indicating a potential therapeutic strategy for future endodontic treatment.

Production of Stem Cell Antigen 1 Sca-1/Ly-6A/E by Freshly Isolated NK Cells Cultured with Relevant Cytokines.

Activated NK cells in appropriate conditions are known to express stem cell antigen 1 (Sca-1/Ly-6A/E). To investigate its production, NK cells isolated from mouse spleens were incubated ex vivo in the presence of different combinations of cytokines (IL-12, IL-15, IL-18, and IFNγ). Expression of Sca-1 was found to be considerably higher in NK cells incubated in the presence of IL-18, IL-15, and IL-12 than in those treated with IL-15 and IL-18 only. To test the hypothesis that the effect of IL-12 was due to stimulation of IFNγ production, we replaced IL-12 with IFNγ in some samples and added specific anti-IFNγ antibody to some samples cultured with IL-15/IL-18+IL-12. In the subpopulations incubated in the presence of IL-15/IL-18 with added IFNγ instead of IL-12, the expression of Sca-1 was not increased. By contrast, in samples treated with IL-15/IL-18+IL-12 and anti-IFNγ antibody, the expression of Sca-1 was activated to a similar extent as in those stimulated by IL-15/IL-18+IL-12 combination without the antibody. The obtained data suggest that IL-12 activates the production of Sca-1 by NK cells through an IFNγ-independent mechanism.

Effects of IL-18 on the proliferation and steroidogenesis of bovine theca cells: Possible roles in the pathogenesis of polycystic ovary syndrome.

Interleukin 18 (IL-18) is a pleiotropic pro-inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL-18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL-18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL-18 on folliculogenesis. This work revealed that at 300-1000 pg/mL, IL-18 led to a time- and dose-dependently increase in cell proliferation (P < .05). IL-18 increased 17-hydroxyprogesterone (17OHP4) and androstenedione (A2) secretion with up-regulation of key steroidogenesis-related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL-18R protein is predominantly expressed in small-follicle (3-6 mm) TCs than large follicles (8-22 mm) by immunohistochemistry. We also found that the stimulation effects of IL-18 on TCs can be reversed with the addition of IL-18BP as early as at 4 hours of culture and reached the peak at 16 hours. We conclude that IL-18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL-18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL-18 in theca cells as well as its precise role in folliculogenesis.

High-efficient generation of natural killer cells from peripheral blood with preferable cell vitality and enhanced cytotoxicity by combination of IL-2, IL-15 and IL-18.

Natural killer (NK) cells are pivotal effector lymphocytes characterized for the innate immune response to pathogenic microorganism and tumor cells without priming and sensitization. Despite emerging knowledge has highlighted the rosy prospects in tumor immunosurveillance, yet the large-scale clinical application of NK cell-based therapy is hindered largely attributes to the defects in generating sufficient and high-quality cellular products. Herein, on the basis of 16 kinds of candidate combinations, we investigated the feasibility of cytokine cocktail-based strategy for convenient and standardized NK cell cultivation as well as the multifaceted characteristics and cytotoxicity against tumor cells. Our results revealed that joint utilization of Interleukin (IL)-2, IL-15, IL-18 manifested the optimal facilitation upon the ex vivo expansion and proportion of NK cells in peripheral blood mononuclear cells (PBMCs). Meanwhile, the obtained NK cell population expressed high levels of activating molecules (CD16 and NKG2D) and exhibited splendid cytotoxicity against K562 cell line. Collectively, with the aid of cytokine-based programming, we established an alternative strategy for facilitating the large-scale persistence and activation of NK cells from peripheral blood, which would benefit the NK cell- and chimeric antigen receptor-modified NK (CAR-NK) cell-based autologous or allogeneic tumor immunotherapy.