Purpose Increasing evidence suggested that microRNAs (miRs) are implicated in the regulation of the inflammatory response and autophagy in multiple diseases. The present study aimed to explore the effect of miR-223-3p on inflammation and autophagy in fungal keratitis (FK). Methods An FK mouse model was established, and primary corneal stromal cells were isolated by inoculation with Fusarium solani . The expression of miR-223-3p was determined by quantitative RT-PCR. Subsequently, the target gene of miR-223-3p was identified by a dual-luciferase reporter assay. The levels of miR-223-3p were altered by transfecting miR agomir/antagomir to evaluate its effects. Slit-lamp biomicroscopy and hematoxylin and eosin staining were employed to detect corneal damage. The levels of autophagy were assessed by immunofluorescence, Western blotting, mRFP-GFP-LC3 fluorescence microscopy, and electron microscopy. In addition, inflammation was demonstrated by determining the proinflammatory mediators IL-1β and TNF-ɑ. Results Our data suggested that miR-223-3p was increased and that autophagic flux was impaired in mouse FK. Then, we confirmed that autophagy-related gene 16L1 (ATG16L1) was a potential target of miR-223-3p and that this miR negatively regulated the expression of ATG16L1. The inhibition of miR-223-3p attenuated inflammation in FK, reduced P62 expression, and increased the ratio of LC3-II/LC3-I, whereas the overexpression of miR-223-3p displayed the opposite results. Conclusions Taken together, miR-223-3p might regulate autophagy via targeting ATG16L1 in experimental F. solani keratitis and is associated with the inflammatory response. MiR-223-3p might be a potential therapeutic target for FK.
Purpose Our previous investigations revealed a significant role of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m 6 A) modification in the development of corneal inflammation in Fusarium infection, but the exact mechanism is unknown. Therefore, this research aimed to explore how METTL3 affects the inflammatory process of fungal keratitis (FK) in mice. Methods We established in vitro and in vivo models by inoculating mice and primary corneal stromal cells with F. solani . METTL3 expression was confirmed by real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. After that, siRNAMETTL3 and AAV-sh-METTL3 were transfected into cells and mice to explore the role of METTL3 in the PI3K/AKT signaling pathway and inflammation. PI3K, p-PI3K, AKT, and p-AKT expression was analyzed by western blotting. Viability of corneal stromal cells was measured using a Cell Counting Kit-8 (CCK-8). Additionally, we detected interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels in corneal tissues and analyzed the role of METTL3 in inflammation in FK using slit-lamp biomicroscopy and hematoxylin and eosin staining. Results Here, our results show that METTL3 increased in mouse FK, and the expression of p-PI3K and p-AKT decreased when METTL3 was downregulated. We also found that knockdown of METTL3 expression attenuated the inflammatory response and decreased TNF-α, IL-1β, and IL-6 expression in corneal-infected mice. Furthermore, inhibition of the PI3K/AKT pathway attenuated the inflammatory response of FK, decreased the expression of the above inflammatory factors, and enhanced the viability of corneal stromal cells. Conclusions Based on the study results, METTL3 downregulation attenuates Fusarium -induced corneal inflammation via the PI3K/AKT signaling pathway.
Purpose The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK). Methods We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani . The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT–PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT–PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1 β (IL-1 β ), interleukin-6 (IL-6) and TNF- ɑ . Results Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani -induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK. Conclusions Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.
The chemokine (C-X3-C motif) ligand 1 (CX3CL1), also named fractalkine (FKN), has been implicated in psychiatric disorders and functions as a novel adipocytokine. However, no attention has been paid to the role of FKN in anorexia nervosa (AN). The current study was performed to explore FKN levels in AN to determine its role in the involvement of AN. A total of 96 girls aged 11-18 years with AN (n=34), healthy controls (HC; n=32) and simple obesity (OB, n=30) were enrolled in the cross-sectional study. Blood samples were collected during the fasting state. Serum FKN concentrations were determined using ELISA. The skinfold thickness (TSF) of the biceps and triceps as well as mid-arm muscle circumference (MAMC) were used to determine the nutritional status. Our results showed that serum FKN levels were significantly lower in the AN group than in the control and OB groups. After adjusting for body mass index (BMI), FKN concentrations in the AN group were statistically higher than in the HC and OB groups. Significant correlations between serum FKN and body weight, BMI, Cole index and serum insulin were observed. In addition, serum FKN levels were positively related to TSF and MAMC in all subjects. Serum FKN concentrations are attenuated in girls with AN compared with healthy adolescents and are positively related to nutritional status. The lower FKN levels may be regulated by nutrition status and response to starvation. After adjusting for BMI, higher FKN levels may reflect that persistent inflammation is present in patients with AN.
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