Objective:To investigate the possible mechanisms underlying the involvement of the gut microbiota metabolite trimethylamine oxide (TMAO) in the pathogenesis of chronic spontaneous urticaria (CSU) .Methods:From June 2023 to June 2024, 67 CSU patients were enrolled from the Dermatology and Cosmetic Center, the Third Affiliated Hospital of Chongqing Medical University, and 69 age-matched healthy controls were also collected at the same time. Serum TMAO levels in both groups were measured using enzyme-linked immunosorbent assay (ELISA) , and D-dimer levels were collected from the CSU patients. A degranulation model was established in rat basophilic leukemia RBL-2H3 cells using anti-DNP IgE/DNP-BSA (IgE/Ag group) ; these cells were additionally grouped to be treated with different concentrations of TMAO (IgE/Ag+10 μmol/L TMAO group, IgE/Ag + 50 μmol/L TMAO group, IgE/Ag + 100 μmol/L TMAO group) ; untreated RBL-2H3 cells served as a blank control group. To investigate the effect of the extracellular signal-regulated kinase (ERK) phosphorylation inhibitor U0126 on the action of TMAO, RBL-2H3 cells were divided into another 5 groups: blank group, IgE/Ag group, IgE/Ag + 1 μmol/L U0126 group, IgE/Ag + 100 μmol/L TMAO group, and IgE/Ag + 100 μmol/L TMAO + 1 μmol/L U0126 group. In vivo, a localized allergic reaction model was established in the ears of C57BL/6 mice using anti-DNP IgE/DNP-BSA (IgE/Ag group) , and additional groups included blank group, IgE group, IgE/Ag + solvent (DMSO) group, and IgE/Ag + 10 μg/μl TMAO group. ELISA was performed to detect levels of inflammatory mediators in cell culture supernatants and mouse serum. Toluidine blue staining was employed to observe mast cell degranulation in the cell experiment and mouse ear tissue samples, Evans blue staining to assess vascular permeability in mouse ear tissue samples, and Western blot analysis to detect the ERK phosphorylation levels. The t test was used for comparisons between two groups, and one-way analysis of variance for multiple comparisons. Results:Serum TMAO levels were significantly higher in the 67 CSU patients than in the 69 healthy controls ( t = 13.27, P < 0.001) . Among the 32 CSU patients with available data about D-dimer, serum TMAO levels were positively correlated with D-dimer levels ( r = 0.62, P < 0.001) . In RBL-2H3 cell experiments, degranulation rates were significantly higher in the IgE/Ag + 10, 50, and 100 μmol/L TMAO groups than in the IgE/Ag group; morphologically, RBL-2H3 cells treated with 10, 50, and 100 μmol/L TMAO became increasingly rounded; 50 and 100 μmol/L TMAO significantly promoted the production of β-hexosaminidase (β-Hex) , interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) (all P < 0.01) , and upregulated ERK phosphorylation levels ( P < 0.01) ; the levels of ERK phosphorylation, IL-6, TNF-α, and β-Hex were significantly lower in the IgE/Ag + U0126 group than in the IgE/Ag group, as well as lower in the IgE/Ag + TMAO + U0126 group than in the IgE/Ag + TMAO group (all P < 0.001) . In the mouse model of localized allergic reaction, the IgE/Ag + TMAO group showed increased vascular permeability, edema degree, and mast cell degranulation, as well as significantly elevated ERK phosphorylation levels and TNF-α expression in mouse ear tissues compared with the IgE/Ag + DMSO group (both P < 0.05) . Conclusion:Elevated serum TMAO may participate in the pathogenesis of CSU by upregulating ERK phosphorylation levels in mast cells and skin tissues, thereby promoting IgE/Ag-mediated degranulation of effector cells and production of inflammatory mediators.

Objective:To investigate the possible mechanisms underlying the involvement of the gut microbiota metabolite trimethylamine oxide (TMAO) in the pathogenesis of chronic spontaneous urticaria (CSU) .Methods:From June 2023 to June 2024, 67 CSU patients were enrolled from the Dermatology and Cosmetic Center, the Third Affiliated Hospital of Chongqing Medical University, and 69 age-matched healthy controls were also collected at the same time. Serum TMAO levels in both groups were measured using enzyme-linked immunosorbent assay (ELISA) , and D-dimer levels were collected from the CSU patients. A degranulation model was established in rat basophilic leukemia RBL-2H3 cells using anti-DNP IgE/DNP-BSA (IgE/Ag group) ; these cells were additionally grouped to be treated with different concentrations of TMAO (IgE/Ag+10 μmol/L TMAO group, IgE/Ag + 50 μmol/L TMAO group, IgE/Ag + 100 μmol/L TMAO group) ; untreated RBL-2H3 cells served as a blank control group. To investigate the effect of the extracellular signal-regulated kinase (ERK) phosphorylation inhibitor U0126 on the action of TMAO, RBL-2H3 cells were divided into another 5 groups: blank group, IgE/Ag group, IgE/Ag + 1 μmol/L U0126 group, IgE/Ag + 100 μmol/L TMAO group, and IgE/Ag + 100 μmol/L TMAO + 1 μmol/L U0126 group. In vivo, a localized allergic reaction model was established in the ears of C57BL/6 mice using anti-DNP IgE/DNP-BSA (IgE/Ag group) , and additional groups included blank group, IgE group, IgE/Ag + solvent (DMSO) group, and IgE/Ag + 10 μg/μl TMAO group. ELISA was performed to detect levels of inflammatory mediators in cell culture supernatants and mouse serum. Toluidine blue staining was employed to observe mast cell degranulation in the cell experiment and mouse ear tissue samples, Evans blue staining to assess vascular permeability in mouse ear tissue samples, and Western blot analysis to detect the ERK phosphorylation levels. The t test was used for comparisons between two groups, and one-way analysis of variance for multiple comparisons. Results:Serum TMAO levels were significantly higher in the 67 CSU patients than in the 69 healthy controls ( t = 13.27, P < 0.001) . Among the 32 CSU patients with available data about D-dimer, serum TMAO levels were positively correlated with D-dimer levels ( r = 0.62, P < 0.001) . In RBL-2H3 cell experiments, degranulation rates were significantly higher in the IgE/Ag + 10, 50, and 100 μmol/L TMAO groups than in the IgE/Ag group; morphologically, RBL-2H3 cells treated with 10, 50, and 100 μmol/L TMAO became increasingly rounded; 50 and 100 μmol/L TMAO significantly promoted the production of β-hexosaminidase (β-Hex) , interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) (all P < 0.01) , and upregulated ERK phosphorylation levels ( P < 0.01) ; the levels of ERK phosphorylation, IL-6, TNF-α, and β-Hex were significantly lower in the IgE/Ag + U0126 group than in the IgE/Ag group, as well as lower in the IgE/Ag + TMAO + U0126 group than in the IgE/Ag + TMAO group (all P < 0.001) . In the mouse model of localized allergic reaction, the IgE/Ag + TMAO group showed increased vascular permeability, edema degree, and mast cell degranulation, as well as significantly elevated ERK phosphorylation levels and TNF-α expression in mouse ear tissues compared with the IgE/Ag + DMSO group (both P < 0.05) . Conclusion:Elevated serum TMAO may participate in the pathogenesis of CSU by upregulating ERK phosphorylation levels in mast cells and skin tissues, thereby promoting IgE/Ag-mediated degranulation of effector cells and production of inflammatory mediators.

Enterovirus (EV) includes a group of important RNA viruses with similarity in viral structure and pathogenesis. They can cause various diseases in humans and pose a great threat to human health. The biological events that occur in EV-host interactions are the core topics to be investigated for better understanding the pathogenesis of EV and developing related prevention and treatment strategies. With the development of genomics technologies, RNA modifications and protein post-translational modifications (PTM) in EV and its hosts have drawn much attention and some achievements have been made, which may shed light on developing precise strategies for the prevention and control of EV-associated diseases. In this paper, to better understand the pathogenesis of EV infection and provide reference for clinical translational research, the progress in the roles of RNA modifications and protein PTM in EV-host interactions are summarized.

Objective:To study the role and possible molecular mechanism of neural precursor cell-expressed developmentally down-regulated gene 4-like ( NEDD4 L) in the replication of hepatitis B virus (HBV). Methods:Small interfering RNA (siRNA) targeting NEDD4 L, plasmid expressing NEDD4 L with hemagglutinin(HA) C-terminal tag (pcDNA3.1- NEDD4 L-HA), plasmid expressing 1.3×HBV genome (pGEM-HBV1.3) and poly (dAT: dAT) were respectively transfected into HepG2 cells using Lipofectamine2000. HepG2.2.15 cells, a cell line that can stably express HBV, were used as control. The mRNA levels of NEDD4 L, interferon (IFN)-α, IFN-β, interferon-stimulated gene 56 ( ISG56), myxovirus resistance protein A ( MxA), oligoadenylate synthetase ( OAS), and the levels of HBV DNA or 3.5 kb HBV RNA were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the silence and over-expression of NEDD4 L, and the protein levels of the related signaling molecules. The amount of IFN-β in the cellular supernatant was measured by enzyme linked immunosorbent assay (ELISA). Student t test was used for comparison of continuous data between groups. Results:The levels of NEDD4L mRNA and protein in HepG2.2.15 cells were 10.53±0.47 and 4.17±0.43, respectively, which were both statistically higher than those in HepG2 cells (1.00±0.05, t=3.27, P=0.008 and 1.26±0.25, t=1.68, P=0.030, respectively). In HepG2 cells with knockdown of NEDD4 L, the expression level of HBV DNA in cellular supernatant was 0.32±0.09, which was statistically lower than that in the control (1.00±0.05, t=-0.93, P=0.020), and the expression level of 3.5 kb HBV RNA was 0.49±0.11, which was statistically lower than that in the control (1.00±0.05, t=-0.68, P=0.040), while the mRNA levels of IFN-β and downstream effector molecules ( ISG56, MxA and OAS) were all significantly increased compared with the control ( t=4.66, 9.38, 7.29 and 7.01, respectively, all P<0.01). With poly (dAT: dAT) treatment and vesicular stomatitis virus (VSV) stimulation, the levels of IFN-β in HepG2 cells with knockdown of NEDD4 L were (776.41±115.49) ng/L and (961.21±130.19) ng/L, respectively, which were both statistically higher than those of the control group ((320.15± 56.05) ng/L, t=2.43, P=0.020; (440.17±67.82) ng/L, t=2.85, P=0.030, respectively). With poly (dAT: dAT) treatment and VSV stimulation, the levels of IFN-β in HepG2 cells with overexpression of NEDD4 L were (156.18±26.47) ng/L and (176.67±34.51) ng/L, respectively, which were both statistically lower than those of the control group ((320.38±49.39) ng/L, t=-2.03, P=0.040; (440.59±68.83) ng/L, t=-1.93, P=0.030, respectively). Western blot showed that the replication of HBV reduced the protein level of melanoma differentiation-associated protein 5 (MDA5), a key molecule in upstream of IFN-β, but the down-regulation was not obvious in cells with the knockdown of NEDD4 L. Conclusion:The replication of HBV could promote the up-regulation of NEDD4L protein and subsequently reduce the protein level of MDA5, thereby inhibiting the production of IFN-β, which facilitates HBV to escape the innate immune response.