Objective: This study aims to investigate the therapeutic effects and underlying mechanisms of Xuanfei Baidu Decoction (XFBD) in a mouse model of dampness-heat toxin pneumonia. By exploring how XFBD exerts its effects, we seek to deepen our understanding of its role in treating pulmonary diseases and to address the current knowledge gap regarding its mechanisms of action, thereby supporting its clinical application. Methods: Ultra-high-performance liquid chromatography and high-resolution mass spectrometry (HRMS) were employed to analyze the chemical constituents of XFBD. The protective effects of XFBD were evaluated using a dampness-heat toxin-induced mouse model, established through dampness-heat exposure and HCoV-229E infection. XFBD was administered orally, followed by assessments including lung index measurement, micro-CT imaging, viral load quantification, cytokine analysis, and histological evaluation via hematoxylin-eosin staining. Proteomics and single-cell transcriptomic analyses were conducted to explore the potential mechanisms underlying XFBD’s pharmacological effects. A cellular model of HCoV-229E infection was developed to investigate changes in the cAMP/PKA signaling pathway. Molecular docking and surface plasmon resonance (SPR) experiments confirmed the strong binding affinity between key XFBD components and PKA. Finally, PKA activators and inhibitors were applied in vitro to validate these mechanistic findings. Results: In vivo studies demonstrated that XFBD significantly reduced the lung index, improved the structural integrity of lung and tongue tissues, and decreased levels of proinflammatory mediators, including IL-6, IL-8, and TNF-α. Proteomic and single-cell transcriptomic analyses showed that the differentially expressed proteins after XFBD treatment were primarily associated with inflammatory responses and immune regulation. The cAMP/PKA signaling pathway was identified as a key mechanism underlying these therapeutic effects. Notably, Western blot, ELISA, molecular docking, and SPR analyses confirmed that XFBD elevated cAMP levels and p-PKA expression, thereby activating the cAMP/PKA signaling pathway in vitro. Conclusion: This study demonstrated that XFBD significantly alleviates symptoms in mice with dampness-heat toxin pneumonia. Its therapeutic effects are mediated, at least in part, through activation of the cAMP/PKA signaling pathway. These findings provide compelling evidence that XFBD is an effective herbal remedy against HCoV-229E infection.

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Alanine ; chemistry ; metabolism ; Amino Acid Motifs ; Aspartic Acid ; chemistry ; metabolism ; Autophagy ; genetics ; Autophagy-Related Proteins ; Carrier Proteins ; chemistry ; metabolism ; Gene Expression Regulation, Fungal ; Membrane Proteins ; chemistry ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrogen ; deficiency ; Phagosomes ; chemistry ; drug effects ; metabolism ; Phosphorylation ; Protein Transport ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism ; Serine ; chemistry ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology

Objective To investigate the effect of sevoflurane preconditioning combined with postconditioning (Spost) on anoxia/reoxygenation (A/R) injury to neonatal rat cardiomyocytes.Methods Primary cultured neonatal rat cardiomyocytes were isolated from SD rats aged 1-3 days and cultured in DMEM liquid culture medium.The cells were seeded in 24-well plates (1 ml/hole),35 mm diameter dishes (5 ml/dish) or in 50 mm culture flasks (8 ml/flask) with a density of 3 × 105/ml and randomly divided into 9 groups (n =24 each):control group (group C),A/R group,Spre group (group S1),Spre + SB203580 group (group S1 + SB),sevoflurane postcon-ditioning (Spost) group (group S2),Spost + SB203580 group(group S2 + SB),Spre + Spost group (group S3),Spre + Spost + SB203580 group (group S3 + SB),and group SB203580 (group SB).The cells were cultured routinely for 160 min in group C and the cells were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 120 min followed by reoxygenation for 20 min in the other groups.The cells were incubated with 2.5 ％ sevoflurane for 20 min before anoxia in groups S1,S1 + SB,S3 and S3 + SB and in addition SB203580 (specific p38MAPK inhibitor) 5 μmol/L was added simultaneously in groups S1 + SB and S3 + SB.The cells were incubated with 2.5％ sevoflurane for 20 min after beginning of reoxygenation in groups S2,S2-SB,S3 and S3 + SB,and in addition SB203580 5 μmol/L was added simultaneously in groups S2 + SB and S3 + SB.The cells were incubated with SB203580 5 μmol/L for 20 min before anoxia and after beginning of reoxygenation in group SB.The lactate dehydrogenase (LDH) activity,cell survival rate and apoptotic rate were measured at the end of reoxygenation.The levels of phosphor-p38MAPK (p-p38MAPK) was detected at the end of Spre and Spost.Results Compared with group C,the LDH activity and apoptotic rate were significantly increased,while the cell survival rate was significantly decreased in the other groups (P ＜ 0.05).Compared with group A/R,the LDH activity and apoptotic rate were significantly decreased,while the cell survival rate was significantly increased in groups S1,S2 and S3 (P ＜ 0.05).There was no significant difference in the LDH activity,cell survival rate and apoptotic rate between groups S1,S2and S3 (P ＞ 0.05).The myocardial protective effect of Spre or Spost alone or in combination was eliminated by SB203580 (P ＜ 0.05).Spre or Spost alone up-regulated the expression of p-p38MAPK,Spre combined with Spost offered no additional benefit over Spre or Spost alone,and the up-regulative effect was eliminated by SB203580 (P ＜ 0.05).Conclusion Spre combined with Spost produces similar myocardial protective effect with that of either alone and it may because that both Spre and Spost attenuate A/R-induced injury to cardiomyocytes through p38MAPK signaling pathway.