BACKGROUND:N6-Methyladenosine (m6A) methylation, a common form of RNA modification, play an important role in the pathogenesis of various diseases and in the ontogeny of organisms. Nevertheless, the precise function of m6A methylation in photoaging remains unknown. OBJECTIVES:This study aims to investigate the biological role and underlying mechanism of m6A methylation in photoaging. METHODS:m6A dot blot, Real-time quantitative PCR (RT-qPCR), western blot and immunohistochemical (IHC) assays were employed to detect the m6A level and specific m6A methylase in ultraviolet ray (UVR)-induced photoaging tissue. The profile of m6A-tagged mRNA was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis. Finally, we investigated the regulatory mechanism of KIAA1429 by MeRIP-qPCR, RNA knockdown and immunofluorescence assay. RESULTS:m6A levels were increased in photoaging and were closely associated with the upregulation of KIAA1429 expression. 1331 differentially m6A methylated genes were identified in the UVR group compared with the control group, of which 1192 (90%) were hypermethylated. Gene ontology analysis showed that genes with m6A hypermethylation and mRNA downregulation were mainly involved in extracellular matrix metabolism and collagen metabolism-related processes. Furthermore, KIAA1429 knockdown abolished the downregulation of TGF-bRII and upregulation of MMP1 in UVR-irradiated human dermal fibroblasts (HDFs). Mechanically, we identified MFAP4 as a target of KIAA1429-mediated m6A modification and KIAA1429 might suppress collagen synthesis through an m6A-MFAP4-mediated process. CONCLUSIONS:The increased expression of KIAA1429 hinders collagen synthesis during UVR-induced photoaging, suggesting that KIAA1429 represents a potential candidate for targeted therapy to mitigate UVR-driven photoaging.

To the Editor: Hepatocellular carcinoma (HCC) is a prominent contributor to cancer-related mortality on a global scale. While therapeutic approaches, such as surgical resection, liver transplantation, and radiofrequency ablation, have demonstrated effectiveness for specific patient populations, the 5-year overall survival (OS) rate in HCC is discouragingly low, estimated at only 18%. This unfavorable outcome can primarily be attributed to the high recurrence and metastasis rates.[1] Therefore, there is an urgent need for novel therapeutic strategies or targets to surmount the challenges in treating HCC. It has been postulated that tumor resistance to pyroptosis and an immunosuppressive tumor microenvironment significantly contribute to a suboptimal therapeutic response in various types of cancer interventions.[2] Pyroptosis, a form of programmed cell death triggered by inflammation, is distinguished by the formation of membrane pores mediated by gasdermin (GSDM) proteins. This process results in the release of cellular contents and cytokines, thus inciting a robust inflammatory response.[3] The intricate connections between pyroptosis and HCC have been unveiled. On the one hand, pyroptosis demonstrates the ability to inhibit tumorigenesis and tumor progression, presenting a favorable effect on anticancer therapy.[4] On the other hand, the release of inflammatory molecules prompted by pyroptosis creates environments conducive to the proliferative, invasive, metastatic, and drug-resistance potential of tumor cells.[5] Key regulators of pyroptosis have also been implicated in the establishment of an immunosuppressive microenvironment and linked to poorer prognosis. Given the conflicting roles of pyroptosis in HCC, investigating the underlying molecular mechanistic actions of pyroptosis in tumorigenesis and progression holds paramount importance in unearthing novel biomarkers and therapeutic targets. Epigenetic abnormalities play pivotal roles in HCC development and progression, and alterations in RNA N6-methyladenosine (m6A) modifications are involved in pyroptosis.[6] However, the potential connection between m6A modification and pyroptosis in HCC has not yet been extensively investigated. Epigenetic abnormalities play crucial roles in HCC development and progression. Additionally, alterations in RNA m6A modifications have been proposed to be linked with pyroptosis. In our analysis of the The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset, we examined the expression patterns of m6A writers and erasers in both cancerous and adjacent normal tissues. Interestingly, we discovered that among all the modifying enzymes, Wilms' tumor 1-associated protein (WTAP) exhibited the highest expression specifically in tumor tissues [Supplementary Figure 1A, https://links.lww.com/CM9/C298]. Furthermore, patients exhibiting high expression of WTAP had a poorer prognosis compared to those with low expression levels [Supplementary Figure 1B, https://links.lww.com/CM9/C298]. WTAP can serve as an independent prognostic factor, as suggested by both univariate and multivariate clinical analyses [Supplementary Figures 1C, D, https://links.lww.com/CM9/C298]. Additionally, there is a strong correlation between WTAP and the malignancy degree of HCC [Supplementary Figure 1E, https://links.lww.com/CM9/C298]. We assessed the expression level of WTAP in both normal hepatocytes and HCC cell lines, revealing markedly higher levels in cancer cells [Supplementary Figure 2A, https://links.lww.com/CM9/C298]. Following the transfection of Hep3B and PLC cells with synthetic small interfering RNA (siRNA) targeting WTAP [Supplementary Figure 2B, https://links.lww.com/CM9/C298], we observed a consistent reduction in the m6A level of HCC cells when the expression of WTAP was silenced [Supplementary Figure 2C, https://links.lww.com/CM9/C298]. We categorized the expression of WTAP and generated a heatmap of the differentially expressed genes [Supplementary Figure 2D, https://links.lww.com/CM9/C298]. Pathway enrichment analysis revealed that WTAP was involved in various inflammation and immune pathways. Notably, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway caught our attention due to its involvement in the caspase-1-gasdermin D (GSDMD) pyroptosis pathway [Supplementary Figure 2E, https://links.lww.com/CM9/C298]. To further confirm the involvement of WTAP in the apoptosis-related pathway in HCC, we conducted experiments where Hep3B cells were treated with lipopolysaccharide (LPS) and nigericin individually. Intriguingly, knocking down WTAP exhibited a remarkable reduction in cell pyroptosis [Figure 1A]. Additionally, we observed a noticeable downregulation in the protein levels of caspase-1 and GSDMD [Figure 1B]. Although WTAP has been reported to function in m6A modification through its interaction with methyltransferase-like 3 (METTL3) and METTL14, WTAP knockdown in HCC cells did not lead to a significant change in the expression levels of METTL3 and METTL14. This suggests that there might be an alternative pathway through which WTAP influences m6A modification [Supplementary Figure 3A, https://links.lww.com/CM9/C298]. Afterward, we utilized Cytoscape to construct the regulatory network of WTAP and its target genes.[7] Intriguingly, we found a correlation between WTAP and four target genes: JUNB, HK2, CCNB1, and ENO1 [Figure 1C]. The mRNA levels of HK2, JUNB, and ENO1 were significantly reduced after WTAP knockdown [Figure 1D]. However, through the methylation immunoprecipitation combined with quantitative real-time polymerase chain reaction (MeRIP-qPCR) assay, a notable reduction in m6A modification was observed only in JUNB after WTAP knockdown [Figure 1E]. Consequently, we predicted potential m6A sites on JUNB (http://www.cuilab.cn/sramp; Supplementary Figures 3B, C, https://links.lww.com/CM9/C298) and subsequently confirmed them through a RNA immunoprecipitation (RIP) assay. Finally, we established that WTAP could bind to a novel m6A site at position + 1456 on JUNB [Figure 1F], thereby maintaining the stability of JUNB mRNA [Figure 1G]. JUNB enhances the mRNA and protein expression of NLRP3.[8] To further investigate whether WTAP could regulate HCC pyroptosis through JUNB, we established a stable Hep3B cell line expressing JUNB-short hairpin RNA (shRNA) to analyze the role of JUNB in WTAP-involved pyroptosis [Supplementary Figure 3D, https://links.lww.com/CM9/C298]. Our findings demonstrated that the reduction of JUNB significantly inhibited the pyroptotic pathway of NLRP3-caspase-1-GSDMD, and this activity could not be restored by WTAP overexpression [Figures 1H, I]. This finding indicates that JUNB is required for WTAP-involved pyroptosis.Figure 1: WTAP promotes HCC cell pyroptosis through the JUNB-NLRP3-GSDMD pathway. (A) Microscope images (scale bar: 100 μm) and histogram showing relative pyroptotic cells in Hep3B cells transfected with si-NC or si-WTAP1 and treated with 0.1% LPS and 10 μmol/L nigericin. (B) Western blot analysis of WTAP, caspase-1, and GSDMD protein levels in Hep3B cells transfected with si-NC or si-WTAP1 after treatment with 10 μmol/L nigericin and 0.1% LPS. (C) Networks depicting the relationship between WTAP and its targets. (D) qRT-PCR results indicating the relative mRNA levels of WTAP, HK2, CCNB1, JUNB, and ENO1 in Hep3B cells transfected with si-NC or si-WTAP1. (E) MeRIP-seq showing m6A enrichment of HK2, ENO1, JUNB, and CCNB1 in Hep3B cells after si-NC or si-WTAP1 transfection. (F) RIP-RT-PCR assays detecting m6A sites in Hep3B cell lysates immunoprecipitated with WTAP antibody. (G) qPCR results showing JUNB mRNA levels at different time points after treatment with actinomycin D in Hep3B cells transfected with si-NC or si-WTAP. (H) Western blot analysis of WTAP, JUNB, NLRP3, Caspase-1, and GSDMD protein levels in Hep3B cells transfected with shNC, shJUNB, or shJUNB plus WTAP after treatment with 0.1% LPS and 10 μmol/L nigericin. (I) Microscope images (scale bar: 100 μm) and histogram depicting relative pyroptotic cells in Hep3B cells transfected with shNC, shJUNB, or shJUNB plus WTAP and treated with 0.1% LPS and 10 μmol/L nigericin. "ns" indicates not significant, * P <0.05, † P <0.01, ‡ P <0.001. HCC: Hepatocellular carcinoma; LPS: Lipopolysaccharide; m6A: N6-methyladenosine; meRIP-seq: Methylated RNA immunoprecipitation sequencing; qPCR: Quantitative polymerase chain reaction; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; WTAP: Wilms' tumor 1-associated protein.To investigate the clinical significance of WTAP, we analyzed tissue sections from 56 patients with HCC. immunohistochemical (IHC) staining and the resulting IHC scores suggested a higher expression level of WTAP in tumor tissues than in adjacent non-tumor tissues [Supplementary Figures 4A, B, https://links.lww.com/CM9/C298]. Using IHC scoring of WTAP, the tissue sections of the 56 patients were categorized into two groups. Although no correlation existed between WTAP expression and patient age, sex, tumor size, or clinical stage, a negative association was observed between WTAP and HCC differentiation [Supplementary Figure 4C, https://links.lww.com/CM9/C298]. Furthermore, patients with lower WTAP expression levels exhibited better OS [Supplementary Figure 4D, https://links.lww.com/CM9/C298]. To explore potential connections between WTAP and the immune microenvironment, we analyzed the correlation of WTAP with JUNB, CD8, and FOXP3. Patients with high expression of WTAP showed increased levels of JUNB and FOXP3, as well as decreased CD8 staining. This indicates an immunosuppressive microenvironment characterized by enhanced infiltration of regulatory T cells and a reduced number of cytotoxic T cells in patients with high expression of both WTAP and JUNB [Supplementary Figure 4E, https://links.lww.com/CM9/C298]. In conclusion, we validated the pivotal role of WTAP in HCC pyroptosis. WTAP enhances the NLRP3-associated GSDMD pyroptosis pathway by binding to and stabilizing JUNB mRNA, creating an immune-suppressive microenvironment that fosters HCC development. As a crucial m6A writer involved in pyroptosis, WTAP emerges as a critical determinant of immune suppression in HCC and holds promise as a potential target for HCC immunotherapy. Acknowledgments The authors thank Professor Guangjin Guo from the Laboratory of Air Force Medical Center for his assistance with the immunohistochemistry experiments. Funding This work was supported by grants from the Capital's Funds for Health Improvement and Research (No. 2022-25121) and Project of the Eighth Medical Center of PLA General Hospital (No. 2021MS003). Conflicts of interest None.

Background: The continuous clinical use of cisplatin is prevented by gastrointestinal toxicity. Methods: Cisplatin was used to treat THP-1-derived macrophages to see its differential effects on different subtypes of macrophages. Wild-type and Gsdme−/− mice models were used to examine the effect of cisplatin and metformin on intestinal inflammation in vivo. The effect of GSDME on macrophage polarization was further confirmed by GSDME knockdown. Results: We found that M2 macrophages, with more cell blebbing and GSDME cleavage, were more sensitive to cisplatin-induced pyroptosis than M1 macrophages. Cisplatin was capable of enhancing the M1 phenotype, which was reversed by GSDME knockdown. GSDME contributed to M1 polarization and GSDME knockdown promoted M2 phenotype via STAT6 activation. Reduced intestinal inflammation and increased M2 macrophage numbers was detected in cisplatin-treated GSDME-knockout mice. Furthermore, metformin alleviated cisplatin-induced intestinal inflammation by reducing M2 pyroptosis and enhancing M2 phenotype through GSDME inhibition. Conclusion: This is the first study to reveal the non-pyroptotic role of GSDME in macrophage polarization, revealing that metformin could be used in combination with cisplatin to reduce intestinal toxicity.

The potential of human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) in wound healing is promising, yet a comprehensive understanding of how fibroblasts and keratinocytes respond to this treatment remains limited. This study utilizes single-cell RNA sequencing (scRNA-seq) to investigate the impact of hucMSC-EVs on the cutaneous wound microenvironment in mice. Through rigorous single-cell analyses, we unveil the emergence of hucMSC-EV-induced hematopoietic fibroblasts and MMP13+ fibroblasts. Notably, MMP13+ fibroblasts exhibit fetal-like expressions of MMP13, MMP9, and HAS1, accompanied by heightened migrasome activity. Activation of MMP13+ fibroblasts is orchestrated by a distinctive PIEZO1-calcium-HIF1α-VEGF-MMP13 pathway, validated through murine models and dermal fibroblast assays. Organotypic culture assays further affirm that these activated fibroblasts induce keratinocyte migration via MMP13-LRP1 interactions. This study significantly contributes to our understanding of fibroblast heterogeneities as well as intercellular interactions in wound healing and identifies hucMSC-EV-induced hematopoietic fibroblasts as potential targets for reprogramming. The therapeutic targets presented by these fibroblasts offer exciting prospects for advancing wound healing strategies.

Front Cover Caption: The cover image is based on the Research Article Baicalin inhibits influenza A (H1N1)-induced pyroptosis of lung alveolar epithelial cells via caspase-3/GSDME pathway by Zhenqiao Wei et al., https://doi.org/10.1002/jmv.28790.

Baicalin (7-d-glucuronic acid-5, 6-dihydroxyflavone) derived from the root of Scutellaria baicalensis used as Traditional Chinese Medicine (TCM) has been revealed to exert potential antiviral activity via various pathways, while the molecular mechanisms have not been fully understood. Pyroptosis, an inflammatory form of programmed cell death (PCD), is reported to play a crucial role in host cell fate during viral infection. In this study, transcriptome analysis of mice lung tissue reveals that baicalin reverses the alterations of the mRNA levels of PCD-associated genes upon H1N1 challenge, with a concomitant decrease in the population of H1N1-induced propidium iodide (PI)+ and Annexin Ⅴ+ cells. Intriguingly, we find that baicalin contributes to the survival of infected lung alveolar epithelial cells partly through its inhibition of H1N1-induced cell pyroptosis, which is manifested by reduced bubble-like protrusion cells and lactate dehydrogenase (LDH) release. Moreover, the antipyroptosis effect of baicalin in response to H1N1 infection is found to be mediated by its repression on caspase-3/Gasdermin E (GSDME) pathway. Cleaved caspase-3 and N-terminal fragment of GSDME (GSDME-N) are detected in H1N1-infected cell lines and mice lung tissues, which are markedly reversed by baicalin treatment. Furthermore, inhibition of caspase-3/GSDME pathway by caspase-3 inhibitor or siRNA exerts an antipyroptosis effect equal to that of baicalin treatment in infected A549 and BEAS-2B cells, indicating a pivotal role of caspase-3 in the antiviral activities of baicalin. Conclusively, for the first time, we demonstrate that baicalin could effectively suppress H1N1-induced pyroptosis of lung alveolar epithelial cells via caspase-3/GSDME pathway both in vitro and in vivo.

Influenza A virus (IAV) infection causes a frenzied host response, which promotes acute lung inflammation. Andrographolide, an active component extracted from Andrographis paniculate, exerts significant inhibitive effects on acute inflammation induced by IAV. However, the molecular mechanism of the anti-inflammatory and antiviral effects remains poorly understood. Here, we found that andrographolide reduced mortality, alleviated body weight loss, and decreased lung index and inflammatory cytokines secretion rather than inhibiting viral replication. Further study with RNA-seq analysis revealed that the PI3K/AKT signaling pathway is significantly activated in the lungs of andrographolide-treated mice. The phosphorylated AKT and PI3K were significantly increased after andrographolide intervention by Western blot. Moreover, [pyroptosis](javascript:;)-related proteins, and downstream pathways of the PI3K/AKT signaling pathway, including cleaved-caspase 3 and GSDME-N, were decreased. The protective effect of andrographolide was significantly reduced after treatment with an AKT inhibitor. In summary, our findings suggested that andrographolide exerts a protective effect on IAV-induced [pneumonia](javascript:;) by activating the PI3K/AKT signaling pathway, which may represent a novel therapeutic strategy for IAV infection.