ObjectiveTo investigate the effect of isoacteoside on the replication process of respiratory syncytial virus （RSV） and its underlying mechanism. MethodsRSV-infected HEp-2 cells （control group） were treated with isoacteoside （isoacteoside-treated group）. Real-time quantitative PCR （qPCR） was used to analyze the mRNA expression levels of the RSV fusion protein （F） gene and matrix protein （M） gene in both groups. Viral titers were determined by plaque assay to assess the inhibitory effect of isoacteoside on viral replication. The number of RSV inclusion bodies formed was determined using cell transfection and laser confocal imaging to evaluate the role of isoacteoside in the formation of inclusion bodies required for RSV replication. 18 seven-week-old female BALB/c mice were randomly divided into three groups： uninfected control group （n=6）， RSV-infected group （n=6）， and RSV-infected group treated with isoacteoside （n=6）. 5 days after infection， the mice were euthanized and lung tissues were collected for pathological analysis to assess the therapeutic effects of isoacteoside on RSV-induced lung tissue injury. ResultsCompared to the control group， the isoacteoside-treated group exhibited the reduced mRNA levels of the RSV F gene （t=17.13， P<0.001） and M gene （t=18.22， P<0.001）， as well as a decrease in viral titer （t=15.32， P<0.001）. The number of inclusion bodies was also significantly reduced （t=16.12， P<0.001）. In the RSV-infected mouse experiment， compared to the RSV-infected group， the isoacteoside-treated group showed a decrease in the levels of inflammatory cytokines IL-4 （t=14.76， P<0.01） and IL-6 （t=21.13， P<0.001） in lung tissues， as well as decreased mRNA levels of the viral load-associated genes F （t=19.52， P<0.001） and M （t=18.76， P<0.001） in lung tissues. Pathological damage to lung tissue caused by RSV infection was alleviated. ConclusionIsoacteoside inhibits RSV replication by interfering with the formation of RSV inclusion bodies and alleviates lung tissue injury induced by RSV infection in mice.

ObjectiveTo investigate the molecular mechanism by which SAC1-like phosphatidylinositide phosphatase （SAC1） regulates NOD-like receptor protein 3 （NLRP3） inflammasome activation in macrophages and to explore its role in the pathological progression of metabolic dysfunction-associated steatohepatitis （MASH）. MethodsSingle-cell RNA sequencing （scRNA-seq） was utilized to analyze the differential expression levels of SACM1L in liver macrophage subpopulations between control and MASH mice. Bone marrow-derived macrophages （BMDMs） from control and high-fat diet-fed mice were isolated and cultured， and the expression levels of SACM1L and NLRP3 were determined via RT-qPCR and Western blot. CRISPR/Cas9 technology was employed to establish SACM1L-knockout THP-1 macrophage and HeLa cell lines. The phosphorylation of p65 and the activation status of the NLRP3 inflammasome were compared between wild-type and SACM1L-knockout THP-1 cells. Immunofluorescence assays were conducted to evaluate the spatial recruitment capacity of NLRP3 to the dispersed trans-Golgi network （dTGN） in wild-type and SACM1L-knockout HeLa cells. Furthermore， a MASH mouse model was established by feeding mice a choline-deficient high-fat diet containing 0.1% methionine （CDA-HFD） for 10 weeks， and the expression levels of SACM1L and NLRP3 in liver tissues were compared between the control and MASH groups. ResultsBioinformatics analysis and in vivo experiments consistently demonstrated that the expression levels of SACM1L and NLRP3 were significantly upregulated in both liver tissues and macrophages of MASH mice. SACM1L deficiency attenuated the increase in p-p65 levels induced by tripalmitoyl cysteinyl seryl tetra-lysine （Pam3CSK4）， inhibited the spatial recruitment of NLRP3 to the dTGN， and attenuated the subsequent maturation and release of downstream Caspase-1 and IL-1β. ConclusionSAC1 promotes NLRP3 expression by facilitating p65 signaling activation and mediates the spatial recruitment of NLRP3 to the dTGN. This process exacerbates the macrophage inflammatory response， thereby accelerating the pathological progression of MASH.

Objective : To explore the mechanism of Golgi phosphoprotein 3 (GOLPH3) regulating lysosomal biogenesis via mTORC1 signaling. Methods : GOLPH3 knockout (GOLPH3 KO) stable cell line was constructed by CRISPR Cas9. mTORC1 activity and the levels of TFEB and p ⁃TFEB in the control cells and GOLPH3 KO cells were compared through Western blot. Further, lysosome⁃associated membrane protein LAMP1 was labelled by the means of immunofluorescence and the number of lysosomes in the control cells and GOLPH3 KO cells was com⁃pared. Results : GOLPH3 KO suppressed mTORC1 activity significantly , decreased the cytoplasm level of p ⁃TFEB , increased the nuclear localization of active TFEB , and promoted the lysosome biogenesis. Conclusion Golgi protein GOLPH3 regulates lysosome biogenesis through mTORC1 signaling.

Objective : To explore the role of Golgi protein ACBD3 knockdown in ferroptosis. Methods : ACBD3 knockdown via shRNA , combined with treatment of ferroptosis inducer RSL3 , cell viability , iron levels , reactive oxygen species (ROS) levels , and lipid peroxide levels in different treatment groups were measured to comprehensively analyze the role of ACBD3 in ferroptosis. Results : Compared to control cells , the levels of ROS increased in ACBD3 knockdown cells. The total iron content was significantly reduced (P < 0. 001) , while the Fe2 + concentration in ACBD3 knockdown cells increased compared to control cells (P < 0. 001) . In the presence of ferroptosis inducer RSL3 , compared to control cells , cell viability significantly decreased ( P < 0. 001) , and lipid peroxide greatly increased in ACBD3 knockdown cells ( P < 0. 001) . Conclusion Golgi protein ACBD3 knockdown in mammalian cells changes iron homeostasis and promotes ferroptosis.