Correction: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics
Year: 2026
Type: erratum
Abstract: Influenza remains a formidable global health challenge, caused by the influenza virus, and responsible for an estimated 3 to 5 million severe cases and 290 to 650 thousand deaths annually worldwide (1). Influenza A virus (IAV), in particular, is a major etiological agent capable of crossing species barriers and is frequently associated with severe clinical outcomes like pneumonia and acute respiratory distress syndrome (2). While vaccination serves as the primary strategy for prevention, its efficacy is often compromised by antigenic mismatch with circulating strains (3). Antiviral drugs, though available, face escalating challenges due to the evolution of drug-resistant influenza viruses (4). This underscores the urgent imperative to develop novel therapeutic strategies that can effectively mitigate influenza pathogenesis.A hallmark of severe IAV infection is the onset of a dysregulated immune response characterized by a cytokine storm, which correlates strongly with disease severity and mortality (5). Emerging evidence indicates that IAV reprograms host metabolic pathways, including sphingolipid metabolism, to fuel excessive inflammation (6,7). Sphingolipids, once regarded primarily as structural membrane components, are now recognized as dynamic signaling molecules regulating viral infection and inflammatory processes. Key plays include sphingomyelin (SM), ceramide (Cer), sphingosine (Sph) and sphingosine-1-phosphate (S1P). The enzymes acid sphingomyelinase (ASMase) and sphingosine kinase-1 (SPHK1) catalyze the formation of Cer and S1P, respectively, which in turn activate the NF-kB, MAPK, and PI3K/AKT signaling cascade (8)(9)(10)(11). Notably, SPHK1 has become an important target against respiratory virus infection (8,12), and its inhibition has been shown to protect mice from IAV infection (13). Despite these insights, no sphingolipid-targeting therapies have yet transitioned to clinical use, highlighting a critical gap in current treatment options.Traditional Chinese medicines (TCMs) have been widely used to treat respiratory diseases in China (14)(15)(16). Reflecting the TCM principle of "clearing heat and detoxifying," many formulations target inflammatory excess and immune dysregulation (17,18). Liu Shen Wan (LSW), a classical prescription comprising Bezoar (the gall-stone of Bos taurus domesticus Gmelin), Musk (the excretion of Moschus), cinobufagin venom toad (the excretion of Venenum Bufonis), pearl (the shell of Pernulo), realgar, and borneol, is officially recommended in China for influenza treatment due to its potent heat-clearing and detoxifying properties (19). Multiple studies also indicate that LSW can suppress overactivated inflammatory response induced by IAV or Staphylococcus aureus (18,20,21). A recent clinical study further associated LSW's antiinflammatory effects with modulation of sphingolipid signaling (22). However, the precise mechanistic basis, especially its impact on sphingolipid metabolism remains unexplored.Given the multi-component nature of TCM formulations, systems-level approaches are essential to decipher their mechanisms (23). Network pharmacology offers a powerful framework for identifying potential drug-disease interactions and mapping complex mechanisms of action (24,25). Furthermore, lipidomics has emerged as a pivotal tool for elucidating how TCMs modulate lipid metabolic networks, including sphingolipids, in the context of infectious diseases (26). Integrating these methods can bridge traditional knowledge with contemporary molecular science, offering holistic insights into formula efficacy.In this study, we employed an integrated strategy combining network pharmacology and lipidomics to investigate whether LSW ameliorates IAV-induced inflammation via regulation of the sphingolipid signaling pathway. We specifically focused on the SPHK1/S1P axis, which was a key nexus of inflammatory signaling, using in vitro and in vivo models of IAV infection. Our findings reveal that LSW significantly attenuates dysregulated sphingolipid metabolism and suppresses inflammatory responses, underscoring its potential as a multi-target agent against influenzaassociated inflammation.The active components and corresponding targets of LSW were identified in TCMSP databases as previously mentioned (24). All the compounds were screened using the criteria of Drug Likeness (DL) ≥ 0.18 and Oral Bioavailability (OB) ≥ 30%. In addition, the active components and targets were supplied in PubMed and HERB database.Influenza-related targets were collected from Genecard, Drugbank, DisGeNET and CTD databases. The target name was corrected by the UniProt database. The potential targets of LSW against influenza were obtained via the online Venny 2.1 website (24).Potential therapeutic targets of LSW for influenza were analyzed in the DAVID database with the parameters set to Homo sapiens species and a significance threshold of P ≤ 0.05.To explore the key ingredients that exert pharmacological effects, the binding sites and binding activity of ingredients with SPHK1 were determined by the CB-Dock website. The compounds were selected according to the degree analyzed by Cytoscape software. SPHK1 (PDB format) was obtained from the PDB database and three-dimensional (3D) structures of key ingredients were acquired from the PubChem database. The vina scores and cavity size were used to evaluate the binding activity of ingredients with key targets, which could be obtained on the CB-Dock website. PyMOL software was used to analyze the molecular binding sites.LSW (lot: SA01004C) was offered by Suzhou Leiyunshang Medicine Pharmaceutical Co., Ltd. (Suzhou, China) and prepared as previously mentioned (27). In the previous study, the index components in LSW were detected by high-performance liquid chromatography (28). SPHK1 (lot: Ab262679) was purchased from Abcam. GAPDH (lot: 5174S) and b-Actin (lot: 3700) were purchased from CST. PF-543 hydrochloride (lot: S7177), a SPHK1 inhibitor, was purchased from Selleck.A549 cells were purchased from American Type Culture Collection (ATCC, USA). A549 cells were cultured in DMEM/ F12 (1:1) medium (Gibco, USA) with 10% fetal bovine serum. A549 cells were exposed to PR8 at a MOI of 1 for 60 min at 37 °C. After removing the inoculum, A549 cells were washed with PBS, then replaced with DMEM/F12 containing LSW(H) (1.5 mg/mL), LSW (M) (0.75 mg/mL), LSW(L) (0.375 mg/mL) or oseltamivir (OSE). During this process, A549 cells were infected with PR8 for 24 h to activate the sphingolipid signaling pathway. In addition, recombinant human TNF-a (Peprotech, lot:021825) was also used to stimulate A549 cells to activate the sphingolipid signaling pathway. A549 cells were stimulated with TNF-a (50 ng/mL) and treated with different doses of LSW for 24 h to detect the sphingolipid signaling pathway and cytokine expression.SPHK1 overexpression was achieved by transfecting A549 cells with plasmids containing the full-length human SPHK1 cDNA, which was designed and synthesized by AmyJet Scientific Inc. A549 cells (1.7 × 10 5 ) were seeded into 12-well plates in an incubator for 24 h, followed by further transfection with the plasmidstransfection reagent mixture using Lipofectamine 3000 ™ (Thermo Fisher Scientific, Pittsburgh, PA) for 6 h. After that, the media were refreshed with DMEM/F12 containing 2% FBS. After transfection for 16 h, A549 cells were incubated with LSW or PF-543 hydrochloride for 24 h. The sequence of SPHK1 can be obtained in Supplementary Table 1.Specific pathogen-free female BALB/c mice (Certificate No. GZL0008) were purchased from Guangdong medical laboratory animal center (Guangdong, China). Mice weighing 18-20 g and aged 6 to 8 weeks were used for animal experiments. They were housed in collective cages at 22 ± 1°C with a relative humidity of 50 ± 10% and a 12-h light/dark cycle. Mice were randomly allocated into 6 groups including normal control (NC) group, PR8 group, OSE group, LSW(H) (100 mg/kg), LSW(M) (50 mg/kg) and LSW (L) (25 mg/kg). All the mice were anesthetized and inoculated intranasally with 50 μL of PR8 (1LD 50 ) or PBS as previously described (24). After being infected with PR8 for 2 h, the infected mice were orally administered different doses of LSW, OSE or water daily for 5 days. All the mice were anesthetized and euthanized on day 6 after PR8 infection. The experiments involving in animals were carried out following the guidelines of the Ethics Committee of Guangzhou Medical University for the management of experimental animals and were approved by the Ethics Committee of Guangzhou Medical University (20230234).The rAAV9 expressing 3Flag (rAAV9-3Flag) and SPHK1 (rAAV9-SPHK1) were provided by PackGene Technology (Guangzhou, China). Mice were randomly allocated into 3 groups including rAAV9-3Flag, rAAV9-SPHK1 and LSW(H) treatment group. Mice were intranasally challenged with the rAAV9-3Flag or rAAV9-SPHK1 (50 mL; 2 × 10¹¹ GC/mL). After 15 days of infection, mice were administered LSW(H) or PBS via gavage for 5 days. The body weight was monitored daily for 6 consecutive days. Lung tissues were obtained to analyze the mRNA expression of SPHK1 and inflammatory mediators. In addition, the histological changes were analyzed by hematoxylin and eosin (H&E) reagents.The total RNA from cell samples and the lung tissues were extracted by TRIzol reagent. Complementary DNA was synthesized from RNA samples by reverse transcription, followed by amplification using the SYBR Premix Ex Taq kit (Vazyme, Nanjing, China). The sequences of primers were shown in Table 1.A549 cells were exposed to PR8 for 60 min at 37°C. After that, the inoculum was removed and replaced with DMEM/F12 containing LSW or OSE. After incubation for 24 h, A549 cells were washed with PBS and fixed with 4% paraformaldehyde for 25 min. Subsequently, A549 cells were permeabilized by 1% Triton for 20 min, which were then blocked with 5% BSA for 40 min. After that, A549 cells were stained with anti-SPHK1 antibody overnight at 4°C, followed by incubation with secondary antibodies (lot: SA00003-2, Proteintech, USA). The nuclei were stained with proLong antifade mountant and reagents containing DAPI (Thermo Fisher Scientific, USA) for 10 min. The images were acquired using a microscope (Nikon, Japan).A549 cells were infected with PR8 and treated with LSW for 24 h. After that, the culture medium was collected and centrifuged at 11,000 rpm at 4°C for 6 min to obtain the supernatant. Subsequently, the concentrations of Cer were detected using ELISA kits (Shanghai, mlbio). Experiments were conducted following the manufacturer's instructions. The lung tissues were homogenized and then were centrifuged at 11,000 rpm at 4°C for 15 min. The supernatants were obtained for detecting S1P (JONLNBIO, China). Experiments were conducted under the manufacturer's instructions.Samples were lysed on ice with RIPA lysis buffer (lot: P0013B, Beyotime, Shanghai, China). Protein concentrations were measured using BCA protein assay kits (Thermo Fisher Scientific, USA). Western blots were conducted as previously mentioned (27). The primary antibodies, including SPHK1, GAPDH or b-Actin, were incubated overnight at 4°C, followed by incubation with the secondary antibodies for 1 h.After being deparaffinized with xylene and rehydrated with gradient ethanol, lung tissue sections were soaked in 3% H 2 O 2 for 10 min. Subsequently, the lung sections were incubated with 5% BSA at room temperature for 60 min. After that, the lung sections were stained with SPHK1 antibody for 16 h. After being washed with PBST, the lung sections were incubated with the corresponding secondary antibody at room temperature for 60 min. The immunoreactivity of SPHK1 protein was visualized by a DAB substrate kit and stained with hematoxylin. Finally, the images we re obtained by inverted fl uorescence microscopy (Leica, Germany).Each sample was spiked with 20 μL of an internal standard mixture containing representative lipid classes (SPLASH ® LIPIDOMIX MASS SPRCSTANDARD, AVANTI, 330707-1EA), followed by the addition of 200 μL of water. The mixture was then vortexed for 10 s. Subsequently, 240 μL of precooled methanol was introduced, and the mixture was vortexed for an additional 30 s. Following this, 800 μL of pre-chilled methyl tert-butyl ether was added. Lipid extraction was performed by sonicating the mixture in an ice-cooled sonication bath for 20 min. The resulting mixture was then incubated at room temperature for 30 min and subsequently subjected to centrifugation at 14,000 g for 15 min at 10°C, after which the upper organic solvent layer was collected and evaporated to dryness under a nitrogen stream. Reverse-phase chromatography was employed for liquid chromatography (LC) separation using a CSH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters). Prior to analysis, the lipid extracts were dried under a gentle nitrogen stream and reconstituted in 200 μL of 90% isopropanol in acetonitrile. After centrifugation at 14,000 g for 20 min at 4°C, the clear supernatant was transferred to an LC-MS vial. A 3 μL aliquot was injected for analysis. The chromatographic separation employed a binary gradient with the following mobile phases: Solvent A, composed of acetonitrile and water (60:40, v/v) containing 0.1% formic acid and 0.1 mM ammonium formate; and Solvent B, composed of acetonitrile and isopropanol (10:90, v/v) containing 0.1% formic acid and 0.1 mM ammonium formate. The initial mobile phase was set at 40% solvent B with a flow rate of 300 mL/min for 3.5 min, which was then linearly increased to 75% solvent B over 9.5 min and further increased to 99% solvent B over 6 min, followed by reequilibration at 40% solvent B for 5 min. Mass spectra were acquired using the Q-Exactive Plus instrument in both positive and negative ionization modes. The electrospray ionization parameters were optimized and standardized for all measurements as follows: the source temperature was maintained at 300°C, the capillary temperature was set at 350°C, the ion spray voltage was set to 3000 V, the S-Lens RF level was adjusted to 50%, and the scan range of the instrument was configured to m/z 200 -1800.The raw LC-MS data were annotated using LipidSearch software. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal partial least squares-discriminant analysis (OPLS-DA), were conducted using SIMCA software (Umetrics, Sweden). Differential metabolites were identified by combining the Variable Importance in Projection (VIP) value with the P-value (VIP > 1.0 and P < 0.05).Statistical analysis was analyzed using GraphPad Prism 8. All quantitative data are reported as mean ± SD. To compare differences among groups, one-way ANOVA (Bonferroni or Dunnett's test) was applied depending on the outcome of homogeneity of variance testing. The statistical significance was defined as P < 0.05.LSW has previously been shown to suppress influenza virusinduced inflammatory response via inhibiting signaling pathway. on clinical evidence that LSW sphingolipid metabolism in by of and we applied network pharmacology to its The that potential targets were by LSW of key including NF-kB, and the sphingolipid signaling pathway these targets, and SPHK1, emerged as of a signaling network using Cytoscape SPHK1 as a critical the sphingolipid signaling pathway its pivotal in the effects of pharmacology analysis that the sphingolipid signaling pathway a key which LSW its the primary of infection, the lung is LSW ameliorates metabolic which in turn modulate sphingolipid in To this, we employed quantitative lipidomics to whether LSW sphingolipid metabolism in the clear separation among the and normal control groups modulation of lung lipid by We identified major lipid in including sphingolipids, and Table Notably, LSW significantly the of lipid species were and The effects of LSW on and Cer were to the effects in of the of In addition, LSW a range of dysregulated in inflammatory and signaling pathways, including that LSW attenuates lipid metabolic with effects on sphingolipid and pivotal in the sphingolipid signaling the of to and SPHK1 sphingosine to S1P, a key signaling of PR8 infection this mRNA of both and with LSW significantly this their expression with LSW also Cer induced by PR8 infection Furthermore, analysis that LSW SPHK1 protein expression in cells that influenza infection TNF-a and that SPHK1 is essential for of inflammatory as and we whether LSW this Notably, network pharmacology previously identified the signaling pathway as a target of LSW this LSW treatment SPHK1 mRNA also the expression of and to the of in vitro the protein expression of SPHK1 also in PR8 infection, which could also be by LSW LSW could the of S1P in or PR8 infection that LSW attenuates the inflammatory cascade by To the of SPHK1 in we employed the SPHK1 PF-543 hydrochloride in experimental shown in PF-543 treatment significantly the expression of SPHK1, and SPHK1 as a critical of IAV-induced inflammation and its therapeutic potential as a pharmacological with LSW(H) also the corresponding protein induced by SPHK1 overexpression further that LSW attenuates inflammation specifically the sphingolipid signaling we SPHK1 in A549 SPHK1 overexpression the expression of SPHK1, and an that was significantly by both LSW and the SPHK1 PF-543 in vitro findings were in Mice administered rAAV9-SPHK1 SPHK1 expression to the rAAV9-3Flag control group. treatment with LSW(H) significantly the expression of SPHK1 and the of key inflammatory and in the of these animals underscoring the therapeutic potential of LSW in the molecular by which LSW sphingolipid signaling, the molecular was to analyze the binding of key compounds with the CB-Dock binding was on scores and cavity size Multiple compounds binding potential to SPHK1 with and acid the binding The precise binding and molecular interactions of these compounds the SPHK1 active were in their as critical of the effects of LSW inhibition of virus is of the virus strains mortality and The overactivated inflammatory are hallmark of influenza virus infection. inflammation can in the formation of a which can a of inflammatory cells to sites of infection (2). inflammatory cells as and can normal lung tissues by is of the key to mortality in studies have that the sphingolipid signaling pathway is associated with the overactivated inflammatory response and in disease respiratory viral infection metabolites as S1P, have as key in inflammatory signaling and immune S1P can to to activate the signaling pathway. In addition, S1P can be to activate the signaling via the the pivotal of S1P SPHK1 is in overactivated inflammatory response viral infection. SPHK1 in S1P metabolic The of S1P signaling pathways, which a of excessive inflammation of SPHK1 can the of inflammatory in A549 cells stimulated by TNF-a IAV infection can the expression of SPHK1, by increased of S1P, and SPHK1 and overexpression of SPHK1 that inhibition of SPHK1 also the expression of and in A549 cells PR8 infection. Our that both pharmacological inhibition and overexpression of SPHK1 modulate the expression of inflammatory underscoring the pivotal of the SPHK1/S1P in influenza is important to that LSW, as a is to exert effects SPHK1 The therapeutic from a modulation of targets the inflammatory often to mitigate excessive inflammation in severe LSW, a traditional Chinese formula for its has shown efficacy in induced by and Staphylococcus primarily via inhibition integrated network pharmacology and we identified the sphingolipid signaling pathway as a key target of SPHK1 emerged as a critical by We further that LSW SPHK1 expression and S1P to of and in A549 cells and in that and acid as potent of SPHK1, in regulating sphingolipid of lipid are also important of inflammatory response influenza virus infection in addition to S1P Cer are that a in the as the The of Cer can the of cells by the signaling pathway by the of inflammatory of the of Cer also to the effects of LSW or the of remains to be further In addition, recent findings strongly that RNA viruses as influenza virus, have to or for or the of virus The modulation of or is a strategy to the of influenza A previous study has that LSW could the of influenza virus in cells and in mice LSW could the of (SM), and and and in PR8 infection. The effects of LSW on viral be associated with of the of or on the nature of current studies on To that SPHK1 is a key mechanistic pathway which LSW its effects, we both pharmacological and S1P experiments be conducted to can reverse the outcomes of LSW treatment in infected cells and animal cells or mice the of this these experiments whether the in SPHK1 activity and S1P signaling is essential for the therapeutic action of The of the key with The of with SPHK1 The of with SPHK1 The of with SPHK1 The of acid with SPHK1 findings SPHK1 as a critical of inflammation and that LSW potent effects by inhibiting the SPHK1/S1P signaling This study mechanistic evidence that LSW lung modulation of sphingolipid Furthermore, by lipidomics with we a systems-level on how traditional can host to viral infection, highlighting the value of approaches in
Source: Frontiers in Immunology
Authors: Biao Lei, Zhenyang Liu, Peifang Xie, Xuanxuan Li, Zhanyu Cui, Ruihan Chen, Bin Liu, Shihua Chen, Yaxin Li, Min Liang, Hao Liang, Ai Li, Fanghao Zheng, Zifeng Yang, Qinhai Ma
Institutions: First Affiliated Hospital of Guangzhou Medical University, TCM-Intigrated Cancer Center of Southern Medical University, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, Southern Medical University, Guangzhou Medical University, Guangzhou University of Chinese Medicine, Macau University of Science and Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W7130415843
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