Journal: Journal of Cellular and Molecular Medicine

Article Title: Jianpi Jiedu Xiaozheng Fang Regulates Hepatocellular Carcinoma Proliferation and Metastasis Based on Network Pharmacology

doi: 10.1111/jcmm.71040

Figure Lengend Snippet: In vitro experiments to explore the effects of JPJDXZF on HCC cell proliferation and migration. (A) IC50 determination of JPJDXZF‐containing serum. IC50 values with 95% confidence intervals were calculated using a four‐parameter logistic model in GraphPad Prism. (B–E) Cell viability, apoptosis, and migration abilities of HepG2 and MHCC97‐H cells were evaluated using CCK‐8 assay, flow cytometry, Transwell assay, and wound healing (scratch) assay, respectively. (F) Relative mRNA expression levels of BIRC5, YAP, and TAZ were measured by qRT‐PCR. (G) Protein expression levels of BIRC5, YAP, and TAZ were assessed by Western blot analysis, normalised to GAPDH. Mean ± SD ( n = 3); unpaired two‐tailed Student's t ‐test. *** p < 0.001, **** p < 0.0001.

Article Snippet: The membrane was then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: BIRC5 (Proteintech, 10778‐1‐AP, 1:1000), YAP (Proteintech, 13663‐1‐AP, 1:1000), TAZ (Proteintech, 10670‐1‐AP, 1:1000), GAPDH (Proteintech, 60004‐1‐Ig, 1:5000), Phospho‐LATS1 (Thr1079) (Proteintech, 28998‐1‐AP, 1:1000), Phospho‐YAP1 (Ser127) (Proteintech, 80694‐2‐RR, 1:1000), Phospho‐MST1 (Thr183)/MST2 (Thr180) (Proteintech, 80093‐1‐RR, 1:1000), Phospho‐WWTR1 (Ser89) (Invitrogen, PA5‐105066, 1:1000), MST1 (Proteintech, 22245‐1‐AP, 1:1000), and STK3/MST2 (Proteintech, 12097‐1‐AP, 1:1000).

Techniques: In Vitro, Migration, CCK-8 Assay, Flow Cytometry, Transwell Assay, Wound Healing Assay, Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Jianpi Jiedu Xiaozheng Fang Regulates Hepatocellular Carcinoma Proliferation and Metastasis Based on Network Pharmacology

doi: 10.1111/jcmm.71040

Figure Lengend Snippet: Animal experiments to evaluate the effects of JPJDXZF on tumour growth in nude mice. (A) Subcutaneous tumour‐bearing mice, tumour images, and tumour weight ( n = 5). (B and C) Relative expression levels of BIRC5, YAP, and TAZ assessed by qRT‐PCR and Western blot, both normalised to GAPDH. (D) IHC analysis of the proliferation marker Ki67 in tumour tissues. Data are presented as mean ± SD ( n = 3). Statistical significance was assessed using unpaired Student's t ‐test. * p < 0.05, **** p < 0.0001 and ** p < 0.01.

Article Snippet: The membrane was then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: BIRC5 (Proteintech, 10778‐1‐AP, 1:1000), YAP (Proteintech, 13663‐1‐AP, 1:1000), TAZ (Proteintech, 10670‐1‐AP, 1:1000), GAPDH (Proteintech, 60004‐1‐Ig, 1:5000), Phospho‐LATS1 (Thr1079) (Proteintech, 28998‐1‐AP, 1:1000), Phospho‐YAP1 (Ser127) (Proteintech, 80694‐2‐RR, 1:1000), Phospho‐MST1 (Thr183)/MST2 (Thr180) (Proteintech, 80093‐1‐RR, 1:1000), Phospho‐WWTR1 (Ser89) (Invitrogen, PA5‐105066, 1:1000), MST1 (Proteintech, 22245‐1‐AP, 1:1000), and STK3/MST2 (Proteintech, 12097‐1‐AP, 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Jianpi Jiedu Xiaozheng Fang Regulates Hepatocellular Carcinoma Proliferation and Metastasis Based on Network Pharmacology

doi: 10.1111/jcmm.71040

Figure Lengend Snippet: Effects of siMST1/2 interference on the regulatory role of JPJDXZF in HCC cells via the Hippo pathway. HepG2 and MHCC97‐H cells were transfected with si‐NC or siMST1/2 and treated with JPJDXZF‐containing serum as indicated. (A) qRT‐PCR and Western blot analyses verifying the knockdown efficiency of siMST1/2 in HepG2 and MHCC97‐H cells, both normalised to GAPDH. (B) Cell viability was assessed by CCK‐8 assay following different treatments. (C) Flow cytometric analysis of apoptosis. (D and E) Cell migratory ability was evaluated by Transwell and Wound healing assays. (F) Western blot analysis of nuclear and cytoplasmic YAP/TAZ, p‐TAZ, p‐LATS1, p‐MST1/2, and BIRC5, with GAPDH as the loading control. (G) Quantitative densitometric analysis of Western blot results, normalised to GAPDH. (H) TEAD luciferase reporter assay evaluating transcriptional activity downstream of the Hippo pathway. Data are presented as mean ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membrane was then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: BIRC5 (Proteintech, 10778‐1‐AP, 1:1000), YAP (Proteintech, 13663‐1‐AP, 1:1000), TAZ (Proteintech, 10670‐1‐AP, 1:1000), GAPDH (Proteintech, 60004‐1‐Ig, 1:5000), Phospho‐LATS1 (Thr1079) (Proteintech, 28998‐1‐AP, 1:1000), Phospho‐YAP1 (Ser127) (Proteintech, 80694‐2‐RR, 1:1000), Phospho‐MST1 (Thr183)/MST2 (Thr180) (Proteintech, 80093‐1‐RR, 1:1000), Phospho‐WWTR1 (Ser89) (Invitrogen, PA5‐105066, 1:1000), MST1 (Proteintech, 22245‐1‐AP, 1:1000), and STK3/MST2 (Proteintech, 12097‐1‐AP, 1:1000).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Control, Luciferase, Reporter Assay, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Jianpi Jiedu Xiaozheng Fang Regulates Hepatocellular Carcinoma Proliferation and Metastasis Based on Network Pharmacology

doi: 10.1111/jcmm.71040

Figure Lengend Snippet: In vitro experiments investigating the regulation of BIRC5 expression by JPJDXZF via the Hippo pathway in HCC. (A) Verification of BIRC5 overexpression by qRT‐PCR and Western blot analysis, normalised to GAPDH. (B–E) Cell viability, apoptosis, and migratory capacity of HepG2 and MHCC97‐H cells were assessed using CCK‐8 assay, flow cytometry, Transwell assay, and wound healing assay, respectively. (F) Relative mRNA expression levels of BIRC5, YAP, and TAZ determined by qRT‐PCR. (G and H) Western blot analysis of total, nuclear, and cytoplasmic YAP/TAZ, phosphorylated YAP/TAZ, phosphorylated LATS1, phosphorylated MST1/2, and BIRC5 (GAPDH was used as internal reference). (I) The transcriptional activity downstream of the Hippo pathway was evaluated by the luciferase reporter assay. Data are presented as mean ± SD ( n = 3). Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membrane was then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies: BIRC5 (Proteintech, 10778‐1‐AP, 1:1000), YAP (Proteintech, 13663‐1‐AP, 1:1000), TAZ (Proteintech, 10670‐1‐AP, 1:1000), GAPDH (Proteintech, 60004‐1‐Ig, 1:5000), Phospho‐LATS1 (Thr1079) (Proteintech, 28998‐1‐AP, 1:1000), Phospho‐YAP1 (Ser127) (Proteintech, 80694‐2‐RR, 1:1000), Phospho‐MST1 (Thr183)/MST2 (Thr180) (Proteintech, 80093‐1‐RR, 1:1000), Phospho‐WWTR1 (Ser89) (Invitrogen, PA5‐105066, 1:1000), MST1 (Proteintech, 22245‐1‐AP, 1:1000), and STK3/MST2 (Proteintech, 12097‐1‐AP, 1:1000).

Techniques: In Vitro, Expressing, Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry, Transwell Assay, Wound Healing Assay, Activity Assay, Luciferase, Reporter Assay

Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: Gut microbiota induces sphingolipid pathway disturbance in the peripheral and brain. ( A ) Representative metabolites classified and counted based on their chemical taxonomy in shScr and shCry2 mice. The size of the pie chart corresponded to the relative abundance levels of metabolites, (n = 10 mice/group)., (B) Volcano plot of differentially expressed metabolites from shScr versus shCry2 mice. Blue, red, and grey represented downregulated, upregulated, and no significantly expressed metabolites, respectively, (n = 10 mice/group)., ( C ) Unsupervised hierarchical clustering of differentially expressed metabolites from shScr and shCry2 mice, (n = 10 mice/group)., ( D ) Pathway enrichment analysis of differentially expressed metabolites between shScr and shCry2 mice, (n = 10 mice/group)., ( E ) Representative Western blot and quantification of Sphk1 and S1PR1 in the hippocampus of shScr and shCry2 mice., ( F ) LC/MS analysis of Cer(18:1/18:0) and Cer(18:2/18:0) in shScr and shCry2 mice., ( G ) Linear regression analyses between sphingosine and Akkermansia, (left plot) and Ruminococcaceae, (right plot) expression levels from data integrated from 16 S rRNA sequencing and metabolomic sequencing. All data were expressed as mean ± SEM. Data were considered significant if * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Means were compared using the Student’s t-test in panel, ( E , F ). shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing, Sequencing, Metabolomic, IF-P

Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: FTY720 ameliorated the cognitive decline, impairment of BBB and tau pathology. ( A ) An overview of the experimental design. After receiving stereotaxic brain injections, three-month-old mice were housed for six weeks, followed by intraperitoneal injections of saline or FTY720 for two weeks. Behavioral and molecular biology tests were then conducted., ( B - E ) Escape latency of the shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice for 5 consecutive days in the MWM test, ( B ). Percentage of time spent in the target quadrant in the spatial probe trail of the MWM test, ( C ). Number of platform crossings in the spatial probe trail of the MWM test, ( D ). Representative heatmaps of the swimming path in the spatial probe trail of day 6 in the MWM test, ( E ), (n = 11–12 mice/group)., ( F - H ) Representative Western blot and quantification of Occludin, ZO-1, ( F ), Sphk1, S1PR1, ( G ), Tau1, pS396-tau, Tau5, and pT231-tau, ( H ) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice., ( I , J ) Representative immunofluorescence staining and quantification of pS396-tau, (red) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice. Magnification × 10. Scale bar = 100 μm. All data were expressed as mean ± SEM. Data were considered significant if *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Means were compared using a repeated measures 2-way ANOVA test with Bonferroni post hoc comparison in panel, ( B ) and one-way ANOVA, ( C - D , F - I ). FI: Fluorescence intensity. shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Saline, Western Blot, Immunofluorescence, Staining, IF-P, Comparison, Fluorescence

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: MAFG was highly expressed in the activated A1 astrocyte. (A) In rat astrocytes, MAFG positive cells were checked using immunofluorescence. (B and C) In rat astrocytes, MAFG mRNA expression was detected by RT‐PCR ( n = 3). (D and E) In rat astrocytes, the protein expression of MAFG, Serping1, C3, Sphk1, and S100A10 was detected using Western blot ( n = 3). Data in (B, C, D, and E) were analyzed using the unpaired t ‑test. ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation in rat astrocytes. (A and B) In rat astrocytes, the positive cells of C3 and S100A10 were checked using immunofluorescence. (C and D) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 were checked using RT‐PCR ( n = 3). (E and F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 were checked using Western blot ( n = 3). (G and H) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (C, D, E, F, G, and H) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Activation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the methylation of CRYAB in vitro which exerted anti‐inflammatory effects. (A) BSP was performed to detect CRYAB methylation (white represented unmethylation, black represented methylation). (B) In rat astrocytes, Western blot was applied to check the changes of key factors in DNA methylation (DNMT1, DNMT3a, and DNMT3b) ( n = 3). (C) In rat astrocytes, the positive cells of CRYAB were checked using immunofluorescence. (D) In rat astrocytes, CRYAB protein expression was checked using Western blot ( n = 3). (E) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (G) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (B, D, E, F, and G) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Methylation, In Vitro, Western Blot, DNA Methylation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation via CRYAB methylation. (A and B) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (C and D) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (E and F) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). All data were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Activation Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: LSW inhibited sphingolipid metabolism in vitro . (A) The mRNA expression of ASMase in A549 cells during PR8 infection. (B) The mRNA expression of SPHK1 in A549 cells during PR8 infection. (C) The protein expression of SPHK1 in A549 cells during PR8 infection. (D) The relative expression of SPHK1 analyzed by Image J. (E) The protein expression of SPHK1 in A549 cells during PR8 infection; Scale bar = 50 μm. (F) The relative expression of SPHK1 analyzed by Image-Pro Plus 6.0. (G) The mRNA expression of SPHK1 in A549 cells stimulated by TNF-α. (H) The mRNA expression of MCP-1 in A549 cells stimulated by TNF-α. (I) The mRNA expression of CXCL10 in A549 cells stimulated by TNF-α. (J) The mRNA expression of IL6 in A549 cells stimulated by TNF-α. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). *, p < 0.05 or ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: In Vitro, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: LSW inhibited SPHK1/S1P axis in vivo during PR8 infection. (A) The protein expression of SPHK1 in lungs during PR8 infection. (B) The protein expression and localization of SPHK1 in lungs during PR8 infection; Scale bar = 100 μm. (C) The relative expression of SPHK1 analyzed by Image-Pro Plus 6.0. (D) The production of S1P in lungs during PR8 infection. (E) The production of S1P in sera during PR8 infection. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). *, p < 0.05; **, p < 0.01 or ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: In Vivo, Infection, Expressing

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: Inhibition of SPHK1 reduced the expression of cytokines and chemokines during PR8 infection. (A) The mRNA expression of MCP-1 in A549 cells during PR8 infection. (B) The mRNA expression of CXCL10 in A549 cells during PR8 infection. (C) The mRNA expression of TNF-α in A549 cells during PR8 infection. (D) The mRNA expression of IL6 in A549 cells during PR8 infection. (E) The protein expression of SPHK1 in A549 cells during PR8 infection; Scale bar = 50 μm. (F) The relative expression of SPHK1 analyzed by Image-Pro Plus 6.0. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: Inhibition, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: LSW inhibited the expression of SPHK1, cytokines and chemokines induced by SPHK1 overexpression. (A) The mRNA expression of SPHK1 induced by SPHK1 overexpression in A549 cells. (B) The mRNA expression of CXCL10 induced by SPHK1 overexpression in A549 cells. (C) The mRNA expression of MCP-1 induced by SPHK1 overexpression in A549 cells. (D) The mRNA expression of IL6 induced by SPHK1 overexpression in A549 cells. (E) H&E staining of lungs after delivery of rAAV; Scale bar = 500 μm. (F) The expression of SPHK1 in lungs after delivery of rAAV. (G) The expression of TNF-α in lungs after delivery of rAAV. (H) The expression of IFN-γ in lungs after delivery of rAAV. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). **, p < 0.01 or ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: Expressing, Over Expression, Staining

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: The interaction of the key compound with SPHK1. (A) The interaction of bufalin (golden) with SPHK1 (gray); (B) The interaction of bufotalin (golden) with SPHK1 (gray); (C) The interaction of decamine (golden) with SPHK1 (gray); (D) The interaction of ursolic acid (golden) with SPHK1 (gray).

Article Snippet: 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).

Techniques:

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: Schematic representation of the mechanisms of LSW on influenza virus infection. The sphingolipid signaling pathway was activated during influenza virus infection, accompanied with the release of cytokines and chemokines. LSW alleviated virus-induced overactivated inflammatory response by inhibiting SPHK1/S1P axis.

Article Snippet: 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).

Techniques: Virus, Infection

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: LSW inhibited sphingolipid metabolism in vitro . (A) The mRNA expression of ASMase in A549 cells during PR8 infection. (B) The mRNA expression of SPHK1 in A549 cells during PR8 infection. (C) The protein expression of SPHK1 in A549 cells during PR8 infection. (D) The relative expression of SPHK1 analyzed by Image J. (E) The protein expression of SPHK1 in A549 cells during PR8 infection; Scale bar = 50 μm. (F) The relative expression of SPHK1 analyzed by Image-Pro Plus 6.0. (G) The mRNA expression of SPHK1 in A549 cells stimulated by TNF-α. (H) The mRNA expression of MCP-1 in A549 cells stimulated by TNF-α. (I) The mRNA expression of CXCL10 in A549 cells stimulated by TNF-α. (J) The mRNA expression of IL6 in A549 cells stimulated by TNF-α. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). *, p < 0.05 or ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: In Vitro, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: Inhibition of SPHK1 reduced the expression of cytokines and chemokines during PR8 infection. (A) The mRNA expression of MCP-1 in A549 cells during PR8 infection. (B) The mRNA expression of CXCL10 in A549 cells during PR8 infection. (C) The mRNA expression of TNF-α in A549 cells during PR8 infection. (D) The mRNA expression of IL6 in A549 cells during PR8 infection. (E) The protein expression of SPHK1 in A549 cells during PR8 infection; Scale bar = 50 μm. (F) The relative expression of SPHK1 analyzed by Image-Pro Plus 6.0. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: Inhibition, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Liu Shen Wan regulates the SPHK1/S1P axis to ameliorate influenza-induced inflammation via integrated network pharmacology and lipidomics

doi: 10.3389/fimmu.2025.1764754

Figure Lengend Snippet: LSW inhibited the expression of SPHK1, cytokines and chemokines induced by SPHK1 overexpression. (A) The mRNA expression of SPHK1 induced by SPHK1 overexpression in A549 cells. (B) The mRNA expression of CXCL10 induced by SPHK1 overexpression in A549 cells. (C) The mRNA expression of MCP-1 induced by SPHK1 overexpression in A549 cells. (D) The mRNA expression of IL6 induced by SPHK1 overexpression in A549 cells. (E) H&E staining of lungs after delivery of rAAV; Scale bar = 500 μm. (F) The expression of SPHK1 in lungs after delivery of rAAV. (G) The expression of TNF-α in lungs after delivery of rAAV. (H) The expression of IFN-γ in lungs after delivery of rAAV. The data were shown as mean ± SD and analyzed by one-way ANOVA Bonferroni or Dunnett’s multiple comparisons tests (n=3). **, p < 0.01 or ***, p < 0.001. vs . PR8 group.

Article Snippet: 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).

Techniques: Expressing, Over Expression, Staining