Journal: Journal of Functional Foods

Article Title: Astragalin protects against lipopolysaccharide-triggered acute liver injury through suppression of necroptosis and inflammation and improvement of energy metabolism

doi: 10.1016/j.jff.2024.106298

Figure Lengend Snippet: Fig. 7. Astragalin (AST) eliminated lipopolysaccharide (LPS)-induced inflammatory response by inhibiting glycolysis enhancement mediated by the hypoxia- inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) signaling pathway. (A) LPS changed the concentrations of adenosine triphosphate (ATP) and lactic acid (LA) in liver and serum, and AST reversed these changes. Student’s t-test was performed, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001. (B) The activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and serum were enhanced by LPS, whereas AST inhibited the enhancement. Student’s t-test was performed, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001. (C) Glycolysis-related genes, including HK2, LDHA, glucose-6-phosphate dehydrogenase X-linked (G6pdx), and glucose transporter 1 (Glut-1), were upregulated by LPS but AST prevented the upregulation. Similar results also showed in the expression of HIF-1α and PKM2 genes. Student’s t-test was performed, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001. (D-E) Expressions of HIF-1α and PKM2 proteins were increased by LPS treatment, while AST decreased their expression. Student’s t-test was performed, n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: Adenosine triphosphate (ATP) content detection kit (#BC0305), lactic acid (LA) content assay kit (#BC2235), lactate dehydrogenase (LDH) activity assay kit (#BC0685), hexokinase (HK) activity assay kit (#BC0745), pyruvate kinase (PK) activity assay kit (#BC0545) were obtained from Beijing Solarbio Science & Technology Co., Ltd. Trizol (total RNA extraction reagent, #R0016), RIPA lysis buffer (strong, #P0013B), and BCA protein assay kit (#P0011) were provided by Beyotime Biotech Inc. Plus All-in-one 1st strand cDNA synthesis supermix (gDNA Purge, #E047-01B) and SYBR qPCR supermix plus (#E096-01B) were obtained from Novoprotein Scientific Inc. We got TNF-α polyclonal antibody (#17590–1-AP), TNFR1associated death domain protein (TRADD) polyclonal antibody (#15468–1-AP), HIF-1α polyclonal antibody (#20960–1-AP), PKM2, muscle2-specific monoclonal antibody (#60268–1-Ig), β-actin recombinant antibody (#81115–1-RR), and TANK-binding kinase 1 (TBK1) polyclonal antibody (#28397–1-AP) from Proteintech Group, Inc. Phospho-TBK1 (P-TBK1, Ser172) polyclonal antibody (#BD-PP1527) was provided by Biodragon Co. Ltd. Anti-phospho-RIP family of serinethreonine kinases (Ser166, P-RIPK1) polyclonal antibodies (#31122S), RIPK1 (D94C12) monoclonal antibody (#3493S), RIPK3 (D8J3L) monoclonal antibody (#15828S), and anti-phospho-RIPK3 (Thr231/ Ser232, E7S1R, #91702) were obtained from Cell Signaling Technology, Inc. Anti-phospho-MLKL (Ser345, P-MLKL) monoclonal antibody (#MABC1158) was provided by Merck Millipore Corporation, while MLKL polyclonal antibody (#PA5-71886) was provided by Thermo Fisher Scientific Inc. Bio-Rad Laboratories Co. Ltd provided CD68 monoclonal antibody (#MCA1957).

Techniques: Expressing

Journal: Journal of Functional Foods

Article Title: Astragalin protects against lipopolysaccharide-triggered acute liver injury through suppression of necroptosis and inflammation and improvement of energy metabolism

doi: 10.1016/j.jff.2024.106298

Figure Lengend Snippet: Fig. 8. The involved molecular mechanisms of astragalin (AST) in the treatment of lipopolysaccharide (LPS)-induced acute liver injury (ALI). LPS promotes the M1 type macrophage activation, causing inflammatory responses and the release of inflammatory cytokines, including tumor necrosis factor (TNF)-α. By binding to its receptor, TNF-α activated the receptor-interacting protein kinase 1 (RIPK1)/RIPK3/ mixed lineage kinase domain-like protein (MLKL) signal axis and mediated necroptosis. Meanwhile, LPS triggered the hypoxia-inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) signaling pathway, which mediates glycolysis-enhanced inflammatory response and oxidative stress. However, AST inhibited the transition of macrophages into M1 type, reduced the expression of pro-inflammatory cy tokines, which further decreased the activation of related signaling pathways, thereby preventing the liver from acute injury. This figure is created by Figdraw.

Article Snippet: Adenosine triphosphate (ATP) content detection kit (#BC0305), lactic acid (LA) content assay kit (#BC2235), lactate dehydrogenase (LDH) activity assay kit (#BC0685), hexokinase (HK) activity assay kit (#BC0745), pyruvate kinase (PK) activity assay kit (#BC0545) were obtained from Beijing Solarbio Science & Technology Co., Ltd. Trizol (total RNA extraction reagent, #R0016), RIPA lysis buffer (strong, #P0013B), and BCA protein assay kit (#P0011) were provided by Beyotime Biotech Inc. Plus All-in-one 1st strand cDNA synthesis supermix (gDNA Purge, #E047-01B) and SYBR qPCR supermix plus (#E096-01B) were obtained from Novoprotein Scientific Inc. We got TNF-α polyclonal antibody (#17590–1-AP), TNFR1associated death domain protein (TRADD) polyclonal antibody (#15468–1-AP), HIF-1α polyclonal antibody (#20960–1-AP), PKM2, muscle2-specific monoclonal antibody (#60268–1-Ig), β-actin recombinant antibody (#81115–1-RR), and TANK-binding kinase 1 (TBK1) polyclonal antibody (#28397–1-AP) from Proteintech Group, Inc. Phospho-TBK1 (P-TBK1, Ser172) polyclonal antibody (#BD-PP1527) was provided by Biodragon Co. Ltd. Anti-phospho-RIP family of serinethreonine kinases (Ser166, P-RIPK1) polyclonal antibodies (#31122S), RIPK1 (D94C12) monoclonal antibody (#3493S), RIPK3 (D8J3L) monoclonal antibody (#15828S), and anti-phospho-RIPK3 (Thr231/ Ser232, E7S1R, #91702) were obtained from Cell Signaling Technology, Inc. Anti-phospho-MLKL (Ser345, P-MLKL) monoclonal antibody (#MABC1158) was provided by Merck Millipore Corporation, while MLKL polyclonal antibody (#PA5-71886) was provided by Thermo Fisher Scientific Inc. Bio-Rad Laboratories Co. Ltd provided CD68 monoclonal antibody (#MCA1957).

Techniques: Activation Assay, Binding Assay, Expressing, Protein-Protein interactions

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 1 SNL induces elevated S100A4 expression in the SDH, and niclosamide attenuates NP. Western blot analysis revealed the temporal profile of S100A4 expression in both sham and SNL rats (A). SNL induced elevated S100A4 levels in ipsilateral L4/5 spinal cord segments from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Real-time PCR (C) revealed similar trends in S100A4 mRNA expression at 3, 5, 7 and 10 days in SNL rats. The data are presented as the means ± SEMs. ****p < 0.0001 versus the sham group. Immunofluorescence assays revealed the distribution of S100A4 (green) protein in the L4/5 SDH after sham or SNL surgery on POD 10 (D1, D2, and D3; scale bar, 500 μm). The fluorescence in tensity significantly increased at 14 days after SNL and remained high at 21 dpi (D4, D5) (scale bar, 500 μm). Data analysis further confirmed these results, as shown in E. Data are presented as the mean ± SEM. **p < 0.01, ****p < 0.0001, ipsilateral SDH versus sham SDH; ####P < 0.0001, ipsilateral SDH versus contralateral SDH. Double immunofluorescence staining images (F1, F2, and F3) revealed extensive coexpression of S100A4 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (scale bar, 500 μm). More details are shown at higher magnification for F1-1, F2-1 and F3-1 (scale bar, 100 μm). Single intrathecal administration of niclosamide (15–45 µg in 20 µl) in the later phase (POD 7) partially suppressed mechanical hypersensi tivity in the ipsilateral hind paw for up to 5 days after SNL, and the effect was observed 12 h after injection. Moreover, 45 µg of niclosamide had a greater effect than 15 µg of niclosamide (G). The data are presented as the means ± SEMs. ****p < 0.0001 for 15 µg of niclosamide versus DMSO. ####p < 0.0001, 45 µg of niclosamide versus DMSO. ψψψp < 0.001, ψψψψp < 0.0001, 15 µg of niclosamide versus 45 µg of niclosamide. Repeated administration of ni closamide in the early phase (POD 1) partially attenuated the development of mechanical hypersensitivity in SNL rats as well (I), which was maintained until POD 12. The data are presented as the means ± SEMs. ****p < 0.0001 for niclosamide versus DMSO. However, niclosamide treatment did not affect mechanical pain sensitivity in the contralateral hind paw (H, J). As shown in K, a single intrathecal injection of the rat S100A4 peptide (200 ng or 800 ng in 20 µl) rapidly decreased the PWT in the bilateral hind paws of naive rats from 2 h to 7 d. Moreover, 800 ng S100A4 induced more severe hyperalgesia than did 200 ng S100A4. On the other hand, saline did not affect the PWT of the hind paw. The data are presented as the means ± SEMs. ****p < 0.0001, 200 ng of S100A4 versus saline. ####p < 0.0001 800 ng S100A4 versus saline. ψψψψp < 0.0001 200 ng S100A4 versus 800 ng S100A4

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Double Immunofluorescence Staining, Injection, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 3 Exogenous S100A4 significantly contributes to A1 astrocyte activation in vitro. Western blot analysis revealed that GFAP and C3 protein levels were significantly increased after stimulation of 70% fusion primary astrocyte cultures with different concentrations of rat recombinant S100A4 protein (A), whereas 80 ng/ml S100A4 stimulation induced the greatest increase in GFAP and C3 expression (B). The data are presented as the means ± SEMs. *p < 0.05, **p < 0.01, ****p < 0.0001, normal versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus 80 ng/ml S100A4. RT‒PCR analysis revealed changes in the mRNA levels of A1-specific astrocyte genes after 80 ng/ml S100A4 stimulation, similar to the effects of the commonly known “cytokine cocktail” containing TNF-α, IL-1α and C1q (C). The data are presented as the means ± SEMs. *p < 0.05, ****p < 0.0001 S100A4 versus normal. #p < 0.05, ###p < 0.001, ####p < 0.0001 TNF-α, IL-1α and C1q versus normal. Moreover, incubation with 80 ng/ml S100A4 resulted in higher OD values than those of normal cells and cells incubated with other concentrations of S100A4 (D). The data are presented as the means ± SEMs. ****p < 0.0001 versus 80 ng/ml S100A4. Im munofluorescence revealed that 70% of the fusion primary astrocytes (GFAP, green; DAPI, blue) incubated with TNF-α, IL-1α and c1q and S100A4 (80 ng/ ml) for 24 h presented stronger GFAP (green) and C3 (red) fluorescence intensities and, accordingly, showed higher GFAP+C3+ fluorescence intensities (E) (scale bar, 100 μm). Quantitative analysis further confirmed these results (F). The data are presented as the means ± SEMs. ****p < 0.0001, normal versus S100A4. ####p < 0.0001, normal versus TNF-α, IL-1α and C1q

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Incubation, Fluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 4 S100A4 inhibition reduces A1 astrocyte activation following SNL. Western blot analysis revealed significantly reduced levels of GFAP and C3 at 10 and 14 days post-SNL in rats treated with successive niclosamide injections, starting from POD 1, along with decreased S100A4 expression (A and B). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001, normal versus SNL + DMSO. #p < 0.05, ##p < 0.01, ####p < 0.0001, SNL + DMSO versus SNL + niclosamide. In addition, fluorescence images confirmed that astrocytes were remarkably activated in the SDH after SNL, and the fluorescence in tensity of GFAP (C3) (green) was much greater than that in the sham group (C1) (scale bar, 500 μm). Successive intrathecal injection of niclosamide begin ning on POD1 for 3 days, but not DMSO, suppressed SNL-induced activation of astrocytes (C3 and C4). However, niclosamide had no effect on sham rats (C2) (scale bar, 500 μm). The data summary further confirmed these results, as shown in D. Moreover, niclosamide treatment reduced the fluorescence intensity of GFAP+C3+ (yellow) in SNL rats (E and F). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO group versus SNL + DMSO group. ###p < 0.001, SNL + DMSO versus SNL + niclosamide. ELISA (G) revealed that the administration of niclosamide also decreased the protein levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), while RT‒PCR data confirmed that the injection of niclosamide decreased the mRNA levels of A1 astrocyte-specific factors, including CCL2, CXCL1, MMP2, and tPA (H). The data are presented as the means ± SEMs. ****p < 0.0001, sham + DMSO versus SNL + DMSO. ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + niclosamide. Finally, pain behavioural assessment revealed that the ad ministration of recombinant S100A4 protein decreased the PWT of the bilateral hind paws from 2 h to 7 days, whereas fluorocitrate partially reversed S100A4-induced mechanical hyperalgesia, which persisted for up to 7 days (I). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + fluorocitrate

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Injection, Enzyme-linked Immunosorbent Assay, Recombinant, Saline

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 5 Exogenous S100A4 induces A1 astrocyte activation via the TLR4/NF-κB signalling pathway in vitro. Western blot analysis revealed that TLR4 and p-NF-κB p65 protein levels were also significantly increased after stimulation with 80 ng/ml rat recombinant S100A4 protein in primary astrocyte cultures, whereas total NF-κB p65 expression was not affected (A, B). The data are presented as the means ± SEMs. **p < 0.01, ****p < 0.0001, normal versus S100A4. Pretreatment with PDTC decreased the S100A4-induced upregulation of GFAP and C3, as well as p-NF-κB p65 expression (C). The selective TLR4 inhibitor Resatorvid significantly decreased the S100A4-mediated upregulation of GFAP and C3, along with the protein levels of p-NF-κB p65 and TLR4 (D). Quanti tative analysis confirmed these results, as shown in E and F. Immunofluorescence staining confirmed that, after confluence, adherent astrocytes activated by S100A4 presented significantly greater fluorescence intensities of both GFAP and C3 than did control cells. PDTC ammonium or Resatorvid treatment reduced the fluorescence intensity of GFAP and C3 (G) (scale bar, 100 μm). Quantitative analysis further confirmed these results, as shown in H. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 normal versus S100A4. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Activation Assay, In Vitro, Western Blot, Recombinant, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 6 SNL upregulates spinal TLR4/NF-κB signalling, whereas S100A4 inhibition attenuates A1 astrocyte activation via this pathway. Western blot show ing the time courses of TLR4 and p-NF-κB p65 expression in sham and SNL rats (A). SNL induced a progressive increase in TLR4 and p-NF-κB p65 levels in the spinal cord, especially during the maintenance period of NP, and then tended to decrease from Days 1 to 21 after surgery (B). The data are presented as the means ± SEMs. For TLR4, ***p < 0.001, ****p < 0.0001 SNL versus sham. For NF-κB, ##p < 0.01, ###p < 0.001, ####p < 0.0001 SNL versus sham. Dual- label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group (scale bar, 500 μm). Immunofluorescence images (C1-C, C2-C and C3-C) revealed intense colo calization of p-NF-κB p65 (green) with NeuN (red) and limited coexpression with GFAP (red) but not with iba-1 (red) (scale bar, 500 μm). More details are shown at higher magnification in C1-D, C2-D and C3-D (scale bar, 100 μm). WB analysis revealed that successive intrathecal niclosamide injections beginning at 1 dpi induced significantly lower levels of TLR4 and p-NF-κB p65 expression at 10 and 14 days in SNL rats (D and E). The data are presented as the means ± SEMs. ***p < 0.001, ****p < 0.0001 SNL versus sham. ##p < 0.01, ###p < 0.001 SNL versus SNL + niclosamide. Moreover, the administration of Resatorvid caused a decrease in GFAP and C3 expression, as well as p-NF-κB p65 protein levels, at 10 and 14 days in SNL rats (F and G). Repeated ad ministration of the NF-κB inhibitor PDTC decreased the expression of GFAP and C3 (H and I). The data are presented as the means ± SEMs. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001, ####p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium or Resatorvid. Dual-label fluorescence colocalization analysis revealed that the intensity of GFAP/C3 double-positive fluorescence in the ipsilateral L4/5 SDH was significantly greater on POD 10 after SNL than in the sham group. Repeated administration of PDTC ammonium or Resatorvid decreased the intensity of GFAP+C3+ fluorescence (J and K) (scale bar, 500 μm). Quantitative analysis further confirmed these results, as shown in L and M. Data are presented as the mean ± SEM. ****p < 0.0001 sham versus SNL + DMSO. ###p < 0.001 SNL versus SNL + PDTC ammonium or Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Fluorescence, Immunofluorescence

Journal: The journal of headache and pain

Article Title: Inhibition of S100A4 decreases neurotoxic astrocyte reactivity and attenuates neuropathic pain via the TLR4/NF-κB pathway in a rat model of spinal nerve ligation.

doi: 10.1186/s10194-025-02045-9

Figure Lengend Snippet: Fig. 7 Blocking TLR4/NF-κB signalling alleviates neuropathic pain after SNL and impacts exogenous S100A4-induced hyperalgesia. Repeated administra tion of PDTC ammonium (50 µg/20 µl) in the early postoperative phase (POD 1) partially attenuated the development of mechanical hypersensitivity in the ipsilateral hind paw of SNL rats (A), which was maintained until POD 14. Moreover, after receiving repeated intrathecal injections of 20 µg Resatorvid, the PWT of the ipsilateral hind paw was significantly greater than that of the saline group from Day 3 to Day 14 after SNL (A). Nonetheless, the treatment did not influence the PWT of the contralateral hind paws (B). The data are presented as the means ± SEMs. ****p < 0.0001 SNL + DMSO versus SNL + PDTC ammonium. ####p < 0.0001 SNL + DMSO versus SNL + Resatorvid. Moreover, pain behavioural assessment revealed that PDTC ammonium and Resator vid could partially reverse S100A4-induced mechanical hyperalgesia in the bilateral hind paws of naive rats, with the effect lasting up to 5 days (left, C; right, D). The data are presented as the means ± SEMs. ****p < 0.0001, saline versus S100A4. ####p < 0.0001 S100A4 versus S100A4 + PDTC ammonium. ѱѱѱp < 0.001, ѱѱѱѱp < 0.0001 S100A4 versus S100A4 + Resatorvid

Article Snippet: Antibodies against S100A4 (16105-1-AP), C3 (21337- 1-AP), NF-κB p65 (10745-1-AP), and TLR4 (19811-1- AP) were purchased from Proteintech (Hubei, Wuhan, China).

Techniques: Blocking Assay, Saline