Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Electroacupuncture Attenuates Cerebral Ischemia–Reperfusion Injury via Vagus Nerve–α7 Nicotinic Acetylcholine Receptor–Dependent Microglial Polarization

doi: 10.1161/JAHA.126.049344

Figure Lengend Snippet: A , qRT‐PCR analysis of α7nAChR mRNA levels in the ischemic penumbra at 1, 3, and 7 d after MCAO (n=3 per group). B , C , Representative Western blots and quantification of α7nAChR protein levels in the penumbra at the indicated time points (n=3 per group). D , E , Representative immunofluorescence images showing α7nAChR (red) and Iba1 (green) in the penumbra at d 3 post MCAO, and quantification of their fluorescence colocalization intensity (n=3 per group; scale bar, 100 μm). F , G , Representative immunofluorescence images showing α7nAChR (red) and NeuN (green) in the penumbra, and quantification of co‐localization intensity (n=3 per group; scale bar, 100 μm). H , I , Neurological deficit scores (modified Garcia, Petullo) at d 3 after MCAO following α‐BGT administration (n=6 per group). J , K , TTC staining and quantitative analysis of infarct volume (n=6 per group). In ( A ) and ( C ), values in the Sham group were normalized to 1 as a reference to present relative expression changes. In ( H , I , and K ), the identical values in the Sham group reflect normal physiological baselines by design. α7nAChR indicates α7 nicotinic acetylcholine receptor; α‐BGT, α‐bungarotoxin; EA, electroacupuncture; Iba‐1, ionized calcium binding adaptor molecule 1; MCAO/R, middle cerebral artery occlusion/reperfusion; NeuN, neuronal nuclei; qRT‐PCR, quantitative real‐time polymerase chain reaction; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Article Snippet: The following primary antibodies were used: Arg‐1 (arginase 1; 1:30 000; Proteintech, #16001‐1‐AP), TGF‐β1 (transforming growth factor beta 1; 1:1000; Abclonal, #A18692), iNOS (inducible nitric oxide synthase; 1:1500; Affinity, #AF0199), TNF‐α (tumor necrosis factor alpha; 1:1500; Wanleibio, #WL01581), TLR4 (1:1000; Cell Signaling Technology, #14358T), MyD88 (1:500; Abclonal, #A0980), α7nAChR (1:500; Abclonal, #A1588), β‐tubulin (1:100000; Proteintech, #80713‐1‐RR), p‐IκBα (phospho‐IkappaB alpha; 1:800; Abclonal, #AP0999), IκBα (1:1000; Abclonal, #A11168), NF‐κB p65 (1:1000; Cell Signaling Technology, #8242S), histone (Proteintech, #17168‐1‐AP), and β‐actin (1:1000; Wanleibio, #WL01372).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Fluorescence, Modification, Staining, Expressing, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Electroacupuncture Attenuates Cerebral Ischemia–Reperfusion Injury via Vagus Nerve–α7 Nicotinic Acetylcholine Receptor–Dependent Microglial Polarization

doi: 10.1161/JAHA.126.049344

Figure Lengend Snippet: Tissues were collected from the ischemic penumbra at d 3 after MCAO. A through C , Representative Western blots and quantification of anti‐inflammatory markers Arg‐1 and TGF‐β1 (n=3 per group). D through F , Representative Western blots and quantification of proinflammatory markers iNOS and TNF‐α (n=3 per group). G through L , Representative Western blots and quantification of Arg‐1, TGF‐β1, iNOS, and TNF‐α following α7nAChR antagonism with α‐BGT (n=3 per group). M , N , Representative immunofluorescence images showing colocalization of Arg‐1 (red) and iNOS (red) with the microglial marker Iba‐1 (green) in the penumbra (scale bar, 100 μm). O through R , ELISA analysis of anti‐inflammatory markers (CD206, IL‐10) and proinflammatory markers (CD86, IL‐1β) (n=6 per group). α7nAChR indicates α7 nicotinic acetylcholine receptor; α‐BGT, α‐bungarotoxin; Arg‐1, arginase 1; EA, electroacupuncture; Iba‐1, ionized calcium binding adaptor molecule 1; IL, interleukin; iNOS, inducible nitric oxide synthase; MCAO/R, middle cerebral artery occlusion/reperfusion; TGF‐β1, transforming growth factor beta 1; and TNF‐α, tumor necrosis factor alpha.

Article Snippet: The following primary antibodies were used: Arg‐1 (arginase 1; 1:30 000; Proteintech, #16001‐1‐AP), TGF‐β1 (transforming growth factor beta 1; 1:1000; Abclonal, #A18692), iNOS (inducible nitric oxide synthase; 1:1500; Affinity, #AF0199), TNF‐α (tumor necrosis factor alpha; 1:1500; Wanleibio, #WL01581), TLR4 (1:1000; Cell Signaling Technology, #14358T), MyD88 (1:500; Abclonal, #A0980), α7nAChR (1:500; Abclonal, #A1588), β‐tubulin (1:100000; Proteintech, #80713‐1‐RR), p‐IκBα (phospho‐IkappaB alpha; 1:800; Abclonal, #AP0999), IκBα (1:1000; Abclonal, #A11168), NF‐κB p65 (1:1000; Cell Signaling Technology, #8242S), histone (Proteintech, #17168‐1‐AP), and β‐actin (1:1000; Wanleibio, #WL01372).

Techniques: Western Blot, Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Electroacupuncture Attenuates Cerebral Ischemia–Reperfusion Injury via Vagus Nerve–α7 Nicotinic Acetylcholine Receptor–Dependent Microglial Polarization

doi: 10.1161/JAHA.126.049344

Figure Lengend Snippet: A , Representative Western blots of total TLR4, MyD88, p‐IκBα, IκBα, and nuclear NF‐κB p65 in the ischemic penumbra at day 3 after MCAO. B through E , Quantitative analysis of protein levels for total TLR4, MyD88, p‐IκBα/IκBα ratio, and nuclear NF‐κB p65 (n=3 per group). F , G , Representative immunofluorescence images showing TLR4 and NF‐κB p65 expression in the penumbra (scale bar, 100 μm). α7nAChR indicates α7 nicotinic acetylcholine receptor; α‐BGT, α‐bungarotoxin; EA, electroacupuncture; MCAO/R, middle cerebral artery occlusion/reperfusion; MyD88, myeloid differentiation primary response 88; NF‐κB, nuclear factor kappa B; p‐IκBα, phospho‐IkappaB alpha; and TLR4, toll‐like receptor 4.

Article Snippet: The following primary antibodies were used: Arg‐1 (arginase 1; 1:30 000; Proteintech, #16001‐1‐AP), TGF‐β1 (transforming growth factor beta 1; 1:1000; Abclonal, #A18692), iNOS (inducible nitric oxide synthase; 1:1500; Affinity, #AF0199), TNF‐α (tumor necrosis factor alpha; 1:1500; Wanleibio, #WL01581), TLR4 (1:1000; Cell Signaling Technology, #14358T), MyD88 (1:500; Abclonal, #A0980), α7nAChR (1:500; Abclonal, #A1588), β‐tubulin (1:100000; Proteintech, #80713‐1‐RR), p‐IκBα (phospho‐IkappaB alpha; 1:800; Abclonal, #AP0999), IκBα (1:1000; Abclonal, #A11168), NF‐κB p65 (1:1000; Cell Signaling Technology, #8242S), histone (Proteintech, #17168‐1‐AP), and β‐actin (1:1000; Wanleibio, #WL01372).

Techniques: Western Blot, Immunofluorescence, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Electroacupuncture Attenuates Cerebral Ischemia–Reperfusion Injury via Vagus Nerve–α7 Nicotinic Acetylcholine Receptor–Dependent Microglial Polarization

doi: 10.1161/JAHA.126.049344

Figure Lengend Snippet: A and B , Representative immunofluorescence images showing c‐Fos (red) and ChAT (green) in the DMV at d 3 post MCAO, and quantitative analysis of their co‐localization (n=3 per group; scale bar, 200 μm). C and D , Representative Western blots and quantification of α7nAChR protein levels in the penumbra following SDV (n=3 per group). E , F , Neurological deficit scores (modified Garcia, Petullo) at d 3 after MCAO (n=6 per group). G , H , TTC staining and quantitative analysis of infarct volume (n=6 per group). The identical values in the Sham group reflect normal physiological baselines by design. α7nAChR indicates α7 nicotinic acetylcholine receptor; α‐BGT, α‐bungarotoxin; ChAT, choline acetyltransferase; EA, electroacupuncture; MCAO/R, middle cerebral artery occlusion/reperfusion; SDV, subdiaphragmatic vagotomy; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Article Snippet: The following primary antibodies were used: Arg‐1 (arginase 1; 1:30 000; Proteintech, #16001‐1‐AP), TGF‐β1 (transforming growth factor beta 1; 1:1000; Abclonal, #A18692), iNOS (inducible nitric oxide synthase; 1:1500; Affinity, #AF0199), TNF‐α (tumor necrosis factor alpha; 1:1500; Wanleibio, #WL01581), TLR4 (1:1000; Cell Signaling Technology, #14358T), MyD88 (1:500; Abclonal, #A0980), α7nAChR (1:500; Abclonal, #A1588), β‐tubulin (1:100000; Proteintech, #80713‐1‐RR), p‐IκBα (phospho‐IkappaB alpha; 1:800; Abclonal, #AP0999), IκBα (1:1000; Abclonal, #A11168), NF‐κB p65 (1:1000; Cell Signaling Technology, #8242S), histone (Proteintech, #17168‐1‐AP), and β‐actin (1:1000; Wanleibio, #WL01372).

Techniques: Immunofluorescence, Western Blot, Modification, Staining

Journal: Inflammation

Article Title: GTS-21 Alleviates Acute Lung Injury by Enhancing GLP-1 Secretion and Regulating Alveolar Surfactant Proteins via α7nAChR Activation

doi: 10.1007/s10753-026-02455-0

Figure Lengend Snippet: Kaplan-Meier survival analysis of pneumonia. GTS-21 (4 mg/kg, i.p.) or vehicle was administered 2 h prior to the induction of P. aeruginosa pneumonia. Mice were monitored for 24 h post-infection to assess survival and mortality outcomes. WT mice were used to assess GTS-21’s impact on mortality in P. aeruginosa pneumonia (A, n: Control = 8, Pneu = 17, Pneu/GTS-21 = 14) WT and α7nAChR KO mice were used to investigate α7nAChR’s role in mortality under P. aeruginosa infection (B, n: Control = 5, α7nAChR KO = 14, WT = 14). Log-Rank Test was used to compare the difference in survival probability (Fig. 1A: WT control vs. WT Pneu/GTS-21 P = 0.041. Figure 1B: WT Pneu vs. α7nAChR KO Pneu P = 0.036). Pneu: pneumonia.

Article Snippet: Eight-week-old C57BL/6 mice (Strain #: 000664) and α7nAChR KO mice (Strain #: 003232) were obtained from Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Control

Journal: Inflammation

Article Title: GTS-21 Alleviates Acute Lung Injury by Enhancing GLP-1 Secretion and Regulating Alveolar Surfactant Proteins via α7nAChR Activation

doi: 10.1007/s10753-026-02455-0

Figure Lengend Snippet: Histological assessment of lung injury in pneumonia mice: Mice were treated with GTS-21 (4 mg/kg, i.p.) or vehicle 2 h before induction of pneumonia by P. aeruginosa or sham pneumonia (by saline). Mice were sacrificed 24 h after induction, and then lung tissues were collected for histological analysis using H&E staining. Lung injury was evaluated using the lung injury scoring system in WT and α7nAChR KO mice. Quantification of lung injury was characterized by neutrophil infiltration, hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening. Bar graph with overlapping dots plot represents mean values and standard error of mean (SE) ( n = 4/group). WT Control vs. WT Pneu P = 0.001, WT Pneu vs. WT Pneu/GTS-21 P = 0.015. KO control vs. KO Pneu P = 0.001, KO Pneu vs. KO Pneu/GTS-21 P = 0.513. WT Pneu vs. KO Pneu P = 0.038. *: P < 0.05, **: P < 0.01. Pneu: pneumonia.

Article Snippet: Eight-week-old C57BL/6 mice (Strain #: 000664) and α7nAChR KO mice (Strain #: 003232) were obtained from Jackson Laboratory (Bar Harbor, ME).

Techniques: Saline, Staining, Control

Journal: Inflammation

Article Title: GTS-21 Alleviates Acute Lung Injury by Enhancing GLP-1 Secretion and Regulating Alveolar Surfactant Proteins via α7nAChR Activation

doi: 10.1007/s10753-026-02455-0

Figure Lengend Snippet: BALF cytology and inflammatory mediator in pneumonia mice. Mice were treated with GTS-21 (4 mg/kg, i.p.) or vehicle 2 h before induction of pneumonia by P. aeruginosa or sham pneumonia (by saline). Mice were sacrificed 24 h after induction, then BALF and lung were collected for examining neutrophils and macrophages by Hema 3 staining. Quantification of macrophages (A) and neutrophils (B) in WT and α7nAChR KO mice per slide were counted at ×400 magnification under light microscopy. Representative images are shown in supplemental file 3. The levels of HMGB1 in serum (C) and BALF (D) were examined by ELISA. Bar graph with overlapping dots plot represents mean values and standard error of mean (SE) ( n = 4/group). Figure 3A: WT sham vs. WT Pneu P = 0.02, WT Pneu vs. WT Pneu/GTS-21 P = 0.042, KO sham vs. KO Pneu P = 0.044, KO Pneu vs. KO Pneu/GTS-21 P = 0. 284. Figure 3B: WT sham vs. WT Pneu P < 0.0001, WT Pneu vs. WT Pneu/GTS-21 P = 0.034, KO sham vs. KO Pneu P < 0.0001, KO Pneu vs. KO Pneu/GTS-21 P = 0. 696. Figure 3C: WT sham vs. WT Pneu P = 0.013, WT Pneu vs. WT Pneu/GTS-21 P = 0.046, KO sham vs. KO Pneu P = 0.019, KO Pneu vs. KO Pneu/GTS-21 P = 0. 906. Figure 3D: WT sham vs. WT Pneu P = 0.026, WT Pneu vs. WT Pneu/GTS-21 P = 0.048, KO sham vs. KO Pneu P = 0.015, KO Pneu vs. KO Pneu/GTS-21 P = 0. 859. Figure 3E: WT sham vs. WT Pneu P = 0.008, WT Pneu vs. WT Pneu/GTS-21 P = 0.037, KO sham vs. KO Pneu P = 0.007, KO Pneu vs. KO Pneu/GTS-21 P = 0. 804.*: P < 0.05, **: P < 0.01. Pneu: pneumonia.

Article Snippet: Eight-week-old C57BL/6 mice (Strain #: 000664) and α7nAChR KO mice (Strain #: 003232) were obtained from Jackson Laboratory (Bar Harbor, ME).

Techniques: Saline, Staining, Light Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: GTS-21 Alleviates Acute Lung Injury by Enhancing GLP-1 Secretion and Regulating Alveolar Surfactant Proteins via α7nAChR Activation

doi: 10.1007/s10753-026-02455-0

Figure Lengend Snippet: Inflammatory cytokine levels in murine LPS-induced lung injury models. WT and α7nAChR KO mice were treated with GTS-21 (4 mg/kg, i.p.) or vehicle 2 h before induction of acute lung injury by LPS (2.5 mg/kg) or sham lung injury (by saline) using noninvasive tracheal installation. Mice were sacrificed 24 h after LPS or saline, then BALF was collected for IL-6 and HMGB1 in WT (A&B) and α7nAChR KO (C&D) mice. Bar graph with overlapping dots plot represents mean values and standard error of mean (SE) (n: Fig. 5A: Control = 6, LPS = 6, LPS/GTS-21 = 6; Fig. 5B: Control = 3, LPS = 4, LPS/GTS-21 = 4; Fig. 5C: Control = 4, LPS = 4, LPS/GTS-21 = 4; Fig. 5D: Control = 4, LPS = 6, LPS/GTS-21 = 5; Fig. 5E: Control = 3, LPS = 6, LPS/GTS-21 = 5; Fig. 5E: Control = 4, LPS = 4, LPS/GTS-21 = 4). Figure 5A: Control vs. LPS P = 0.021, LPS vs. LPS/GTS-21 P = 0.037. Figure 5B: Control vs. LPS P = 0.034, LPS vs. LPS/GTS-21 P = 0.045. Figure 5C: Control vs. LPS P = 0.026, LPS vs. LPS/GTS-21 P = 0.041. Figure 5D: Control vs. LPS P = 0.03, LPS vs. LPS/GTS-21 P = 0.485. Figure 5E: Control vs. LPS P = 0.035, LPS vs. LPS/GTS-21 P = 0.615. Figure 5F: Control vs. LPS P = 0.027, LPS vs. LPS/GTS-21 P = 0.557. *: P < 0.05.

Article Snippet: Eight-week-old C57BL/6 mice (Strain #: 000664) and α7nAChR KO mice (Strain #: 003232) were obtained from Jackson Laboratory (Bar Harbor, ME).

Techniques: Saline, Control

Journal: Inflammation

Article Title: GTS-21 Alleviates Acute Lung Injury by Enhancing GLP-1 Secretion and Regulating Alveolar Surfactant Proteins via α7nAChR Activation

doi: 10.1007/s10753-026-02455-0

Figure Lengend Snippet: The induction of GLP-1 by GTS-21 in a murine model of LPS-Induced lung injury. WT, α7nAChR KO and GLP-1R KO mice were treated with GTS-21 (4 mg/kg) or vehicle 2 h before induction of acute lung injury by LPS (2.5 mg/kg) or sham lung injury (by saline) using noninvasive tracheal installation. Mice were sacrificed 24 h after LPS or saline, then serum was collected for the measurement of GLP-1 levels in WT (A), α7nAChR KO (B) and GLP-1R KO (C) mice. Bar graph with overlapping dots plot represents mean values and standard error of mean (SE) (n: Fig. 6A: Control = 6, LPS 8, LPS/GTS-21 = 9, GTS-21 = 4; Fig. 6B: Control = 3, LPS = 5, LPS/GTS-21 = 5, GTS-21 = 4; Fig. 6C: Control = 4, LPS = 4, LPS/GTS-21 = 4, GTS-21 = 4). Figure 6A: Control vs. LPS P = 0.04, Control vs. LPS/GTS-21 P = 0.009, Control vs. GTS-21 P = 0.035, LPS vs. LPS/GTS-21 P = 0.036. Figure 6B: Control vs. LPS P = 0.043, Control vs. GTS-21 P = 0.769, LPS vs. LPS/GTS-21 P = 0.672. Figure 6C: Control vs. LPS P = 0.042, Control vs. LPS/GTS-21 P = 0.026, Control vs. GTS-21 P = 0.042, LPS vs. LPS/GTS-21 P = 0.048. *: P < 0.05, **: P < 0.01.

Article Snippet: Eight-week-old C57BL/6 mice (Strain #: 000664) and α7nAChR KO mice (Strain #: 003232) were obtained from Jackson Laboratory (Bar Harbor, ME).

Techniques: Saline, Control