Tui Na Acupressure Modulates Treg Immunosuppression via FoxP3 / mTORC1 Signalling in ALS Mice methods
Aim. Evidence-backed execution summary for Tui Na Acupressure Modulates Treg Immunosuppression via FoxP3 / mTORC1 Signalling in ALS Mice methods from Tui Na Acupressure Modulates Treg Immunosuppression via FoxP3 / mTORC1 Signalling in ALS Mice.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Mechanical Stimulation via Tui Na Enhances Treg Proliferation and Immunosuppressive Function Through the FoxP3 / mTOR...
reagent used in the protocol.
- Use
- Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat group, whereas both markers were significantly downregulated in the FoxP3 or mTORC1 inhibitor groups (Figure ).
Tui Na Acupressure Enhances Treg Function by Activating the FoxP3 / mTORC1 Pathway, Affecting Cell Cycle and Immune R...
reagent used in the protocol.
- Use
- Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhibitor treatment markedly attenuated this upregulation (Figure ).
Animal Grouping
reagent used in the protocol.
- Use
- Animal grouping for subsequent pathway validation experiments was as follows: (1) WT group: no treatment; (2) ALS model group: ALS model mice that did not receive any intervention; (3) Riluzole group: ALS mice treated with Riluzole (10 mg/kg, intraperitoneal injection) combined with 2% sodium butyrate in filte...
Sample Collection
reagent used in the protocol.
- Use
- Blood Samples: Approximately 3 mL of cardiac blood was collected into heparinized vacuum tubes and centrifuged at 4°C for 10 min at 3000 rpm to separate serum for enzyme-linked immunosorbent assay (ELISA)-based cytokine analysis.
Flow Cytometry Analysis
reagent used in the protocol.
- Use
- T Cell Subset Analysis: Flow cytometry assessed T cell subsets in the spleen, lymph nodes, and peripheral blood, focusing on the proportion and number of Tregs (CD4 + CD25 + FoxP3 + ). Single-cell suspensions were prepared in PBS containing 10% fetal bovine serum (FBS; Gibco, USA), and incubated in the dark at...
Cytokine Level Measurement
reagent used in the protocol.
- Use
- Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention. Peripheral blood samples were collected in hepariniz...
Immunohistochemistry
reagent used in the protocol.
- Use
- After deparaffinisation, the antigen was retrieved by microwaving the sections in citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide (1.08600, Sigma-Aldrich, USA) for 10 min. To reduce nonspecific binding, 10% norm...
Western Blot
reagent used in the protocol.
- Use
- Tissue homogenates were prepared using RIPA lysis buffer (P0013B, Beyotime, China) and centrifuged at 12 000 rpm for 15 min at 4°C to collect the supernatant. Protein concentrations were determined using a BCA protein assay kit (23 225, Thermo Fisher Scientific, USA). A total of 30...
Tui Na Intervention
Acupoint selection was based on three criteria: Traditional theory: According to Huangdi Neijing, BL23 has the function of "governing bones and generating marrow" and is associated with neurological regulation [ ]. Anatomical correspondence: Vertebral morphology was used to establish human-to-...
- Use
- Acupoint selection was based on three criteria: Traditional theory: According to Huangdi Neijing, BL23 has the function of "governing bones and generating marrow" and is associated with neurological regulation [ ]. Anatomical correspondence: Vertebral morphology was used to establish human-to-...
Behavioural Assessments
Rotarod Test: Motor coordination and balance were assessed using a rotarod apparatus (Ugo Basile, Italy) at weeks 11, 14, 16, 18, and 20, as well as at the end stage of the disease. The rotation started at 4 rpm and gradually increased to 40 rpm. The time until the mouse fell was recorded. Each mouse und...
- Use
- Rotarod Test: Motor coordination and balance were assessed using a rotarod apparatus (Ugo Basile, Italy) at weeks 11, 14, 16, 18, and 20, as well as at the end stage of the disease. The rotation started at 4 rpm and gradually increased to 40 rpm. The time until the mouse fell was recorded. Each mouse und...
Cognitive Function Assessments
Novel Object Recognition Test: Mice were tested at 11, 14, 16, 18, and 20 weeks. Each mouse was placed in a 50 cm × 50 cm testing chamber with two identical objects (blocks of the same height and shape). After 24 h, one object was replaced with a novel one. The time spent exp...
- Use
- Novel Object Recognition Test: Mice were tested at 11, 14, 16, 18, and 20 weeks. Each mouse was placed in a 50 cm × 50 cm testing chamber with two identical objects (blocks of the same height and shape). After 24 h, one object was replaced with a novel one. The time spent exp...
Cognitive Function Assessments
Y-Maze Test: The Y-maze test assessed spatial memory and exploratory behaviour. Each arm of the maze measured 30 cm in length and 7 cm in width, forming 90-degree angles. Mice were placed in the starting arm, and spontaneous alternation behaviour-entering a different arm on each c...
- Use
- Y-Maze Test: The Y-maze test assessed spatial memory and exploratory behaviour. Each arm of the maze measured 30 cm in length and 7 cm in width, forming 90-degree angles. Mice were placed in the starting arm, and spontaneous alternation behaviour-entering a different arm on each c...
Sample Collection
Cell Suspensions: Spleen and lymph node tissues were mechanically dissociated in phosphate-buffered saline (PBS), and the resulting suspensions were filtered for flow cytometry analysis.
- Use
- Cell Suspensions: Spleen and lymph node tissues were mechanically dissociated in phosphate-buffered saline (PBS), and the resulting suspensions were filtered for flow cytometry analysis.
Flow Cytometry Analysis
T Cell Subset Analysis: Flow cytometry assessed T cell subsets in the spleen, lymph nodes, and peripheral blood, focusing on the proportion and number of Tregs (CD4 + CD25 + FoxP3 + ). Single-cell suspensions were prepared in PBS containing 10% fetal bovine serum (FBS; Gibco, USA), and incubated in the dark at...
- Use
- T Cell Subset Analysis: Flow cytometry assessed T cell subsets in the spleen, lymph nodes, and peripheral blood, focusing on the proportion and number of Tregs (CD4 + CD25 + FoxP3 + ). Single-cell suspensions were prepared in PBS containing 10% fetal bovine serum (FBS; Gibco, USA), and incubated in the dark at...
Flow Cytometry Analysis
Interleukin-4 (IL-4) expression was measured using IL-4-PE (12-7041-41, eBioscience, Thermo Fisher Scientific, USA) to assess immune function. After staining, cells were washed twice with PBS and resuspended in 500 µL PBS. Flow cytometry was performed using a BD FACSCan...
- Use
- Interleukin-4 (IL-4) expression was measured using IL-4-PE (12-7041-41, eBioscience, Thermo Fisher Scientific, USA) to assess immune function. After staining, cells were washed twice with PBS and resuspended in 500 µL PBS. Flow cytometry was performed using a BD FACSCan...
Cytokine Level Measurement
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention. Peripheral blood samples were collected in hepariniz...
- Use
- Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention. Peripheral blood samples were collected in hepariniz...
Oxidative Stress Markers
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All experiments were performed in triplicate. Images were captured using the ChemiDoc imaging system (Bio-Rad, USA), and data for each marker were quantified using ImageJ software (NIH, USA). The final results were normalized to protein concentration and subjected to statistical analysis.
Gene Ontology/Kyoto Encyclopedia of Genes and Genomes ( GO / KEGG ) Functional Enrichment Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- GO and KEGG pathway enrichment analyses were performed on the DEGs and proteins to identify genes and pathways significantly affected by the Tui Na intervention. These analyses were conducted using the ClusterProfiler package (version 4.0.2). An enrichment threshold of q < 0.05 was applied. The results...
Data Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data was analysed using GraphPad Prism 9 software (GraphPad Software, USA). Data is presented as mean ± SEM. Differences among groups were analysed by one-way ANOVA with Tukey's post hoc test for multiple comparisons. Statistical significance was indicated as follows: * p < 0.05;...
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Mechanical Stimulation via Tui Na Enhances Treg Proliferation and Immunosuppressive Function Through the FoxP3 / mTOR...
Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat group, whereas both markers were significantly downregulated in the FoxP3 or mTORC1 inhibitor groups (Figure ).
Tui Na Acupressure Enhances Treg Function by Activating the FoxP3 / mTORC1 Pathway, Affecting Cell Cycle and Immune R...
Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhibitor treatment markedly attenuated this upregulation (Figure ).
Animal Grouping
Sixty mice were randomly divided into three groups: a wild-type (WT) control group, an ALS model group (ALS mice without any treatment), a Riluzole positive control group (ALS mice treated with Riluzole), a Tui Na intervention group (Treat, ALS mice receiving point-pressing massage at BL23), a non-acupoint massage group (Treat-non, receiving identical stimulation at a muscle-rich region of the hip), and a sham stimulation group (Sham, skin contact only without pressure). Throughout the intervention period, body weight and general health were monitored daily to ensure the smooth progression of the experiment.
Animal Grouping
Animal grouping for subsequent pathway validation experiments was as follows: (1) WT group: no treatment; (2) ALS model group: ALS model mice that did not receive any intervention; (3) Riluzole group: ALS mice treated with Riluzole (10 mg/kg, intraperitoneal injection) combined with 2% sodium butyrate in filtered drinking water [ ]; (4) Treat group: ALS mice receiving Tui Na intervention; (5) Treat-non group: ALS mice receiving identical mechanical stimulation at the hip muscle site; (6) Sham group: ALS mice with skin contact only, no pressure applied; (7) Treat + GSK126 group: received Tui Na intervention in combination with the FoxP3 signalling inhibitor GSK126 (5.00580, 150 mg/kg, Sigma, USA); (8) Treat + Rapa group: received Tui Na intervention in combination with the mTORC1 inhibitor rapamycin (S1039, 4 mg/kg, Selleckchem, USA). All...
Tui Na Intervention
Mice in the Treat group received manual Tui Na acupressure treatment. The procedure was performed by professionally trained Tui Na practitioners using a thumb-and-index finger pressing technique, targeting the Shenshu acupoint (1.5 cm lateral to the L2-L3 vertebrae). The applied pressure was controlled at approximately 10 pounds. Each manipulation lasted for 60 s with 5-s intervals, at a frequency of approximately 200 presses per minute. Each session lasted 5 min. The Treat-non group received identical stimulation parameters at the hip muscle site, while the Sham group received skin contact only without pressure, at the same frequency of 200 presses/min.
Behavioural Assessments
Tail Suspension Test: Mice were suspended by the tail, and their struggle behaviour was observed to assess muscle strength and neurological function. The total duration of active struggle (in seconds) was recorded, and the average muscle tension was calculated. The test lasted 5 min.
Cognitive Function Assessments
Y-Maze Test: The Y-maze test assessed spatial memory and exploratory behaviour. Each arm of the maze measured 30 cm in length and 7 cm in width, forming 90-degree angles. Mice were placed in the starting arm, and spontaneous alternation behaviour-entering a different arm on each consecutive trial-was recorded. The test duration was 8 min. A higher alternation percentage indicated better cognitive performance.
Cytokine Level Measurement
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention. Peripheral blood samples were collected in heparinized BD Vacutainer tubes (367 525, Becton Dickinson, USA) and centrifuged at 3000 rpm for 10 min at 4°C to isolate serum. Mouse-specific ELISA kits (M4000B, M1000B, DB100C, QK285, MTA00B, MLB00C, D6050B; R&D Systems, USA) were used according to the manufacturer's instructions. For each assay, 50 µL of serum per sample was used. The kits included standards and all necessary detection antibodies and enzyme conjugates. Samples were incubated on a shaker at room temperature in microplates for 2 h, then enzyme conjugates and washing st...
Measurement outputs
What raw and processed outputs should exist?
Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat g...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
We applied Tui Na acupressure intervention to the abdominal surface of ALS mice, after which CD4 + T cells were isolated from the spleen and lymph nodes and induced to different...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhi...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Acupoint selection was based on three criteria: Traditional theory: According to Huangdi Neijing, BL23 has the function of "governing bones and generating marrow" an...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat g...; We applied Tui Na acupressure intervention to the abdominal surface of ALS mice, after which CD4 + T cells were isolated from the spleen and lymph nodes and induced to different...; Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhi...; Acupoint selection was based on three criteria: Traditional theory: According to Huangdi Neijing, BL23 has the function of "governing bones and generating marrow" an....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood wer...; All experiments were performed in triplicate. Images were captured using the ChemiDoc imaging system (Bio-Rad, USA), and data for each marker were quantified using ImageJ...; GO and KEGG pathway enrichment analyses were performed on the DEGs and proteins to identify genes and pathways significantly affected by the Tui Na intervention. These analyses...; Data was analysed using GraphPad Prism 9 software (GraphPad Software, USA). Data is presented as mean ± SEM. Differences among groups were analysed by one-...
from paperReporting output
Report representative outputs alongside summary comparisons for Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat g..., We applied Tui Na acupressure intervention to the abdominal surface of ALS mice, after which CD4 + T cells were isolated from the spleen and lymph nodes and induced to different..., Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhi..., Acupoint selection was based on three criteria: Traditional theory: According to Huangdi Neijing, BL23 has the function of "governing bones and generating marrow" an....
inferred from protocolStructured statistical methods
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood wer...; All experiments were performed in triplicate. Images were captured using the ChemiDoc imaging system (Bio-Rad, USA), and data for each marker were quantified using ImageJ...; GO and KEGG pathway enrichment analyses were performed on the DEGs and proteins to identify genes and pathways significantly affected by the Tui Na intervention. These analyses...; Data was analysed using GraphPad Prism 9 software (GraphPad Software, USA). Data is presented as mean ± SEM. Differences among groups were analysed by one-...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Flow cytometric analysis of Treg functional markers revealed that the expression levels of CTLA-4 and IL-2RA were significantly upregulated in Tregs from the Treat group, whereas both markers were significantly downregulated in the FoxP3 or mTORC1 inhibitor groups (Figure ).
Western blot results showed that Tregs from the Treat group had significantly upregulated Cyclin D1 and CDK4 protein expression levels compared with the ALS group. However, inhibitor treatment markedly attenuated this upregulation (Figure ).
Sixty mice were randomly divided into three groups: a wild-type (WT) control group, an ALS model group (ALS mice without any treatment), a Riluzole positive control group (ALS mice treated with Riluzole), a Tui Na intervention group (Treat, ALS mice receiving point-pressing massage at BL23), a non-acupoint massage group (Treat-non, receiving identical stimulation at a muscle-rich region of the hip), and a sham stimulation group (Sham, skin contact only without pressure). Throughout the intervention period, body weight and general health were monitored daily to ensure the smooth progression of the experiment.
Animal grouping for subsequent pathway validation experiments was as follows: (1) WT group: no treatment; (2) ALS model group: ALS model mice that did not receive any intervention; (3) Riluzole group: ALS mice treated with Riluzole (10 mg/kg, intraperitoneal injection) combined with 2% sodium butyrate in filtered drinking water [ ]; (4) Treat group: ALS mice receiving Tui Na intervention; (5) Treat-non group: ALS mice receiving identical mechanical stimulation at the hip muscle site; (6) Sham group: ALS mice with skin contact only, no pressure applied; (7) Treat + GSK126 group: received Tui Na intervention in combination with the FoxP3 signalling inhibitor GSK126 (5.00580, 150 mg/kg, Sigma, USA); (8) Treat + Rapa group: received Tui Na intervention in combination with the mTORC1 inhibitor rapamycin (S1039, 4 mg/kg, Selleckchem, USA). All mice were housed in a standard clean environment with a 12-h light/dark cycle, temperature maintained at 22°C ± 2°C, humidity at 50% ± 5%, and were given free access to food and water.
Mice in the Treat group received manual Tui Na acupressure treatment. The procedure was performed by professionally trained Tui Na practitioners using a thumb-and-index finger pressing technique, targeting the Shenshu acupoint (1.5 cm lateral to the L2-L3 vertebrae). The applied pressure was controlled at approximately 10 pounds. Each manipulation lasted for 60 s with 5-s intervals, at a frequency of approximately 200 presses per minute. Each session lasted 5 min. The Treat-non group received identical stimulation parameters at the hip muscle site, while the Sham group received skin contact only without pressure, at the same frequency of 200 presses/min.
Tail Suspension Test: Mice were suspended by the tail, and their struggle behaviour was observed to assess muscle strength and neurological function. The total duration of active struggle (in seconds) was recorded, and the average muscle tension was calculated. The test lasted 5 min.
Y-Maze Test: The Y-maze test assessed spatial memory and exploratory behaviour. Each arm of the maze measured 30 cm in length and 7 cm in width, forming 90-degree angles. Mice were placed in the starting arm, and spontaneous alternation behaviour-entering a different arm on each consecutive trial-was recorded. The test duration was 8 min. A higher alternation percentage indicated better cognitive performance.
Cytokine levels of IL-4, interleukin-10 (IL-10), TGF-β, IFN-γ, TNF-α, IL-1β, and IL-6 in peripheral blood were measured using ELISA to evaluate the immunomodulatory effects of Tui Na intervention. Peripheral blood samples were collected in heparinized BD Vacutainer tubes (367 525, Becton Dickinson, USA) and centrifuged at 3000 rpm for 10 min at 4°C to isolate serum. Mouse-specific ELISA kits (M4000B, M1000B, DB100C, QK285, MTA00B, MLB00C, D6050B; R&D Systems, USA) were used according to the manufacturer's instructions. For each assay, 50 µL of serum per sample was used. The kits included standards and all necessary detection antibodies and enzyme conjugates. Samples were incubated on a shaker at room temperature in microplates for 2 h, then enzyme conjugates and washing steps were added. A substrate solution was then added for colour development, and absorbance was measured at 450 nm using a microplate reader (BioTek Synergy HTX, BioTek, USA). A standard curve for each cytokine was generated using serial dilutions of the standard solution to quantify cytokine c...
Machine-readable layer
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"name": "Tui Na Acupressure Modulates Treg Immunosuppression via FoxP3 / mTORC1 Signalling in ALS Mice methods",
"item": "https://replicatescience.com/experiments/tui-na-acupressure-modulates-treg-immunosuppression-via-foxp3-mtorc1-signalling-in-als-mice-methods-jie-han-pmc12867591/tui-na-acupressure-modulates-treg-immunosuppression-via-foxp3mtorc1-signalling-i"
}
]
}
]