Tui Na Acupressure Modulates Treg Immunosuppression via FoxP3 / mTORC1 Signalling in ALS Mice methods
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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...
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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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