Electroacupuncture protects gastric Cajal cells by reducing macrophage pyroptosis in diabetic gastroparesis

Figure 1 Proposed mechanistic hypothesis in diabetic gastroparesis. In the diabetic gastroparesis setting, M2 macrophages usually recognize and phagocytose apoptotic interstitial cells of Cajal (ICCs). However, upregulation of Gasdermin D triggers pyroptosis, impairing mitochondrial function and reducing phagocytic efficiency. The resulting accumulation of damaged DNA from incompletely cleared ICCs activates the cyclic GMP-AMP synthase-stimulator of interferon genes inflammatory pathway, leading to the release of proinflammatory mediators and further apoptosis of ICCs. The dashed section represents assumptions based on the literature. ICC: Interstitial cells of Cajal; IL: Interleukin; GSDMD-N: Gasdermin D-N terminal domain; NLRP3: NOD-like receptor family pyrin domain containing 3; mtDNA: Mitochondrial DNA; cGAS: Cyclic GMP-AMP synthase; STING: Stimulator of interferon genes; IRF3: Interferon regulatory factor 3; NF-κB: Nuclear factor-κB; TBK1: TANK-binding kinase 1; GSDMD: Gasdermin D.

Figure 2 Experimental design and electroacupuncture intervention illustration. A: Experimental workflow: Rats were subjected to a 9-week modeling protocol, which included a high-fat, high-sugar diet from week 8 to week 0, followed by streptozotocin injection between week 0 and week 1. Electroacupuncture treatment was administered from week 1 to week 9, for a total of 8 weeks; B: Schematic representation of the transverse section of the tibia-fibula plane at the Zusanli acupoint in rats. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; EA: Electroacupuncture.

Figure 3 Effects of electroacupuncture on blood glucose, whole-gut transit time, and gastric emptying rate. A: Changes in blood glucose levels following electroacupuncture (EA) treatment; B: Effects of EA on gastric emptying rate; C: Effects of EA on whole-gut transit time. Error bars indicate SEM in A and C. The box plot in B depicts the median (center line), interquartile range (box bounds), and whiskers extending to the furthest data point within 1.5 × interquartile range. ^aP < 0.05 vs control group, ^bP < 0.05 vs diabetic gastroparesis group, ^cP < 0.01 vs diabetic gastroparesis group, and ^dP < 0.05 vs high-frequency electroacupuncture group at 9 weeks. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; WGTT: Whole-gut transit time.

Figure 4 Effect of electroacupuncture on interstitial cells of Cajal apoptosis in the gastric muscle layer of diabetic gastroparesis rats. A: Representative immunofluorescence images showing transferase dUTP nick-end labeling (green), c-kit+ cells (red), and DAPI (blue) staining in each group (scale bar: 50 μm); B and C: Quantitative analysis of the relative fluorescence areas of transferase dUTP nick-end labeling and c-kit+ cells. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; TUNEL: Transferase dUTP nick-end labeling.

Figure 5 Effect of electroacupuncture on pyroptosis of M2 macrophages in the gastric muscle layer of diabetic gastroparesis rats. A: Representative immunofluorescence images showing Gasdermin D (green), CD206+ cells (red), and DAPI (blue) in each group (scale bar: 50 μm); B and C: Quantitative analysis of the relative fluorescence areas of CD206+ cells and Gasdermin D. Data are expressed as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; GSDMD: Gasdermin D.

Figure 6 Effects of electroacupuncture on proteins involved in the Gasdermin D-mediated pyroptosis pathway. A-D: Representative western blot images and quantitative analysis of Gasdermin D, NOD-like receptor family pyrin domain containing 3, and caspase-1 protein expression in each group. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; NLRP3: NOD-like receptor family pyrin domain containing 3; GSDMD: Gasdermin D; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 7 Effects of electroacupuncture on inflammatory mediators. A-C: Representative western blot images and quantitative analysis of interleukin-1β and interleukin-18 protein expression across groups. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. IL: Interleukin; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture.

Figure 8 Effects of electroacupuncture on the cyclic GMP-AMP synthase-stimulator of interferon genes signaling pathway. A: Representative western blot images; B-F: Quantitative analysis of cyclic GMP-AMP synthase, stimulator of interferon genes, p- stimulator of interferon genes, interferon regulatory factor 3, and p-interferon regulatory factor 3 protein expression across groups. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; cGAS: Cyclic GMP-AMP synthase; STING: Stimulator of interferon genes; IRF3: Interferon regulatory factor 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; DM: Diabetes mellitus.

Figure 9 Immunofluorescence analysis of stimulator of interferon genes expression in gastric tissues. A: Representative immunofluorescence images showing stimulator of interferon genes+ cells (red) and DAPI nuclear staining (blue) in each group (scale bar: 50 μm); B: Quantitative analysis of the relative fluorescence area of stimulator of interferon genes+ cells. Data are presented as mean ± SD (n = 6). ^aP < 0.05 vs control group, and ^bP < 0.05 vs diabetic gastroparesis group. DGP: Diabetic gastroparesis; SEA: Sham-electroacupuncture; LEA: Low-frequency electroacupuncture; HEA: High-frequency electroacupuncture; STING: Stimulator of interferon genes.