Role and mechanism of Roux-en-Y gastric bypass in the treatment of diabetic urinary bladder hyperactivity by reducing TRPV1 and P2X3

Figure 1 Body weight and blood glucose of mice after Roux-en-Y gastric bypass treatment. A: Body weights of mice before and after treatment; B and D: Changes in oral glucose tolerance test (OGTT) values and area under the glucose curve (AUC); C and E: Changes in insulin tolerance test (ITT) values and AUC. All statistical results were expressed as mean ± SD (n = 8). ^aP < 0.05, overactive bladder (OAB) compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) compared with the OAB group.

Figure 2 Urine stain test results in mice. A: Representative image for void spot assay analysis, B: Urine spot count; C: Total area of urine spot; D: The ratio of urine spot area in central area. All the statistical results were expressed as mean ± SD (n = 8). ^aP < 0.05, overactive bladder (OAB) compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) compared with the OAB group.

Figure 3 Bladder manometry of mice in the awake state. A: Images of bladder manometry of mice in the sham group; B: Vesical images of mice in the overactive bladder (OAB) group; C: Bladder images of mice in the Roux-en-Y gastric bypass (RYGB) group; D: The number of urination contractions; E: The quantity of non-urination contractions; F: Bladder capacity; G: Maximum peak urination pressure. All the statistical results were expressed as mean ± SD (n = 8). ^aP < 0.05, OAB group compared with the sham group; ^bP < 0.05, RYGB group compared with the OAB group.

Figure 4 Morphological analysis results of mouse bladder. A: Representative images of bladder hematoxylin and eosin (H&E) staining; B: The thickness of bladder epithelial layer; C: Representative images of Masson’s trichrome staining of the bladder; D: The ratio of collagen fibers in the bladder muscle layer; E: Western blot representative image of bladder tissue alpha-smooth muscle actin (α-SMA); F: Statistical results of bladder α-SMA (n = 8). ^aP < 0.05, overactive bladder (OAB) group compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) group compared with the OAB group. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5 Expression and localization of transient receptor potential vanilloid type 1 and purinergic receptor P2X3 in bladder tissue. A: Immunohistochemistry of transient receptor potential vanilloid type 1 (TRPV1) and purinergic receptor P2X3 in bladder tissue (n = 8); B: Immunofluorescence colocalization of TRPV1 and P2X3 in bladder tissue (the green indicates TRPV1, the red indicates P2X3, and the yellow indicates coexpression of TRPV1 and P2X3; n = 8). RYGB: Roux-en-Y gastric bypass; OAB: Overactive bladder.

Figure 6 Detection of the mRNA and protein levels of transient receptor potential vanilloid type 1 and purinergic receptor P2X3 in bladder tissues. A: The mRNA expression level of transient receptor potential vanilloid type 1 (TRPV1); B: The mRNA expression level of purinergic receptor P2X3; C: Western blot representation of TRPV1 and P2X3; D: Protein expression level of TRPV1; E: Protein expression level of P2X3 (n = 8). ^aP < 0.05, overactive bladder (OAB) group compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) group compared with the OAB group.

Figure 7 Detection of inflammatory factors in mouse bladder tissue. A: Western blot representative image of inflammatory proteins; B: Statistical results of tumor necrosis factor (TNF-α); C: Statistical results of interleukin (IL)-6; D: Statistical results of IL-1β; E: Statistical results of nuclear factor (NF)-κB (n = 8). ^aP < 0.05, overactive bladder (OAB) group compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) group compared with the OAB group.

Figure 8 Detection of ATP and energy metabolism signaling pathways in mouse urine. A: Urine ATP standard curve; B: Statistical results of urine ATP content; C: Western blot representative images of energy signaling pathway proteins; D: Statistical results for phosphorylated AMP-activated protein kinase (p-AMPK); E: Statistical results for phosphorylated mammalian target of rapamycin (p-mTOR) (n = 8). ^aP < 0.05, overactive bladder (OAB) group compared with the sham group; ^bP < 0.05, Roux-en-Y gastric bypass (RYGB) compared with the OAB group.