Inhibition of the microglial voltage-gated proton channel 1 channel ameliorates diabetes-associated cognitive dysfunction by regulating axon demyelination

Figure 1 The voltage-gated proton channel 1 gene deletion alleviates early cognitive impairment induced by diabetes. A: Blood glucose levels during glucose tolerance tests (n = 4 per group); B: Blood glucose levels during insulin tolerance tests (n = 4 per group); C: Working memory errors (n = 6 per group); D: In the eight-arm radial maze test, the voltage-gated proton channel 1-/- diabetic group mice made more different arm choices in the first eight entries than DB mice (n=6 per group). GTT: Glucose tolerance tests; ITT: Insulin tolerance tests; DB: Diabetic group; Hv1: The voltage-gated proton channel 1.

Figure 2 The voltage-gated proton channel 1 gene knockout improves the abnormal secretion of interleukin-1β and tumor necrosis factor-α in the corpus callosum of diabetic mice. A-F: The levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the control group at P2; G-L: The levels of IL-1β and TNF-α in microglial cells were significantly elevated in the diabetic group; M-R: This response was effectively reversed by voltage-gated proton channel 1 gene knockout treatment; S-X: There was no significant difference in the IL-1β and TNF-α levels in the voltage-gated proton channel 1 gene knockout group compared with the control group. The scale bar = 50 μm. ^dP < 0.05. Iba-1: Ionized calcium-binding adaptor molecule 1; IL: Interleukin; TNF: Tumor necrosis factor; DB: Diabetic group; Hv1: The voltage-gated proton channel 1.

Figure 3 The voltage-gated proton channel 1 gene deletion hinders the proliferation of M1-type microglia in the corpus callosum of diabetic mice. Immunofluorescence images showing ionized calcium-binding adaptor molecule 1 + microglia in the corpus callosum at P2. ^dP < 0.05. The scale bar = 50 μm. Iba-1: Ionized calcium-binding adaptor molecule 1; EdU: 5-ethynyl-2’-deoxyuridine; DB: Diabetic group; Hv1: The voltage-gated proton channel 1.

Figure 4 The voltage-gated proton channel 1 gene knockdown improved oligodendrocyte progenitor cell apoptosis in diabetic mice. A-L: The terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay results showing the apoptosis levels of neuro-glia antigen 2-positive oligodendrocyte precursor cells (OPCs) compared with the control group. The number of OPCs significantly decreased after five weeks; The scale bar = 50 μm; M: The voltage-gated proton channel 1 gene knockdown reduced OPC apoptosis. ^dP < 0.05. NG2: Neuro-glia antigen 2; DB: Diabetic group; Hv1: The voltage-gated proton channel 1.

Figure 5 The voltage-gated proton channel 1 gene knockout improves demyelination in diabetic mouse axons. A-D: Transmission electron microscopy images of the myelin sheath; E and F: The G-ratios showing the levels of demyelination in different groups. ^dP < 0.05. The scale bar = 1 μm. DB: Diabetic group; Hv1: The voltage-gated proton channel 1.

Figure 6 The voltage-gated proton channel 1 gene is involved in the high glucose and high osmolarity-induced production of interleukin-1β and tumor necrosis factor-α. The interleukin (IL)-1β levels decreased in microglial cells after adding siRNA-the voltage-gated proton channel 1 but remained the same after treatment with control RNA. This finding aligns with the observed alterations in IL-1β levels in the animal corpus callosum, where the expression of IL-1β was unaffected by using control RNA. ^dP < 0.05. The scale bar = 50 μm. Hv1: The voltage-gated proton channel 1; Iba-1: Ionized calcium-binding adaptor molecule 1; HG: High glucose; PBS: Phosphate buffer saline; IL: Interleukin.

Figure 7 High glucose affects M1-type microglia by activating glucose-regulated protein 78 in microglia. The levels of ferroptosis markers, including ferritin heavy chain, ferritin light-chain, CCAAT/enhancer-binding protein homologous protein, and glucose-regulated protein 78, were significantly increased in high glucose cultured microglia, which were restored in the voltage-gated proton channel 1 (Hv1) si-RNA-treated cells. In addition, when the YUM70 was added, the effect of siRNA-Hv1 was enhanced, while the control RNA did not affect the expression of these markers. ^dP < 0.05. Hv1: The voltage-gated proton channel 1; FTH: Ferritin heavy chain; FTL: Ferritin light-chain; GRP78: Glucose-regulated protein 78; CHOP: CCAAT/enhancer-binding protein; HG: High glucose; PBS: Phosphate buffer saline.

Figure 8 The voltage-gated proton channel 1 gene knockout inhibits interleukin-1β in diabetic mice by suppressing the glucose-regulated protein 78 pathway. The microglial cells in the diabetic group exhibited elevated interleukin (IL)-1β levels compared to the control, which were restored the voltage-gated proton channel 1 (Hv1) knockout. Treatment with YUM70 enhanced the effect of Hv1 knockout, indicating that Hv1 knockout inhibits IL-1β by suppressing the glucose-regulated protein 78 pathway. ^dP < 0.05. The scale bar = 50 μm. Hv1: The voltage-gated proton channel 1; Iba-1: Ionized calcium-binding adaptor molecule 1; IL: Interleukin; DB: Diabetic group.