p38 mediates mechanical allodynia in a mouse model of type 2 diabetes

Abstract

Background: Painful Diabetic Neuropathy (PDN) affects more than 25% of patients with type 2 diabetes; however, the pathogenesis remains unclear due to lack of knowledge of the molecular mechanisms leading to PDN. In our current study, we use an animal model of type 2 diabetes in order to understand the roles of p38 in PDN. Previously, we have demonstrated that the C57BLK db/db (db/db) mouse, a model of type 2 diabetes that carries the loss-of-function leptin receptor mutant, develops mechanical allodynia in the hind paws during the early stage (6-12 wk of age) of diabetes. Using this timeline of PDN, we can investigate the signaling mechanisms underlying mechanical allodynia in the db/db mouse.

Results: We studied the role of p38 in lumbar dorsal root ganglia (LDRG) during the development of mechanical allodynia in db/db mice. p38 phosphorylation was detected by immunoblots at the early stage of mechanical allodynia in LDRG of diabetic mice. Phosphorylated p38 (pp38) immunoreactivity was detected mostly in the small- to medium-sized LDRG neurons during the time period of mechanical allodynia. Treatment with an antibody against nerve growth factor (NGF) significantly inhibited p38 phosphorylation in LDRG of diabetic mice. In addition, we detected higher levels of inflammatory mediators, including cyclooxygenase (COX) 2, inducible nitric oxide synthases (iNOS), and tumor necrosis factor (TNF)-alpha in LDRG neurons of db/db mice compared to non-diabetic db+ mice. Intrathecal delivery of SB203580, a p38 inhibitor, significantly inhibited the development of mechanical allodynia and the upregulation of COX2, iNOS and TNF-alpha.

Conclusions: Our findings suggest that NGF activated-p38 phosphorylation mediates mechanical allodynia in the db/db mouse by upregulation of multiple inflammatory mediators in LDRG.

Figure 1

Phosphorylation of p38 during the course of mechanical allodynia. A: Representative immunoblots of pp38, p38 and GAPDH using LDRG extracts from db+ and db/db mice at 5, 8, 10, and 12 wk of age. Increased levels of pp38 were detected in db/db mice when compared to db+ mice at 5, 8 and 10 wk of age. At 12 wk of age, p38 phosphorylation returned to the control level. In addition, no change in expression levels of p38 was detected in LDRG of db/db mice and db+ mice. GAPDH served as the loading control. B: Densitometric analysis of immunoblots using LDRG of db+ and db/db mice at 5, 8, 10 and 12 wk of age during the development of mechanical allodynia. Significantly enhanced pp38 levels were detected in db/db mice at 5, 8 and 10 wk of age but not at 12 wk of age. N = 4, *p < 0.05.

Figure 2

pp38 immunohistochemistry in LDRG of db+ and db/db mice. A, B: Representative pictures of pp38 immunohistochemistry in DRG from db+ (A) and db/db (B) mice at 8 wk of age. Increased nuclear immunoreactivity was detected in db/db (arrow) but not db+ (arrowhead) LDRG neurons. Bar = 15 μm. C: Quantitation of pp38-positive LDRG neurons in db+ and db/db mice. Significantly increased numbers of pp38-positive LDRG neurons were detected in db/db mice in comparison with db+ mice. N = 6, *p < 0.05, **p < 0.01. At 8 wk of age, the maximum increase in the percentage of pp38-positive neurons was detected in comparison to 5 and 10 wk LDRG. *p < 0.05, one-way ANOVA test. D: Cell size distribution of pp38-positive LDRG neurons at 8 wk of age. Significant increase of pp38-positive LDRG neurons were detected in neurons <20 μm, 20-30 μm and 30-40 μm in diameter. Small numbers of large DRG neurons (<40 μm) were also positive for pp38 in db/db mice but not db+ mice. N = 4, *p < 0.05.

Figure 3

Anti-NGF treatment inhibits the phosphorylation of p38 in db/db mice. A: Representative immunoblots of pp38, p38 and actin using LDRG from db+ and db/db mice treated with either control (IgG) or an anti-NGF antibody for 2 wk. Anti-NGF treatment decreased the intensity of pp38 signals in db/db mice but not db+ mice at 8 wk of age. B: Densitometric studies of pp38 immunoblots using LDRG from 8 wk old db+ and db/db mice. A significant decrease in p38 phosphorylation was detected in db/db mice after anti-NGF treatment. N = 4, **p < 0.01

Figure 4

SB203580 inhibits p38 phosphorylation, mechanical allodynia, and the percentage of SP-positive LDRG neurons in db/db mice. A: Representative immunoblots of pp38, p38 and actin from db+ and db/db LDRG at 8 wk of age after 1 wk of intrathecal treatments of vehicle (control CSF with 10% DMSO) (C) or vehicle with SB203580. SB203580 treatment decreased levels of p38 phosphorylation in db/db mice but not in db+ mice. B: Densitometric studies of pp38 immunoblots demonstrated significant inhibition of p38 phosphorylation in db/db mice. C: SB203580 treatment reversed the decrease in mechanical thresholds (allodynia) in db/db mice. D: SB203580 treatment also lowered the elevated percentages of SP-positive LDRG neurons in control db/db mice compared to control db+ mice. E: SB203580 treatment had no effect on the percentage of IB4-labelled LDRG neurons in db+ and db/db mice. N = 4, **p < 0.01.

Figure 5

SB203580 inhibits the upregulation of COX2, iNOS and TNF-α gene expression. RT-PCR analysis of COX2 (A), iNOS (B) and TNF-α (C) in LDRG of db+ and db/db mice. The gene expression of all three inflammatory mediators was upregulated in LDRG of db/db mice. SB203580 treatment significantly decreased the elevated levels of gene expressions in db/db mice. In addition, SB203580 treatment lowered the gene expression of TNF-α in db+ mice. N = 4, *p < 0.05.

Figure 6

Immunolocalization of iNOS, TNF-α, CD68, and COX2. Immunohistochemistry studies were performed on LDRG of db+ (A, B, E, F, I, J, M, N ) and db/db mice (C, D, G, H, K, L, O, P). The mice were treated with vehicle control (A, C, E, G, I, K, M, O) or vehicle with SB203580 (B, D, F, H, J, L, N, P). In vehicle control treated mice, an increased percentage of iNOS-positive DRG neurons were detected in both large (arrow) and small to medium (arrowhead)-sized DRG neurons in db/db (C) in comparison with db+ (A) mice. Double immunofluorescent studies demonstrated that TNF-α immunoreactivity was detected in the iNOS-positive neurons of db/db mice (G) with minimal immunoreactivity detected in db+ mice (E). In LDRG of db/db mice, many CD68-positve macrophages were detected but not in db+ LDRG (compare K and I, arrows). COX-2 expression was also detected in both small and large neuronal populations (O, arrowhead and arrow respectively) in db/db but not db+ mice. SB 203580 treatment did not change the patterns of immunoreactivity of iNOS, TNF-α, CD68, and COX2 in db+ mice (compare A to B, E to F, I to J, and M to N). In comparison, SB203580 treatment significantly decreased the immunoreactivity of iNOS, TNF-α, CD68 and COX2 in LDRG of db/db mice (compare C to D, G to H, K to L, and O to P). N = 4, Bar = 30 μm.

Figure 7

SB203580 treatment decreases the expression of iNOS, COX2, and TNF-α in db/db LDRG neurons. The percentages of immunopositive LDRG neurons for iNOS (A), COX2 (B), and TNF-α (C) were measured in db+ and db/db mice. The mice were treated with vehicle control (artificial CSF containing 10% DMSO) or vehicle with SB203580. Significant increases in the percentages for iNOS, COX2, and TNF-α were detected in vehicle-treated db/db mice, in comparison to db+ mice. SB203580 treatment significantly blocks the increased percentages of iNOS, COX2, and TNF-α in db/db mice to levels comparable with the nondiabetic control (db+) mice. N = 4, ***p < 0.001.

Figure 8

SB203580 treatment inhibits protein expression of inflammatory mediators in db/db mice. A: Representative immunoblots of LDRG from db+ and db/db mice. After 1 wk treatments with vehicle control or vehicle with SB203580, immunoblots were performed for iNOS, COX2, and TNF-α. Increased protein levels for all three inflammatory mediators were demonstrated in LDRG of db/db mice compared to db+ littermates. This upregulation was inhibited by treatment with SB203580. Actin served as a loading control. B, C, D: Densitometric analysis of COX2 (B), iNOS (C), and TNF-α (D) demonstrated significant upregulation of each respective protein in db/db mice that is inhibited by SB203580. N = 4, *p < 0.05, **p < 0.01.