Neural-Inflammation Mechanism of Spinal Palmitic Acid Promoting Atopic Dermatitis in Mice

Abstract

Objective: To profile spinal medium- and long- chain fatty acids (ML-CFAs) and itch-related gene expressions (IRGEs) in dorsal root ganglion (DRG), and investigate the role of spinal palmitic acid (PA) in atopic dermatitis (AD), and its relationship with DRG and spinal extracellular signal-regulated kinase (ERK).

Methods: MC903 was applied topically to the nape of C57BL/6 mice to induce AD. Two doses of PA were administered intrathecally during MC903 treatment, and several antagonists were administered intrathecally one day before PA challenge. Transcriptome sequencing was performed on DRGs, and 36 ML-CFAs in the spinal cord were analyzed.

Results: A global upregulation of IRGEs in DRGs and increases in major ML-CFAs including PA in the spinal cord were observed in adult AD model. MC903 resulted in less severe dermatitis with weaker IRGEs in DRGs and lower spinal ML-CFAs in senile than adult mice. In adult mice, intrathecal PA injection caused acute scratches, aggravated AD, and induced stronger IRGEs in DRGs. Intrathecal injection of transient receptor potential vanilloid-1 channel (TRPV1) antagonist capsazepine or Mas-related G protein-coupled receptor D (MRGPRD) antagonist d-Pro7-ANG-(1-7) remarkably halted PA/MC903-induced dermatitis and PA-induced scratching. Administration of histamine h4 receptor antagonist JNJ7777120 only moderately alleviated dermatitis, with no notable effect on scratches. Intrathecal pan-palmitoylation inhibitor 2-Bromopalmitate moderately alleviated MC903/PA-induced lesions and spinal ERK phosphorylation. Intrathecal lidocaine markedly suppressed both lesions and ERK phosphorylation, along with a global reduction in IRGEs in DRGs. Finally, PA-induced scratches were significantly improved by intrathecal lidocaine but not 2-Bromopalmitate.

Conclusion: MC903-induced AD develops more readily in adult than senile mice, with consistent changes in IRGEs in DRG and spinal ML-CFA levels, including PA. Spinal PA promotes AD involving spinal TRPV1 and MRGPRD signaling, and IRGEs increments in DRG. Intrathecal lidocaine suppresses AD aggravated by PA via inhibiting spinal ERK phosphorylation and reducing IRGEs in DRG.

Keywords: Atopic dermatitis; Dorsal root ganglion; Extracellular signal-regulated kinase; Palmitic acid; Spinal cord; Transcriptome sequencing.

Figure 1

Difference in MC903-treated adult and senile mice. MC903 (45 μM; 20 μL/cm2) was topically applied to the exposed skin areas of 8–10-week-old mice (n = 6 per group) for 14 consecutive days, and skin lesions were photographed. Mice were then anesthetized via intraperitoneal pentobarbital sodium, and the skin lesions and DRGs were obtained. (A) Skin lesions and HE and TB staining results (scale bar = 100 μm). DRGs (n = 4 per group) were used for transcriptome sequencing. (B) DEGs identified between mock and MC903-treated adult mice are shown. The numbers are equal to the difference (obtained by subtracting the data in column one from that in column two) divided by that in column one; the minus sign represents a decrease and the corresponding numbers of the down-regulated genes are marked in red. (C) The DEGs between adult and senile mice in the context of MC903 treatment are shown. Numbers are represented the same as those in Figure 1B; the minus sign represents a decrease and the corresponding numbers of the up-regulated genes are marked in red. Student’s t-test was used for statistical analysis, * P < 0.05, #P < 0.01, not labelled P < 0.001.

Figure 2

Comparisons of spinal ML-CFAs levels in the three groups. Fresh spinal cords from the mice (n = 4 per group) in Figure 1 were collected to identify 36 ML-CFAs using GC–MS. (A) Principal component analysis. (B) Heatmap analysis of each fatty acid in each sample of the three groups. Comparisons of the average levels of the four fatty acids with the highest abundance in the three groups were performed, and C18:1n9t (C), C18:0 (D), C16:0 (E), and C20:1t (F) are shown. One-way ANOVA with Bonferroni’s test was used for statistical analysis, *** P < 0.001.

Figure 3

Effect of intrathecal PA injection on AD-like dermatitis and spontaneous scratches. MC903 (45 μM; 20 μL/cm²) was topically applied to the exposed skin areas of 8–10-weeks-old mice (n = 6 per group) for 10 consecutive days, and PA (5 μg in 10 μL) or corresponding solvent (SVT) in equal volume was intrathecally injected on days 3 and 7. (A) Skin lesions and histological staining results at day 10 (scale bar = 100 μm). (B) Skin IL-13 levels at day 10. (C) Skin TSLP levels at day 10. DRGs (n = 4 per group) were used for transcriptome sequencing. (D) The DEGs between the two groups are shown, and the numbers are represented same as those in Figure 1B; the minus sign represents a decrease and the corresponding numbers of the down-regulated genes are marked in red. Student’s t-test was used for statistical analysis, * P < 0.05, ** or # P < 0.01, not labelled P < 0.001.

Figure 4

Effect of several itch receptor antagonists on PA/MC903-induced dermatitis. MC903 (45 μM; 20 μL/cm²) was topically applied to the exposed skin areas of 8–10 weeks-old mice (n = 6 per group) for 10 consecutive days. On days 2 and 6, intrathecal injection of solvent, chlorpheniramine (2 μg in 10 μL saline), GSK189254 (5 μg in 10 μL saline), JNJ7777120 (2 μg in 10 μL saline), HC-030031(5 μg in 10 μL saline), capsazepine (5 μg in 10 μL saline), or d-Pro7-ANG-(1-7) (5 μg in 10 μL saline) was administered. On days 3 and 7, all the mice received intrathecal PA injections. Skin lesions and histological staining results are presented (scale bar = 100 μm).

Figure 5

Effect of intrathecal 2BP or lidocaine on dermatitis, scratches, and spinal ERK phosphorylation. MC903 (45 μM; 20 μL/cm²) was topically applied to the exposed skin areas of 8–10 weeks-old mice (n = 6 per group) for 10 consecutive days. On days 2 and 6, intrathecal injection of SVT, 2BP (5 μg in 10 μL saline), or lidocaine (Lid, 1%, 10 μL) was administered. On days 3 and 7, intrathecal injection SVT or PA was administered. (A) Skin lesions and histological staining results at day 10 (scale bar = 100 μm). (B) Spinal phosphorylated-ERK levels at day 10. (C) 2BP (5 μg in 10 μL saline) or lidocaine (1%, 10 μL) was intrathecally pre-administered to 8–10-week-old mice (n = 6 per group). Intrathecal PA (5 μg in 10 μL) or corresponding SVT in equal volume was administered 24 h later. The scratching behaviors within 10 min after PA injection were monitored. One-way ANOVA followed by Dunnett’s test was used for statistical analysis, ***P < 0.001, n.s. P > 0.05.

Figure 6

DEGs between the two groups. DRGs from MC903 + PA (n=2) and MC903 + PA/lidocaine (n = 4) groups were used for transcriptome sequencing. The DEGs between the two groups are shown, and the numbers are represented the same as those in Figure 1B; the minus sign represents a decrease and the corresponding numbers of the up-regulated genes are marked in red. Student’s t-test was used for statistical analysis, *P < 0.05, #P < 0.01, not labelled P < 0.001.