Involvement of Substance P (SP) and Its Related NK1 Receptor in Primary Sjögren's Syndrome (pSS) Pathogenesis

Abstract

Primary Sjögren's Syndrome (pSS) is a systemic autoimmune disease that primarily attacks the lacrimal and salivary glands, resulting in impaired secretory function characterized by xerostomia and xerophthalmia. Patients with pSS have been shown to have impaired salivary gland innervation and altered circulating levels of neuropeptides thought to be a cause of decreased salivation, including substance P (SP). Using Western blot analysis and immunofluorescence studies, we examined the expression levels of SP and its preferred G protein-coupled TK Receptor 1 (NK1R) and apoptosis markers in biopsies of the minor salivary gland (MSG) from pSS patients compared with patients with idiopathic sicca syndrome. We confirmed a quantitative decrease in the amount of SP in the MSG of pSS patients and demonstrated a significant increase in NK1R levels compared with sicca subjects, indicating the involvement of SP fibers and NK1R in the impaired salivary secretion observed in pSS patients. Moreover, the increase in apoptosis (PARP-1 cleavage) in pSS patients was shown to be related to JNK phosphorylation. Since there is no satisfactory therapy for the treatment of secretory hypofunction in pSS patients, the SP pathway may be a new potential diagnostic tool or therapeutic target.

Keywords: minor salivary gland (MSG); neurokinin receptor 1 (NK1R); primary Sjögren’s syndrome (pSS); sicca syndrome; substance P (SP).

Figure 1

(A,B) Representative Western blot of SP (A) and NK1R (B) levels in MSGs from sicca and pSS patients. Data are expressed as mean optical density in arbitrary units (a.u.) and are given as mean ± SD of n = 9 independent experiments. Statistical significance calculated using one-way analysis of variance (ANOVA) for repeated measures followed by Tukey’s post hoc test, indicated with ** p < 0.01; *** p < 0.001. β-actin expression was used to normalize sample variability. Molecular weight markers (kDa) are shown on the left.

Figure 2

(A,B) SP expression and localization was severely affected in MSG sections from pSS and sicca patients. (A) Representative images of immunofluorescence staining (red) of SP in pSS and sicca patient MSG sections. (B) Quantitative analysis showing lower expression of SP in pSS compared to sicca patients. n = 5 independent experiments and statistical significance calculated using t-Student test. *** p < 0.001; t = 6.302. Scale bar 25 µm; 40× magnification. First row: negative control (ctl neg).

Figure 3

SP and ECAD expression and localization was severely affected in MSG sections from pSS and sicca patients. Representative images of double immunofluorescence staining of substance P (SP, red) and e-cadherin (ECAD, green) in pSS and sicca patients MSG sections. Scale bar 25 µm; 40× magnification.

Figure 4

(A,B) Representative Western blot of c-PARP-1 (A) and pJNK (B) levels in MSGs from sicca and pSS patients. Data are expressed as mean optical density in arbitrary units (a.u.) and are given as mean ± SD of n = 9 independent experiments. Statistical significance was calculated using one-way analysis of variance (ANOVA) for repeated measures followed by Tukey’s post hoc test, indicated with * p < 0.05. β-actin expression was used to normalize sample variability. Molecular weight markers (kDa) are shown on the left.