Acupuncture Alleviates Corneal Inflammation in New Zealand White Rabbits with Dry Eye Diseases by Regulating α 7nAChR and NF- κ B Signaling Pathway

Abstract

Purpose: The purpose of this study is to determine the mechanism of improvement in dry eye diseases (DEDs) treated by acupuncture. The inflammatory molecules and related pathways will be analyzed in our study.

Methods: In order to establish the animal model for DEDs, healthy New Zealand white rabbits were treated with scopolamine (Scop) hydrobromide for 21 consecutive days. After 21 days, acupuncture, fluorometholone (Flu), and α7nAChR antagonist (α-BGT) treatments were performed, and the Scop injections were continued until day 35. The therapeutic effect of acupuncture on DED inflammation was evaluated by corneal fluorescence staining, tear film rupture time, tear flow measurement, in vivo confocal microscopy (IVCM), corneal histopathology, and cytokine protein chip technology. The influence of acupuncture on the corneal pathology and inflammatory factors ACh, α7nAChR, and NF-κB was detected by enzyme-linked immunosorbent assay (ELISA) and western blot.

Results: Compared with the group Scop, acupuncture can significantly reduce corneal staining and increase the tear film rupture time and tear flow, which are accompanied by a decrease in corneal epithelial detachment and lymphocyte infiltration. Acupuncture can relieve the inflammation of corneal stroma and mitigate the expression of proinflammatory factors and chemokines. Acupuncture can upregulate the expression of ACh and α7nAChR and downregulate the expression of NF-κB.

Conclusion: Our findings demonstrate that acupuncture can alleviate corneal inflammation in New Zealand white rabbits with DEDs via α7nAChR and NF-κB signaling pathway regulation. The expression indicates that α7nAChR/NF-κB signaling pathway may be active and that acupuncture is a potential therapeutic target for dry eye.

Figure 1

(a) Experimental design for acupuncture treatment and (b) the locations of acupuncture points. (c) Acupuncture operation and (d) the Schirmer I test.

Figure 2

The effect of acupuncture treatment on tear flow and tear film rupture time in DED induced by scopolamine hydrobromide. (a) Tear fluid flow; (b) tear film rupture time. Quantitative data are shown as mean ± SEM. ^∗∗P < 0.01 vs group Con; ^#P < 0.05, ^##P < 0.01 vs group Scop; ^&P < 0.05, ^&&P < 0.01 vs group Scop + Sham.

Figure 3

The effect of acupuncture treatment on corneal fluorescence staining and the histopathological images of the cornea (hematoxylin-eosin staining, ×10 times) on the 35th day in DED induced by scopolamine hydrobromide. (a) Corneal fluorescence staining score and (b) corneal fluorescence staining and the histopathological images of the cornea. Quantitative data are expressed as mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 vs group Con. ^#P < 0.05, ^##P < 0.01 vs group Scop; ^&P < 0.05, ^&&P < 0.01 vs group Scop + Sham.

Figure 4

The IVCM images of the anterior and posterior stromal layers under the cornea on the 35th day. Quiet stromal cells, a small number of globular immune cells, and nerve fibers (Con). A large number of globular immune cells as shown by the arrow and activated stromal layer cells with unclear borders and irregular sizes. Areas with irregular intercellular spaces were seen. A change in nerve width was seen with the decreasing nerve branch reflectivity (Scop). Disordered stromal cells and intermittently thinner nerve fibers with high tortuous (Scop + Sham). The morphology of the stromal layer cells was improved, the cells were slightly activated, and there was no obvious abnormality in nerve reflexes. This indicates that the signs of inflammation in the corneal stromal layer were significantly reduced (Scop + Acup). Quiet stromal cells and nerve fibers (Scop + Flu). The image size is 400 × 400 microns.

Figure 5

Changes in the cytokines and the chemokines in rabbit corneal on day 35. Quantitative data are expressed as mean ± SEM. Acupuncture stimulus significantly decreased the levels of IL-1b, MMP-9, IL-17A, and IL-21 when compared with the Scop group. The Scop + Flu group significantly decreased the levels of IL-1b, MMP-9, IL-21, MIP-1b, NCAM-1, and TNF-a when compared with the Scop group. ^∗P < 0.05, ^∗∗P < 0.01 vs. group Con; ^#P < 0.05, ^##P < 0.01 vs. group Scop; ^&P < 0.05, ^&&P < 0.01 vs. group Scop + Sham.

Figure 6

The influence of acupuncture on the changes of corneal ACh and α7nAChR was analyzed by ELISA. (a and c) The expression of ACh in each group of corneas and (b and d) the α7nAChR expression in each group of corneas. Western blot analysis of the effect of acupuncture on the activation of NF-κB, with GAPDH as control (e–h). Quantitative data are expressed as mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 vs. group Con; ^#P < 0.05, ^##P < 0.01 vs. group Scop; ^&P < 0.05, ^&&P < 0.01 vs. group Scop + Sham; %P < 0.05 vs. group Scop + Acup; $P < 0.05 vs. group Scop + α-BGT + Flu.

Figure 7

Dependence of the regulation of the acupuncture stimulation corneal cytokines through α7nAChR. The α7nAChR antagonist α-BGT reverses the inhibitory effect of acupuncture stimulation on corneal cytokines. Quantitative data are expressed as mean ± SEM. The group Scop + Acup + α-BGT had upregulated IL-1b, IL-21, MMP-9, and TNF-a expression levels when compared with the group Scop + Acup. ^#P < 0.05 and ^##P < 0.01 vs. group Scop; %P < 0.05 vs. group Scop + Acup.