Acupuncture Alleviates Corneal Inflammation in New Zealand White Rabbits with Dry Eye Diseases by Regulating α7nAChR and NF-κB Signaling Pathway

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.


Introduction
Dry eye diseases (DEDs) are multifactor ocular surface diseases, characterized by unbalanced tear flm homeostasis and accompanied with eye discomfort. Te main pathophysiological mechanisms of DEDs include unstable tear flm, increased tear osmotic pressure, ocular surface infammatory response, and neurological abnormalities [1]. As DEDs develop, the life quality of patients with DEDs gradually declines, with some even experiencing psychological illnesses such as depression and anxiety. Infammation has been identifed as the key mechanism of the physiological and pathological pathogenesis of DEDs [2]. Te excessive production of proinfammatory cytokines and chemokines causes the disruption of physiological homeostasis [3], leading the DEDs to progress. Terefore, infammatory suppression in the ocular surface is the key point in the treatment of DEDs. It is reported that DEDs are closely related to the central nervous system [4]. Previous studies have also shown that the cholinergic anti-infammatory pathway (CAP) can stimulate infammatory molecular signaling by integrating the aferent vagus nerve into the central nervous system. Moreover, eferent nerve endings are near the immune cells of acetylcholine (ACh) and release into the reticular endothelial tissue, which can bind with α7nAChR receptors on the surface of macrophages to transmit vagal signals. CAP plays an important anti-infammatory role by regulating the production of cytokines in cells, which is mainly related to the NF-κB signaling pathway [5,6].
It is demonstrated that acupuncture has a certain efect in the treatment of DEDs [7], which is to prolong the tear flm rupture time and reduce conjunctival infammation [8,9]. Some published studies have shown that acupuncture can change the morphology of lacrimal glands in rabbits with DEDs and increase the secretion of tears; however, no mechanistic explanation has been given [10]. Several recent studies have shown that acupuncture can increase the expression of α7nAChR while attenuating tissue infammation, further reducing tissue damage [11][12][13][14]. In this study, systemic administration of the muscarinic cholinergic inhibitor scopolamine hydrobromide could inhibit tear secretion in New Zealand rabbits to mimic DEDs [15]. We evaluated the treatment efcacy of acupuncture on DED manifestations in the experimental DEDs rabbit model, including tear secretion, ocular surface stability, and infammatory markers. Te aim of this research is to explore whether acupuncture is able to inhibit the corneal infammation associated with DEDs by regulating α7nAChR, which could subsequently reduce the infammatory response caused by the NF-κB pathway activation.

Experimental Animals.
Healthy New Zealand rabbits (male and female, 2-3 months old, weighing about 1.5 kg) were purchased from Qinglong Mountain Experimental Animal Farm (Nanjing, China) and raised in the pharmacology laboratory of Jiangsu Provincial Hospital of Traditional Chinese Medicine. Te experimental animals were housed in ambient conditions of room temperature (22 ± 2°C), at a relative humidity of 60% ± 5%, with an alternating 12-hourlight-dark cycle, and with water and standard feed provided constantly available. Te experimental protocol was approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine (Approval ID: 201809A018).

Experimental Procedures.
Before the experiment, all animals were required to have no abnormality in the anterior segment of the eye, and the tear fow was required to be greater than 10 mm/5 min. In our frst experiment, 30 healthy adult New Zealand white rabbits were randomly divided into the following fve groups: group control (Con), group scopolamine (Scop), group sham acupuncture (Scop + Sham), group acupuncture (Scop + Acup), and group fuorometholone (Scop + Flu). Group Con received no treatment, the other fve groups were given 2.0 mg/mL of scopolamine hydrobromide, and injected subcutaneously four times a day (8 : 00, 11 : 00, 14 : 00, and 18 : 00) for 35 days. Treatment began after the DEDs model was completed, on the 22nd day (Figure 1(a)). Group Scop + Sham: sham acupuncture treatment (Jingming BL1, Cuanzhu BL2, Sizhukong SJ23, Taiyang Ex-HN5, and Tongziliao GB1) was initiated, with a blunt needle pointing at the acupoints but not inserting into the skin, once a day for 14 consecutive days. Group Scop + Acup: the acupuncture point was the same as that of group Scop + Sham. Te needle was retained for 15 minutes, once a day, for 14 days (Figures 1(b) and 1(c)). Group Scop + Flu : fuorometholone eye drops were administered three times a day (8 : 00, 13 : 00, and 18 : 00) for 14 days after successful modeling. Te tear amount (Schirmer I test, SIt), fuorescein staining score (FL), and tear break-up time (BUT) were measured at 1, 7, 14, 21, 28, and 35 days, and the experimental animals were euthanized by air embolism after confocal microscope examination on day 35. In the second experiment, 30 healthy adult New Zealand white rabbits were randomly divided into fve groups: Scop, Scop + Acup, Scop + α-BGT, Scop + α-BGT + Flu, and Scop + α-BGT + Acup. Te specifc α7nAChR antagonist α-BGT was injected into the ear vein from day 22, 4.0 μg/kg daily until the animals were euthanized by air embolism on day 35.

Schirmer I Test (SIt).
A tear detection flter paper strip was folded at one end and put into the conjunctival sac of the outer third of the rabbit's lower eyelid. After 5 minutes, the flter paper was taken out and the wetting length was measured from the folding point (Figure 1(d)). Evidence-Based Complementary and Alternative Medicine 2.5. Corneal Fluorescein Staining Score (FL). Te corneal staining flter paper strip was put into the lower eyelid fornix of the rabbit, the staining flter paper strip was wetted, and the fuorescein was rapidly and evenly distributed on the cornea through the eye blink. Te corneal epithelial injury was graded with a cobalt blue flter. Te cornea was classifed into four quadrants and the scores were made, respectively. Te modifcations were scored between 0 and 3 points as follows: 0 points if absent, 1 point if fewer than fve spots, 2 points if more than fve spots, and 3 points if a large area of fuorescein plaque was evident. Finally, the score of each grade was added up, giving a total potential score of 12 [16].

Tear Break-Up Time (BUT).
Te time from the corneal fuorescence staining to the appearance of the frst corneal dry spot was measured. Te testing time was recorded under a slit lamp, and the BUT test was repeated three times for each rabbit.

In Vivo Confocal Microscopy (IVCM).
Te rabbit's head was fxed in a corneal laser confocal microscope, and obuvacaine hydrochloride eye drops were used for topical anesthesia. After setting the blepharostat, transparent Vidisci gel was applied on the surface of a 40-foldwater-immersed conical objective lens. Te lens was moved forward slowly, and the recording button was pressed when the corneal cells were visible and the image was clearly in the center of the display screen. Photographs of the stroma layer were taken in the center of the cornea in the microscopic feld.

Optical Microscope.
After euthanasia, corneas were collected and fxed in 4% paraformaldehyde for 24 hours. Te corneal epithelial specimens were separated without contact with the epithelium, and the corneal size was 2 × 2 mm. Ten, the dissection, parafn embedding, RM2135 slicing, H&E staining, and DMLS2 optical microscope observation were performed.

Cytokine Quantifcation Array.
Rabbit cytokine quantifcation arrays were performed using the Qal-CYT-1 kit. All protein was extracted from the cornea using the tissue protein extraction kit, and protein concentration was determined by the BCA method (Pierce, No. 23227).

Evidence-Based Complementary and Alternative Medicine
InnoScan 300 Microarray Scanner was applied to scan signals using Cy3 excitation curves.

ELISA.
Te cornea of the rabbit was immediately taken out after euthanasia. After rinsing with normal saline, the cornea was fully homogenized in an ice bath and diluted with 300 μ salines. After centrifugation, the supernatant was taken and stored at −80°C for further experiments. Te ACh and α7nAChR were detected using the ELISA kit, and an enzyme-labeled instrument was used for the determination of ACh and α7nAChR.
2.11. Western Blot. RIPA lysis bufer was used to extract corneal proteins from each group. Te supernatant was centrifuged, the total protein concentration was determined by the BCA method, and the supernatant was calculated.
Electrophoresis separation was carried out in SDS-PAGE gel with quantifed protein samples. After electrophoresis, the gel and the membrane were cut into small strips according to the molecular weight of the protein. After the membrane was completed, 5% milk was blocked for 1 h, then the membrane was incubated in phospho-NF-κB P65, NF-κB p65, and GAPDH at 4°C overnight. Te next day, it was washed three times with 0.05% Tween-20 Tris bufer saline for 10 minutes each time, then incubated in the hrP-conjugated goat antirabbit IgG for 1 hour. Te membrane was washed in TBST three times, for 10 minutes each time. Te membrane was covered by ECL liquid, then detected by using an Imager.

Statistical Analyses
Data were represented as mean ± SEM. Statistical signifcance was evaluated by two-way ANOVA with Boferroni's post hoc test or the Mann-Whitney test using graphing software (GraphPad Prism 8.0; GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically signifcant.

Acupuncture Treatment Can Increase Tear Flow and BUT.
Compared with the group Con, the SIt of the group Scop and the group Scop + Sham decreased signifcantly and had a continuous reduction from day 14. After 21 days of treatment, compared with the group Scop and the group Scop + Sham, SIT in the group Scop + Acup signifcantly increased (Day 35:7.1 ± 3.6 vs. 2.9 ± 1.2 mm, P < 0.05; 7.1 ± 3.6 vs. 2.9 ± 1.2 mm, P < 0.05, Figure 2(a)). Compared with the group Scop and the group Scop + Sham, tear fow in the group Scop + Flu also signifcantly increased (P < 0.01, Figure 2(a)). Compared with the group Con, the BUT of the group Scop and the group Scop + Sham was decreased 14 days (P < 0.05, Figure 2(b)). Compared with the group Scop and the group Scop + Sham, the BUT in the group Scop + Acup was increased (Day 35:2.7 ± 0.9 vs. 0.9 ± 1.0 s; 2.5 ± 0.9 vs. 0.8 ± 0.8 s, P < 0.05, Figure 2(b)). Compared with the group Scop and the group Scop + Sham, the BUT in the group Scop + Flu was also increased (P < 0.05).

Acupuncture Treatment Can Reduce Corneal Epithelial
Damage. Compared with the group Scop and the group Scop + Sham, the FL score was signifcantly reduced in the group Scop + Acup on day 35 (4.1 ± 1.4 vs 7.1 ± 2.4 points, 4.1 ± 1.4 vs 7.3 ± 2.3 points, P < 0.05, Figure 3(a)). Meanwhile, compared with the group Scop and the group Scop + Sham, FL of the group Scop + Flu also signifcantly reduced the FL (P < 0.01, Figure 3(a)). Te corneal fuorescence staining on the 35th day is shown in Figure 3(b). Te corneal epithelium of the rabbits in the group Con had almost no staining, whereas those in the group Scop and the group Scop + Sham were signifcantly stained. After treatment, the ocular surface staining of the group Scop + Acup and the group Scop + Flu decreased, and the staining was scattered as dots.
H&E staining results showed that hyperkeratotic squamous epithelial cells, lymphocyte infltration, increasing focal epithelial cell layers, and shedding surface epithelial cells were seen on the surface of corneal tissues in the group Scop and the group Scop + Sham. Compared with these groups, the shedding corneal epithelial and lymphocyte infltration decreased in the group Scop + Acup, and the group Scop + Sham (Figure 3(b)).

Acupuncture Treatment Improves Corneal Stromal
Infammation. As the New Zealand rabbit is used as an animal model, it is difcult to observe the corneal epithelium, which is mainly seen in the IVCM image of the corneal stromal layer (Figure 4). Compared with the other groups, the group Scop and the group Scop + Sham showed a large number of globular immune cells 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 refectivity. Te branches were intermittent, which indicates a state of infammation Compared with the group Scop, the group Scop + Acup showed that the morphology of the stromal layer cells was improved, the cells were slightly activated, and there was no obvious abnormality in nerve refexes. Tis indicates that the signs of infammation in the corneal stromal layer were signifcantly reduced. Compared with the group Scop, the cell shape and size of the group Scop + Flu gradually became regular.

Discussion
Acupuncture has a history of more than 2000 years as a means of treatment in traditional Chinese medicine. Te therapeutic efects of acupuncture have been recognized and applied in 183 countries [17]. Nowadays, acupuncture is widely used to treat many ophthalmic diseases, such as glaucoma, ophthalmoplegia, nystagmus, and DEDs [18][19][20][21]. DEDs are common worldwide. According to the survey carried out by the Tear Film and Ocular Surface Society DEDs Workshop II Epidemiology subcommittee, the prevalence rate of DEDs is around 5% to 50%. Artifcial tears are recommended as the frst line of treatment for DEDs [21]. Moreover, anti-infammatory drugs and physical therapy are also used. A large number of clinical reports state that acupuncture can be used for the treatment of DEDs [9,22,23]. Interestingly, clinical studies have shown that the efect of acupuncture treatment is not reduced by the use of artifcial tears. Acupuncture can improve midterm outcomes for patients [24], so we can speculate that acupuncture be used as an efective complementary therapy to the conventional clinical treatments of DEDs. According to the literature, acupuncture has anti-infammatory and neuroprotective efects [25]. We used the rabbit DEDs model induced by scopolamine hydrobromide as the observation object. Tis study found the presence of corneal epithelial damage, tear reduction, shortened tear flm rupture time, infammatory cell infltration in corneal epithelial and squamous epithelial metaplasia, and corneal cells activated in the stromal layer. Te abnormal corneal nerve morphology is in line with previous studies [26][27][28] and has a strong similarity with the infammation and damage caused by human DEDs [15]. In the current study, we provided new evidence of acupuncture treatment for DEDs, indicating that acupuncture can reduce the severity of corneal damage in rabbits and can inhibit the formation of DEDs.
Our study demonstrated that the treatment of DEDs with acupuncture may be mediated by the downregulation of proinfammatory factors and chemokines. Infammation plays a key role in the progress of DEDs. Damage in the ocular surface can cause activation of NF-κB and other related pathways [29], leading to the production of infammatory cytokines and chemokines, such as IL-1, IL-8, TNF-α, IL-17A, and MMP-9 [2,30,31]. Infammatory factors and chemokines could aggravate and amplify the infammation through their interaction. For example, the degree of corneal epithelial damage is related to the expression of IL-1. IL-1 can induce the expression of several infammatory cytokines and chemokines [32,33]. Our data show similar results to previous studies [2,30,31]. Te concentration of IL-1, IL-8, TNF-α, IL-17A, and MMP-9 in  the cornea in the DEDs rabbit model were all increased. In addition, we also detected IL -21, leptin, MIP-1b, and NCAM-1 expression, and found they were all increased to varying degrees. Paiva et al. [34] indicated that IL-21 and mRNA transcription levels of the cornea in CD25KO DEDs mice were signifcantly higher than those in wild mice. Choi et al [35]. detected an increasing MIP-1b expression of the ocular surface in DED patients, especially in Sjogren syndrome (SS) patients; however, no studies of increasing leptin or NCAM-1 in DED patients have been reported. In our present research, we found that acupuncture can improve the expressions of IL-1b, IL-21, IL-17A, and MIP-1b on the cornea in DEDs. Tis shows that acupuncture can protect the cornea by inhibiting infammation of the cornea in DED patients.
NF-κB is a transcription factor that cannot be replaced in the infammatory response. NF-κB can bind to the promoters of a variety of regulatory genes in the infammatory response, such as IL-1b, IL-6, IL-8, and TNF-α. Our data proved that acupuncture can reduce the nuclear metastasis of NF-κB p65, indicating that anti-infammatory acupuncture can work by inhibiting the NF-κB signaling pathway.
Previous studies have found that CAP is a neural signaling pathway, which relies on the infammatory refex of the vagus nerve in the nervous system. It can simultaneously connect the nervous system and the immune system to create an anti-infammatory efect, releasing ACh and binding to the macrophage surface receptor α7nAChR, inhibiting the synthesis of cytokines. In addition, CAP also plays an important role in preventing the release of cytokines [36][37][38]. Activation of α7nAChR in macrophages, monocytes, and other immune cells may downregulate the production of proinfammatory cytokines and attenuate the infammatory responses by several possible mechanisms: NF-κB activation and JAK-STAT3 pathway [39]. Among these, the key receptor for transmitting cholinergic antiinfammatory signals is α7nAChR. Studies have found that the expression of α7nAChR in the lung tissue of acute lung injury rat models is signifcantly reduced. Pretreatment with electroacupuncture (EA) can prevent the decrease of α7nAChR expression and reduce the release of serum and lung infammatory cytokines after CPB, and the release of protein concentration in BALF and HMGB1. α-BGT reduces its benefcial efects [11]. Another study found that EA pretreatment can activate the expression of α7nAChR in ischemic brain tissue, thereby reducing the content of iNOS, IL-1β, CD86, and proinfammatory cytokines (TNF-α and IL-6) and increasing Arg-1, TGF-β1, and CD206 and the expression of anti-infammatory cytokines (IL-4 and IL-10) [12]. Previous experiments have shown that the mechanism of activating α7nAChR receptors is through the inhibition of NF-κB [40]. We hypothesized that acupuncture can inhibit the activation of the NF-κB pathway by regulating α7nAChR, and thereby the infammation of the cornea in the DEDs model could be suppressed. Recent studies have reported that electroacupuncture can minimize the damage   to the central cholinergic system caused by ischemia, and upregulate the expression of α7nAChR in neurons after cerebral ischemia [14]. Consequently, we used scopolamine hydrobromide as a cholinergic inhibitor in this study, which efectively reduced the levels of corneal ACh and α7nAChR in the group Scop. After acupuncture treatment, the expressions of corneal ACh and α7nAChR were increased.
Meanwhile, fuorometholone treatment also upregulated the expressions of corneal ACh and α7nAChR. When the α7nAChR antagonist α-BGT was used, the expression of α7nAChR in the mesial cornea of fuorometholone + α-BGT was inhibited; however, the NF-κB pathway was still suppressed, suggesting the existence of other anti-infammatory mechanisms or signaling pathways. Te NF-κB pathway of the middle cornea of acupuncture + α-BGT was activated.
Te experimental results prove that acupuncture can inhibit corneal infammation through the NF-κB pathway and α7nAChR regulation. Our study provides a new therapeutic target for DEDs and an important theoretical basis for the anti-infammatory efect of acupuncture.

Data Availability
Te data used to support the fndings of this study can be obtained from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.