Analyze interleukin-1β, interleukin-6, and tumor necrosis factor-α levels in dry eye and the therapeutic effect of cyclosporine A

Abstract

BACKGROUND

Dry eye is a common eye disease. Artificial tears supplements are widely used for the treatment of dry eyes. However, multiple adverse effects have been observed in patients receiving long-term treatment with artificial tears, which may affect the therapeutic effect.

AIM

To analyze the characteristics of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels in patients with dry eye and the therapeutic effect of artificial tears combined with cyclosporine A.

METHODS

A total of 124 dry eye patients treated at The First People’s Hospital of Xining from April 2020 to April 2022 were selected as the observation group, while 20 healthy individuals served as the control group during the same period. Levels of inflammatory markers, including IL-1β, IL-6, and TNF-α, were analyzed. The observation group was further divided into a study group and a control group, each consisting of 62 patients. The control group received artificial tears, whereas the study group received a combination of artificial tears and cyclosporine A. Inflammatory markers, Schirmer’s test (SIT), tear break-up time (TBUT), corneal fluorescein staining (CFS), National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25) scores, and adverse events (AEs) were compared between the two groups.

RESULTS

The observation group exhibited significantly elevated serum levels of IL-1β, IL-6, and TNF-α in comparison to the healthy group. Following treatment, the study group demonstrated substantial reductions in IL-1β, IL-6, and TNF-α levels relative to the control group. Moreover, after treatment, the study group experienced a marked decrease in CFS scores and significant increases in both SIT and BUT levels when compared to the control group. Additionally, significant improvements were observed in the primary symptom of dry eye and secondary symptoms such as photophobia, foreign body sensation, fatigue, red eye, and burning sensation within the study group. Furthermore, post-treatment NEI-VFQ-25 scores across all dimensions exhibited significant enhancements in the study group compared to the control group (P < 0.05). It is noteworthy that significant AEs were reported in both groups throughout the treatment period.

CONCLUSION

Cyclosporine A combined with artificial tears is effective in treating dry eye, yielding enhanced outcomes by improving SIT and TBUT levels, reducing CFS scores, and ameliorating vision-related quality of life.

Key Words: Artificial tears, Dry eye syndrome, Cyclosporine, Eye inflammation, Interleukin-1β, Interleukin-6, Tumor necrosis factor-α, Cyclosporine A

Core Tip: Few studies have explored the expression of inflammatory factors in dry eye disease and the impact of cyclosporine A on these factors. This study confirmed that the levels of tear inflammatory factors in patients with dry eye syndrome were significantly higher than those in healthy individuals. Moreover, intervention with artificial tears substantially reduced inflammatory factors and alleviated symptoms, an effect that was significantly enhanced when combined with cyclosporine A.

INTRODUCTION

Dry eye disease is a prevalent clinical ophthalmic condition. With the aging population, widespread use of electronic devices, and lifestyle changes, the prevalence of dry eye is increasing[1]. Dry eye is a chronic ocular surface disease caused by multiple factors, including tear film instability and ocular surface microenvironment imbalance due to abnormalities in tear quality, quantity, and dynamics. This condition can be accompanied by ocular surface inflammation, tissue damage, and nerve abnormalities, leading to various symptoms of ocular discomfort and/or visual dysfunction[2]. Emerging evidence has elucidated a significant correlation between the magnitude of the inflammatory response and the clinical phenotype of dry eye disease[3]. Dry eye is characterized by damage to the ocular surface epithelium, resulting in exposure of corneal nerve endings and chronic irritation due to the hypertonic state of tears[4]. The stress response occurs due to gradual corneal desensitization, which eventually leads to widespread destruction of the ocular surface and the release of pro-inflammatory factors, further aggravating ocular surface injury[5]. Therefore, in addition to traditional symptomatic treatment, anti-inflammatory therapy is now considered an essential part of the dry eye treatment regimen[6].

Artificial tear supplementation is a widely adopted approach for the management of dry eye. However, patients undergoing prolonged artificial tear therapy have been observed to experience various adverse effects, potentially compromising treatment efficacy[7]. Cyclosporine A is an immunomodulatory drug with anti-inflammatory properties that selectively inhibits the early stages of T lymphocyte activation, thereby reducing the production of interferon by lymphocytes[8]. As a commonly utilized immunosuppressant, cyclosporine A can effectively augment tear secretion through the inhibition of T-lymphocytes and inflammatory cytokines[9]. Studies have shown that topical 0.05% cyclosporine A is effective for the prevention and treatment of cataract surgery-related dry eye symptoms, can slow or prevent disease progression in patients with severe dry eye disease, does not inhibit wound healing, and does not cause adverse changes in the lens or systemic adverse reactions[10,11]. It has been confirmed that in patients treated with topical cyclosporine A, the number of goblet cells increases, and tear film balance is restored, improving dry eye symptoms[12]. One prospective study showed that 0.05% cyclosporine A improved symptoms and signs in patients with dry eye, with a significant difference compared to conventional artificial tears[13]. However, the effect of cyclosporine A on inflammatory factor levels in patients with dry eye disease has not been reported.

It has been reported that most secretory pro-inflammatory factors are overexpressed in dry eye, with medium- to high-level cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) stimulating the maturation of antigen-presenting cells[14,15]. In addition to its pro-inflammatory properties, IL-1β is also involved in apoptosis and pain hypersensitivity[16]. Elevated levels of IL-1β can inhibit neurotransmitter secretion, affecting the neuromodulation of tear secretion, reducing tear production, and ultimately leading to dry eye syndrome[17]. TNF-α plays a key role in the occurrence and progression of various autoimmune diseases and is widely involved in the induction of protein synthesis in the acute phase of hepatocytes, the differentiation of myeloid leukemia cells into macrophages, cell proliferation and differentiation, neutrophil phagocytosis, anti-infection responses, and the killing and inhibition of tumor cells[18]. IL-6 is a cytokine that regulates the inflammatory response, modulates the function of B cells and T cells, and interacts synergistically with TNF-α[19]. In this study, cyclosporine A was used to treat patients with dry eye disease, and changes in IL-1β, IL-6, and TNF-α levels were analyzed to discuss the characteristics of inflammatory markers and the intervention effect of cyclosporine A in patients with dry eye disease.

MATERIALS AND METHODS

Study population

The observational cohort for this study included patients diagnosed with dry eye disease who were admitted to The First People’s Hospital of Xining from April 2020 to April 2022. The inclusion criteria were: (1) Symptoms of foreign body sensation, eye fatigue, visual fluctuations, dryness, and/or burning, with or without positive corneal fluorescein staining (CFS), and a tear break-up time (TBUT) ≤ 5 s; (2) TBUT less than 10 s; (3) complete clinical data without missing information; and (4) Schirmer test results less than 5 mm/5 min. Exclusion criteria were: (1) Concomitant ocular diseases; (2) liver or kidney dysfunction, or coagulation disorders; and (3) concurrent infection. Based on these criteria, 124 patients were enrolled and divided equally into a control group and a study group, each consisting of 62 patients.

The control group had a mean age of 63.53 ± 3.13 years (range: 45-78 years) and included 34 males and 28 females. The study group had a mean age of 64.01 ± 2.69 years (range: 42-79 years) and included 33 males and 29 females. Additionally, 20 healthy individuals who underwent routine physical examinations at The First People’s Hospital of Xining during the same period were recruited as the healthy control group. This group had a mean age of 64.13 ± 3.12 years (range: 46-67 years) and included 11 males and 9 females. No significant differences were observed in age or sex distribution among the healthy control, control, and study groups (F = 0.54, P = 0.584).

Treatment methods

The control group received artificial tear treatment (Registration No. H20140945; Allergan Pharmaceuticals, Dublin, Ireland), administered as 1-2 drops three times daily. The study group received the same artificial tear regimen, with the addition of 0.05% cyclosporine A (Chinese Medicine Approval No. H20203239; Shenyang Xingqi Pharmaceutical Co., Ltd., Shenyan, Liaoning, China), applied as 1 drop three times daily. The two medications were administered at a 15-min interval, with artificial tears applied first, followed by cyclosporine A. After an 8-week treatment period, therapeutic outcomes were compared between the two groups.

Outcome measures

Inflammatory indicators: After the 8-week treatment period, 15 μL of tear fluid was collected from all participants, and the levels of IL-6, IL-1β, and TNF-α were measured using enzyme-linked immunosorbent assay.

Schirmer test: Before any anesthesia, a 5 mm folded end of a standard filter paper was placed in the middle and outer one-third of the lower conjunctival sac. The length of tear film wetting was recorded before and after treatment. The normal range was defined as 10-25 mm/5 min.

Tear film break-up time: With the patient in a seated position, 0.25% sodium fluorescein was instilled into the conjunctival sac, and participants were instructed to blink gently. The time interval between a blink and the first appearance of a dry spot in the tear film was recorded. The average of three measurements was used, with a TBUT value of ≥ 10 s considered normal.

Corneal fluorescent staining score: To facilitate the analysis of corneal lesions, the cornea was divided into five equal regions. A 0.25% sodium fluorescein solution was instilled into the patient’s eye, and staining patterns in each of the five regions were observed under a slit lamp. Positive staining appeared as a yellow-green discoloration.

The staining in each region was scored from 0 to 3 points based on the following criteria: (1) 0 points: No staining; (2) 1 point: 1-30 punctate stains; (3) 2 points: More than 30 punctate stains; and (4) 3 points: Filamentary or diffuse staining.

The final CSC score was calculated as the sum of the scores for the five regions, with a maximum possible score of 15 points.

Subjective symptom improvement analysis: Patients’ subjective symptoms were evaluated both prior to and following the treatment period. The primary symptom assessed was dry eye, rated on a scale from 0 to 6. Secondary symptoms, such as eye fatigue, foreign body sensation, photophobia, eye redness, and burning sensation, were each rated on a scale from 0 to 3.

National Eye Institute Visual Function Questionnaire-25: The National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25), a 25-item questionnaire, measures three dimensions: visual impairment, mobility impairment, and general health status. Higher scores on this questionnaire reflect a lesser impact of the disease on the patient’s quality of life.

Safety assessment: Adverse events (AEs) occurring during the intervention period were meticulously recorded and categorized into systemic, ocular, and non-ocular AEs. Serious adverse events (SAEs) are defined as new or prolonged hospitalization, disability, inability to work, life-threatening medical events, death, or congenital anomalies.

Statistical analysis

The normality of continuous data was tested. For normally and partially normally distributed data, the mean ± standard deviation was utilized. Comparisons between groups were performed using the independent samples t-test, while within-group comparisons employed the paired samples t-test. One-way ANOVA was applied for multiple group comparisons. For other continuous data, the median and interquartile range were used, with the Mann-Whitney U test for inter-group comparisons. Categorical data were expressed as counts and percentages [n (%)] and analyzed using the χ² test. Data analysis was conducted using SPSS version 22.0, with a P value < 0.05 deemed statistically significant.

RESULTS

Detection of inflammatory markers

In comparison to the healthy control group, the levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α were significantly elevated in the dry eye observation group (P < 0.05). This finding supports the hypothesis that inflammatory mediators, including IL-1β, IL-6, and TNF-α, are crucial in the pathogenesis and persistence of dry eye disease.

Before treatment initiation, no significant differences were observed in the concentrations of IL-1β, IL-6, and TNF-α between the study cohort and the control group. However, following the treatment period, the levels of these cytokines significantly decreased in the study group compared to the control group (P < 0.05). This suggests that artificial tear supplementation can mitigate the inflammatory state, with cyclosporine A therapy further enhancing this anti-inflammatory effect. The profiles of inflammatory markers for the study and control groups are detailed in Tables 1 and 2, respectively.

Table 1 Inflammatory indexes in the observation and healthy groups.

Group	n	IL-6 in pg/mL	TNF-α in ng/L	IL-1β in ng/L
Observation group	124	153.67 ± 4.12	46.34 ± 1.54	42.23 ± 2.12
Healthy group	20	36.34 ± 2.53	12.32 ± 1.34	11.42 ± 1.82
t value	-	13.232	14.232	14.722
P value	-	< 0.05	< 0.05	< 0.05

Data are mean ± standard deviation. IL-1β: Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha.

Table 2 Inflammatory indicators in the study and control groups.

Group	n	Time	IL-6 in pg/mL	TNF-α in ng/L	IL-1β in ng/L
Control group	62	Before treatment	152.34 ± 3.67	46.35 ± 2.09	42.42 ± 2.21
		Aftertreatment	89.43 ± 4.12a	28.43 ± 1.25a	23.25 ± 2.08a
Study group	62	Before treatment	154.11 ± 4.12	46.32 ± 2.31	42.15 ± 2.18
		After treatment	65.34 ± 3.19a,c	16.57 ± 1.11a,c	18.42 ± 1.84a,c

Data are mean ± standard deviation.

^aP < 0.05 vs before treatment.

^cP < 0.05 vs control group at the same time.

IL-1β: Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha.

Comparison of Schirmer’s test, TBUT, and FL

Post-treatment, the CFS score in the study group was significantly lower than that of the control group (P < 0.05). Additionally, the TBUT and Schirmer’s test (SIT) values were significantly higher in the study group compared to the control group (P < 0.05). These results indicate that artificial tears improve tear film stability and restore corneal/conjunctival epithelial integrity. Furthermore, the combination of cyclosporine A therapy can amplify these beneficial effects on the ocular surface. The objective ocular surface parameters are summarized in Table 3.

Table 3 Comparison of Schirmer’s test, tear break-up time and corneal fluorescein staining.

Group	n	Time	SIT in mm/5 min	TBUT in s	FL points
Control group	62	Before treatment	6.41 ± 1.32	7.47 ± 1.43	5.18 ± 0.45
		After treatment	9.24 ± 1.97a	7.43 ± 1.65a	2.53 ± 0.35a
Study group	62	Before treatment	6.38 ± 2.14	7.04 ± 1.25	5.14 ± 0.51
		After treatment	12.21 ± 2.41a,c	11.54 ± 1.58a,c	1.25 ± 0.36a,c

Data are mean ± standard deviation.

^aP < 0.05 vs before treatment.

^cP < 0.05 vs control group at the same time.

TBUT: Tear break-up time; FL: Corneal fluorescein staining; SIT: Schirmer’s test.

Comparison of subjective symptom scores

Compared to the control group, the study group exhibited significantly reduced scores for the primary symptom of dry eye, as well as the secondary symptoms of photophobia, foreign body sensation, eye fatigue, ocular redness, and burning sensation after the treatment period (P < 0.05). This indicates that artificial tear supplementation can alleviate subjective dry eye-related symptoms, with cyclosporine A further enhancing symptom improvement. The subjective symptom scores are presented in Table 4.

Table 4 Comparison of subjective symptoms scores.

Group	n	Time	Dry eyes	Photophobia	Foreign body sensation	Feeling tired	Redeyes	Burning sensation
Control group	62	Before treatment	3.82 ± 0.34	1.69 ± 0.24	1.75 ± 0.43	1.93 ± 0.21	1.67 ± 0.21	1.26 ± 0.25
		After treatment	2.18 ± 0.32a	0.77 ± 0.23a	0.72 ± 0.31a	0.89 ± 0.17a	0.67 ± 0.16a	0.56 ± 0.15a
Study group	62	Before treatment	3.97 ± 0.42	1.71 ± 0.32	1.78 ± 0.25	1.91 ± 0.35	1.64 ± 0.32	1.27 ± 0.23
		After treatment	1.54 ± 0.41a,c	0.53 ± 0.14a,c	0.51 ± 0.32a,c	0.70 ± 0.34a,c	0.53 ± 0.15a,c	0.46 ± 0.21a,c

Data are mean ± standard deviation.

^aP < 0.05 for comparison of before and after treatment in the same group.

^cP < 0.05 for comparison to the control group and study group at the same time.

Comparison of the NEI-VFQ-25 score

Following treatment, the study group exhibited significantly higher scores across all dimensions of the NEI-VFQ-25 compared to the control group (P < 0.05). This suggests that the use of artificial tears, potentially enhanced by cyclosporine A therapy, can effectively improve patients’ vision-related quality of life. The NEI-VFQ-25 scores are detailed in Table 5.

Table 5 NEI-VFQ-25 score detection.

Group	n	Time	Mobility barriers	Visual impairment	Health conditions
Control group	62	Before treatment	80.34 ± 3.12	85.12 ± 4.35	55.23 ± 3.54
		After treatment	92.12 ± 3.52a	90.23 ± 5.12a	61.23 ± 4.12a
Study group	62	Before treatment	80.34 ± 4.11	85.45 ± 3.25	56.23 ± 4.12
		After treatment	99.67 ± 3.64a,c	99.34 ± 3.25a,c	68.84 ± 3.81a,c

Data are mean ± standard deviation.

^aP < 0.05 for comparison of before and after treatment in the same group.

^cP < 0.05 for comparison to the control group and study group at the same time.

Comparison of adverse reactions

No SAEs, systemic AEs, or non-ocular AEs were reported in either the study or control group during the treatment period. However, ocular AEs were observed, with 6 cases in the study group and 7 cases in the control group. The primary manifestations included ocular pain, increased ocular discharge, and dry eye symptoms. All ocular AEs were mild in severity and resolved with or without appropriate management.

DISCUSSION

The pathogenesis of dry eye disease is multifactorial, characterized primarily by tear film deficiency and/or persistent instability, which can lead to ocular discomfort and even affect patient vision. These changes are often accompanied by varying degrees of inflammation, epithelial lesions on the ocular surface, or sensory abnormalities of the nerves[20]. The development of dry eye is typically closely related to environmental factors. Exposure to ultraviolet rays, pollutants, and ozone has been implicated in the causation of dry eye[21]. However, the precise pathogenesis remains unclear. Several studies have indicated that inflammation plays a central role in the onset and progression of dry eye disease. Levels of inflammatory mediators, including cytokines (e.g., IL-1β, IL-6, TNF-α), are elevated in the tears and ocular surface tissues of patients with dry eye[6,22,23]. This inflammatory state can disrupt the structure and function of the ocular surface and tear film. The theory of inflammation is the predominant and comprehensive hypothesis, followed by theories involving apoptosis and hormonal imbalance[24]. In recent years, artificial tear supplementation has become a common treatment approach for dry eye disease. These lubricating agents are formulated to mimic the pH and mucin composition of healthy human tears, effectively improving corneal permeability and relieving dry eye symptoms[23,25,26]. Interventions with artificial tears have been shown to increase tear secretion, promote corneal epithelial repair, and prolong tear TBUT in patients with dry eye[27]. Cyclosporine A, an immunosuppressant drug, is also utilized in the clinical management of dry eye disease. It can inhibit the aggregation of inflammatory cells, reduce the release of inflammatory factors, improve the infiltration of conjunctival tissue and the lacrimal gland, alleviate the degree of inflammatory reaction, and inhibit the apoptosis of conjunctival goblet cells and the lacrimal gland[28].

The results of the present study showed that the levels of IL-6, TNF-α, and IL-1β in the observation group were significantly higher than in the healthy control group, suggesting the presence of substantial ocular inflammation in patients with dry eye disease. After comparing different treatment regimens, the levels of IL-6, TNF-α, and IL-1β in the study group were significantly lower than in the control group (P < 0.05), indicating that combined treatment with cyclosporine A can effectively reduce ocular inflammation. SIT, tear TBUT, and CFS are commonly used evaluation indicators closely related to the symptoms of dry eye disease[29]. The results showed that cyclosporine A can reduce the CFS score and increase the levels of SIT and TBUT. This further confirmed that combined treatment with cyclosporine A can increase tear secretion and prolong TBUT in patients with dry eye compared to artificial tears alone. The combination of cyclosporine A and artificial tears likely promotes corneal repair, improves the ocular surface and tear film environment, and increases tear secretion[30]. Dry eye is the main clinical manifestation of dry eye disease, and patients may also experience fatigue, photophobia, foreign body sensation, and other symptoms of ocular discomfort[31]. The results showed that the main symptoms of dry eye and secondary symptoms of photophobia, foreign body sensation, fatigue, ocular redness, and burning sensation were significantly reduced in the study group after treatment, suggesting that combined cyclosporine A treatment can effectively alleviate the symptoms of dry eye. The findings indicated that IL-6, TNF-α, and IL-1β were highly expressed in patients with dry eye, and cyclosporine A had a better effect in inhibiting the development of inflammation, preventing the aggregation of inflammatory cells, and reducing the release of inflammatory factors. Therefore, combined treatment with cyclosporine A and artificial tears could alleviate the symptoms of dry eye[32,33]. To further analyze the therapeutic effects of different treatment regimens, this study assessed the vision-related quality of life in patients. The findings demonstrated that combined treatment with cyclosporine A significantly increased the scores across all dimensions of the NEI-VFQ-25, suggesting that this combination therapy can effectively improve patients’ vision-related quality of life. No SAEs were reported, and all AEs were mild and resolved with or without appropriate treatment. Studies have indicated that cyclosporine A can cause greater irritation to the ocular surface, potentially increasing adverse reactions in patients. In this study, patients were treated with artificial tears first, followed by cyclosporine A after 15 min, effectively reducing the incidence of adverse reactions.

CONCLUSION

Dry eye syndrome exhibits a strong pathogenic relationship with inflammatory processes. The administration of 0.05% cyclosporine A has been established as an effective pharmacologic intervention for managing dry eye disease. When used concurrently with artificial tear supplementation, cyclosporine A therapy can enhance patient benefits by increasing SIT values, extending TBUT, reducing CFS scores, and improving vision-related quality of life metrics.

To further elucidate the therapeutic potential of the cyclosporine A and artificial tears combination for treating this chronic, multifactorial ocular condition, additional investigation through a larger, multicenter clinical trial conducted over an extended treatment duration is warranted. Such an approach would facilitate the confirmation of the long-term efficacy of this combinatorial modality.