Changes in tear cytokine and lactoferrin levels in postmenopausal women with primary acquired nasolacrimal duct obstruction complicated with obstructed meibomian gland dysfunction

Abstract

Background

Epiphora and secondary ocular surface damage are increasingly impairing the quality of life of people, particularly elderly women. We aimed to investigate the changes in tear cytokine and lactoferrin levels in postmenopausal women with primary acquired nasolacrimal duct obstruction (PANDO) complicated with obstructed meibomian gland dysfunction (OMGD) and preliminary explore the pathological mechanisms of OMGD in patients with PANDO.

Methods

The prospective study involved 43 and 41 postmenopausal women with and without PANDO, respectively. Based on the presence or absence of OMGD, the participants were subdivided. Tear fluid was collected from affected eyes of all participants using Schirmer I test and tested for cytokine concentrations (interleukin (IL)-6, IL-8, IL-1β, interferon-gamma, tumour necrotic factor (TNF)-α, IL-10, epidermal growth factor (EGF)) using the Multiplex Luminex Assay. Tear lactoferrin levels was determined using ELISA.

Results

In the PANDO with OMGD group, the IL-8, IL-1α, IL-1β, and macrophage inflammatory protein-1α levels were significantly higher than those in the other groups. The IL-6 and TNF-α levels were significantly higher than those in the controls. The lactoferrin level in the PANDO with OMGD group was lower than that in the Non-PANDO with OMGD group. The IL-10 and EGF levels were not significantly different among the groups.

Conclusion

The level of pro-inflammatory factors in the tears of patients with PANDO complicated with OMGD was significantly increased, which is likely to cause further damage to ocular surface. Anti-inflammatory therapy may help protect ocular surface in postmenopausal women with PANDO.

Background

Primary acquired nasolacrimal duct obstruction (PANDO) is a common ophthalmic condition, mainly affecting perimenopausal and postmenopausal women. The primary pathological processes include chronic inflammation and fibrosis of nasolacrimal duct (NLD) tissue, leading to the manifestation of epiphora with or without purulent discharge [1–3]. Tear analysis has revealed a substantial increase in the levels of inflammatory factors and matrix metalloproteinases in the tears of patients with PANDO, increasing the risk of ocular surface diseases [4, 5]. PANDO can induce structural and functional alterations in the meibomian glands (MGs) [6]. However, the pathophysiology of MG impairment in patients with PANDO remains unknown owing to the lack of molecular evidence.

Tear cytokine analysis is efficient for not only investigating the pathophysiology of ocular surface illnesses, such as dry eye and keratoconus, but also monitoring their therapy progress [7–9]. Inflammatory diseases of the ocular surface, such as conjunctivitis, Stevens–Johnson syndrome, and dermatological disorders, can cause structural changes in MGs, leading to tear dysfunction [10]. According to tear analyses, inflammatory cytokines such as interleukin (IL)−1, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-α) can promote the development of MG dysfunction (MGD) via a harmful inflammatory cycle [11, 12]. Consequently, inflammatory factors in the tears of patients with PANDO may also lead to structural and functional changes in MGs through an inflammatory cascade reaction.

Reportedly, inflammatory cytokines exist in the tears of patients with PANDO and MGD and play a key role in their respective pathogenesis [4, 13–18]. Changes in the tear cytokine levels during MGD in the context of certain systemic and eye diseases have also been reported [19–21]. However, studies on tears of patients with PANDO complicated with OMGD are scant. Here, we aimed to investigate the changes in cytokine and lactoferrin levels in the tears of patients with PANDO complicated with OMGD to preliminary explore the mechanism of ocular surface damage in patients with PANDO, provide clues on the pathogenesis of ocular surface damage, and identify new treatment methods for ocular surface damage in patients with PANDO.

Material and methods

Study participants

The study participants visited Wuhu Eye Hospital between January 2022 and February 2023. Postmenopausal women who complained of epiphora and were diagnosed with PANDO by lacrimal irrigation and dacryocystography were recruited into the PANDO group. Postmenopausal women with no complaints of epiphora and with a normally functioning lacrimal system, confirmed by slit-lamp examination and lacrimal irrigation, visited the hospital to improve their vision due to cataracts, and healthy volunteers were recruited in the Non-PANDO group. The exclusion criteria included premenopausal women, Sjögren’s syndrome, or systemic diseases affecting tear film quality and stability; ocular infammation; diabetes mellitus; continuous use of eye drops or other treatments; long-term contact lens wear; and a history of ocular trauma or surgery. Diagnostic criteria for PANDO include epiphora with or without purulent discharge, obstruction located in the NLD, no trauma, and other secondary factors. Diagnostic criteria for OMGD include ocular surface discomfort symptoms such as burning sensation, foreign body sensation, ocular pain or fatigue, and visual acuity fluctuation; the ocular surface disease index (OSDI) score ≥ 13; and palpebral margin abnormalities, such as MG orifice occulsion, eyelid margin irregularity, mucocutaneous junction anterior/posterior, and vascular engorgement. Due to the limited sample size for tear testing, participants with SIT ≥ 10 mm could only be included in this study. In this prospective study, 43 postmenopausal women with PANDO were included in the PANDO group and 41 postmenopausal women with normal structure and function of the lacrimal passage were included in the Non-PANDO group. Based on the presence or absence of PANDO and OMGD, all participants were further divided: 21 individuals without PANDO and without OMGD (Non-PANDO without OMGD group, age range: 51–69 years), 20 individuals without PANDO and with OMGD (Non-PANDO with OMGD group, age range: 49–74 years), 21 individuals with PANDO and without OMGD (PANDO without OMGD group, age range: 48–79 years), and 22 individuals with PANDO and with OMGD (PANDO with OMGD group, age range: 48–79 years). The affected eye was selected as the study eye for the PANDO group, and the more severely affected eye was preferred when both eyes were affected. In the Non-PANDO group, the right eye of the participants was selected. The study was approved by the Ethics Committee of Wuhu Eye Hospital Committees for Medical and Health Research Ethics (No. 20210107), and it adhered to the principles of the Declaration of Helsinki. Participants voluntarily provided written consent after receiving information about the study orally and in writing.

Ocular parameters examinations

Both the participants and the professional operator were blind to the allocation. All measurements were performed between 9 and 11 a.m. Temperatures in the examination room ranged from 22 to 28 °C, with relative humidity values between 40 and 50%. The parameters of the ocular surface have been measured in order. Ocular discomfort was evaluated using the ocular surface disease index (OSDI) questionnaire. A higher score on the questionnaire denotes more severe symptoms of ocular discomfort. The questionnaire has a total score range of 0–100 [22]. The MG loss was measured with a Keratograph 5 M (Oculus GmbH, Wetzlar, Germany). Grades of MG loss were allocated to each eyelid: grade 0 (no loss), grade 1 (MG loss < 1/3 of total MGs), grade 2 (MG loss 1/3–2/3 of total MGs), and grade 3 (MG loss > 2/3 of total MGs) [23, 24]. Fuorescein sodium test strips (Tianjin Jingming New Technology Development Co. Ltd., China) were used to determine the tear break-up time (TBUT). The average of the three tear break-up time measurements was obtained for analysis. A total MG loss score was calculated for both the upper and lower eyelids. A score of 0 (no staining), 1 (one to thirty dots of staining), 2 (staining between one and three), or 3 (filaments, confluent stains, or ulcers) was given to each of the four corneal zones: superior nasal, inferior nasal, superior temporal, and inferior temporal. The corneal fluorescein staining (CFS) score was the total of these four scores [25]. The anterior/posterior shifting of the mucocutaneous junction, vascular engorgement, occluded MG orifices, and irregular eyelid margin on each eyelid were all assessed using slit-lamp diffused light and given a 0 (absent) or 1 (present) score [26]. The total score ranged among 0 and 4. Five MGs were digitally pressed in the middle of the upper eyelid to assess MG expressibility [27]. On a scale of 0–3, the number of MGs that could express meibum was measured as follows: 0 (all 5 MGs), 1 (3–4 MGs), 2 (1–2 MGs), and 3 (0 MGs). Meibum quality was assessed in the upper and lower eyelids from the center of the eight MGs [28]. The meibum quality score was divided into four categories: clear (0), cloudy (1), cloudy with debris (2), and inspissated (3). The meibomian gland orifices (capping, narrowing, cufng loss, obliteration level, opaque/scarred, and pouting) were assessed as follows: 0 = normal; 1 = fat cap; 2 = obstruction or stenosis with protuberance; 3 = severe obstruction or atrophy.

Sample collection and storage

Tear fluid was collected using test strips (Whatman No. 41 Strips; Tianjin Jingming New Technology Development Co., Ltd., Tianjin, China), without local anaesthesia. While wearing gloves, the examiner positioned the test strip along the lateral border of the eyelid. The participants kept their eyes closed, and both eyes were examined simultaneously. After 5 min, the strip was removed, and the wet portion was evaluated up to the indentation line on a millimetre scale. Subsequently, the strip was placed in a cuvette filled with 500 mL of phosphate-buffered saline and placed in a deep freezer at − 80 °C.

Assessment of tear cytokine levels using the multiplex luminex assay

The level of nine cytokines, namely IL-6, IL-8, IL-1α, IL-1β, interferon-gamma (IFN-γ), macrophage inflammatory protein 1α (MIP-1α), TNF-α, IL-10, and epidermal growth factor (EGF), was measured using the Multiplex Luminex Assay (Luminex 200TM; USA R&D Systems, Inc., Minneapolis, MN, USA) with a commercial software, Luminex 200™ (USA R&D Systems). The assay was performed in accordance with the manufacturer’s instructions. All participants had a Schirmer I test score (SIT) of ≥ 10 mm, indicating that there was enough tear fluid for analysis.

The Eppendorf tube containing the tear sample was thawed to room temperature after removal from the − 80 °C refrigerator. Thereafter, 150 μL of ammonium bicarbonate (100 mmol/L) was added. The mixture was left undisturbed for 1 h, and then centrifugation at 14,000 × g for 10 min. Subsequently, the cytokine levels were determined. All reagents, standards, and samples were prepared per the manufacturer’s instructions. Briefly, 50 μL of standard and 50 μL of configured Luminex beads prebound with specific capture antibodies were added to the wells. The plate was incubated on a horizontal shaker at 800 ± 50 rpm for 2 h, with temperatures ranging from 22–28 °C and a relative humidity of 40 – 50%. After three washes with washing buffer, a biotin-labelled detection antibody was added to each well, and the plate was incubated for 1 h on a horizontal shaker. After repeating the washing step, fluorescein phycoerythrin (Streptacidin-PE) was added to the wells, and the plate was incubated for 30 min on a horizontal shaker. The washing step was repeated three times, and microspheres were resuspended in the wells with 100 μL of washing buffer. The plate was placed on a horizontal shaker 2 min after being subjected to shock on the machine. The level of the cytokines was determined using Luminex® MAGPIX®. The conversion formula for sample volume is as follows: a 30 ul sample is required for the full absorption of the tear filter paper strip (35 mm), and a 200 ul sample is required for the tweenelution protein. Assuming that the scale of the filter paper strip for the not-full absorption sample is Xmm, the concentration measured by the instrument is Mpg/ml, and the final concentration is Y, Y = M*200/(30*X/35).

Assessment of tear lactoferrin level using enzyme-linked immunosorbent assay

The tear level of lactoferrin was measured using an enzyme-linked immunosorbent assay kit (Abcam, UK) per the manufacturer’s instructions. All reagents, standards, and samples were prepared according to the instructions provided. Briefly, 50 μL of tear sample was added to the wells, following which, 50 μL each of specific antibody-trapping and antibody-detection reagents were added. The plate was incubated in a horizontal shaker at 400 ± 50 rpm for 1 h at room temperature. Thereafter, the wells were washed with washing buffer three times, 100 μL of substrate TMB was added to each well, and the plate was incubated again in the horizontal shaker for 8 min. This step was followed by the addition of 100 μL of the reaction-termination solution; the plate was then placed in the horizontal shaker for 1 min. The absorbance of the samples at 450 nm was determined. Lactoferrin level was calculated using a standard curve and the absorbance value of the sample.

Statistical analyses

Statistical analyses were performed using the SPSS software package (version 22.0; SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. Age; menopause time; and levels of IL-6, IL-8, IL-10, EGF, IL-1β, IFN-γ, MIP-1α, IL-1α, TNF-α, and lactoferrin were tested for normality using Shapiro–Wilk test. Except for the lactoferrin level, none of the other parameters conformed to normal distribution. Kruskal–Wallis H test was used to compare these parameters among the four study groups. The level of lactoferrin was consistent with normal distribution and homogeneity of variance, and a one-way analysis of variance was used for its comparison among the four study groups. Results with P < 0.05 were considered significant.

Ethics approval and consent to participate

This prospective study was approved by the Ethics Committee of Wuhu Eye Hospital (No. 20210107) and adhered to the principles of the Declaration of Helsinki. After explaining the nature and potential consequences of the study, written consent was obtained from the participants.

Results

Clinical characteristics of the participants

There were no significant differences in age (H = 3.878, P = 0.275) or menopause time (H = 5,931, P = 0.115) among the four groups. There was no significant difference in the menopause duration between the PANDO without OMGD group and PANDO with OMGD group (Z = 0.906, P = 0.365). The demographic characteristics of the four groups are shown in Table 1.

Table 1.

Characteristics of the study participants

Group	Age (years)	Menopause time (years)	Duration of PANDO (years)
Non-PANDO without OMGD (n = 21)	59.62 ± 5.94	8.52 ± 6.87
Non-PANDO with OMGD (n = 20)	60.75 ± 6.41	11.93 ± 7.55
PANDO without OMGD (n = 21)	58.38 ± 9.15	7.93 ± 9.59	5.27 ± 5.07
PANDO with OMGD (n = 22)	61.59 ± 7.90	10.27 ± 8.91	6.76 ± 6.25
P	0.275	0.115	0.365

There were no significant differences in age or menopause time among the four groups. There was no significant difference in the menopause duration between the PANDO without OMGD group and PANDO with OMGD group. Kruskal–Wallis H test was used for comparison. *P < 0.05; **P < 0.01; ***P < 0.001

PANDO primary acquired nasolacrimal duct obstruction, OMGD obstructed meibomian gland dysfunction

Ocular surface disease index and meibomian gland expression combined with eyelid evaluation

The OSDI score in the PANDO with OMGD group (41.52 ± 16.41) was significantly higher than those in the PANDO without OMGD group (27.64 ± 23.42, p = 0.046) and Non-PANDO with and without OMGD groups (23.71 ± 13.35, p = 0.012; 5.52 ± 5.52, p < 0.001). The OSDI score in the PANDO without OMGD group was significantly higher than that in the control group (p = 0.001). The OSDI score in the Non-PANDO with OMGD group was significantly higher than that in the control group (p = 0.001). The NITMH in the PANDO with OMGD group (0.66 ± 0.32) was significantly higher than those in the Non-PANDO with OMGD group (0.23 ± 0.07, p < 0.001) and Non-PANDO with and without OMGD groups (0.25 ± 0.08, p < 0.001). The NITMH in the PANDO without OMGD group (0.48 ± 0.25) was significantly higher than those in the Non-PANDO with OMGD group (0.23 ± 0.07, p < 0.001) and Non-PANDO with and without OMGD groups (0.25 ± 0.08, p = 0.001). The MG loss score in the PANDO with OMGD group (3.23 ± 1.31) was significantly higher than that in the PANDO without OMGD group (2.29 ± 1.31, p = 0.009) and Non-PANDO with and without OMGD groups (2.30 ± 0.86, p = 0.022; 2.48 ± 1.29, p = 0.035). The eyelid margin score in the PANDO with OMGD group (3.05 ± 0.95) was significantly higher than those in the PANDO without OMGD group (2.19 ± 1.33, p = 0.028) and Non-PANDO without OMGD groups (2.00 ± 1.30, p = 0.009). The MG orifce score in the Non-PANDO without OMGD group (1.05 ± 1.20) was significantly lower than those in the PANDO with OMGD group (2.05 ± 0.58, p = 0.009) and Non-PANDO with OMGD group (1.95 ± 1.32, p = 0.032). MG expressibility score, meibum quality score, CFS score, and TBUT have no significant differences in these four groups (Table 2).

Table 2.

Comparison of the ocular surface parameters among the study groups

Ocular surface parametersa	Non-PANDO without OMGD (n = 21)	Non-PANDO with OMGD (n = 20)	PANDO without OMGD (n = 21)	PANDO with OMGD (n = 22)	P
OSDI score	5.52 ± 5.52	23.71 ± 13.35	27.64 ± 23.42	41.52 ± 16.41	< 0.001
NITMH (mm)	0.25 ± 0.08	0.23 ± 0.07	0.48 ± 0.25	0.66 ± 0.32	< 0.001
MG loss score	2.48 ± 1.29	2.30 ± 0.86	2.29 ± 1.31	3.23 ± 1.31	0.036
eyelid margin score	2.00 ± 1.30	2.50 ± 1.32	2.19 ± 1.33	3.05 ± 0.95	0.045
MG expressibility score	1.57 ± 0.93	1.95 ± 0.89	2.05 ± 0.67	2.05 ± 0.58	0.192
meibum quality score	2.90 ± 1.67	3.45 ± 0.42	3.52 ± 0.34	3.59 ± 1.22	0.616
MG orifces score	1.05 ± 1.20	1.95 ± 1.32	1.33 ± 1.28	2.05 ± 0.58	0.036
CFS score	0.62 ± 0.80	1.20 ± 1.15	0.86 ± 1.11	0.82 ± 0.73	0.323
TBUT(s)	10.50 ± 7.61	6.22 ± 4.77	12.25 ± 7.27	9.92 ± 7.92	0.046

The OSDI score, NITMH, MG loss score, eyelid margin score, and MG orifice score differ significantly among these four groups. There are no significant variations in MG expressibility, meibum quality score, CFS score, or TBUT amongst these four groups. ^aKruskal–Wallis H-test, ^aKruskal–Wallis H test and Holm–Bonferroni method were used for the ocular surface parameters comparison

PANDO primary acquired nasolacrimal duct obstruction, MGD meibomian gland dysfunction, OSDI ocular surface disease index, NITMH non-invasive tear meniscus height, MG meibomian gland, CFS corneal fluorescein staining, TBUT tear break-up time

Tear cytokine levels in the study groups

The levels of IL-8, IL-1α, IL-1β, and MIP-1α in the PANDO with OMGD group were significantly higher than those in the PANDO without OMGD group and Non-PANDO with and without OMGD groups (all P < 0.05; Table 3). There was no significant difference among the other groups. The level of interferon-gamma (IFN-γ) was significantly higher in the PANDO with OMGD group than in the without OMGD group and Non-PANDO without OMGD group (all P < 0.05). There was no significant difference among the other groups. The levels of IL-6 and TNF-α in the PANDO with OMGD group were significantly higher than those in the Non-PANDO with and without OMGD groups (all P < 0.05). However, the levels of IL-6 and TNF-α in the PANDO with OMGD group were not significantly different from those in the PANDO without OMGD group. There was no significant difference among the other groups. There was no significant difference in the IL-10 level among the four groups (Table 3 and Fig. 1). The level of lactoferrin in the PANDO with OMGD group was significantly lower than that in the Non-PANDO with OMGD group (P < 0.05). There were no significant differences in the EGF level among the four groups (P > 0.05) (Table 3 and Fig. 2).

Table 3.

Tear cytokine levels among the study groups

Cytokinea	Non-PANDO without OMGD (n = 21)	Non-PANDO with OMGD (n = 20)	PANDO without OMGD (n = 21)	PANDO with OMGD (n = 22)	P
IL-6 (pg/mL)	4.01 ± 2.68	5.92 ± 5.32	12.20 ± 14.40	45.25 ± 105.36	< 0.001†
IL-8 (pg/mL)	121.45 ± 75.32	179.22 ± 160.58	381.71 ± 513.50	827.67 ± 654.36	< 0.001†
TNF-α (pg/mL)	0.23 ± 0.34	0.40 ± 0.58	0.88 ± 0.94	1.98 ± 2.47	< 0.001†
IL-1α (pg/mL)	3.89 ± 2.58	3.50 ± 1.59	4.29 ± 3.21	10.77 ± 8.40	< 0.001†
IL-1 β (pg/mL)	3.25 ± 7.77	1.42 ± 2.10	7.83 ± 13.63	49.85 ± 104.56	< 0.001†
MIP-1α (pg/mL)	170.49 ± 49.68	180.32 ± 47.71	215.20 ± 87.30	298.04 ± 97.85	< 0.001†
IFN-γ (pg/mL)	1.54 ± 1.31	2.05 ± 1.50	2.49 ± 3.38	24.50 ± 89.01	0.001†
IL-10 (pg/mL)	3.05 ± 1.44	3.05 ± 1.55	2.15 ± 0.93	2.89 ± 1.02	0.065†
EGF (pg/mL)	335.61 ± 285.76	374.50 ± 253.92	217.20 ± 110.80	317.32 ± 255.12	0.219†
Lactoferrin (ng/mL)	11.61 ± 0.87	11.87 ± 0.63	11.68 ± 0.80	11.18 ± 0.62	0.019a

The levels of IL-8, IL-1α, IL-1β, and MIP-1α in the PANDO with OMGD group were significantly higher than those in the PANDO without OMGD group and Non-PANDO with and without OMGD groups. The level of interferon-gamma (IFN-γ) was significantly higher in the PANDO with OMGD group than in the without OMGD group and Non-PANDO without OMGD group.The level of lactoferrin in the PANDO with OMGD group was significantly lower than that in the Non-PANDO with OMGD group. †Kruskal–Wallis H test and Holm–Bonferroni method were used for cytokine level comparison. ^aOne-way analysis of variance was used for lactoferrin level comparison.*P < 0.05; **P < 0.01; ***P < 0.001

Mean ± SD. EGF epidermal growth factor, IL interleukin, IFN interferon, MIP macrophage inflammatory protein, TNF tumour necrosis factor

Fig. 1.

Tear inflammation cytokine levels among the study groups. The levels of IL-8 (A), IL-1α (B) , IL-1β (C), and MIP-1α (D) in the PANDO with OMGD group were significantly higher than those in the PANDO without OMGD group and Non-PANDO with and without OMGD groups.The level of IFN-γ (E) was significantly higher in the PANDO with OMGD group than in the without OMGD group and Non-PANDO without OMGD group.The levels of IL-6 (F) and TNF-α (G) in the PANDO with OMGD group were significantly higher than those in the Non-PANDO with and without OMGD groups.There was no significant difference in the IL-10 (H) level among the four groups. Kruskal–Wallis H test and Holm–Bonferroni method were used for cytokine level comparison. *P<  0.05; **P<  0.01; ***P <  0.001. IL, interleukin; PANDO, primary acquired nasolacrimal duct obstruction; Non-PANDO, without primary acquired nasolacrimal duct obstruction; OMGD, obstructed meibomian gland dysfunction; IL-interleukin, MIP, macrophage inflammatory protein, TNF, tumour necrosis factor

Fig. 2.

Levels of tear (a) EGF and (b) lactoferrin among the study groups. There were no significant differences in the EGF (A) level among the four groups.The level of lactoferrin (B) in the PANDO with OMGD group was significantly lower than that in the Non-PANDO with OMGD group. Kruskal–Wallis H test and Holm–Bonferroni method were used for cytokine level comparison. One-way analysis of variance was used for lactoferrin level comparison. *P  <  0.05; **P  <  0.01; ***P  <  0.001. EGF, epidermal growth factor; PANDO, primary acquired nasolacrimal duct obstruction; Non-PANDO, without primary acquired nasolacrimal duct obstruction; OMGD, obstructed meibomian gland dysfunction

Discussion

In this study, we found that the levels of pro-inflammatory cytokines (IL-8, IL-1α, IL-1β, and MIP-1α) significantly increased, and ocular surface discomfort, MG loss, palpebral margin abnormality, and MG orifice occulsion were more serious in patients with PANDO complicated with OMGD. This result suggests that inflammation plays an important role in the pathogenesis of postmenopausal women with PANDO complicated with OMGD, consistent with the findings of previous studies, which have reported that inflammation plays a key role in the pathogenesis of MGD in ocular surface diseases [13, 21, 29]. The pathogenesis of MGD can be a self-perpetuating vicious cycle of inflammation [13]. We speculated the following three reasons for the increased levels of these pro-inflammatory cytokines. First, when the NLD is blocked, the mucosal epithelial cells or fibroblasts of the tear drainage system may produce cytokines in an inflammatory state. Although the tear drainage system is a one-way system controlled by a pressure gradient, locally produced cytokines may diffuse back into the tear film and alter the cytokine profile [16, 30, 31]. Second, inflammatory factors produced by NLD tissue may promote the development of OMGD through a vicious cycle of inflammatory cascade reactions. In turn, OMGD may further increase the level of pro-inflammatory cytokines in tears. Finally, the accumulation effect caused by delayed tear clearance leads to an increase in the level of pro-inflammatory factors in tears [32].

Interestingly, we observed significantly higher IL-6 and TNF-α levels in the PANDO with OMGD group than in the control group but not in the PANDO without OMGD group. Yoon reported an increase in the levels of IL-6 and TNF-α in the tears of patients with dry eyes and suggested that reduced tear production in these patients likely leads to increased IL-6 levels, stimulating ocular surface epithelial cells to produce inflammatory cytokines [29]. Delayed tear clearance is a common feature of tear dynamics in NLD and dry eye. Tear dysfunction leads to tear accumulation, increasing IL-6 and TNF-α levels due to water evaporation. Lee and Kim [16] found that the levels of the inflammatory cytokines IL-2, IL-6, IL-10, VEGF, and FGF-2 in the tears of patients with PANDO were higher than those in the contralateral eyes after dacryocystorhinostomy; however, the inflammatory cytokine levels decreased rapidly upon the removal of silicone scaffolds compared with those in the controls. Tear dysfunction, causing delayed tear clearance, maybe a key factor for elevated cytokine levels in the tears of patients with blocked NLD. Therefore, we speculate that the higher IL-6 and TNF-α levels in patients with PANDO are likely the result of delayed tear clearance and accumulation, rather than an inflammatory cascade.

A high osmotic pressure in tears has been associated with higher levels of the pro-inflammatory cytokine IFN-γ, which can be used as a specific biomarker for evaporative dry eye [33]. In this study, the level of IFN-γ in the PANDO with OMGD group was significantly higher than that in the PANDO without OMGD group, consistent with the findings of a previous study [33]. Hence, IFN-γ can be used as an effective biological marker for the diagnosis of OMGD in patients with PANDO. As tear osmolarity was not examined in the present study, we cannot positively conclude whether the increase in the IFN-γ level was induced by hypertonic stress. Additionally, the IL-8 level in the tears of patients in the PANDO with OMGD group was significantly higher than that in the other three groups. The increase in the IL-8 level in patients with PANDO complicated with OMGD may effectively signal the recruitment of T lymphocytes to the ocular surface, resulting in ocular surface damage. IL-8 is a potent attractant of T cells and neutrophils, and a high level of IL-8 has been identified as a cause of inflammatory diseases and intraocular inflammation [34, 35]. The increase in inflammatory factor levels in patients with PANDO complicated with OMGD is clinically significant for diagnosis and treatment.

In this study, no significant difference was observed in the levels of all cytokines between the Non-PANDO without OMGD group and the PANDO without OMGD group. These results were not consistent with the findings of a previous study, which reported that the levels of pro-inflammatory factors in patients with PANDO was higher than those in healthy controls [16]. This discrepancy in the findings could be attributed to some differences between the studies. The previously published study grouped participants based on the occurrence of PANDO only, and the OMGD prevalence in participants was not taken into consideration. Hence, the PANDO group could have included many patients with OMGD. Furthermore, our study focused on only a few of the cytokines that had been previously discovered in classic MGD studies, and some important cytokines may have been missed. Hence, the results do not completely reflect the differences in inflammatory response between the Non-PANDO without OMGD group and PANDO without OMGD group. Alternatively, it may be assumed that there was no significant difference in these cytokine levels in this study. In this study, no significant difference was observed in the levels of all cytokines between the Non-PANDO without OMGD group and the Non-PANDO with OMGD group. Owing to the limited sample size of tear testing, all participants in the present study had SIT ≥ 10 mm, which reduced the increased levels of inflammatory factors caused by delayed tear clearance in dry eye patients, which may explain the absence of differences in tear cytokines in patients with dry eye disease accompanied by MGD, regardless of PANDO.

Lactoferrin, mainly secreted by the acinus of the lacrimal glands, has antibacterial and immunomodulatory effects. We selected lactoferrin as the target protein as it is a representative protein secreted from lacrimal glands [36]. Our study revealed that the levels of lactoferrin in the tears of the PANDO with OMGD group were significantly lower than those in the Non-PANDO with OMGD group. This finding is consistent with the results of Yaginuma et al. who reported a decrease in the levels of total protein and albumin in the tears of patients with PANDO. They also reported the total protein and albumin significantly increased to levels similar to those of the control group after lacrimal duct intubation. These results suggest that the change in tear protein levels is caused by delayed tear clearance due to NLD [32]. EGF has a potential regulatory role in maintaining the ocular surface and controlling corneal wound healing and ocular surface diseases [37]. The level of tear EGF significantly correlated with the Schirmer 1 test score, indicating that it is a marker of lacrimal gland function [38].We found no significant difference in the level of EGF among the four groups, suggesting that NLD, regardless of its complication with OMGD, had no significant effect on the function of the lacrimal gland on the obstructed side. Similarly, the control group with normal lacrimal duct function, regardless of whether complicated with OMGD or not, did not significantly affect the secretory function of the lacrimal gland. Notably, owing to the limited sample size of tear testing, all participants in the present study had SIT ≥ 10 mm, which may explain the absence of lacrimal gland damage.

In this study, the PANDO with OMGD group exhibited significantly higher levels of pro-inflammatory factors, whereas the level of IL-10, which has anti-inflammatory effects, did not significantly increase, suggesting that the inflammation level in the PANDO with OMGD group was higher than that in the other groups. IL-10, which is secreted by regulatory B cells, reduces Th1 immunity and is considered to have anti-inflammatory effects [39]. In patients with PANDO, the pro-inflammatory cytokine levels in tears increased, whereas IL-10 was detected only in tears on the side of NLD, and its level rapidly decreased to undetectable levels 2 months after surgery [16]. IL-10 exhibited a distinct pattern compared to other inflammatory cytokines, hinting at its potential as a biomarker for NLD. In our study, IL-10 was detectable in the tears of all participants, which is consistent with the finding of a previous study, that is, IL-10 is found in the tears of healthy volunteers [40]. The inflammatory state is determined by the balance between pro-inflammatory and anti-inflammatory factors [41, 42].

The present study had a few limitations. First, owing to the limited sample size for tear testing, all participants in this study had SIT ≥ 10 mm. Second, this study focused only on nine classic tear cytokines and lactoferrin, including pro-inflammatory and anti-inflammatory cytokines and those that reflect lacrimal gland function, and some important cytokines may have been missed. Hence, future studies should aim to include male participants to provide a more comprehensive analysis. Furthermore, improved detection technologies should be employed for tear analysis of individuals with SIT < 10 mm. We plan to supplement the results with follow-up study results using molecular biology techniques to assess the changes in the levels of other tear cytokines and proteins and comprehensively analyse the pathological mechanism of OMGD damage caused by PANDO.

Conclusions

Our study showed that the level of pro-inflammatory factors in the tears of postmenopausal women with PANDO complicated with OMGD was significantly increased, which is likely to cause further damage to the ocular surface. Anti-inflammatory therapy may help protect the ocular surface in postmenopausal women with PANDO. Increased ocular surface inflammation may partially explain the impairment of MG structure and function in postmenopausal women with PANDO. It is crucial that future studies focus on developing improved treatment options that specifically target these pathways.

Abbreviations

EGF

Epidermal growth factor

IFN-γ

Interferon-gamma

IL

Interleukin

MIP-1α

Macrophage inflammatory protein 1α

MG

Meibomian gland

MGD

Meibomian gland dysfunction

OMGD

Obstructed meibomian gland dysfunction

NLD

Nasolacrimal duct

PANDO

Primary acquired nasolacrimal duct obstruction

SIT

Schirmer I test

TNF

Tumour necrotic factor

CFS

Corneal fluorescein staining

TBUT

Tear break-up time

PE

Phycoerythrin

Authors’ contributions

HJ, XC, and FJ designed the study; HJ, XC, and FJ analysed the results, and drafted and revised the manuscript. YL, YS, GW, LH, XW, HD, JL, and QF selected the participants and obtained the relevant cases. All authors have read and approved the final manuscript.

Funding

This research was funded by the Wuhu Municipal Science and Technology Bureau, grant number 2022cg15.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Committee of Wuhu Eye Hospital (protocol code 20210107). Informed consent was obtained from all participants of the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.