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. 2023 Apr 5;71(4):1582–1586. doi: 10.4103/IJO.IJO_2855_22

Modulation of mucin secretion using combined polyethylene glycol–propylene glycol topical formulation in a hyperosmotic stress-based explant model

Trailokyanath Panigrahi 1,✉, Edwin James 1, Pooja Khamar 2, Bhavya Gorimapalli 2, Sharon D’Souza 2
PMCID: PMC10276745  PMID: 37026305

Abstract

Purpose:

Ocular surface discomfort and dry eye disease are caused by a dysfunctional tear film. The efficacy of lubricating eye drops on the human eye is known, but the compositions may show differential effects on rescuing the tear film. Mucins form a critical layer of the tear film, a reduction of which may be causative for ocular surface conditions. Therefore, it is essential to develop relevant human-derived models to test mucin production.

Methods:

Human corneoscleral rims were obtained from a healthy donor (n = 8) post-corneal keratoplasty and cultured in DMEM/F12 media. Hyperosmolar stress mimicking dry eye disease was induced by exposing the corneoscleral rim tissues to +200 mOsml NaCl-containing media. The corneoscleral rims were treated with polyethylene glycol–propylene glycol (PEG–PG)-based topical formulation. Gene expression analysis was performed for NFAT5, MUC5AC, and MUC16. Secreted mucins were measured by enzyme-linked immunosorbent assay (ELISA) (Elabscience, Houston, TX, USA) for MUC5AC and MUC16.

Results:

The corneoscleral rims responded to hyperosmolar stress by upregulating NFAT5, a marker for increased osmolarity, as observed in the case of dry eye disease. The expression of MUC5AC and MUC16 was reduced upon an increase in hyperosmotic stress. The corneoscleral rim tissues showed induction of MUC5AC and MUC16 expression upon treatment with PEG–PG topical formulation but did not show significant changes in the presence of hyperosmolar treatments.

Conclusion:

Our findings showed that PEG–PG-based topical formulation slightly alleviated hyperosmolar stress-induced decrease in MUC5AC and MUC16 gene expression that is encountered in DED.

Keywords: Dry eye disease, ELISA, hyperosmotic stress, mucin, PEG–PG

Dry eye disease (DED) is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.[1-3] Global estimates of DED vary from 5.5% to 33.7%, with a significantly higher prevalence in India of 18%–54%.[4-9] Disturbances of the lacrimal functional unit (LFU) that comprises the ocular surface (cornea, conjunctiva, accessory lacrimal gland, and meibomian glands), the main lacrimal gland, and the interconnecting innervation and disruptions in tear film homeostasis are implicated in the pathophysiology of DED. DED is commonly classified as aqueous deficient (reduced tear production) and evaporative (increased tear film evaporation).[10,11] Various factors such as aging, autoimmune disease, hormonal variation, use of contact lenses, laser-assisted in situ keratomileusis (LASIK), blepharitis, medication, alcohol and smoking, environmental factors, and occupational hazards are associated with the development DED.[12]

Tear film continuously covers the ocular surface and is composed of three layers: the outermost lipid layer, the middle aqueous tears, and the innermost mucus layer, which are vital for ocular surface health.[3,13] The aqueous layer protects and lubricates the ocular surface and contains soluble mucin and microvilli. Mucins are high-molecular-weight glycoproteins that are secreted by the conjunctival goblet cells and lacrimal glands and are also expressed at the apical membrane of the squamous corneal and conjunctival epithelia.[13,14] They have been ascribed several roles: stabilizing the tear film, providing a smooth and refractive surface of high optical quality over the cornea, lubricating the corneal and conjunctival epithelial surfaces during blinking, and preventing desiccation of the ocular surface through water retention.[14-16] There are two types of ocular mucins that contribute to ocular surface homeostasis: 1) cell surface–associated mucins (MUC1/4/16), which are expressed on the ocular surface epithelium, and 2) gel-forming mucin (MUC5AC), which is secreted by the conjunctival goblet cells. Alterations in both cell surface–associated and gel-forming mucins occur in dry eye–related ocular diseases.[17-20]

Lacrimal acinar destruction or dysfunction causes reduced tear secretion and volume, resulting in dryness and tear hyperosmolarity, which stimulates a cascade of inflammatory processes, involving mitogen-activated protein kinases and NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. The hyperosmolar environment results in upregulation of human leukocyte antigen – antigen D related (HLA-DR), expression of proinflammatory cytokines, chemokines, and matrix metalloproteinases, epithelial cell death by apoptosis, and a loss of conjunctival goblet cells, leading to mucin–glycocalyx disruption.[21,22] Nuclear factor of the activated T cells-5 (NFAT5) is the predominant transcription factor activated in response to cellular hyperosmotic stress.[23] Lee et al.[24] demonstrated an increase in NFAT5 expression upon NaCl-based hyperosmotic stress in human corneal cells. An increase in NFAT5 expression has also been reported in patients suffering from DED. Increased NFAT5 level shows that the ocular surface cells undergo hyperosmotic stress due to an increase in tear film osmolarity in the case of DED. Thus, to mimic this condition ex vivo, we have used NaCl to induce hyperosmotic stress in the corneal tissues.

Human corneal epithelial cells (HCE) and human conjunctival epithelial cells are commonly chosen for in vitro studies of dry eye as the corneal epithelium is involved in the discomfort and pain associated with dry eye. Human-derived models such as ex vivo corneoscleral rims (CRs) can mimic the response of human eye tissues to DED treatments such as lubricating eye drops.

DED management involves the treatment of symptoms and varies according to disease severity. Treatment modalities include artificial tears, anti-inflammatory agents, topical corticosteroids, immunosuppressants (cyclosporine A), antibiotics (tetracyclines, macrolides), omega fatty acids, punctal plugs, and eyelid hygiene.[3,25] Artificial tears are the first-line treatment of DED and reduce ocular surface stress, improve contrast sensitivity and optical quality of the surface, and increase the quality of life. Artificial tears contain demulcents such as carboxymethyl cellulose (CMC) and propylene glycol, which are water-soluble polymers that protect and lubricate mucus membrane surfaces and relieve dryness and irritation, or emollients such as mineral oil and petrolatum that retard tear evaporation.[26]

Polyethylene glycol and propylene glycol (PEG–PG) combination lubricating eye drops been effectively used as a treatment option in case of DED patients. It has been reported that it alleviate the symptoms related to DED, protects goblet cell loss and reduces the squamous metaplasia.[27-29] PEG–PG combination has been reported to work better than CMC in case of DED patients by adding moisture to the eyes and keeping them lubricated.[30,31] But till now, no research has been carried out to find out how PEG–PG combination improves the mucin secretion in case of DED and whether it can be used as a secretagogue in DED condition.

The objective of this study was to analyze the effect of PEG–PG, an artificial tear formulation, on the molecular recognition and expression patterns of hyperosmolar stress-related factors and mucin secretion in ex vivo human CR explant culture. To the best of our knowledge, this study is the first of its kind to investigate the effect of lubricating eyedrops containing PEG–PG on hyperosmotic stress-induced ex vivo (CR) DED model.

Methods

CR explant culture (ex vivo model)

Human CRs from healthy donors (n = 8) were collected post-corneal keratoplasty in MK (McCarey-Kaufman medium) medium and washed using 1× sterile phosphate-buffered saline (PBS) containing 1% of antibiotic/antimycotic solution (Gibco, Waltham, MA, USA), followed by two times of wash with 1× PBS. Each CR was dissected into multiple pieces of equal size and immediately transferred into a 12-well plate with DMEM (Dulbecco’s Modified Eagle Medium)/F-12 media (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) in a humidified CO2 incubator at 37°C.

Hyperosmolar stress induction and drug treatment

Hyperosmolar stress was induced in CRs by adding 100 mM sodium chloride to DMEM/F12 media (Gibco) by increasing the osmolarity to +200 mOsm. Briefly, CRs were cultured in DMEM/F-12 media without serum supplementation for 12 h, followed by replacement of serum-free hyperosmotic stress media for another 48 h. 0.4% PEG and 0.3% PG (Cipla Ltd, Himachal Pradesh, India)[32] were added to the CRs 2 h before the addition of hyperosmotic media. Quantitative real-time polymerase chain reaction (PCR) was used to determine the levels of mucin (MUC5AC and MUC16) and hyperosmolar stress marker (NFAT5).

RNA extraction, cDNA synthesis, and quantitative PCR

The mRNA expression of NFAT5 (hyperosmotic stress marker), MUC5AC, MUC16, and b-actin was measured in CR samples (n = 3) by quantitative PCR (qPCR). RNA extraction was performed using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesize using iScript® cDNA synthesis kit (Bio-Rad, Philadelphia, PA, USA). Quantitative real-time PCR was performed using SYBR Green dye and CFX Connect real-time PCR detection system (Bio-Rad).

Enzyme-linked immunosorbent assay mucin assay

Mucin concentration in the CR culture supernatant was measured using enzyme-linked immunosorbent assay (ELISA). Explant culture supernatants with or without treatment (n = 5) were collected and used to estimate MUC5AC (E-EL-H2279; Elabscience, Houston, TX, USA) and MUC16 (E-EL-H0636, Elabscience) according to the manufacturers’ protocols.

Statistical analysis

Descriptive analysis was performed to summarize the data for continuous and quantitative variables. Continuous data are presented as frequency count (N), mean, standard deviation, minimum, maximum, and median values. Data were checked for normality using the one-sample Kolmogorov–Smirnov test before performing comparative analysis. The mean difference between different groups for NFAT5, MUC5AC, and MUC16 gene expression was assessed using an independent sample t-test or Mann–Whitney U test based on the normality of the variables. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 27.0. A P value of <0.05 was considered as statistically significant for all comparative analyses.

Results

The purpose of this study was to develop an ex vivo DED model by inducing hyperosmotic stress in donor CRs to study the mucin expression and secretion using qPCR and ELISA, respectively. Secondly, we aimed to check and validate the secretagogue effect of PEG–PG on the above experimental model.

PEG–PG treatment rescues hyperosmotic stress-induced reduction in MUC5AC and MUC16 gene expression in CRs ex vivo culture

We investigated the effect of PEG–PG treatment on mucin 5AC and mucin 16 in an ex vivo CR culture model under hyperosmolar stress. NFAT5 gene expression was measured using qPCR to assess the induction of hyperosmotic stress. In a similar way, we also assessed the MUC5AC and MUC16 gene expression in CR under hyperosmotic stress in the presence and absence of PEG–PG. An increase in NFAT5 gene expression was observed following treatment of CRs with +200 mOsm sodium chloride compared to control [Fig. 1a]. We observed a reduction in mucin 16 gene expression upon hyperosmotic stress (+200 mOsm), though it was not significant [Fig. 1b and c]. There was no significant change observed in the gene expression level of MUC5AC. A comparative analysis of results among the treatment groups indicated a significant increase in NFAT5 gene expression in the presence of PEG–PG alone or in combination with +200 mOsm hyperosmotic stress [Fig. 1a]. Higher expression of MUC5AC and MUC16 was observed in the CRs treated with 1% PEG–PG alone. Similarly, we found a significant increase of MUC5AC gene expression in CRs under +200 mOsm osmotic stress and PEG–PG, and in similar treatment conditions, increase in MUC16 gene expression was observed [Fig. 1b and c]. Though the increase in mucin gene expression (except +200 mOsm +2% PEG–PG) was not significant in other treatment groups, we noticed an increase in gene expression of MUC5AC and MUC16 in +200 mOsm stress with PEG–PG treatment groups compared to the +200 mOsm condition, showing the rescue effect of PEG–PG under hyperosmotic stress condition [Fig. 1b and c].

Figure 1.

Figure 1

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Effect of PEG–PG on NFAT5, MUC5AC, and MUC16 gene expression in the presence or absence of hyperosmotic stress in ex vivo CR culture. Bar graph indicates the mean mRNA expression levels of NFAT5 (a), MUC5AC (b), and MUC16 (c) normalized to the expression of b-actin (housekeeping gene) in CRs, following exposure to hyperosmotic stress (+200 mOsm) with or without PEG–PG for 48 h. The treatment categories include control (without hyperosmotic stress and PEG–PG), CR under hyperosmotic stress (+200 mOsm), and CR under hyperosmotic stress in the presence of PEG–PG (1% and 2%) (+200 mOsm + PEG–PG). The bar graph indicates mean ± SD from three individual CRs with two technical replicates. *P < 0.05, **P < 0.01, Mann–Whitney U test. CR = corneoscleral rim, PEG–PG = polyethylene glycol–propylene glycol, SD = standard deviation

PEG–PG treatment resists hyperosmotic stress-induced reduction in secreted MUC5AC and MUC16 protein expression in CRs ex vivo culture

The secreted mucin (MUC5AC and MUC16) levels in the culture supernatants of all treatment groups were measured using ELISA and the fold difference compared to the control was calculated. As seen in Fig. 2a and b, increase in hyperosmotic stress (+200 mOsm) reduced the secreted MUC5AC and MUC16 levels in the culture supernatant. An increase in mucin secretion was observed only in the supernatant collected from 1% PEG–PG-treated CRs. The supernatant collected from CRs under hyperosmotic stress treated with PEG–PG did not show a significant increase in mucin secretion when compared to that of CRs with +200 mOsm hyperosmotic stress. Collectively, these observations indicate the beneficial role of PEG–PG on resisting the reduction of mucin level due to hyperosmotic stress.

Figure 2.

Figure 2

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Status of secreted MUC5AC and MUC16 proteins in the CR culture following hyperosmotic stress in the presence of PEG–PG. Bar graph indicates average fold change for the secreted MUC5AC (a) and MUC16 (b) proteins normalized to their respective control. The treatment categories include control (without hyperosmotic stress and PEG–PG), CR under hyperosmotic stress (+200 mOsm), and CR under hyperosmotic stress in the presence of PEG–PG (1% and 2%) (+200 mOsm + PEG–PG). The bar graph indicates mean ± SD from supernatants collected from five individual CR explant cultures. CR = corneoscleral rim, PEG–PG = polyethylene glycol–propylene glycol, SD = standard deviation

Discussion

DED is one of the most common ocular diseases with a prevalence of up to one third in some countries, making it a major public health concern. With the rising prevalence of DED and its negative impact on patients’ daily and social activities, it is imperative that DED is managed appropriately. Tear deficiency, increased tear film osmolarity, ocular surface inflammation, and altered mucin secretion are implicated in the pathogenesis of DED, and therefore, treatment modalities are focused on improving or restoring any of these factors. In addition to patient education and avoidance of aggravating factors (cigarette smoke, dry heating air, air conditioning), artificial tears remain the first line of treatment for DED for providing immediate, symptomatic relief. Artificial tears function by improving the aqueous layer of the eye by increasing water retention.[33] Diquafosol, a P2Y2 (Purinergic receptor P2Y2) receptor agonist that promotes the secretion of mucin from conjunctival goblet cells as well as fluid secretion from conjunctival epithelial cells, was approved for use as eye drops in Japan in 2010. Rebamipide is a quinolinone approved for the treatment of dry eye as an ophthalmic suspension and increases the number of goblet cells in normal rabbit conjunctiva and lid wiper of human conjunctiva.[34-38] A study by Nakamura et al.[17] showed that JBP485, a dipeptide isolated from placental extract, promoted the expression and secretion of gel-forming mucin (Muc5ac) in rabbit conjunctival epithelium and also elevated the expression of cell surface–associated mucin (Muc1/4/16) in rabbit corneal epithelium.

Our work studied the effect of artificial tears containing PEG–PG on mucin secretion following induction of hyperosmolar stress. We observed a reduction in MUC16 gene expression upon hyperosmotic stress [Fig. 1c] and both MUC5AC and MUC16 levels were reduced with hyperosmotic stress [Fig. 2a and b]. PEG–PG was able to maintain hyperosmolar stress-induced decrease in both secretory mucin, MUC5AC, and cell surface–associated mucin, MUC16, though the increase was not significant, thereby helping to establish tear film stability. However, drug treatment alone caused a decrease in mucin production, but an increase was seen under drug treatment coupled with hyperosmolar conditions. We observed an increase in NFAT5 expression upon hyperosmotic stress induction in CR explant culture [Fig. 1a]. Similar results were observed previously in impression cytology samples collected from clinically diagnosed DED patients.[39] We also observed increased NFAT5 compared to the study control following treatment with PEG–PG, which could indicate prolonged inflammation or was caused by unknown factors related to the drug. The osmoprotective role of NFAT5 is also well studied and reported in literature.[40,41] Hence, we cannot ignore the osmoprotective side of NFAT5 transcription factor, though it has not been studied here. In case of PEG–PG 1% and 2%, the mean NFAT5 gene expression was 2.42 ± 1.35 and 3.58 ± 1.72, respectively, indicating reduced gene expression in the case of PEG–PG 1%.

PEG–PG 1% showed increased MUC5AC gene expression than PEG–PG 2%. Likewise, MUC16AC gene expression was higher in the PEG–PG 1% group compared to the PEG–PG 2% group. Future work should include in vivo determination of ocular surface mucin production and hyperosmolar marker level production following treatment with PEG–PG in a DED animal model or patients with DED.

There was a reduction in secreted mucin levels (both MUC5AC and MUC16) upon treatment with PEG–PG hyperosmotic stress, as estimated using ELISA, but to a varied degree. The effect of PEG–PG was also varied across the CRs tested. PEG–PG treatment was able to slightly alleviate the hyperosmotic stress induced decrease in MUC5AC and MUC16 gene expression in explant culture system. However, no significant difference in the reduction of MUC5AC and MUC16 secretion was observed upon hyperosmolar stress when comparing control versus PEG–PG.

Conclusion

Our findings show that artificial tears containing 0.4% PEG and 0.3% PG eye drops slightly alleviate the hyperosmolar stress-induced decrease in MUC5AC and MUC16 levels that is encountered in DED. Further, long-term treatment of PEG–PG using this model system may give a better understanding of its effect on NFAT5 and mucin expression.

Financial support and sponsorship

This work was supported by Cipla Ltd, India and Narayana Nethralaya Foundation, Bangalore, India. The funders had no role in the study design, data collection, analysis, or decision to publish.

Conflicts of interest

There are no conflicts of interest.

Acknowledgements

The authors acknowledge Cipla Ltd, India and Narayana Nethralaya Foundation, Bangalore, India for funding this research.

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