Protective effects of pituitary adenylate cyclase-activating peptide (PACAP) on high glucose-induced damage in human corneal epithelial cell

Abstract

Background: Diabetic keratopathy (DK), a vision-threatening complication of diabetes mellitus, remains a significant clinical challenge. Pituitaries adenylate cyclase-activating peptide 38 (PACAP38) has been demonstrated to have neuroprotective effects. This study aimed to investigate whether PACAP38 mitigates high glucose (HG)-induced damage in human corneal epithelial cells (HCECs) and to elucidate the underlying mechanisms.

Methods: HCECs were exposed to HG to simulate diabetic injury. Cell viability, apoptosis, migration, and autophagy were evaluated using Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell assays, and Western blotting, respectively.

Results: Compared to the normal control (NC) group, HG significantly suppressed cell proliferation (p < 0.01), whereas PACAP38 treatment restored proliferative capacity (p < 0.01). PACAP38 enhanced cell migration, counteracting HG-induced impairment. At the molecular level, HG downregulated Ki-67 and Bcl-2 mRNA expression, while PACAP38 markedly upregulated these markers (p < 0.01). Notably, the HG impaired the autophagy in HCEC cells, while the PACAP38 significantly increased the autophagic ability. Mechanistically, PACAP38 increased the expression of p-AMPK, p-ERK, and Bcl-2 while reducing p62 (p < 0.01). Crucially, these protective effects were abolished by the autophagy inhibitor 3-MA or the AMPK inhibitor compound C (p < 0.05), confirming pathway dependency.

Conclusions: Our findings demonstrate that HG compromises HCEC proliferation and migration while promoting apoptosis. PACAP38 counteracts these detrimental effects by activating AMPK/ERK signaling, thereby enhancing cell survival and autophagy under hyperglycemic conditions. These results highlight PACAP38 as a promising therapeutic candidate for diabetic keratopathy.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12886-025-04309-z.

Keywords: Diabetic complications.; Diabetic keratopathy; High glucose; Human corneal epithelial cells; Peptides activated by pituitary adenylate cyclase.

Fig. 1

The rate of apoptosis in HCEC cells treated with different concentrations of HG Apoptosis was detected with flow cytometry. Quantification of the percentage of apoptosis is shown (A). Histograms of four groups were exhibited (B). The upper left was designed as necrosis (Annexin V-/PI+), the upper right was late-stage apoptosis (Annexin V+/PI+), the lower left was normal cells (Annexin V-/PI-), the lower right was early-stage apoptosis (Annexin V+/PI-). Total apoptosis = late apoptosis + early apoptosis. All experiments were repeated 3 times. ***p < 0.001, versus NC group, ^### p < 0.001, versus 50 mM group, ⁺⁺⁺ p < 0.001, versus 100 mM group

Fig. 2

Comparison of the rate of total, early and late apoptosis in HCEC cells treated with different concentrations of HG The total, early and late apoptosis were measured. The rates of apoptosis with different concentrations of HG treatment were compared. All experiments were repeated 3 times. ** p < 0.01, *** p < 0.001, versus NC group

Fig. 3

HCEC proliferation activity treated with different concentrations of PACAP38 Cell proliferation activity with different concentrations of PACAP38 was examined using the CCK8 assay. Detection absorbance at 450 nm was determined on day 1, 2, 3, 4, and 5. All experiments were repeated 3 times. ***p < 0.001, versus NC group

Fig. 4

PACAP38 improved HG decreased cell proliferation in HCEC cells To investigate the effect of PACAP38 on HG treated cell proliferation activity, HCEC proliferation activity was examined by CCK8 assay. Detection absorbance at 450 nm was determined on day 1, 2, 3, 4, and 5. The fold changes of cell proliferation activity were defined as OD450/Fold. All experiments were repeated 3 times. ^***p < 0.001 represents to compared with NC, ^###p < 0.001 represents compared to the HG group, ⁺⁺p < 0.01 represents compared to the HG + PACAP38 group

Fig. 5

Cell wound healing under different conditions Cell wound healing was assessed by the cell scratch test. Distance of digital images was collected and measured at initial and 12 h after scratch. scale bar = 200 μm. All experiments were repeated 3 times. ^*p < 0.05, ^***p < 0.001 versus the NC group, ^#p < 0.05, ^###p < 0.001 versus the HG group, ⁺p < 0.05⁺ versus the HG + PACAP38 group

Fig. 6

The mRNA and protein expression in HCEC with different treatment. A The protein expression of P62, Beclin1 and LC3B in HCEC cells with different treatment was detected by WB. B The protein expression of KI67 and Bcl2 in HCEC cells with different treatment was detected by WB. C The mRNA level of KI67 and Bcl2 in HCEC cells with different treatment was detected by qRT-PCR. The RNA expression was calculated and analyzed. GAPDH was a reference gene used for normalization. All experiments were repeated 3 times. ^*p < 0.05, ^**p < 0.01 versus the NC group, ^#p < 0.05, ^##p < 0.01 versus the HG group, ⁺⁺p < 0.01 versus the HG + PACAP38 group

Fig. 7

The protein expression in HCEC cells with different treatments. A-B The pathway-related proteins AMPK, p-AMPK, ERK, and p-ERK with different treatments were detected by Western blot. The protein expression level was measured. GAPDH was used as a reference for normalization. The protein expression level was measured as the gray level of protein bands and quantified with image J. All experiments were repeated 3 times. ^*p < 0.05, ^**p < 0.01 versu the NC group, ^#p < 0.05, ^##p < 0.01 versus the HG group, ⁺p < 0.05, ⁺⁺p < 0.01 versus the HG + PACAP38 group

Fig. 8

Inhibition of the AMPK pathway reversed PACAP38 function in HCEC cells. A The pathway-related proteins AMPK, p-AMPK, ERK, and p-ERK with different treatments were detected by Western blot. B HCEC proliferation activity was examined by CCK8 assay. Detection absorbance at 450 nm was determined on day 1, 2, 3, 4, and 5. C Cell wound healing was assessed by the cell scratch test. Distance of digital images was collected and measured at initial and 12 h after scratch. scale bar = 200 μm. D The protein expression of P62, Beclin1 and LC3B in HCEC cells with different treatment was detected by WB. E The protein expression of KI67 and Bcl2 in HCEC cells with different treatment was detected by WB. All experiments were repeated 3 times. ^*p < 0.05, ^***p < 0.001 versus the NC group, ^###p < 0.001 versus the HG group, ⁺⁺⁺p < 0.001 versus the HG + PACAP38 group