Analysis of the effect of recombinant collagen eye drops on repairing corneal injury in rabbits

Objective  To explore the therapeutic effect of recombinant collagen eye drops (RCED) on corneal injury repair in a rabbit corneal alkali burn model.

Methods  L929 cells were divided into a control group, a positive control group, a negative control group, and an RCED group, and cultured with normal cell culture medium, 10% dimethyl sulfoxide solution, high-density polyethylene membrane extract and RCED for 24 hours, respectively. The cytotoxicity on L929 cells in each group was detected by thiazolyl blue assay. Human corneal epithelial cells-transformed (HCE-T) were divided into a control group and an RCED group and were cultured in normal cell culture medium and RCED for 24 hours, respectively. The cell counting kit-8 (CCK-8) assay and cell scratch assay were used to detect the effects of RCED on the proliferation and migration abilities of HCE-T cells. Twelve New Zealand rabbits were randomly divided into a normal control group, a model control group, a positive control group and an RCED group by the random number table method, with 3 rabbits in each group. The normal control group received no treatment. Corneal alkali burn models were established in the model control group, positive control group and RCED group. On the day of successful modeling, the rabbit eyes in the model control group, positive control group and RCED group were treated with normal saline solution, chondroitin sulfate eye drops and RCED for 14 continuous days, respectively. After 14 days of treatment, corneal turbidity score and sodium fluorescein staining were performed in all groups, and corneal healing was recorded by photographing under a slit lamp, and the corneal epithelial healing rate was calculated. Hematoxylin-eosin staining was performed to observe the pathological staining of all groups and the expression differences of α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were detected by immunohistochemistry. This experiment was conducted at the Center for New Drug Safety Evaluation and Research, Hebei Medical University, and the experimental protocol was reviewed and approved by the Laboratory Animal Ethics Committee of the Center for New Drug Safety Evaluation and Research, Hebei Medical University (No. IACUC-Hebyd AP-2023018).

Results  The results of in vitro cell experiments confirmed that there was no significant difference in cell absorbance values between the blank control group and the negative control group ( P>0.05); the cell proliferation rate and cell migration rate in the RCED group were (135.17±11.75)% and (35.18±7.83)%, significantly higher than the blank control group (100.00±0.00)% and (16.03±3.96)%, showing statistically significant differences ( t=7.33, 3.78; both P<0.05). In vivo animal experiments confirmed that after 14 days of treatment, the healing rates of the positive control group and the RCED group were both higher than those of the model control group, with statistically significant differences (both P<0.05). The corneal transparency score of the model control group was higher than that of the normal control group, and the corneal transparency score of the positive control group and the RCED group were both lower than those of the model control group, with statistically significant differences (all P<0.05). The hematoxylin-eosin staining results confirmed that RCED treatment could increase the integrity of corneal epithelial cells in rabbits, make the arrangement of matrix collagen fibers more regular, and reduce corneal congestion and inflammatory cell infiltration. The immunohistochemical staining results confirmed that the expressions of α-SMA, TNF-α and IL-6 in the model control group were significantly upregulated compared with the normal control group, and the expressions of α-SMA, TNF-α and IL-6 in the positive control group and the RCED group were significantly decreased compared with the model group. Among them, the expressions of α-SMA, TNF-α and IL-6 in the RCED group decreased more significantly, and the differences were statistically significant (all P<0.05).

Conclusions  RCED has no potential cytotoxicity to L929 cells, and can significantly promote the proliferation and migration of HCE-T cells. In the treatment of corneal alkali burn in rabbits, it can significantly promote corneal healing, improve corneal transparency, and reduce the level of fibrosis and inflammation in the tissue.