[Stiffness of scleral fibroblasts and extracellular matrix remodeling in models of cellular senescence].

Abstract

Objective: To explore the effects of aging on the stiffness of human scleral fibroblast (HSF) and the remodeling of the extracellular matrix. Methods: This experimental study was conducted from January 2022 to June 2024. HSFs were cultured, and after cell passage, β-galactosidase staining was conducted. The expression levels of aging markers P16 and P21 were detected through Western blotting. To establish a replicative aging model of HSF, cells were divided into two groups: the 4th generation cells as the young control group and the 16th generation cells as the aging experimental group. Atomic force microscopy was used to measure the stiffness of the cell bodies and the extracellular matrix produced by both groups, expressed as Young's modulus, a measure of stiffness. The biological characteristics, including cell contractility, and the expression of extracellular matrix-related proteins in both groups were assessed through collagen gel contraction experiments and Western blotting. The expression levels of protein cross-linking indicators-lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1), and transglutaminase (TGM2)-were measured at both mRNA and protein levels using quantitative real-time PCR and Western blotting. Statistical analysis was performed using independent samples t-test and Mann-Whitney U test. Results: HSF successfully established a replicative senescence model after passage, and when the cells were passed to the 16th generation, the positive rate of β-galactosidase staining significantly increased, the cell doubling time was prolonged, and the expression of p16 protein in the senescence experimental group (1.40±0.05) was higher than that in the young control group (0.87±0.11); the expression of p21 protein in the senescence experimental group (1.13±0.12) was higher than that in the young control group (0.79±0.04), with statistically significant differences (all P<0.05). The Young's modulus of the cell bodies in the senescent group [2.259 (1.971, 2.745) kPa] was higher than that in the young control group [1.268 (1.068, 1.489) kPa]. Likewise, the Young's modulus of the extracellular matrix in the senescent group [13.598 (9.073, 17.352) kPa] was higher than that in the young control group [10.050 (6.633, 14.999) kPa], with statistically significant differences (both P<0.001). The collagen gel contraction ability of the senescent group was lower than that of the young control group after 12, 24, 36, and 48 hours of culture, with values of 0.803±0.015, 0.773±0.021, 0.713±0.015, and 0.697±0.015 compared to 0.880±0.010, 0.833±0.015, 0.820±0.010, and 0.803±0.006, respectively. These differences were statistically significant (all P<0.05). After 48 hours of culture, the migration distance of cells in the senescence experimental group [(73.33±4.16) μm] was lower than that in the young control group [(187.33±3.06) μm], with a statistically significant difference (P<0.001). The expression levels of type Ⅰ collagen, anti-adhesion protein, and anti-fibronectin in the senescence experimental group were lower than those in the young control group [(0.153±0.009) vs. (0.250±0.012), (0.233±0.010) vs. (0.315±0.016)and (0.510±0.020) vs. (0.616±0.050)], with statistically significant differences (all P<0.05). The mRNA expression of LOX, LOXL1, and TGM2 in the senescence experimental group was higher than that in the young control group [(2.250±0.060) vs. (1.033±0.059), (2.565±0.153) vs. (1.002±0.019)and (2.985±0.138) vs.(1.023±0.050)], with statistically significant differences (all P<0.001). The protein expression of LOX, LOXL1, and TGM2 in the senescence experimental group was also higher than that in the young control group [(0.682±0.022) vs. (0.279±0.025), (0.602±0.012) vs. (0.230±0.027)and (0.723±0.010) vs. (0.331±0.036)], with statistically significant differences (all P<0.001). Conclusions: In the HSF senescence model, the stiffness of senescent HSF and their associated extracellular matrix increased, while the expression levels of extracellular matrix component proteins decreased, but the expression levels of cross-linking proteins increased.