Inhibitory effects of α-crystallin on optic nerve astrocytes proliferation, activation and secretion stimulated by lipopolysaccharide

Background

Glial scaring induced by the activation and proliferation of astrocytes after optical nerve damage is one of causes of neural axons difficult to regeneration.Researches showed that α-crystallin can promote the regeneration and pass through scaring zone of retinal ganglion cells (RGCs) axons, and we speculate α-crystallin protect optical nerve tissue against scaring process.

Objective

This study was to investigate the influence of α-crystallin for the activation and secretion of inflammatory factors of astrocytes.

Methods

Optical nerver tissue was isolated from 3-5 day-old SPF Long Evans rats to culture and purify astrocytes.The cells were identified by detecting the expression of glial fibrillary acidic protein (GFAP) with immunofluorescence technique.The cells were cultured with regular culture medium in the normal control group, and 5 μg/ml lipopolysaccharides (LPS) was added in the LPS group, while 5 μg/ml LPS and 1×10^-4 g/L α-crystallin were added in the α-crystallin group, and the cells were consecutively cultured for 24 hours.The proliferation (absorbance, A) of the cells was assayed by cell counting kit-8 (CCK-8). The expression of GFAP in the cells was detected by immunofluorescence technique and quantitated by Western blot.The contents in the cell supernatants of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA.

Results

The morphology and size were well-proportioned in 3-4 generation of cells with the GFAP positive rate over 95%.The A values were 1.335±0.070, 1.643±0.069 and 1.390±0.004 in the normal control group, LPS group and α-crystallin group, and the A values in the LPS group were significantly higher than those in the normal control group and α-crystallin group (t=3.315, 3.681, both at P<0.05). Immunofluorescence examination showed that the fluorescence intensity was evidently enhanced in the LPS group compared with the normal control group and α-crystallin group and presented the largest cell bodies in the LPS group.The relative expressions of GFAP in the cells were 0.851±0.076 in the LPS group, which were higher than those in the normal control group and α-crystallin group (0.786±0.091, 0.569±0.049). Compared between the LPS group and α-crystallin group, there is a significant difference between the two groups (t=3.115, P<0.01). In addition, compared with the LPS group, the contents of TNF-α and IL-1β in the suspensions were significantly reduced in the normal control group and α-crystallin group (all at P<0.05).

Conclusions

α-Crystallin protein can inhibit the activation and secretion of optic nerve astrocytes stimulated by LPS.