Effects Of Sodium Salicylate On The Expression Of HSP 27 Protein In Tissue-cultured Human Lens Epithelial Cells

【Objective】To investigate the effects of Sodium Salicylate on the expression of heat shock protein 27(HSP 27) during oxidative stress in tissue-cultured human lens epithelial cells. And explore a new drug to inhibit the development of cataract.【Materials and Methods】Cultured human lens epithelial cells(HLB-3) were divided into three groups: control group, oxidation injury group and sodium salicylate group. A poptosis of human lens epithelial cells cultured in vitro was induced by 150μmol/L H2O2. Sodium salicylate at concentrations in sodium salicylate group from 200 to 1000 mg/L each 200 mg/L was added in cell suspension respectively.1. Methyl thiazol tetrazolium(MTT) chrom atom etry : Lens endothelial cells were cultured in 96-well plates for 2, 6 and 16 h. There were 6 parallel wells for each group. Afterwards, the absorbance value (A) at 490 nm was measured by using a microplate reader. The proliferation activity of cells was positively correlated with A value.2. Immunocytochemistry: The cover-slips crawled with cells were glued to the slide with transparent scotch tape. Cells with brown yellow substance on the surface were considered to be positively stained with Immunocytochemistry.3. RT-PCR: After incubation, the cells in each group were collected for RNA isolation. The PCR product were analyzed by 1.5 % agarose gel electrophoresis and scanned by GDS8000 gel-imagine analysis system. The absorbency (A) of each band was taken and the the ratio of A between HSP 27 mRNA andβ-action mRNA was calculated.【Results】1. MTT assay: According to the value of optical density (OD) data, the proliferation activity of lens epithelial cells in oxidation injury group was significantly decreased as compared with control group (P<0.01), and the proliferation activity in sodium salicylate group was significantly increased as compared with oxidation injury group (P<0.01), especially in 600 mg/L sodium salicylate group of 6h.2. Immunocytochemistry: Immunocytochemistry staining revealed that there were many brownish yellow granules in three groups, suggesting the positive staining. The HSP 27 expression was increased significantly in sodium salicylate group as compared with that in oxidation injury group (P<0.01). This difference was the most manifest in 600 mg/L sodium salicylate group of 6h .3. RT-PCR: The total RNA in each group was extracted with Trizol-reagent guanidine isothiocyanate centrifugation. Semi-quantitative RT-PCR was performed to detect the expression of the HSP 27 by usingβ-action as control. RT-PCR indicated that the HSP 27 expression in sodium salicylate group was stronger than that in oxidation injury group. This result coincided with the immunocytochemistry.【Conclusion】It is known that the decline of the proliferation activity in lens epithelial cells is a cellular basis for the early development of cataract .The proliferation activity of lens epithelial cells was significantly decreased when oxidation injury is inflicted, it follows that oxidation injury is one of the pathological mechanisms in the development of cataract. There is an increase of HSP 27 expression in lens epithelial cells when oxidation injury is appended, inferring that there is a close relationship between HSP 27 and cataract development. Our studies have shown that as the expression of HSP 27 was increased significantly in the presence of sodium salicylate, the proliferation activity of lens epithelial cells increased accordingly. The increased expression of HSP 27 by Sodium Salicylate could play an important role in the protection on hydrogen peroxide induced injury of human lens epithelial cells, suggesting that sodium salicylate could suppress, at least in part, the development of cataract.