The Effect Of Astaxanthin On Inflammation In Hyperosmolarity Of Experimental Dry Eye Model In Vitro And In Vivo

Purpose: To investigate the anti-inflammatory effect of Astaxanthin(AST)in the hyperosmotic induced dry eye model in vitro and in vivo.Methods: 1.Human corneal epithelial cells(HCECs)were cultured exposed to different hyperosmotic saline solution(450,500 and 550mOsm/L).The mRNA and protein expression of HMGB1 and inflammatory factors TNF-?,IL-1? were detected by qRT-PCR and Western blot.2.After pretreated with 1,2.5 and 5 ?M Astaxanthin for 2 h then placed into hyperosmotic media,HCECs were collected to detect the gene and protein levels of HMGB1,TNF-? and IL-1? by qRT-PCR and Western blot.The levels of HMGB1 were also detected by immunofluorescence staining.3.BALB/c mice were randomly divided into four groups: normal control group,dry eye(DE)group,DE+AST,DE+dimethyl sulfoxide(DMSO).Corneal flouorescein staining and the mRNA and protein expression of HMGB1,TNF-? and IL-1? were detected the ocular surface damage of each group.4.HCECs were pretreated with LY294002,a PI3 K inhibitor,combined with astaxanthin,and then stimulated with hyperosmotic media.The mRNA and protein expression of HMGB1,Akt and phospho-Akt(p-Akt)were detected by qRT-PCR and Western blot.Results: 1.Concentration gradient of osmotic pressure: compared with 310 mOsm/L group,the mRNA expression of HMGB1 and inflammatory factors TNF-?,IL-1? in 500 and 550 mOsm/L group increased significantly,among which the expression of HMGB1 mRNA in 500 mOsm/L group obviously increased.Meanwhile,compared with 310 mOsm/L group,the protein expression of HMGB1,TNF-? and IL-1? was significantly increased in 450,500 and 550 mOsm/L group,especially in the 500 mOsm/L group.Therefore,we chose 500 mOsm/L as the better experimental stimulus osmotic pressure in our experiment.2.The addition of AST significantly reduced the expression of HMGB1,as well as significantly inhibited the increases of TNF-?,IL-1? in a dose-dependent manner.3.Compared with the dry eye mice group,the corneal fluorescein sodium staining score in AST treated group decreased significantly,and the mRNA and protein expression of HMGB1,TNF-?,IL-1? decreased significantly.4.AST preconditioning could increase the production of p-Akt compared to that in the DE group,but not affected the Akt expression.Pretreatment with LY294002 abolished the effects of AST preconditioning on the decrease of HMGB1 levels compared to that in AST+DE group.Conclusions: Hyperosmolarity induced the production of HMGB1 and inflammatory cytokine TNF-? and IL-1? in dry eye model.Astaxanthin significantly decreased the expression of HMGB1,TNF-? and IL-1? in the hyperosmotic induced dry eye model in vitro and in vivo.In addition,astaxanthin can activate the PI3K/Akt pathway to inhibit the expression of HMGB1,so as to reduce the inflammation of the ocular surface and protect the hyperosmotic dry eye model.