The Effect And Mechanism Of α-MSH Combined With CMC On The Human Corneal Cells Stressed In Hyper-Osmolarity

Objective Previously,the protective effect of α-melanocyte stimulating hormone(α-MSH)combined with sodium carboxymethylcellulose(CMC)on scopolamine-induced dry eye model in rats was demonstrated in vivo.Based on this,we explored whether α-MSH combined with CMC could protect human corneal epithelial cells under hyperosmotic condition in vitro and its specific mechanism.Methods 1.Different concentrations of sodium chloride were added to the culture medium,and osmotic pressure was measured by freezing point osmometer.The relationship between the concentration of sodium chloride and osmotic pressure was established.Human corneal epithelial cells were cultured in the osmotic pressure model,and the optimal osmotic pressure was screened by CCK-8.2.Screening the optimal concentration of α-MSH and CMC.The cells were divided into five groups: normal osmotic pressure group,hyper-osmolarity group,hyperosmolarity + CMC group,hyperosmolarity + α-MSH group,hyperosmolarity + CMC + α-MSH group.3.After hyperosmolarity and drug treatment,the changes of cell biological behavior,including cell vitality,cell migration ability,cell epithelial transmembrane resistance(TEER),were detected.4.To study the mechanism of human corneal epithelial cells after hyperosmolarity and drug treatment.Apoptotic level by Annexin V kit,ROS level by fluorescence detection,NLRP3-related gene transcription level,NLRP3-related protein expression level by western-blot and Caspase1 activity were detected.5.Using NLRP3 agonist LPS and inhibitor MCC950,the gene and protein expression of NLRP3 of human corneal cells stressed in hyperosmotic environment were detected.Results 1.The cellular hyperosmotic model was successfully established.With the increase of osmotic pressure,the activity of corneal epithelial cells decreased.After 24 hours of stimulation in 400,450 and 500 m Os M hyperosmotic environment,the morphology of corneal epithelial cells remained unchanged.The corneal epithelial cells were irregular in shape and increased in dead cells after 24 hours of stimulation in 550 m Os M hyperosmotic environment.After 24 hours of stimulation at 400,450,500 and 550 m Os M,the cell vitality decreased to(84.99 5.78)%,(78.18 4.79)%,(64.83 4.32)% and(40.52 2.09)% in 295 m Os M culture,respectively.The difference was statistically significant.Therefore,500 m Os M hyperosmotic environment was chosen as the stimulation concentration for the follow-up study.2.After 4 hours of stimulation with 500 m Os M,the cell migration ability decreased,the number of apoptotic cells increased from 28.5% to 41.67%.The ROS level increased significantly compared with 312 m Os M group,(5.14 0.33)fold compared with(4.09 0.11).The m RNA expression level of NLRP3,IL-1 increased to(1.90 0.07)fold,and(1.35 0.13)fold,respectively after 24 hours of stimulation.The Caspase1 activity was(1.4 0.0)fold compared with 312 m Osm group.3.When pre-treated with 0.01,0.1,1 μM of α-MSH and 0.2,0.5,1,2.5 mg/m L of CMC 30 minutes earlier,the cell vitality increased after stimulation by 500 m Os M for 24 hours,but there was no significant difference among the three groups of α-MSH.CMC increased the cell viability most significantly at the concentration of 0.2 and 0.5 mg/m L,but there was no difference between the groups.Therefore,0.01 μM α-MSH and 0.2 mg/m L CMC were selected as the final treat concentration.4.When using 0.01μM α-MSH and 0.2 mg/m L CMC alone under 500 m Os M stimulation,the migration ability of corneal epithelial cells,transepithelial cell resistanc were increased compared with the hyperosmolarity group;apoptotic cell number,ROS level,gene transcription level of NLRP3 and IL-1,protein expression level of NLRP3 and IL-1,Caspase1 activity were decreased compared with in hyperosmolarity group.And combined both,the effect was more significant,compared with CMC group,there was significant difference(P < 0.05).5.After agonism and antagonism with LPS and MCC950,the transcription and translation levels of NLRP3 were detected.Compared with hyperosmotic group,LPS + hyperosmolarity group had higher m RNA level and protein expression level of NLRP3,higher activity of Caspase1.Moreover,when given 0.01μM α-MSH and 0.2 mg/m L CMC,the m RNA and protein expression level of NLRP3 and the activity of Caspase1 decreased.The inhibitory effect of MCC950 on the transcription level of NLRP3 was similar to the combination of CMC and α-MSH.Conclusions In hyperosmolar environment,the combination of CMC and α-MSH can increase cell viability,promote cell repair and increase TEER.These changes in biological behavior may be related to the reduction of ROS level,the decrease of transcription and protein expression of NLRP3,IL-1,the decrease of Caspase1 activity and apoptotic cells after the combination of CMC and α-MSH.