Effect Of Polysaccharide From Dendrobium Candidum On Cell Proliferation,Apoptosis And Expression Of Alsode Reductase,Laminin In Human Corneal Epithelial Cells Cultured With High Glucose

Objective:To observe the effect of polysaccharide from dendrobium candidum(PDC) on cell activity, apoptosis and the expression of aldose reductase,laminin in human corneal epithelial cells(HCEC)cultured under the high glucose and explore the probable protective mechanism of PDC against diabetic corneal epitheliopathy.Methods:1. To screen the optimal concentration and processing time of high glucose(HG): HCEC were used as an experimental model in vitro and cultured in DMEM wtih low glucose. Access to pertinent literature, delineate the scope of glucose concentration, experiment as follows: HCEC cells were treated with glucose solution(5.5mmol/l-50mmol/l) for 12 h, 24 h, 48 h or 72 h and cell viability was measured by MTT to determine the best concentration of glucose and time.2.To screen the best concentration and processing time of PDC: According to the results of optimal glucose concentration in experiment 1, cells were divided into HG and C, both of them were treated with different PDC concentrations(0?g/ml, 100?g/ml, 200?g/ml, 400?g/ml)for 12 h, 24 h, 48 h or 72 h, cell viability was measured by MTT to determine the best concentration of PDC and time.3. According to the high glucose concentration?PDC concentration and treatment times identified in method 1 and 2, cells were divided into 4 groups, namely control group(C),high glucose group(HG), PDC group and HG+PDC group. And investigated the migration, proliferation, apoptosis and the expressions of aldose reductase(AR)and LN of HCEC.(1)Cell migration was determined using cell scratch assay(2)Cell proliferation was determined by MTT(3)Cell apoptosis were dealt with Annexin V-FITC/PI double staining method and tested by flow cytometry to identify the best treatment time, and bax m RNA and bcl-2 m RNA were tested through RT-q PCR(4)Expressions of AR and LN were identified by ELISA.Results:1.For experiments, the proper high glucose concentration was 50mmol/l, the treatment time was 48 h. The best PDC concentration was 200?g/ml, and the treatment time was 72 h.2.Compared wtih the control group,in the high glucose group,the cellular proliferation activity?the cell migration and the expression of bcl-2 m RNA and LN decreased significantly((49)<0.05). The rate of cell apoptosis?the expression of bax m RNA and AR increased significantly((49)<0.05).3.Compared with the control group,in the PDC group(200?g/ml),the cellular proliferation activity?the cell migration and the expression of bcl-2 m RNA and LN increased significantly((49)<0.05). The expression of bax m RNA and AR decreased significantly((49)<0.05).While it had no significant influence on the rate of cell apoptosis((49)>0.05).4. Compared with the high glucose group,in the high glucose plus PDC(200?g/ml)group,the cellular proliferation activity?the expression of bcl-2 m RNA and LN increased significantly((49)<0.05).The rate of cell apoptosis and the expression of bax m RNA and AR decreased significantly((49)<0.05).While both of the proliferation activity and rate of cell apoptosis had no significant difference with control group((49)>0.05).Conclusion:1. High glucose could inhibit the migration, proliferation and expression of LN of HCEC, induce cell apoptosis, enhance expression of AR.2.PDC could significantly elevate the migration, proliferation and expression of LN of HCEC under the high glucose, decrease cell apoptosis, suppress expression of AR.3. The polysaccharide from dendrobium candidum(PDC)could protect the HCEC, and the mechanism would be related to the signaling pathway of AR and protein LN.