Effect Of AFDX-116on Human Fetal Scleral Fibroblasts In Vitro

As the most common eye disease in the wordwide, there are about one thirdadult in America have varying degrees of myopia, in recent years, the morbidity rateof myopia continued to rise in Asia, even gradually surpasses the rate of European. Asa tissue that connected other organization, scleral was formed with fibroblasts andcollagen fiber sheath, it plays a very important role in the growth of the eyeballdevelopment and the scleral remodeling in the myopia progression. The scleralremodeling was accomplished by the scleral fibroblasts, the scleral fibroblasts play avery important role in the sclera structure and biological characteristics, the scleralfibroblast can reduces the collagen fibers and the sclera get thinner in the near-sightedperson. Several studies shows that, in recent years, in the progression of myopia, thechange of the collagen level of the scleral fibroblast have an important effect in thescleral remodeling. Therefore, study the change of the scleral fibroblasts proliferationand the collagen level in the myopia and find the medicine to control it plays a veryimportant role in the treatment of myopia.In the people who has myopia, atropine, as the nonspecific M receptor antagonist,and pirenzepine, as the specificity antagonist of the M1receptor, were effectively usedto slow the progression of myopia. Five M receptor subtypes(M1-M5) are expressed inboth mouse and human sclera tissues. In the years not long before, some studiesshowed that, except atropine and pirenzepine, the specificity antagonist of the M4receptor can affect the scleral fibroblasts and scleral collagen and delay the development of myopia, the M2receptor also plays an important role in theprogression of the myopia. This experiment testing the expression of the M2receptorin the human fetal scleral fibroblasts to research the effect of the AFDX-116, the M2receptor antagonist, on the expression of the M2receptor, on the scleral fibroblastsproliferation and on the compound of the type I collagen in the scleral fibroblasts andto explore the role of M2receptor in myopia development and the mechanism in stopthe development of myopia by AFDX-116.ObjectiveTo observe the expression of the M2receptor in the human fetal scleralfibroblasts, study the the effect of the AFDX-116on the expression of the M2receptor,on the scleral fibroblasts proliferation and on the compound of the type I collagen inthe scleral fibroblasts and to explore the role of M2receptor in myopia developmentand the mechanism in stop the development of myopia by AFDX-116.MethodsTake the five to seven generation human fetal scleral fibroblasts and divided intotwo groups, the control group: don’t accept the drug stimulation, the experimentalgroup: accept the AFDX-116stumulus, the concentrations were0.1,1,10,100μmol/L.After the completed the drug stimulation, the relative expression levels of M2mRNAwas analyzed by RT-PCR, the change of proliferation of the human fetal scleralfibroblasts of the control group and experimental group were analyzed by CCK8, theexpression of COL1A1of human fetal scleral fibroblasts of the control group andexperimental group were affirmed by immunofluorescence.Results1．The expression and change of mRNA of M2receptor in HFSFsThe transcript level of mRNA of muscarinic M2receptor of the control group is0.372±0.022in the human fetal scleral fibroblasts, the cell was trained in the nutrient solution of different concentration included AFDX-116for5days, theexpression of the mRNA of muscarinic M2receptor in the0.1μmol/L AFDX-116treated group was0.391±0.018, in the1μmol/L AFDX-116treated group was0.418±0.007, in the10μmol/L AFDX-116treated group was0.579±0.035and in the100μmol/L AFDX-116treated group was0.768±0.042, we found that there was nosignificant diffence between the control group and the0.1μmol/L AFDX-116treated group(P=0.327), the results between the control group and the1μmol/L,10μmol/L,100μmol/L AFDX-116treated group were apparently difference, theexpression of the mRNA of muscarinic M2receptor in the AFDX-116treated groupwas obviously increased(P=0.029,0.001,0.000).2The change of cell proliferationThe OD value of the human fetal scleral fibroblasts in the control group is0.820±0.048, the OD value of the cells in the0.1μmol/L AFDX-116treated group is0.802±0.078, in the1μmol/L AFDX-116treated group is0.725±0.078, in the10μmol/L AFDX-116treated group is0.690±0.053and in the100μmol/L AFDX-116treated group is0.612±0.042. There was no significantly difference between thecontrol group and the experimental group which has the concenetration of0.1μmol/L(P=0.745), and there were significantly difference between the control group and theexperimental group which have the concentration of1μmol/L,10μmol/L and100μmol/L, the proliferation of the human fetal scleral fibroblasts was obviouslyreduced in the AFDX-116treated group(P=0.049,0.004,0.000).3The change of expression of COL1A1The transcript level of type I collagen in human fetal scleral fibroblasts wasapparently increased in the AFDX-116treated group. The expression of the type Icollagen can be detected in the control group and it were obviously increased in the1μmol/L AFDX-116treated group,10μmol/L AFDX-116treated group and100μmol/L AFDX-116treated group. Conclusion1. The acetylcholine M2receptor expressed stably in the human fetal scleralfibroblasts in vitro.2. The AFDX-116can affect directly on human fetal scleral fibroblasts, it canpromote the relative expression of the M2receptor mRNA, inhibit the proliferation ofthe human fetal scleral fibroblasts and promote the composition of the COL1A1.3. The M2receptor may participate in the development process of the occurrenceof myopia.