| Objective:Through the observation of the inhibitory effect of pirfenidone on the proliferation of cultured rabbit Tenons fibroblasts(RTFS)in vitro,and to explore its possible mechanism,in order to provide the molecular biological basis for the future application of pirfenidone in the experimental study of anti scarring of filtering areas after glaucoma surgery.Methods:Culture and identification of RTFs in vitro: Taking the tissue of Tenons capsule from the rabbit eyes,using the simplified cell culture method to culture the rabbit Tenons fibroblasts in vitro and identifying the cells with microscope and immunohistochemistry methods.CCK-8 methods were used to detect the effect of different concentrations of pirfenidone(0.01?0.1?0.15?0.2?0.25?0.3?0.5?1mg/ml)compared with control groups(untreated group containing only cells and culture medium)on RTFS.The results showed that there was no significant difference between the experimental groups(0.01?0.1?0.15?0.2?0.25?0.3?0.5mg/ml)and the control groups(P>0.05),while the experimental group(1mg/ml)and the control group(P<0.05),which initially confirmed that1mg/ml might be the initial concentration of pirfenidone in inhibiting the proliferation of RTFS;Then CCK-8 methods were used to detect the effect of different concentrations of pirfenidone(0.5?1?1.5?2?2.5mg/ml groups;1?1.2?1.5?1.6?1.8?2mg ml groups)compared with control groups(untreated group containing only cells and culture medium)after 24 hours,and to further determine the initial action concentration and the maximum non-toxic concentration of pirfenidone;The results showed that there was no significant difference between the experimental group(0.5mg/ml)and the control group(P<0.05).The cell survival rate of 2mg/ml and 2.5mg/ml groups were 0.32051 and 0.07692 respectively.The cell survival rate was lower than 50%,which may have toxic effect on the cells.Therefore,1.5mg/ml may be the maximum non-toxic concentration of pirfenidone.In order to further determine the maximum non-toxic concentration of pirfenidone on RTFS,CCK-8 methods were used to detect the effect of different concentrations of pirfenidone(1?1.2?1.5?1.6?1.8?2mg/ml)on RTFS after 24 hours.The results showed that the experimental groups had statistical significance compared with the control groups(P<0.05),while the cell survival rates of 1.8mg/ml and 2mg/ml groups were 0.46429 and 0.45238 respectively The cell survival rate was less than 50%.Therefore,the initial concentration and the maximum non-toxic concentration of pirfenidone were 1mg/ml and 1.6mg/ml respectively.Immunofluorescence methods were used to detect the expression of TGF-?1,TGF-?2 and Smad3 in RTFS;Western blot analysis were used to detect the effects of pirfenidone on TGF-?1,TGF-?2 and Smad3's proteins expression;RT-PCR were used to detected the gene expression changes of TGF-?1,TGF-?2 and Smad3.Results: Successfully Cultured the RTFs in vitro,most of the cells were long fusiform,with large nucleus and rich cytoplasm.The cells grew fast and most of the cells grew in a whirlpool or radial shape when space was sufficient;RTFs was positive in Vimentin staining and negative in Keratin staining,which could be used to estimate that the cultured cells were fibroblasts rather than conjunctival cells or epithelial cells;CCK-8 methods were used to detect the effect of different concentrations of pirfenidone(0.01?0.1?0.15?0.2?0.25?0.3?0.5?1mg/ml)on RTFS.The results showed that there was no significant difference between the experimental groups(0.01?0.1?0.15? 0.2?0.25?0.3?0.5mg/ml)and the control group(P > 0.05),but there was significant difference between the experimental group(1mg/ml)and the control groups(P<0.05).In order to determine the initial action concentration of pirfenidone and screen the maximum non-toxic concentration of the drug,CCK-8 methods were used to detect different concentrations of pirfenidone(0.5?1?1.5?2?2.5mg/ml)after 24 hours of RTFS.The results showed that there was no significant difference between the experimental group(0.5mg/ml)and the control group(P<0.05).There was significant difference between the experimental groups(1?1.5?2?2.5mg / ml)and the control group(P <0.05).The cell survival rate of 2 mg/ml and 2.5mg/ml groups were 0.32051 and 0.07692 respectively.The cell survival rate was lower than 50%,which may have toxic effect on cells.Therefore,1.5mg/ml may be the maximum non-toxic concentration of pirfenidone.In order to further determine the maximum non-toxic concentration of pirfenidone,CCK-8 method was used to detect different concentrations of pirfenidone(1?1.2?1.5?1.6?1.8?2mg/ml).The results showed that there was significant difference between the experimental groups and the control groups(P<0.05),while the cell survival rate of 1.8mg/ml and 2mg/ml groups were 0.46429 and 0.45238 respectively,and the cell survival rate was lower than 50%.Therefore,the initial action concentration and the maximum non-toxic concentration of pirfenidone were 1mg/ml and 1.6mg/ml.The results of immunofluorescence showed that the expression of TGF-?1,TGF-?2 and Smad3 in RTFS of the experimental groups decreased compared with that of the control groups(P<0.05),and the inhibition of pirfenidone increased with the increase of drug concentration;Western blot and RT-PCR showed that the inhibition of pirfenidone(1?1.6mg/ml),compared with the control groups,the protein expression and the relative gene expression of TGF-?1,TGF-?2 and Smad3 in the experimental groups decreased(P<0.05),and the inhibition increased with the increase of drug concentrations.Conclusions(1)Pirfenidone can inhibit the proliferation of RTFs in vitro.1mg/ml and1.6mg/ml are the initial and the maximum non-toxic concentrations of pirfenidone;(2)The possible mechanism of pirfenidone to inhibit the proliferation of RTFS is that it can inhibit the expression of TGF-?1,TGF-?2 and Smads which are important factors in TGF-?/Smad pathway and then play a role in inhibiting the proliferation of RTFS. |
PDF Full Text Request