| Objective: 1.To investigate the anti-fibrosis effect of pirfenidone on TGF-β/Smad pathway,After being treated with pirfenidone at different concentrations,Protein and gene expression of important factors in TGF-β /Smad signaling pathway and cell proliferation were detected.2.Molecular biological experimental methods were used to explore the possible mechanisms by which pirfenidone should inhibit scar formation in the TGF-β/Smad pathway.3.In order to find a new anti-fibrosis drug that is effective and has little toxic and side effects for treating ocular fibroproliferative diseases,It provides the theoretical basis of molecular biology for clinical application.Methods: 1.Rabbit eye Tenons fibroblasts(RYTF)were cultured in vitro,The Tenons capsule tissue from the rabbit eye was obtained from a fresh donor,The tissue was cultured by tissue block culture method,Active and active primary cells were successfully cultured in vitro.The morphology of the cells was observed by microscope and the cells were identified by immunohistochemical method.2.CCK-8 detection method was used to detect different concentrations of pirfenidone(0.01,0.1,0.2,0.3,0.5,1.0mg/ml)acting on RYTF,OD value was measured after 24 h.Comparing the experimental group with the control group(the untreated group),The initial concentration and optimum concentration of pirfenidone were determined.3.In the experimental group compared with control subjects RYTF TGF-β3,collagenⅠ,collagenⅢ fluorescence staining expression of collagen.4.Western blot was used to detect pirfenidone at different concentrations(0.1,0.26610mg/ml)for 24 h after treatment.The experimental group than the control group of TGF-β3,collagenⅠ,collagenⅢ protein expression.5.The possible mechanism of inhibiting RYTF proliferation by pirfenidone was explored by RT-PCR detection.Different concentrations of pirfenidone(0.1,0.26610mg/ml)were detected after 24 h of treatment.The experimental group than the control group of TGF-β3,collagenⅠ,collagenⅢ m RNA expression changes.6.Results SPSS22.0statistical software was used for statistical analysis,P<0.05 was considered statistically significant.Results:1.Normal RYTF was cultured in vitro,most cells are long and fusiform,the nuclei are large and full,the cytoplasm is filled,cells have a strong ability to grow.They were feathered in petri dishes with plenty of room to grow.Vimentin staining for RYTF was positive,negative results of keratin staining for RYTF.It can be proved that the cultured cells are fibroblasts,not epithelial cells or conjunctival cells.2.CCK-8 method was used to detect different concentrations of pirfenidone(0.01,0.1,0.2,0.3,0.5,1.0mg/ml)acting on RYTF,changes of absorbance value(OD value)after 24 h.The results showed that there was no statistical difference between the experimental group(0.01mg/ml)and the control group(P > 0.05).It was preliminarily confirmed that 0.1mg/ml might be the initial concentration of pirfenidone inhibiting the proliferation of RYTF.In order to further determine the initial action concentration of pirfenidone and screen the maximum non-toxic concentration of the drug,different concentrations of pirfenidone(0.1,0.2,0.3,0.5,1.0mg/ml)were detected by CCK-8method on RYTF.OD value was measured after 24 h.The inhibition rates of each concentration group were respectively 0.05436,0.18544,0.30426,0.36838 and0.51527,and the IC50 was 0.26610 mg/m L.The results showed that the experimental group(0.1,0.2,0.3,0.5,1.0mg/ml)had statistical differences compared with the control group(P < 0.05).In the 1.0mg/ml group,the cell survival rate was less than50%,It has a toxic effect on cells,The cell survival rate of other groups was above50%.IC50 value is 0.26610 mg/ml,Therefore,0.26610mg/ml may be used as the maximum non-toxic concentration of pirfenidone.In this experiment,the initial action concentration and maximum non-toxic concentration of pirfenidone to inhibit RYTF proliferation were determined to be 0.1mg/ m L and 0.26610mg/ml,respectively.3.Immunofluorescence test results showed that treatment with different concentrations of pirfenidone(0.1,0.26610mg/ml)for 24 h,the experimental group was compared with the control group in the experimental group RYTF TGF-β3,collagenⅠand collagen Ⅲ fluorescence expression of collagen were reduced(P < 0.05).With the increase of pirfenidone concentration,the inhibitory effect of RYTF was enhanced.4.Western blot results,compared with the control group,the experimental group was treated with different concentrations of pirfenidone(0.1,0.26610mg/ml)for 24 h.In the experimental group RYTF TGF-β3,CollagenⅠand Collagen Ⅲ amount of collagen protein expression were reduced(P < 0.05).With the increase of pirfenidone concentration,its inhibitory effect on RYTF was enhanced.5.RT-PCR results showed that compared with the control group,the experimental group was treated with different concentrations of pirfenidone(0.1,0.26610mg/ml)for 24 h.In the experimental group RYTF TGF-β3,collagenⅠand collagen Ⅲ relative expression of collagen gene are reduced(P < 0.05).With the increase of pirfenidone concentration,its inhibitory ability to RYTF was enhanced.Conclusions:(1)Pirfenidone had a significant inhibitory effect on the proliferation of RYTF cultured in vitro.The inhibition was concentration dependent.(2)Within the action concentration range of pirfenidone(0.01-0.5mg/ml),Inhibition of RYTF proliferation in vitro was not associated with drug cytotoxicity.The initial action concentration and maximum non-toxic concentration of pirfenidone to inhibit RYTF proliferation were 0.1mg/ ml and 0.26610mg/ml,respectively.(3)The possible mechanism by which pirfenidone inhibits RYTF proliferation is affecting the gene and protein expression of effector factors such as TGF-β3 in RYTF,thus inhibiting the TGF-β/Smad pathway signaling pathway.Impact collagenⅠand collagen Ⅲ synthesis and then play the role of inhibiting the proliferation of RYTF.The decreased expression of growth factor and collagen may be the direct cause of inhibition of RYTF proliferation and migration. |
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