Effects Of Chinese Herbal Compound On Signal Transduction Pathways Of Retroocular Fibroblast In Patients With TAO

Objective: the study on the clinical and experimental effective basis,explore the mechanism of purging fire conflicts powder,Huoxue conflicts powder,raising goal conflicts powder in the treatment of TAO.And to observe the effect of three kinds of Chinese herbal medicine compound on proliferation of RFs.To observe the effect of stimulation of IFN-? and IL-4 on ERK1/2?JNK?P38MAPK?ERK5?NF-?B in RFs.Methods:1.Identification of primary cultured RFs.Retrobulbar connective tissue is from ocular trauma or other eye operation patients.Cells were used in the 2-8 generation.Identification of cultured RFs was carried out using an inverted phase contrast microscope and immunocytochemical staining method.2.After IFN-?,IL-4 and the drug-containing serum(20%)stimulation,RFs Vigor is detected by MTT colorimetric assay.3.RFs and IFN-?(200U / m L),IL-4(200U / m L)and three drug-containing serum were incubated for 48 hours.Using immunocytochemistry staining method detected IFN-?,IL-4 and three kinds containing Effect of ERK1/2?JNK?P38MAPK?ERK5?NF-?B expression in serum.Results:1.Retroocular fibroblasts of normal human were cultivated in vitro successfully.The forms of living cells were observed under light microscope.The cell shape is shuttle shape and polygon.After about 6-10 days,fibroblast cells grew confluent monolayer.The cell identification results show that fibroblast cell marker vimentin was positive and cytokeratin was negative.The results suggested that the cultured cells were pure RFs.2.The results of Preliminary experiments suggested that compound Chinese medicine containing serum can inhibit the proliferation of RFs,and the medium dose(20%)was the best dosage.3.After IFN-?,IL-4 and the drug-containing serum stimulation,RFs Vigor is detected by MTT colorimetric assay.The blank control group showed no significant difference(P>0.05)in rat serum control group.Compared with the blank control group and rat serum control group,rat serum added IFN-? group OD values were significantly increased.And the difference was statistically significant(P<0.05).The results of rat serum added IL-4 group and rat serum added IFN-?and IL-4 group were same as rat serum added IFN-? group.The prescription 1,the prescription 2 and the prescription 3 interfered groups OD values were significantly lower than rat serum added IFN-? group,rat serum added IL-4 group and rat serum added IFN-?and IL-4 group.The difference was statistically significant(P<0.05).4.Using immunocytochemistry staining method detected effect of ERK1/2?JNK?P38MAPK?ERK5?NF-?B expression in serum.The blank control group showed no significant difference(P>0.05)in rat serum control group.Compared with the blank control group and rat serum control group,the activity of ERK1/2 in rat serum added IFN-? group were significantly increased.And the difference was statistically significant(P<0.05).The results of rat serum added IL-4 group and rat serum added IFN-?and IL-4 group were same as rat serum added IFN-? group.The activity of ERK1/2 in the prescription 1,the prescription 2 and the prescription 3 interfered groups were significantly lower than rat serum added IFN-? group,rat serum added IL-4 group and rat serum added IFN-?and IL-4 group.The difference was statistically significant(P<0.05).So were JNK,P38 MAPK,ERK5,NF-?B.Conclusion:IFN-? and IL-4 can Promote the proliferation of RFs.Traditional Chinese medicine medicated serum in the medium dose(20%)can obviously inhibit the proliferation of RFs.Traditional Chinese medicine containing serum also can inhibit the expression on activity of ERK1/2,JNK,P38 MAPK,ERK5,NF-?B in RFs after IFN-?and IL-4 stimulation.So block the signal transduction pathways and reduce the immune injury of TAO.