| ObjectiveTo explore the molecular mechanism of emodin interfering with RA through affecting the TNF-a-HIF-1?-iNOS-NO signaling pathway by study the effects of emodin on the expression of tumor necrosis factor-alpha(TNF-?),hypoxia-inducible factor-1 alpha(HIF-1?),inducible nitric oxide synthase(iNOS)and nitric oxide(NO)in fibroblast-like synoviocytes(RA-FLS)of human and collagen-induced arthritis(CIA)rats.Finally,it will provide experimental evidence for new drug development and clinical application of RA.Methods1.High performance liquid chromatography(HPLC)was used to extract and determine emodin in Polygonum cuspidatum,and then the corresponding concentration of emodin solution was prepared according to the results.2.The 60 SPF male Wistar rats were randomly divided into normal group,model group,high dose group,medium dose group,low dose group,positive control group.In the normal control group,the rats were given ordinary feed.For the other five groups,in the first 20 days,the rats were uesd to build a Collagen induced arthritis model and accept pharmacologic intervention in the following 20 days.Emodin would be divided into three different dose groups,with 80mg.kg-1,40mg.kg-1 and 20mg.kg-1 respectively,each group was given a gavage daily.For the dexamethasone group,7.5 mg.kg-1 dexamethasone was given for gavage once a day.In terms of the model group,the rats were perfused with the same volume of saline(2 mL)once daily.The weight and foot swelling degree of rats in each group were measured once a week.Then,the animals were killed at the end of 40 days to analyze the appeareance of paws by X-ray and macroscopic observation,measure the content of TNF-?,HIF-1?,iNOS and NO protein in blood serum,spleen and synovial tissue by enzyme-linked immunosorbent assay(ELISA),nitrate reductase method,immunohistochemistry and Western blot,measure the content of TNF-?,HIF-1? and iNOS mRNA in spleen by reverse transcription-polymerase chain reaction(RT-PCR).Rrespectively,observe the tissue morphology of of liver,spleen and synovial tissue of the knee joint after HE staining under microscope.3.Human rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)and normal fibroblast-like synoviocytes were cultured.The experiment were designed normal group,model group,high dose group,medium dose group,low dose group,positive control group.The normal group was treated with normal FLS,while the other 5 groups were treated with RA-FLS.Emodin would be divided into three different dose groups,with 80?mol.L-1,40?mol.L-1 and 20?mol.L-1.For the dexamethasone group,0.1mg.L-1 dexamethasone was given.The duration of drug intervention was set at 24 hours,48 hours,72 hours,96 hours.Rrespectively,observe the growth and morphological changes of FLS after HE staining and toluidine blue staining under microscope.The cell culture medium were collected and the content of TNF-?,HIF-1?,iNOS and NO protein were measured by enzyme-linked immunosorbent assay and nitrate reductase method.The cell were collected and the content of TNF-?,HIF-1? and iNOS protein were measured by Western blot.Results1.The extraction rate of emodin in Polygonum cuspidatum was 1%by HPLC.2.(1)After modeling,the rats gradually developed symptoms of burnout,depilation,diarrhea,claudication,redness and swelling of hind feet,deformity and osteolysis.On the twentieth day of modeling,the symptoms were most acute.The weight of rats in model group was significantly lower than that in normal group(P<0.05),and the degree of toe swelling in model group was significantly higher than that in normal group(P<0.05).After 20 days of administration,the above symptoms were relieved in varying degrees.Compared with the model group,the degree of toe swelling decreased gradually in each group,especially in the middle dose group(P<0.05).(2)HE staining showed that the liver,spleen and synovium of knee joint in normal group were normal and clear.In the model group,synovial cells were damaged,inflammatory cells were increased,and pannus was proliferated;red and white pulps of spleen were obviously proliferated,the structure was blurred and granuloma was formed;liver cytoplasm staining was deepened,nuclei were significantly increased and unevenly distributed.After 20 days of drug intervention,the above conditions were relieved in all the administration groups,especially in the middle dose group.The infiltration of inflammatory cells in synovial tissue and the proliferation of pannus were inhibited,the proliferation of red and white pulp and granuloma in spleen were decreased,and the structure of liver became clearer,the staining of cytoplasm became lighter,and the proliferation of nucleus was decreased.(3)Immunohistochemical,ELISA,nitrate reductase,Western blot and RT-PCR showed that the expression of TNF-?,HIF-1?,iNOS,NO protein and mRNA in blood serum,spleen and synovium of knee joint in model group was significantly higher than that in normal group(P<0.05).In the synovial tissue of knee joint of rats,the levels of TNF-?,HIF-la and iNOS were decreased in the middle dose group(P<0.05);In the blood serum of rats,the expression of TNF-? in the positive group decreased the most,and the low dose group was the second(P<0.05).However,HIF-1?,iNOS and NO all decreased most in the middle dose group(P<0.05);In spleen of rats,the expression of TNF-?,HIF-1? and iNOS mRNA genes all decreased most in the middle dose group(P<0.05).The level of HIF-1? protein decreased most in the middle dose group(P<0.06).However,the expression of TNF-? and iNOS protein all decreased most in high dose group(P<0.05).3.(1)The results of FLS toluidine blue and HE staining showed that the FLS grew on the wall,most of them are spindle shaped,and they also have polygonal,dendrite shaped and so on.The nucleus is ovoid and is located in the center of the cell.RA-FLS grew faster than normal FLS.(2)The results of ELISA and nitrate reductase showed that the expressions of TNF-?,HIF-1?,iNOS and NO in cell culture medium of model group were significantly higher than those of normal group at 24 hours,48 hours,72 hours and 96 hours(P<0.05).After RA-FLS was treated with different concentrations of emodin,the expressions of TNF-?,HIF-1?,iNOS and NO in RA-FLS were all decreased at 4 time periods,especially when the dosage of emodin was 80 mol.L-1 and the intervention time was 96 hours(P<0.05);(3)The results of Western blot showed that after 96 hours of emodin intervention,the expression of TNF-?,HIF-1? and iNOS in RA-FLS cells of emodin groups and positive group were lower than that of model group,with the highest decrease in high dose group and the lowest decrease in low dose group(P<0.05).Conclusions1.With the development of arthritis,the expression levels of TNF-?,HIF-1?,iNOS,NO protein and mRNA in serum,spleen and synovial tissue of knee joint in CIA rats increased significantly.Emodin can down-regulate the expression of TNF-?,HIF-1?,iNOS,NO protein and mRNA in blood serum,spleen and synovium of CIA rats.Moreover,when the dosage of emodin was 40 mg.kg-1,the effect of emodin was the most significant,which could control inflammation,reduce bone and cartilage injury,inhibit angiogenesis and alleviate the development of CIA rats.2.With the development of arthritis,the expression of TNF-?,HIF-1?,iNOS and NO in human RA-FLS would gradually increase,especially in 96 hours of cell culture.Emodin could down-regulate the expression of TNF-?,HIF-1?,iNOS and NO protein in human RA-FLS in a dose-dependent manner,especially when the dosage of emodin was 80 mol.L-1 and the intervention time was 96 hours.3.Emodin can inhibit the opening of TNF-?-HIF-1?-iNOS-NO signaling pathway,down-regulate the expression of TNF-?,HIF-1?,iNOS,NO protein and mRNA in related cells and tissues,control inflammatory development,reduce bone and cartilage joint injury,reduce angiogenesis,play a therapeutic role in RA. |
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