ABSTRACT
Objective To explore the potential effects of neutrophil extracellular traps (NETs) on rheumatoid arthritis synovial fibroblasts (RA-FLSs).Methods The synovial tissues of RA patients were isolated and cultured in vitro.Peripheral blood neutrophils were extracted from healthy volunteers and used to stimulate NETs' formation,following with NETs' extraction.MTS proliferation assay was used to evaluate the effect of NETs on the proliferation of RA-FLSs.QRT-polymerase chain reaction (q-PCR) was used to determine the expression of connective tissue growth factor (CTGF) mRNA in cells treated with NETs-stimulated RA-FLSs for 60 h.The results were processed using paired sample t-test and one-way analysis of variance (ANOVA).Results The isolated and purified neutrophils could form NETs by in vitro stimulation.The concentration of extracted NETs-DNA was 58.5 ng/μl (1×106 cells).Compared with the control group (0 μl NETs),NETs could promote the proliferation of RA-FLSs.With the increase of NETs' concentration,the proliferation of RA-FLSs was also enhanced (F=99.519,P＜0.05).Compared with the control group (0 μl NETs),10 μl NETs could significandy promote the proliferation of RA-FLSs (t=-12.226,P＜0.01).Pretreatment of NETs with DNase Ⅰ inhibited its effect on promoting the proliferation of RA-FLSs (t=-2.376,P=0.049),NETs stimulated the upre-gulation of CTGF mRNA expression in RA-FLSs [(30.7±0.5),t=12.13,P＜0.01].Conclusion NETs can promote the proliferation of RA-FLSs and stimulate the up-regulation of CTGF mRNA in RA-FLSs in vitro.
ABSTRACT
OBJECTIVE:To optimize the best crushing particle size of Agkistrodon decocting powder,and study its effect on apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes. METHODS:Pre-column derivatization RP-HPLC was adopted. Using the total decoction amounts of 4 main amino acids (aspartic acid,glutamic acid,L-hydroxyproline,glycine) that decocted once as index,the Agkistrodon decoction pieces and those through No.1-8 seive were screened,and the best crushing particle size of Agkistrodon decocting powder was optimized. The human rheumatoid arthritis fibroblast-like synoviocytes were divided into nega-tive control group,positive control group (1 μmol/L methotrexate) and the best crushing particle size of Agkistrodon decocting powder group(2.0 mg/mL),flow cytometry was used to determine the cell apoptosis after cultured for 48 h. RESULTS:The best crushing particle size was through No.6 seive,when the total amount of 4 main amino acids was(61.27±0.02)mg/g(n=3). Com-pared with negative control group,the apoptosis rate in the best crushing particle size of Agkistrodon decocting powder group was significantly increased(P<0.05),which was slightly higher than positive control group. CONCLUSIONS:The best crushing parti-cle size is through No.6 seive;and Agkistrodon decocting powder shows effect on reducing the apoptosis of human rheumatoid ar-thritis fibroblast-like synoviocytes.
ABSTRACT
OBJECTIVE:To optimize the best crushing particle size of Agkistrodon decocting powder,and study its effect on apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes. METHODS:Pre-column derivatization RP-HPLC was adopted. Using the total decoction amounts of 4 main amino acids (aspartic acid,glutamic acid,L-hydroxyproline,glycine) that decocted once as index,the Agkistrodon decoction pieces and those through No.1-8 seive were screened,and the best crushing particle size of Agkistrodon decocting powder was optimized. The human rheumatoid arthritis fibroblast-like synoviocytes were divided into nega-tive control group,positive control group (1 μmol/L methotrexate) and the best crushing particle size of Agkistrodon decocting powder group(2.0 mg/mL),flow cytometry was used to determine the cell apoptosis after cultured for 48 h. RESULTS:The best crushing particle size was through No.6 seive,when the total amount of 4 main amino acids was(61.27±0.02)mg/g(n=3). Com-pared with negative control group,the apoptosis rate in the best crushing particle size of Agkistrodon decocting powder group was significantly increased(P<0.05),which was slightly higher than positive control group. CONCLUSIONS:The best crushing parti-cle size is through No.6 seive;and Agkistrodon decocting powder shows effect on reducing the apoptosis of human rheumatoid ar-thritis fibroblast-like synoviocytes.