Abstract

To investigate the effect of pristimerin, an effective component of maple leaf, on the cell cycle and apoptosis of adjuvant arthritis fibroblast like synoviocytes adjuvant arthritis fibroblast like synoviocytes cells. Adjuvant arthritis fibroblast like synoviocytes cells were cultured in vitro, the effect of pristimerin on the proliferation of adjuvant arthritis fibroblast like synoviocytes cells was analyzed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide method, and the effect of pristimerin on adjuvant arthritis fibroblast like synoviocytes cell cycle was analyzed by flow cytometry. Hoechst 33258 staining experiment and flow cytometry were used to detect the effect of pristimerin on cell apoptosis of adjuvant arthritis fibroblast like synoviocytes cells. The effect of pristimerin on mitochondrial membrane of adjuvant arthritis fibroblast like synoviocytes cells was analyzed by flow cytometry. Western Blotting was used to detect the effect of pristimerin on the expression of adjuvant arthritis fibroblast like synoviocytes mitochondrial pathway regulators Bax, B-cell lymphoma 2 and Caspase-3. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide experiments showed that pristimerin had a significant inhibitory effect on the proliferation of adjuvant arthritis fibroblast like synoviocytes cells in a dose-dependent manner. The half inhibitory concentration of pristimerin on adjuvant arthritis fibroblast like synoviocytes cells was 2.97 μM. Cell cycle results showed that 1.5 μM and 3 μM pristimerin could significantly increase the number of cells in the Gap 0/Gap 1 phase (p<0.05) and decrease the number of cells in the synthesis phase (p<0.01). Hoechst 33258 staining experiment and flow cytometry detection of apoptosis showed that the rate of early apoptotic cells in adjuvant arthritis fibroblast like synoviocyteswas 17.307±2.519 % in the 0.75 μM scopolamine group, which was not statistically different from the control group (p>0.05). The apoptosis rate of adjuvant arthritis fibroblast like synoviocytes cells in the 1.5 μM group was 47.413±2.503 %, and the apoptosis rate of adjuvant arthritis fibroblast like synoviocytes cells in the 3 μM group was 70.970 ±4.205 %, which were significantly higher than those in the control group (p<0.01). Cell membrane potential test results showed that the proportion of adjuvant arthritis fibroblast like synoviocytes depolarized cells in the pristimerin 1.5μM group and the 3 μM group were 54.550±3.056 % and 91.373±3.163 %, which were significantly higher than the control cell depolarization rate of 38.480±7.447 % (p<0.01), pristimerin 0.75 μM had no significant effect on adjuvant arthritis fibroblast like synoviocytes mitochondrial membrane potential (p>0.05). Western Blotting results showed that compared with normal fibroblast like synoviocytes cells, the relative protein levels of B-cell lymphoma 2 , Pro-Caspase-3 and Cleave-Caspase-3 in adjuvant arthritis fibroblast like synoviocytes cells were significantly increased (p<0.01), while the expression of Bax in adjuvant arthritis fibroblast like synoviocytes cells was decreased but not statistically different (p>0.05). The relative expression of B-cell lymphoma 2 , Pro-Caspase-3 and Cleave-Caspase-3 in pristimerin-1 μM and 3 μM groups was significantly lower than that of adjuvant arthritis fibroblast like synoviocytes group (p<0.01), while the expression of Bax was higher than that of adjuvant arthritis fibroblast like synoviocytes group (p<0.05). Pristimerin can induce adjuvant arthritis fibroblast like synoviocytes apoptosis through the cell cycle arrest and regulate the mitochondrial apoptotic pathway to inhibit the adjuvant arthritis fibroblast like synoviocytes cell hyper proliferation and achieve Anti-rheumatoid arthritis synovial hyperplasiay.