Pristimerin Inhibits Rheumatoid Arthritis Fibroblasts-like Synoviocytes Proliferation And Underlying Mechanisms

Rheumatoid arthritis (rheumatoid arthritis, RA) is a chronic inflammatory disease characterized by persistent inflammatory in joints and systemic cartilage and bone destruction. Although the etiology of RA remains is unclear, but the synovitis and the proliferation of synovial is the main pathological features. RA can cause joints cartilage and bone destruction, eventually leads to joints deformity and dysfunction. It is one of the main causes of disability in humans. Fibroblasts-like synoviocytes (FLS) playes a key role in the proess of RA. The proliferation of the rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLS) is similar with the characteristic of cancer cells in clinical pathology. The expression of inflammatory cytokines in RA-FLS is a high level and the cells apoptosis is inhibited. All of the stage of RA demans the RA-FLS to join in. In the first place, immune and inflammation reaction is regulated by RA-FLS through the cells secrete inflammatory medium, inflammatory cytokines and anti-inflammatory cytokines. Then a growing body of evidence points to the hyperplasia of synovium tissue is closely related to the massive proliferation of FLS. The high level of matrix metalloproteinases(MMPs) are produced by RA-FLS lead to the cartilage degradation, even the bone and joints destruction. Besides, RA-FLS also involves in the angiogenesis. More and more evidence suggests that inhibiting the vitality of RA-FLS could delay the disease devepopment of RA, which can protect joints and improve the quality of life to RA patients. Thus, inhibiting of the proliferation of RA-FLS may be is a viable and effective clinical treatment of RA, that has a broad prospects method.Adjuvant Arthritis Rats (AA rats) are induced by Mtb (Heat-killed M.tuberculousis H37Ra), have a similarities with clinical RA patients in clinical manifestations andpathology changes, is a perfect animal model on RA that convenient way to copy and repeat. Compared with AA rats fibroblasts-like synoviocytes (AA-FLS) and rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLS), AA-FLS is more easy to isolated. If AA-FLS can be used as RA-FLS in vitro, we can get a lot of culture cell model in the short term. Therefore, we studied the characteristics of AA-FLS and evaluated the possibility of AA-FLS was used to the test in vitro by contrasted the resemblance of AA-FLS and RA-FLS.Celastrus aculeatus Merr. Belongs to the family named Celastrus orbiculatus, is a traditional Chinese medicine has been used in China for centuries to treat various rheumatoid diseases. We previously reported that ethanol extracts of Celastrus aculeatus Merr. exhibits the anti-inflammatory activity and reduces bone destruction in AA rats. Pristimerin is a natural activities, extracted by the effective part in Celastrus aculeatus Merr. It showed biological activities, just as anti-inflammatory and anti-tumor activity. We have suggested that pristimerin could inhibit synovial inflammation and synovial angiogenesis and could protect bone joints in RA.Based on previous study, we surveyed the effect of pristimerin on AA rats model and AA-FLS and explored the activity of pristimerin to inhibit a proliferation of AA-FLS. Further more, we studied related mechanisms of pristimerin to reduce rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLS) in RA.Objectives:1. To establish the methods of primary culture of fibroblast-like synoviocytes in rats with adjuvant arthritis(AA-FLS) and analyze the feature.2. To investigate the effects of pristimerin inhibits hyperplasia of synovium tissue in AA rats and reduces the proliferation of AA-FLS.3. To explore the underlying mechanisms of pristimerin on anti-proliferative in AA-FLS.Methods:1. Primary culture of fibroblast-like synoviocytes in rats with adjuvant arthritis(AA-FLS) and analyze the feature.The synovial cells were obtained from the SD rats were immunized the Mtb and identified by morphology and immunocytochemistry. The viability of AA-FLS was assessed by Cell Counting Kit-8. ELISA was applied to detect TNF-α and IL-1βin cell media. Apoptosis was measured by Hochest 33258 stain. The expressions of mitochondrial apoptosis-related molecules, including Bcl-2, Bax, Pro-caspase-3 and Cleave-caspase-3 were determined by Western Blot.2. Pristimerin inhibits the hyperplasia of synovial tissue and the proliferation of AA-FLS2.1 Animal levelAdjuvant arthritis (AA) rats model:SD rats after randomization, all of the rats were injected Mtb in the tail except for the control group rats. The volume of inflamed hind paws were determined by using the small animal limbs swelling instrument in order to observed the effect of pristimerin improved swelling joints on AA rats.2.2 Organization levelThe sections were stained with hematoxylin and eosin to evaluated pathological and histological characteristics of synovial tissue.2.3 Cells levelCCK-8 and Trypan blue exclusion assay the effect of different concentrations pristimerin inhibited the proliferation of AA-FLS.3 The mechanisms of pristimerin reduces the proliferation of AA-FLS3.1 Pristimerin blockades of AA-FLS cell cycleAA-FLS is treated with different concentrations pristimerin for 24h, assayed with the flow cytometry. Counted the differences in the proportion of the distribution of the cell cycle in each group.3.2 Pristimerin induces AA-FLS apoptosisAA-FLS is treated with different concentrations pristimerin and stained with Hoechst 33258. Counted cells apoptosis rate in each group. Explored the effect of pristimerin induced AA-FLS apoptosis3.3 The effect of pristimerin on the expression of Bcl-2, Bax and Caspase-3 proteinThe expression of Bcl-2, Bax and Caspase-3 protein in AA-FLS were detected by western blot method after treated with the different concentrations pristimerin for 24h.Results:1. Primary culture of AA-FLS and its characteristic.1.1 Primary culture of AA-FLSIn isolated primary synovial cells, more than 95% AA-FLS were fusiform Immunocytochemistry result showed that positive expression of vimentin and negative expression of CD68 in AA-FLS.1.2 The characteristic of AA-FLS(1) The cell proliferation of AA-FLSAA-FLS and FLS after subculture from the 1st day to the 4th day, two group cells grew slowly. The growth rate of AA-FLS was higher than the rate of FLS after four days ago. The number of AA-FLS spent less time for doubling than FLS. Cell proliferation of AA-FLS was higher than FLS.(2) The expression of cytokines in AA-FLSThe expression of cytokines in AA-FLS, such as TNF-a and IL-1β, is higher than FLS.(3) The cells apoptosis rate of AA-FLSThe cells were stained with Hoechst 33258 and observed with a microscope. The results showed that the apoptosis of AA-FLS was inhibited.(4) The expression of Bcl-2, Bax and Caspase-3 in AA-FLSWestern Blot data demonstrated that the protein expression of Bcl-2 was increased and the expression of Bax was reduced, at the same time the expression of Caspase-3 was activated in AA-FLS.2 Pristimerin reduces the hyperplasia of synovial tissueThe foot volume of AA rats were reduced by treated with pristimerin(0.40mg/kg and 0.80 mg/kg) in a dose-dependent manner. The sections showed that pristimerin inhibited the hyperplasia of synovial tissue.3 Pristimerin inhibits the proliferation of AA-FLSThe results of tests assay suggested that the cells vitality of AA-FLS was inhibited and the mortality rate raised by treated with pristimerin (0.75μM to 10μM) in a dose-dependent manner.4 Pristimerin induces AA-FLS apoptosisThis result showed that pristimerin treatment influenced the rate of AA-FLS apoptosis. Pristimerin treatment at concentration 0.75μM to 3μM increased the number of apoptotic AA-FLS.5 Pristimerin causes AA-FLS G0/G1 arrestBased on the flow fytometry assay, we evaluated that pristimerin inhibited AA-FLS proliferation via blockade of the cell cycle in G0/G1 period.6 The effect of pristimerin on the expression of Bcl-2, Bax and Caspase-3 proteinWe studied the change in the expression level of Bcl-2, Bax and Caspase-3 in the AA-FLS by treated with pristimerin. Western Blot analysis showed that treated with pristimerin decreased the anti-apoptotic protein Bcl-2 and increase pro-apoptotic protein Bax with increasing concentrations of pristimerin. Thus, pristimerin treatment could increase Caspase-3 activation significantly in a does-dependent.Conclusions:We studied pristimerin activation based on AA rats and AA-FLS, obtained following conclusions:1 AA-FLS is biological characteristics by the high level proliferation activity and inflammatory cytokines and apoptosis suppression. AA-FLS can be uesed as the model for the RA in vitro.2 Pristimerin can inhibit the proliferation of AA-FLS and induce AA-FLS apoptosis.3 Pristimerin inhibits the proliferation of AA-FLS by inducecell apoptosis through regulating the expression of Bcl-2, Bax and Caspase-3 in the AA-FLS. Thus, pristimerin causes AA-FLS cell cycle arrest by blockading of the cell cycle in the G0/G1 phase.