Studies On The Antirheumatic Material Basis And The Mechanism Of Action Of Securidaca Inappendiculata

Securidaca inappendiculata (SI) is a traditional antirheumatic medicine used in China. Although it has been used to cure rheumatoid arthritis for hundreds of years, the obtained knowledge about the relative material basis and the underlying mechanism of action is still poor.Objective:To screen out and isolate the potential active compounds from SI contributing the most importance to the antirheumatic activity, explore the underlying mechanism of action concerning the antirheumatic activity.Methods:1. The therapeutic effects of aqueous and alcohol extract of SI were investigated on adjuvant induced arthritis (AA) rats. Arthritis severity was evaluated by paw weight, histological changes and hyperplasia of lymphatic tissues.2. The potential antirheumatic activity of the different fractions from SI was assessed by their anti-inflammatory, analgesic and immune regulatory activities, and these activities were investigated via carrageenan induced paw edema test, hot plate test, carbon clearance test in vivo and lymphocyte transformation test in vitro test.3. The therapeutic efficacy of dichloromethane fraction of SI (SID) was investigated on A A rats, and assessed by the following indexes, arthritic score, body weight loss, paw circumference, histological changes, hyperplasia of lymphatic tissues, levels of SOD, GSH, OH·, NO, MDA, NAG, SA, ALP, AST and ALT in serum and (or) liver, levels of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) in serum, and total and differential leucocyte counts.4. Compounds from the active fractions were isolated by using different chromatographies, mainly silica gel MPLC and LH-20 column chromatography. The chemical structures of obtained compounds were determined by various spectrometers. The composition analysis of the fatty and essential oil was performed using GC-MS, and the identification of components was carried out by comparing with built in libraries and the calculated retention indices relative to n-alkanes.5. The therapeutic effects of three typical xanthones isolated from SI were investigated on AA mice. Arthritic score, body weight loss, paw circumference, histological changes and hyperplasia of lymphatic tissues were investigated to assess the arthritis severity. Levels of IL-1,TNF-α, MCP-1 and VEGF in serum were determined using ELISA method, and levels of GSH, MDA, NAG and SA in liver were assessed by colorimetric method.6. The anti-proliferatory and anti-inflammatory activities of 1,7-dihydroxy-3,4-dimethoxyxanthone (X-3) on MH7A cells were assessed by MTT and ELISA methods. The pro-apoptosis and cell cycle arrest activities were analyzed by flow cytometry. The underlying mechanism of action was mainly analyzed based on levels of hallmark kinases by W-B method.Results:1. Aqueous and alcohol extract of SI significantly both ameliorated the AA severity, suggested by the modulatory effects of paw swelling, hyperplasia of lymphatic tissues and synovial membrane.2. SID was sreened out with significant anti-inflammatory, analgesic and immumodepressive activities, and deemed as the potential active fraction. It inhibited both of the paw edema and increased PGE2 level induced by carrageenan, prolonged the reaction times in hot plate test, suppressed the carbon clearance rate, and hindered the transformation and proliferation of the lymphocyte.3. SID significantly modulated the inflammatory changes such as body weight loss, paw swelling, hyperplasia of lymphatic tissues and synovial membrane in AA rats. It also exerted the restorative modulation on the levels of NO, MDA, OH-, SOD, GSH, AST, ALT, ALP, NAG, SA, IL-1, TNF-α, MCP-1 and VEGF in serum and (or) liver. These results suggest SID exerted the anti-arthritis activity on AA rats via the immumodepressive, cytokines regulatory, and antioxidantive activities.4.25 compounds were isolated and identified from the active fraction of SI. Among them 12 already known xanthones and a new xanthone was identified.34 from 55 signals were identified in the fatty oil, which together contributed 97.18% to the total area. The major compounds were hexadecanoic acid (36.89%) and 9-octadecenoic acid(E)-(31.12%).29 with 83.71% relative area rate from 48 peaks from the essential oil were identified. The results indicated that the essential oil was mainly comprised of mono-terpenes (23.12%) and fatty acids (21.70%).5. All the xanthones exerted significant therapeutic effects on AA mice. They dramatically reduced the levels of pro-inflammatory cytokines IL-1, TNF-α, MCP-1 and VEGF in serum, and restored abnormal changed levels of MDA, GSH, NAG and SA in liver.6. 1,7-Dihydroxy-3,4-dimethoxyxanthone (X-3) efficiently inhibited the proliferation of MH7A cells via cell cycle arrest at Gi/S phase and pro-apoptosis, and suppressed the secretion of IL-1β and IL-6 of MH7A cells. It up-regulated levies of p-p65, casepase-3, casepase-9, p-p38, p-ERK, bax and p21, and down-regulated levies of p-JNK, cyclin D1 and bcl-2.Conclusion:SI was validated with substantial therapeutic effects on AA in vivo, and the sreening assay found SID from it may be the most important material basis contributing to the antirheumatic activity. The isolation of SID afforded 25 compounds. Among them, xanthones were deemed as the most important potential antirheumatic agents based on the results from AA rats assay. The therapeutic effects of xanthones on AA were at least partly due to the anti-proliferatve and anti-inflammatory activities on RA-FLS. In this course, xanthones selectively modulated MAPKs signaling, which induced the Gi/S cell cycle arrest, and suppressed the pro-inflammatory cytokines secretion. The up-regulation of NF-κB also contributed to the inhibition on the proliferation of MH7A cells via the pro-apoptosis activity.