Effect of TFC on Ultrastructure and Secretory Function of Synoviocytes in Joint of Adjuvant Arthritis Rats

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Author: Xiaoyu Chen a, b, Jin Li a﹡, Xuefeng Xie a, Wenming Chenga, Hui Jianga

Effect of TFC on Ultrastructure and Secretory Function of Synoviocytes in Joint of Adjuvant Arthritis Rats

Xiaoyu Chen a, b, Jin Li a﹡, Xuefeng Xie a, Wenming Chenga, Hui Jianga a School of pharmacy, b Department of Histology and Embryology, Anhui Medical University, Hefei 230032,China

* To whom correspondence should be addressed. E-mail: lijunayd@yahoo.com.cn 

Abstract Total flavonoids Chrysanthemum indicum (TFC) is an effective composition from a Chinese medicinal herb the wild chrysanthemum of the dried bud. It is used in the therapy of anti-inflammation, analgesia and immunoloregulation action, but the therapeutic mechanism is unclear. This study explain the mechanism of TFC of therapeutical effect from ultrastructure and secretory function of synoviocytes in joint of adjuvant arthritis (AA) rats. The complete Freund's adjuvant was subcutaneously injected into the right hind feet pads of the SD rats. 12 days after immunity, TFC 84，168，336 mg/kg continuous intragastrice injected administration for 16days. Synoviocytes were derived from explant culture of tissue fragment, transmission electron microscope was used to analysis the morphological alteration of secretory function organell. Radioimmunoassay was used to detect level of IL-1β,TNF-α in secretory synoviocytes. RT-PCR observed mRNA expressions of IL-1β,TNF-αin the synovial membrane to explore the probable mechanism of TFC in the treatment of RA. We found TFC 84，168，336 mg/kg could recovery not only secretory function of A, B type synoviocytes, but also morphologic abnormality of golgi body, mitochondrion, rough endoplasmic reticulum. TFC corrected the high level of IL-1β,TNF-α in secretory synoviocytes. TFC showed a dose-dependent inhibition on the mRNA expressions of the IL-1βand TNF-αin a certain range of concentrations. It may be one of pathways to therapy AA that TFC could recovery the morphology of secretory function organell of synoviocytes in joint of AA rats and modify synoviocytes excessive inflamed medium. Key words adjuvant arthritis; total flavonoids of Chrysanthemum indicum (TFC); synoviocytes; rats

SUMMARY

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint swelling, synovial membrane inflammation, and cartilage destruction. Adjuvant arthritis（AA）is one of the main experimental models that shares some features with human RA at present[1,2]. Chrysanthemum is the dried inflorescence or bud of wild herb of Compositae (Chrysanthemum indicum L.), it has a long history using as a Chinese traditional medicine mainly for the treatment of inflammation. Previous studies have reported that Chrysanthemum indicum possesses the anti-bacteria, anti-virus, anti-oxidant and immunomodulatory properties. However, the relevant pharmacology research of active components from this plant remain to be investigated. Total flavonoids of Chrysanthemum indicum (TFC) was the effective composition from the wild Chrysanthemum indicum that separated and drew getting from the dried bud which explored anti-inflammation, analgesia and immunoloregulation action, the experiment indicates it had obvious prevention and therapeutical effect on AA rats [3,4]. In the present study, to further revealed the TFC function mechanism to AA rats, we adopt electron microscope technology and immunology method to observe the effect of TFC on AA rat's synoviocytes ultrastructure and secrete inflammation cytokine.

MATERIALS

Animals SD male rats, weighing 180~200g, were purchased from Animal center, Anhui medical University, Hefei, China. (Certificate No 01). All rats were housed for 1 week before experiment. All animal procedures were followed the approved protocol for use of experimental animals set by the committee on animal care at Anhui medical University. Drug and reagent The bud of chrysanthemum indicum was purchased from a crude drug market in Bozhou, Anhui Province, China, on March 2006, and authenticated classified by Dr. Weng-ming Cheng. A voucher specimen has been deposited at School of Pharmacy, Anhui Medical University, Hefei,China. The dried bud of chrysanthemum indicum(8kg) was macerated with 95%ethanol at room temperature for 2 weeks. The ethanol extract (CIEE) was suspended in water, then extracted by petroleum ether, ethyl acetate and butanol successively, obtained by elution, purified. includes total flavonoids of chrysanthemum indicum(TFC, brown powder) content beyond 5%. The fraction with obvious anti-inflammatory activity was analyzed on the thin layer chromatography(TLC) plate of RP-18 F254s. A mixture of methanol and water(60∶40) was used as mobile phase. The flavoniods was detected at 365nm as yellow fluorescent spots with the solution of 1% AlCl3 in ethanol. Phytochemistry studies of TFC have revealed the presence of several flavonoids, and its flavone purity is above 70%. TFC contains the cyanidenon, acacetin and linarin etc. compounds through the thin layer chromatography, high-efficient liquid phase chromatography[3]. Bacillus Calmette Guerin (BCG) was obtained from Shanghai Biochemical Institute. The RPMI-1640 medium was purchased from Gibco Company (CA, USA), supplemented with Hepes 2.5 mmol/L, pyruvic acid sodium 1 mmol/L, L-glutamine 2 mmol/L, 2-mercaptoethanol 50µmol/L, penicillin sodium 100ku/L，streptomycin 100iu/L, and 10 % new born bovine serum and pH was adjusted to 7.2. 125I-IL-1β, 3H-TNF- RIA kits were the product of Beijing Northern Biomedicine company (China). All other reagents were of analytical purity. METHODS Animals groupings and drug treatment Male Sprague-Dawley (SD) rats weighing 200-210 g, were divided randomly into six groups of 10 each, which were TFC（84，168，336mg/kg）and positive drug triperygium wilfordii polycoride (TPT) 150mg/kg given intragastrically, for the normal and AA model, rats were given an equal amount of vehicle. Besides normal group, AA was induced as previously described[5]. Briefly, rats were immunization d 0 by intradermal injection of Complete Freund's adjuvant (FCA), containing 10 mg 800C heat 1h inactive BCG in 1 mL paraffin oil, into the right hind feet pads of the rats in 0.1 mL for each rat. Given continuous intragastrically (ig 10ml/kg) from d 12 to d 24 after immunization. For the normal and AA model, rats were given an equal amount of 0.5% CMC-Na. AA evaluation AA was evaluated as previously described[6].Briefly, Left hind paw volume was determined with YLS-TA volume meter (Shandong Medicine academy, China) before immunization (basic value, d 0) and repeated on d 16, d 20, d 24, d 28. The paw swelling ( mL) was defined as the paw volume evaluated after inflammation. The polyarthritis severity was graded on a scale of 0-4, 0=no swelling; 1=isolated phalanx joint involvement; 2=involvement of phalanx joint and digits; 3=involvement of the entire region down to the ankle; 4=involvement of entire paw, including ankle. The maximum joint score was 12 including 3 secondary arthritis paws for each rat. Synoviocytes culture Rats were killed on d 28 by the subaxillary exsanguinations under the consciousness after immunization. The synovial tissues of the bilateral knee joints of rats were taken under the sterile condition, and then the synoviocytes culture was done using the method specified in the reference literature[7]. Synoviocytes were resuspended in RPMI-1640 medium at a concentration of 1×10 9 cells/L，the cell suspension 500 μL and LPS 500 μL with the concentration of 10 mg/Lwere added to six-well plate. After incubation at 37 ºC in 5 % CO2 atmosphere for 48 h, the supernatant containing IL-1β,TNF- was collected and stored at -20 ºC. Ultrastructure of synoviocyte Harvested synoviocytes were pre-fixed with 4 % glutaral Milloning buffer overnight. Synoviocytes were washed with PBS after fixing with 1 % osmic acid for 2 h. Being embedded in an Epon/Araldite mixture and stained with uranyl acetate and lead citrate, ultrathin section, the cells were observed under 1230 type transmission electron microscope (Electron Co, Japan) and photographed. IL-1, TNF- radioimmunoassay (RIA) IL-1β, TNF- produced by cultured synoviocytes were measured according to the procedures offered in IL-1β, TNF- 125I RIA kit. Measurement of mRNA expressions of the IL-1β,TNF-α The general RNA of IL-1β, TNF-α in the synovial membrane were extracted using the routine method as the reverse transcription-PCR（RT-PCR）assay [8]. The gel image assay apparatus was used to scan and the Lab Image 3.2 gel image analysis software was used for the grey ratio of the object band and internal control β-actin standard band.

Statistical analysis

All the data were expressed as Mean±SD, and SPSS12.0 software was used for t test and the related analysis between groups. A P value less than 0.05 were considered to be significant. RESULT Synoviocytes Culture After synovial membrane fibroblast smeared and Giemsa dyed, we examined 300 cells with microscope, its purity above 95%. Effects of TFC on secondary inflammatory Inflammatory polyarthritis were induced in all immunized rats. The peak incidence occurred on d 20 after immunization. Treatment with TFC and TPT diminished the left hind paw swelling and polyarthritic symptoms on d 16 after immunization. The suppressive effect of TFC 336 mg/kg was the most prominence (Tab 1). Tab1 Effect of TFC on secondary inflammatory reaction in AA rats（Mean±SD, n=10）

Group Dose Hind paws swelling (ml) (mg/kg) 12d 16d 20d 24d 28d Normal 0.12±0.07 0.13±0.05 0.14±0.06 0.17±0.08 0.18±0.04 AA model 0.33±0.07ΔΔ 0.37±0.08ΔΔ 0.46±0.06ΔΔ 0.41±0.05ΔΔ 0.38±0.07**ΔΔ AA+TFC 84 0.33±0.05 0.36±0.09 0.36±0.06* 0.33±0.08** 0.31±0.05** AA+TFC 168 0.30±0.06 0.34±0.06 0.35±0.05 0.31±0.05** 0.29±0.04** AA+TFC 336 0.31±0.08 0.33±0.04 0.29±0.06 0.25±0.06** 0.22±0.05** AA+TPT 150 0.27±0.04 0.28±0.07* 0.26±0.08** 0.23±0.05** 0.20±0.04** Compared with normal groupΔΔpEffect of TFC on ultrastructure of synoviocytes There were two types of synoviocytes，Type A synoviocyte derives from macrophage, Type B synoviocyte derives from fibroblast. Type B synoviocytes were major in normal synovial membrane which was characterized by developed rough endoplasmic reticulum (RER) and free ribosomes in physiological state. It was found that the number of type A synoviocytes which had plentiful Golgi body and the vesicle dispersed in the cytoplasm were increased significantly in AA rats compared with the normal. In contrast, the number of type B synoviocytes was decreased. Furthermore, the intracellular changes were observed in AA rats, including that Golgi bodies reduced in size and curled, mitochondria swelled with ridges decreasing, RER increased and dilated, dense bodies and vesicle increased. TFC（168, 336 mg/kg）and TPT repaired the injury mentioned above to some extent (Fig 1). In tissue culture, we could see the phenomena of the type A cells and type B cells conversion each other.

Fig. 1-1 Normal type A synoviocyte（×12 000）, much sporadic vesic was seen in cytoplasm. Fig. 1-2 Normal type B synoviocyte（×12 000）, several RER and Ri were seen in cytoplasm. Fig. 1-3 AA model rats type A synoviocyte（×6 000）, much sporadic vesic and golgiosome were seen in cytoplasm, RER was few, mitochondria swelled and cristae destroyed. Fig. 1-4 TFC（168mg•kg-1）ig rats type B synoviocyte（×24 000）, a few mitochondria swelled and cristae destroyed, partial recovery. IL-1β, TNF-α content assay Adopt radioimmunoassay. Synoviocytes suspension containing IL-1β, TNF- were collected for assay. Synoviocytes from AA rats released a higher level of IL-1β, TNF- than that from the normal rats. TFC significantly inhibited the production of IL-1β, TNF- of synoviocytes (Tab 3). TPT also reduced the content of IL-1β and TNF-. Tab 3 Effect of TFC on IL-1β, TNF-α in secretory synoviocytes（Mean±SD, n=10） Group Dose（mg•kg-1） IL-1β(ng•L-1) TNF-α(ng•L-1) Normal － 60.12±17.32 361.63±99.63 AA model － 125.41±25.87△△ 514.17±112.03△△ AA+TFC 84 101.75±27.72* 463.88±102.81 AA+TFC 168 83.48±10.63** 412.88±79.23* AA+TFC 336 68.26±15.23** 403.13±76.17* AA+TPT 150 64.75±11.63** 407.75±119.01* Compared with normal group ** pEffect of TFC at different concentrations on mRNA expressions of the IL-1β, TNF-α in the synovial membrane In the normal control group, there were weak mRNA expressions of the IL-1β，TNF-α,while the expressions of cytokines were significantly increased in the AA group (P IL-1β (377 bp)

M 1 2 3 4 5 6 Fig 2a Representative gels stained for RT-PCR products of IL-1β，TNF-α mRNA expression of PMФ in AA rats treated with TFC. Steady-state expression of GAPDH was used to control for equal loading of PCR product onto gels. M:Marker; 1:Normal; 2:AA model group; 3 TFC 84mg•kg-1 group; (4) TFC168mg•kg-1 group; (5)TFC336mg•kg-1 group; (6) TPT 150mg•kg-1 group Fig 2b Semi-quantitation of IL-1β,TNF-α mRNA expression of PMФ in AA rats treated with TFC. Compared with normal groupΔΔPDiscussion RA is a chronic autoimmune disease characterized by joint swelling, synovial membrane inflammation, and cartilage destruction. AA in rat is an experimental model that shares some features with human RA[3], such as swelling, cartilage degradation and loss of joint function. Up to date, the cause of RA is not fully understood. Although there are a few anti-rheumatic drugs showing effectiveness on treating RA, their side effects and toxicity call for new and more effective natural drugs. Under the project'Primary Study of National Basic Research Programme'(No.2002CCC02900), we extracted and separated from the wild chrysanthemum indicum, and carried on orthogonal design to several pieces of effective component and dose of administration through the pharmacology experiment, selected the active component of anti-inflammation and dose of administration. Appraised by the department of natural medicine of School of Pharmacy, Anhui Medical University, Hefei, China, had confirmed effective component of the wild chrysanthemum indicum as TFC, its flavone purity was above 70%, And it contained the cyanidenon, acacetin and linarin etc compound through the thin layer chromatography, high-efficient liquid phase chromatography [3,4]. Type A, B synoviocytes were final effector cells in RA joint damage in synovial membrane and articular cavity. Analysis from function, Golgi bodies in type A synoviocytes mainly take part in secretory function, while the mitochondrion is the energy provider to cell metabolism; meanwhile, the rough endoplasmic reticulum (RER) locating to type B synoviocytes is the place where proteins are processed and secreted[9-13]. Our results showed that the secretion and metabolism of synoviocytes of AA rats became hyperfunctional at the d 28 after immunization, which were improved by TFC. The morphologic changes of synoviocytes contributed to its secretory function which was related to the over-production of IL-1, TNF-. Golgi bodies, mitochondrion, RER, vesicle in TFC treated decrease than AA model group. The effects of TFC on synoviocytes secretory function might provide an explanation for the mechanism of its action in AA rats. These pharmacological effects of TFC strongly suggested its potential therapeutic role for the autoimmune disease, especially RA[14]. Under the normal situation, expressed and secreted of IL-1, TNF-α were controlled strictly by the organism. In RA, the higher secretion of synovial cells activated by proinflammatory factors such as IL-1, TNF- is thought to be a key step in the destruction of cartilaginous and bony tissues in RA joints[15-17]. IL-1, TNF- overproduction play potential pathogenic roles in the establishment of rheumatoid synovitis, in the formation of pannus tissue and in the process of joint destruction. Studies in experimental models revealed that IL-1, TNF- were indeed pivotal cytokines in joint swelling and cartilage destructive[18]. It was also found IL-1, TNF- receptor antagonists were recently backed up with similar data in arthritis models in IL-1 and TNF- gene knockout mice [19,20]. In our study, synoviocytes of AA rats released a higher level of IL-1 and TNF- than that of the normal rats. TFC inhibited the production of IL-1 and TNF-. The RT-PCR also showed that the mRNA expressions of the IL-1, TNF- in AA rats were significantly increased compared with those of the normal control group, TFC had down-regulated the mRNA expressions of the inflammatory factors IL-1, TNF-. TFC might ameliorate the secondary inflammatory reaction of AA via influencing secretory function of activated synoviocytes. Results were identical of inhabiting synoviocytes secreted hyperfunction and transmission electron microscope observed organ secreted function. Therefore, while treating RA, we will try to inhibiting yield of the cytokine and reduce its activity, block the course of arthritis, induce the condition to relieve[21]. TFC could be an effective and useful candidate drug in treating RA. Acknowledge Financial support for the project (1) Primary Study of National Basic Research Programme (No.2002CCC02900) by the Ministry of Science and Technology of China; (2) Youth Teacher Science and Technology Educational Fundation (No.2006jql100zd) of Anhui Province, China.

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