The Effect Of Hydrostatic Pressure On Synovial Fibroblasts And Screening Of Moutan Cortex For Anti-inflammatory Activities And Chemical Constituents

Part one:Effects of hydrostatic pressure on cytoskeleton in rat temporomandibular synovial fibroblastsTemporomandibular disorders(TMD)are common syndromes in dental.clinic and are characterized by orafacial pain,joint sounds,and limited mandibular movement.Several studies have demonstrated that the increased pressure in synovium of the bilaminar zone, chiefly caused by disc displacement,may be contributed to the progression of chondrogenesis in bilaminar zone and remodeling in joint.As a physiological factor,the pressure in joint cavity takes an important role in keeping intra-articular fluid and maintaining the stability of the join.In part one of this study,the double condyles of 3 months old Sprague-Dawley rats(～200 g)were excised.Surface parts of synovial tissues were isolated from TMJ of rats,followed by washing extensively with phosphate-buffered saline,then minced into 1mm³ pieces and plated onto tissue culture dishes with DMEM supplemented with 15%fetal calf serum.SFs were confirmed by immunocytochemical staining to detect marker proteins CD68 and vimentin,which are macrophage marker and fibroblasts marker,respectively.SFs were seeded onto the culture dishes at a concentration of 2×10⁶ cells/dish.When the cells reached 70%confluence,the culture dishes were transferred to a columned stainless-steel vessel.The vessel was then placed in the cell-culture incubator.HP was applied at levels of 30 kPa,60 kPa,90 kPa for 12 hrs.Our results showed that the primary cultured cells were found to proliferate with a fibroblastic character,whilst no spontaneous changes in shape were found even in repeated culture.When grown to confluence,the cells looked spindly and polygonal.The cells were confirmed by immunocytochemical staining.All cells were negative for CD68.Moreover, they were all positive for vimentin,a fibroblasts marker and confirmed to be synovial fibroblasts(SFs).In part two of this study,Changes of proliferation,cell cycle,ultrastructure and cytoskeleton were observed by MTT assay,flow cytometry transmission electron microscope and fluorescent microscope after loading different HP.Our results showe that the proliferation rates in HP treated groups were lower than 100%, but there were no statistical differences in comparison of cells in normal condition.The result of cell cycle of SFs is the similar.The ultrastructure of SFs at 0kPa was normal and intact.At 30 kPa,the ultrastructure of SFs mostly shows that the chromatin was condensated lightly and ruptured to the nuclear margin.At 60 kPa,the karyon takes on crescent and the mitochondria seem varicose.At 90 kPa,the apoptosis-like body was wrapped by membrane and embedded in the high density chromatin.Compared with the untreated control,the cellular actin configuration of SFs became elongated and more intense F-actin stress fiber staining was observed after HPloading.The SFs primary cell culture and HP loading were successfully established in this study. Our results suggested that HP could impact on the change of ultrastucture and cytoskeleton of SFs.Meanwhile,SFs could response the HP and might be occurring some molecular eventswhich relative to the new biomechanicai environment.Part two:Effects of hydrostatic pressure on BMP-2,TGF-βand SOX-9 production in rat temporomandibular synovial fibroblastsRecent experimental evidence has suggested that pressure may play an important role in the pathogenesis of arthritic diseases such as temporomandibular disorders,rheumatic diseases and osteoarthritis.This study examines the effects of hydrostatic pressure(HP)on the protein production of BMP-2,TGF-βand SOX-9 in SFs of rat TMJ.The primary SFs culture and loading profile were the same as the part one.Production of TGF-β,BMP-2 and SOX-9 was examined by immunocytochemical assay and Western blot.Exposure of SFs to HP for 12 hrs resulted in significant up-regulation of BMP-2 by 46%,54%,66%at 30,60,90 kPa,whilst TGF-βincreased by 11%,19%and 28%at 30,60, 90kPa.HP also induced the increase of SOX-9 by 72%at 30 kPa and 83%at 60 kPa,but only 54%at 90 kPa.The obtained data suggests that HP induced the alteration of cytoskeleton and bone-morphogenetic-related proteins production of SFs,which may influence the pathological condition of temporomandibular disorders.Part three:Effects of hydrostatic pressure on MAPK and TGF-βin rat temporomandibular synovial fibroblastsVarious data obtained mitogen-activated protein kinases(MAPKs)in many types of cells argue that activation of a pathway occurs after loading stimulation.The purpose of the present work is to detect the relationship between ERK,JNK and p38 activation and the magnitude of HP by SFs culture,and examine the change of TGF-βwith or without ERK inhibitor(PD 98059)with time.The primary SFs culture and loading profile were the same as described before.The first part in this study,protein extracts were analyzed by western blot for the activation of ERK,JNK and p38. Our results showed that the production and activation of ERK,JNK and p38 were changed with the magnitude of HP.SFs exposured to 30kPa HP showed low expression of ERK,and then returned to normal.Finally,the expression of ERK decreased.On the other way,the activation of ERK shows highest on 30kPa and shows lowest on 90kPa. Meanwhile,30 kPa HP can also change the expression of JNK.But HP has less effect on the activation of JNK and p38.The second part in this study,SFs were cultured in the HP for a constant time point such as 5 mins,30 mins,1 hr,2 hrs,4 hrs,8 hrs and 12 hrs with or without 15μM PD 98059, an inhibitor of ERK.Cells were pre-incubated with PD 98059 for 1 hr before the mechanical stimulation began.The inhibitor was reconstituted in dimethylsulphoxide (DMSO),and control cells were pre-incubated with equivalent amounts of 0.04%DMSO alone.The TGF-βconcentration was tested by ELISA method.The assay procedure was performed accordi ng to the manufacturer's instructions.Our results showed that HP especially 30 kPa HP could increase the expression of TGF-β,But the ERK inhibitor PD98059 suppressed HP-induced expression of TGF-β.These results demonstrate that mechnical pressure induced by HP involved activation of ERK,JNK and p38 with different level.Mechanical stimulation induced a rapid activation of ERK,resulting in a visible phosphorylation at 30 kPa.On contract,the production of JNK and p38 changed along with the different HP,but the change of both phosphorylations is not visible.It indicates that JNK and p38 are not sensitive to the HP. There is a relationship between ERK and TGF-βassociated with HP.which may be play a key rolein remodeling of injured SF. Part four:screening of Moutan Cortex for anti-inflammatory activities and chemical constituentsMoutan Cortex,a widely used traditional Chinese medicine for the treatment of various diseases,is the root bark of Paeonia suffruticosa Andrews(Paeoniaceae).Most of the pharmacological investigations of Moutan Cortex have been addressed to its central nervous system activities,antioxidant and sedative actions.Otherwise,there are few reports about the active compounds with anti-inflammatory activity of Moutan Cortex.The aim of the present study was to screen and identify bioactive compounds with anti-inflammatory effect from Moutan Cortex.With the aid of preparative HPLC technique,ethyl acetate and ethanol extract of Moutan Cortex were isolated into twenty-two fractions.Bioactivities of these fractions were evaluated by measuring expression of tumor necrosis factor-alpha(TNF-α)on rat synovial fibroblasts subjected to interleukin-1beta(IL-1β).Eight compounds were isolated from six active fractions and identified by HPLC/MSⁿ.Purified compounds,paeoniflorin,paeonol, and pentagalloylglucose resulted in dose-dependent inhibition of TNF-αand IL-6 synthesis in synoviocytes treated with proinflammatory mediator.Traditionally,temporomandibular disorder(TMD)is treated with conservative therapy. Some alternative and complementary therapy for TMD patients have also been introduced, including intra-articular injection of superoxide dismutase(SOD)and sodium hyaluronate (SH).Evaluation of cell viability indicated that incubation of synoviocytes with fractions of Moutan Cortex for 1 d at concentration of 10^-5g/ml dose not reduce the percent of viable cells.These results suggested that paeonol,paeoniflorin,glycosides and pentagalloylglucose has potential to be used as anti-inflammatory drug for patients with TMD.Condusion:1.HP could impact on the change of cytoskeleton and ultrastructure of SFs after exposure in HP,and the direct proportion along with HP.2.TGF-βand BMP-2 might regulate the remodeling of the mandibular condyle induced by the mechanical stimulation during the disc displacement.SOX-9 would be a valuable parameter of cartilage regeneration3.The production and activation of ERK1/2 could be changed by different magnitude of HP and HP has less effect on activation of JNK and p38.ERK1/2 plays an important role in SFs suffering HP and transfer the signal by TGF-β.4.Paeonol,paeoniflorin,glycosides and pentagalloylglucose contribute to the anti-inflammatory effect of Moutan Cortex to SFs.