| Background:Rheumatoid arthritis(RA)is a chronic systemic inflammatory disease and its etiology remains unclear,which is a universal disease on a global scale and the number of case accounts for 0.01 percent of the total population around the world.The clinical symptom of patients with RA is the inflammation of the symmetry facet joint damaging to the structure and function of joint with the development of the disease and ultimately lead to disability.RA is described as "undead cancer" because of the long process and the high deformity,resulting in great agony to the patients.The most prominent pathological features of RA includes chronic hyperplasia of synovium and sustainability synovitis.With a better understanding of pathogenesis of RA,chronic synovitis is attracting more and more attention and research because it play a critical role in promoting the disease progression.The synovial tissue abnormal hyperplasia was observed in the early stage of RA,and then the more and more hyperplasia of synovial membrane occupy the joint-space,leading to stiffness and inconvenient stretching in the joints of the patients with RA that causes an illness aggravated;in the middle stage,narrowing even disappearing of joint space because of the hyperplasia of synovial membrane occupying the joint-space affecting joint function,which aggravates synovitis and lead to joints functional disorder for the patients of RA;in the late stage,angiogenesis occurs in the synovial membrane,which maintain inflammatory state by transporting inflammatory cells to the synovium and by supplying nutrients to the pannus that invades cartilage and bone;in this manner,joint damage is exacerbated and ultimately lead to disability,resulting in the patients of RA lost or partially lost his capacity to work.In a short,the sustainability chronic synovitis is a key factor to aggravate the degree of RA.Therefore,relieving or inhibiting the chronic synovitis is a realistic therapy for treatment of RA.Synovitis is a complex process and there aren't any clearly reasons,but it it is widely believed that regulated by external environmental factor,cytokine stimulating and others factors.A growing body of evidence points to the hyperplasia of synovial membrane and chronic inflammatory reaction are closely related to the biological characteristics change of the synovial tissues,especially aberrant activation and massive proliferation of fibroblast-like synoviocyte(FLS).FLS in the lining layer of the synovial of the RA patients possess the ability of the tumor-like hyperplasia and erosion,which can produce large amounts of pro-inflammatory cytokines,chemokines and adhesion factors and widespread participate in the process of the synovitis,inflammatory cell infiltration,the erosion of cartilage and the formation of pannus,leading to the severity of synovitis,which will accelerate the disease progresses.Therefore,inhibiting the proliferation of RA-FLS could delay the disease development of RA.The abnormal proliferation of RA-FLS is a complex process and its mechanism remains unclear,but it is commonly considered that might be caused by the change of inherent genetic,external environment and the stimulation of cytokines.Clinical studies have confirmed that the sera and joint fluid of RA patients contain a large number of tumor necrosis factor-a(TNF-?),one of the important pro-inflammatory cytokines,which can not only directly induce synovitis but also promote the proliferation of RA-FLS and inhibit apoptosis of RA-FLS.Therefore,TNF-a makes a key role in promoting the excessive proliferation of RA-FLS.The self-anti-apoptotic features of RA-FLS is the directly cause of excessive proliferation except for the external factors.Apoptosis is a form of programmed cell death(PCD)that the destroy process of cell self results from multi-channel signal transduction on condition that physiology or pathology.Apoptosis is programmed active death process controlled by genes,which involved in a variety of enzymes.Researches show that the antiapoptotic property of RA-FLS was strongly associated with the inhibition of endogenous apoptosis pathway.The endogenous apoptosis pathway,known as the mitochondrial pathway,is a vital organs for cell life activities regulating by Bcl-2 protein family that is located in mitochondrial membrane,nuclear membranes and endoplasmic reticulum membrane and is divided into two categories:anti-apoptotic and pro-apoptotic proteins.One of the anti-apoptotic proteins,Bcl-2 and Bax,one of the pro-apoptotic proteins,are the key regulator of the signaling pathways.Bcl-2 regulates the permeability of mitochondrial membrane,opening and closing of mitochondrial membrane and stability of the mitochondria membrane potential with the combination of BAX.Bcl-2 bind to BAX forming a dimmer and all of the two is comparatively stable in normal cells.However,imbalance expression of Bcl-2(express increases abnormally)and Bax(express decreases obviously)may be the major causes of excess proliferation of RA-FLS.Celastrus aculeatus Merr.,a traditional Chinese herbal medicine that belonged to the family Celastraceae and named Celastrus orbiculatus.or Hemiboea subcapitata.,has been used in China for centuries to treat various rheumatoid diseases and is widespread across China such as Taiwan,Fujian,Guangdong,etc.We previously reported that ethyl acetate extracts of Celastrus aculeatus Merr.exhibits anti-inflammatory activity and reduces the severity of clinical arthritis and bone destruction in a rat adjuvant-induced arthritis by regulating the function of immunity,anti-inflammatory and inducing apoptosis of the immune activated cells.Pristimerin is a bioactive natural triterpenoid quinine methide for the ethyl acetate extracts of Celastrus aculeatus Merr.,which showed various pharmacological activities.At present,the anti-RA ability of pristimerin hadn't been idented.Therefore,in this present research,rats with adjuvant arthritis were immunized by Mtb in order to evaluating the effect on anti-synovitis of pristimerin and understanding the therapeutic basis matter and action mechanism of the effect on anti-RA of Celastrus aculeatus Merr.Objective:The aim of this study is to evaluate the therapeutic effect of pristimerin on joint synovial membrane inflammation and its underlying mechnism in rat with AA,to investigate the major target of pristimerin on antirheumatic of A A,in order to testify the effectiveness and material basis for anti-RA drug development from Celastrus aculeatus Merr..Methods:1.Study of pristimerin on suppress joints inflammation in rat with AA1.1 Induction of rats with adjuvant arthritis(AA)by Mtb60 mg of MTB(heat-killed Mycobactertum tuberculosis H37Ra)powders was fully grinded under a more sterile procedure,adding mineral oil to diluting into the MTB revulsant solution with final concentration of 7.5 mg/mL.The SD rats was injected with the revulsant of 0.1 mL(1.5 mg/each)at the tail and left hind paw.A total of 32 rats was received the injection.1.2 Subgrouping and administration28 AA rats was divided into Model group,methotrexate positive control group(MTX),pristimerin high dose group(Pris H)and pritimerin low dose group(Pris L)randomLy,with 8 rats in each group.8 normal SD rats was served as blank control.Model group and control group was given 0.5 mL normal saline per day by intraperitoneal injection;MTX was suspended in normal saline and intraperitoneal injection to AA rats with dosage of 1.5 mg/kg per week;Pristimerin was given to rats(low-dose group,0.50 mg/kg/d of body weight;high-dose group,1 mg/kg/d of body weight)daily by intraperitoneal injection.All the rats was continuous treated for 14 days.1.3 Changes of general physiological signs and weight fluctuation of ratsDuring the administration period,general physiological signs,including the rat's state of mind,independent activity,ear swelling,tail nodules and ankle swelling and inflammation,was regular observed.The body weight of each group was regular recorded,the first record of each rat body weight was obtained at 7 d,then the second record was obtained at 15 d,since then,the weighting of each group rats was taken by every 2 days.1.4 Arthritis index score of ratsArthritis index score was regular recorded in terms of signs observed in rats with AA.1.5 Measure of the rat's foot volumeAt the day of 7,15,18,21,24 and 27 after SD rat was immuned with MTB,a straight line draw upon 0.5 cm on rats ankle as the standard,the rat's foot volume was measured by the paw swelling test instrument in order to observe the anti-arthritis effect of pristimerin,then calculate swelling rate as follow the formula:swelling degree=(the foot volume after immunization-the foot volume pro-immunization)/the foot volume pro-immunization1.6 Histopathology and histologic scoring of syno-vial in ratsH&E staining method was used to detect the pathological changes of knee joint synovial tissue pathology,scoring criteria as follows:(1)invasion degree of lymphocytes,plasma cells,and neutrophils:0:no invasion;1:a small amount of inflammatory cells invasion;Two points:inflammatory cells are scattered in;Three points:a large number of inflammatory cells invasion.(2)the degree of synovial cell hyperplasia:0:1-3 layer;1:3-5 layer;Two points:5-10 layer;Three points:10 layer above.(3)cartilage or bone erosion conditions:0:no damage,articular cavity structure is normal,no obvious synovial tissue holder;1:yeah,destruction,articular cavity structure is normal,synovial tissue mild placeholder,but no erosion of cartilage;Two points:destroy insect damage samples,synovial membrane of articular cavity placeholder,direct erosion of cartilage;Three points:destruction accompanied by all the granulation tissue formation,articular cavity gap,synovial tissue erosion of cartilage and bone and pannus is formed.Each rat knee joint pathological scoring a total of 9 points.2.Effect of pristimerin on immune organ index and cytokine secretion of synovium and serum in rats with AA2.1 Determination of immune organ indexAfter the treatment,thymus and spleen was taken from the rats and weighted,thymus and spleen index was recorded for analyze.2.2 Detection of IL-6 and IL-17 levels in synovial tissue and TNF-ain serumThe expression levels of tumor necrosis factor-a in serum of rats were assayed using an ELISA kit.The expression levels of IL-6 and IL-17 in synovial group homogenate of rats with AA were assayed using an ELISA kit.3.The effect of pristimerin on the proliferation of AA FLS and its mechanism3.1 AA FLS cell vibility assayAfter treated with different final concentrations(6?M,3?M,1.5 ?M,0.8?M and 0.4?M)(including final concentration)for 24 h,cell viability was quantified by MTT assay and calculate the IC50 of pristimerin on AA FLS.3.2 Detection of cell apoptosisThe apoptotic cells was detected by the Hoechst 33258 staining assay or by flow cytometry to detect labeled Annexin V/7ADD.AA FLS were treated various pristimerin concentrations(3 ?M,1.5 ?M and 0.75 ?M)for 24 h before the detection.3.3 Detection of mitochondrial membrane potentialAfter AA FLS were treated various pristimerin concentrations(3 ?M,1.5 ?M and 0.75 ?M)for 12 h,mitochondrial membrane potential of cells was measured by flow cytometry with JC-1 stain.3.4 Mitochondrial apoptotic pathways regulate proteins detectionThe expression of Bcl-2,Bax and Caspase 3 protein in AA FLS were detected by western blot method after treated with the concentration of 3?Mor 1.5?M pristimerin for 24 h.Results:1.Pristimerin inhibits joint inflammation and regulates immunity in rats with AAAfter treated with pristimerin for 14 days,the general physical condition of rat with AA was improved in both high-dosage group and low-dosage group.From the 15 d,both high and low dosage pristimerin group interfered with increasing joint inflammation index score in AA rats(F=5.100,P=0.000<0.05).Consistent with the arthritis scoring and compared with model group,the the foot volume of rats also showed high and low dose of pristimerin both to be highly effective(F =6.446,P=0.000<0.05).The synovial of knee joints were evaluated by histologically.Compared with the model group rats,apparent adverse effects,including the joint synovial hyperplasia,inflammatory cells infiltration,and inhibit the synovial tissue erosion and destruction of bone structure were not markedly reduced in rats treated with pristimerin in a dose-depended manner(F=4.446,P=0.013<0.05).2.Pristimerin immune organ index of rat and cytokine levelsStudy results showed that pristimerin inhibited the abnormal incerease of thymus index with dosage depend mannar(F=4.190,P=0.000<0.05),at the same time pristimerin high dose group also can down-regulate spleen index with statistical difference(F=4.009,P=0.010<0.05).Both high and low dosage groups of pristimerin strongly reduced the TNF-a secreion in serum of AA rats(F=53.694,P=0.000<0.01).At same time,high and low dosage groups of pristimerin can significantly inhibit the secretion of IL-6(F=64.147,P=0.000<0.01)and IL-17(F=71.276,P=0.000<0.01)in synovial membrane compared with model group.3.Pristimerin inhibits the proliferation of AA FLSThe results showed that pristimerin inhibit the proliferation of AA FLS with statistical difference from the concentration of 0.8 ?M(F=596.877,P=0.000<0.01),in a does dependent manner.The IC50 of pristimerin on AA FLS for 24 h was 2.97?M.4.Pristimerin induces AA FLS apoptosis and affects the change of action potentials of cell membranesThe results of Hoechst 33258 stain assay showed that AA FLS apoptosis were induced by pristimerin(0.75 ?M to 3.0?M)by the change of the nuclear structure and the rupture of the nuclear in a dose-dependent manner(F=418.733,P?0.000<0.01).Annexin V/7AAD stain assay results showed that the percentage of AA FLS in early apoptosis stage were higher than control with a dose-dependent manner(F=326.784,P=0.000<0.01).Furthermore,JC-1 test results found that 1.5?M and 3?M concentration of pristimerin induce depolarization of mitochondrial membrane potential in AA FLS(F=59.493,P =0.000<0.01).5.Pristimerin modifies the protein expression level of apoptosis in AA FLSWestern blot results showed that both 1.5?M and 3?M pristimerin reduced the expression of Bcl-2 expresion in AA FLS compared with FLS control group(F=28.429,all P<0.05),at same time,increased the expression of Bax(F= 10.216,P=0.000<0.01)and cleave-caspase-3(F=43.780,P=0.000<0.05)in AA FLS.ConclusionPristimerin,one main effective component of extracted from ethyl acetate fraction of Celastrus aculeatus Merr.,did not reported the effect on anti-RA yet.At present study,we assess the effect of pristiemrin on anti-inflamation of joints and synovial membrane in AA rat model induced by Mtb,and we obtain the following conclusions:1.Pristimerin can suppress joint inflammation by inhibiting synovitis and the hyperplasia of synovial as well as regulating the balance of cytokine network.2.Pristimerin can inhibit AA FLS proliferation and regulate the abnormalities of cytokine network by reducing the secretion of proinflammatory factor IL-6 and IL-17 to suppress synovial tissue hyperplasia and inflammation in joints.3.Pristimerin can inhibit AA FLS proliferation by introducing AA FLS apoptosis thought regulating the expression of Bcl-2 and Bax in mitochondrial pathway,promoting the activation of Pro-Caspase-3 and increase the expression of Cleave-Caspase-3. |
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