Celastrol Inhibits Heterotopic Ossification In Ankylosing Spondylitis Based On Interactions Between Wnt/β-catenin Pathway And Its Inhibitor Dickkopf-1 With Prostaglandin E2

BackgroundAnkylosing Spondylitis (AS) is a kind of chronic inflammatory and autoimmune disease, which develops ankylosis in the sacroiliac joint, spine,and peripheral joints (especial the hip joint) in some cases,leading to functional disability and impaired quality of life.Traditional Chinese Medicine believes that the essential reason for pathogenesis of AS is attributed for congenital deficiency, lack of Liver and Kidney as well as malnutrition of Du Meridian. Invasion of the meridian by external evil is the manifestation cause. There are two structural damage phases in AS including inflammation induced erosion and subsequently abnormal new bone formation at entheses. Ossification of the entheses is the major cause of functional disability in AS patients.To inhibit or slow the process of abnormal bne forming plays a crucial role in the treatment of AS. For decade years,research works related to treating AS are mainly focused on the aspects of inflammation or immune field.Although the potential mechanism remains unclear. So far, there has heen a concensus among the scholars that inflammation and new bone formation in AS is not linked directly but in some indirectly ways, the potential mechanism remains unclear.Various studies have shown that Wnt signaling plays a pivotal role in the development of bone formation,which serves as the most important signal way in endochondral ossification process. Wnt/β-cantenin is the classical signal.The Wnt proteins play important roles in the role of bone metabolisms,especially the osteoblasts guided new bone formation.The effect of Wnt signaling could be blocked by Dickkopf-1(DKK-1).DKK-1 is one of the DKK family numbers, which could be soluble proteins to function as natural inhibitor of Wnt signaling to further suppress the osteogenesis activity.It has been demonstrated that Wnt signaling play a great part in the AS progreesion (heterotopic ossification),especially the DKK-1,serving as a key regulator in AS process,and also playing as an important role in the regulation of osteogenesis process..Studies have also shown that functional DKK-1 expression is decreased in AS patients.However,studies related to whether DKK-1 is downregulated in the AS tissues or correlated with radiographic progression are not available.PGE-2 is the most important inflammation mediator and participates various inflammation process as well as anabolic/catabolic procedure of bone formation in vivo. PGE-2 expression is the highest in osteoblasts among all the prostaglandins. PGE2 could induce osteogenesis to conduct bone remodeling and play an important role of heterotopic ossification.Up to the present,works on PGE-2 in AS are few. One GWAS indicated that one of the PGE-2 receptor PGER4 is associated with AS progression.In addition,PGE-2 could activate Wnt signal and inhibit expression the DKK-1 through series of reactions. So far,most works targeting AS factors are most focused on Tumor Necrosis Factor-a(TNF-a)However,recent studies have shown that although TNF-α is the major factor of AS pathogenesis, TNF-a blockade seems not to slow the radiographic progression of AS,indicating TNF-a may not serve as a bridge factor of inflammation and bone forming.Up to now, western medicine used for treating AS includes Non-steroidal anti-inflammatory drugs (NSAIDs), Disease-modifying anti-rheumatic drugs (DMARDs) and TNF-a blockade. NSAIDs are the first-line treatment drugs. Various studies demonstrated that NSAIDs could prevent abnormal bone formation in AS, supporting the hypothesis that PGE-2 may serve as the connective factor between inflammation and new bone formation. However, potential gastrointestinal and cardiovascular adverse effects have restricted its long use clinically. Application of biological agents such as TNF-a could relieve symptoms and systemic inflammation reactions. But in recent years, adverse effects gradually increased under safety treatment window,short or long term use may produce serious adverse events. In addition, TNF-a could was not able to prevent radiographic progression in two years. Therefore, finding new drugs to target novel AS related osteogenesis genes or proteins is an urgent task for researchers.The traditional Chinese medicine accumulates lots of experience and favorable Chinese herbs when treating AS with various methods, certain effects and unique advantages. To clarify the active ingredients and molecular targets of Chinese herbs is an important research task in modern Chinese medicine. So far, many studies reported that glucosidorum tripterygll totorum is effective in treating AS in clinical studies.As one of the most important ingredients of glucosidorum tripterygll totorum, celastrol is a kind of natural product isolated from the Thunder God Vine with various biological components. Celastrol is endowed with strong anti-oxidative function. And also celastrol could perform its anti-cancer effects though inhibition of angiogenesis. There are no studies available illustrating the effect of celastrol on abnormal bone formation in AS patients. Previous reports demonstrated that celastrol strongly suppressed lipopolysaccharide-induced expression of PGE-2 at low concentrations, via the downregulation of COX-1 and -2 activation. This provided a theoretical basis for understanding the inhibitory effects of celastrol against PGE-2-induced osteogenesis.In the current study, we used AS serum, hip capsule tissues and isolated cultured AS hip synovial fibroblasts, through modern biological technics such as ELISA, western blot analysis, QRT-PCR, flow cytometric analysis and lentivirus transfection, etc. First, we explored whether serum total or functional DKK-1 levels in AS patients differs from the normal controls, and functional DKK-1 levels are associated with radiographic progression. The we investigated the difference of Wnt/β-catenin related proteins and DKK-1 in AS and normal control hip capsule tissues as well as in isolated fibroblasts. And next,based on the Wnt/β-catenin signaling we observed fibroblasts proliferation and osteogenesis ability altered following DKK-1 overexpression or silencing using lentivirus. transfection And finally, we explored celastrol could inhibit AS fibroblasts osteogenesis and slow the process through the cross-interactions between PGE-2 and Wnt/β-cantenin as well as DKK-1, further inhibiting AS bone forming from the molecular transcription aspectObjective1.1 To study whether serum total or functional DKK-1 levels in AS patients differs from the normal controls, and functional DKK-1 levels are associated with radiographic progression.1.2 To investigate whether DKK-1 is downregulated in the AS tissues and isolated fibroblasts compared with controls. And the AS fibroblasts.1.3 To explore fibroblasts proliferation and osteogenesis ability altered following DKK-1 overexpression or silencing using lentivirustransfection.1.4 To explore whether celastrol could inhibit AS fibroblasts osteogenesis and slow the process through the cross-interactions between PGE-2 and Wnt/β-cantenin as well as DKK-1, further inhibiting AS bone forming from the molecular transcription aspect.Thus providing basis for the work of further pharmacokinetic,animal and clinical studies.Methods2.1Total Dkkl levels were assessed by commercial sandwich enzyme-linked immunosorbent assay and functional DKK-1 levels of serum were measured using functional enzyme-linked immunosorbent assay method in 62 patients with AS and 60 healthy controls.Pelvic and whole spine plain radiography were taken when each participant was lying in supine position and the X-ray beam was centered at the level of the pelvis and then the whole spine. The radiographic progression of AS was classified according to the radiographic events of modified New York Criteria for sacroiliac joints evaluation and modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) system for spine assessment. Statistical analyses were performed utilizing the Statistical Package for Social Sciences (SPSS 18.0 for Windows). Normally distributed continuous variables are presented as mean±SD, whereas non-normally distributed continuous variables are presented as median(interquartile range).Differences for total DKK-1 levels between the two groups were analyzed using the unpaired t-test, Differences for functional DKK-1 levels between AS and normal control as well as different groups according to modified New York Grading were analyzed using Mann-Whitney U test.The Spearman rank correlation coefficient were performed to evaluate the correlation between functional DKK-1 levels and modified New York Grading as well as mSASSS scores.2.2 Hip capsule tissues were obtained from six AS patients (aged between 30-50 years old)undergoing total hip replacement in our hospital, according to the modified New York criteria.Six cases of hip capsule tissues were also obtained with the informed consent of the patients(aged between 30-40 years old), following traumatic femoral neck fracture.Isolation of tissues was performed in a sterile operation environment. Primary fibroblasts from hip capsules in the two groups were obtained for analysis and then cultured, utilizing an explant culture method.In brief, fibroblasts were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 20% penicillin,20% streptomycin and 10% fetal bovine serum. The culture medium was changed after 24 h and every other day thereafter.DKK-1 expression was assessed by western blotting, real time-polymerase chain reaction (RT-PCR), immunohistochemistry as well as cell Immunofluorescence analysis of hip synovial tissues and cultured isolated fibroblasts obtained from AS and control patients.All data are presented as the means ± SD. Statistical analyses were performed using SPSS 18.0. Student’s t test was used to analyze the difference between AS and normal control groups. P<0.05 (two-tailed) indicates statistical significance.2.3 The third generation of cultured AS fibroblasts were obtained. Fibroblasts were transfected with lentiviral vectors for overexpressing human DKK-1 or an shRNA for silencing DKK-1; empty vector was used as a negative control. Ascorbic acid (AA), P-glycerophosphate, and dexamethasone were added to osteogenic medium to induce osteogenesis. Flow cytometric analysis and a 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay were used to detect AS fibroblast proliferation after transfection. The expression levels of P-catenin, phosphorylated β-catenin, c-Myc, cyclin D1, and the osteogenesis markers ALP, OCN, and Runx2 were then examined by western blot analysis. Alizarin red staining (ARS) was also used to observe biomineralization activity.One-way ANOVA followed by the LSD multiple-range test was used to analyze the differences between groups. P<0.05 (two-tailed) indicates statistical significance.2.4 The previous cultured AS fibroblasts were retreated with PGE-2 for proliferation and osteogenic induction. Different doses of celastrol and indometacin were added at day 12 to observe their effects on proliferation and osteogenic differentiation. Cell proliferation was performed at day 12,14,17,20.The Edu analysis for each group was performed at day 14.RT-PCR for osteogenic markers BMP-2,OCN,Runx2 and Type I collagen was performed at day 14,21,28,the activity of alkalineas well as phosphatase alizarin red staining for each group was performed at day 14,21,28. Western blot analysis was tested for the proteins of PGE-2,AKT,PI3K,GSK-3β,DKK-1,SOST and β-cantenin at day 14,21,28.One-way ANOVA followed by the scheffe’s test was used to analyze the differences between groups. P<0.05 (two-tailed) indicates statistical significance.Results3.1 No significant difference was seen between AS patients and healthy controls for total Dkkl levels. Decreased levels of functional DKK-1 in serum were found in AS patients compared with healthy controls. Functional DKK-1 levels in serum of AS patients were significantly associated with the disease radiographic severity evaluated by modified New York grading criteria and mSASSS system.3.2 DKK-1 was downregulated in hip sy no vial tissues from AS patients compared to that observed in controls. AS fibroblasts exhibited excessive proliferation, a higher growth rate, and a decreased apoptotic rate.3.3 EdU assay demonstrated that DKK-1 suppressed the growth of AS fibroblasts. Downregulation of DKK-1 decreased the phosphorylation of β-catenin and upregulated the expression of β-catenin, c-Myc, cyclin D1, and osteogenesis markers. Overexpression of DKK-1 had the opposite effect, resulting in the inhibition of the Wnt/β-catenin pathway. ARS showed an increase in biomineralization activity after the inhibition of DKK-1.3.4 Celastrol significantly inhibits cell proliferation of isolated AS fibroblasts and in vitro osteogenic differentiation compared with control groups in a time-and dose-dependent manner.Conclusions4.1 Serum functional DKK-1 levels showed an independent and negative correlation with radiographic severity of the disease in patients with AS. Functional DKK-1 in serum might serve as a potential biomarker for reflecting the progression of ankylosing spondylitis.4.2 AS fibroblasts are characterized by an imbalance between proliferation and apoptosis. The proliferation and osteogenesis of fibroblasts in AS combine to contribute to abnormal new bone formation. DKK-1 expression is attenuated in AS patients.4.3 Downregulation of DKK-1 enhances fibroblast proliferation and osteogenic ability through Wnt/β-catenin signaling, which indicates that DKK-1 may play a role in switching to new bone formation in AS progression.4.4 Our results demonstrated that celastrol could inhibit isolated AS fibroblast proliferation and in vitro osteogenic differentiation. The interaction of PGE-2,PI3K/AKT signaling and Wnt protein may be involved in the process. Further studies should be performed in vivo and animal models to identify the potential effect of celastrol on the bone metabolism of AS patients.