A combination of the "disease-syndrome-symptom" approach was used to study the syndrome characterization and features of dampness-heat obstruction syndrome in papain-induced knee osteoarthritis(KOA) model rats during the disease process. Forty-eight male SD rats were randomly divided into sham and model groups. The KOA model was established by injecting a mixture of papain and L-cysteine into the joint cavity on days 1, 3, and 5. During the 8 weeks following model establishment, the rats were assessed weekly for the plantar mechanical pain threshold, knee joint diameter, local skin temperature of the knee joint, weight-bearing difference between the two hind feet, and the modified Lequesne MG score of the knee joint. Samples were collected at 1, 2, 4, 6, and 8 weeks after model establishment to observe the gross lesions in cartilage and synovium. Histopathological changes in joint tissues were examined using hematoxylin-eosin, Masson's trichrome, and Senna red O-solid green staining. ELISA and immunohistochemical analysis were performed to detect the levels of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, prostaglandin E2(PGE2), and the expression of aquaporins(AQP) 1 and 3 in serum and synovium. The results showed that the ink score of articular cartilage in the model group significantly increased from 4 to 8 weeks, the cartilage Mankin's score and the percentage of Masson-positive area in cartilage increased significantly from 1 to 8 weeks. The percentage of red-stained area for cartilage proteoglycans decreased significantly from 1 to 8 weeks. The synovitis score from 1 to 6 weeks and the percentage of blue-stained collagen fibers in the synovium from 1 to 8 weeks increased significantly, with statistically significant differences compared to the sham group. The mechanical pain threshold in the model group significantly decreased from 1 to 8 weeks, the knee joint diameter significantly increased from 1 to 6 weeks, and the local skin temperature of the knee joint, the weight-bearing difference between the two hind feet, and the modified Lequesne MG score from 1 to 5 weeks significantly increased, all with statistically significant differences compared to the sham group. The levels of IL-1β, IL-6, TNF-α, and PGE2 in serum and synovium of the model group significantly increased from 1 to 6 weeks. Serum TNF-α and PGE2, and synovial IL-1β, also significantly increased at 8 weeks. The levels of cartilage AQP1 and AQP3 significantly increased from 1 to 4 weeks, while synovial AQP1 and AQP3 increased significantly from 1 to 6 weeks, with all differences statistically significant compared to the sham group. In conclusion, papain-induced KOA rats exhibited pathological changes, including articular cartilage degeneration and synovial inflammation, within 1 week of induction. The KOA rats showed characteristics of dampness-heat obstruction syndrome, such as joint pain, swelling, elevated skin temperature, and decreased function, as well as increased inflammatory factors and AQP1、AQP3 in serum and joint tissues within 5 to 6 weeks of disease onset. These results provide an experimental model for studying the syndromes of KOA with dampness-heat obstruction syndrome.
Animals ; Male ; Rats, Sprague-Dawley ; Rats ; Osteoarthritis, Knee/physiopathology* ; Disease Models, Animal ; Humans ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Knee Joint/pathology*

Objective Based on 25 indicators including immune factors, cell count classification, and smear results of the knee joint fluid, machine learning models were established to distinguish between osteoarthritis (OA) and rheumatoid arthritis (RA). Methods 100 OA and 40 RA patients scheduled for total knee arthroplasty were enrolled respectively. Each patient's knee joint fluid was collected preoperatively. Nucleated cells were counted and classified. The expression levels of immune factors, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-8, IL-15, matrix metalloproteinase 3 (MMP3), MMP9, MMP13, rheumatoid factor (RF), serum amyloid A (SAA), C-reactive protein (CRP), and others were measured. Smears and microscopic classification of all the immune factors were performed. Independent influencing factors for OA or RA were identified using univariate binary logistic regression, Lasso regression, and multivariate binary logistic regression. Based on the independent influencing factors, three machine learning models were constructed which are logistic regression, random forest, and support vector machine. Receiver operating characteristic curve (ROC), calibration curve and decision curve analysis (DCA) were used to evaluate and compare the models. Results A total of 5 indicators in the knee joint fluid were screened out to distinguish OA and RA, which were IL-1β(odds ratio(OR)=10.512, 95× confidence interval (95×CI) was 1.048-105.42, P=0.045), IL-6 (OR=1.007, 95×CI was 1.001-1.014, P=0.022), MMP9 (OR=3.202, 95×CI was 1.235-8.305, P=0.017), MMP13 (OR=1.002, 95× CI was 1-1.004, P=0.049), and RF (OR=1.091, 95×CI was 1.01-1.179, P=0.026). According to the results of ROC, calibration curve and DCA, the accuracy (0.979), sensitivity (0.98) and area under the curve (AUC, 0.996, 95×CI was 0.991-1) of the random forest model were the highest. It has good validity and feasibility, and its distinguishing ability is better than the other two models. Conclusion The machine learning model based on immune factors in the knee joint fluid holds significant value in distinguishing OA and RA. It provides an important reference for the clinical early differential diagnosis, prevention and treatment of OA and RA.
Humans ; Arthritis, Rheumatoid/metabolism* ; Machine Learning ; Male ; Female ; Middle Aged ; Aged ; Synovial Fluid/immunology* ; Osteoarthritis, Knee/metabolism* ; Knee Joint/metabolism* ; ROC Curve ; Diagnosis, Differential

OBJECTIVE: To observe the effect of needle-knife on the chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis (KOA) based on the CircSERPINE2-miR-1271-5P-E26 specific transformation-related gene (ERG) axis, and to explore the mechanism of needle-knife for KOA. METHODS: Thirty-six New Zealand white rabbits were randomly divided into a normal group, a model group, a needle-knife group and a sham needle-knife group, 9 rabbits in each group. The rabbits in the model group, the needle-knife group and the sham needle-knife group were treated with modified Videman method to prepare KOA model. After successful modeling, the rabbits in the needle-knife group were treated with needle-knife at cord adhesion and nodules near quadriceps femoris tendon and internal and external collateral ligament on the affected knee joint; the rabbits in the sham needle-knife group were treated with sham needle-knife baside the needle insertion point of the needle-knife group (needle-knife was only inserted, without any operation). The treatment was given once a week, 3 times in total. The Lequesne MG behavioral score was used to evaluate the knee joint damage in each group before and after intervention. After intervention, HE staining and transmission electron microscopy were used to observe the cartilage tissue morphology and ultrastructure of chondrocytes in the knee joint in each group; TUNEL method was used to detect the level of chondrocyte apoptosis in the knee joint; real-time fluorescence quantitative PCR was used to detect the expression of CircSERPINE2, miR-1271-5P and ERG mRNA in knee cartilage tissue in each group. RESULTS: After intervention, compared with the normal group, the Lequesne MG behavioral score in the model group was increased (P<0.01). Compared with the model group and the sham needle-knife group, the Lequesne MG behavioral score in the needle-knife group was decreased (P<0.01). In the model group and the sham needle-knife group, the number of chondrocytes and organelles was decreased, the cell nucleus was shrunk, mitochondria was swelling or disappeared; in the needle-knife group, the number of chondrocytes and organelles was increased, the cell nucleus was not obviously shrunk and the mitochondria was not obviously swelling. Compared with the normal group, the level of chondrocyte apoptosis in the model group was increased (P<0.01); compared with the model group and the sham needle-knife group, the level of chondrocyte apoptosis in the needle-knife group was decreased (P<0.01, P<0.05). Compared with the normal group, the expression of CircSERPINE2 and ERG mRNA in the model group was decreased (P<0.01), and the expression of miR-1271-5P mRNA was increased (P<0.01); compared with the model group and the sham needle-knife group, the expression of CircSERPINE2 and ERG mRNA in the needle-knife group was increased (P<0.01), and the expression of miR-1271-5P mRNA was decreased (P<0.01). CONCLUSION Needle-knife could reduce the knee joint damage and chondrocyte apoptosis in KOA rabbits, which may be related to up-regulating the expression of CircSERPINE2 and ERG mRNA, and inhibiting the expression of miR-1271-5P mRNA.
Rabbits ; Animals ; Osteoarthritis, Knee/metabolism* ; Chondrocytes/metabolism* ; Knee Joint/surgery* ; Apoptosis ; MicroRNAs/genetics*

OBJECTIVE: To investigate the changes in the expression of voltage-gated potassium channel subunit KCNA2 in the dorsal root ganglion (DRG) neurons of rats with osteoarthritis (OA) pain induced by sodium monoiodoacetate and explore the mechanism. METHODS: A total of 156 adult male Sprague-Dawley rats were randomly divided into blank control group, saline group and intra-articular monoiodoacetate injection-induced OA group. The paw withdrawal mechanical threshold (PWMT) was measured before and at 1, 2, 4, and 6 weeks after monoiodoacetate injection. At 4 weeks after the injection, the pathological changes in the knee joints were analyzed using HE staining and Safranin O-Fast Green staining, and the expression of activating transcription factor 3 (ATF-3) and inducible nitric oxide synthase (iNOS) in the DRG neurons were detected by immunofluorescence staining. The expression of mRNA in the DRG neurons was detected by RT-qPCR at 1, 2, 4 and 6 weeks after the injection. The expression of KCNA2 in the DRG was measured by Western blotting, and the methylation level of promoter region was measured by MSPCR at 4 weeks after the injection. RESULTS: The PWMT of the rats in OA group was significantly decreased at 2, 4, and 6 weeks after the injection as compared with the baseline ( < 0.05 or < 0.001) as well as the control group ( < 0.05 or < 0.001). Four weeks after the intra-articular injection, fractures and defects on the surface of the articular cartilage, bone hyperplasia, and blurred tidal line were observed in the rats in OA group, but no obvious pathological changes were detected in the control or saline groups. Compared with those in the control group, the expressions of ATF-3 and iNOS were significantly increased ( < 0.01) at 4 weeks after injection; the expression of mRNA at 2, 4 and 6 weeks and the expression of KCNA2 protein at 4 weeks were all significantly decreased ( < 0.05 or < 0.01), and the methylation level of gene was significantly increased at 4 weeks after the injection in OA group ( < 0.01). CONCLUSIONS The expression of KCNA2 is decreased in the DRG neurons of rats with OA pain likely as a result of enhanced methylation of promoter region.
Animals ; Disease Models, Animal ; Ganglia, Spinal ; Knee Joint ; Kv1.2 Potassium Channel ; metabolism ; Male ; Osteoarthritis ; complications ; metabolism ; Pain ; etiology ; metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1control group (group A, n=6); SIRT1osteoarthritis group (group B, n=6); SIRT1control group (group C, n=6); SIRT1osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1osteoarthritis group and SIRT1control group, SIRT1 protein expression was not obviously changed in the SIRT1osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.
Animals ; Cartilage ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Humans ; Knee Joint ; metabolism ; pathology ; Mice ; Mice, Knockout ; Oncogene Protein v-akt ; genetics ; Osteoarthritis ; genetics ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; Signal Transduction ; genetics ; Sirtuin 1 ; genetics ; Sterol Regulatory Element Binding Protein 2 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo compare and estimate the diagnostic value and characteristic of different diagnostic methods (blood laboratory test, histological analysis, synovial fluid cytological test and microbiological examination) in detecting the presence of periprosthetic joint infection.

METHODSData of 52 patients underwent hip or knee joint revision in Peking University People's Hospital Arthritis Clinic and Research Center between July 2013 and March 2015 were analyzed retrospectively. For each patient, results of blood laboratory tests(peripheral-blood white blood cell, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (Hs-CRP)), histological analysis, synovial fluid white cell count (SWCC), microbiological examinations (synovial fluid, tissue and prosthetic joint sonication fluid) were collected. Data were analyzed by t-test, independent sample median test or χ(2) test, respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for each method were calculated and compared by receiver operating characteristic curve.

RESULTSThere were 30 female and 22 male patients. Twenty-one patients (40.4%) were diagnosed as PJI. The levels of CRP, ESR, IL-6 and Hs-CRP in patients with PJI were higher than that in aseptic failure patients (Z=23.084, 13.499, 5.796, 17.045, all P<0.05). The sensitivities of CRP, ESR, IL-6 and Hs-CRP were 90.5%, 81.0%, 95.0% and 90.0%. The sensitivities of histological analysis and SWCC were 55.0% and 70.6%, while they had high specificity as 89.7% and 85.7%. The sensitivity of sonication fluid culture was 90.0%, which was higher than that of tissue culture (71.4%) and synovial fluid culture (65.0%) (χ(2) = 5.333, 6.400, all P<0.05).

CONCLUSIONSThe tests of CRP, ESR, IL-6 and Hs-CRP have good value in detecting PJI preoperatively. Histological analysis and SWCC have high specificity, which could help to exclude PJI. Sonication fluid culture has a higher sensitivity than tissue culture and synovial fluid culture.

Arthroplasty, Replacement, Hip ; Arthroplasty, Replacement, Knee ; Blood Sedimentation ; C-Reactive Protein ; metabolism ; Female ; Humans ; Interleukin-6 ; blood ; Knee Joint ; Male ; Prosthesis-Related Infections ; diagnosis ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity ; Synovial Fluid ; cytology

PURPOSE: The aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilage samples from OA knee joints was examined at mRNA and protein levels. MATERIALS AND METHODS: Knee joints were examined using plain radiographs, and OA severity was assessed using the Kellgren and Lawrence (KL) grading system. Specimens were collected from 29 patients (31 knees) who underwent total knee replacement because of severe medial OA of the knee (KL grades 3 and 4), 16 patients who underwent knee arthroscopy (KL grade 2), and 5 patients with traumatic knees (KL grade 0). HIF-2α and VEGF expression was quantified by real-time polymerase chain reaction and western blotting. RESULTS: Cartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05). CONCLUSION: In OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expression of both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.
Adult ; Aged ; Arthroplasty, Replacement, Knee ; Arthroscopy ; Basic Helix-Loop-Helix Transcription Factors/*metabolism ; Biomarkers/*blood ; Cartilage/*metabolism ; Female ; Humans ; Knee Joint/*diagnostic imaging ; Male ; Middle Aged ; Osteoarthritis, Knee/*blood/diagnostic imaging/physiopathology ; RNA, Messenger ; Radiography ; Real-Time Polymerase Chain Reaction ; Severity of Illness Index ; Vascular Endothelial Growth Factor A/*metabolism

OBJECTIVETo investigate the effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9, vascular endothelial growth factor (VEGF) and type X collagen (ColX) in impaired cartilage of rabbits with knee osteoarthritis (KOA) and explore its possible mechanisms.

METHODSForty New Zealand rabbits were randomized equally into normal control group, KOA model group, eletroacupuncture with close-to-bone needling group (CN group), and normal thrust needing group (NTN group). In the latter 3 groups, KOA was induced by Hulth-Telhag treatment and evaluated with X-ray examination, and 6 weeks after the modeling, eletroacupuncture for 20 min was administered in CN and NTN groups at the acupoints "Zusanli", "Waixiyan", "Neixiyan", "Liangqiu" and "Yinlingquan" in the left knee joints once daily for 5 days as a treatment cycle. After 5 treatment cycles, the rabbits were examined for behavioral changes, cartilage morphology, and Mankin scores; The protein and mRNA expressions of S0x9, VEGF, and ColX were examined using Westen blotting, immunohistochemistry, and RT-PCR as appropriate.

RESULTSThe rabbits in the model, CN and NTN groups showed significant changes in behaviors and cartilage histomorphology after the modeling and after the treatments. HE staining showed that cartilage injury was repaired and tended to recovery in CN and NTN groups. The cartilage pathologies was severer in the model group than in the normal control, CN and NTN groups (P<0.01); Sox9 protein increased and VEGF mRNA level decreased in CN and NTN groups after treatment as compared with those in the model group (P<0.01).

CONCLUSIONEletroacupuncture with close-to-bone needling can effectively improve KOA in rabbits probably by enhancing Sox9 and reducing VEGF and ColX expressions in the cartilage to inhibit hypertrophic differentiation of the chondrocytes, maintain chondrogenic phenotype and repair cartilage cells.

Acupuncture Points ; Animals ; Cartilage, Articular ; metabolism ; pathology ; Cell Differentiation ; Chondrocytes ; cytology ; Chondrogenesis ; Collagen Type X ; metabolism ; Electroacupuncture ; Knee Joint ; physiopathology ; Osteoarthritis, Knee ; therapy ; Rabbits ; SOX9 Transcription Factor ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis (OA) patients, and explore the relationship between the miRNA-140 expression and OA severity.Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence (KL) grade 1-2], moderate (KL grade 3) and severe (KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 snRNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant (F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well (F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.
Adult ; Aged ; Cells, Cultured ; Chondrocytes ; metabolism ; Female ; Humans ; Knee Joint ; metabolism ; Male ; MicroRNAs ; analysis ; Middle Aged ; Osteoarthritis, Knee ; metabolism ; Real-Time Polymerase Chain Reaction ; Synovial Fluid ; metabolism

OBJECTIVETo explore the effect of Bushen Gujin Recipe (BGR) on serum and synovial expression of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) in knee osteoarthritis (KOA) model rabbits.

METHODSTotally 36 8-month-old healthy male New Zealand white rabbits were randomly divided into 4 groups, i.e., the normal control group, the model group, the Western medicine group (Meloxicam, at the daily dose of 6 mg/kg), and the TCM group (BGR, at the daily dose of 53 g/kg), 9 in each group. Modeling was performed in all rabbits except those in the normal control group by using Hulth A method. All medication was performed for 8 consecutive weeks. Contents of IL-1 and TNF-α were detected using ELISA from serum, partial synovial tissue of the front knee joint, cartilage and subchondral bone of the medial femoral condyle.

RESULTSThe joint space became narrowed in the Western medicine group, ranging between the model group and the TCM group. The articular surface was rough with obvious osteophytes. The joint space was slightly narrower in the TCM group; the articular surface was slightly rough with mild osteophytes. Compared with the normal control group, contents of IL-1 and TNF-α in serum and synovial increased in the model group (P < 0.01). Compared with the model group, contents of IL-1 and TNF-α in serum and the synovial fluid decreased in the two treatment groups (P < 0.01). There was no statistical difference in contents of IL-1 and TNF-α between the Western medicine group and the TCM group.

CONCLUSIONBGR promoted the synthesis of cartilage matrix and carti- lage repair through inhibiting the secretion of IL-1 and TNF-α, and prolonging cartilage degeneration.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interleukin-1 ; metabolism ; Knee Joint ; Male ; Osteoarthritis, Knee ; drug therapy ; metabolism ; Rabbits ; Synovial Fluid ; Synovial Membrane ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism