Objective To review the research progress on protease-activated receptor 2 (PAR-2) in the pathogenesis of osteoarthritis (OA). Methods The relevant literature about the mechanism of PAR-2 in the occurrence and development of OA in recent years was extensively reviewed and comprehensively analyzed. Results Abnormal activation of PAR-2 plays an important role in responses to occurrence and development of OA. Through regulating production and releasing of a variety of cytokines (such as inflammatory factors, metabolic factors, pain factors, etc.), the PAR-2 can involve in pathophysiological progression of OA articular cartilage, subchondral bone, and synovial membrane, as well as occurrence and transmission of pain. Conclusion PAR-2 participation in the development of OA has been confirmed. However, since PAR-2 is complicated and widespread, it is necessary to study the specific role of PAR-2 and the interaction between various signal pathways in the progression of OA, and to elucidate the potential pathophysiological mechanisms of PAR-2 participating in the process of OA, in the hope of exploring the new targets for the effective control of OA.
ObjectiveTo summarize the active changes of Wnt signaling pathway in osteoarthritis (OA) as well as the influence and mechanism of dual-targeted regulation on cartilage and subchondral bone and the role of crosstalk between them on OA process.MethodsThe relevant literature concerning the articular cartilage, subchondral bone, and crosstalk between them in OA and non-OA states by Wnt signaling pathway in vivo and vitro experimental studies and clinical studies in recent years was reviewed, and the mechanism was analyzed and summarized.ResultsWnt signaling can regulate the differentiation and function of chondrocytes and osteoblasts through the classic β-catenin-dependent or non-classical β-catenin-independent Wnt signaling pathway and its cross-linking with other signaling pathways, thereby affecting the cartilage and bone metabolism. Moreover, Wnt signaling pathway can activate the downstream protein Wnt1-inducible-signaling pathway protein 1 to regulate the progress of OA and it also can be established gap junctions between different cells in cartilage and subchondral bone to communicate molecules directly to regulate OA occurrence and development. Intra-articular injection of Wnt signaling inhibitor SM04690 can inhibit the progress of OA, and overexpression of Wnt signaling pathway inhibitor Dickkopf in osteoblasts can antagonize the role of vascular endothelial growth factor work on chondrocytes and inhibit the catabolism of its matrix.ConclusionThe regulation of metabolism and function of cartilage and subchondral bone and crosstalk between them is through interactions among Wnt signaling pathway and molecules of other signaling. Therefore, it plays an vital role in the occurrence and development of OA and is expected to become a new target of OA treatment through intervention and regulation of Wnt signaling pathway.
Objective Simvastatin has been reported to be effective on stimulation of bone formation. To investigate the effects of simvastatin on bone formation relative factors of proximal tibia trabecular bone and on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Fourty 1-week-old male Sprague Dawley rats were divided randomly into 2 groups, 20 rats per group. Rats in experimental group received subcutaneous injection of simvastatin [(5 mg/ (kg• d)], and the rats in control group received injection of normal sal ine at the same dose. The expressions of bone morphogenetic protein 2 (BMP-2), matrix metalloproteinase 13 (MMP-13), and vascular endothel ial growth factor (VEGF) of trabecular bone were analyzed in the tibia by immunohistochemical staining at 1 and 3 weeks after injection. BMSCs from the rat femur at 1 and 3 weeks after injection were cultured under condition of osteogenic induction. ALP staining wasperformed on the 14th day after culture; real-time fluorescent quantitative PCR was used to detect the mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 on the 21st day after culture; and von Kossa staining was performed on the 28th day after culture. Results There was no significant difference in the expressions of BMP-2, MMP-13, and VEGF betweenthe experimental group and control group at 1 and 3 weeks after injection (P gt; 0.05). There was no significant difference in the percentages of ALP positively-stained cells between the experimental group and the control group on the 14th day after culture (P gt; 0.05). The mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 in osteogenic differentiation-inducedBMSCs had also no significant difference between the experimental group and the control group at 1 and 3 weeks after culture (P gt; 0.05). No significant difference in biomineral ization was found between the experimental group and control group at 1 and 3 weeks after culture (P gt; 0.05). Conclusion Subcutaneous injection of simvastatin [(5 mg/(kg•d)] for 1 or 3 weekscan affect neither the expressions of bone formation relative factors of proximal tibia trabecular bone nor the osteogenic differentiation of the BMSCs.
Objective To investigate the feasibil ity of alendronate (ALN) in treating osteoarthritis (OA) by observing the effects of ALN on interleukin 1β (IL-1β) induced chondrocytes of rat in vitro. Methods The chondrocytes of knee articular surface from 15 SD rats (1-month-old, female or male, weighing 100-150 g) were cultured. The chondrocytes were observed by inverted phase contrast microscope and identified by toluidine blue staining and HE staining. The third passage chondrocytes were divided into 3 groups: the chondrocytes were cultured with DMEM for 5 days in group A, with 10 ng/mL IL-1β for 2 days and with DMEM for 3 days in group B, and with 10 ng/mL IL-1β for 2 days and with 1 × 10-6 mol/L ALN for 3 days in group C. Immunocytochemistry and real-time PCR were performed to determine the expression levels of collagen type II (Col II), matrix metalloproteinase 13 (MMP-13), and β-catenin. Results Toluidine blue staining proved that the cultured cells were chondrocytes. The integrated absorbency (IA) value of Col II in group C (10.290 7 ± 0.499 2) was lower than that in group A (15.377 0 ± 0.571 8) and higher than that in group B (5.463 2 ± 0.450 4), showing significant differences (P lt; 0.05). The IA value of MMP-13 in group C (3.068 6 ± 0.205 6) was significantly lower than that in group B (6.998 1 ± 0.329 7, P lt; 0.05), but there was no significant differenc when compared with group A (2.777 5 ± 0.199 6, P gt; 0.05). The IA value of β-catenin in group C (6.611 7 ± 0.381 8) was lower than that in group B (11.799 9 ± 0.348 7) and higher than that in group A (4.390 3 ± 0.551 9), showing significant differences (P lt; 0.05). The mRNA expression of Col II in group C was significantly higher than those in groups A and B (P lt; 0.05), the mRNA expression of MMP-13 in group C was significantly lower than that in group B (P lt; 0.05) but there was no significant difference when compared with group A (P gt; 0.05). The mRNA expression of β-catenin in group C was significantly lower than that in group B (P lt; 0.05) and higher than that in group A (P lt; 0.05). Conclusion ALN can protect rat chondrocyte from OA induced by IL-1β in vitro possibly by upregulating Col II and inhibiting the expression of MMP-13 and β-catenin in the chondrocytes.
Objective To investigate the effects of xianl inggubao (XLGB) on subchondral bone and articular cartilage in the rat osteoarthritis model induced by anterior cruciate l igament transection (ACLT). Methods Twentyfour 3-month-old female SD rats were divided randomly into 3 groups (n=8): Sham group (group A), ACLT group (group B) and XLGB group (group C). The osteoarthritis model was made by ACLT in groups B and C, the joint cave was sutured after exposure of ACL in group A. After 4 days, XLGB was given at 250 mg/(kg·d) in group C and the equivalent amount of sal ine was given in groups A and B. After 12 weeks, the gross appearance of femoral condyles was observed, the degree of cartilagedegeneration was scored by Mankin scoring system. The immunostaining for MMP-13 was performed to investigate the effect of XLGB on prevention of cartilage matrix loss. The bone mineral density (BMD) measurement and bone histomorphometric analysis were done in subchondral bone of right distal femur and proximal tibia after 12 weeks. Results The gross appearance of femoral condyles showed that ulcer in the group C was smaller than that in group B after 12 weeks. The Mankin’s scale and IA value for MMP-13 in group C were markedly lower than those in group B (P lt; 0.05). BMD of the subchondral bone in the group B was significantly lower than those in the groups A and C (P lt; 0.05). The bone mass in group C were significantly higher than that in group B (P lt; 0.05). Conclusion Oral administration of XLGB (250 mg/ kg per day) for 12 weeks could prevent the cartilage degeneration of rats after ACLT, down-regulating MMP-13 and increasing subchondral bone mass might participate in this process.
To evaluate the effects of XiangLingGuBao (XLGB) on femoral fracture heal ing in ovariectomized (OVX) rats. Methods Forty 12-week-old female SD rats weighing (258 ± 14) g were divided randomly into 4 groups (n=10 per group): group A, sham operation by opening the abdominal cavity; group B, bilateral ovariectomy; group C, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and normal sal ine by gavage; group D, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and 250 mg/(kg•d) XLGB by gavage. The weight of rabbits in groups A and B was measured 0, 1, 2, 3, 4 and 5 weeks after operation. The right femur of each rat was obtained 5 weeks after operation. Total femur bone mineral density (tBMD), distal femur bone mineral density (dBMD) and middle femur bone mineral density (mBMD) were measured by double energy X-ray absorptiometry CR filming, HE staining and immunohistochemistry staining of groups C and D were performed. Results The weight of rats in group B was obviously higher than that of group A at 3, 4 and 5 weeks after operation (P lt; 0.05), indicating the animal model of postmenopausal osteoporosis was establ ished successfully. CR films showed more callus and obscure fracture l ine in group D, while less callus and distinct fracture l ine in group C. The tBMD and the dBMD of group B were far less than that of group A, the mBMD of group D was significantly higher than that of group C(P lt; 0.05), the tBMD and the dBMD of group D were higher than that of group C, but no significant difference was evidentbetween two groups (P gt; 0.05). Histology observation showed, when compared with group C, most fracture ends in groupD reached bone union, and the introduction of capillaries was evident in the marrow cavity. Immunohistochemistry staining demonstrated that the BMP-2 integrated absorbance (IA) value in groups C and D was 2.236 6 ± 0.181 8 and 3.727 3 ± 0.874 2, respectively, the VEGF IA value in groups C and D was 2.835 5 ± 0.537 0 and 3.839 6 ± 0.223 0, respectively, indicating there were significant differences between two groups (P lt; 0.05). Conclusion XLGB can obviously promote the femoral fracture heal ing in OVX rats, and speed the transformation of woven bone into lamellar bone, which may rely on its role of enhancing expression of BMP-2 and VEGF.
Objective To confirm the stimulating effect of simvastatin on BMSCs of SD rats osteogenic differentiation, and to further study the role of Wnt signal ing pathway in this process. Methods BMSCs derived from the tibia and femur of 6-week-old female SD rats were cultured in vitro．Two groups were establ ished: control group and experimental group. After the 2nd passage, the cells of experimental group were treated with simvastatin (1 × 10-7mol/L) and the cells of control group with absolute ethyl alcohol and PBS. ALP staining was used at 7 days and von Kossa staining was appl ied at 28 days to assess osteoblastic differentiation and mineral ization. Real-time quantitative PCR was performed to evaluate theexpressions of Axin2, β-catenin, osteocalcin (OC), frizzled-2, Lef-1, and Wnt5a mRNA at 7 days and 14 days after simvastatin treatment. Results The observation of inverted phase contrast microscope showed that the majority of cells were polygonal and triangular in the experimental group, and were spindle-shaped in the control group at 7 days. The ALP staining showed blue cytoplasm, the positive cells for ALP staining in the experimental group were more than those in the control group at 7 days. The von Kossa staining showed that mineral ization of extracelluar matrix at 28 days in two groups, but the mineral ization in the experimental group was more obvious than that in the control group. The expression of Axin2 mRNA was significantly lower, and frizzled-2, Lef-1 mRNA were significantly higher in the experimental group than in the control group (P lt; 0.05) at 7 days, while the mRNA expressions of Axin2, OC, frizzled-2, Lef-1, and Wnt5a were significantly higher in the experimental group than in the control group at 14 days (P lt; 0.05). Conclusion Simvastatin can promote the osteogenic differentiation of BMSCs and change the expression of mRNA of some components of Wnt signal ing pathway.
Objective To examine the effects of alendronate (ALN) on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate l igament transection (ACLT). Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: thechondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days, subsequently with (ALN group, group A1) orwithout (IL-1β group, group B1) 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n=8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/(kg·d) for 8 weeks. Rabbits of group B2 and C2 received equal normal sal ine treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was appl ied to subchondral bone of proximal tibia. Results In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was sl ightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was sl ightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lowerthan that of group B2 (P lt; 0.05), but significantly higher than that of group C2 (P lt; 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was sl ightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P gt; 0.05..........