| Part one Effects of calcitonin on articular cartilage of knee joints in ACLT-induced rat modelsObjective:To investigate the effect of calcitonin (CT) on articular cartilage degeneration in the anterior cruciate ligament transection (ACLT)-induced rat knee joint models of osteoarthritis (OA).Methods:Thirty3-month-old male Sprague-Dawley (SD) rats were randomly into three groups, as follow:Sham group, ACLT+Normal saline (ACLT+NS) group and ACLT+CT group. The ats in ACLT+NS group and ACLT+CT group were received ACLT surgery on the right knees. In Sham group, the rats were deal with Sham-operation. After the operation, rats in ACLT+CT group were subcutaneously injected CT with5IU/kg-2d dosage. The rats in ACLT+NS group and in Sham group were subcutaneously administrated with normal saline. All animals were killed after the surgery at12weeks. Right knee joints spaces were opened. Then, cartilage surfaces of femur and tibia were observed and recorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na (pH7.4, at4℃). Tissues were regularly dehydrated, embedded in paraffin, and cut into6-8um-thick sections. The sections were stained with Safranin-O/fast green and hematoxylin-eosin (HE). Using with light microscope (Olympus BX61, Japan), three color digital images were recorded to analyze articular cartilage lesions from each section in different regions. For morphological changes of cartilage analysis, Mankin’s score system was properly adjusted and applied. The expressions of type Ⅱ collagen, ADAMTS-4and MMP-3antibody in cartilage were detected using immunohistochemistry method. Positive expression was quantified by optical density method, using with Image pro-Plus (IPP) software.Results:1. Sham-operation did not affect cartilage surface. We noted that cartilage was rather integrity and smooth. The remarkable lesions were observed in ACLT+NS group, local ulceration and erosion occurred in the part of cartilage surface. In ACLT+CT group, articular cartilage surfaces showed that there was no significant damage. Cartilage showed slight rough appearances.2. Safranin-O/fast green staining showed that cartilage and subchondral bone were stained with different color, respectively. In Sham group, distribution of cell and staining was in order. The boundary between calcified cartilage and subchondral bone was integrity. In the ACLT+NS group, cartilage lesions were severe. Cartilage displaced surface fissuring, cell loss, cell cloning and staining disarrangement. The thickness of cartilage significantly lessened. Subchondral bone invaded calcified cartilage. In the ACLT+CT group, there was not significant crack, slight erosion was observed in cartilage surface.3. Mankin scores in ACLT+NS group was higher than that in sham group and in ACLT+CT group (P<0.05). There was a significant difference of Mankin scores between in Sham group and in ACLT+CT group (P<0.05).4. Immunohistochemical staining displaced that expression of type II collagen was significantly suppressed in ACLT+NS group and in ACLT+NS group compared to Sham group (P<0.05). The levels of ADAMTS-4and MMP-3expressions in ACLT+NS group and in ACLT+NS group were markedly lower than that in Sham group. CT treatment significantly stimulated synthesis of type II collagen and markedly inhibited ADAMTS-4and MMP-3expressions compared to ACLT+NS group (P<0.05).Conclusion:Early intervention with CT stimulated type II collagen synthesis and inhibited expressions of ADAMTS-4and MMP-3in ACLT-induced rat models of OA.Part two Effects of calcitonin on subchondral trabecular bone of knee joints in ACLT-induced rat modelsObjective:CT was a potent bone-turnover agent and widely applied into abnormal bone metabolic diseases treatments. In this study, we tested the effect of early intervention with CT on subchondral bone mineral density and subchondral bone microstructure in ACLT-induced rat models.Methods:Three-month-old male SD rats were included in this study. All the rats were randomly divided into three groups (n=10per group):(1) Sham-operation group (Sham),(2) ACLT+normal saline group (ACLT+NS), and (3) ACLT+calcitonin group (ACLT+CT). Rats in ACLT+NS group and ACLT+CT group were received ACLT surgery. After that, rats in ACLT+CT group were subcutaneous injected with CT at a dosage of5units/kg-2d, rats in ACLT+NS group were subcutaneous injected with normal saline (NS) according to CT dosage. The other ten rats were received Sham-operation as control. All the rats were labeled with10mg/kg of calcein and10mg/kg tetracycline injected subcutaneous at10d and4d before they were sarcificed, respectively. All animals were treated for12weeks after surgery. After soft tissues removed, femurs were placed under the probe and followed same position. The right distal femur was scanned by dual energy X-ray absorptometry (DEXA). Tibia were fixed for72hours in70%ethanol and regularly dehydrated. Proximal tibiae were embedded in methylmethacrylate. Static parameters and dynamic parameters, including bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS), were measured from Gemisa-stained7-8μm-thick sections and unstained7-8μm-thick sections, respectively. For quantifying histologic parameters, we applied to IPP software.Results:1. BMD in Sham group was significant higher than that in ACLT+NS group and in ACLT+CT group (P<0.05). CT treatment attenuated BMD loss. In ACLT+CT group, BMD was significant increase when compared to that in ACLT+NS group(P<0.05).2. In ACLT+NS group, subchondral trabecular microstructure was markedly damaged, static parameters including BV/TV, Tb.Th and Tb.N were significant decrease compare to Sham group (P<0.05). In contrast, trabecular separation (Tb.Sp) value was obviously higher than Sham groups (P<0.05). The values of BV/TV, Tb.Th and Tb.N in ACLT+CT group were significant higher than that in ACLT+NS group, and lower value in Tb.Sp than ACLT surgery animals (P<0.05). Compared to Sham group, static parameters including BV/TV, Tb.Th and Tb.N were significant lower in ACLT+CT group, whereas Tb.Sp value was markedly higher than Sham groups (P<0.05).3. Analysis of dynamic parameters including MS/BS, MAR and BFR/BS showed a markedly difference among three groups (P>0.05). In ACLT+NS group, dynamic parameters including MS/BS、MAR and BFR/BS were significant decrease compare to Sham group (P<0.05). Compared to ACLT+NS group, CT treatment showed a great bone formation according by dynamic parameters. However, the dynamic parameters in ACLT+CT group was lower than that in Sham group (P<0.05).Conclusion:In ACLT-induced rat models of OA, CT inhibited subchondral bone mass loss and markedly improved subchondral trabecular microstructure, which maintained biomechanics functions of subchondral bone.Part three Effects of calcitonin on MMP-13expression via MAPKs signal pathway in IL-1/3-induced chondrocytesObjective:CT is not only well-known as anti-resorptive agent but also showed a chondroprotective effect in experimental OA. In this study, we discussed the role of CT on MAPK pathway in IL-1/3-inducd chondrocytes.Methods:Chondrocytes from1-month-old SD rat cartilage were cultured in Dulbecco’s modified eagle’s medium (DMEM) with10%fetalbovine serum at37℃with5%CO2. The second passage of chondrocytes was used for the following treatment. Chondrocytes were divided into6groups and cultured for24hours:(a) blank (0.5%-FBS DMEM);(b) vehicle (0.5%-FBS DMEM)+IL-1/3(lOng/ml);(c) CT (5ng/ml)+IL-1/3(10ng/ml);(d) CT (500ng/ml)+IL-1/3(lOng/ml);(e) CT (50ng/ml)+DMEM;(f) CT (500ng/ml)+DMEM. The cells in group c and d were incubated with IL-1/3for15min before the vehicle and CT pretreated. The protein expressions of MAPKs including p38, ERK1/2, JNK and MMP-13were detected by immunocytochemistry and western blot in chondrocytes. The IOD scores of positive expression was quantified by Image pro-Plus (IPP) softwareResults:1. Immunochemistry staining of phospho-p38, phospho-ERK1/2, phospho-JNK and MMP-13were observed in different groups. There were strong expressions of phospho-p38, phospho-ERK1/2, phospho-JNK and MMP-13in chondrocytes following incubating with IL-1/3for15minutes compared to normal cells. However, significant inhibitions of phospho-p38, phospho-ERK1/2and MMP-13were found in the chondrocytes pretreated with CT for24hours subsequent to IL-1/3-induced for15minutes when compared to the chondrocytes induced with IL-1/3. In addition, there was no significant different of phospho-JNK expression between IL-1/3-induced chondrocyts and CT pretreated chondrocytes, although a high level expression of phospho-JNK stimulated by IL-1/3was observed compared to normal chondrocytes.2. MAPKs pathway was analyzed by Western blot. The productions of phospho-p38, phospho-ERK1/2and MMP-13were increased significantly in IL-1/3-induced chondrocytes compared to control (P<0.05). The protein expressions of phospho-p38, phospho-ERK1/2and MMP-13were lower in pretreated with CT than IL-1/3-induced cells (P<0.05). However, there was no significant dose-depend inhibition of CT on phospho-p38, phospho-ERK1/2and MMP-13. In addition, CT pretreatment did not show influence on MAPKs in normal chondrocytes, and there was no effect of CT pretreatment on JNK protein. Conclusion:CT pretreatment inhibited MMP-13expression in IL-1/3-induced chondrocytes, which the protential mechanism of cell responses for CT pretreatment might be associated with phospho-p38and phospho-ERK1/2pathway in this study.Conclusion:Early intervention with CT attenuated articular cartilage lesions and protected subchondral bone microstructure in experimental rat models of OA, which delayed the development of OA. CT treatment could a protential therapeutic strategy of OA. |
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