| Part 1:The study on protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by cyclic strainObjective:To investigate the protective effects of Salubrinal on mandibular condylar chondrocytes (All chondrocytes are specific to rat mandibular condylar chondrocytes) apoptosis induced by cyclic strain.Methods:1. Chondrocytes from 3-week-old Sprague-Dawley rats were isolated and cultured in vitro. We used third-generation chondrocytes in order to maintain their original characteristics.2. Chondrocytes were subjected to 20% elongation of cyclic strain for 24 hours (rectangular wave) or with a combination of 40mmol/L Salubrinal for 30 minutes. Chondrocytes were divided into three groups:Control group,20% cyclic strain group, Salubrinal+20% cyclic strain group. Flow cytometer was used to detect cell apoptosis after 24h treatment. The expression of GRP78, GRP94, CHOP, Caspase-12 was detected by real-time quantitive PCR and Western Blot.Results:1. Chondrocytes apoptosis increased (P<0.05) after 24 hours treatment of 20% cyclic strain. Pretreatment of Salubrinal could decrease chondrocytes apoptosis induced by cyclic strain (P<0.05).2.20% Cyclic strain increased the expression of CHOP, Caspase-12 on transcription level and Caspase-12 expressionon protein translation level in chondrocytes. Salubrinal+20% Cyclic strain down regulated their expression.Conclusion:1.20% Cyclic strain triggered chondrocytes apoptosis through ER stress.2. Salubrinal protected chondrocytes from apoptosis induced by 20% cyclic strain through down regulated ER stress signaling proteins.Part 2:The study on protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by hypoxiaObjective:To investigate the protective effects of Salubrinal on mandibular condylar chondrocytes apoptosis induced by hypoxia.Methods:1. In vivo experiment:we used an established animal model for loading compressive mechanical stress to mandibular condylar cartilage of 6-week-old Sprague-Dawley rats for 7 days, Salubrinal pretreatment was local intra-articular injection. Control group SD rats do not wear any equipment. Detect the expression of hypoxia-inducible factor-1α of mandibular condylar cartilage through immunohistochemistry.2. Invitro experiment:Mandibular Condylar chondrocytes from 3-week-old Sprague-Dawley rats were isolated and cultured in vitro. We used third-generation mandibular condylar chondrocytes in order to maintain their original characteristics.3. Chondrocytes were divided into three groups:Control group, Hypoxia group, Salubrinal+hypoxia group. For hypoxia treatment, cells were cultured in a hypoxia chamber flushed with 5% O2 and 5% CO2 with balance of 90% nitrogen at 37℃ for 24h or 1% O2 and 5% CO2 with balance of 94% nitrogen for 30h. Control cells were placed in a 5% CO2 and 95% air incubator (20% O2) at 37℃. Salubrinal pretreatment (40 mmol/L) should be 30 mins prior to hypoxia culture. Cell apoptosis was detected by flow cytometry.4. Select the appropriate hypoxia stimulation magnitude of chondrocytes according to the results of flow cytometry.The expression of GRP78, GRP94, CHOP, Caspase-12 was detected by real-time quantitive PCR and Western Blot. The expression of Hif-1 a was also detected by Western Blot.Results:1. The expression of hypoxia-inducible factor-1a (Hif-1 a) in chondrocytes was increased after applying compressive mechanical stress to mandibular condylar cartilage(P<0.05). Pretreatment of Salubrinal could decreased the expression of Hif-1 a.2.5% 02,24h hypoxia did not induce chondrocytes apoptosis while 1% 02,30h hypoxia induced chondrocytes apoptosis(P<0.05). Pretreatment of Salubrinal could decrease chondrocytes apoptosis induced by hypoxia (1% 02,30h).3. Under normoxia (20% 02) condition, Hif-1 a was not expressed.1% 02,30h hypoxia induced Hif-1 a protein expression and Salubrinal pretreatment would reduce the increase of hypoxia-induced Hif-1 a expression (P<0.05).1% 02,30h hypoxia increased the expression of GRP78, GRP94, CHOP, Caspase-12 on both transcription and protein translation level in chondrocytes. Salubrinal+1% 02,30h hypoxia down regulated their expression.Conclusion:1. Condyle cartilage of SD rats under compressive mechanical stress for 7 days was in hypoxia state.2. Hypoxia (1%,30h) triggered chondrocytes apoptosis through ER stress.3. Salubrinal protected chondrocytes from apoptosis induced by hypoxia (1%,30h) through down regulated ER stress signaling pathway. |
