Ca²⁺-dependent Ndoplasmic Reticulum Stress Correlates With Pathological Changes In Rat Mandibular Condylar Cartilage Under Compressive Mechanical Stress

Partl:The effect of Ca2+-dependent endoplasmic reticulum stress on rat mandibular condylar chondrocytesObjective:To study the effect of Ca2+-dependent endoplasmic reticulum stress on mandibular condylar chondrocytes after Tunicamycin (Tm, ERS inducer) treatment.Methods:Mandibular condylar chondrocytes from 3-week-old Sprague-Dawley rats were isolated and cultured in vitro. The chondrocytes were cultured, when the third-generation condylar chondrocytes grow over 80% treated with Tm which the final concentration is 4μg/ml. Groups of experiment presented here included control, Tm(4μg/ml) group, Tm+2APB group, Tm+Rya group, Tm+2APB+Rya group. The cells were incubated for 24h, and then the [Ca2+]i was measured by Fluorescence spectrophotometer. The flow cytometry was used to detect cell apoptosis and the protective role of 2APB and Ryanodine after 24h treatment. The expression of GRP78, GRP94, CHOP, Caspase-12 was detected by real-time quantitive PCR and Western blot.Results:1. The [Ca2+]i of chondrocytes increased after 24 hours treatment of tunicamycin, which has significant deviation (P<0.01). Pretreatment of 2APB and Ryanodine can reduce the [Ca2+]i(Tm+2APB+Rya group>Tm+Rya group>Tm+2APB group). There is not significant different among the control,2APB group, Rya group, 2APB+Rya group.2. Chondrocytes apoptosis increased after 24 hours treatment of Tm (P<0.01) Pretreatment of 2APB and Ryanodine decreased chondrocyte apoptosis induced by Tm (P<0.01), while There is not significant different from the control.3. Tm increased the expression of GRP78, GRP94, Caspase-12, CHOP on both transcription and protein translation level in chondrocytes. Pretreatment of 2APB and Ryanodine can reduce their expression. Besides Caspase-12, the Tm+2APB+Rya group’s effect is the most significant.Conclusion:1. Tm induced ER stress in chondrocytes and triggered apoptosis.2. Tm increased the [Ca2+]i in chondrocytes.3.2APB and Ryanodine protected condylar chondrocyte apoptosis induced by Tm through down regulated ER stress signaling proteins.Part2:Research on the Ca2+-dependent endoplasmic reticulum stress on pathological changes in rat mandibular condylar cartilage under compressive mechanical stressObjective:To investigate the histological change, the ultrastructure, the proliferation and apoptosis, the alteration of ERS related proteins such as GRP78 and GRP94, CHOP, Caspase-12 in mandibular cartilage of SD rats under the effect of calcium antagonist after loading compressive mechanical force treatment.Methods:We chose 90 male SD rats of 6 weeks.The rats in experiment groups wore force appliance we designed by ourselves (Patent number:201120210396.4).80g of compress mechanical force was exerted on cartilage to induce ER stress. All animals were divided into eight groups at random for 3 days and 7days:control group, mechanial force (MF) group, MF+2APB group, MF+Rya group, MF+2APB+Rya group,2APB group, Rya group,2APB+Rya group.The change of [Ca2+]i was investigated by GENMED organization calcium ion concentration colorimetric method. Histological change of mandibular condyles was investigated by hematoxylin and eosin stain (H&E); ultrastructural change of chondrocytes was observed by transmission electron microscope (TEM); GRP78, GRP94, Caspase-12 were observed by immunohistochemisty; and apoptosis of chondrocytes was detected by TUNEL analysis.Results:The mandibular condylar cartilage of calcium ion concentration was increased with time after loading force. The histological change of condyles revealed that cartilage became thin under heavy force. The expansion of endoplasmic reticulum, vacuolar change within the chondrocytes were observed by TEM under compressive mechanical force stimulation. The expression of GRP78, GRP94 and Caspase-12 increased in the condylar chondrocytes under compressive mechanical force stimulation. The apoptosis of chondrocytes was observed in the force treatment during the early time. But using the calcium ion antagonist, the apoptosis was reduced and ERS related proteins decreased. In addition, simple dosing and the control group have no significant difference in all of the above.Conclusion:Ca2+-dependent endoplasmic reticulum stress signaling pathway plays an important role in pathological changes of the rat condylar cartilage. Using the calcium ion antagonist, the calcium ion concentration, thickness of condylar cartilage and apoptosis were increased. The calcium ion antagonist can lower ERS marker molecules expression to provide protection.