| Part Ⅰ. The study of BET inhibitors JQ1’s effect on the inflammation of rat mandibular condylar cartilage induced by compressive mechanical stress[Objective]To investigate whether JQ1 can inhibit inflammation of rat mandibular condylar cartilage induced by compressive mechanical stress and explore the underlying mechanism.[Method]Ninety-six 7-wk-old male Sprague-Dawley rats were used in this study. According to the animal model of rat temporomandibular joint compressive loading device (patent N.O.:201120210396.4), which was designed by our research group, an upward and backward compressive stress (80 g) was exerted on rat condylar cartilage of the force-applying groups for 4 days,7 days or 14 days. For the non-force applying groups, the same operation was performed, but force was not applied. We chose 14 days for following experiments because the subchondral bone resorption was more obvious in day 14 than day 7. JQ1 was injected into the temporomandibular joint region in different concentrations(5μM、10μM、50μM). Hematoxylin-eosin (HE) staining was used to observe the thickness and morphological changes of rat condylat cartilage after applying strength and(or) drug. And Polymerase Real-time Chair Reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to detect the expression changes of inflammatory factors. We chose 50μM as the JQ1 concentration for following experiments because its effect was most apparent. Chromatin Immunoprecipitation (chIP) was utilized to detect the binding of Brd4 with TNF-α and IL-8 promoter regions in different conditions.[Results]1. HE staining results showed that there were no significant changes of the mandibular condylar cartilage thickness between the control group and the JQ1-only group, but the cartilage thickness of the force-applied group decreased obviously, as the time extended, the degree increased, while there is no significant difference between 7 days loading group and 14 days(4 days,34%, P<0.01; 7 days,58%, P<0.01; 14 days,60%, P<0.01). When JQ1(5μM、10μM、50μM) and force were both used, the thinning cartilage recovered, and the effect of the 50μM group was more significant (5μM,29%, P>0.05;10μM,36%, P<0.01; 50μM,93%, P<0.01).2. The results of qRT-PCR showed that the expression of TNF-a, IL-1(3, IL-6. of rat condylar cartilage increased significantly after 14 days loading. JQ1 can inhibit the increase of inflammatory factors expressions induced by compressive mechanical stress and the effect of the 50μM JQ1 was most obvious. The results of IHC showed that JQ1 can inhibit the increase of TNF-a and IL-1β induced by compressive mechanical stress. The expression levels of the inflammatory factors showed no significant differences between the control and the JQl-only group.3. The results of chIP showed that the binding of Brd4 with TNF-a and IL-8 promoter regions increased in the force applied group compared to the control group, while in the force+JQ1 group these bindings were inhibited. There was no significant difference between the control and the JQ1-only group.[Conclusion]BET inhibitors JQ1 can alleviate the thinning of condylar cartilage under compressive stress and it can reduce compressive mechanical stress-mediated inflammatory factors expressions by inhibiting the binding of Brd4 with the relevant inflammatory factor promoters. This effect is concentration-dependent.Part Ⅱ The study of BET inhibitors JQ1’s effect on the bone resorption of rat mandibular condylar subchondral bone induced by compressive mechanical stress[Objective]To investigate whether JQ1 can inhibit osteoclast differentiation and alleviate the bone resorption of rat mandibular condylar subchondral bone induced by compressive mechanical stress[Method]The rat models and experiment groups was the same as those in part Ⅰ. The experiment period was 14 days. The concentration of JQ1 was 50μM. Tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number changes of osteoclasts in rat condylar subchondral bone after applying strength and (or) drug. Micro-CT was utilized to make three-dimensional reconstruction and to compare the morphological changes of trabecular bone with parameters includes BV/TV, Tb.N, Tb.Th and Tb.Sp. Polymerase Real-time Chain Reaction (qRT-PCR) was applied to detect the expression changes of osteoclast differentiation relevant factors, such as RANKL, RANK, NFATc1, TRAP and Cathepsin K.[Results]1. Tartrate-resistant acid phosphatase (TRAP) staining results showed that there was no significant difference between the control and the JQ1-only group. Compared to the control group, there was more TRAP positive cells in the force applied group. JQ1 can reduce the number of TRAP positive cells after overloading (25%, P<0.05).2. Micro-CT analysis showed that there was no significant difference in condylar subchondral bone morphology between the control and the JQ1-only group. In the force applied group, BV/TV, Tb.N and Tb.Th decreased while Tb.Sp increased. JQ1 can reverse the change of BV/TV, and Tb.Th induced by mechanical stress (BV/TV,27%, P<0.01; Tb.Th,9%, P<0.01).3. The results of qRT-PCR showed that the expression of osteoclast differentiation relevant factors (RANKL, RANK, NFATc1, TRAP and Cathepsin K) in rat condylar subchondral bone increased significantly after 14 days loading. JQ1 can inhibit these increases. There was no significant difference between the control and the JQ1-only group.[Conclusion]BET inhibitor JQ1 can reduce the overload-induced osteoclast number increase in condylar subchondral bone and inhibit bone resorption. The underlying mechanism can be related the inhibition of osteoclast differentiation by JQ1. |
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