| Part Ⅰ.The Mechanism of BRD4 In The IL-1β-induced Inflammation of Rat Condylar Chondrocytes[Objective]To test whether the inhibition of BRD4 can reduce the expression of the inflammatory factors in rat condylar chondrocytes stimulated by IL-1β,and to explore the underlying mechanism.[Method]The condylar chondrocytes were extracted from 6 male SD rats aged 3 weeks,and the third generation of the chondrocytes were used in the experiment.The first part is the experiment of BRD4’s inhibitor JQ1.We designed four experiment groups in this part.They are blank control group,IL-1β group,JQ1 group and IL-1β+JQ1 group.10ng/ml IL-1β and 400nM JQ1 were selected to act for 24 hours respectively.The second part was the experiment of small interfering RNA(siBRD4).Four experiment groups include the blank control group,the IL-1β group,the IL-1β+siNC group and the IL-1β+siBRD4 group.After siBRD4 or siNC transfection for 48 hours,the chondrocytes were treated with IL-1β for 24 hours to see whether the expression of inflammatory genes decreased after the silencing of Brd4 gene.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression changes of Brd4、Il-1β,Il-6,Nrf2 and Nlrp3 mRNA.The expression of BRD4,NRF2 and NLRP3 were detected by western-blot.[Results]1.BRD4 inhibitor JQ1 can inhibit the inflammatory response in rat condylar chondrocytes induced by the IL-1β.The mRNA level of Brd4,Il-1β,Il-6 and Nlrp3 in the IL-1β+JQ1 group decreased to 29.6%,25.3%,11.8%and 13.0%of those in the IL-1β group.The mRNA level of Nrf2 in the IL-1β+JQ1 group increased to 1.6 times of that in the IL-1β group.The protein level of BRD4 and NLRP3 in the IL-1β+JQ1 group were decreased to 7.3%and 80.2%of those in the IL-1β group,while the protein level of NRF2 was increased to 1.4 times of that in the IL-1β group.The mRNA level of Brd4,Il-1,Il-6 and Nlrp3 in the JQ1 group were 42.4%,57.8%,18.1%and 14.3%of those in the Blank group,respectively.The protein level of BRD4 and NLRP3 in the JQ1 group were 19.6%and 75.7%of those in the Blank group.2.Small interfering RNA-siBRD4 inhibited the inflammatory response in rat condylar chondrocytes induced by IL-1β.The mRNA level of Brd4,Il-1β,Il-6,and Nlrp3 in the IL-1β+siBRD4 group decreased to 30.0%,9.7%,6.0%,and 64.1%of those in the IL-1β+siNC group.The mRNA level of Nrf2 in the IL-1β+siBRD4 group increased to 2.8 times of that in IL-1β+siNC group.The protein level of BRD4 and NLRP3 in the IL-1β+siBRD4 group were decreased to 30.6%and 75.1%,while NRF2 was increased to 1.3 times.[Conclusion]In the inflammation,Brd4 and Nlrp3 are pro-inflammatory factors.BRD4 inhibition can cause Nrf2 increased and Nlrp3 decreased and the inflammatory response was suppressed.PartⅡ The Identification of BRD4’s Target Genes Related to the Inflammatory Response Induced by the Overloaded Mechanical Stress in the Rat Condylar Cartilage[Objective]To screen out the inflammatory response associated genes in rat condylar cartilage which bind to BRD4 using the ChIP-seq experiment.To clarify the molecular mechanism of BRD4 in the process of rat condylar cartilage inflammation induced by the overloaded mechanical stress.[Method]Eight weeks old male SD rats were selected to establish an animal model of mechanical stress loading on the temporomandibular joint in rats.According to the animal model device designed by our research group(Patent No.201120210396.4),the upward and backward mechanical stress(80g)was applied to the condylar cartilage of the rats in the mechanical stress group for 7 days.The same operation was performed for the rats in the control group,but no stress was loaded.After 7 days,the condylar cartilage of the control group and the mechanical stress group were taken for the Realtime fluorescence quantitative polymerase chain reaction and chromatin immunoprecipitation sequencing(ChIP-seq)experiment.Venn diagram analysis was performed on the genes which bind to BRD4 in the promoter region.With Venny diagram,we screened out the genes whose binding with BRD4 increased after overloading.UCSC was used to visualize the results and to further screen out the target genes.The Real-time fluorescence quantitative polymerase chain reaction were used for the verification.[Results]1.qPCR results showed that the expressions of Brd4 and Il-1β mRNA in the mechanical stress group were 1.8 times and 4.2 times of those in the control group.2.ChIP-seq results showed that the genes whose promoter regions binding with BRD4 increased after overloading includes Il-1β,Il-6st,Hmg20a,Voppl,Cpxml,Ypel3,Arfip1,Fktn,ler5,P2ry1,Taar3,Klkb1,Clic2,Lipn,Agtrap,Ppmlk,Ints2,Polg and Mall.Among them,Il-1β,Il-6st,Ebf2,Voppl are the pro-inflammatory genes,and Cpxml is an anti-inflammatory gene.The mRNA level of Il-6st,Ebf2,and Voppl in the mechanical stress group were 2.0,3.9,and 5.0 times of those in the control group.The mRNA level of Cpxml in the mechanical stress group were only 24.9%of the control group.[Conclusion]After overloading,the binding of BRD4 with the promoter regions of I1-1β,Il-6st,Ebf2,Voppl and Cpxml increased,which promoted the inflammatory response.Part Ⅲ.The Identification of BRD4 Target Gene Related to The Growth and Development of The Rat Condylar Cartilage[Objective]To screen out the growth and development associated genes in condylar cartilage which bind to BRD4 using the ChIP-seq experiment.To clarify the molecular mechanism of BRD4 in the process of rat condylar cartilage growth and development.[Method]Male SD rats aged 3 or 8 weeks were selected and condylar cartilage were taken for the Real-time fluorescence quantitative polymerase chain reaction chromatin to detect the Brd4 mRNA expression.Male SD rats aged 8 weeks were selected and condylar cartilage were taken for chromatin immunoprecipitation sequencing(ChIPseq).GO analysis was performed on the sequencing results to find the BRD4’s target genes related to the biological growth and to screen out the condylar cartilage growth and development associated DNA fragments which bind to BRD4.UCSC was used to visualize the results.The genes related to condylar cartilage growth and development were verified by q-PCR using the SD rats aged 3 weeks and 8 weeks.[Results]1.q-PCR results showed that the mRNA level of Brd4 in the condylar cartilage of 8-weeks-old rats was 2.0 times higher than that of 3-weeks-old rats.2.GO analysis showed that BRD4 can bind to the promoter regions of Cited 1,Foxcl,Grem1,Egfl7,D114,Mmp14,Mmp19,Smad6,Coll3a1,Wnt8a in the condylar cartilage of rats.These genes are related to the growth and development of cartilage.Among them,the Foxcl,Cited1 were positively correlated with cartilage formation,the Mmp14,Mmp19,Col13a1,Egfl7 and Grem1 were related to cartilage matrix remodeling and Wnt8a,D114,and Smad6 were related to signal pathway.3.The mRNA level of Foxcl,Citedl,Wnt8a,Col13a1,Mmp19,D114,Smad6 in the 8 weeks-old rat group is 3.1,1.8,2.5,2.1,3.3,3.0,4.4 times of those in the 3 weeksold rat group.The mRNA level of Grem1 and Egfl7 in the 8 weeks-old rat group decreased to 30.6%and 43.2%of those in the 3 weeks-old rat group.The mRNA level of Mmp14 showed no difference between 3 and 8 weeks-old rat groups.[Conclusion]BRD4 may be involved in the growth and development of rat condylar cartilage by binding to the Citedl,Foxc1,Grem1,Egfl7,D114,Mmp14,Mmp19,Smad6,Col13a1,Wnt8a. |
