Daphnetin Combined With 5-aza-Dc And Bcl-2-miRNA Interference Plasmid Induces Apoptosis In The Collagen-Induced Arthritis Rats Synovial Cells

Objective:To Investigate Daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmid induces apoptosis in the Collagen-Induced Arthritis Rats Synovial Cells and further explore the possible mechanism of daphnetin combined with 5-aza-Dc and Bcl-2-miRNA to the treatment of RA.These findings may provide a systematic and scientific theoretical basis for the medicinal application of daphnetin and the treatment of RA.Methods:1.To build and identification of the Bcl-2-miRNA interference plasmid,respectively using cloning method and enzyme cutting and gene sequencing method.2.To daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmid induces apoptosis in the Collagen-Induced Arthritis Rats Synovial Cells: Experimental groups:(1)The CIA rats synovial cells(Control);(2)triptolide(40?g/ml)treatment group(TP);(3)daphnetin(40?g/ml)treatment group(DAP);(4)5-aza-Dc(20?M)treatment group(5-aza-Dc);(5)Bcl-2-miRNA interference plasmid(8?g/ml)treatment group(MR-Bcl-2);(6)daphnetin(40?g/ml)joint 5-aza-Dc(20?M)treatment group(DAP+5-aza-Dc);(7)daphnetin(40?g/ml)joint Bcl-2-miRNA interference plasmid(8?g/ml)treatment group(DAP+MR-Bcl-2);(8)5-aza-Dc(20?M)joint Bcl-2-miRNA interference plasmid(8?g/ml)treatment group(5-aza-Dc+MR-Bcl-2);(9)daphnetin(40?g/ml)joint5-aza-Dc(20?M)and Bcl-2-miRNAinterference plasmid(8?g/ml)treatment group(Combination);(10)Empty plasmid(8?g/ml)treatment group(Mock)According to experimental groups,synovial fibroblasts were transfected by liposome method.Detection of following indicators:(1)mRNA relative expression of Bcl-2?Bax?Bid and Related pathways proteins(Caspase-3?Caspase-8?Caspase-9)were detected by RT-PCR;(2)The methylation of Bax?Bid was measured by the methylationspecific polymerase chain reaction(MSP);(3)The apoptosis rate of synovial cells was detect using Annexin V/PI double dyeing method;(4)The protein expression of Bcl-2?Bax?Caspase-3?Caspase-8?Caspase-9 were detected by Cell-based ELISA(5)transmission electron microscopy were used to observe the change of CIA rats joint synovial cells' ultrastructural structure.Results:1.Successful build the Bcl-2-plasmid miRNA interference.2.To daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmid induces apoptosis in the Collagen-Induced Arthritis Rats Synovial Cells:? The effect of daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmidm on the mRNA relative expression of Bcl-2?Bax?Bid and Related pathways proteins(Caspase-3?Caspase-8?Caspase-9)in the CIA rats synovial fibroblasts:Bcl-2 mRNA relative expression in each experimental groups was significantly lower than in CIA group and Mock group(p<0.05),Bcl-2 mRNA relative expression in Combination group was lowest levels,by 0.074±0.025?Bax? Bid?Caspase-3?Caspase-8?Caspase-9 mRNA relative expression were significantly higher than CIA group and Mock group(p<0.05).? The effect of daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmidm on the methylation status of Bax?Bid in the CIA rats synovial fibroblasts:non-methylation primer and the methylation primer amplification specific strips of the proapoptotic genes Bax?Bid were all in the untreated CIA rats synovial cells,the promoter were all in some high methylation status.For the bid gene of Group 6(daphnetin+5-aza-Dc group)after teated by DAP and 5-aza-Dc,the brightness of the methylated amplified bands were all decreased,the corresponding unmethylated amplified bands were all co-brightness.While for the bid gene of Group 3(daphnetin group)after teated by DAP,the brightness of the methylated amplified bands and the corresponding unmethylated amplified bands had no significant change.It can be deduced from the above that 5-aza-Dc has some demethylation on the bid gene.For the gene Bid of other groups,methylated amplified bands and its corresponding unmethylated amplified bands had no significant changes in brightness.For gene Bax,each experimental group methylated and unmethylated primers were amplified specific bands.? The effect of daphnetin combined with 5-aza-dC and Bcl-2-miRNA interference plasmidm on the apoptosis rate of CIA synovial fibroblasts:the apoptosis rate of each experimental groups were significantly higher than CIA group and Mock group(P<0.05);the 67.08% apoptosis rate of the combination group was significantly higher than other experimental groups(P<0.05).? The effect of daphnetin combined with 5-aza-dC and Bcl-2-miRNA interference plasmid on protein expressions of Caspase-3?Caspase-8?Caspase-9 ?Bcl-2?Bax in the CIA rats synovial fibroblasts:the protein expression of Bcl-2 in each experimental groups were significantly lower than in CIA group and Mock group(P<0.05).the protein expression of Bax?Caspase-3?Caspase-8?Caspase-9 in each experimental groups were significantly higher than in CIA group and Mock group(P<0.05).?The result of transmission electron microscopy are showed as bellow.Control: Cells were oval,the nuclear membrane structure were clear,large nuclear cytoplasm ratio,prominent nucleoli;TP: chromatin margination,a small amount of vacuoles in cytoplasm,the cytoplasm was lost;DAP: chromatin margination,a large vacuole-like structures appeared in the cytoplasm,the cytoplasm serious lost,and further development of apoptosis;5-aza-Dc: cell morphology was irregular,with chromatin condensated,and there were a small amount of cytoplasmic vacuoles denaturation;MR-Bcl-2: The were cytoplasmic serious degeneration,and the nuclear membrane disappeared.Apoptotic bodies formed;DAP+5-aza-dC: cell morphology was irregular,with chromatin condensated,and there were a large amount of cytoplasmic vacuoles denaturation;DAP+MR-Bcl-2: Cells lost their normal structure,with serious cytoplasmic vacuolar degeneration and considerable cytoplasm lost.The nuclear membrane disappeared.Apoptotic bodies formed;5-aza-dC+MR-Bcl-2: There were a small amount of cytoplasmic vacuoles denaturation,with chromatin condensated;Combination: Cells lost their normal structure,with serious loss of cytoplasm.Apoptotic bodies formed.They showed typical apoptotic changes;Mock: Cells were oval,and structure of the nuclear membrane was clear.Nucleolus were clear.Conclusion:1.Respectively acting on CIA rats synovial fibroblasts,daphnetin?5-aza-Dc?Bcl-2-miRNA interference plasmidm down-regulated both mRNA relative expressions and protein expressions of Bcl-2,and up-regulated both mRNA relative expressions and protein expressions of Bax?Bid?Caspase-3?Caspase-8?Caspase-9;2.Daphnetin collaborated 5-aza-Dc could make proapoptotic genes Bid demethylated,expression increased and promoted synovial cells apoptosis.3.Daphnetin cooperated with 5-aza-Dc and Bcl-2-miRNA interference plasmid down-regulated both mRNA relative expressions and protein expressions of Bcl-2 in the CIA rats synovial fibroblasts,up-regulated both mRNA relative expressions and protein expressions of Bax?Bid?Caspase-3?Caspase-8?Caspase-9,and induced apoptosis in the CIA Rats Synovial Cells;One of possible inducing apoptosis molecular mechanisms might be related to which daphnetin down-regulating Bcl-2?up-regulating Bax ? Bid,and activating the apoptosis channels of Caspase-3 ?Caspase-8?Caspase-9 induced FLS apoptosis.These findings were expected to provide a scientific and systematic theoretical basis with a new treatment way of RA and a medicinal application of daphnetin combined with 5-aza-Dc and Bcl-2-miRNA interference plasmidm.