The Molecular Mechanism And The Effect Of Daphnetin Combined With Bci-2-siRNA And SFas-siRNA On Anti-apoptotic Genes In The Collagen-induced Arthritis Rats Synovial Fibroblasts

Objective:To Investigate the molecular mechanism and the effect of daphnetin combinedwith Bcl-2-siRNA and sFas-siRNA on the anti-apoptotic genes of synovial fibroblastsin the collagen-induced arthritis rats and further explore the possible mechanism ofdaphnetin combined with small interfering RNA to the treatment of RA. Thesefindings may provide a systematic and scientific theoretical basis for the medicinalapplication of daphnetin and the treatment of RA.Methods:1. To design and to synthesis in chemical way three pieces of si-Bcl-2andthree pieces of si-sFas2. To select the best siRNA:Experimental groups:①the first Bcl-2-siRNA treatment group(si-Bcl-2.1group);②the second Bcl-2-siRNA treatment group(si-Bcl-2.2group);③the thirdBcl-2-siRNA treatment group(si-Bcl-2.3group);④the first sFas-siRNA treatmentgroup(si-sFas.1group);⑤the second sFas-siRNA treatment group(si-sFas.2group);⑥the third sFas-siRNA treatment group(si-sFas.3group);⑦negative controlgroup(NC group);⑧negative fluorescence control group(NC-Cy3group);⑨healthWistar rats synovial fibroblasts control group(health group). All siRNAs weretransfected into CIA rats synovial fibroblasts by liposome method(siRNAs’ finalconcentrations for100nM). Synovial cells of CIA rats in NC-Cy3group wereconventionally cultured for24hours and48hours, then the red fluorescence wasrespectively observed under the inverted microscope with the purpose of confirmingwhether the transfection was successful. CIA rats synovial fibroblasts in other groupswere collected after cultured for48hours, then anti-apoptotic genes called Bcl-2andsFas mRNA relative expression, both of which came from CIA rats synovialfibroblasts, were detected by real-time fluorescent quantitative PCR(RT-PCR). 3. To optimize the role of siRNA: Six concentration grads of the bestsi-Bcl-2and si-sFas selected at first step were arranged:10nM、20nM、30nM、50nM、100nM、200nM, respectively acting on synovial cells of CIA rats for24hours、48hours、72hours. And anti-apoptotic genes called Bcl-2and sFas mRNA relativeexpression, both of which came from CIA rats synovial fibroblasts, were detected byRT-PCR.4. To optimize the role of Daphnetin: Daphnetin(40μg/mL) respectivelyacts on CIA rats synovial fibroblasts for24hours、48hours、72hours. Andanti-apoptotic genes Bcl-2and sFas mRNA relative expression, both of which camefrom CIA synovial fibroblasts, were detected by RT-PCR.5. To investigate the molecular mechanism and the Effect of Daphnetincombined with Bcl-2-siRNA and sFas-siRNA on the Anti-apoptotic Genes in theCollagen-Induced Arthritis Rats synovial cells:experimental groups:①health Wistar rats synovial fibroblasts controlgroup(health group);②CIA rats synovial fibroblasts control group(CIA group);③Daphnetin (40μg/mL)treatment group(E003group);④Bcl-2-siRNA treatmentgroup(si-Bcl-2group);⑤sFas-siRNA treatment group(si-sFas group);⑥Bcl-2-siRNA combined with sFas-siRNA treatment group(si-B-F group);⑦Daphnetin (40μg/mL)combined with Bcl-2-siRNA treatment group(E003-Bgroup);⑧Daphnetin (40μg/mL)combined with sFas-siRNA treatment group(E003-Fgroup);⑨Daphnetin (40μg/mL)combined with Bcl-2-siRNA and sFas-siRNAtreatment group(E003-B-F group). According to experimental groups, synovialfibroblasts were transfected by liposome method. Detection of following indicators:⑴mRNA relative expression of anti-apoptotic genes Bcl-2、sFas and signal proteinsSTAT3、 caspase8in apoptosis pathway were detected by RT-PCR;⑵proteinexpression of anti-apoptosis genes Bcl-2and sFas were detected by flow cytometry;⑶apoptosis of synovialfibroblasts were detected by Annexin V/PI double stainingflow cytometry.Results:1. The result of selecting the best siRNA: Bcl-2mRNA relative expression in si-Bcl-2.1group(0.960±0.030) was significantly lower than in si-Bcl-2.2group(1.352±0.023)、si-Bcl-2.3group(1.418±0.024) and NC group(1.428±0.061)(P<0.05);sFas mRNA relative expression in sFas.1group(0.829±0.015) was significantlylower than in sFas.2group(1.238±0.021)、sFas.3group(1.260±0.020) and NCgroup(1.276±0.030)(P<0.05).2. The result of optimizing daphnetin: After daphnetin respectively acted onsynovial fibroblasts for48hours and72hours, Bcl-2mRNA relative expression wassignificantly lower than24hours(P<0.05), but there was no significant differencebetween48hours and72hours(P>0.05). After daphnetin respectively acted on CIArats synovial fibroblasts for48hours and72hours, sFas mRNA relative expressionwas significantly lower than24hours(P<0.05), and sFas mRNA relative expressionof72hours was lower than48hours with statistically significant difference(P<0.05).3. The result of investigation the molecular mechanism and the effect ofdaphnetin combined with Bcl-2-siRNA and sFas-siRNA on anti-apoptotic genesin Collagen-Induced Arthritis rats synovial fibroblasts⑴The effect of daphnetin combined with Bcl-2-siRNA and sFas-siRNA onmRNA relative expressions of anti-apoptotic genes Bcl-2and sFas in CIA ratssynovial fibroblasts: Via daphnetin and siRNA alone/combination treatment for48hours, Bcl-2and sFas mRNA relative expressions in all treatment groups weresignificantly lower than in CIA group(P<0.05); Both of them in E003-Bgroup/E003-F group and E003-B-F group were significantly lower than in E003group、si-Bcl-2group/si-sFas group、si-B-F group (P<0.05), but there was nosignificant difference between E003-B group/E003-F group and E003-B-Fgroup(P>0.05).⑵The effect of daphnetin combined with Bcl-2-siRNA and sFas-siRNA onprotein expressions of anti-apoptotic genes Bcl-2and sFas in the CIA rats synovialfibroblasts: Via daphnetin and siRNA alone/combination treatment for72hours, theprotein expression of Bcl-2and sFas in all treatment groups were significantly lowerthan in CIA group(P<0.05); Both of them in E003-B group/E003-F group andE003-B-F group were significantly lower than in other treatment groups(P<0.05), butthere was no significant difference between E003-B group/E003-F group and E003-B-F group(P>0.05).⑶The effect of daphnetin combined with Bcl-2-siRNA and sFas-siRNA on theapoptosis rate of CIA synovial fibroblasts: The apoptosis rate of synovial cells inE003-B-F group was significantly higher than in other groups(P<0.05). The apoptosisrate of synovial fibroblasts in E003group was significantly higher than in si-Bcl-2group、 si-sFas group、 si-B-F group(P<0.05); The apoptosis rate of ssynovialfibroblasts in si-Bcl-2group was significantly higher than in si-sFas group; but theywere significantly lower than in E003-B group and E003-F group(P<0.05).⑷The effect of daphnetin combined with Bcl-2-siRNA and sFas-siRNA onmRNA relative expressions of signal proteins STAT3(mitochondrial pathway) andcaspase8(death receptor pathway) in CIA rats synovial fibroblasts: STAT3mRNArelative expression in all treatment groups were significantly lower than in CIAgroup(P<0.05); STAT3mRNA relative expression in E003-B group and E003-B-Fgroup were significantly lower than in E003group、 si-bcl-2group、 si-B-Fgroup(P<0.05). caspase8mRNA relative expression in all treatment groups weresignificantly higher than in CIA group(P<0.05); caspase8mRNA relative expressionin E003-F group and E003-B-F group were significantly higher than in E003group、si-sFas group、si-B-F group(P<0.05).Conclusion:1. Respectively acting on CIA rats synovial fibroblasts, daphnetin、si-Bcl-2andsi-sFas down-regulated both mRNA relative expressions and protein expressions ofanti-apoptotic genes Bcl-2and sFas.2. Daphnetin cooperated with si-Bcl-2and si-sFas down-regulated both mRNArelative expressions and protein expressions of anti-apoptotic genes Bcl-2and sFas inCIA rats synovial fibroblasts apoptosis.3. Daphnetin cooperated with si-Bcl-2and si-sFas promoted CIA rats synovialfibroblasts apoptosis.4. One of possible molecular mechanisms that daphnetin combined withBcl-2-siRNA and sFas-siRNA promoted CIA rats synovial fibroblasts apoptosis wasthat combined effects down-regulated levels of Bcl-2and sFas expressions,simultaneously down-regulated the level of signal protein STAT3, which was located upstream of Bcl-2in mitochondrial pathway, and up-regulated the level of signalprotein caspase8in death receptor pathway, finally leading to promote CIA ratssynovial fibroblasts apoptosis as the result of enhancing the signal transduction in twoapoptosis pathways.These findings were expected to provide a scientific and systematic theoreticalbasis with a new treatment way of RA and a medicinal application of daphnetincombined with siRNA.