| Background: Plasmocytoid dendritic cells(p DCs), one subtype of dendritic Cells(DCs), is mainly derived from the marrow, peripheral blood and lymph tissues that are abundant in T cell. In human body p DCs have the strongest ability to secrete type I interferon(IFN I) and if dysfunction of p DCs may lead to autoimmune disorders(such as systemic lupus erythematosus,SLE). SLE can result in injury of tissues and organs in many systems, which has characteristics of affluent activated T, B cells, alexins, accumulational immune complexes, autoantibodies, and abnormal immunoregulation. The incidence of SLE is between 0.3‰ and 0.8‰ in the world. Recently, more and more studies reveal genetic factor has an important role in disease susceptibility of SLE and many genes participate in the onset of SLE. However, the pathogeny of SLE has not been clear by now.At present, Glucocorticoids(GCs) is still the main therapeutic drug of SLE, which has many side effects like osteoporosis, fracture, femoral head necrosis, gastrointestinal bleeding, secondary infection, and so on. Sometimes side effects of GCs is so serious that the use of GCs must be restricted. Toll-like receptor(TLR) can activate p DCs, which can resist the process of GCs-induced apoptosis of p DCs and thus weaken the treatment effect of GCs on SLE. Therefore, any substance accelerating p DCs apoptosis may contribute to promote the treatment effect of GCs on SLE.mi RNAs that can bind to the target m RNA 3’ untranslated regions(UTR) according to the principle of base pairing are able to negatively regulate gene expression during the course of post-transcription. Many studies have shown that mi RNAs have important roles in immune response and signal conductive processes of inflammatory factors. Some documents reported in SLE the overactive IFN I path may be relative with abnormal expression of some mi RNAs.GCs treat SLE through a series of mi RNAs pro-apoptotic effect on p DCs, which course can be remarkably weakened by TLR. So the mechanism about TLR resistance of GCs-induced apoptosis of p DCs demands further study in order to improve the effect of GCs on SLE.Methods: This study selected Dex, Cp G, p DCs, and so on to carry out cytological tests.(1) Fresh peripheral blood mononuclear cells(PBMCs) were obtained from 76 healthy individuals. p DCs were isolated by a cell-sorting technic using the Human Diamond Plasmacytoid Dendritic Cell Isolation Kit. Purity of the isolated p DCs was over 95%, which was tested by flow cytometry(FCM). Then p DCs were cultured in 37 incubator for 2 hours. Next,℃ p DCs were treated with Dex alone(Dex Group) or the combination of Dex and Cp G(Dex+Cp G Group) for 4-16 hours, meanwhile control group was established without any stimulation. Additionally, 20 ng/m L IL-3, which promotes p DCs survival and activation, was added to the culture medium.(2) Mi RNA profiles of Dex Group,Dex+Cp G Group, and control group were detected by using mi RNA microarrays. Mi RNAs that could bind Bcl-2 family members(BCL-2, Bcl-xl, Mcl-1, Bcl-w) were selected as candidate ones by target prediction analysis.(3) After p DCs were stimulated by Cp G, western blot and q RT-PCR assays were used to measure the proteins and mi RNAs expressions of Bcl-2, Bcl-xl, and Mcl-1.(4) After the knockdown of Mcl-1 by transfecting p DCs with si-Mcl-1, the apoptosis of p DCs was tested through staining p DCs with annexin V-FITC and PI, and then was analyzed by FCM.(5) After having transfected p DCs with these candidate mi RNAs mimics to realize their overexpression, stimulated p DCs with Dex+Cp G and measured the apoptosis of p DCs.(6) Further, p DCs were transfected with some of these candidate mi RNAs inhibitors to knock down these related mi RNAs and then the apoptosis of p DCs was detected.(7) Last, the dynamic expressions of Bcl-2 and Mcl-1 proteins and mi R-29 were detected to further confirm their relationship during Dex induced- Cp G inhibited p DCs apoptosis.Results:(1) A total of 120 mi RNAs were induced by Dex, including many known pro-apoptotic mi RNAs, such as mi R-16, mi R-29, and so on. Further analysis showed that 36 of the 120 mi RNAs were inhibited by Cp G stimulation. 83 mi RNAs were inhibited by Dex, and among these, 20 mi RNAs were induced by Cp G stimulation. In total, 56 mi RNAs could be involved in TLR inhibited GCs-induced apoptosis of p DCs.(2) By target prediction analysis selected 6 mi RNAs(mi R-29 c, mi R-29 b, mi R-101, mi R-148 a, mi R-1, mi R-let-7a) that could bind Bcl-2 family members(BCL-2, Bcl-xl, Mcl-1, Bcl-w). Those selected 6 mi RNAs as 6 candidate mi RNAs would be studied specially.(3) These proteins’ expressions of Bcl-2, Mcl-1, and Bcl-xl were increased significantly after stimulating p DCs with Cp G. Further, after stimulating p DCs with Cp G, the expressions of Bcl-2 m RNA and Bcl-xl m RNA were found increase significantly, the expression of Mcl-1 m RNA was decreased slightly, while the expression of Bcl-w m RNA was not affected at all.(4) The rate of p DCs apoptosis after transfecting p DCs with si-Mcl-1 increased to 59.1% from 18.9% in control group, while the rate of viable p DCs decreased to 24.4% from 49.2 % in control group.(5) In addition, after transfected with 5 candidate mi RNAs mimics, the rates of p DCs apoptosis induced by mi R-29 b and mi R-29 c were 58.4% and 52.7% respectively while the corresponding rate in Dex+Cp G group was 21.1%; the statuses of p DCs apoptosis induced by 3 other mi RNAs had no significant change.(6) Moreover, after transfected p DCs with mi R-29 b inhibitor or mi R-29 c inhibitor, p DCs apoptosis rate decreased significantly.(7) Finally, this study discovered the expression of Mcl-1 protein was inhibited significantly at the point of the sixth hour after p DCs being transfected with mi R-29 b mimic, while the expression of Bcl-2 protein was inhibited significantly at the point of the twelfth hour. When Cp G simulated p DCs, the expressions of mi R-29 b and mi R-29 c were inhibited most significantly at the point of the sixth hour and then increased gradually after that point but could not return to the level at beginning of stimulation until the twelfth hour.Conclusion:(1) Glucocorticoids(GCs) including Dex can definitely induce p DCs apoptosis,however, TLR can certainly weaken the type of apoptosis.(2) GCs-induced p DCs apoptosis takes place mainly through the increase in mi R-29 b and mi R-29 c expression in p DCs stimulated by GCs.(3) Mi R-29 b and mi R-29 c of pro-apoptotic effect on p DCs is produced by inhibiting Bcl-2 and Mcl-1; And mi R-29 b has continuous pro-apoptotic effect on p DCs. Mi R-29 b pro-apoptotic effect on p DCs mainly depends on inhibiting Mcl-1 in 6 hours of mi R-29 b mimic stimulation of p DCs and then on inhibiting Bcl-2.(4) According to the result in this study that the expression of Bcl-w m RNA was not affected by Cp G, we consider that Bcl-w may not participate in the process of TLR resistance of GCs-induced p DCs apoptosis.(5) TLR resistance of GCs-induced p DCs apoptosis mainly relies on TLR inhibition of mi R-29 b and mi R-29 c expression.(6) If one new developed drug can weaken TLR inhibition of mi R-29 b and mi R-29 c expression, then this drug can boost GCs-induced p DCs apoptosis and would probably promote the treatment effect of GCs on SLE. |
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