The Effect Of A20 Gene On H₂O₂-mediated Vascular Smooth Muscle Cells Proliferation And Related Molecular Mechanisms And The Emodin Intervention On Them

Object:Vascular smooth muscle cells proliferation is the major cause leading to Atherosclerosis AS and restenosis RS after percutaneous coronary artery intervention PCI. Proliferation mechanism and its inhibiting experiments are intensively studied by cardiovascular researchers in recent years. Hydrogen peroxide H2O2 is an invivo oxygen free radical, which belongs to the active oxygen family. Recent in vitro study has showed that H2O2 may affect cell proliferation, differentiation and transcription of multiple genes through the intracellular signal conduction pathway. The transcription factor NF-κB is susceptible to reactive oxygen, which has an important effect on proinflammatory factor; Reactive oxygen can activate NF-κB through oxidating regulation. Then the active transcription factor NF-kB translates to the nucleus, where it controls the transcription of several protein involving in inflammation and proliferation. Present researches have showed that taking H2O2 as the primal representative of the reactive oxygen mediates VSMC proliferation through NF-κB signal pathway which is participate the development of AS and RS after PCI. Varied growth factor, cytokine, adhesion molecule and so on are taking part in the pathophysiological processes. Pure inhibition of some specific factor can not totally prevent focus from forming, so it has become the research orientation towards effectively preventing the vascular proliferation– associated diseases to block the key gene NF-κB of such final common target as cellular proliferation, differentiation, migration and inflammatory reaction and to find the endogenous conserving strategies.Zink finger protein A20 is an important ubiquitinating modification enzyme, which can exert feedback regulation on the NF-κB signal system through ubi- quitination and deubiquitination In recent years, Many studies showed that A20 can restrain vascular smooth muscle cells proliferation originated from NF-kB mediated by many kinds of inflammatory cell molecule, as a very key endogenous self-protective protein, while there is very few researches about its anti-oxidative damage. The present study was designed to regulate A20 expression through using zink finger A20 RNA interfering and transfecting measures. We observed Human Umbilical Artery Smooth Muscle Cells HUASMc proliferation and NF-Κb, tyrosine protein kinase Syk, CyclinD1, P21 and NF-κB downstream inflammatory factor TNF-a protein expression in order to investigate the protective effect of A20 gene on cell oxidation reduction side and to seek a new way to prevent and cure the vascular proliferation–associated diseases.Methods:1) Tree pairs of siRNAs directed against human A20 were designed and were chemically synthesized, the pair of siRNA with the best efficiency to inhibit the expression of A20 was picked out by evaluate the expression of A20mRNA and protein. HUASMc was divided into six groups: (1) blank group; (2) A20siRNA group; (3) scramble siRNA group; (4) H2O2 group;(5) A20siRNA +H2O2 group;(6) scramble siRNA +H2O2 group. The cell proliferation was determined by MTT. The cell cycle was detected by flow cytometry. A20mRNA was measured by RT-PCR; The protein expression of A20, NF-κB p65, Syk, CyclinD1, P21 were detected by Western blot;2) Smearing the obtained E.coli carrying pCAGGS-GFP/A20 plasmid on ampicillin resistance plate, overnight cultured. Puting single colony from plate into 3ml ampicillin resistance LB medium and overnight cultured on 37℃shaking table. After collecting bacteria, extracting recombinant plasmid DNA in small quantity and identificating by using EcoRⅤ/SmaⅠdouble restriction enzyme digestion. The production 1% agarose gel electrophoresis was performed. Endo-free plasmid DNA was extracted in large quantity from E.coli carrying plasmid. Plasmid DNA extraction was diluted in suitable proportion and detected the concentration. HUASMc was randomly divided into 4 groups: (1) blank group; (2) H2O2 group;3) A20 group; (4) A20+ H2O2 group. The cell proliferation was determined by MTT. The cell cycle was detected by flow cytometry. The protein expression of NF-κB p65, Syk, CyclinD1, P21were detected by Western blot;4) Cultured HUASMc in vitro. H2O2 was used as stimulating factor and different concentration emodin were used as therapeutic agent. Cells was randomly divided into 4 groups Blank group: no drug administered; H2O2 group: 100umol/L H2O2 added for 6h; H2O2+EMD group: H2O2 (100μmol/L) and different concentrations (10, 20, 40 and 80μmol/L) of EMD. The cell proliferation was determined by MTT and BrdU; A20mRNA was measured by RT-PCR; the protein expression of A20 and NF-κB were detected by Western blot.Results:1) Compared with control group, three pair of siRNA reduced A20 mRNA and protein expression level of HUASMc,and the maximum inhibition sequence was found in siRNAc, whose interfering efficiency was about 55%. The results of MTT assay showed that H2O2 can stimulate HUASMc proliferation and A20RNAi had a synergistic effect on the HUASMc proliferation activity after inducing by H2O2 (P<0.05). Shown by flow cytometry,constituent ratio of HUASMc S G2/M cell cycle and proliferation index of H2O2 group were higher obviously than those of blank group (P<0.05). In comparison with H2O2 group, A20RNAi + H2O2 could remarkably heighten the constituent ratio of S cell cycle and proliferation index (P<0.05). After H2O2 stimulation, the expression of A20 mRNA,A20 protein, NF-κB p65 protein, Syk protein, and CyclinD protein were increased while the expression of P21 protein was decreased (P<0.05). These changes stimulated by H2O2 were more significant after A20RNAi (P<0.05). From the results of double antibody sandwich ELISA, we found that A20RNAi enhanced evidently the level of NF-κB p65 downstream cell inflammatory factor TNF-αof HUASMc supernatant liquid (P<0.05);2) After the pCAGGS-GFP/A20 plasmid was digested by EcoRⅤ/SmaⅠdouble restriction enzyme, the production was identified by 1% agarose gel electrophoresis, plasmid size and enzyme digested result was right. After HUASMc was transfected with plamid containing A20 gene, the cell proliferation was suppressed effectively by reducing the activity of proliferation and inhibiting the progression of cell cycle from quiescent G0/G1 to S phase. The transfection of A20 gene strongly inhibited the improvement of NF-κB p65 protein, Syk protein, CyclinD protein and TNF- protein expression and significantly inversed the down regulation of P21 protein expression stimulated by H2O2 (P<0.05);3) Within the range of 10-80μmol/L, as the EMD concentration increased, OD values of MTT and BrdU incorporation assays both decreased gradually, EMD could exert the inhibitory effect on H2O2 induced HUASMc proliferation in a concentration- dependent manner. At the same time, the expression of A20 mRNA and A20 protein was increased gradually with the increasing of EMD in a concentration- dependent manner; on the contrary, the expression of NF-κB p65 protein was decreased.Conclusion:1) H2O2 can stimulate the proliferation of HUASMc cultured in vitro and inflammatory reaction, activate the Syk /NF-κB p65 signal pathway, up-regulate Syk, NF-κB p65 and CyclinD1 protein expression, down-regulate P21 protein expression and promote the generation of inflammatory factor TNF-α;2) A20siRNA may inhibit endogenous expression of A20mRNA and A20 protein level in HUASMc, which has an effect of RNAi;3) A20 interference may decrease feedback inhibition of H2O2/Syk/NF-κB signal transduction pathway mediate by endogenous A20, up-regulate the expression of Syk, NF-κB and CyclinD1, down-regulate P21 protein expression, increase the expression of inflammatory factor TNF-αand strengthen proliferation activity and inflammatory reaction stimulated by H2O2;4) Transfection of A20 gene may up-regulate the expression of A20 in vivo, enhance feedback inhibition of H2O2/Syk/NF-κB signal transduction pathway, down-regulate CyclinD1 protein expression, up-regulate P21 protein expression, reduce the generation of inflammatory factor TNF-α, thereby, inhibit the proliferation of VSMC and inflammatory reaction;5) Interfering A20 may become a new target point of preventing and curing As and up-regulating A20 may be a new method of preventing and curing As;6) EMD could inhibit H2O2-mediated HUASMc proliferation. The possible mechanism was that EMD could further suppress activation of NF-κB via increasing the expression of endogenous protective protein A20.