| BackgroundAtherosclerosis is a long-term chronic disease characterized by the accumulation oflipids and fibrous connective tissue in the large arteries, accompanied by a local inflammatory response. Inflammatory cells, like T lymphocytes and macrophages, play an important role in all stages of atherosclerotic lesion development. Moreover, cells from the vessel wall, like endothelial cells, smooth muscle cells, adventitial fibroblasts and mast cells, are able to act as immunological cells that produce proinflammatory cytokines. The triggers and pathways of initiation and regulation of the immune responses in atherosclerotic disease are largely unknown, In general, the human immune system has two closely related pathways to respond to potentially harmful agents: the innate and the adaptive immune recognition systems. The adaptive immune system involves dynamic adaptation to unique antigenic epitopes that are present in the environment. The innate immune response is the first line of defense in which highly conserved pathogen motifs, entitled pathogen-associated molecular patterns (PAMPs), are recognized. The receptors capable of recognizing these PAMPs are toll-like receptors (TLRs). Until now, 10 TLRs have been identified in humans. Different members of this receptor family are activated by different PAMPs. Ligand binding of the extracellular domain induces dimerization of the TLRs and activating immune system, which leads to the activation of signals. Subsequently, TLR activation induces the expression of a wide variety of genes encoding proinflammatory proteins. Moreover, TLR4 has been shown to be involved in modulating the recruitment and adhesion of leukocytes and monocytes to atherosclerotic lesions, even in plaque stabilizing and embolism. Thus, TLR4 is an interesting gene therapeutic target for the treatment of diseases related to atherosclerosis. Although in recent yeas, many scholars had made great progress in the realationg of TLR4 and AS,there are many question waiting for answer. Can we block the gene expression of TLR4 then prevent the progression of atherosclerosis and stable the plaque of AS. Now there are many ways to inactive genes, for example antisense oligonucleotides, nucleic acid technique, monoclonal antibody,et al.Although antisense technology can inactive the expression of harmful gene ,the design of antisense RNA is difficult. The chemical force of antisense RNA and mRNA and the specificity of their binding of are affected by second order or tertiary structure of ambi- sequential binding sites. The polyanion character of antisense oligonucleotides also cause a series of side effects. Triplex DNA technique applies through binding deoxy-oligonucleotide and double helix double strands DNA to TFO.This technique is short of stability and half life. Morever,monoclonal antibody treatment also exist the short half life. Whether can find an effect method block the gene? Which treatment gene interference or drug treatment play the key point in inflammation signal transduction and the rupture of vulnerable plaque?So we develop the research about block TLR4 to prevent AS progress and stable vulnerable plaque.Aims1. To construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones successfully;2. To compare TLR4-RNAi with atorvastatin treatments on influence of cell biology in Ecv304 cells and explores their mechanism;3. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4 induced the effects of mmune and inflammation in order to provide experience in vivo; 4. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4blocking effects in orde to reveal the mechanism that prevent AS progress and stablevulnerable plaque.Methods1. selection of effective siRNA vector for TLR41.1 Construction and identification of siRNA vector for TLR4According to the design principles, two pairs of siRNA sequences were design At the same time design the negatve control fragment.All the siRNA oligos were ligated with pRNAT-U6.lneo. The positive recombinants were sequenced and called pRNAT-TLR4-1, pRNAT-TLR4-2 and pRNAT-Neg.1.2 screening of positive clonesTransfected with plasmid,most of ECV304 cells was lyzed and died in G418-containing medium.The positive clones stably grew in G418-containing medium and subcultrued for every 4 days.The G418-resistant clones ,named as ECV304-pTLR4-1 , ECV304-pTLR4-2 and ECV304- pNeg respectively,were obtained according to the transfected vectors.1.3 selection of efficient siRNA vector for TLR4Total RNA from cells were extracted.Real time PCR, western blot showed the expression of TLR4 and select the efficient siRNA.2. Comparation TLR4-RNAi with atorvastatin treatments on mechanism of TLR4 induced by LPS and retroconversion of inflammatory action induced by TLR4 in vivo2.1 Cells were divided into the following groups: control group, LPS group, LPS+RNAi group and LPS+atorvastatin group. We observe the change of TLR4 and the downstream signal protein NF-κB and explore the difference of mechanism between gene treatment and atorvastatin treatment.2.2 SN50,Ly294002 ,PD98059 or SB203580 was added into LPS+RNAi group or LPS+atorvastatin group respectively in order to explore the inner mechanism between gene treatment and atorvastatin treatment.2.3 Cells were divided into the following groups: control group, LPS group, LPS+RNAi group and LPS+atorvastatin group. We observe the relationship between downstream effector including IL-6 , IL-10 and TNFα.In this way ,we can explore and compare the reverse effect of immune and inflammation between gene treatment and atorvastatin.3. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4 blocking effects in orde to reveal the mechanism that prevent AS progress and stable vulnerable plaque.3.1 Proliferation ,titration and activity detection of TLR4 RNAi adenovirus HEK293 cells were infected with pSuppressorAd-TLR4 ,pSuppressorAd-Neg.72h later,the cytopathic effects were observed and the culture supernant and cells were collected respectively.The collected cells were fully frozen and thawed for 3 times in order to obtain the viral paticles.Titer of virus was detected by plaque-forming assay.Then RAW247.5 cells were treated with Real-time PCR to detect TLR4 mRNA.3.2 The research of TLR4 RNAi adenovirus treatment and atorvastatin treatment on ApoE-knock out mice.ApoE-knock out mice with 8 week old were fed on western-diet. At the same time either pSAd-TLR4 or pSAd-Neg diluted to a total volume of of 100μL was injected into the tail vein of each group of mice (n=27). Mice (n=27) injected with pSAd-Neg (100μL) served as control. Mice(n=22) in atorvastatin group were intervened with atorvastatin (dissolved in ultrapure water, 10mg/kg/d,ig) . Mice (n=22) were intervened with placebo served as control. All the animals sacrificed 10 weeks later. The lipid core and composition of plaque were characterized with oil red O staining. The vulnerable plaque was characterized with vulnerability index.The expression of antigens in brachiocephalic trunk and their plaques among 5 groups such as TLR4, SM-actin and MAMO-2 were compared semi-quantitatively using immunohistochemical technology. The differential displays of mRNAs for TLR4, IL-6, IL-10, IL-12, TNF-α VCAM-1 MMP-9 and ICAM-1 were determined using relative quantitative real-time PCR analysis.Results 1. We construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones successfully; Real time PCR and Westemblot displayed that the expression of TLR4 mRNA and protein were significantly downregulated in ECV304-pTLR4-1 cells when compared with those of other groups.2. The effection of atorvastatin on LPS upregulated TLR4 in ECV304 cells Western blot showed that LPS+atorvastatin group reversed the LPS-inducedincreases in TLR4 dose dependently. Maximal effects were obtained after 12h of exposure to atorvastatin (1 μmol/L)3. The effection of TLR4-RNAi and atorvastatin on the NF-κB activation induced by LPS-TLR4ELISA showed that relative to the basal activity detected in control cells(0.67±0.002) , NF-κB binding activity was distinctly enhanced in cells treated for30 min with LPS(1.89±0.04) (P<0.05) , and this augmented NF-κB binding activitywas significantly inhibited in RNAi+ LPS group (0.72±0.02) and atorvastatin +LPS group (0.96±0.12 ) .Western blot showed that comared with control group ,IkBa levels obtained after 30 min of LPS stimulation have a rapid disappearance(P<0.05) .Comared with LPS group, IkBa levels were significantly increase inatorvastatin( 1 μmol/L , 12h)+ LPS(30min)group.While compared with LPS group,IkBa levels have no significant difference in RNAi+LPS group. (P>0.05)4. The effection of TLR4-RNAi on the PI3K-Akt induced by LPS -TLR4 Western blot showed that comared with LPS (30min) group, p-Akt/Akt(0.73±0.07 vs 0.2±0.02) and p65 (43.4±1.29 vs 23.78 ±0.26) levels were significantly decrease in RNAi+ LPS (30min) group. Comared with LPS ( 30min) group, p-Akt/Akt (0.73±0.07 vs 0.13±0.008) and p65 levels (43.4±1.29 vs 12.16 ±0.33) were significantly decrease in RNAi+ LPS (30min) group.While IxB-α level had no significant difference among these groups (10.00±0.06 vs 9.89±0.19) (P >0.05) .5. The effection of atorvastatin on the MAPKs activation induced by LPS -TLR4 in ECV304 cell Western blot showed that comared with control group, p-ERK/ERK levels were significantly increase in LPS (100 ng/mL, 30min) group (0.018±0.0001 vs 0.786±0.0276) (P<0.01) .Comared with LPS group, p-ERK/ERK levels were significantly decrease in (1μmol/L , 12h)+LPS (100 ng/mL, 30min) group(0.215±0.0060 vs 0.786±0.0276) (P<0.05) . Comared with control group, p-P38/P38 levels were significantly increase in LPS (100 ng/mL, 15min) group(P<0.01) .Comared with LPS group, p-P38/P38 levels were significantly decrease in atorvastatin (1 μmol/L , 12h)+LPS (100ng/mL, 15min) group (0.690±0.008 vs 0.782±0.015 ) (P<0.05) . Comared with control group, p-JNK/JNK levels were significantly increase in LPS (100 ng/mL, 30min) group (0.053±0.004 vs 0.584±0.011 ) .There was no significant difference in the expression of p-JNK/JNKlevels between LPS group and atorvastatin(1 μmol/L , 12h)+LPS (100 ng/mL, 15min) group.6. The effection of LPS-TLR4- MAPKs induced by atorvastatin on the activity of NF-κB(1) ELISA showed that comared with atorvastatin (1 μmol/L , 12h)+LPS (100 ng/mL, 30min) group group, NF-κB activitions were significantly decrease in atorvastatin ( 1μmol/L , 12h) +PD98059+LPS ( 100 ng/mL , 30min ) group(0.75±0.049 vs 0.96±0.12) (P<0.05) .Westernblot showed that there was no significant difference in the expression of p-ERK/ERK between atorvastatin( 1μmol/L , 12h) + SN50+LPS ( 100 ng/mL , 30min ) group and atorvastatin(1μmol/L , 12h)+LPS (100 ng/mL, 30min) group( 0.221 ±0.0049 vs 0.215±0.0060) (P<0.05) .(2) ELISA showed that there was no significant difference in the expression of NF-κB activitions between atorvastatin (1μmol/L , 12h) + SB203580+LPS (100 ng/mL, 30min) group and atorvastatin(1 μmol/L , 12h)+LPS( 100 ng/mL, 30min) group(1.08±0.21 vs 0.96±0.12) (P>0.05) . Westernblot showed that there was no significant difference in the expression of p-P38/P38 between atorvastatin( 1 μmol/L , 12h) + SN50+LPS ( 100 ng/mL , 30min ) group and atorvastatin(1 μmol/L , 12h)+LPS (100 ng/mL, 30min) group( 0.695±0.0011 vs 0.690±0.0079) (P>0.05) .(3) Westernblot showed that compared with atorvastatin (1μmol/L , 12h)+LPS (100 ng/mL, 30min) group group, p-ERK/ERK levels were significantly increase in atorvastatin (1 μmol/L , 12h) + SB203580+LPS (100 ng/mL, 30min) group (0.240±0.0051 vs 0.215±0.0060) (P<0.05),but there was no significant difference between the two groups.There was no significant difference in the expression of p-P38/P38 between atorvastatin (1μmol/L , 12h) + PD98059+LPS (100ng/mL, 30min) group and atorvastatin(1 μmol/L , 12h)+LPS (100 ng/mL, 30min) group( 0.695±0.0023 vs 0.690±0.0079) (P>0.05) .7. The effection of TLR4-RNAi and atorvastatin on the downstream factors induced by LPS -TLR4IL-6: 12h:ELISA showed that there was no significant difference between atorvastatin group and LPSgroup. Compared with LPS group , IL-6 levels were significantly decrease in RNAi group. 24h: Compared with LPS group , IL-6 levels were significantly decrease in atorvastatin group. Compared with atorvastatin group, IL-6 levels were significantly decrease in RNAi group. 48h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , IL-6 levels were significantly decrease in RNAi group.IL-10: 24h: Compared with LPS group , IL-10 levels were significantly increase in RNAi group. Compared with atorvastatin group , EL-10 levels were significantly increase in RNAi group.TNF-α: 6h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , TNF-α levels were significantly decrease in RNAi group. 12h: Compared with LPS group , TNF-α levels were significantly decrease in atorvastatin group. Compared with atorvastatin group , TNF-α levels were significantly decrease in RNAi group. 24h: Compared with LPS group , TNF-α levels were significantly decrease in atorvastatin group. Compared with atorvastatin group , TNF-α levels were significantly decrease in RNAi group. 48h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , TNF-α levels were significantly decrease in RNAi group.8. Proliferation ,titration and activity detection of TLR4 RNAi adenovirus pSuppressorAd-TLR4 and pSuppressorAd-Neg were amplified in scale.Plaque-forming assay showed that viral titer was 6×1011 pfu/mL . Real-time PCR showed TLR4 mRNA in pSAd-TLR4 group was suppressed.9. The research of TLR4 RNAi adenovirus treatment and atorvastatin treatment on ApoE-knock out miceAfter 10 week treatment, there was no significant difference between RNAi and control group (p>0.05). Moreover, no significant differences in either body weight or total cholesterol and triglyceride concentrations in plasma among four groups were observed. Compared with control group, the area of plaque lipid contents have no significant difference in RNAi group,but there is a thinker fiber cap in RNAi group. Compared with placebo group, the area of plaque has no significant difference but lipid contents were decreased in atorvastatin group. Compared with control or placebo group, the vulnerability index was decreased in RNAi or atorvastatin group. Compared with atorvastatin group, the vulnerability index was decreased in RNAi group. Upregulation of SM-actin antigen activity and downregulation of TLR4 and MAMO-2 antigen activity in RNAi group was observed (P<0.05). Downregulation of TLR4 and MAMO-2 antigen activity in atorvastatin group was observed (P<0.05). Real-time detection of PCR showed increasing of mRNAs for IL-10, while TLR4,IL-6,IL-12,TNF-α,VCAM-1,MMP-9 and ICAM-1 gene expressions decreasing in RNAi group.There is an increasing of mRNAs for IL-10, while TLR4,IL-6,IL-12, VCAM-1 and ICAM-1 gene expressions decreasing in RNAi group.Conclusions1. Construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones (ECV304-pTLR4-1, ECV304-pTLR4-2 and ECV304- pNeg cell) successfully.2. ECV304-pTLR4-1 can effective inhibit the expression of TLR4 mRNA and TLR4 protein3. NF-κB is the key point in immunity reaction. So we study the effection of TLR4-RNAi or atorvastatin on NF-κB and found RNAi may be inhibit the activity of NF-κB better.The mechanism maybe the negative feedback of P38 in atorvastatin treatment.4. TLR4-RNAi prevents NF-κB transactivation independently of the IκB-α pathway.The mechanism is inhibiting PI3K/AKT then p-p65 and then NF- κB.5. Atorvastatin may block NF-κB activation by causing abolition the TLR4 pathway then stabilization of the IkBa in cellular cytoplasm.The mechanism maybe as follows: First, atorvastatin regulate the NF-κB translocation dependent pathway through TAK1- IKKα dependent IκBα stabilization. Second, the inaction of phosphorylation of ERK leads to the IκBα stabilization and then the inhibition of NF-kB translocation. While the suppression of p38MAPK may exert slightly up regulation on ERK and NF-κB.6. Atorvastatin may supprss the expression of IL-6 and TNF-α, and increase the expression of IL-10.Howerver, RNAi had better effect.7. In vivo, TLR4-RNAi treatment and atorvastatin treatment can decrease the vulnerability of plaque. However RNAi had better effect.8. The anti-atheroscleris of TLR4-RNAi and atorvastatin treatment may be attributed to balance of proinflammation/antiinflammation, synthesis and degradation of collagen and immunoregulation...
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