| Research background and aimThe Toll-like receptors（TLRs） exist on the surface of mammalian immune cells and are regulated by germline-encoded system, when they are provoked by structural motifs from bacteria, viruses and fungi, belong to PAMPs. Of all TLRs, TLR4 is deemed to a crucial factor, activation of which regulates not only the release of pro-inflammatory cytokines but also the functional maturation of antigen presenting cells of the innate immune system. After recognizing and binding to TLR4 in the membrane, LPS transmits signals through My D88（myeloid differentiation primary response gene 88） dependent pathway and TRAM-TRIF（also called as MyD88 independent pathway） pathway. In this process, adapter proteins TIRAP and TRAM raise aptamer proteins My D88 and TRIF respectively, and initiate two molecular signaling pathways from membrane and early endosome. Then signal information cascade is transmitted into cells, and activated nuclear transcription factor results in releasing inflammatory factor and I interferon. Over activation of this pathway will cause severe inflammation and sepsis, therefore several negative regulatory protein molecules are involved in signal transduction feedback inhibition. These negative regulatory molecules have interactions with effector molecules in inflammatory signaling pathways, constituting precise and complicated network regulatory system, which determines the activation degree of inflammatory cells ultimately. Nowadays LPS/TLR4 signal transduction pathway is still under investigation, and some regulatory mechanisms and molecules are to be studied.As the most important nuclear transcription factor in LPS/TLR4 inflammatory signal transduction pathway, NF-κB participates in the signal transduction of two pathways, and has a key role in regulating transcription synthesis of inflammatory mediators. In mmon state, it forms a complex with its inhibitor IκB and is the inactive state. When stimulus occur in cells, external messages are passed on into cytoplasm, and IκB is degraded by phosphorylation, thus NF-κB is released and translocated into cell nucleus, activates related genes’ transcription, and releases inflammatory cells factors TNF-α、IL-6 and IFN-γ. Therefore, the research of new ways to control the NF-κB is helpful to improve the level of the understanding about the mechanism of LPS/TLR4 signal transduction pathways. Ubiquitin can be linked to target molecules covalently to form poly-ubiquitin chain mediated proteins, which can be degraded. Ubiquitin serves a critical function for transcriptional modulation, signal transduction, inflammation, and immunity and so on. Recent researches show that SUMO-specific protease 6 has some similarities to ubiquitin, can make target de SUMOylation, and participate all NF-κB related signal pathways, among which it has negative regulatory effects. Since SENP6 is a newly found inhibitory molecule, its expression and regulation mechanisms in macrophages are still unknown. Therefore if regulatory molecule of SENP6 can be found in macrophages, we can inhibit the important transcription factor NF-κB to regulate LPS/TLR4 signal pathways at the same time. Small molecule RNA,namely the non-coding RNA, it is consist of about 20-24 nucleotides. Small molecule RNA can have base complementary with target molecule’s 3’-untranslated region, thus it can degrade target molecule’s m RNA or inhibit the expression of target molecule’s translation levels. Several experiments indicate that mi RNAs are an important regulatory element of protein post-translation levels and have a very broad influence. Current research results show that several proteins related to infection, inflammatory immune, etc are under mi RNAs’ control. Besides, mi RNAs perform a vital function in adjusting signal transduction of TLRs. The question that whether mi RNAs can regulate SENP6’s up-regulated expression needs to be confirmed by future researches.In addition to the transcription factor NF-κB, its upstream effector TLR4 has an important function for the signal delivery in My D88/TRAM-TRIF pathways. TLR4’s activation plays important initiation and regulatory functions in two pathways’ signal transduction. Thus TRAM-TRIF signal pathway and MyD88 signal pathway are closely connected by TLR4, and two pathways can be regulated by the distribution of TLR4 on membrane and organelles. Both the MyD88 and the TRAM-TRIF signal transduction pathways play an important role in the macrophages activation which be mediated by LPS/TLR4. Studies indicated that in LPS mediated cell activation, the degree of cellular activation and the delivery of signal are largely influenced by the amount of TLR4 on the plasma membrane and the transport efficiency after internalization; the inhibition of TLR4-LPS complex internalization can promote the activation of MyD88 signal pathway. In eukaryotic cells, Rab protein（Ras related in brain） act as a key regulatory molecule on intracellular transport, it is not only involved in the activity of TLR4 transport, its transport efficiency but also may have an important influence on the distribution of the TLR4. In summary, the Rab molecule is likely to be involved in the regulation and control of LPS/TLR4 mediated monocyte and macrophage activation. And the role of Rab protein in this process remains to be studied in further experiments.From previous study we learnt that, chloroquine could enhance survival ratio of pyemic mice caused by LPS; chloroquine pretreatment has a significant negative regulatory effect on the activation（several cell factors like TNF-α、IL-6 are released） of h PBMC and ANA-1 caused by LPS stimulation. CQ could not bind and antagonize directly LPS’s viability, thus its inhibitory effect on inflammation can only be taken on the effector molecules of LPS/TLR4 signal pathways. Research indicates that chloroquine, as a kind of anti-inflammatory drug, can inhibit excessive activation of monocytes and macrophages mediated by LPS/TLR4. However its potential mechanism of action is not yet clear. These remind us that the transcription factor NF-κB is able to control the release of inflammatory cytokines, CQ is probably to inhibit cell factor’s upstream transcription factor NF-κB. So we consider that whether SENP6 is related to CQ’s inhibition on LPS/TLR4 signal pathways. In addition, the research group also found in previous experiments, CQ may change TLR4 amount of cell membrane. These hints that, TLR4 may be affected by the efficacy of CQ, resulting in the changes of intracellular translocation efficiency. Thus there are changes of the number of TLR4 in cell plasma membrane and TLR4’s distribution, making TLR4 is degraded in endosome and does not participate in signal transduction, and negatively regulate the activation of LPS induced monocyte macrophage. In view of the regulation of CQ on multiple effector molecules in LPS/TLR4 signal pathways, we employ this mechanism as a tool to further study LPS/TLR4 signal transduction pathways.This study is going to study the inhibitory effect of CQ on two signal transduction pathways of mediated LPS/TLR4 monocyte and macrophage activation, by studying the different inflammatory effector molecules experiments, the possible molecular mechanism of inhibition of LPS/TLR4 mediated macrophage activation is described in two aspects. On the one hand, study the regulatory effect and mechanism of SENP6/miRNAs on the most important transcription factors NF-κB in the inflammatory signaling pathway; on the other hand, study the key intracellular transport molecules Rab5/7 by adjusting the distribution and expression of TLR4 cells, the effect and possible mechanism of the two TLR4 mediated downstream signal pathways activation. Altogether, through the research on the inhibitory effect of different molecules in LPS-induced inflammatory signaling pathways, this study provides some experimental experience for explaining some key regulatory mechanisms in LPS/TLR4-mediated macrophage activation and discovering new regulatory molecules of inflammation.Methods1. Study on the inhibiting function of chloroquine on the LPS/TLR4 mediated signal transduction pathways.1.1 One hour later after pretreated mouse macrophage RAW264.7 with 20μg/ml CQ, LPS（100ng/ml） was added to provoke 24 hours, then detected the levels of inflammatory cytokines TNF-α, IL-6 and IFN-β with ELISA method;1.2 Western blot（WB）was applied to affirm the change of signal molecular in TLR4 signal pathway: IκB-α, inhibitor of NF-κB, IRF3, the level of JNK、ERK1/2、p38 and protein phosphorylation in MAPKs signal transduction pathway;1.3 Observe the change of transposition into the nuclear of NF-κB p65 and IRF3 by means of laser confocal imaging.2. Study on the mechanism of miR-669 n by regulating and controling SENP6 to inhibit the LPS/TLR4 mediated signal transduction pathways.2.1 Detect if the expression of SENP6 is related to CQ and the effect of SENP6 on the LPS mediated macrophage activation:（1） One hour later after pretreated mouse macrophage RAW264.7 with different doses of CQ（0、5、10、20μg/ml）, LPS（100ng/ml） was added to provoke 24 hour, SENP6 gene and protein level were respectively detected by means of real time fluorescent quantitative PCR（RT-PCR） and Western blot（WB）;（2） 24 hours later after construction of the over expression of SENP6 and shRNA interference vector（pcDNA3.1） transfection RAW264.7 cells, to confirm SENP6 gene and protein change and assess the effect on the transfection via RT-PCR and WB;（3） RAW264.7 cells were transfected with SENP6 over expression and interference vector. Then LPS was treated in 24 h, then detected the expression change of inflammatory cytokines TNF-α, IL-6 and IFN-β with ELISA method.2.2 Prediction and identification miRNAs in RAW264.7 cells which take SENP6 as the target molecule with Bioinformatics.（1） Predicted by using the target analysis software TargetScan, Miranda, and miRecords, and select the miR-669 n complementary of the 3’UTR sequence of the target gene SENP6;（2） One hour later after pretreated mouse macrophage RAW264.7 with different doses of CQ（0、5、10、20μg/ml）, LPS（100ng/ml） was added to provoke 24 hour,then collected the sample, to affirm the mRNA change of miR-669 n throught RT-PCR;（3） Construct the over expression of miR-669 n and antisense expression vector（antisense of miR-669 n,as-miR-669n） to transfect RAW264.7 cells, to confirm the gene change of miR-669 n and assess the effect on the transfection via RT-PCR;（4） Build luciferase reporter gene for SENP6, together with the over expression of miR-669 n and antisense expression vector to transfect RAW264.7 cells,according to the number of fluorescence, judge if miR-669 n can affect the expression of target gene.2.3 The effect of miR-669 n by regulating and controlling SENP6 on the LPS mediated macrophage activation:（1） 24 hours later after the over expression of miR-669 n and interference vector of miR-669 n transfect RAW264.7 cells separately, to confirm the effect of miR-669 n on SENP6 gene and protein change via RT-PCR and WB;（2） 24 hours later after the over expression of miR-669 n and interference vector of miR-669 n transfect RAW264.7 cells separately, LPS（100ng/ml） was added to process 24 hour, then detected the expression change of inflammatory cytokines TNF-α, IL-6 and IFN-β with ELISA method;（3） Establish the Septic mice model on miR-669 n over expression and inhibition expression through tail vein injection of MiR-669 n and as-miR-669 n vectors: Divided the female mice（10 weeks old） into control group, miR-669 n over expression group, and the inhibition expression group, 10 in each group. Inject the carrier of physiological brine preparation through the tail vein separately, make LPS（20mg/kg） intraperitoneal injection 48 hour later, collect peripheral blood and detect cell factor by ELISA method 3 hour later, separate liver cells and extract mRNA and protein, RT-PCR and WB were used to affirm the gene and protein change of miR-669 n and SENP6, then assesss the effect on transfection; the status of mice were observed and recorded during 40 hours, to ensure the effect on the mice with sepsis with the expression change of miR-669 n.3. Study on the mechanism of Rab5/7 inhibiting the LPS/TLR4 mediated signal transduction pathways by adjusting TLR4’s distribution3.1 Study on the adjusting function of chloroquine on the expression and distribution of TLR4（1） The effect of CQ on the expression of TLR4 protein in cells by Western blot detection method（2） The effect of CQ on the expression of cytomembrane TLR4 by flow cytometry detection method;（3） Detect the localization of TLR4 cells by confocal laser scanning: Label with Immunofluorescence respectively on TLR4（Cy3）, early stage endosome（EEA1, FITC）, late stage endosome / lysosomal LPS（LAMP1,FITC）, Golgi apparatus（Giantin, FITC）, Rab11（Circulating endosome, FITC） were used to observe the distribution of TLR4 in the cells before and after CQ process.3.2 Study on the mechanism of Rab5/7 inhibiting LPS/TLR4 mediated signal transduction pathways3.2.1 Effect of CQ on the macrophages activation influence after silencing Rab5 and 7（1） Combined with the known function of Rab, select Rab5 and Rab7 which produce a significant effect on the distribution of TLR4. RT-PCR and WB were used to examine the effect of CQ on the mRNA and protein level of Rab5/7.（2） SiRNA technique was used to detect the expression of Rab5 and Rab7 respectively, 24 hours later following the transfection of RAW264.7, to detect the change of gene and protein level of Rab5、7 to evaluate the interference effect via RT-PCR and WB;（3） With the interference via siRNA, RAW264.7 was pretreated with CQ in 1 hour, and LPS（100ng/ml） was added to provoke 24 hours, then detected the levels of inflammatory cytokines TNF-α, IL-6 and IFN-β with ELISA method;（4） After the siRNA interfere on Rab5、7, used Western blot to detect the Molecular changes of inflammatory signaling pathway in the lower reaches of the lower reaches of TLR4: JNK、ERK1/2、p38 and the protein phosphorylation level in the signal transduction pathway of IκB-α、IRF3、MAPKs;（5） After the siRNA interfere on Rab5、7, detect the subunit p65 and the transposite of the nuclear IRF3 of the transcription factor NF-κB by laser confocal localization.（6） Construct luciferase reporter vector of the target NF-κB and IRF3 responsive genes（IFN-β）, RAW264.7 cells were respectively interfered by siRNA Rab5/7 for 24 hours, CQ performed for 1 hour, added LPS（100ng/ml） 6 hours for provoking, used double fluorescent pigment enzymatic assays detection to reflect the change levels of NF-κB and IRF3 activity.3.2.2 Influence of acid inhibitor NH4 Cl on LPS induced macrophage activation（1） One hour later after pretreated mouse macrophage RAW264.7 with 500μg/ml NH4 Cl, LPS（100ng/ml） was added to provoke 24 hours, then detected the levels of inflammatory cytokines TNF-α, IL-6 and IFN-β with ELISA method;（2） WB was applied to assess the effect of NH4 Cl on Rab5/7 via protein level.4. Statistical analysis: the experimental data were expressed by mean±standard deviation（mean±SD）. Data processing software SPSS 13.0 was applied to analysis and contrast the single factor of variance（One-way ANOVA）, P<0.05 is considered as existing significant difference.Results1. Study on the inhibiting function of chloroquine on the LPS/TLR4 mediated signal transduction pathways.1.1 ELISA results showed that CQ could significantly make the expression of inflammatory cytokines TNF-α、IL-6、IFN-β down（P<0.05）;1.2 ELISA results showed that CQ could significantly make phosphorylation levels of multiple inflammatory signaling pathways in the lower reaches（pIκB-α、p-IRF3、p-JNK、p-ERK1/2、p-p38）down, significantly make the expression of IκB-α up（P<0.05）.1.3 The laser confocal results showed that CQ can significantly inhibit the translocation of subunit p65 and IRF3 of nuclear transcription factor NF-κB.2. Study on the mechanism of miR-669 n by regulating and controling SENP6 to inhibit the LPS/TLR4 mediated signal transduction pathways.2.1 Detect if the expression of SENP6 is related to CQ and the effect of SENP6 on the LPS mediated macrophage activation（1）There is a positive adjusting relationship between CQ and SENP6, but the gene expression had no correlated with CQ;（2） The expression of SENP6 was confirmed through RT-PCR and WB, found that a good effect occurred in transfection by the SENP6 over expression and shRNA interference vector;（3） After the over expression of SENP6, significant fall existed in inflammatory cytokines（P<0.05）, and vice versa.2.2 Prediction and identification miRNAs in RAW264.7 cells which take SENP6 as thetarget molecule with Bioinformatics.（1） MiR-669 n was obtained with SENP6 as the target gene by bioinformatics prediction Combined with literature.（2） The expression of mi R-669 n molecules has a negative effect on CQ;（3） The construction of miR-669 n over expression and antisense expression after the vector transfection cells had an obvious influence and effect on the expression;（4） MiR-669 n can be combined with mRNA in the prediction site by using the double luciferase reporter vector with the target SENP6.2.3 The effect of miR-669 n by regulating and controlling SENP6 on the LPS mediated macrophage activation:（1） The results showed that miR-669 n could significantly inhibit the expression of SENP6 protein level in RAW264.7 cells（P<0.05）, but had no significant impact at the genetic level;（2） The over expression of mi R-669 n increased the expression of macrophage inflammatory factor（P<0.05）, but the inhibition expression of miR-669 n decreased the expression of macrophage inflammatory factor（P<0.05）;（3） In the sepsis mice model of miR-669 n over expression and inhibition expression, the over-expressed miR-669 n make significant rise occur to inflammatory factor of peripheral blood（P<0.05）, and the inhibition expression make the peripheral blood cell inflammation factor expression significantly decreased（P<0.05）. The mice dead cells results showed that the transfection effect was obvious, and miR-669 n could significantly inhibit the expression of SENP6 protein level（P<0.05）, but no significant effect was found on the mRNA level, mi R-669 n over expression could significantly reduce the survival rate of mice with sepsis, but the survival rate was significantly increased（P<0.05）.3. Study on the mechanism of Rab5/7 inhibiting the LPS/TLR4 mediated signal transduction pathways by adjusting TLR4’s distribution.3.1 Study on the adjusting function of chloroquine on the expression and distribution of TLR4.（1） The results of WB indicated that there was no significant difference on the protein level of TLR4 following CQ processing.（2） Flow cytometry showed that significant decline occurred to the amount of membrane TLR4 following CQ pretreatment;（3） Laser confocal localization results showed that co localization（Yellow fluorescence co localization） of TLR4 was significantly enhanced together with early stage endosome（EEA1） and late stage endosome/ lysosomal（LAMP1） after CQ pretreatment.3.2 Study on the mechanism of Rab5/7 inhibiting LPS/TLR4 mediated signal transduction pathways.3.2.1 Effect of CQ on the macrophages activation influence after silencing Rab5 and 7（1） The result of real time fluorescence quantitative PCR and Western blot showed that CQ could significantly increase mRNA and protein levels of Western and protein levels of early stage endosome transport molecules（Rab5）and late stage endosome transport molecules（Rab7）（P<0.05） respectively;（2） The RT-PCR and WB detection results showed that it had good interfere effects on the Rab5、7 respectively, after siRNA was silenced with the two molecules;（3） The ELISA results showed that the expression of inflammatory cytokines TNF-α、IL-6、IFN-β was significantly increased（P<0.05） after siRNA was silenced with the two Rab5、7 molecules;（4） WB results indicated that significant decline were happened to phosphorylation levels of various inflammatory signaling molecules（eg. pIκB-α、p-IRF3、p-JNK、p-ERK1/2、p-p38）, the expression of IκB-α decreased significantly（P<0.05） after si RNA was silenced with the two Rab5、7 molecules.（5） The laser confocal results showed that the translocation of subunit p65 and IRF3 of nuclear transcription factor NF-κB increased significantly after siRNA was silenced with the two Rab5、7 molecules.（6） Dual luciferase experiment results showed that the NF-κB responsive genes and IFN-β expression was significantly increased, namely NF-κB and IRF3 transcription activity significantly increased（P < 0.05） after siRNA was silenced with the two Rab5、7 molecules.3.2.2 After NH4 Cl treatment on cells, the ELISA results showed that inflammatory cytokines TNF-α、IL-6、IFN-β had no significant changes, and Western blot results showed that the protein expression levels of Rab5 and 7 had no significant changes.Conclusions1.Confirm that CQ has inhibitory effect on LPS/TLR4 mediated MyD88 dependent and signaling pathway of TRIF/TRAM dependent.2. SENP6 protein with high expression can modulate the macrophage activation, confirm that miR-669 n can act on the target SENP6, while the decrease of expression of miR-669 n can promote the level of SENP6 protein increasing; the negative regulatory effect of mi R-669 n on target SENP6 may be a function of inhibiting the translation of proteins by the mRNA combination of the two.3. The LPS-TLR4 complex could be promote to internalize following CQ pretreatment, can gather TLR4 in the early stage endosome and the late stage endosome. The expression of Rab5 and 7 can increase the intracellular transport efficiency, the intracellular layout of TLR4 may be affected. After Rab5, 7 expressions was inhibited, the inhibition effect of CQ on LPS/TLR4 mediated cell activation was significantly decreased. Therefore, the transport molecules Rab5、 7 may regulate the distribution of TLR4 in the cell, thus play a role in inflammation inhibiting effect. Acidification inhibitor NH4 Cl have no significant effect on LPS/TLR4 mediated cell activation, and have no significant effect on Rab5 and 7 protein expression. Thus, the inhibition effect of CQ was not related to the inhibition of its phosphorylation, but was related to the expression of Rab5 and 7 molecules.4. The inhibitory effect of LPS/TLR4 mediated cell activation may require a synergistic effect of multiple regulatory molecules, regulation of different effector molecules in the inflammatory signaling pathway, developed the inflammation inhibition function in many ways. |
PDF Full Text Request