TLR4 Signaling In Monocytes From Unstable Angina Patients With Type 2 Diabetes And Its Regulatory Mechanisms

Background and Objective: Inflammation is the the common soil of atherosclerosis (AS) and type 2 diabetes (T2DM). Coronary heart disease (CHD) is the main type of AS. T2DM is CHD risk equialents. Instable plaque is the pathology foundation of acute coronary syndrome (ACS), and inflammation play a key role in plaque rupture, thrombosis, and local vasoconstriction. It has been evident that ACS patients with T2DM have serious coronary artery lesions, bad curative effects, and bad prognosis when compare with those without T2DM owing to the enhanced infla- mmation.It has been demonstrated the innate and the adaptive immunity participate systematic inflammatory response. Pattern-recognition receptors (PRRs) paly a key role in the innate immunity, which may be a bridge linkage between inflammation and AS. Tolls are the main PRRs, and TLR4 is a member of this family. The activation of TLR4 induces the expression of many inflammatory cytokines. TLR4 was expressed by different cell types in the atherosclerotic vessl wall: endothelial cells, macrophages, adventitional fibroblasts and dendritic cells. The expression of TLR4 is also increased in the peripheral blood monocytes from the patients with ACS. In addition, the IKK activation and the inflammatory cytokines secretion induced by the activation of TLR4 signaling mediate insulin resistance, which is the key pathophysiologic mechanism basis of T2DM. However there is less and vary researches about TLR4 expression in patients of T2DM. In the past researches, we found that the over-activation of TLR4 existed in the peripheral blood monocytes of ACS patient without diabetes; the actived monocytes produced massive inflammatory cytokines. The inflammatory response mediated by TLR4 may play a significant role in the formation, development, and rupture of atherosclerosis plaque. However, it is still unclear whether the enhanced inflammatory response in ACS with T2DM has connections with the over-activation of TLR4 and the regulation effect of TLR4 signal pathway.Researches indicate that various endogenous and exogenous ligands can specifically activate TLR4 , participate further in the inflammatory cytokines generation and inflammatory responses via TLR4/NF-kB signaling. Recently, it is found that the zinc finger protein A20 negativly regulate TLR4 signaling through ubiquitina- tion and de-ubiquitination. A20 can be induced by a wide variety of stimuli including TNF-α,LPS. A20 gene is a NF-κB target gene. A20 protein can inhibit the activation of NF-κB. A20 is essential for the termination of TLR-induced NF-κB active- tion and pro-infla- mmatory cytokines production. It is unclear that how A20 is ex- pressed in patients of CHD, T2DM and CHD with T2DM.Inflammatory cytokines secreted from actived monocytes/macrophages in instable plaque result in plaque rupture, and those mono- cytes/ma- crophages come from circulation. Thus, monocytes in circulation can reflect the situation of the cells in plaque. The present study was to explore changese of TLR4 and A20 in peripheral monocytes from T2DM patients, UA patients and UA patients with T2DM, and to analyze their corelationg with plasma inflammatory cytokines. Further, changese of A20 and cytokines after the activation of TLR4 and the effect of A20 transfection on TLR4 signaling in monocytes from these patients were examined. Finally, the effects of high concentration of glucose on TLR4 signaling and the intervention of atorvastatin or magnolol (Mag) were observed.Methods: Part one: The objects were divided into four groups: controls (group A):20, T2DM group (group B):22, UA group (group C):18, UA with T2DM group (group D):19. TLR4 protein on monocytes was examined by flow cytometry. A20 protein in monocytes was detected by immuno- histochemistry. TLR4mRNA and A20mRNA were analyzed by real time RT-PCR. The concentrations of plasma TNF-αand IL-10 were measured by ELISA. We found that the expression of TLR4 and A20 in monocytes, the level of plasma TNF-αand the ratio of TNF-α/IL-10 were higher in T2DM group and UA group when compared to those in controls(P<0.05). They were highest in UA with T2DM(P<0.05). TLR4 mRNA and TLR4 protein positively correlated with FBG,FIN,HOMA-IR,HbA1c,TNF-αand IL-10. (P<0.05).Part two: (1) Monocytes were isolated from the objects above. Isolated monocytes were stimulated with LPS or PBS for 3h to examine TLR4mRNA and A20 mRNA by real time RT-PCR and for 24h to analyze the level of TNF-αand IL-10 in supernants by ELISA. The results showed that after stimulated by LPS, the expressions of TLR4mRNA and A20 mRNA were upregulated in monocytes from controls, but changes were not obvious in monocytes from type 2 diabetic patiens, UA patients, and UA patients with T2DM. Monocytes from type 2 diabetic patiens, UA patients, and UA patients with T2DM produced more TNF-αbut less IL-10 in response to LPS when compared to those from controls (P<0.05). (2)Monocytes from type 2 diabetic patients, UA patients, and UA patients with T2DM were divided into A20 plasmid transfected group and empty plasmid transfected group respectively. After 48h transfection with plamid, monocytes were stimulated with LPS for 2h to explore the expression of NF-κBp65 by immuno- histochemistry, and for 24h to examine the concentration of TNF-αand IL-10 in supernatants by ELISA. Transfection effects were valued according detecting GFP reporting gene by immunofluorescence, examing exogenous A20 gene by real time RT-PCR and analyzing A20 protein by immunohistochemistry. The results demonstrated enhanced A20 expression by transfection A20 gene into monocytes can reduce NF-κBp65 expression and decrease TNF-αsecretion while increase IL-10 secretion.Part three: Peripheral blood monocytes isolated from health people were divided into four groups: 5.6mM glucose(Glu), 25mM Glu, 25mM Glu+20μM Mag, 25mM Glu+10μM atorvastatin. After 48h incubation, TLR4mRNA and A20mRNA in monocytes were messured by real time RT-PCR, TLR4 and A20 protein were analyzed via western blot. Monocytes were incubated with 5.6mM Glu, 25mM Glu, 25mM Glu+20μM Mag, 25mM Glu +10μM atorvastatin for 48h. Then stimulated with LPS for 2h to examine NF-κBp65 by western blot and stimulated with LPS for 24h to explore the level of TNF-αand IL-10 in supernatants via ELISA. We found that high glucose enhance TLR4 and A20 expression. Pre-exposure of healthy monocytes to high glucose concentrations markedly enhanced the LPS-induced expression of NF-κBp65 in monocyte and TNF-αsecretion from monocytes but impaired LPS- induced IL-10 secretion from monocytes compared with pre- exposure to normal glucose. Aatorvastatin or Mag decreased the expression of TLR4 and A20 in monocytes induced by high glucose, and inhibited high glucose-enhanced NF-κBp65 expression and TNF-αrelease, improved IL-10 release.Conclusions: 1. The imbalance between pro-inflammatory and anti-inflammatory occurs in the patients of UA and T2DM. This imbalance gets more prominent in the patients of UA with T2DM.2. TLR4 signaling in monocytes participates in the inflammatory responses of UA and T2DM, and correlates with enhanced inflammation in UA with T2DM.3. The pro/anti-inflammation imbalance in UA, T2DM and UA with T2DM is related to the deregulation of TLR4 and A20 in monocytes.4. The enhancement of A20 expression can suppress the inflammatory reponses induced by the activation of TLR4 signaling in monocytes from UA, T2DM and UA with T2DM.5. High glucose can enhance the inflammatory responses mediated by TLR4 activation via upregulation of TLR4 expression in monocytes.6. Statins can inhibit the effects of high glucose on TLR4 signaling in monocytes by decreasing the expression of TLR4 and suppressing the activation of NF-κB.7. Magnolol can inhibit the effects of high glucose on TLR4 signaling in monocytes by decreasing the expression of TLR4 and suppressing the activation of NF-κB.