The Effects Of Atorvastatin On Vascular Endothelial Function And TNF-α Level In LPS-stimulated RAW264.7 Macrophages

Background:Sepsis, an overwhelming systemic response to infection characterized by systemic inflammation and multiple organ dysfunction syndrome, is also the primary cause for the death of critical illness. The pathological process of sepsis is very complex, involving activation of generous cells, inflammatory mediator as well as blood coagulation system. Sepsis caused by Gram-negative bacteria, and lipopolysaccharide (LPS), released from the outer membrane of Gram-negative bacteria, are considered to be the major molecule responsible for the endotoxin sepsis. Endotoxin is properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens. It is in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria. It activates the cascade of complement and coagulation, promotes the production of kinin, stimulates macrophage to produce IL-1, IL-6 and tumor necrosis factor-α, as well as other cytokines and mediators, which play important role in the pathologic processes of sepsis. LPS is released and bound to the main effector cells including endothelial cells, monocytes, macrophages, and neutrophil cells to produce a great deal of cytokines, namely "cytokines storm", and then untrolled inflammatory cascade reaction including hemorrhage, leukocytes infiltration, vasodilatation, plasma protein extravasation and edema was originated, which leads to endotoxin shock, tissue damage and multiple organ dysfunction syndrome. Accordingly, it is very important to control inflammatory response in the therapy of sepsis, and also the key study project in this field.Atherosclerosis is a multiple-factor disease. Over the years there have been many theories from different perspectives to elaborate its pathogenesis, including lipid infiltration theory, receptor deficiency theory and endothelial injury theory. According to different theories mentioned above, the different therapy strategies of atherosclerotic disease are developed. At present, it is well accepted that AS is originated and developed due to the endothelial injury caused by the major risk factors, and AS is thought to be the artery inflammatory fibrous proliferative response to vascular endothelial injury. Increasing evidences support that inflammation plays pivotal role in the pathogenesis and development of atherosclerosis. It has been demonstrated that an inflammatory process was involved in all stages of atherosclerosis including the infiltration of inflammatory cells, formation of foam cells, endothelial dysfunction, generation of fatty streak (plaque) and the final coronary events such as acute coronary syndromes (ACS). Vascular endothelium is an important protective barrier of blood vessel. Vascular endothelial dysfunction, as an early systemic change pathological process of atherosclerosis, as well as inflammatory response participate by macrophages through the whole process of occurrence and development of the AS. Vascular endothelial dysfunction and inflammatory response are core links of the course of atherogenesis, and become the main target of preventing and treating atherosclerotic diseases. Vascular endothelial is the key protective barrier. Vascular endothelial dysfunction is an early systemic change during the course of atherosclerosis, and it together with inflammatory response mediated by macrophages participates in all the courses of atherosclerosis, whuch is also the key parts of atheroselerotic diseasesand the main target of preventing and treating atherosclerotic diseases.Cardiovascular and cerebrovascular diseases have already excessed cancer to become the top killer of mankind, while atherosclerosis is the most important basis of cardiovascular and cerebrovascular diseases. Despite of progress of atherosclerosis treatment, the morbility and hospitalization rates remain higher and seem to be increasing, as a result, the patients with sepsis and arteriosclerotic diseases are also increasing. The treatment of sepsis has been the research hot spot of critical illness, while the treatment of the patients with sepsis and arteriosclerotic disease has not been well concerned and studied due to the cross field between critical illness and arteriosclerotic disease. Although some progress was made in the treatment of sepsis in recent years, the mortality of sepsis is still high, and the mortality of the patients with sepsis and arteriosclerotic diseases are much higher. Accordingly, it is worthwhile to search the methods to shorten the duration of hospital and reduce mortality of these patients. However, the new drug research and development needs much longer time and huge amounts of money. Thus, exploring new therapeutic effects by using the existing drugs is becoming a trend in recent years.Statins, namelyβ-hydroxy-β-mathylglutaryl-CoA (HMG-CoA) inhibitor, is one of the most important lipid-lowing drugs widely used in clinic, and it is one of the key drugs to reduce lipids and cardiovascular events. Recent studies showed atherosclerosis is an inflammatory disease, share many similarities with the pathogenesis of sepsis. Recent studies showed that statins could not only reduce blood lipids, but also antagonize inflammation, oxidation, thrombus, which is statins' non-lipid-lowing effects. Increasing studies demonstrated that the cardiac protective effects of statins are not only due to its lipid-lowing effects, but also anti-inflammatory effects. Accordingly, statins is accepted to be the outstanding representative of anti-inflammatory drugs, and the studies about statins in many fields become deeper and deeper. Sepsis being the inflammatory disease and statins possessing wide anti-inflammatory effects, consequently, it is possible that statins antagonizes sepsis by regulating the gene expression in inflammatory network and alleviating inflammatory response. A new retrospective analysis from Toronto showed that statins might help to prevent sepsis for patients with cardiovascular disease. Recent several small-scale clinical studies demonstrated that statins had important protective effects on the prognosis of patients with sepsis and septic shock, but the mechanisms are still unclear. So far, large-scale randomed control trials are lacking due to the characteristic of patients with sepsis and septic shock.Hemeoxygenase-1 (HO-1) is a kind of kinase to degrade hemocrystallin. With the help of NADPH, cytochrome P-450 reductase, and dioxygen, hemocrystallin is degraded to biliverdine, carbon monoxide (CO), and iron ions by HO-l's catalysis. Biliverdine is reduced as hemocrystallin with very strong anti-oxidant ability, and CO is an important messenger molecule. Recent studies showed that the co-operation of HO-1 and its degradation products including hemocrystallin, CO, and transferrin resulted in the effects to antagonize imflammation response as well as oxidation stress, inhibit cell apoptosis and improve tissue microcirculation, participating in antagonizing oxidative stress injury of tissue cells of heart, brain, lung, liver, kidney, and so on. HO-1 and its degradation products is one of the most important endogeneous protective systems, recently, protection of tissues and organs gradually becomes the research hotspot of this field. HO-1 as the protective factor, the effects against inflammation, oxidative stress, apoptosis, hyperplasia have been demonstrated in endothelial cells, cardiomyocytes, smooth muscle cells, and the other cells. Accordingly, the present study aimed to evaluate the effects of atorvastatin on lipopolysaccharide (LPS)-mediated inflammatory response and the associated mechanisms to make certain the possible value of atorvastatin in the treatment of the patients with sepsis combined arteriosclerotic disease and establish the theory base in this field.Part 1 Pivotal role of inflammation in vascular endothelial dysfunction of hyperlipidemic rabbit and atorvastatin's effects on itObjective:To evaluate the role of inflammation in vascular endothelial function of hyperlipidemic rabbits and atorvastatin's effects on it.Methods:16 rabbits were divided into single high-fat diet (CTL) and atorvastatin plus high-fat diet (ATV) group. Basic levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), C-reactive protein (CRP), interleukin-6 (IL-6), nitric oxide (NO), endothelin-1 (ET-1) and endothelial function were respectively measured when grouping. Eight weeks later, all above parameters were measured again and repeated at day 1,4 and 7 after atorvastatin withdrawal.Results:Eight-week high-fat diet could significantly induce increased blood lipids, inflammatory markers, imbalance between ET-1 and NO, and direct endothelial dysfunction determined by transcutaneous noninvasive B-type ultrasound, which could be significantly improved by atorvastatin therapy but could not be well controlled to near baseline. Abrupt withdrawal of atorvastatin caused sharp increase of inflammatory markers, and endothelial dysfunction at day 4 and 7 after atorvastatin withdrawal independent of the changes of blood lipids.Conclusions:High-fat diet could cause endothelial dysfunction associated with inflammation, and atorvastatin could counter-regulate it. Sudden withdrawal of statins could induce rebound of inflammatory response and endothelial dysfunction independent of changes of lipids, which may be responsible for increased cardiovascular events in patients with coronary artery disease after withdrawing statins.Part 2 The effects of Atorvastatin attenuates TNF-alpha production in LPS-stimulated RAW264.7 macrophagesObjective:To evaluate the effects of atorvastatin on the expression and secretion of TNF-αin Lps stimulated RAW264.7 macrophages and related mechanisms.Methods:Resuscitated well-being RAW264.7 macrophages were selected for experiment, and cells'activity was evaluated by trypanblau dying. The secretion of TNF-αwere measured by enzyme linked immunosorbent assay (ELISA), and the mRNA expressions of TNF-αand heme oxygenase-1 (HO-1) were detected by real-time RT-PCR. Protein expression of HO-1 was measured by westernblot.Results:(1) HO-1 expression could be significantly increased by atorvastatin treatment. (2)LPS could significantly increase mRNA expression of TNF-αand its secretion in dose- and time-dependent manners, which could be significantly attenuated by atorvastatin. In addition, Suppressing HO-1 activity by SnPP could significantly attenuate attorvastatin's effects on TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages, while SnPP could not significantly attenuate TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages Conclusion:Atorvastatin can attenuate LPS-induced TNF-αexpression and secretion by activating HO-1.