The Mechanisms And Inhibitory Effects Of Sodium Paeonolsilate On Vascular Inflammation And Foamy Cells Formation

BackgroundChronic inflammation plays a crucial role in the initiation and progression of atherosclerosis (AS) and endothelial disorder plays a major role in the early stage of AS. Under the effects of multiple pathogenic factors, vascular endothelial cells are activated and the expressions of adhesion molecules, pro-inflammatory cytokines and chemokine receptor are increased, which assist the infiltration of inflammatory cells and results in endothelial dysfunction, increase of oxidative stress, apoptosis, lipidosis and finally occurrence of AS.The activation of transcription factor NF-κB is closely correlated with the occurrence and development of inflammatory response of AS. NF-κB is homogenous or heterogeneous dimmer composed of P50, P52, P65, RelB and c-Rel, and is involved in the modulation of pathophysiology processes such as immune, inflammation and proliferation. Under the normal condition of cells, the components of NF-κB are constrained in the cytoplasm. Under the exogenous stimulation, IκBs can be activated by the upstream IκB kinase (IKK) complex, phosphorylated, ubiquitinated and degraded by protease, which finally results in the release of NF-κB (predominantly in dimmer of P65/P50) and translocation from cytoplasm to nuclei to modulate the transcription and expression of target genes.Oxidative stress plays important role in formation process of AS. Oxidative stress is the imbalance between the generation of excessive active oxygen and the anti-oxygenation system clearing active oxygen. Active oxygen can affect many functions of vascular endothelia, such as the phenotype modulation of growth, apoptosis and survival of vascular endothelial cells and smooth muscle cells, the endothelia-dependent vascular dilation, the damage of vascular wall due to lacking NO bioactivity, the adhesion increase of monocytes and the angiogenesis.As a new kind of compound of paeonol extracted from the moutan bark and after sulfonation and salifying, sodium paeonol sulfonate has multiple effects in anti-inflammation, pain-killing, antitumor, anti-lipid peroxidation, and immunity modulation. Recent study indicated that sodium paeonol sulfonate has effect in anti-AS, but the mechanism of which is not clear. Therefore, the present study investigated the effect of sodium paeonol sulfonate on the vascular inflammation and its possible mechanism.Objectives1. To investigate the effect of sodium paeonol sulfonate on the expressions of adhesion molecules, pro-inflammatory cytokines and chemokine receptor in monocytes induced by TNF-a.2. To investigate the effects and possible mechanism of sodium paeonol sulfonate on the NF-κB activation and oxidative stress in endothelial cells induced by TNF-a.Methods1) Culture of human aortic endothelial cellsHuman aortic endothelial cells (HAECs) were cultured with ECM medium containing20%FBS in5%CO2incubator at37℃and changed with medium every3days. Passaging and plating were completed as required.2) Establishment of cell modelHAECs were incubated with5,10and20ng/ml TNF-a, respectively, for24h. The expressions of ICAM-1and VCAM-1were examined with Western Blot.3) Adhesion experiment of monocytesTHP-1cells labeled with calcein-AM at final concentration of5μmol/L were co-cultured with HAECs in dark at37℃for2h. The cells were rinsed with PBS for2-3times to remove un-adhered cells and added2ml PBS to be observed under confocal microscopy. The excitation wavelength and emission wavelength was485nm and530nm, respectively.4) ROS measurementAccording to the manual of ROS kit, DCFH-DA was used to measure the release of intracellular superoxides after treatment with sodium paeonol sulfonate.5) NO,SOD and LDH measurementsNO,SOD and LDH were measured according to the manuals of kits respectively. 6) Protein measurement with Western blotThe expressions of ICAM-1and VCAM-1, the phosphorylation of p-eNOS, and the activities of NF-κB and p-IKKβ were measured with Western blot.7) Establishment of in vivo inflammation of miceThe in vivo inflammation of mice was established by intraperitoneal injection of30μg/kg TNF-a.8) Measurement of serum ICAM-1, VCAM-1, IL-8and MCP-1The levels of serum ICAM-1, VCAM-1, IL-8and MCP-1were measured with artery blood according to the manual of ELISAkit.9) ImmunohistochemistryUnder deep anesthesia, the thoracic aorta was removed, dehydrated in gradient sucrose solution, embedded with paraffin, cut for coronary slices with a Fully Motorized Rotary Microtome at4μm and stored at-80℃. The tissue staining was performed according to the procedure of ICAM-1and VCAM-1staining kit.Results1. Sodium paeonol sulfonate can inhibit the expressions of ICAM-1and VCAM-1, reduce the release of ROS, increase the production of NO and SOD, decrease the vascular inflammatory response, respectively induced by TNF-α.2. Sodium paeonol sulfonate increased the phosphorylation of endothelial eNOS and decreased the activities of NF-κB and the expressions of p-IKKβ, respectively induced by TNF-α.3. Sodium paeonol sulfonate inhibited the vascular inflammation induced by TNF-a in in vivo model.ConclusionSodium paeonol sulfonate can inhibit the expressions of ICAM-1and VCAM-1, reduce the release of ROS, increase the production of NO and SOD, decrease the vascular inflammatory response, induced by TNF-α, and maintain the normal function of endothelial cells. BackgroundFoamy macrophages and inflammatory factors play important roles in atherosclerosis (AS). Endothelial dysfunction is the initial factor of AS while metabolism disorder of lipid is the key step of AS. Oxidative stress can induce endothelial injury and modify LDL to ox-LDL which can aggravate endothelial inflammation and promote macrophages to form foamy cells. After apoptosis or necrosis, the lipid in foamy cells is released to form stripped atherosclerotic plaque. Therefore, the formation of foamy cells plays same important role in the occurrence and development of AS.The extracted paeonol from moutan bark is formed of sodium paeonol sulfonate (Spae) through sulfonation and salifying, and has many effects including anti-inflammation, pain killing, anti-tumor, anti-lipid oxygenation, immune modulation. From previous chapter, we have found that Spae has effects in antagonizing the oxidative stress and vascular inflammation induced by TNF-a. Simultaneously, many studies have confirmed that the formation of foamy cells accompanies the formation of inflammation and the continuity of oxidative stress. In the present section, we further investigated the inhibitory effect of SPae on the foamy macrophages and AS induced by ox-LDL.Therefore, the present study investigated the effect of SPae on the foamy macrophages and AS formation induced by ox-LDL from the three features in cell model and in vivo model:(1) establishing the model of foamy macrophages to observe the effects of SPae on the phagocytosis of ox-LDL by foamy macrophages and on the expressions of scavenge receptor A (SRA), CD36and ATP-binding cassette transport protein A1(ABCA1);(2) using apoE-/-mouse AS model to study the therapeutic effect of SPae on AS;(3) to investigate its potential mechanism at molecular level.Objectives1) To investigate the effect of SPae on the formation of foamy macrophages.2) To investigate the effect of SPae on the early plaque of AS in vector model.Methods1) Establishment and identification of foamy macrophagesTHP-1cells were induced to differentiate for24h by100ng/ml PMA. The differentiated cells were treated with80μg/mL ox-LDL for24h, rinsed with pre-cooled PBS twice, fixed with60%isopropanol for5min, stained with0.3%rathonum red solution for15min. After removal of staining solution, the cells were observed under inverted microscopy. Existence of massive red lipid drops in foamy macrophages demonstrated formation of foamy macrophages from THP-1cells.2) Treatment of cellsThe cells were randomly divided into6groups:normal group, high-dosage control group, model group, high-dosage group, middle-dosage group and low-dosage group. For model group, THP-1macrophages were treated with80μg/mL ox-LDL for24h. For SPae treatment groups, the cells were pre-incubated with SPae for2h, followed by co-incubation with SPae and80μg/mL ox-LDL for24h. The high, middle and low dosage of SPae was40μM,20μM and10μM, respectively.3) Rathonum red stainingThe cells treated as method2) were fixed with60%isopropanol for5min, stained with0.3%rathonum red solution for15min and rinsed with PBS twice. After mounting with aqueous mounting medium, the cells were observed under fluorescent microscopy and taken photos randomly.4) Measurements of intracellular cholesterol, NO,SOD and ROSThe cells treated as method2) were measured of intracellular total cholesterol and cholesterol ester according to the kit manual. NO,SOD and ROS were measured according to the manuals of kits respectively.5) Effect of SPae on the binding of THP-1macrophages with Dil-oxLDLUnder4℃, the cells treated as method2) were incubated with10μg/mL Dil-oxLDL for2h, rinsed3times and taken photos randomly with confocal microscopy (400x) with the excitation wavelength and emission wavelength of520nm and580nm, respectively.6) Effect of SPae on the uptake of Dil-oxLDL in THP-1macrophagesUnder4℃, the cells treated as method2) were incubated with10μg/mL Dil-oxLDL for4h, rinsed3times and taken photos randomly with confocal microscopy (400x) with the excitation wavelength and emission wavelength of520nm and580nm, respectively.7) Effect of SPae on the expressions of CD36and SRA in THP-1macrophagesThe cells treated as method2) were analyzed the effect of SPae on the expressions of CD36and SRA in THP-1macrophages, using Western blot.8) Effect of SPae on the expression of ABCA1in THP-1macrophagesThe cells treated as method2) were analyzed the effect of SPae on the expression of ABCA1in THP-1macrophages, using Western blot.9) Measurement of effects of ox-LDL on PPARγ, NF-κB, pP38, JNK and oxidative stress.Western Blot was used to measure the effects of ox-LDL on the expressions of IKKβ, β-IKKβ, p-IKBα, IkBα, P38, pP38, PPARγ, JNK and P-JNK in THP-1macrophages. NO,SOD and ROS were measured according to the manuals of kits respectively.10) Establishment of apoE-/-mouse AS modelFifteen male apoE knockout (apoE-/-) mice and3wild-type male C57BL/6J mice were used. apoE-/-mice were randomly divided into model group, high-dosage group, middle-dosage group and low-dosage group,3mice for each group. From the beginning, all animal were synchronously administrated with chemical once every day and high-fat food, for consecutively16weeks11) Immunohistochemistry and immunofluorescenceUnder deep anesthesia, the hear and thoracic aorta were removed, dehydrated in gradient sucrose solution, embedded with OCT, cut for coronary slices with cryomicrotome at8um and stored at-80℃. The tissues were stained with rathonum red and observed under inverted microscopy for the red AS plaque in the aortic sinus and aorta. Immunohistochemical method was used to measure the levels of mac-3and VCAM-1in plaque. Immunofluorescent method was used to measure the PEC AM-1to evaluate the integrity of vascular endothelia of aortic root. 12) Serum levels of ICAM-1, VCAM-1, TNF-a, MCP-1, IL-8and LDL-CAfter anesthesia, the eyes of mice were taken out to separate serum. The serum levels of ICAM-1, VCAM-1, TNF-α, MCP-1, IL-8and LDL-C were measured.Results1) Successful establishment of foamy macrophages;2) SPae dose-dependently inhibited THP-1to form foamy macrophages;3) SPae inhibited the expressions of SRA and CD36in THP-1macrophages, decreased the binding of Dil-ox-LDL to and the uptake of Dil-ox-LDL by THP-1macrophages, increased the expression of ABCA1, and enhanced the transportation of intracellular free cholesterol to extracellular space;4) SPae inhibited the accumulation of AS plaque and reduced the inflammatory response in in vivo mice model.ConclusionThrough modulating the activities of PPARy and NF-κB and the oxidative stress, and modulating the expressions of SRA and ABCA1and the formation of inflammation during AS, SPae achieves the anti-AS effect. BackgroundCompared with patients without diabetes, patients with diabetes mellitus (DM) had significantly increased cardiovascular morbidity and mortality. So far,coronary stent had been widely used for revascularization in acute cardiac events and proved to improved prognosis, with a high success rate and favorable early outcome. The traditional bare-metal stent (BMS) had been initially widely used for treatment of coronary heart disease and had considerable efficacy and safety. However, the long term outcome and restenosis rate has been very discouraging. Recently, sirolimus-eluting stents (SES) had been increasingly performed for treating restenosis after BMS as well as the native coronary narrowing, and made therapeutic progress. Efforts to explore the treatment of coronary heart disease patients with diabetes mellitus at home and abroad has never been interrupted.For coronary arterial disease (CAD) patients with diabetes, the outcome, the efficacy and safety of SES and BMS remain controversial in different clinical trials.ObjectiveTo compare the efficacy and safety of sirolimus eluting and bare metal stents in CAD patients with diabetes by meta-analysis.Methods: The clinical trials were searched according to the detailed inclusion and exclusion criteria, The following key words were used in Chinese,"rapamycin eluting stent;,sirolimus eluting stent, bare metal stent, diabetes, coronary heart disease". The following key words were used in English,"rapamycin eluting stent,sirolimus eluting stent, bare metal stent, diabetes, coronary arterial disease". According to modified Jadad score, two independent researchers included in the study evaluated the quality of each article separately. When consensus can not be reached at the controversial point, the third reviewer were asked and resolved the discrepancies. Grade1-3belongs to low quality and4-7to high quality. If the results of the measurement indexes from the included literatures being dichotomous variables, Odds ratio (OR) and95%confidence intervals (CI) were estimated in each study. Statistical results were expressed in the forest plots and publication bias was evaluated with funnel plots, Begger’s and Egger’s test.Results:Meta-analysis was then performed in a total of1764patients of SEM group and1912patients of BMS group with diabetes from18studies. Authenticity was evaluated for all literatures included in the study according to modified Jadad quality assessment form.10literatures of Western populations were scored between2-7, including two low-quality documents and eight the high-quality literatures,while the8literatures of Chinese populations scored between1-2,belonged to the low-quality literatures. In the research of Western populations, compared with those in bare-metal stent group, the subjects in sirolimus-eluting stent group had reduced risk for target lesion revascularization (OR0.26,95%confidence interval (CI)0.16-0.45), and for major adverse cardiac events (OR0.40,95%CI0.28-0.58). There was no difference for myocardial infarction event (OR0.94,95%CI0.66-1.35)or mortality event (OR1.11,95%CI0.72-1.73). In the research of Chinese populations, compared with those in bare-metal stent group, the subjects in sirolimus-eluting stent group had reduced risk for target lesion revascularization (OR0.30,95%CI0.14,0.62), and for major adverse cardiac events (OR0.25,95%CI0.15-0.42). There was no difference for myocardial infarction event (OR0.64,95%CI0.31-1.31) or mortality event (OR0.62,95%CI0.22-1.76). Subgroup analysis showed significant difference for overall risk of major cardiac events between SES and BMS, when the sample size was<90(OR0.30,95%CI0.19～0.47), when it was randomized control trial (RCT)(OR0.30,95%CI0.22～0.40), or when it was performed in Europeans (OR0.42,95%CI0.32～0.52).Conclusion:1、In the research of Chinese and Western populations, target lesion revascularization rate in SEM group was significantly less than that of BMS group.2、In the research of Chinese and Western populations, incidence of myocardial infarction in SEM group was less than that of BMS group, but the difference was not statistically significant.3、In the research of Western populations, mortality event in SEM group were more than the BMS group, and in the research of Chinese populations, mortality event rate in SEM group were less than that of BMS group., but the difference was not statistically significant.4、In the research of Chinese and Western populations, incidence of major cardiac events in SEM group was significantly less than that of BMS group.Our study confirmed that SES is more effective and safer than BMS in CAD patients with diabetes.