| IntroductionRupture of atherosclerotic plaque and secondary thrombosis are the main causes of major adverse cardiovascular and cerebrovascular events (MACCE) during perioperative period.This pathological process depends on the stability of atherosclerotic plaque.Vulnerable plaques (VP) are characterized by thin fibrous cap,large lipid core,low density of smooth muscle cells (SMC),increased infiltration of activated macrophages and the imbalance between the synthesis and degradation of extracellular matrix (ECM),the obvious neovascularization or bleeding in some plaques.Sevoflurane (Sevo) is the most commonly used inhalation anesthetics at home and abroad.Sevoflurane not only plays an anesthetic role,but also has anti-inflammatory,protective effects on heart and brain ischemia-reperfusion injury.At present,some studies suggest that sevo can reduce the incidence of MACCE in general anesthesia,but its specific mechanism is not clear.Rupture of unstable plaque and/or secondary intracavitary thrombosis is the most important link in inducing acute coronary syndrome (ACS) and stroke.So far,whether sevo affects the stability of vulnerable plaque has not been reported.C-Jun N-terminal kinase (JNK) is a member of mitogen activated protein kinase (MAPK) family.A large number of studies have confirmed that JNK plays an important role in myocardial hypertrophy,ventricular remodeling after myocardial infarction,AS and vascular restenosis.Our team's previous study found that tumor necrosis factor alpha (TNF-?) down regulated the expression of 4-hydroxyproline hydroxylase ? 1 (P4H?1) in SMCs by inhibiting the activation of ASK1-JNK-NonO signaling pathway,P4H?1 is the key rate limiting enzyme of collagen synthesis,maturation and secretion.The inhibition of P4H?1 will lead to the decrease of collagen synthesis and secretion,the imbalance of ECM,and the decrease of plaque stability.Previous studies of our team found that sevo can inhibit the contraction of vascular SMC by inhibiting MAPK signal transduction pathway in vascular SMC,which indirectly proved the possibility of interaction between sevo and MAPK signal pathway.In order to explore the possible mechanism of sevo in reducing the occurrence of MACCE during perioperative period,our team put forward the following scientific hypothesis:sevo can inhibit the expression of apolipoprotein E deficiency (ApoE-/-).The above mechanism is related to the inhibition of the activation of ASK1-JNK-NonO signaling pathway by sevo.In order to test this hypothesis,a series of in vivo and in vitro experiments were carefully designed.objective1.To investigate whether sevo can stabilize vulnerable plaque in ApoE-/-mice and its dose effect.2.To explore the pathophysiological mechanism of sevo stabilizing vulnerable plaque in ApoE-/- mice.3.To investigate whether sevo can stabilize vulnerable plaques by inhibiting ASK1-JNK-NonO signaling pathway.MethodsPart one:Experimental study of sevoflurane to stabilize atherosclerotic vulnerable plaque1.Animal models and groupingOne hundred and twenty 8-week-old male ApoE-/-mice were randomly divided into 4 groups after high-fat feeding for 10 weeks.(1) Control group:limited space stress and noise stimulation 6 hours/day;(2) 1% sevo group:inhalation of 1% sevo for 6 hours/day,limited space stress and noise stimulation for 6 hours/day after awake;(3) 2% sevo group:inhalation of 2% sevo for 6 hours/day,limited space stress and noise stimulation for 6 hours/day after awake;(4) 3% sevo group:inhalation of 3% sevo for 6 hours/day,limited space stress and noise stimulation for 6 hours/day.Repeated inhalation of sevo for 4 weeks and stimulation for 4 weeks.At the end of the 14th week,ApoE-/- mice were euthanized.2.Body weight and Blood pressure measurementThe mice were weighed with a small animal weight balance (week 0 and week 14).The blood pressure of mice was measured by mouse tail sphygmomanometer (14th week).The body weight and blood pressure of each mouse were measured three times,and the average value was taken as the final value.3.Tissue specimen collectionThe mice were starved for 6-8 hours before taking the material.After the mice were euthanized by inhaling excessive CO2,blood was taken from the heart immediately,and the whole blood was collected in a separation gel accelerator tube,and the serum was collected by centrifugation and stored in the refrigerator at-80?.Subsequently,the blood vessels were perfused with normal saline,and the heart,complete aortic tissue,liver and kidney were retained;a part of the heart and complete aortic tissue were quick-frozen in liquid nitrogen and then transferred to the-80?refrigerator for storage,and a part was fixed in 4% paraformaldehyde.4.Preparation of tissue specimenThe heart and complete aortic tissue were cut from the outlet of the ascending aorta of the cardiac outflow tract perpendicular to the apical direction,and the base of the heart,namely the aortic root,was taken to make frozen sections.Three complete aortic rings were preserved in each aortic root tissue section;the complete aorta (ascending aorta to common iliac artery branch) was reserved for oil red O staining of the whole aorta.Paraffin sections of liver and kidney were made.5.Blood lipid testingDetection of total cholesterol (TC),triglyceride (TG),low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL) in serum of mice level6.Histopathological examinationThe lipid content of aortic plaques was detected by oil red O staining.Hematoxylin and eosin (H&E) staining of liver and kidney were was used to observe the morphology of liver and kidney.Frozen section histopathological staining of aortic root included:H&E staining was used to observe plaque morphology and cross-sectional area;perls staining was used to observe cross-sectional plaque rupture;oil red O staining was used to observe lipid content in cross-sectional plaque;sirius red staining was used to observe the collagen content in cross-sectional plaques;immunohistochemical staining was used to observe the contents of macrophages and SMCs,and the expression of monocyte chemoattractant protein-1 (MCP-1),TNF-? and interleukin-6 (IL-6).Image Pro Plus 6.0 software was used to measure and analyze the pathological staining tissues.7.Calculation of plaque vulnerability indexAccording to the formula of vulnerability index=(macrophage relative content+lipid relative content)/(smooth muscle cell relative content+collagen relative content),the vulnerability index of aortic root plaque was calculated.8.Statistical analysis of dataSPSS 13.0 was used for statistical analysis.The measurement data were expressed as mean ± standard deviation (SD),and the count data were expressed as value and percentage.The measurement data of two groups were analyzed by independent sample t-test,the measurement data of multiple groups were analyzed by one-way ANOVA Tukey's post test.The count data were analyzed by chi square test.P <0.05 was considered as statistical difference.Part two:Molecular Mechanism of Sevoflurane Stabilizing Vulnerable Atherosclerotic Plaques by Inhibiting ASK1-JNK-NonO Pathway.1.Isolation and culture of vascular smooth muscle cells (SMCs)The aorta was obtained from 6-week-old male ApoE-/- mice.The adventitia of the aorta was removed under general microscope and cut into 1 mm long tissue fragments with sterile ophthalmic scissors.The tissue fragments were mixed in 300-500 ?l serum,and evenly planted in a 25cm2 culture bottle with a straw.The culture bottle was turned upside down and incubated in a 5% CO2,37? saturated humidity incubator for about 1.5-2 h.Then,put the culture bottle in the right position and add 3-5ml DMEM containing 20% fetal bovine serum (FBS).Change the medium every 3 days.The cells with purity of more than 90% were obtained,and passage 3-9 cells were used in the experiment.2.Preparation of saturated medium of sevoSaturated sevo medium was used and prepared on each experimental day.Sevo was added to a high glucose-DMEM containing 20% FBS,and the mixed solution was stored in a sealed glass container at room temperature (22-24?) for at least 2 hours.After standing,the saturated sevo medium containing serum formed the upper layer,and the liquid sevo deposited at the bottom of the container.Saturated sevo medium are stored in sealed glass containers until use.3.Grouping and stimulation of vascular SMCsVascular SMCs were randomly divided into 5 groups(1) Control group:without any treatment;(2) TNF-? group:TNF-? (100 ng/ml) was added to cell culture medium at the same time point as sevo group;(3) 1:33 sevo group:the mixture of saturated sevo medium and cell culture medium with the concentration ratio of 1:33 was treated for 4 hours,then TNF-? (100 ng/ml) was added for 8 hours;(4) 1:10 sevo group:the mixture of saturated sevo medium and cell culture medium with the concentration ratio of 1:10 was treated for 4 hours,then TNF-? (100 ng/ml) was added for 8 hours;(5) 1:3 sevo group:the mixture of saturated sevo medium and cell culture medium with the concentration ratio of 1:3 was treated for 4 hours,then TNF-? (100 ng/ml) was added for 8 hours.The medium containing sevo was changed every 4 hours.After 4 hours of sevo treatment,TNF-? (100 ng/ml) was added to TNF-? group,1:33 sevo group,1:10 sevo group and 1:3 sevo group for another 8 hours.4.Histopathological examinationThe expression of MMP-2 and MMP-9 in aortic root plaques was detected by immunohistochemistry.5.ImmunocytochemistryThe expression of MMP-2 and MMP-9 in vascular SMCs was detected by immunofluorescence staining.6.Quantitative real-time polymerase chain reaction (qRT-PCR)The expression of MMP-2 and MMP-9 mRNA in aorta was detected.The mRNA expressions of MCP-1,VCAM-1,ICAM-1,P4H?1,MMP-2 and MMP-9 in vascular SMCs were detected.7.Western blotThe expressions of MMP-2,MMP-9 and P4Hal in the aorta were detected by Western blot.The expressions of MMP-2,MMP-9,P4H?1,type ? collagen (Col ?),type? collagen (Col ?),ASK1,P-JNK and NonO in vascular SMCs were detected by Western blot.8.Statistical analysis of dataThe method is the same as the part one.ResultsPart one:Experimental study of sevoflurane to stabilize atherosclerotic vulnerable plaque1.Body weight,blood pressure and serum lipid profilesThe ApoE-/- mice in control group and sevo treatment group (1% sevo group,2%sevo group and 3% sevo group) completed the experiment.There was no significant difference in body weight between the control group and the sevo treated group at week 0 and week 14.There was no significant difference in blood lipid levels (TC,TG,LDL-C and HDL-C) and blood pressure between the control group and the sevo treated group at week 14.These results indicated that sevo had no effect on body weight,blood pressure and blood lipid level of ApoE-/- mice.2.Effects of sevo on the liver and kidneys of miceH&E staining of mouse liver and kidney showed:liver cell fatty degeneration,central vein dilation;glomerulus hypertrophy,mesangial cells increased,mesangial matrix showed mild proliferation,and the structure of renal tubules was disordered.There was no significant difference in the pathological changes of liver and kidney between the Control group and the Sevo treatment group (1% Sevo group,2% Sevo group and 3% Sevo group).These above results indicate that long-term use of Sevo does not cause liver and kidney toxicity.3.Effect of sevo on the lipids of aorta and aortic root plaqueGross oil red O staining was used to detect the percentage of lipid content in aortic plaque to show plaque load.Compared with the control group,the percentage of lipid content in the 1%,2%,and 3% Sevo groups were significantly reduced,and with the increase of Sevo concentration,the percentage of lipid content in aortic plaque decreased in a dose-dependent manner.Aortic root plaque H&E staining and oil Red O staining were used to detect the cross-sectional area of the plaque.Quantitative analysis showed that compared with the control group,the plaque cross-sectional area of each dose of sevo treatment group (1% sevo group,2% sevo group and 3% sevo group) was significantly reduced.These above results indicate that Sevo can reduce the lipid content of the aorta and aortic root plaque in a dose-dependent manner.4.Effect of sevo on plaque compositionCompared with the control group,the contents of macrophages and lipids in aortic root plaques of 1%,2% and 3% sevo groups were decreased,while the contents of collagen and SMCs were increased.The vulnerable index of aortic root plaque in sevo groups was significantly lower than that in control group.These results indicate that sevo intervention can reduce the content of macrophages and lipids in plaque,increase the content of collagen and SMCs,and reduce the vulnerability index of plaque.5.Effect of sevo on the expression of inflammatory factorsImmunohistochemical staining results showed that compared with the control group,the contents of MCP-1,TNF-? and IL-6 in 1% sevo group,2% sevo group and 3% sevo group decreased in a dose-dependent manner.These results indicate that sevo intervention can inhibit the inflammation level in plaque.6.Effect of sevo on plaque rupture rateThe results of H& E staining and Perls staining showed that 60% (6/10),40%(4/10),30% (3/10) and 10% (1/10) of as plaques ruptured in control group,1% sevo group,2% sevo group and 3% sevo group,and the rupture rate of each dose of sevo group was significantly lower than that in control group.These results suggest that sevo intervention can prevent vulnerable plaque rupture.Part two:Molecular Mechanism of Sevoflurane Stabilizing Vulnerable Atherosclerotic Plaques by Inhibiting ASK1-JNK-NonO Pathway.1.Effect of sevo on the expression of inflammatory factors in vascular SMCsWe detected the expression of MCP-1,VCAM-1 and ICAM-1 mRNA in vascular SMCs of control group,TNF-? group and sevo treated group (1:33 sevo group,1:10 sevo group and 1:3 sevo group) by qPCR.The results showed that compared with TNF-a group,the expression of MCP-1,VCAM-1 and ICAM-1 mRNA in 1:33 sevo group,1:10 sevo group and 1:3 sevo group decreased.These results indicate that sevo inhibits the expression of MCP-1,VCAM-1 and ICAM-1 in vascular SMCs.2.Effect of sevo on the expression of MMP-2 and MMP-9In vivo,we used immunohistochemical method to detect the effect of sevo on the expression of MMP-2 and MMP-9 in ApoE-/- mice.The results showed that compared with the control group,the expressions of MMP-2 and MMP-9 in aortic root plaques in 1 % sevo group,2% sevo group and 3% sevo group were decreased.Western blot and qPCR showed the same trend.These results suggest that sevo inhibits the expression of MMP-2 and MMP-9 in aortic plaques of ApoE-/- mice.In vitro,we detected the effect of sevo on the expression of MMP-2 and MMP-9 in vascular SMCs by cell immunofluorescence.The results showed that compared with the control group,the expression of MMP-2 and MMP-9 in vascular SMCs stimulated by 100 ng/ml TNF-? increased.Compared with TNF-? group,the expressions of MMP-2 and MMP-9 in vascular SMCs pretreated with sevo at the concentrations of 1:33,1:10 and 1:3 were decreased.The results of Western blot and qPCR were consistent with those of immunofluorescence.These results suggest that sevo inhibits the expression of MMP-2 and MMP-9 in vascular SMCs.3.Effect of sevo on the expression of P4H?1In vivo,we detected the effect of sevo on P4Ha1 in ApoE-/- mice by Western blot.The results showed that compared with the control group,the expression of P4H?1 in aortic root plaques was increased in 1 % sevo group,2% sevo group and 3% sevo group.These results suggest that sevo promotes the expression of P4H?1 in aortic plaques of ApoE-/- mice.In vitro, we detected the effect of sevo on the expression of P4H?1 in vascular SMCs by Western blot.The results showed that compared with the control group,the expression of P4H?1 in vascular SMCs stimulated with 100 ng/ml TNF-? decreased.Compared with TNF-? group,the expression of P4H?1 in vascular SMCs pretreated with sevo at the concentrations of 1:33,1:10 and 1:3 was increased.The same trend was also confirmed by qPCR.These results suggest that sevo promotes the expression of P4H?1 in vascular SMCs.4.Effect of sevo on collagen synthesisIn vitro,the effect of sevo on collagen synthesis in vascular SMCs was detected by Western blot.The results showed that compared with the control group,the expression of type ? collagen and type ? collagen in SMCs stimulated with 100 ng/ml TNF-? decreased.Compared with TNF-? group,the expression of type ? collagen and type ? collagen increased in vascular SMCs pretreated with sevo at the concentrations of 1:33,1:10 and 1:3. These results suggest that sevo promotes collagen synthesis in vascular SMCs.5.Effect of sevo on ASKI/JNK/NonO pathwayWe detected the expression of ASK1,P-JNK and NonO in vascular SMCs by Western blot.The results showed that the expressions of ASK1,P-JNK and NonO in SMCs stimulated with 100 ng/ml TNF-? were higher than those in control group.Compared with TNF-? group,the expressions of ASK1,P-JNK and NonO protein in vascular SMCs pretreated with sevo at the concentrations of 1:33,1:10 and 1:3 were decreased.These results suggest that sevo can inhibit the activation of ASK1-JNK-NonO signaling pathway in vascular SMCs.6.Sevo promotes the expression of P4H?1 by inhibiting the activation of the ASK1/JNK/NonO pathwayIn order to verify whether sevo can promote the expression of P4H?1 by inhibiting the activation of ASK1-JNK-NonO pathway,selonsertib (10 ?m) was added to the cell culture medium 1 hour before the stimulation of vascular SMCs with 100 ng/ml TNF-?.The results showed that compared with TNF-? group,the expressions of ASK1,P-JNK and NonO in vascular SMCs in selonsertib group and sevo treated groups (1:33 sevo group,1:10 sevo group and 1:3 sevo group) were decreased,while the expression of P4H?1 was increased.These results suggest that both sevo and selonsertib can promote the expression of P4H?1 by inhibiting the activation of ASK1-JNK-NonO signaling pathway.Conclusion1.In the ApoE-/- mouse vulnerable plaque model,Sevo can stabilize the vulnerable plaque in a dose-dependent manner and reduce the rupture rate of the plaque.2.The mechanism of sevo stabilizing as vulnerable plaque is to inhibit or reduce local inflammatory response,reduce the release of inflammatory factors and MMP-2,MMP-9,increase the level of P4H?1 and increase collagen deposition.3.Sevo increases the expression of P4H?1 by inhibiting ASK1-JNK-NonO signaling pathway.Innovation and significance1.It is the first time that the inhaled anesthetic Sevo can stabilize atherosclerotic vulnerable plaque in a dose-dependent manner.2.It was verified that Sevo stabilizes atherosclerotic vulnerable plaques by increasing collagen deposition and inhibiting inflammation.3.Clarify the important role of ASK1-JNK-NonO signaling pathway in Sevo's stabilization of atherosclerotic vulnerable plaques.Limitations1.There is no ideal model for atherosclerotic vulnerable plaques that are simple to manufacture and have a high plaque rupture rate.2.In vitro,the concentration of Sevo in the cell culture medium is difficult to maintain constant due to volatilization.3.It has not been further proved that Sevo can inhibit inflammation by inhibiting the ASK1-JNK-NonO pathway.4.It has not been further verified that Sevo stabilizes the rupture of vulnerable plaques by inhibiting the activation of the ASK1-JNK-NonO pathway by ApoE-/-JNK-/-mice. |
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