| 1.BackgroundAbdominal aortic aneurysm(AAA)is a chronic inflammatory vascular disease,which is more common in men over 65 years old.Patients often come to the emergency department with severe chest pain as the first symptom.Once the aortic aneurysm ruptures,it has a higher mortality rate.Histologically,abdominal aortic aneurysms are characterized by infiltration of inflammatory cells in the adventitia and middle layer of blood vessels,destruction of extracellular matrix and formation of new blood vessels.At present,the pathogenesis of AAA is still poorly understood.Surgery is the only widely used therapy for the disease.Therefore,more researches are urgently needed for the treatment of AAA.More and more evidences show that regulating the expression of inflammation-related genes is an effective strategy to delay the progression of AAA.Poly(ADP-ribose)polymerase 1(PARP-1)is a ribozyme that can be activated by DNA damage and promote DNA repair.A large number of studies have shown that PARP-1 is activated in inflammatory diseases,such as myocardial reperfusion injury,stroke and shock.Inhibiting the expression of PARP-1 can reduce inflammation-related reactions.However,whether PARP-1 inhibition can prevent angiotensin ?(Ang ?)-induced AAA and the mechanism remains to be studied.Based on the above knowledge,we hypothesized that inflammation in AAA may induce PARP-1 activation,and inhibition of PARP-1 may slow down the formation of AAA by inhibiting the inflammatory response.Therefore,we tried to explore the therapeutic effect of PARP-1 inhibition on Ang ?-induced AAA and its possible mechanism.2.Objectives(1)To verify whether the expression and activity of PARP-1 in mice AAA tissue increase;(2)To explore whether inhibition of PARP-1 can reduce the formation and severity of AAA;(3)To clarify the mechanism of inhibiting PARP-1 on reducing AAA.3.Methods3.1 Experimental Animals(1)Animal strainsWild-type C57BL/6J mice were purchased from BeijingVital River Laboratory Animal Co.,Ltd.ApoE-/-and PARP-1-/-mice were generated in BeijingVital River Laboratory Animal Co.,ApoE-/-PARP-1-/-mice were crossed and bred by ApoE-/-and PARP-1-/-mice.The nursing and laboratory procedures complied with the "Guidelines for the Protection and Use of Laboratory Animals".In this study,all experiments were approved by the ethics committee of Qilu Hospital of Shandong University.(2)Grouping of experimental animalsMice aged 6?8 weeks were randomly selected and divided into four groups:ApoE-/-mice infused with PBS(control)group,ApoE-/-mice infused with Ang ?(Ang ?)group,ApoE-/-PARP-/-mice infused with PBS(PARP-1-/-)group,ApoE-/-PARP-1-/-mice infused with Ang ?(Ang ?+PARP-1-/-)group.3.2 Establishment of AAA modelPBS or Ang ? was injected into 6-8 weeks old male ApoE-/-mice or ApoE-/-PARP-1-/-mice through a subcutaneous osmotic pump,and the release constant concentration was 1000 ng/kg/min.The 28-day survival rates of the mice in each group were observed.After 28 days,the AAA formation rates and diameters of aneurysm were recorded.At the end of the experiments,the tissues were fixed or stored in-80? refrigerator after quenched in liquid nitrogen.3.3 RNA extraction,reverse transcription and real-time fluorescent quantitative PCR(qRT-PCR)The RNA from the abdominal aortic tissue or AAA tissues of the four groups was extracted,and the mRNA was reverse-transcribed into cDNA using a reverse transcription kit.The Ct value of the target gene was detected by qRT-PCR,and?-actin was used as an internal parameter.Ct values were calculated by 2-??CT.3.4 Western blotting analysisThe protein of the abdominal aortic tissue or AAA of the four groups was extracted,and the protein concentration was determined by BCA,then SDS-PAGE gel electrophoresis was performed to detect the protein expression of PARP-1.3.5 Histological examination and immunohistochemical examinationThe tissues fixed in 4%paraformaldehyde were dehydrated and embedded into wax blocks,and 4 ?m paraffin sections were prepared,and hematoxylin-eosin staining(H?E)was used to analyze the morphology of the tissues.Masson staining was used to detect the content of tissue collagen,immunohistochemistry was used to analyze the composition of vascular smooth muscle cells and the infiltration of inflammatory cells.3.6 Cell cultureHuman aortic endothelial cells(HAECs)are cultured in endothelial cell medium(ECM),which is contains 10%fetal bovine serum(FBS),100 IU/mL penicillin and 100 ?g/mL Streptomycin.Human aortic vascular smooth muscle cells(VSMCs)were cultured in DMEM supplemented with 10%FBS,100 IU/mL penicillin and 100?g/mL streptomycin.All cells are cultured in a 37? incubator containing 5%CO2.3.7 Dihydroethidium staining(DHE)In vitro,HAECs were divided into 3 groups:PBS control group,Ang ?stimulation group,and Ang ?+PARP-1 siRNA.5 ?M DHE was added to each group,incubated at 37? for 30 min in the dark,and then washed 3 times with ECM without FBS.Fluorescence microscopy was used to detect the production of reactive oxygen species(ROS)in HAECs.3.8 Transwell experimentIn vitro,VSMCs were divided into 3 groups:PBS control group,Ang ? group,and Ang ?+PARP-1 siRNA.100 ?L VSMCs suspension of each group were added into the Transwell chamber;then 600 ?L serum-free medium were added into the lower chamber,the Transwell chamber were taken out 24 hours later,and the medium in the well were discarded,then they were washed twice with calcium-free PBS,and fixed with methanol for 30 min,0.1%crystal violet was stained for 20 min;after washing with PBS 3 times,five fields of view were randomly taken under the microscope to observe and count the cells.3.9 EdU staining experimentThe cells of the control group,the Ang ? group and the Ang?+siRNA-PARP-1 group were inoculated into a 12-well plate previously placed on a sterile small round glass slide.After the cells adhered to the wall,EdU solution was added to each well.After fixation with cell fixative and treatment with 0.1%Triton X-100,200 ?L Click-iT reaction mixture were added into each slide,incubated for 30 min at room temperature and in dark,after stained the nucleus with DAPI;pictures were taken under a confocal microscope for observation,The results were analyzed with ImageJ software.3.10 Annexin-V/PI experimentVSMCs were divided into 3 groups in vitro:PBS control group,Ang ? group,Ang?+siRNA-PARP-1 group.1?L Annexin V-APC dye was added in VSMCs suspension of each group and mixed well.After that,it was placed at 37? for 20 minutes,and 1?L PI was added into each tube for 2 minutes,then flow cytometry was performed.3.11 Data Statistics and AnalysisAll the above experimental results were obtained from three or more independent repeated experiments,statistical software Graphpad Prism 8 was used for data statistics and graph drawing.Kaplan-Meier survival curve was used to observe the survival rates between each group,and the Log-Rank method was used for comparative analysis.First,the Shapiro-Wilk test was used to test the normality of the data distribution.For normally distributed univariate data,unpaired Student-t test analysis was used between the two groups;for three groups and more than three groups one-way analysis of variance(ANOVA)test was used,and then Newman-Keuls was used.For data that was not normally distributed,the non-parametric Kruskal-Wallis H test was used to analyze the non-parametric statistics.P<0.05 was considered statistically significant.4.Results4.1 Increased expression and activity of PARP-1 in AAA tissueAfter the AAA model was established by Ang II infusion,compared with the PBS infusion group,the expression of PARP-1 mRNA and protein in the AAA tissue of mice induced by Ang ? was significantly increased,and the activity was significantly enhanced.4.2 PARP-1 knockout reduced the formation rate of AAA and mortalityCompared with the PBS infusion group,Ang ? infusion successfully induced the formation of AAA in ApoE-/-mice.Compared with the Ang ? group,the AAA formation rate of mice in the Ang ?+PARP-1-/-group decreased,and the mortality rate due to aortic rupture decreased.4.3 PARP-1 knockout reduced the diameter and thickness of AAACompared with the control group,the Ang ? infusion group significantly increased the aortic diameter and wall thickness;and compared with the Ang ? group,the aneurysm diameter of the AAA mice in the ApoE-/-PARP-1-/-group was lower than that of the Ang ? group.The wall thickness was significantly reduced.4.4 PARP-1 knockout reduced the infiltration of inflammatory cells in blood vesselsCompared with the control group,the contents of monocytes and macrophages in the aortic endothelium were significantly increased after Ang ? stimulation;compared with the Ang ? group,the Ang ?+PARP-1-/-group had less intravascular monocytes and macrophages.4.5 PARP-1 knockout increased smooth muscle cell in blood vesselsCompared with the control group,the expression of the smooth muscle cell marker ?-SM actin was significantly decreased after Ang ? stimulation.Compared with the Ang ? group,PARP-1 knockout could increase the expression of ?-SM actin.4.6 PARP-1 knockout increased collagen fibers in blood vesselsCompared with the control group,the content of collagen fibers was significantly reduced after Ang ? stimulation;compared with the Ang ? group,PARP-1 knockout increased the expression of collagen fibers in blood vessels.4.7 Inhibiting the expression of PARP-1 in HAECs can reduce the oxidative stress induced by Ang ?In HAECs,Ang ? induced cell oxidative stress and DNA damage.After reducing PARP-1 expression,cell oxidative stress and DNA damage were alleviated.4-8 Inhibiting the expression of PARP-1 in VSMCs reduced migration,proliferation and apoptosisIn VSMCs,Ang ? promoted cell migration,proliferation and apoptosis,while inhibitng of PARP-1 improved cell migration,proliferation and apoptosis.5 Conclusions(1)The expression and activity of PARP-1 in AAA tissues were significantly increased compared with normal abdominal aorta;(2)PARP-1 knockout can reduce the formation rate and mortality of AAA,and improve the diameter and thickness of AAA;(3)PARP-1 knockout can increase the composition of smooth muscle cells and content of vascular collagen fibers in blood vessels while decrease the infiltration of monocytes and macrophages;(4)Inhibiting the expression of PARP-1 can alleviate the oxidative stress induced by Ang ? in HAECs;(5)Inhibiting the expression of PARP-1 can reduce the migration,proliferation and apoptosis of VSMCs.In conclusion,inhibiting PARP-1 expression may be a feasible strategy for the treatment of AAA.1.BackgroundSepsis is a systemic inflammatory response syndrome caused by the imbalance of the host's response to infection,and the incidence has been increasing year by year.In sepsis,vascular endothelial cells transfer to the pro-apoptotic,pro-inflammatory,pro-adhesion and pro-coagulant phenotype,release cytokines,chemokines,and procoagulant factors,resulting in increased permeability to proteins and fluids,causing intermittent qualitative leakage.Therefore,studying the activation pathways that lead to vascular endothelial dysfunction caused by sepsis is an effective way to limit organ failure caused by sepsis.Remifentanil is an effective ?-opioid receptor agonist.A number of studies have proved that remifentanil plays a protective role in a variety of inflammatory diseases,such as acute lung injury,ischemia/reperfusion injury and excitotoxic brain injury.However,the potential role and mechanism of remifentanil in sepsis have not been fully studied.Studies have shown that remifentanil can inhibit the release of inflammatory factors and oxidative stress in cells induced by hypoxia/reoxygenation.In the above studies,we have shown that inhibiting PARP-1 can inhibit the oxidative stress response.In this study,we aimed to investigate whether remifentanil can play a protective role in sepsis by regulating the expression of PARP-1 and the underlying mechanism of the effects.2.Objectives(1)To explore whether remifentanil has a protective effect on lipopolysaccharide(LPS)-induced inflammation in human aortic endothelial cells(HAECs);(2)To explore whether remifentanil can reduce the expression and the activity of PARP-1 induced by LPS in HAECs;(3)To explore whether remifentanil can inhibit the LPS-induced inflammatory response in HAECs through the PARP-1/NF-?B signaling pathway.3.Methods3.1 Cell culture and cell treatmentHAECs were cultured in endothelial cell medium(ECM),which contained 10%fetal bovine serum(FBS),100 IU/mL penicillin and 100 ?g/mL Streptomycin.All cells were cultured in a 37? incubator containing 5%CO2.HAECs were seeded in a 24-well plate at a density of 105/well,and they were divided into three groups:control group,LPS+PBS group and LPS+remifentanil group.HAECs were pretreated with an equal volume of PBS or 2.5 ?M remifentanil for 30 min,and then stimulated with 10 ?g/mL LPS for 24 h.Cells were collected for subsequent experiments.3.2 Dihydroethidium staining(DHE)stainingThe HAECs were divided into three groups:control group,LPS+PBS group and LPS+ remifentanil group.The production of O2-in each group of HAECs was measured according to the DHE instructions.The cells were incubated in 5 ?M DHE at 37? for 30 min in the dark,and then washed 3 times with ECM without FBS.Results were obtained at 535 nm using a fluorescent microscope.3.3 Comet assayTo detect DNA damage,we followed the instructions of the Comet Assay Kit,and observed the length of the comet tail through a fluorescence microscope,and then we used the CaspLab Comet Assay software to analyze the cells of each group.3.4 RNA extraction,reverse transcription and real-time fluorescent quantitative PCR(qRT-PCR)The RNA of the above three groups of HAECs was extracted,and the mRNA was reverse-transcribed into cDNA using a reverse transcription kit.The Ct value of the target gene was detected by qRT-PCR,?-actin was used as an internal parameter,and 2-??CT was used to calculate the relative expression of targets genes such as iNOS,ICAM-1 and PARP-1.3.5 Western blotting analysisThe proteins of the above three groups of HAECs were extracted,and the protein concentration was determined by BCA,and then SDS-PAGE gel electrophoresis was performed to detect the expression of PARP-1,iNOS,ICAM-1,PARP-1,PAR and NF-?B p65.3.6 Data Statistics and AnalysisThe data was expressed as the meanąstandard deviation(meanąSD),and Graphpad 8.0 was used for statistical analysis of the data.All the above experimental results were obtained from three or more independent repeated experiments.First,the Shapiro-Wilk test was used to test the normality of the data distribution.For normally distributed univariate data,unpaired Student-t test analysis was used between the two groups of data;three groups and more than three groups were analyzed by one-way analysis of variance(ANOVA)test,and then Newman-Keuls was used between multiple groups comparative test analysis.For the data not belonging to the normal distribution,the non-parametric Kruskal-Wallis H test was used to analyze the non-parametric statistics.P<0.05 was considered statistically significant.4.Results4.1 Remifentanil reduced LPS-induced superoxide anion production in HAECsCompared with the control group,LPS stimulation increased the production of superoxide anions in HAECs,and remifentanil pretreatment significantly reduced the production of superoxide anions.4.2 Remifentanil reduced LPS-induced DNA damage in HAECsCompared with the control group with little DNA in the tail,in LPS group,DNA content was significantly increased in the tail of HAECs,and compared with the LPS group,remifentanil pretreatment significantly reduced the DNA content in the tail of HAECs.4.3 Remifentanil reduced the expression of iNOS and ICAM-1 induced by LPS in HAECsThe results of qRT-PCR and Western blotting showed that LPS stimulation significantly increased the expression of iNOS and ICAM-1 mRNA and protein in HAECs compared with the Control group;compared with the LPS group,remifentanil pretreatment significantly reduced LPS-induced the expression of iNOS and ICAM-1 mRNA and protein in HAECs.4.4 Remifentanil reduced the expression and activity of PARP-1 induced by LPS in HAECsThe results of qRT-PCR and Western blotting showed that compared with Control group,LPS stimulation significantly increased the expression of PARP-1 mRNA and protein in HAECs,and significantly increased the expression of PAR,the active form of PARP-1.Compared with the LPS group,remifentanil can reduce the expression of PARP-1 mRNA and protein induced by LPS in HAECs,and significantly reduced the expression of PAR,the active form of PARP-1.4.5 Remifentanil inhibited LPS-induced nuclear translocation and expression of NF-?B p65Immunofluorescence results showed that NF-?B p65 was mainly located in the cytoplasm of HAECs before LPS stimulation.After LPS stimulation,NF-?B p65 was mainly localized in the nucleus.In the remifentanil pretreatment group,p65 remained mainly in the cytoplasm after LPS stimulation.In addition,Western blotting results showed that compared with the control group,LPS can significantly increase the expression of NF-?B p65,while PARP-1 siRNA or remifentanil can reduce the expression of NF-?B p65 induced by LPS5 Conclusions(1)Remifentanil can reduce LPS-induced superoxide anion production and DNA damage in HAECs.(2)Remifentanil can reduce the increase in the expression and activity of PARP-1 induced by LPS in HAECs.(3)Remifentanil can reduce the LPS-induced inflammatory response in HAECs through the PARP-1/NF-?B signaling pathway.1 BackgroundVascular endothelial cell glycocalyx is a layer of villous polyglycoprotein composite structure located on the apical membrane of endothelial cells,between the blood vessel wall and the blood,and is a dynamic natural barrier on the surface of endothelial cells.In patients with severe sepsis,the endothelial cell glycocalyx is significantly thinned.The degradation of glycocalyx is also considered to be one of the causes of microcirculation dysfunction in sepsis.The above research results suggested that inhibiting the expression of PARP-1 had a protective effect on vascular endothelial cells in sepsis.PJ34 is a new specific inhibitor of PARP-1.Cecal ligation and puncture(CLP)is a classic animal model of sepsis.In this part of the study,we will establish a CLP-induced sepsis model and continue to explore whether inhibiting the expression of PARP-1 can act on glycocalyx to protect vascular endothelial cells from sepsis on the basis of previous studies.2 Objectives(1)To verify that inhibition of PARP-1 expression has a protective effect on sepsis mice induced by CLP;(2)To verify that inhibiting the expression of PARP-1 can reduce the release of inflammatory factors in septic mice;(3)To clarify that in the CLP-induced sepsis model,the glycocalyx of aortic vascular endothelial cells falls off;(4)To determine whether inhibition of PARP-1 can protect septic mice by regulating the glycocalyx of vascular endothelial cells.3 Methods3.1 Experimental animalsWild-type male C57BL/6J mice aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.The experimental mice were randomly divided into four groups:normal control group(Control group),PJ34 group,Control+CLP group and PJ34+CLP group.Among them,the PJ34 group and PJ34+CLP group were injected intraperitoneally with 25 mg/kg PJ34 for 3 consecutive days before establishing the model.In the Control group and Control+CLP group,an equal volume of DMSO was injected intraperitoneally for 3 consecutive days before the model was established.3.2 Construction of CLP modelIn the Control+CLP group and PJ34+CLP group,after anesthetizing the mice,their chest and abdomen were sterilized;sterile scissors were used to cut an incision in the abdominal wall of the mice,then we entered the abdomen through the incision and separated the distal end of the ileocecal valve and ligated 1/3 cecum with silk thread;the syringe needle was ligated and perforated at the colon end,and a little of feces were squeezed out,and the peritoneum and skin were sutured intermittently.In the Control group and PJ34 group,only abdominal incision was performed,and cecal ligation and perforation were not performed.The survival rates of the mice were observed,and the blood and aortic tissue of the mice were obtained for the follow-up experiments.3.3 ELISA was used to detect the expression of inflammatory factors in serumWe detected the expression of IL-6,IL-1? and TNF-? in serum of mice according to the ELISA kit instructions.3.4 ELISA was used to detect glycocalyx components in serumWe detected the expressions of Syndecan-1,heparan sulfate and hyaluronic acid in serum according to the ELISA Kit instructions.3.5 Data Statistics and AnalysisAll the above experimental results were obtained from three or more independent repeated experiments,statistical software Graphpad Prism 8 was used for data statistics and graph drawing.Kaplan-Meier survival curve was used to observe the survival rate between each group,and the Log-Rank method was used for comparative analysis.First,the Shapiro-Wilk test was used to test the normality of the data distribution.For normally distributed univariate data,unpaired Student-t test analysis was used between the two groups of data;three groups and more than three groups were analyzed by one-way analysis of variance(ANOVA)test,and then Newman-Keuls was used between multiple groups for comparative test analysis.For data that was not normally distributed,the non-parametric Kruskal-Wallis H test was used to analyze the non-parametric statistics.P<0.05 was considered as statistically significant.4 Results4.1 Inhibition of PARP-1 expression improved the survival rate of CLP miceAfter constructing a CLP-induced sepsis model,the mortality of mice was significantly higher than that of the control group.Compared with the Control+CLP group,the survival rate of the PJ34+CLP group was significantly improved.4.2 Inhibiting PARP-1 reduced the expression of inflammatory factors in the serum of CLP miceAfter constructing a CLP-induced sepsis model,the expression of inflammatory factors IL-6,IL-1? and TNF-? in the serum was significantly higher than that in the Control group.Compared with the Control+CLP group,the levels of IL-6,IL-1? and TNF-? in the PJ34+CLP group were significantly lower.4.3 The protective effects of inhibiting PARP-1 on the glycocalyx of CLP-induced vascular endothelial glycocalyx in sepsis miceAfter constructing a CLP-induced sepsis model,the levels of Syndecan-1,heparan sulfate and hyaluronic acid in the serum of mice were significantly increased.Compared with the Control+CLP group,the expressions of Syndecan-1,heparan sulfate and hyaluronic acid were significantly lower.5 Conclusions(1)Inhibition of PARP-1 expression can reduce the mortality of CLP sepsis mice.(2)Inhibiting the expression of PARP-1 can reduce the release of inflammatory factors in septic mice.(3)The glycocalyx of the aortic vascular endothelial cells in CLP sepsis mice was severely shed.(4)Inhibition of PARP-1 expression can reduce glycocalyx shedding of aortic endothelial cells in mice with CLP-induced sepsis. |
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