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Objective @#To investigate the effect of miR-141-3p on LPS induced A549 cell injury by targeting high mobility group protein 1 (HMGB1) ．@*Methods @#A549 cells derived from type Ⅱ alveolar epithelial cells were taken as the study object，miR-141-3p mimics，mimics NC，HMGB1 gene overexpression plasmid (pcDNA3. 1-HMGB1) and empty Vector were transfected into A549 cells respectively or co-transfected，then 10 μg / ml LPS was used for 24 h．Cell proliferation activity was detected by cell counting kit-8 ( CCK-8) ．The activity of lactate dehydrogenase ( LDH) in the supernatant of cell culture was detected by colorimetry．The apoptosis level of each group was detec- ted by flow cytometry．The levels of interleukin (IL) -1 β , IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (Elisa) ．Dual luciferase reporter gene assay verified the targeted regulatory relationship between miR-141-3p and HMGB1 . @*Results @#After treatment with LPS ，the proliferative activity of A549 cells and the expression level of miR-141-3p decreased ( P ＜0. 05 ) ，the apoptosis rate increased ( P < 0. 05) ，the levels of IL-1 β , IL-6，TNF-α and the activity of LDH in supernatant increased (P＜0. 05) ．Overex- pression of miR-141-3p increased the proliferation activity of A549 cells treated with LPS (P ＜0. 05 ) ，and de- creased the apoptosis rate and the levels of IL-1 β , IL-6，TNF-α in cells and LDH activity in supernatant (P < 0. 05) ．However，overexpression of HMGB1 gene could reverse the ameliorative effect of miR-141-3p on LPS-in- duced A549 cell injury．Dual luciferase reporter gene experiment confirmed that HMGB1 was the downstream target gene of miR-141-3p．@*Conclusion @# miR-141-3p can inhibit LPS-induced apoptosis，reduce the expression level of inflammatory factors，and improve the damage of A549 cells，which may be related to the targeted regulation of HMGB1 expression.
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Objective To investigate the effect and mechanism of miR-141-3p on pulmonary fibrosis in rats with acute respiratory distress syndrome(ARDS).Methods Rats were divided into the control group,the model group,the agomir negative control group and the miR-141-3p agomir group according to random number table,with 10 rats in each group.In addition to the control group,the ARDS rat model was established by lipopolysaccharide(LPS)infusion.Rat alveolar typeⅡepithelial cells RLE-6TN cells were divided into the NC group,the LPS group,the miR-NC group,the miR-141-3p mimics group,the miR-141-3p mimics+pcDNA group and the miR-141-3p mimics+NRF2 and Kelch-like ring associated protein 1(Keap1)group.LPS cell model was established in all groups except the NC group.The mRNA expression levels of miR-141-3p and Keap1 in lung tissue and cells were detected by qPCR.Western blot assay was used to analyze lung tissue and cell epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),microtubule associated protein light chain 3B(LC3B),autophagy associated gene Beclin-1,α-smooth muscle actin(α-SMA),type I collagen(Col-Ⅰ),Keap1 and nuclear factors E2 related factor 2(NRF2)and heme oxygenase 1(HO-1).HE staining and Masson staining were used to observe pathological changes of lung tissue and to estimate the area of lung tissue injury and pulmonary fibrosis.Hydroxyproline(Hyp)in lung tissue was detected by the kit.Levels of inflammatory factor interleukin-1β,tumor necrosis factor(TNF-α)and oxidative stress index malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.Dual luciferase reporting experiment was used to verify the targeting relationship between miR-141-3p and Keap1.Results The expression of miR-141-3p was down-regulated and the expression of Keap1 was up-regulated in lung tissue and cells(P＜0.05).Overexpression of miR-141-3p can reduce the degree of pathological damage and fibrosis of lung tissue in rats,Hyp content,and up-regulate expression levels of SOD,E-cadherin,LC3B,Beclin-1,NRF2 and HO-1 in lung tissue and cells,and down-regulate the expression levels of IL-1β,TNF-α,MDA,N-cadherin,α-SMA,Col-I and Keap1(P＜0.05).Overexpression of Keap1 was able to reverse the improvement effect of overexpression of miR-141-3p on alveolar epithelial cell damage in ARDS rats(P＜0.05).Double Luciferase reporter gene experiment confirmed that miR-141-3p and Keap1 may have a targeted regulatory relationship.Conclusion Overexpression of miR-141-3p may activate the Keap1-NRF2/ARE signaling pathway,activate autophagy,inhibit inflammatory response,oxidative stress,and EMT progression,and improve pulmonary fibrosis in ARDS rats.
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Objective:To establish a modified controlled abciximab and device investigation to lower late angioplasty complication (CADILLAC) score, and to compare the predictive value of modified CADILLAC score, the global registry of acute coronary event (GRACE) score and the thrombolysis in myocardial infarction (TIMI) score in predicting the risk of short-term death after percutaneous coronary intervention (PCI) in patients with acute ST segment elevation myocardial infarction (STEMI).Methods:A retrospective study was conducted. The clinical data of 169 STEMI patients under going PCI admitted to the department of cardiology of Guizhou Provincial People's Hospital from September 2019 to December 2020 through emergency chest pain fast track were enrolled. A multivariate Logistic regression analysis was used to screen the factors closely related to the mortality risk within 30 days of STEMI, and a modified CADILLAC scoring system was established by referring to CADILLAC scoring settings. The score of modified CADILLAC, GRACE and TIMI scores of patients were calculated after admission, and the number of deaths due to cardiovascular disease (CVD) within 30 days after onset was recorded. The receiver operating characteristic curve (ROC curve) was used to evaluate the predictive value of three scoring systems on the risk of death within 30 days after PCI in patients with STEMI.Results:In 169 STEMI patients, 16 patients died of CVD within 30 days after PCI, and the actual case mortality was 9.47%. Multivariate Logistic regression analysis showed that age > 75 years old, cardiac function Killip ≥ Grade Ⅲ, ventricular arrhythmia, ST segment elevation ≥ 0.2 mV, cardiac troponin I (cTnI) increase, systolic blood pressure (SBP) < 90 mmHg (1 mmHg ≈ 0.133 kPa) were all independent predictors of death after PCI in STEMI patients. The improved CADILLAC scoring system was constructed based on the above predictive factors combined with left ventricular ejection fraction (LVEF) less than 0.40. The GRACE, TIMI and modified CADILLAC scores of dead patients were significantly higher than those of survival patients (GRACE score: 197.60±31.83 vs. 149.81±36.72, TIMI score: 11.21±2.13 vs. 7.27±1.97, modified CADILLAC score: 12.60±2.52 vs. 6.96±2.17, all P < 0.05). The higher the risk stratification of the three scores, the higher the mortality of patients with CVD within 30 days after PCI [the mortality of patients with low, medium and high risk in GRACE score were 2.41% (2/83), 9.61% (5/52) and 26.47% (9/34); the mortality of patients with low, medium and high risk in TIMI score were 3.12% (3/96), 12.82% (5/39) and 23.53% (8/34); and the mortality of patients with low, medium and high risk in modified CADILLAC score were 3.19% (3/94), 7.69% (4/52) and 39.13% (9/23), respectively, all P < 0.01]. The area under the ROC curve (AUC) of the GRACE, TIMI and the modified CADILLAC scores predicting the risk of death 30 days after PCI in STEMI patients were 0.855 [95% confidence interval (95% CI) was 0.702-0.923], 0.725 (95% CI was 0.666-0.812) and 0.882 (95% CI was 0.732-0.936), respectively, all P = 0.000; the sensitivity of its prediction accuracy were 81.59%, 78.65% and 89.26%, and the specificity were 78.62%, 57.12% and 75.54%, respectively. Conclusions:The GRACE and the modified CADILLAC scores have predictive value for the short-term mortality risk of STEMI patients after PCI, and the modified CADILLAC score is more accurate. But the TIMI score has a poor predictive effect on the short-term mortality risk of STEMI patients after PCI.
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Objective To investigate the effects of α-lipoic acid(ALA)on 6-hydroxydopamine(6-OHDA)-induced autophagy in human neuroblastoma(SH-SYSY)cells and its possible mechanisms.Methods SH-SYSY cells were divided into 5 groups:blank control group (group A),ALA group (group B),6-OHDA group(group C),ALA+6-OHDA group(group D),and rapamycin(RAPA)group (group E).The cell viability,cell apoptosis,and oxidative stress were assayed and analyzed in A-D group.The expression of autophagy-related proteins LC3-Ⅱ,AMP-activated protein kinase(AMP-K),phosphorylated AMPK (p-AMPK),the mammalian target of rapamycin (mTOR) and p-mTOR were detected by Western blot in A-E group.Results Compared with the blank control group,the 6-OHDA group significantly reduced the cell viability(P ＜ 0.01) and p-mTOR protein expression (P ＜0.05),and increased the cellular apoptosis rate(P＜0.01),oxidative stress level(P ＜0.01),LC3-Ⅱ protein expression(P＜0.05,with the highest level at 6 h after treatment),and p-AMPK protein expression(P＜＜0.05).There was no significant difference in these indices between ALA group and the blank control group.Compared with 6-OHDA group,ALA+ 6-OHDA group showed that the cell viability(P ＜ 0.01) and p-mTOR protein expression (P ＜ 0.05) were increased,while the cellular apoptosis rate(P＜0.01),oxidative stress level(P＜0.01),LC3-Ⅱ protein expression(P ＜0.05),and p-AMPK protein expression (P ＜ 0.05)were decreased.Conclusions The 6-OHDA can induce oxidative stress and autophagy in SH-SY5Y cells and decrease the cell viability.ALA can alleviate the 6-OHDA-induced cell injury possibly by inhibiting autophagy via AMPK/mTOR pathway.
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Objective To study the protective effect of erythropoietin (EPO) on brain tissue with cardiac arrest-cardiopulmonary resuscitation (CA-CPR) and its mechanism.Methods 120 male Sprague-Dawley (SD) rats were randomly divided into three groups (each n =40),namely:sham group,routine chest compression group,and conventional chest compression + EPO group (EPO group).The rats in each group were subdivided into CA and 6,12,24,48 hours after restoration of spontaneous circulation (ROSC) five subgroups (each n =8).The model of CA was reproduced according to the Hendrickx classical asphyxia method followed by routine chest compression,and the rats in sham group only underwent anesthesia,tracheostomy intubation and venous-puncture without asphyxia and CPR.The rats in EPO group were given the routine chest compression + EPO 5 kU/kg (2 mL/kg) after CA.Blood sample was collected at different time points of intervention for the determination the content of serum S100 β protein by enzyme linked immunosorbent assay (ELISA).All the rats were sacrificed at the corresponding time points,and the hippocampus was harvested for the calculation of the number of S100 β protein positive cells,and to examine the pathological changes and their scores at 24 hours after ROSC by light microscopy.Results With prolongation of ROSC time,the serum levels of S100 β protein (μg/L) in the routine chose compression group and the EPO group were significantly elevated,peaking at 24 hours (compared with CA:305.7 ± 29.2 vs.44.4 ± 6.2 in routine chest compression group,and 276.7±28.9 vs.44.7±5.6 in the EPO group,both P ＜ 0.05),followed by a fall.The levels of S100β protein at each time point after ROSC in EPO group were significanthy lower than those of the routine chest compression group (83.2 ± 7.5 vs.114.3 ± 15.3 at 6 hours,123.9 ± 20.2 vs.184.9 ± 22.2 at 12 hours,276.7 ± 28.9 vs.305.7 ± 29.2 at 24 hours,256.3 ± 26.6 vs.283.2 ± 23.6 at 48 hours,all P ＜ 0.05).With the prolongation of ROSC time,the S100 β protein positive cell number in brain (cells/HP) in the routine chest compression group and the EPO group was significantly increased,peaking at 24 hours (compared with CA:14.3±2.2 vs.6.7±0.7 in the routine chest compression group,11.3± 1.3 vs.6.8±0.9 in the EPO group,both P ＜ 0.05),then it began to fall.The S100 β protein positive cell number in brain at each time point after ROSC in the EPO group was significantly lower than that of the routine chest compression group (7.0±0.9 vs.7.9± 1.9 at6 hours,8.4± 1.1 vs.10.2±2.2 at 12 hours,11.3± 1.3 vs.14.3±2.2 at24 hours,8.3±0.8 vs.10.8±2.0 at48 hours,all P ＜ 0.05).Under the light microscope,a serious brain cortex injury was found after reproduction of the model,and the degree of injury was reduced after EPO intervention.The pathological score at 24 hours after ROSC in EPO group was lower than that of routine chest compression group (3.83±0.73 vs.4.17±0.75,P ＜ 0.05).Conclusions The S100β protein level in serum and brain tissue was increased early in asphyxia CA-CPR rats.EPO intervention can reduce the expression of S100 protein and reduce the degree of brain injury.
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Objective To study the regulation of aquaporin-1(AQP-1)changes in the heart of septic rats, compare the correlations of the AQP-1 with myocardial cytokines tumor necrosis factor-α(TNF-α),interleukin-6 (IL-6),and myocardial tissue water content,and to investigate the dexmedetomidine protective effect on myocardia in septic rats and its possible mechanism. Methods According to the random number table methods,90 male Sprague-Dawley(SD)rats were divided into sham operation group,sepsis model group and dexmedetomidine group, 30 rats in each group. The rat sepsis model was established by cecal ligation and puncture(CLP). In the sham operation group,the animal abdomen was only opened and closed without CLP. Half hour before operation in dexmedetomidine group,dexmedetomidine 1μg/kg(2μg/mL)was injected into the vein,while in the model and sham groups,saline 5 mL/kg was subcutaneously injected into the rat after the operation. At 2,12,24,48,72 hours after operation,6 rats were sacrificed and their hearts removed at one time point in a group. Enzyme linked immunosorbent assay(ELISA)was used to detect the content of AQP-1 and the levels of the TNF-α,IL-6 in the myocardial tissue homogenate at all time points,the myocardial tissue water content was detected by dry wet weight,and the correlations between AQP-1 and TNF-α,IL-6 and between AQP-1 and myocardial tissue water content were compared. Results From 2 hours after operation,the levels of the AQP-1,TNF-αand IL-6 in model group were significantly higher than those in the sham operation group;with prolongation of time,the level of AQP-1 and myocardial tissue water content were decreased, but the levels of TNF-α and IL-6 were persistently increased. From 2 hours after operation in dexmedetomidine group,all the above indexes except myocardial tissue water content at 72 hours after operation were significantly lower than those in the model group〔AQP-1(ng/g):9.29±0.15 vs. 9.73±0.26,TNF-α(pg/g):109.47±8.41 vs. 128.13±7.36,IL-6(pg/g):232.95±20.56 vs. 279.71±22.24,myocardial tissue water content:(74.82±6.37)%vs.(75.62±6.39)%,all P<0.05〕,but still higher than those of the sham operation group. The correlation analyses for the septic group showed that the change of AQP-1 was positively correlated to the myocardial water content in early stage(r=0.418,P=0.001)and later stage(r=0.235,P=0.022),and the changes of the AQP-1 in early stage (at post-operative 2 hours)were positively correlated to the concentration changes of the cytokines TNF-α(r=0.235,P=0.021)and IL-6(r=0.345,P=0.003),but in the later stage(at post-operative 72 hours)were negatively correlated with the changes of TNF-α(r=-0.408,P=0.037)and IL-6(r=-0.276,P=0.002). Conclusions In the early stage of septic rats,there is obvious myocardial injury,resulting in the over expression of AQP-1 and the occurrence of myocardial edema,dexmedetomidine can play a role in myocardial protection in such rats and its mechanism is possibly related to the reduction of the expression of AQP-1 and the levels of inflammatory cytokines, and in turn the alleviation of myocardial cell edema.