ABSTRACT
Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.
ABSTRACT
Objective:To investigate the diagnostic value of independent and combined subtests of the psychometric hepatic encephalopathy score (PHES) in mild hepatic encephalopathy(MHE) of patients with liver cirrhosis, so as to optimize the PHES.Methods:This was a prospective, multicenter and real-world study which was sponsored by the National Clinical Research Center of Infectious Diseases and the Portal Hypertension Consortium. Twenty-six hospitals from 13 provinces, autonomous regions and municipalities countrywide participated in this study, induding Tianjin Third Central Hospital, the Fourth People′s Hospital of Qinghai Province, the Second Affiliated Hospital of Baotou Medical College, the Third People′s Hospital of Taiyuan, the Fifth Medical Center of PLA General Hospital and so on. From October 2021 to February 2022, outpatients and hospitalized patients with liver cirrhosis and no obvious hepatic encephalopathy were consecutively enrolled. All patients received 5 PHES subjects in the same order: number connection test(NCT)-A, NCT-B, digit symbol test(DST), line tracing test(LTT) and serial dotting test(SDT), and the scores were calculated. The total score of PHES <-4 was taken as the cut-off value for diagnosing MHE. Compare the differences in each subtest between MHE group and non-MHE group. Receiver operating characteristic curve(ROC) and area under the curve(AUC) was performed to assess the diagnostic value of independent and combined subtests in MHE. Mann-Whitney U test and DeLong test were used for statistical analysis. Results:A total of 581 patients with liver cirrhosis were enrolled, 457 were diagnosed as MHE, and the incidence of MHE was 78.7%. The results of NCT-A, NCT-B, SDT, LTT, DST of MHE group were 60.00 s(47.01 s, 88.00 s), 90.45 s(69.32 s, 125.35 s), 74.00 s(57.65 s, 96.60 s), 74.72(60.00, 98.61) and 27.00(20.00, 36.00), respectively. Compared those of non-MHE group(34.00 s(29.15 s, 44.48 s), 50.00 s(40.98 s, 60.77 s), 50.00 s(41.07 s, 63.03 s), 46.23(38.55, 59.42) and 42.00(34.00, 50.75)), the differences were statistically significant( Z=12.37, 12.98, 9.83, 11.56, 10.66; all P<0.001). The AUC(95% confidence interval(95% CI)) of subtests of PHES NCT-B, NCT-A, LTT, DST and SDT alone in MHE diagnosis were 0.880(0.849 to 0.910), 0.862(0.828 to 0.896), 0.838(0.799 to 0.877), 0.812(0.772 to 0.851) and 0.788(0.743 to 0.832), respectively. The combination of 2 PHES subtests significantly increased the diagnostic efficacy. Among them the diagnostic efficacy of the combination of NCT-B and LTT was the best, the AUC(95% CI) was 0.924(0.902 to 0.947), the specificity was 91.9% and the sensitivity was 79.2%, which was better than a single PHES subtest (NCT-A, NCT-B, SDT, LTT and DST) and the combination of NCT-A and DST(AUC was 0.879, 95% CI0.847 to 0.910) which was recommended by guidelines on the management of hepatic encephalopathy in cirrhosis, the differences were statistically significant ( Z=3.78, 3.83, 5.57, 5.51, 5.38, 2.93; all P<0.01). Furthermore, compared between the combination of NCT-B and LTT and the combination of 3 subests of PHES, only the diagnostic efficacy of combination of NCT-B, LTT and SDT (AUC was 0.936, 95% CI 0.916 to 0.956) was better than that of the combination of NCT-B and LTT, the difference was statistically significant( Z=2.32, P=0.020). Conclusion:Based on the diagnostic efficacy and clinical feasibility of PHES subtests and their combinations, the combination of NCT-B and LTT is recommended for the diagnosis of MHE.
ABSTRACT
Objective:To evaluate the differences in organ dysfunction between gram-positive and gram-negative bacteria-induced sepsis in rats.Methods:Fifty-two adult male Sprague-Dawley rats, weighing 340-380 g, aged 15-18 weeks, were divided into 3 groups using a random number table method: control group (C group, n=12), G - bacteria group ( n=20), and G + bacteria group ( n=20). The G + and G - septic models were developed by intraperitoneal injection of inactivated Staphylococcus aureus and Escherichia coli suspension in G + and G - bacteria groups, respectively.The survival status within 36 h after injection of inactivated bacteria was recorded.Mean arterial pressure (MAP), platelet count (Plt), oxygenation index (OI) and serum concentrations of tumor necrosis factor α (TNF-α), cardiac troponin T (cTnT), total bilirubin (TBIL), creatinine (Cr), neuron-specific enolase (NSE) and lactic acid (Lac) were measured at 6, 12, 24 and 36 h after injection of inactivated bacteria.Fear conditioning test and open field test were performed at 12 and 36 h after injection of inactivated bacteria.The apoptosis rate of neurons in hippocampal CA1 area was measured by TUNEL, and the permeability of blood-brain barrier was measured by Evans blue method.The histopathological changes of lung, heart, kidney and brain tissues were examined at 36 h after injection of inactivated bacteria. Results:Compared with C group, the number of crossing grids and percentage of time spent freezing were significantly decreased, the apoptosis rate of neurons and permeability of blood-brain barrier were increased, and the survival rate was decreased in G - bacteria group and G + bacteria group ( P<0.05); the serum TNF-α concentration was significantly increased in G + bacteria group, and the serum cocentration of OI was significantly decreased, and the serum concentrations of cTnT, Cr, TNF-α and Lac were increased in G - bacteria group at 6 h after injection of inactivated bacteria ( P<0.05); the serum concentrations of cTnT, Cr, NSE, TNF-α and Lac in G + bacteria group and serum concentrations of cTnT, Cr, TNF-α and Lac in G - bacteria group were increased, and the OI, MAP and Plt were decreased at 12 h after injection of inactivated bacteria in both groups ( P<0.05); the serum concentrations of cTnT, TBIL, Cr, NSE, TNF-α and Lac were increased, and OI, MAP and Plt were decreased at 24 and 36 h after injection of inactivated bacteria in G + bacteria group and G - bacteria group ( P<0.05). Compared with G + bacteria group, the serum concentrations of TNF-α and cTnT and apoptosis rate of neurons were significantly decreased at 12 h after injection ( P<0.05); the serum concentrations of TNF-α and Lac were significantly increased, the serum concentrations of cTnT, OI, MAP and Plt were decreased at 24 h after injection of inactivated bacteria ( P<0.05); the serum concentrations of Cr, NSE, TNF-α and Lac were significantly increased, OI, MAP and Plt were decreased, and the percentage of time spent freezing, apoptosis rate of neurons and permeability of blood-brain barrier were increased at 36 h after injection of inactivated bacteria in G - bacteria group ( P<0.05). Conclusions:Early respiratory dysfunction and multiple organ failure often occur in G - bacteria sepsis, while G + bacteria sepsis is more likely to cause early circulatory and neurological dysfunction, and G - sepsis presents with more serious organ damage and high fatality rate as the disease progresses.
ABSTRACT
Objective:To evaluate the effect of melatonin preconditioning on hepatic ischemia-reperfusion (I/R) injury in rats with non-alcoholic fatty liver disease (NAFLD).Methods:Forty-eight SPF male Sprague-Dawley rats, aged 10-12 weeks, weighing 200-230 g, were divided into 4 groups ( n=12 each) using a random number table method: control group (Con group), NAFLD group, NAFLD + hepatic I/R group (NAFLD+ HIR group), and NAFLD + hepatic I/R + melatonin treatment group (NAFLD+ HIR+ MT group). The NAFLD model was developed by a high-fat and high-glucose diet (10% glucose, 10% fat) for 8 consecutive weeks in NAFLD, NAFLD+ HIR and NAFLD+ HIR+ M groups, and rats were fed with common chow and freely drank water in the other groups.Melatonin 10 mg/kg was given intragastrically daily for 2 consecutive weeks before developing the model in group NAFLD+ HIR+ MT.The model of liver I/R injury was developed by clipping the hepatic artery and portal vein for 20 min, opening for 5 min, and re-clamping for 20 min followed by restoration of perfusion.Blood samples from inferior vena cava were collected and liver tissues were obtained at 6 h of reperfusion to detect serum levels of insulin, blood glucose, free fatty acid (FFA), triglyceride (TG), alanine aminotransferase (ALT), aspartate amino transferase (AST) and ferritin, and insulin resistance index was calculated.The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reactive oxygen species (ROS) and Fe 2+ in liver tissues were detected by enzyme-linked immunosorbent assay, the pathological changes of liver tissues were examined with a light microscope after hematoxylin-eosin staining.The expression of nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), long-chain fatty acyl-CoA synthase 4 (ACSL4) and glutathione peptide peroxidase 4 (GPX4) was detected by Western blot. Results:Compared with Con group, the levels of serum FFA, TG, ALT, AST and ferritin and insulin resistance index were significantly increased, the levels of ROS and Fe 2+ in liver tissues were increased, the levels of GSH-Px and SOD were decreased, the expression of ACSL4 and LPCAT3 was up-regulated, and the expression of Nrf2 and GPX4 was down-regulated in NAFLD group ( P<0.05). Compared with NAFLD group, the serum levels of FFA, TG, ALT, AST and ferritin and insulin resistance index were significantly increased, the levels of ROS and Fe 2+ were decreased, the levels of GSH-Px and SOD were increased, the expression of ACSL4 and LPCAT3 was up-regulated, and the expression of Nrf2 and GPX4 was down-regulated in NAFLD+ HIR group ( P<0.05). Compared with NAFLD+ HIR group, the serum levels of FFA, TG, ALT, AST and ferritin and insulin resistance index were significantly increased, the levels of ROS and Fe 2+ were decreased, the levels of GSH-Px and SOD were increased, the expression of ACSL4 and LPCAT3 was down-regulated, and the expression of Nrf2 and GPX4 was up-regulated in NAFLD+ HIR+ MT group ( P<0.05). Conclusions:Melatonin preconditioning can alleviate hepatic I/R injury in rats with NAFLD, and the mechanism may be related to activation of Nrf2 signaling pathway, reduction of lipid peroxidation and inhibition of ferroptosis.
ABSTRACT
Objective:To evaluate the effect of chicoric acid on oxidative stress during myocardial injury in sepsis rats and the relationship with nuclear factor E2-related factor 2 (Nrf2) signaling pathway.Methods:Forty healthy male Sprague-Dawley rats, aged 8-12 weeks, weighing 220-250 g, were divided into 5 groups ( n=8 each) using a random number table method: control group (group C), lipopolysaccharide (LPS) group (group LPS), LPS+ chicoric acid group (group LPS+ CA), LPS+ Nrf2 inhibitor ML385 group (group LPS+ ML) and LPS+ chicoric acid+ ML385 group (group LPS+ CA+ ML). LPS 15 mg/kg was intraperitoneally injected to induce sepsis.Immediately after intraperitoneal injection of LPS, chicoric acid 10 mg/kg or ML385 15 mg/kg (in dimethyl sulfoxide) was intraperitoneally injected in group LPS+ CA and group LPS+ ML, respectively, and ML385 15 mg/kg and chicoric acid 10 mg/kg were intraperitoneally injected in LPS+ CA+ ML group.The equal volume of dimethyl sulfoxide was given instead in group C. At 48 h after establishment of the model, blood samples were collected from the aorta for measurement of concentration of serum interleukin-6 (IL-6) and the activities of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) (by enzyme-linked immunosorbent assay). The animals were then sacrificed, and myocardial tissues were obtained for microscopic examination of pathological changes (by HE staining), for determination of activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and contents of reactive oxygen species(ROS) and iron (by colorimetry), for calculation of the ratio of oxidized nicotinamide adenine 2 nucleotides to reduced nicotinamide adenine 2 nucleotides (NAD + /NADH), and for detection of the expression of Nrf2, NADPH quinone oxidoreductase 1 (NQO1), glutathione peroxidase 4 (GPX4) and nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) (by Western blot). Results:Compared with C group, the activities of serum LDH and CK-MB and concentration of IL-6 were significantly increased, the contents of ROS and iron and the ratio of NAD + /NADH were increased, activities of GSH-Px and SOD were decreased, expression of Nrf2, NQO1 and GPX4 was down-regulated, and NOX1 expression was up-regulated in the other four groups ( P<0.05). Compared with group LPS, the activities of serum LDH and CK-MB and concentration of IL-6 were significantly decreased, the contents of ROS and iron and the ratio of NAD + /NADH were decreased, activities of GSH-Px and SOD were increased, expression of Nrf2, NQO1 and GPX4 was up-regulated, NOX1 expression was down-regulated ( P<0.05), and the pathological changes of cardiomyocytes were significantly reduced in group LPS+ CA, and the activities of serum LDH and CK-MB and concentration of IL-6 were significantly increased, the ratio of NAD + /NADH were increased, activities of GSH-Px and SOD were decreased, expression of Nrf2, NQO1 and GPX4 was down-regulated, NOX1 expression was up-regulated ( P<0.05), and the pathological changes of cardiomyocytes were accentuated in group LPS+ ML.Compared with group LPS+ CA, the activities of serum LDH and CK-MB and concentration of IL-6 were significantly increased, the contents of ROS and iron and the ratio of NAD + /NADH were increased, activities of GSH-Px and SOD were decreased, expression of Nrf2, NQO1 and GPX4 was down-regulated, NOX1 expression was up-regulated ( P<0.05), and the pathological changes of cardiomyocytes were accentuated in group LPS+ CA+ ML. Conclusion:The mechanism by which chicoric acid reduces myocardial injury in sepsis rats may be related to activating Nrf2 signaling pathway and inhibiting oxidative stress.
ABSTRACT
Objective:To investigate the effect of ferroptosis on hepatic ischemia reperfusion injury in diabetic rats model.Methods:Forty Sprague Dawley rats were randomly divided into four groups: sham operation group (Sham), hepatic ischemia reperfusion group (HIRI), diabetes mellitus group (DM), diabetes mellitus + hepatic ischemia reperfusion group (DM+ HIRI). The diabetic rat model was established by feeding the rats with high-fat and high-sugar feed for four consecutive weeks combined with intraperitoneal injection of 50 mg/kg 1% streptozotocin, and on this basis, the hepatic ischemia reperfusion injury model was established by local hepatic blood flow occlusion. ELISA assay was used to detect insulin content, liver function and serum lipid metabolism biomarkers. Chemiluminescence method was used to detect liver superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), polyunsaturated fatty acids (PUFA) and lipoxygenase (LOX-1) contents. HE staining was used to evaluate the pathological changes of liver tissue structure, and Western blotting was used to detect the ferroptosis-related protein expression of ACSL4 and GPX4.Results:Compared with Sham group, the level of fasting blood glucose, insulin, insulin resistance index, serum TC, TG and liver PUFA content of DM group were increased significantly ( P<0.05), HE staining showed there were a large number of fatty degeneration of hepatocytes in DM group, and extensive ballooning and necrosis of hepatocytes in DM+ HIRI group. Compared with HIRI group, level of serum TC, TG, ALT, AST and the liver PUFA and LOX-1 in DM+ HIRI group were significantly increased [TC (5.87±0.76) vs (1.34±0.2) mmol/L, TG (2.93±0.47) vs (0.71±0.34) mmol/L, ALT (339.5±40.09) vs (155.17±18.53) U/L, AST (325.50±37.52) vs (102.39±22.68) U/L, PUFA (21.58±3.01) vs (8.12±0.94) mg/g, LOX-1 (200.81±26.03) vs (73.34±10.66) U/m ] ( P<0.05). Compared with HIRI group, the protein expression of ACSL4 in DM+ HIRI group was up-regulated [(0.46±0.06) vs (1.02±0.11)], while the expression of GPX4 was down-regulated [(0.43±0.07) vs (0.14±0.02)] significantly ( P<0.05). Conclusion:The tolerance of DM rats to hepatic ischemia reperfusion injury was significantly reduced, which may be related to hepatic abnormal lipid metabolism and ferroptosis.
ABSTRACT
Metabolic related fatty liver disease(MAFLD) is a kind of fatty liver disease caused by metabolic dysfunction closely related to insulin resistance and genetic susceptibility, also known as nonalcoholic fatty liver disease (NAFLD). Chronic hepatitis C (CHC) is a worldwide curable viral hepatitis caused by hepatitis C virus (HCV) infection.If MAFLD is combined with CHC, CHC can lead to insulin resistance, increase the oxidative stress of hepatocytes, accelerate the steatosis of hepatocytes, and affect the therapeutic effect of MAFLD.
ABSTRACT
Objective@#To evaluate the effects of ferroptosis in hippocampal neurons on cognitive dysfunction in rats with sepsis-associated encephalopathy (SAE) and its potential molecular mechanisms.@*Methods@#① Screening experiment of SAE modeling conditions: 42 healthy male Sprague-Dawley (SD) rats were divided into normal saline (NS) control group (n = 6) and lipopolysaccharide (LPS) 5, 15, 30 mg/kg groups (each n = 12) according to the random number table method. The SAE modeling conditions were determined by survival and the changes in mean arterial pressure (MAP) and heart rate (HR) within 72 hours, the percentage of stiffness status, the levels of serum inflammatory factors including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), neuron specific enolase (NSE, a marker of neuronal injury), serum iron and lactic acid (Lac) contents, and the morphological changes in CA1 of hippocampus after 72 hours. ② Deferoxamine (Def) intervention experiment: according to the results of screening experiments, 28 healthy male SD rats were divided into NS control group (n = 8), SAE group (n = 10) and Def+SAE group (n = 10) according to the random number table method. In the Def+SAE group, 100 mg/kg Def was injected intraperitoneally 12 hours before the modeling, once every 12 hours, with a total of 7 times; the rats in the NS control group and SAE group were injected with the same amount of NS. Then the cognitive function of rats was evaluated by fear conditioning test for the percentage of stiffness status; serum IL-6, TNF-α and NSE levels were determined by enzyme-linked immunosorbent assay (ELISA); the levels of serum Lac, serum iron and hippocampal malondialdehyde (MDA) and iron contents were determined by chemical colorimetric; the protein expressions of nuclear factor E2 related factor 2 (Nrf 2), glutathione peroxidase 4 (GPX4) and NAPDH oxidase 1 (NOX1) in hippocampus were determined by Western Blot; morphological changes in hippocampal CA1 were observed after hematoxylin and eosin (HE) staining.@*Results@#① Compared with the NS control group, intraperitoneal injection of 15 mg/kg LPS could significantly reduce the MAP and HR as time prolonged, and the reduction was most significant at 72 hours. The 72-hour survival rate was significantly reduced and cognitive function was impaired. The levels of serum IL-6, TNF-α, Lac and NSE were increased while the serum iron content was decreased significantly. The morphology of vertebral cells in hippocampal CA1 was irregular and some of the cells were obviously vacuolated. In the LPS 5 mg/kg group, there were no significant changes in vital signs, inflammation, organ function or cognitive dysfunction, while the symptoms of septic shock were apparent in the LPS 30 mg/kg group. Therefore, SAE model was reproduced by intraperitoneal injection of 15 mg/kg LPS for 72 hours. ② Compared with the NS control group, the percentage of stiffness in the SAE group was significantly reduced. The levels of serum IL-6, NSE and hippocampal MDA, iron were significantly increased. The serum iron contents and hippocampal Nrf 2 and GPX4 protein expressions were significantly reduced, while the hippocampal NOX1 protein expression was significantly increased. The morphology of vertebral cells in hippocampal CA1 was irregular and the cytoplasm was deeply stained. The results indicated that the level of oxidative stress in the hippocampus of SAE rats was increased, the neuron degenerations were obvious, and the cognitive function of rats were impaired. Compared with the SAE group, the percentage of stiffness in the Def+SAE group was significantly increased [(63.4±6.4)% vs. (47.6±6.0)%, P < 0.05]. The levels of serum IL-6, NSE, iron and hippocampal MDA, iron were significantly reduced [serum IL-6 (ng/L): 73.14±8.31 vs. 99.86±12.37, serum NSE (μg/L): 3.67±0.51 vs. 5.92±0.79, serum iron (mg/L): 68.43±8.12 vs. 134.60±15.63, hippocampal MDA (mol/g): 4.62±0.90 vs. 6.62±0.84, hippocampal iron (μg/g): 155.32±17.86 vs. 221.54±27.54, all P < 0.05]. The hippocampal protein expressions of Nrf 2 and GPX4 were significantly increased [Nrf 2/β-actin: 0.41±0.07 vs. 0.18±0.03, GPX4/β-actin: 0.74±0.09 vs. 0.40±0.06, all P < 0.05] while the hippocampal NOX1 protein expression was significantly reduced (NOX1/β-actin: 0.62±0.08 vs. 1.11±0.16, P < 0.05). The vertebral cells was significantly improved as compared with the SAE group. These findings showed that the oxidative stress level in hippocampus of the Def+SAE group was reduced, neuron degeneration was significantly alleviated, and the cognitive function of the rats was significantly improved.@*Conclusions@#The cognitive function of rats with SAE was significantly impaired, the hippocampal neurons were obviously damaged and ferroptosis was increased. Def pretreatment could significantly reduce iron deposition and ferroptosis in hippocampal neurons of SAE rats and improve cognitive dysfunction, which may be related to activation of Nrf 2/GPX4 signaling pathway.
ABSTRACT
To evaluate the effects of ferroptosis in hippocampal neurons on cognitive dysfunction in rats with sepsis-associated encephalopathy (SAE) and its potential molecular mechanisms. Methods ① Screening experiment of SAE modeling conditions: 42 healthy male Sprague-Dawley (SD) rats were divided into normal saline (NS) control group (n = 6) and lipopolysaccharide (LPS) 5, 15, 30 mg/kg groups (each n = 12) according to the random number table method. The SAE modeling conditions were determined by survival and the changes in mean arterial pressure (MAP) and heart rate (HR) within 72 hours, the percentage of stiffness status, the levels of serum inflammatory factors including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), neuron specific enolase (NSE, a marker of neuronal injury), serum iron and lactic acid (Lac) contents, and the morphological changes in CA1 of hippocampus after 72 hours. ② Deferoxamine (Def) intervention experiment: according to the results of screening experiments, 28 healthy male SD rats were divided into NS control group (n = 8), SAE group (n = 10) and Def+SAE group (n = 10) according to the random number table method. In the Def+SAE group, 100 mg/kg Def was injected intraperitoneally 12 hours before the modeling, once every 12 hours, with a total of 7 times; the rats in the NS control group and SAE group were injected with the same amount of NS. Then the cognitive function of rats was evaluated by fear conditioning test for the percentage of stiffness status; serum IL-6, TNF-α and NSE levels were determined by enzyme-linked immunosorbent assay (ELISA); the levels of serum Lac, serum iron and hippocampal malondialdehyde (MDA) and iron contents were determined by chemical colorimetric; the protein expressions of nuclear factor E2 related factor 2 (Nrf 2), glutathione peroxidase 4 (GPX4) and NAPDH oxidase 1 (NOX1) in hippocampus were determined by Western Blot; morphological changes in hippocampal CA1 were observed after hematoxylin and eosin (HE) staining. Results ① Compared with the NS control group, intraperitoneal injection of 15 mg/kg LPS could significantly reduce the MAP and HR as time prolonged, and the reduction was most significant at 72 hours. The 72-hour survival rate was significantly reduced and cognitive function was impaired. The levels of serum IL-6, TNF-α, Lac and NSE were increased while the serum iron content was decreased significantly. The morphology of vertebral cells in hippocampal CA1 was irregular and some of the cells were obviously vacuolated. In the LPS 5 mg/kg group, there were no significant changes in vital signs, inflammation, organ function or cognitive dysfunction, while the symptoms of septic shock were apparent in the LPS 30 mg/kg group. Therefore, SAE model was reproduced by intraperitoneal injection of 15 mg/kg LPS for 72 hours. ② Compared with the NS control group, the percentage of stiffness in the SAE group was significantly reduced. The levels of serum IL-6, NSE and hippocampal MDA, iron were significantly increased. The serum iron contents and hippocampal Nrf 2 and GPX4 protein expressions were significantly reduced, while the hippocampal NOX1 protein expression was significantly increased. The morphology of vertebral cells in hippocampal CA1 was irregular and the cytoplasm was deeply stained. The results indicated that the level of oxidative stress in the hippocampus of SAE rats was increased, the neuron degenerations were obvious, and the cognitive function of rats were impaired. Compared with the SAE group, the percentage of stiffness in the Def+SAE group was significantly increased [(63.4±6.4)% vs. (47.6±6.0)%, P < 0.05]. The levels of serum IL-6, NSE, iron and hippocampal MDA, iron were significantly reduced [serum IL-6 (ng/L): 73.14±8.31 vs. 99.86±12.37, serum NSE (μg/L): 3.67±0.51 vs. 5.92±0.79, serum iron (mg/L): 68.43±8.12 vs. 134.60±15.63, hippocampal MDA (mol/g): 4.62±0.90 vs. 6.62±0.84, hippocampal iron (μg/g): 155.32±17.86 vs. 221.54±27.54, all P < 0.05]. The hippocampal protein expressions of Nrf 2 and GPX4 were significantly increased [Nrf 2/β-actin: 0.41±0.07 vs. 0.18±0.03, GPX4/β-actin: 0.74±0.09 vs. 0.40±0.06, all P < 0.05] while the hippocampal NOX1 protein expression was significantly reduced (NOX1/β-actin: 0.62±0.08 vs. 1.11±0.16, P < 0.05). The vertebral cells was significantly improved as compared with the SAE group. These findings showed that the oxidative stress level in hippocampus of the Def+SAE group was reduced, neuron degeneration was significantly alleviated, and the cognitive function of the rats was significantly improved. Conclusions The cognitive function of rats with SAE was significantly impaired, the hippocampal neurons were obviously damaged and ferroptosis was increased. Def pretreatment could significantly reduce iron deposition and ferroptosis in hippocampal neurons of SAE rats and improve cognitive dysfunction, which may be related to activation of Nrf 2/GPX4 signaling pathway.
ABSTRACT
Objective To evaluate the role of sirtuin 1 ( SIRT1)∕nuclear factor kappa B ( NF-κB) signaling pathway in oxygen-glucose deprivation and restoration ( OGD∕R) injury to hippocampal neurons of mice. Methods The HT22 hippocampal neurons were seeded in a culture plate ( 96-well plate, 100 μl∕well; 6-well plate, 2 ml∕well) at the density of 5×104 cells∕ml or in a culture dish (6 cm in diameter), and then divided into 4 groups ( n=24 each) using a random number table method: control group ( group C) , OGD∕R group ( group OGD) , SIRT1 inhibitor EX-527 preconditioning group ( group EX) and SIRT1 agonist SRT1720 preconditioning group ( group SRT) . Neurons were cultured in normal culture atmosphere at 37 ℃ in group C. In OGD, EX and SRT groups, the culture medium was replaced with oxygen-poor flu-id, and neurons were exposed to 5％ CO2-95％ N2 for 12 h in an incubator at 37℃, oxygen-poor fluid was replaced with the culture medium, and neurons were cultured for 24 h in normal culture atmosphere at 37℃. SIRT1 inhibitor EX-5271 μmol∕L and SIRT1 agonist SRT172010 μmol∕L were added at 12 h beforeOGD in EX and SRT groups, respectively. The cell viability was measured by CCK8 assay, the activity of LDH was detected by chemical colorimetry, cell apoptosis rate was determined by flow cytometry, the ex-pression of SIRT1, NF-κB, IκBα, Bcl-2 and Bax was detected by Western blot, and the Bcl-2∕Bax ratio was calculated. Results Compared with group C, the cell viability was significantly decreased, LDH ac-tivity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regula-ted, the expression of NF-κB and Bax was up-regulated, and the Bcl-2∕Bax ratio was decreased in OGD, EX and SRT groups ( P<0. 05) . Compared with group OGD, the cell viability was significantly increased, the LDH activity and cell apoptosis rate were decreased, the expression of SIRT1, IκBαand Bcl-2 was up-regulated, the expression of NF-κB and Bax was down-regulated, and the Bcl-2∕Bax ratio was increased in group SRT, and the cell viability was significantly decreased, LDH activity and cell apoptosis rate were in-creased, the expression of SIRT1, IκBαand Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2∕Bax ratio was decreased in group EX (P<0. 05). Conclusion SIRT1∕NF-κB signaling pathway inhibition is involved in OGD∕R injury to hippocampal neurons of mice.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the role of silent information regulator 1 (SIRT1) signaling pathway in mediating the effect of dexmedetomidine (DEX) to alleviate postoperative cognitive dysfunction (POCD) in aged rats.</p><p><b>METHODS</b>Seventy-two healthy male Sprague-Dawley rats aged 18-20 months (weighing 500-700 g) were randomized equally into normal control group, POCD model group, DEX pretreatment group, and DEX and SIRT1 inhibitor (EX527) pretreatment group. In the latter 2 groups, DEX (25 μg/kg) was injected intraperitoneally in the rats 30 min before the operation, and normal saline was injected instead in the other 2 groups; in EX527 group, EX527 (1 μg/kg) was injected intravenously 5 min before the operation. In all but the control group, the rats were subjected to laparotomy lasting 30 min, and on days 1, 3, and 5 following the operation, 6 rats were randomly selected from each group for Morris water maze test to evaluate their cognitive functions. Immediately after the test, the rats were sacrificed and the hippocampus was collected for determination of the levels of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) using ELISA; Western blotting was used to detect the expression of SIRT1 and nuclear factor- κB (NF-κB) in the hippocampal neurons.</p><p><b>RESULTS</b>Compared with the control rats, the rats in POCD group and EX527 group showed significantly prolonged escape latency, decreased frequency of crossing the original platform, increased TNF- and IL-6 levels, lowered SIRT1 expression in the hippocampal neurons, and increased NF-κB expression ( < 0.05), and these parameters were comparable between POCD group and EX527 group ( > 0.05). DEX pretreatment significantly alleviated cognitive dysfunction and attenuated the changes in TNF-, IL-6, SIRT1, and NF-κB expressions induced by the operation ( < 0.05), and EX527 pretreatment of the rats obviously blocked the effects of DEX ( < 0.05).</p><p><b>CONCLUSIONS</b>DEX alleviates POCD in aged rats probably via SIRT1 signaling pathway.</p>