Objective:To investigate the effect and underlying mechanism of sonodynamic therapy (SDT) on inflammation-related atrial fibrillation (AF) susceptibility.Methods:Lipopolysaccharide (LPS)-stimulated mouse and HL-1 mouse atrial myocyte models were used. (1) In vivo study: experimental groups included control, LPS, LPS+SDT, and SDT groups, with 20 mice in each group. Atrial fibrillation inducibility and duration were assessed by electrical stimulation. Western blot was used to analyze atrial expression of NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-1β, and IL-18. Immunohistochemistry was used to detect calcium voltage-gated channel subunit alpha1 C (CACNA1C) expression. (2) In vitro study: cell counting kit-8 (CCK-8) and Western blot were used to determine the optimal and safe LPS concentration. The safe incubation condition for the sonosensitizer sinoporphyrin sodium was determined by CCK-8 and fluorometry. An LPS-induced inflammatory model in HL-1 atrial myocytes was used, with experimental groups including control, LPS, LPS+SDT, LPS+sinoporphyrin sodium, and LPS+ultrasound groups. NLRP3 was overexpressed using plasmid transfection, with experimental groups including control, NLRP3 plasmid, negative control plasmid, and NLRP3 plasmid+SDT groups. SDT was applied to LPS-stimulated or NLRP3-overexpressing HL-1 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to measure mRNA and protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Cleaved Caspase-1, IL-1β, IL-18, and CACNA1C. The NLRP3 inhibitor MCC950 was used to validate the relationship of NLRP3 and CACNA1C. The experimental groups included control, LPS, LPS+MCC950, and MCC950 groups. Intracellular reactive oxygen species (ROS) levels were detected using the probe DCFH-DA, and the ROS scavenger N-acetyl-L-cysteine (NAC) was used to test if the effects of SDT was ROS-dependent.Results:(1) In vivo: The LPS+SDT group exhibited a lower incidence of atrial fibrillation induction and a shorter duration of atrial fibrillation compared to the LPS group(both P<0.05). Protein expression levels of NLRP3 and IL-1β were lower than those in the LPS group (all P<0.05), while the expression of CACNA1C subunit tended to increase relative to the LPS group ( P>0.05). (2) In vitro: The safe concentration of LPS for administration was ≤20 μg/ml, with an optimal pro-inflammatory concentration of 4 μg/ml. The safe concentration of sinoporphyrin sodium for administration was 0.4 μmol/L, with an optimal incubation time of 4 hours. Compared to the LPS group or NLRP3 plasmid group, the LPS+SDT group or NLRP3 plasmid+SDT group exhibited lower expression levels of NLRP3, ASC, Cleaved Caspase-1, IL-1β, and IL-18, and higher mRNA and protein levels of CACNA1C (all P<0.05). The LPS+MCC950 group had higher CACNA1C protein expression than the LPS group ( P<0.05). SDT increased intracellular ROS levels, and NAC blocked the regulatory effects of SDT on NLRP3 and CACNA1C. Conclusion:SDT reduces atrial fibrillation susceptibility in mice by inhibiting NLRP3 inflammasome activation in atrial cardiomyocytes, thereby upregulating the L-type calcium channel subunit CACNA1C.

Objective:To investigate the effect and underlying mechanism of sonodynamic therapy (SDT) on inflammation-related atrial fibrillation (AF) susceptibility.Methods:Lipopolysaccharide (LPS)-stimulated mouse and HL-1 mouse atrial myocyte models were used. (1) In vivo study: experimental groups included control, LPS, LPS+SDT, and SDT groups, with 20 mice in each group. Atrial fibrillation inducibility and duration were assessed by electrical stimulation. Western blot was used to analyze atrial expression of NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-1β, and IL-18. Immunohistochemistry was used to detect calcium voltage-gated channel subunit alpha1 C (CACNA1C) expression. (2) In vitro study: cell counting kit-8 (CCK-8) and Western blot were used to determine the optimal and safe LPS concentration. The safe incubation condition for the sonosensitizer sinoporphyrin sodium was determined by CCK-8 and fluorometry. An LPS-induced inflammatory model in HL-1 atrial myocytes was used, with experimental groups including control, LPS, LPS+SDT, LPS+sinoporphyrin sodium, and LPS+ultrasound groups. NLRP3 was overexpressed using plasmid transfection, with experimental groups including control, NLRP3 plasmid, negative control plasmid, and NLRP3 plasmid+SDT groups. SDT was applied to LPS-stimulated or NLRP3-overexpressing HL-1 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to measure mRNA and protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Cleaved Caspase-1, IL-1β, IL-18, and CACNA1C. The NLRP3 inhibitor MCC950 was used to validate the relationship of NLRP3 and CACNA1C. The experimental groups included control, LPS, LPS+MCC950, and MCC950 groups. Intracellular reactive oxygen species (ROS) levels were detected using the probe DCFH-DA, and the ROS scavenger N-acetyl-L-cysteine (NAC) was used to test if the effects of SDT was ROS-dependent.Results:(1) In vivo: The LPS+SDT group exhibited a lower incidence of atrial fibrillation induction and a shorter duration of atrial fibrillation compared to the LPS group(both P<0.05). Protein expression levels of NLRP3 and IL-1β were lower than those in the LPS group (all P<0.05), while the expression of CACNA1C subunit tended to increase relative to the LPS group ( P>0.05). (2) In vitro: The safe concentration of LPS for administration was ≤20 μg/ml, with an optimal pro-inflammatory concentration of 4 μg/ml. The safe concentration of sinoporphyrin sodium for administration was 0.4 μmol/L, with an optimal incubation time of 4 hours. Compared to the LPS group or NLRP3 plasmid group, the LPS+SDT group or NLRP3 plasmid+SDT group exhibited lower expression levels of NLRP3, ASC, Cleaved Caspase-1, IL-1β, and IL-18, and higher mRNA and protein levels of CACNA1C (all P<0.05). The LPS+MCC950 group had higher CACNA1C protein expression than the LPS group ( P<0.05). SDT increased intracellular ROS levels, and NAC blocked the regulatory effects of SDT on NLRP3 and CACNA1C. Conclusion:SDT reduces atrial fibrillation susceptibility in mice by inhibiting NLRP3 inflammasome activation in atrial cardiomyocytes, thereby upregulating the L-type calcium channel subunit CACNA1C.

Gut microbiota dysbiosis is an avenue for the promotion of atherosclerosis (AS) and this effect is mediated partly via the circulating microbial metabolites. More microbial metabolites related to AS vascular inflammation, and the mechanisms involved need to be clarified urgently. Paeonol (Pae) is an active compound isolated from Paeonia suffruticoas Andr. with anti-AS inflammation effect. However, considering the low oral bioavailability of Pae, it is worth exploring the mechanism by which Pae reduces the harmful metabolites of the gut microbiota to alleviate AS. In this study, ApoE^-/- mice were fed a high-fat diet (HFD) to establish an AS model. AS mice were administrated with Pae (200 or 400 mg·kg^-1) by oral gavage and fecal microbiota transplantation (FMT) was conducted. 16S rDNA sequencing was performed to investigate the composition of the gut microbiota, while metabolomics analysis was used to identify the metabolites in serum and cecal contents. The results indicated that Pae significantly improved AS by regulating gut microbiota composition and microbiota metabolic profile in AS mice. We also identified α-hydroxyisobutyric acid (HIBA) as a harmful microbial metabolite reduced by Pae. HIBA supplementation in drinking water promoted AS inflammation in AS mice. Furthermore, vascular endothelial cells (VECs) were cultured and stimulated by HIBA. We verified that HIBA stimulation increased intracellular ROS levels, thereby inducing VEC inflammation via the TXNIP/NLRP3 pathway. In sum, Pae reduces the production of the microbial metabolite HIBA, thus alleviating the ROS/TXNIP/NLRP3 pathway-mediated endothelial inflammation in AS. Our study innovatively confirms the mechanism by which Pae reduces the harmful metabolites of gut microbiota to alleviate AS and proposes HIBA as a potential biomarker for AS clinical judgment.
Animals ; Mice ; Atherosclerosis/drug therapy* ; Diet, High-Fat ; Endothelial Cells ; Inflammation/drug therapy* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Reactive Oxygen Species

Objective To study the mechanism of paraquat (PQ)-induced renal injury in rats,the expression changes of ICAM-1 to assess the protective effect of Melatonin in PQ poisoning.Methods Ninety adult healthy Sprague-Dawley (SD) rats were divided into three groups at random.Control group:30 rats;Poisoned group:30 rats;Melatonin group:30 rats.Control group and Poisoned group were treated intragastrically with 1 ml of PQ (50 mg/kg) diluted with normal saline.Control groupwere treated with the same dose of normal saline as Poisoned group and Melatonin group.Melatonin group were given 1 ml of Melatonin at a dose of 10 mg/kg diluted with normal saline (once daily,intraperitoneally) Control and Poisoned group were treated with the same dose of normal saline (once daily,intraperitoneally) as Melatonin group.Pathology of renal tissue were oberserved by HE staining,and electron microscope.The histopathological changes and the expression of ICAM-1 were observed with mmunohistochemistry (IHC).Results (1) There were no obvious pathological changes in Control group.Poisoned group Renal glomerulus had hyperemia and distension.Renal tubule epithelial cell had edema and vacuolar degeneration and renal tubule lumina was narrowing on day 1,There were serious edema exudation and necrosis on day 5,which gradually lessened furthermore;Compared with Poisoned group,the aforementioned pathological lesion was more palliative in Melatonin group.(2) No obvious abnomal changes in ultrastructure of renal tissues in Control group.There were swelling of mitochondrion and rupture of renal tubule epithelial cell and endoplasmic reticulum had extension,lysosome was mult and had much phagocytosis in Poisoned group.(3)There was a very weak expression of ICAM-1 in Control group.while in Poisoned group,there was already a significant higher expression of ICAM-1 of renal tubule on day 1 after PQ poisoning,Immunohistochemistry score (IHS) of Poisoned group on day 1,3,5,7,14 were (0.1561 ±0.0295、0.2572±0.0259、0.3028±0.0153、0.2083 ±0.0227、0.9309 ±0.0059),compared with Control group (P＜0.01);Melatonin group were (0.1259±0.0061、0.2109±0.0280、0.2679±0.0233、0.1771 ±0.0186、0.0791 ±0.0135),compared with Control group (P＜0.01),compared with Poisoned group (P＜0.05);Conclusion ICAM-1 was involved in the procedures of renal injury;MT surely had a protective effect,which might be mediated by ICAM-1 in the paraquat-induced renal injury,but its regulation path still need a further exploration.

Objective To study the mechanism of paraquat (PQ)-induced renal injury in rats,the expression changes of ICAM-1 to assess the protective effect of Melatonin in PQ poisoning.Methods Ninety adult healthy Sprague-Dawley (SD) rats were divided into three groups at random.Control group:30 rats;Poisoned group:30 rats;Melatonin group:30 rats.Control group and Poisoned group were treated intragastrically with 1 ml of PQ (50 mg/kg) diluted with normal saline.Control groupwere treated with the same dose of normal saline as Poisoned group and Melatonin group.Melatonin group were given 1 ml of Melatonin at a dose of 10 mg/kg diluted with normal saline (once daily,intraperitoneally) Control and Poisoned group were treated with the same dose of normal saline (once daily,intraperitoneally) as Melatonin group.Pathology of renal tissue were oberserved by HE staining,and electron microscope.The histopathological changes and the expression of ICAM-1 were observed with mmunohistochemistry (IHC).Results (1) There were no obvious pathological changes in Control group.Poisoned group Renal glomerulus had hyperemia and distension.Renal tubule epithelial cell had edema and vacuolar degeneration and renal tubule lumina was narrowing on day 1,There were serious edema exudation and necrosis on day 5,which gradually lessened furthermore;Compared with Poisoned group,the aforementioned pathological lesion was more palliative in Melatonin group.(2) No obvious abnomal changes in ultrastructure of renal tissues in Control group.There were swelling of mitochondrion and rupture of renal tubule epithelial cell and endoplasmic reticulum had extension,lysosome was mult and had much phagocytosis in Poisoned group.(3)There was a very weak expression of ICAM-1 in Control group.while in Poisoned group,there was already a significant higher expression of ICAM-1 of renal tubule on day 1 after PQ poisoning,Immunohistochemistry score (IHS) of Poisoned group on day 1,3,5,7,14 were (0.1561 ±0.0295、0.2572±0.0259、0.3028±0.0153、0.2083 ±0.0227、0.9309 ±0.0059),compared with Control group (P＜0.01);Melatonin group were (0.1259±0.0061、0.2109±0.0280、0.2679±0.0233、0.1771 ±0.0186、0.0791 ±0.0135),compared with Control group (P＜0.01),compared with Poisoned group (P＜0.05);Conclusion ICAM-1 was involved in the procedures of renal injury;MT surely had a protective effect,which might be mediated by ICAM-1 in the paraquat-induced renal injury,but its regulation path still need a further exploration.

OBJECTIVETo investigate the effects of acetamide at different doses on the expression of inhibitory amino acids (gamma-aminobutyric acid, GABA) and excitatory amino acid (glutamate, Glu) in the cerebral cortex of rats with acute tetramine (TET) poisoning.

METHODSEighty Sprague-Dawley rats (SPF) were randomly divided into five groups, with 16 rats in each group: saline control group, dimethyl sulfoxide (DMSO) control group, TET exposure group, high-dose (2.8 g/kg/d) acetamide treatment group, and super-high-dose (5.6 g/kg/d) acetamide treatment group. Rats in the exposure group and treatment groups were exposed to TET by intragastric administration after fasting, and were then intramuscularly injected with saline or different doses of acetamide in the following 5 days. The cortex of the temporal lobe was collected at 3 h, 12 h, 48 h, or 7 d after treatment. The expression levels of GABA and Glu in the cortex of the temporal lobe were determined by average optical density (OD) values in immunohistochemistry.

RESULTS1) Expression of GABA: The OD value of GABA in TET exposure group started to increase at 12 h after treatment, reached the peak at 48 h, and decreased to the normal level at 7 d. In the high-dose acetamide treatment group, the increase in OD at 12 h was not so significant as that in the TET exposure group, OD value decreased to the normal level at 48 h and was lower than that in the exposure group, and the changes were more like those in the control groups. In the super-high-dose acetamide treatment group, OD value began to increase significantly at 3 h and was significantly higher than that in the TET exposure group (P < 0.01), it reached the peak at 12 h, and was restored to the normal value at 48 h. 2) Expression of Glu: The OD value of Glu in TET exposure group at 3 h after treatment was significantly lower than those in the two control groups, it increased gradually from 12 h to 48 h, and recovered to the normal level at the 7th d. The changes in the high-dose acetamide treatment group were similar to those in the TET exposure group, but became more like those in the control groups after 48 h; the OD value in super-high-dose acetamide treatment group was significantly higher than that in the TET exposure group at 3 h after treatment (P < 0.01), while no significant difference was found at 12 h; it was significantly lower than those of all other groups at 48 h and 7 d (P < 0.01).

CONCLUSIONSTreatment with high dose of acetamide has some curative effect on TET poisoning-induced central nervous lesion, while the effect of super-high-dose acetamide on expression of neurotransmitters is too complex to evaluate.

Acetamides ; pharmacology ; Animals ; Bridged-Ring Compounds ; poisoning ; Cerebral Cortex ; drug effects ; metabolism ; Female ; Glutamic Acid ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; metabolism

OBJECTIVETo observe the effect of different doses of acetamide on the histopathology in the cerebral cortex of rats with tetramine (TET) poisoning and to provide a basis for the treatment of fluoroacetamide poisoning with acetamide.

METHODSEighty clean Sprague-Dawley rats were randomly divided into five groups: saline control group,dimethylsulfoxide water solution control group,TET poisoning group, acetamide (2.88 g/kg/d) treatment group, and acetamide (5.68 g/kg/d) treatment group, with 16 rats in each group. Rats in the poisoning group and treatment groups were poisoned with TET by intragastric administration after fasting; then, saline was injected intramuscularly into rats of the poisoning group, and different doses of acetamide were injected intramuscularly into rats of treatment groups; the course of treatment was 5 d. At 3 h, 12 h, 48 h, and 7 d after treatment, the cerebral cortex was harvested from rats in each group, and the histopathological changes in the cerebral cortex were evaluated under light and electron microscopes.

RESULTSThe light microscopy showed that the TET poisoning group had hypoxia changes in the cerebral cortex, which worsened over time; the treatment groups had reduced hypoxia changes, and the acetamide (2.88 g/kg/d) treatment group had more reduction than the acetamide (5.68 g/kg/d) treatment group. The electron microscopy showed that the apoptosis of neuronal cells were the main pathological changes in the TET poisoning group; the treatment groups had reduced apoptotic changes, and the acetamide (2.88 g/kg/d) treatment group had more reduction than the acetamide (5.68 g/kg/d) treatment group.

CONCLUSIONNo pathological changes associated with the synergistic toxic effect of acetamide and TET are found in the cerebral cortex. Acetamide (2.88 g/kg/d) could reduce central nervous lesions, but the efficacy is not improved after increasing the dose. For patients who cannot be identified with TET or fluoroacetamide poisoning, acetamide could be considered for treatment.

Acetamides ; pharmacology ; Animals ; Bridged-Ring Compounds ; toxicity ; Cerebral Cortex ; drug effects ; pathology ; Disease Models, Animal ; Male ; Rats ; Rats, Sprague-Dawley

Objective To evaluate clinical significance of the grade of ischemia by QRS complex on the admission electrocardiogram(ECG)to predict severe arrithmia in patients with acute ST-segment elevation myocardial infarction(STEMI).Methods Patients with acute ST-segment elevation myocardial infarction(STEMI)admitted to emergency department from July 2003 to April 2008 were enrolled.A total of 223 patients met the criteria(ischemic chest pain ≥ 30 min,2 or more adjacent leads of ST segment elevation and onset time within 12 h).Exclusion criteria were bundle branch block and left ventricular hypertrophy.All enrolled patients were divided into two groups based on the enrollment electrocardiogram:grade 2 ischemia(ST elevation without terminal QRS distortion; n =134)and grade 3 ischemia(ST elevation with terminal QRS distortion; n =89).Patients of the two groups had comparable genderproportion,average age and coronary heart disease risk factors etc.All patients received thrombolytic therapy.The incidence rate of ST segment resolution(STR)and severe arrithmia in hospital stay were observed.Numerical variables were expressed mean ± standard deviation and compared by unpaired Student't test,Categorical variables were expressed percentage and compared by chi square test.Multiple logistic regression analysis was used to determine independent predictors of severe arrithmia.Results Patients with grade 3 ischemia had greater Σ ST on admission and 2 h after thrombolysis ECGs(P ＜ 0.01),the incidence rate of STR in patients with grade 3 ischemia was lower than that in patients with grade 2 ischemia(P ＜0.01).The peak creatine kinase MB fraction was higher in patients with grade 3 ischemia than that in patients with grade 2 ischemia(P ＜ 0.01).There was no significant difference of the incidence of severe arrithmia,such as ventricular premature beat,ventricular tachycardia or fibrillation,second-degree or third-degree atrioventricular block,and sinus arrest between the two groups(P ＞ 0.05),but there was a trend of higher incidence of severe arrithmia in patients with grade 3 ischemia compared with that in patients with grade 2 ischemia.Multiple logistic regression analysis demonstrated that the independent predictors of severe arrithmia were duration from symptom to thrombolysis and initial.Σ ST,whereas grade 3 ischemia remained a strong predictor of severe arrithmia.Conclusions Grade 3 ischemia on admission is associated with lower incidence of STR in patients with ST-segment elevation myocardial infarction(STEMI)after thrombolysis and a strong predictor of severe arrithmia.

Objective To investigate the effects of N - acetylcysteine (NAC) on apoptosis and the expressions of Fas/FasL mRNA in lung tissue of rats with paraquat - induced acute lung injury.Methods Forty five male SD rats were randomly (random number) divided into normal control group,paraquat (PQ) group,and NAC treatment group.The rat model of acute lung injury was made with 2％ PQ induction in dose of 25 mg/kg injected,and NAC was injected into the PQ poisoning rats (200 mg/kg) 30 minutes after PQ administration in NAC treatment group.In the control group,equal amount of saline instead was injected into the rats.Apoptosis was detected by using TUNEL method and the expressions of Fas/FasL mRNA were evaluated by using reverse transcription polymerase chain reaction (RT- PCR),and the levels of Fas/FasL protein were detected by using western blot analysis.Results Compared with control group,cell apoptosis and expressions Fas/FasL mRNA in PQ group were significantly different ( P ＜ 0.05 ).Compared with PQ group,cell apoptosis and expressions Fas/FasL mRNA in NAC group were significantly decreased,were significant lower (P ＜ 0.05).Conclusions NAC inhibited apoptosis in lung tissue of rats with paraquat induction by regulating the activation of Fas/FasL systems.

Objective To observe the expression of tumor necrosis factor-α(TNF-α)in acute lung injury caused by paraquat(PQ)in rats,and investigate the mechanism of the rhubarb in respect of pmteetive effects.Method PQ intragastrically poisoning at the dose of 50 mg/kg made a model of the acute lung injury in Sprague-Dawley(SD)rats.Totally 144 adult healthy SD rats(72 female,72male)were randomly divided into control group (group A,n=24),poisoned group(group B,n=48),rhubarb treated group(group C,n=48)and the shaln poisoning group(group D,n=24).Rats of group B and group C were poisoned intmgastrically with PQ(50 mg/kg).and rats of group C and group D were intervened intragastrieally with 300 mg/(kg·d)of rhubarb in 15 min-utes.The white blood cells and total cells in bronchoalveolar lavage fluid(BALF)were counted by using a blood cell counting plate and the protein content of BALF was measured by using the way of Lowry in order to calculate the neutmphiks pereentage and lung permeability index.A small portion of left lung was stained with HE to observe the pathological changes and the expression oftumor necrosis factor-α in the rest of the left lung was observed with immunohistochemistry.The data were handled by the analysis of variance and NK method using SPSS 14.0.Re-suits Compared with group A,the lungs of rats mainly showed congestion,edema and leukocytes infiltration in group B,and fibrosis was found onlyt in a few rats.And the rate neutrophils percentage,protein content and lung permeability index in BALF increased(P＜0.01).The expression of TNF-α were obviously inereased at 12 hours after PQ poisoning,and immtmohistochemistry score (IHS)was higher,and peaked at 24 hours later(P＜0.05),then remained on a high level for a while and sluggishly declined.Compared with group B,the changes of above mentioned were alleviated obviously,and the expression of TNF-α delayed with the less magnitude of increasing an an obvious tendency of less expression.Compared with group B,delayed,lower increasing extent,obviously re-ducing tendency in group C with statistical difference in IHS(P＜0.05).Conclusions Rhubarb ameliorates a-cute lung injury caused by PQ poisoning in rats by means of inhibiting the expression of TNF-α in turn to alleviate inflammatory reaction.