This paper explored the specific mechanism of ginsenoside Rg_1 in regulating mitochondrial fusion through the neurogenic gene Notch homologous protein 1(Notch1) pathway to alleviate hypoxia/reoxygenation(H/R) injury in HL-1 cells. The relative viability of HL-1 cells after six hours of hypoxia and two hours of reoxygenation was detected by cell counting kit-8(CCK-8). The lactate dehydrogenase(LDH) activity in the cell supernatant was detected by the lactate substrate method. The content of adenosine triphosphate(ATP) was detected by the luciferin method. Fluorescence probes were used to detect intracellular reactive oxygen species(Cyto-ROS) levels and mitochondrial membrane potential(ΔΨ_m). Mito-Tracker and Actin were co-imaged to detect the number of mitochondria in cells. Fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels of Notch1, mitochondrial fusion protein 2(Mfn2), and mitochondrial fusion protein 1(Mfn1). The results showed that compared with that of the control group, the cell activity of the model group decreased, and the LDH released into the cell culture supernatant increased. The level of Cyto-ROS increased, and the content of ATP decreased. Compared with that of the model group, the cell activity of the ginsenoside Rg_1 group increased, and the LDH released into the cell culture supernatant decreased. The level of Cyto-ROS decreased, and the ATP content increased. Ginsenoside Rg_1 elevated ΔΨ_m and increased mitochondrial quantity in HL-1 cells with H/R injury and had good protection for mitochondria. After H/R injury, the mRNA and protein expression levels of Notch1 and Mfn1 decreased, while the mRNA and protein expression levels of Mfn2 increased. Ginsenoside Rg_1 increased the mRNA and protein levels of Notch1 and Mfn1, and decreased the mRNA and protein levels of Mfn2. Silencing Notch1 inhibited the action of ginsenoside Rg_1, decreased the mRNA and protein levels of Notch1 and Mfn1, and increased the mRNA and protein levels of Mfn2. In summary, ginsenoside Rg_1 regulated mitochondrial fusion through the Notch1 pathway to alleviate H/R injury in HL-1 cells.
Ginsenosides/pharmacology* ; Receptor, Notch1/genetics* ; Signal Transduction/drug effects* ; Mice ; Animals ; Mitochondrial Dynamics/drug effects* ; Mitochondria/metabolism* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Oxygen/metabolism* ; Cell Hypoxia/drug effects* ; Cell Survival/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Humans

OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

Objective To explore the role and mechanism of mechanically sensitive ion channel Piezo2 in mechanical allodynia of rats with low back pain induced by simulating the low-frequency vibration of a helicopter.Methods Low-frequency vibration(LFV)model with 3-dimensional 6 degrees of freedom was used to induce low back pain in awake rats in a sitting position.Twenty-four male SD rats(8 weeks old)were randomly divided into control(Ctrl)group and LFV group.HE staining was used to evaluate the injury of the multifidus muscle.Von Frey test was carried out to detect pain sensitivity.Open field test was employed to assess the spontaneous activity and anxiety.ELISA,Western blotting and immunofluorescence staining were performed to detect the expression of NGF,TrkA and downstream molecule Piezo2.Dorsal root ganglia(DRG)neurons was isolated from SD rats and primarily cultured.After identified with immunofluorescence staining,the neurons were divided into the Ctrl group,the LFV group,and the LFV+D-GsMTx4(D-G4,an Piezo2 channel antagonist)group.Western blotting was used to detect the protein expression of Piezo2,and a calcium ion fluorescent probe was utilized to detect the intracellular Ca2+.The DRG neurons were pretreated with 50 ng/mL NGF for 1 h.Calcium ion fluorescent probe was used to observe the changes in intracellular Ca2+in the LFV group,the LFV+NGF group,and the LFV+NGF+D-G4 group.Results The rats of the LFV group showed abnormal morphology in multifidus muscles,accompanied with inflammatory cell infiltration,decreased paw withdrawal reflex threshold(P<0.05),and shortened total active time and active time in the centre,and decreased distance traveled in the centre(P<0.05),while prolonged total stationary time,stationary time in the periphery,and increased distance traveled in the periphery(P<0.05),and moreover,enhanced expression of Piezo2,NGF and TrkA in the DRG tissues(P<0.05).Cell experiments showed that compared with the Ctrl group,the expression of Piezo2 in the neurons was increased(P<0.05),and the intracellular Ca2+level was significantly elevated in the LFV group(P<0.05).Compared with the LFV group,the Ca2+level was higher in the LFV+NGF group(P<0.05),and the sensitization effect of NGF on Piezo2 was reversed after D-G4 treatment(P<0.05).Conclusion Sustained low-frequency vibration induces low back pain and mechanical allodynia in rats through the NGF-TrkA/Piezo2 pathway.

Objective To study the clinical characteristics and bacterial antimicrobial susceptibility testing results of patients with clinically isolated Ralstonia pickettii(R.pickettii),and provide basis for the rational use of antimi-crobial agents.Methods Inpatients with R.pickettii infection who were treated at the Tianjin First Central Hospi-tal from January 2014 to December 2023 were analyzed retrospectively.Clinical characteristics and antimicrobial sus-ceptibility testing results were analyzed.Results A total of 80 strains of Ralstonia spp.were isolated over 10-year period,including 42(52.5％)non-repetitive strains of R.pickettii.Among 42 R.pickettii strains,64.3％were isolated from male patients.The strains isolated from sputum,catheter,blood,throat swabs,and drainage fluid specimens accounted for 38.1％,28.6％,19.0％,4.8％,and 2.4％,respectively.The clinical distribution of R.pickettii was highest in the intensive care unit(ICU),with a proportion of 52.4％.The number of infected patients first increased and then decreased with the years,followed by a slight fluctuation.There was no statistically signifi-cant difference in the number of infected patients in each department over the years(P＞0.05).R.pickettii had higher susceptibility rates to doxycycline,levofloxacin,ciprofloxacin,and minocycline,susceptibility rates were 78.3％-90.9％,but was completely resistant to compound sulfamethoxazole and cefazolin(100％),it also had higher resistance rates to aztreonam,colistin,cefotetan,tobramycin,amikacin,ceftazidime,and gentamicin(80.0％-97.4％).There was no statistically significant difference in the resistance rates to 21 antimicrobial agents among different years(all P＞0.05).Conclusion R.pickettii is mainly from ICU,and the majority of the infected population are adult males.Most strains are isolated from sputum and catheter.R.pickettii presents multidrug re-sistance.Attention should be paid to the changes in the resistance rates of antimicrobial agents,strengthen the dy-namic monitoring of bacterial resistance and guide the rational selection of antimicrobial agents in clinic,implement early and effective treatment to improve the prognosis of the patients.

Zhishi Xiebai Guizhitang is a classical prescription for the treatment of chest impediment with the method of warming Yang. It is included in the Catalogue of Ancient Classical Prescriptions issued by the National Administration of Traditional Chinese Medicine （First Batch）， with the effect of activating Yang， dissipating mass， moving Qi and resolving phlegm. Its main symptoms include chest fullness and pain， or even chest pain radiating to the back， wheezing， coughing， shortness of breath， and Qi reversal from the hypochondrium. In modern traditional Chinese medicine， Zhishi Xiebai Guizhitang is clinically used in the treatment of cardiovascular system， digestive system， respiratory system and other diseases， among which coronary heart disease， unstable angina pectoris， myocardial infarction， sinus bradycardia and other cardiovascular diseases have particularly significant effects. This paper reviewed the pharmacological studies of Zhishi Xiebai Guizhitang in the past 10 years. The results showed that each single medicine and the whole prescription alleviated the above cardiovascular diseases to a certain extent， with the pharmacological effects of improving intravascular environment， myocardial ischemia， myocardial ischemia-reperfusion injury， and myocardial hypoxia， anti-inflammation， plaque stabilisation， etc.， and the pharmacological mechanism involved the regulation of relevant active substances in vivo as well as related signaling pathways and ion channels， mainly including thromboxane B₂ （TXB₂）， prostacyclin I₂（PGI₂） and phosphatidylinositol 3-kinases/protein kinase B/endothelial nitric oxide synthase （PI3K/Akt/eNOS） signaling pathways， and ATP-sensitive potassium channels. In addition， the relationship between the pharmacological effects of some single medicines and transient receptor potential ankyrin 1 （TRPA1） has been reported that TRPA1 is a key to understanding the mechanism of Zhishi Xiebai Guizhitang in treating cardiovascular diseases， which is worth of further study.

Humans ; Coronary Angiography ; Microcirculation ; Coronary Artery Disease/drug therapy* ; Treatment Outcome ; Vascular Resistance ; Coronary Vessels ; Coronary Circulation ; Predictive Value of Tests ; Fractional Flow Reserve, Myocardial

OBJECTIVES: To investigate the risk factors for neonatal asphyxia in Hubei Enshi Tujia and Miao Autonomous Prefecture and establish a nomogram model for predicting the risk of neonatal asphyxia. METHODS: A retrospective study was conducted with 613 cases of neonatal asphyxia treated in 20 cooperative hospitals in Enshi Tujia and Miao Autonomous Prefecture from January to December 2019 as the asphyxia group, and 988 randomly selected non-asphyxia neonates born and admitted to the neonatology department of these hospitals during the same period as the control group. Univariate and multivariate analyses were used to identify risk factors for neonatal asphyxia. R software (4.2.2) was used to establish a nomogram model. Receiver operator characteristic curve, calibration curve, and decision curve analysis were used to assess the discrimination, calibration, and clinical usefulness of the model for predicting the risk of neonatal asphyxia, respectively. RESULTS: Multivariate logistic regression analysis showed that minority (Tujia), male sex, premature birth, congenital malformations, abnormal fetal position, intrauterine distress, maternal occupation as a farmer, education level below high school, fewer than 9 prenatal check-ups, threatened abortion, abnormal umbilical cord, abnormal amniotic fluid, placenta previa, abruptio placentae, emergency caesarean section, and assisted delivery were independent risk factors for neonatal asphyxia (P<0.05). The area under the curve of the model for predicting the risk of neonatal asphyxia based on these risk factors was 0.748 (95%CI: 0.723-0.772). The calibration curve indicated high accuracy of the model for predicting the risk of neonatal asphyxia. The decision curve analysis showed that the model could provide a higher net benefit for neonates at risk of asphyxia. CONCLUSIONS The risk factors for neonatal asphyxia in Hubei Enshi Tujia and Miao Autonomous Prefecture are multifactorial, and the nomogram model based on these factors has good value in predicting the risk of neonatal asphyxia, which can help clinicians identify neonates at high risk of asphyxia early, and reduce the incidence of neonatal asphyxia.
Infant, Newborn ; Humans ; Male ; Pregnancy ; Female ; Nomograms ; Retrospective Studies ; Cesarean Section ; Risk Factors ; Asphyxia Neonatorum/etiology*

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*

Objective:To investigate the changes of cognitive function in non-fatal drowning rats after blast-induced traumatic brain injury (bTBI).Methods:Eighty SD rats were divided into normal group, bTBI group, drowning group and bTBI plus drowning group according to the random number table, with 20 rats per group. Rats in normal group were not injured. In bTBI group, bTBI was established in a BST-I biological shock tube with a pressure of 4.0 MPa in the driving section. In drowning group, rats were subjected to non-fatal drowning by falling into the water with temperature of 18 ℃ and depth of 30 cm from the height of 1 m and were taken out quickly after swimming to exhaustion. After being injured in a biological shock tube, rats in bTBI plus drowning group were immediately forced to drowning using the same method. On day 3 post-injury, the neurocognitive function was evaluated by elevated plus maze and Morris water maze tests. Morphological changes of neurons in CA1 and CA3 regions of hippocampus were observed by Nissl staining, and the number of surviving neurons were counted. The concentrations of hippocampal neurotransmitters glutamate, γ-aminobutyric acid (GABA), glycine and endoplasmic reticulum stress (ERS) related glucose-regulated protein 78 (GRP78) and caspase-12 were examined by ELISA analysis. Levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated protein (Bax) and caspase-3 were detected by Western blotting. The ratio of Bcl-2 to Bax was calculated as well.Results:In elevated plus maze test, the percentage of open arm entry and number of head-dipping behaviour were decreased in bTBI plus drowning group compared with normal and bTBI groups at 3 days after injury ( P<0.05 or 0.01), with no statistical difference from those in drowning group ( P>0.05). The number of head-dipping behaviour in drowning group was lower than that in bTBI group ( P<0.05). In Morris water maze test, bTBI plus drowning group showed increased target latency on the third and fourth days of spatial acquisition training and decreased number of crossing the target area and percentage of swimming time in the target quadrant during probe trials as compared with normal group ( P<0.05 or 0.01), but there was no statistical difference among bTBI, drowning and normal groups (all P>0.05). Nissl staining showed that the neurons in the CA1 and CA3 regions of hippocampus in normal group were arranged neatly with clear Nissl bodies at 3 days after injury, while the other groups showed different degrees of injury. In contrast with normal group, the neurons in the CA1 and CA3 regions of hippocampus in all other groups were decreased with the lowest number in bTBI plus drowning groups ( P<0.05 or 0.01). In ELISA analysis, the level of hippocampal glutamate in bTBI plus drowning group was higher than that in all other groups at 3 days after injury and the level in bTBI injury and drowning groups was higher than that in normal group ( P<0.05 or 0.01); the level of hippocampal glycine in bTBI plus drowning group was lower than that in normal group ( P<0.05), but there was no statistical difference among bTBI, drowning or normal groups (all P>0.05); the concentration of hippocampal GABA had no statistical difference among all groups (all P>0.05). In addition, the concentration of GRP78 in bTBI injury, drowning and bTBI injury plus drowning groups were increased compared with normal group ( P<0.05 or 0.01), but did not statistically differ from each other (all P>0.05). The concentration of caspase-12 in drowning and bTBI plus drowning groups were increased compared with normal group ( P<0.05 or 0.01), but was not statistically different from each other ( P>0.05), and its concentration in bTBI plus drowning group was increased compared with bTBI group ( P<0.05). In Western blotting, the level of Bcl-2 in bTBI plus drowning group was decreased compared with all other groups at 3 days after injury, and the level in bTBI and drowning groups were decreased compared with normal group, but a much lower level was observed in drowning group than that in bTBI group ( P<0.05 or 0.01); the level of Bax in bTBI plus drowning group was increased compared with all other groups at 3 days after injury, and the level in drowning group was increased compared with normal group ( P<0.05 or 0.01), with no statistical difference between bTBI and drowning groups ( P>0.05). The ratio of Bcl-2 to Bax in bTBI plus drowning group was decreased compared with all other groups, while the ratio in bTBI and drowning groups were decreased compared with normal group, showing a much lower level in drowning group than that in bTBI group ( P<0.05 or 0.01). Also, the level of caspase-3 in drowning and bTBI plus drowning groups were increased compared with normal and bTBI groups ( P<0.05 or 0.01), but there was no statistical difference between drowning and bTBI plus drowning groups ( P>0.05). Conclusions:Non-fatal drowning can aggravate hippocampal neuron damage in bTBI rats and cause memory, emotion and other cognitive dysfunction. The mechanism may involve the imbalance of hippocampal neurotransmitters glutamate and glycine, which activates the downstream pro-apoptotic pathway through ERS in the early stage of injury to induce hippocampal neuron apoptosis.

The intestinal absorption characteristics of ten iridoid glycosides and phenolic acids in the Pterocephali Herba were evaluated via rat intestinal valgus model. The intestinal sac fluids at different time after administration of high,medium and low concentrations of Pterocephali Herba extract were collected and ten chemical components in fluid samples were detected by UPLC-PDA. Accumulative absorbed doses( Q) and absorption rate constants( Ka) of ten chemical constituents were calculated,while proportions between Pterocephali Herba extract and intestinal absorption liquid were compared. The results showed that the intestinal absorption of 10 chemical components was linear absorption( R2>0. 9) at different concentrations,which accorded with the zero-order absorption rate. The absorption rate constant was related to the concentration of the drug and the intestinal site,which indicated that intestinal adsorption mechanism of the components were passive diffusion and active transport. Proportions of chemical constituents in intestinal sac fluid were different from those in Pterocephali Herba extract. Therefore,those ten chemical components in Pterocephali Herba extract can be absorbed in whole intestine. Everted intestinal sac model can be used to evaluate intestinal absorption characteristics of ingredients in Pterocephali Herba extract effectively.
Animals ; Caprifoliaceae ; chemistry ; Drugs, Chinese Herbal ; pharmacokinetics ; Intestinal Absorption ; Intestines ; Plant Extracts ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley