Objective To investigate the role of mitochondrial respiratory chain (MRC) in induction of reactive oxygen species (ROS) by hyperbaric oxygen (HBO).Methods Superoxide anion (O·-2) specific fluorescence probe DHE and mitochondrial O·-2 probe Mito-SOX were used to label ROS in human umbilical vein endothelium cells(HUVEC) microscopically after HBO exposure.Results After HBO exposure, O·-2 increased (31±8)% and (137±19) % in whole cells and in mitochondria, respectively (P<0.01).These increments were suppressed by MRC complex Ⅱ inhibitor TTFA for (55±11)% in whole cells (P<0.05)and (61±8) % in mitochondria(P<0.01).Conclusion MRC may be the main source of ROS induced by HBO in HUVECs.

Objective To investigate the effect of Danshen Baoxin Cha (DBC) on a rat model of coronary heart disease combined with cognitive impairment. Methods Male Sprague-Dawley(SD) rats were randomly assigned to two groups:normal group and model group. Streptozotocin was injected into the bilateral ventricles of rats in the model group to establish cognitive impairment model,then isoproterenol hydrochloride was injected subcutaneously to model myocardial ischemia. Behavioral experiments were conducted to verify the success of the model of cognitive dysfunction. The rats of the model group were randomly divided into five groups:model control group,Tongxinluo Capsule group (TXL group,1.6 g·kg-1),and low-(4 g·kg-1),medium-(8 g·kg-1),and high-(16 g·kg-1) dose DBC groups. These groups were received the respective treatments continuously for two weeks. Subsequently,the Y-maze,novel object recognition and Morris water maze experiment were employed to assess the learning and memory abilities of rats. A kit was utilized to quantify the level of oxidative stress in the brain and the adenosine triphosphate (ATP) content in the brain and mitochondria. Hematoxylin-eosin (HE) staining and Nissl staining were employed to observe the pathological changes of neurons in hippocampus CA1 region. Electron microscopy was utilized to observe the pathological changes of mitochondria in hippocampal CA1 region. The expression levels of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),glucose transporter type 4(GLUT4),and optic atrophy 1(OPA1) were quantified by real-time fluorescence quantitative polymerase chain reaction (PCR),and the expression of proteins related to the AMPK/OPA1 signaling pathway was determined by Western Blot analysis. Results Compared with the normal group,the spontaneous alternating reaction rate,the novel object recognition index,number of crossing the original platform,and distance ratio in the model group were obviously decreased (P<0.01). Neuronal density in the CA1 region of the hippocampus was decreased,Nissl bodies were decreased,and nucleus consolidation was increased. The ATP level in mitochondria,and the levels of ATP,SOD,and GSH-PX in brain were significantly decreased(P<0.05,P<0.01),as well as the content of ROS and MDA were significantly increased (P<0.05,P<0.01). The mitochondria of hippocampus in CA1 region were swollen,with sparse and vacuolated cristae. The mRNA expression levels of GLUT4,PGC-1α,and OPA1 were significantly decreased (P<0.01). The protein expression levels of GLUT4,SIRT1,PGC-1α and OPA1,and p-AMPK/AMPK ratio were significantly decreased (P<0.05,P<0.01). Compared with the model group,the behavioral indexes of rats in the DBC groups were significantly improved (P<0.05,P<0.01),the number of neurons in the hippocampal CA1 area,Nissl bodies and nucleus consolidation were improved. The ATP level in mitochondria and the levels of ATP,SOD,and GSH-PX in brain were significantly increased (P<0.05,P<0.01). The levels of ROS and MDA were significantly decreased (P<0.05,P<0.01). The structure of mitochondrial cristae in hippocampal CA1 region were relatively intact. The mRNA expression levels of GLUT4,PGC-1α and OPA1 were increased (P<0.05,P<0.01),and the expression of proteins related to the AMPK/OPA1 signaling pathway was significantly increased(P<0.05,P<0.01). Conclusion DBC can enhance learning and memory abilities,reduce neuronal damage in a rat model of coronary heart disease combined with cognitive impairment. The mechanism may be related to the reduction of oxidative stress damage in the brain,the activation of the AMPK/OPA1 signaling pathway,and the restoration of energy levels.

Objective To explore the mechanism of action of Danshen Baoxin Cha(DBC) on depressed mice with coronary heart disease (CHD) based on network pharmacology and NLRP3 inflammatory pathway. Methods (1) TCMSP and BATMAN-TICAM databases were used to screen the DBC active ingredients and targets. The targets of CHD with depression were screened using the OMIM and Genecards databases. The targets of DBC active ingredients and related targets of CHD with depression were imported into Venny 2.1 online platform to obtain the intersection targets,which was the potential target of DBC in the treatment of CHD with depression. Protein-protein interaction (PPI) analysis was performed on the intersection targets using the STRING platform to screen the key targets. A "drug-active ingredients-disease-targets" network was created to select the main active ingredients and core targets of DBC for the treatment of CHD with depression. Thereafter,the primary targets were examined by GO function and KEGG pathway enrichment using the Metascape database.(2)Kunming mice were split into six groups of eight mice each at random:the control group,the model group,the positive control group (metoprolol tartrate 5.14 mg·kg-1+sertraline hydrochloride 10.3 mg·kg-1),and the DBC high-,middle-,and low-dose groups (30.8,15.4 and 7.7 g·kg-1·d-1). Chronic unpredictable mild stimulation (CUMS)and subcutaneous injection of isoprenaline hydrochloride (ISO) were used to induce a mice model of CHD with depression. Mice were treated orally with the corresponding drug once a day for 18 consecutive days. Behavioral experiments involving forced swimming test,tail suspension test,and open-field test were applied to detect depression levels of mice. Histopathological alterations in hippocampus tissues were noted using HE and Nissl staining. qPCR was used to determine the mRNA expression levels of IL-6,TNF-α,NLRP3,IL-1β,IL-10,and Caspase-1 in hippocampus tissues. Results(1) Sixty-five active components in Salvia and seven active components in green tea were screened out. A total of 1042 potential targets and 2116 CHD complicated with depression-related targets were obtained. The intersection of the targets of active components and disease-related targets was performed by Venny 2.1.0 platform to obtain 299 potential targets (common targets) of DBC in the treatment of CHD with depression. The core targets including IL-1β,AKT1,TNF-α,IL-6,VEGFA,CASP3 and IL-10 were screened through PPI network analysis of potential targets. Key active ingredients including vitamin B,luteolin,salvianolic acid,tanshinone ⅡA and catechin,as well as key targets,such as PTGS2、IL-1β、IL-6、TNF-α and IL-10,were obtained by network analysis of "drugs-active ingredients-disease-targets". The potential targets were correlated with biological processes such as inflammation response,regulation of tumour necrosis factor (TNF),glucocorticoid regulation,regulation of nuclear factor kappa B(NF-κB) transcription factor,as well as major pathways including PI3K-Akt signaling pathway,TNF signaling pathway,apoptosis signaling pathway,and NF-κB signaling pathway.(2) Compared with the control group,mice in the model group showed a significant decrease in the total and center distance of the open field (P<0.01) and a significant increase in the time of forced swimming and immobility time of tail suspension test (P<0.01). The mRNA expression of IL-6,TNF-α,NLRP3,IL-1β,and Caspase-1 was significantly up-regulated(P<0.01) in the hippocampus tissues,but IL-10 mRNA expression was down-regulated (P<0.05). Compared with the model group,the total and center distance in DBC high-,middle-,and low-dose groups were significantly up-regulated(P<0.05,P<0.01),and the time of forced swimming and immobility time of tail suspension test were significantly down-regulated (P<0.01). The mRNA expression of IL-6,TNF-α,NLRP3,IL-1β and Caspase-1 of the DBC high-,middle-,and low-dose groups were significantly down-regulated(P<0.01),IL-10 mRNA expression in mice hippocampus tissue of DBC high-and middle-dose groups was up-regulated (P<0.01). Conclusion The intervention effect of DBC on depressed mice with CHD may be achieved by active ingredients including luteolin,tanshinone,salvianolic acid and catechin acting on the key targets,such as IL-6,TNF-α,IL-1β and IL-10,to regulate the NLRP3 inflammatory signaling pathway.

OBJECTIVETo establish rabbit VX2 colorectal cancer(CRC) model and to compare CT images with gross pathology in order to offer help for TNM staging in patients with CRC.

METHODSVX2 tumor pieces were implanted into colonic wall in 9 New Zealand white rabbits and rectal wall in 2 New Zealand white rabbits. Four weeks after inoculation, Ultravist(370 mg/ml) was injected through ear marginal vein with high pressure injector for stage 3 scanning of chest, abdomen and pelvis, and enhanced CT (collimation 0.5 mm mm × 320, pitch factor 0.828, bulb rotation speed 0.5 s/cycle, 120 kV, automatic ma, range 80 to 100 mAs) was performed to determine the presence of CRC or metastasis once a week for 4-6 weeks. Once inoculated CRC or metastases occurred or 6 weeks after implantation, the rabbits were sacrificed regardless of the presence or absence of CRC or metastasis on the CT images. One rabbit was used for gross anatomy observation. Others were placed in wood boxes with -80centi-degree for 24 hours, then samples of 3 mm thickness were cut using a motorized saw to make macropathology. Each cutting surface of the specimens was photographed in serial number. If certain or suspected lesions were found on the slices, such part was labeled and then placed in 10% phosphate-buffered formaldehyde numbered box for subsequent pathological examination. CT image postprocessing was performed referring to the gross slice specimens and all findings were compared with the pathological reports.

RESULTSAmong 11 rabbits, tumor was successfully established in 8 rabbits. Pathology showed that single lung metastasis (7 to 10 mm) was found in 2 rabbits and liver metastasis (9 mm) in 1 rabbit. Number of lymph node located around the inoculated tumor was 22 and that around mesenteric vessels was 13 with diameter of 2 to 16 mm. Among these 35 lymph nodes from 8 successful rabbits, 9 nodes were positive, including 7 around inoculated tumor and 2 around mesenteric vessels. CT identified above 8 primary inoculated tumors, 2 lung metastatic lesions and 1 liver metastatic lesion, with detection rate of 100%. For the detection of lymph node in CT, 27 nodes were identified in the pericolorectal region (17 nodes) and perimesenteric vessels (10 nodes), in which 6 were positive metastasis (ring-shaped enrichment and central low density necrosis), resulting in a detection rate of 77.1%(27/35 nodes), and positive detection rate of 66.7% (6/9 nodes), respectively.

CONCLUSIONLiving rabbit CT-gross pathological slice(3 mm-cut) of VX2 CRC model can be applied in image evaluation of small metastatic lesion.

Animals ; Colorectal Neoplasms ; Humans ; Liver ; Lung Neoplasms ; Lymph Nodes ; Lymphatic Metastasis ; Rabbits ; Tomography, X-Ray Computed