[Role and related mechanism of resolvin D1 in lung ischemia reperfusion injury in rats].

Objective: To investigate the role and related mechanism of resolvin D1 (RvD1) in lung ischemia-reperfusion injury (LIRI) in rats. Methods: Forty male Sprague-Dawley rats, 7-8 weeks, weighing 220-280 g, were divided into 4 groups using a random number table method: sham operation group, lung ischemia reperfusion control group, normal saline group, and RvD1 group. The rat model of LIRI was produced by 45 min of occlusion of the left hilum of lungs followed by 150 min reperfusion. In sham group, no blocking of the left hilum of lung after thoracotomy; Normal saline 2 ml/kg and RvD1 100 µg/kg were injected respectively at 10 min of reperfusion in normal saline group and RvD1 group. Blood samples were collected from the femoral vein for determination of interleukin (IL)-6, tumor necrosis factor (TNF)-α, soluble inter-cell adhesion molecules (sICAM-1) concentrations at 150 min of reperfusion. The rats were sacrificed after collection of blood samples and then lung tissues were taken for observation of the pathological changes and for measurement of lung wet/dry weight ratio (W/D). The the contents of malondialdehyde (MDA), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2 and the activity of myeloperoxidase (MPO) in lung tissues were determined. The protein relative expression of nuclear factor (NF)-κB in lung tissues was detected by Western blot. Lung tissue cell apoptosis was detected with TUNEL method. Results: The plasma level of IL-6, TNF-α, sICAM-1 in normal saline group and RvD1 group were significantly higher than those in the Sham group [(110±7), (100±4) vs (72±3) ng/L, (151±8), (153±6) vs (104±5) ng/L, (2 690±133), (2 760±167) vs (1 953±125) ng/L]. Besides, NF-κB protein relative expression level of lung tissues up-regulated [(0.681±0.033), (0.664±0.024) vs (0.292±0.011)] (all P<0.05). The W/D, apoptosis index, MDA, MCP-1, MIP-2 contents and MPO activity in lung ischemia reperfusion control group, normal saline group and RvD1 group were significantly higher than those in the Sham group [(5.92±0.31), (5.85±0.24), (5.06±0.08) vs (4.14±0.10), (32.9±1.5)%, (31.9±1.3)%, (17.7±1.8)% vs (8.1±0.6)%, (72.1±2.3), (66.7±3.7), (34.0±1.4) vs (22.0±0.8) nmol/mg, (3.99±0.28), (3.86±0.25), (2.66±0.16) vs (1.47±0.17) pg/mg, (9.45±0.53), (9.68±0.62), (7.62±0.22) vs (4.70±0.41) pg/mg, (3.01±0.18), (2.92±0.19), (1.58±0.11) vs (0.98±0.07) U/g] (all P<0.05). The plasma levels of the cytokines mentioned above, the W/D, the apoptosis index, MDA, MCP-1, MIP-2 contents and MPO activity in RvD1 group were significantly lower than those in the lung ischemia reperfusion control group [(63±4) vs (110±7) ng/L, (90±8) vs (151±8) ng/L, (1 835±182) vs (2 690±133) ng/L, (5.06±0.08) vs (5.92±0.31), (17.7±1.8)% vs (32.9±1.5)%, (34.0±1.4) vs (72.1±2.3) nmol/mg, (2.66±0.16) vs (3.99±0.28) pg/mg, (7.62±0.22) vs (9.45±0.53) pg/mg, (1.58±0.11) vs (3.01±0.18) U/g]. Besides, NF-κB protein relative expression level of lung tissues down-regulated [(0.313±0.012) vs (0.681±0.033)] (all P<0.05). Inflammatory cell infiltration in LIRI groups increased significantly, while it was significantly reduced in RvD1 group. Conclusion: RvD1 can effectively alleviate the tissue damage caused by lung ischemia-reperfusion through down-regulating NF-κB expression, relieving inflammatory reaction and oxidative stress, reducing apoptosis in rats.

[The role of high-mobility group box protein 1 in the signaling pathways of myocardial ischemia-reperfusion injury in rats].

Objective: To investigate the role of high-mobility group box protein 1 (HMGB1) in the signaling pathways of myocardial ischemia-reperfusion injury (MIRI) in rats. Methods: Forty male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 4 groups (n=10) using a random number table: sham operation group (group sham), MIRI group (group IR-C), anti-HMGB1 antibody group (group IR-H-Ig), contrast antibody control group (group IR-Ig). The rat model of MIRI was established by 30 min occlusion of left anterior descending branch (LAD) of coronary artery followed by 180 min reperfusion. In sham group, no blocking of LAD was adopted after thoracotomy. Anti-HMGB1 antibody and contrast antibody immunoglobulin G (IgG) (2 mg/kg) were injected respectively at 30 min of reperfusion in IR-H-Ig and IR-Ig groups. Blood samples were collected from the femoral vein for determination of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), HMGB1, creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) concentrations at 180 min of reperfusion. The rats were then sacrificed after blood samples were taken and the pathological changes of myocardial tissue were observed. The mRNA and protein expressions of HMGB1, toll-like receptor 4(TLR4) and nuclear factor (NF)-κB in myocardial tissues were detected by Western blot and real-time quantitative PCR respectively. Results: Compared with the Sham group, the plasma level of IL-6, TNF-α, HMGB1 increased significantly and HMGB1, TLR4 and NF-κB mRNA and protein levels of myocardial tissues up-regulated in IR-C and IR-Ig groups (all P<0.05). The plasma level of CK-MB and cTnI increased significantly in IR-C, IR-H-Ig, IR-Ig group (all P<0.05). Compared with the IR-C group, the levels of the plasma HMGB1, the cytokines mentioned above, CK-MB and cTnI were significantly decreased, and mRNA and protein expressions of HMGB1, TLR4 and NF-κB of myocardial tissues down-regulated in IR-H-Ig group (all P<0.05). Inflammatory cell infiltration in MIRI groups increased significantly, while it was significantly reduced in IR-H-Ig group. Conclusion: Blocking the combination of HMGB1 and TLR4 can effectively alleviate the tissue damage caused by myocardial ischemia-reperfusion in rats.

Comparison of half- and standard-dose ticagrelor in Chinese patients with NSTE-ACS.

Ticagrelor is a novel direct-acting P2Y12 receptor antagonist used for preventing atherothrombotic events in patients with acute coronary syndromes (ACS). The current recommended dose is 90 mg bid, but a low dose of ticagrelor has not been previously studied in Chinese ACS patients. Therefore, we performed this study to observe the different effects of half- and standard-dose ticagrelor on platelet aggregation in Chinese patients with NSTE-ACS. Sixty-two NSTE-ACS subjects were assigned to half-dose ticagrelor (n = 20), standard-dose ticagrelor (n = 22) and clopidogrel (n = 20) groups. Five days after drug administration, VerifyNow P2Y12 assay was performed to test P2Y12 reaction units (PRU) and inhibition of platelet aggregation (IPA). High-platelet reactivity (HPR) was defined as a PRU > 208. The adverse events, including bleeding events and dyspnoea, were monitored throughout the study. PRU values in the half-dose (44.55 ± 32.88) and standard-dose (39.10 ± 40.02) ticagrelor were dramatically lower than those in the clopidogrel group (189.20 ± 65.22; P < 0.0001). The half-dose (84% ± 10%) and standard-dose (86% ± 13%) ticagrelor both showed greater IPA than clopidogrel (33% ± 20%; P < 0.0001). There were no significant differences in PRU and IPA between the two ticagrelor groups (P = 0.3085 and 0.4028, respectively). HPR rates were significantly lower in the two ticagrelor groups (0% for both) than those in the clopidogrel group (35%). In conclusion, half-dose ticagrelor had a similar inhibitory effect on platelet aggregation as standard-dose ticagrelor in Chinese patients with NSTE-ACS, which was significantly stronger than that of clopidogrel.

Analysis of genetic diversity of Brassica rapa var. chinensis using ISSR markers and development of SCAR marker specific for Fragrant Bok Choy, a product of geographic indication.

Non-heading Chinese cabbage [Brassica rapa var. chinensis (Linnaeus) Kitamura] is a popular vegetable and is also used as a medicinal plant in traditional Chinese medicine. Fragrant Bok Choy is a unique accession of non-heading Chinese cabbage and a product of geographic indication certified by the Ministry of Agriculture of China, which is noted for its rich aromatic flavor. However, transitional and overlapping morphological traits can make it difficult to distinguish this accession from other non-heading Chinese cabbages. This study aimed to develop a molecular method for efficient identification of Fragrant Bok Choy. Genetic diversity analysis, based on inter-simple sequence repeat molecular markers, was conducted for 11 non-heading Chinese cabbage accessions grown in the Yangtze River Delta region. Genetic similarity coefficients between the 11 accessions ranged from 0.5455 to 0.8961, and the genetic distance ranged from 0.0755 to 0.4475. Cluster analysis divided the 11 accessions into two major groups. The primer ISSR-840 amplified a fragment specific for Fragrant Bok Choy. A pair of specific sequence-characterized amplified region (SCAR) primers based on this fragment amplified a target band in Fragrant Bok Choy individuals, but no band was detected in individuals of other accessions. In conclusion, this study has developed an efficient strategy for authentication of Fragrant Bok Choy. The SCAR marker described here will facilitate the conservation and utilization of this unique non-heading Chinese cabbage germplasm resource.

Two-step identification of taro (Colocasia esculenta cv. Xinmaoyu) using specific psbE-petL and simple sequence repeat-sequence characterized amplified regions (SSR-SCAR) markers.

Colocasia esculenta cv. Xinmaoyu is an eddoe-type taro cultivar local to Taicang, Jiangsu Province, China; it is characterized by its pure flavor, glutinous texture, and high nutritional value. Due to its excellent qualities, the Trademark Office of the State Administration for Industry and Commerce of the People's Republic of China awarded Xinmaoyu, a geographical indication certification in 2014. Therefore, there is an urgent need to develop an efficient molecular marker for the specific identification of this cultivar, which would greatly facilitate the conservation and utilization of this unique germplasm resource. In the present study, amplifying the psbE-petL fragment from two dasheen-type and seven eddoe-type taro cultivars revealed three conserved insertions/deletions among sequences from the two taro types. Based on these sequence differences, a pair of site-specific primers was designed targeting the psbE-petL sequence from the dasheen-type taro, which specifically amplified a DNA band in all individuals from cultivars of this type, but not in those from the seven eddoe-type cultivars. To discriminate Xinmaoyu from the other eddoe-type taro cultivars, a pair of simple sequence repeat-sequence characterized amplified region (SSR-SCAR) primers was further developed to specifically amplify a DNA band from all Xinmaoyu individuals, but not from individuals of other eddoe-type taro cultivars. In conclusion, through a two-step-screening procedure using psbE-petL and SSR-SCAR markers, we developed a pair of primers that could specifically discriminate Xinmaoyu from nine taro cultivars commonly cultivated in Jiangsu Province and Fujian Province.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Antioxidant activity of two dibenzocyclooctene lignans on the aged and ischemic brain in rats.

The effects of two dibenzocyclooctene lignans on peroxidative damage of aging and ischemic rat brain were studied. Incubation of eight-month-old rat brain mitochondria and membrane suspension with Fe(2+)-cysteine resulted in the formation of malondialdehyde (MDA) and decrease of ATPase activity. Schisanhenol (Sal) (10(-4) M) completely inhibited the peroxidative damages of brain mitochondria and membrane of rats. The swelling and disintegration of brain mitochondria, as well as the reduction of brain membrane fluidity induced by Fe(2+)-cysteine were also prevented by Sal. The results of imitative experiment of ischemia and reperfusion of brain mitochondria and membrane in vitro indicated that Sal significantly impeded production of MDA and loss of ATPase activity induced by reoxygenation following anoxia. Oral administration of Sal induced increase of cytosol glutathione-peroxidase of brain in mice under the condition of reoxygenation following anoxia. The other compound schizandrin (Sin B) also has similar activity. But its potency is weaker than that of Sal. All these results indicate that Sal and Sin B have protective action against oxidative stress.

[The relation between integrin, type IV collagenase and extracellular matrix in invasion and metastasis of gastric carcinoma].

The expression of integrin Alpha-6-subunit, laminin, type IV collagenase, type IV collagen and ras p21 were studied immunohistochemically in gastric cancer. The results showed that the expression of alpha 6 and laminin (LN) was often in continuous or interrupted linear pattern in the expanding type of gastric carcinoma (GC); while in the infiltrating type of GC, they were expressed either in interrupted spotty or fragmentary pattern, or almost lost. Suggesting that the interaction between the laminin receptor and its ligand may influence the mode of growth in GC. Intense expression of type IV collagenase was often found in GC cells, especially in the infiltrative type of GC and in those with lymph node metastasis, it can therefore be used as a marker for invasion and metastasis of GC cells. A positive correlation was found between the expression of type IV collagenase and ras p21 in GC. The expression of type IV collagen was similar to that of laminin, but in reverse proportion to the expression of type IV collagenase. These investigations provide a better understanding of the molecular pathological basis of tumor invasion and metastasis.

Capsaicin and its analogues: structure-activity relationship study.

Capsaicin, the main ingredient responsible for the hot pungent taste of chilli peppers, is an alkaloid found in the Capsicum family. Capsaicin was traditionally used for muscular pain, headaches, to improve circulation and for its gastrointestinal protective effects. It was also commonly added to herbal formulations because it acts as a catalyst for other herbs and aids in their absorption. In addition, capsaicin and other capsaicinoid compounds showed strong evidence of having promising potential in the fight against many types of cancer. The mechanism of action of capsaicin has been extensively studied over the past decade. It has been established that capsaicin binds to the transient receptor potential vanilloid 1 receptor which was expressed predominantly by sensory neurons. And many analogues of capsaicin have been synthesized and evaluated for diverse bioactivities. In this review, we will attempt to summarize the biology and structure-activity relationship of capsaicinoids.

PPAR signaling pathway may be an important predictor of breast cancer response to neoadjuvant chemotherapy.

PURPOSE: Neoadjuvant chemotherapy for advanced breast cancer may improve the radicality for a subset of patients, but others may suffer from severe adverse drug reactions without any benefit. To predict the responses to chemotherapy, we performed a phase II trial of neoadjuvant chemotherapy using a weekly PCb [paclitaxel (Taxol) plus carboplatin] regimen for stage II/III breast cancer and assessed the correlation between baseline gene expression and the tumor response to treatment. METHODS: A total of 61 patients with stage II-III breast cancer were included and administered four cycles of preoperative PCb. We performed a gene expression analysis using Affymetrix HG-U133 Plus 2.0 GeneChip arrays in 31 breast cancer tissues. Differentially expressed genes (DEGs) were identified by the significance analysis of microarrays (SAM) program using a false discovery rate of 0.05. The Functional Annotation Tool in the DAVID Bioinformatics Resources was used to perform the gene functional enrichment analysis. The other 30 patients (15 pCR and 15 non-pCR patients) were available as an independent validation set to test the selected DEGs by quantitative real-time PCR analysis (qRT-PCR). RESULTS: By analyzing six pathological complete response (pCR) patients and 25 patients with non-pCR, 300 probes (231 genes) were identified as differentially expressed between pCR and residual disease by the SAM program when the fold change was >2. The gene functional enrichment analysis revealed 15 prominent gene categories that were different between pCR and non-pCR patients, most notably the genes involved in the peroxisome proliferator-activated receptor (PPAR), DNA repair and ER signal pathways and in the immune-related gene cluster. The qRT-PCR analysis results for the genes in the PPAR pathway (LPL, SORBS1, PLTP, SCD5, MMP1 and CSTA) in independent validation set were consistent with the results from the microarray data analysis. CONCLUSION: In the present study, we identified a number of gene categories pertinent to the therapeutic response. We believe that the PPAR pathway may be an important predictor of genes that are involved in the chemotherapy response.

[Studies on DNA methylation in transformed mouse liver cells].

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.

Reactive oxygen species participate in peroxynitrite-induced apoptosis in HL-60 cells.

Peroxynitrite (ONOO-) is a physiological product generated by the interaction of superoxide (O2.-) and nitric oxide (.NO). We have previously shown that peroxynitrite induces apoptosis in HL-60 cells. In the present study, we demonstrated that peroxynitrite generates reactive oxygen species (ROS) in HL-60 cells. Brief exposure of HL-60 cells to ONOO- induced elevation of lucigenin chemiluminescence, indicating generation of superoxide anion. Exogenous superoxide dismutase (SOD), a scavenger of O2.-, fully abolished the chemiluminescence response, further supporting this notion. Following O2.- generation, the accumulation of hydrogen peroxide (H2O2) was observed. The addition of SOD exacerbated but that of catalase attenuated peroxynitrite-induced DNA fragmentation, suggesting that this H2O2 production contributes to the apoptotic process. In addition, pre-treatment of HL-60 cells with N-acetyl-L-cysteine (15 mM), a ROS scavenger, fully scavenged peroxynitrite-elicited ROS generation and effectively inhibited (ONOO-)-induced apoptosis, further enforcing this hypothesis. In summary, our results suggest that (ONOO-)-stimulated ROS formation may serve as a mechanism for the propagation of peroxynitrite-mediated apoptotic cell death in an intact cell system.

Peroxynitrite-induced apoptosis in HL-60 cells.

Peroxynitrite (ONOO-), an anion and a potent oxidant, generated by the interaction of nitric oxide (NO) and superoxide is able to induce apoptosis in HL-60 human leukemia cells in a time- and concentration-dependent manner. Characteristic morphology of apoptosis can be observed 3 h after HL-60 cells are exposed to 10 microM ONOO-. Treatment of HL-60 cells with increasing concentrations of ONOO- from 1 to 100 microM confirms the concentration dependence of apoptosis as evidenced by: 1) degradation of nuclear DNA of these cells into integer multiples of approximately 200 base pairs; 2) colorimetric DNA fragmentation assay; and 3) evidence of condensation of chromatin and nuclear fragmentation shown by propidium iodide staining. Under the same conditions, peroxynitrite causes apoptosis in another transformed cell line, U-937 cells, but is ineffective at inducing apoptosis in normal endothelial cells derived from human umbilical cord and normal human peripheral blood mononuclear cells. This direct evidence of peroxynitrite inducing apoptosis implicated a new function of this potent oxidant.

Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease.

Apoptosis is an active process critical for the homeostasis of organisms. Enzymes of the caspase family are responsible for executing this process. We have previously shown that peroxynitrite (ONOO-), a biological product generated from the interaction of nitric oxide and superoxide, induces apoptosis of HL-60 cells. The aim of this study was to elucidate the mechanisms involved in the execution process of peroxynitrite-induced apoptosis. Proteolytic cleavage of poly(ADP-ribose) polymerase, an indication of caspase-3 family protease activation and an early biochemical event accompanying apoptosis, was observed in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 during peroxynitrite-induced apoptosis was substantiated by monitoring proteolysis of the caspase-3 proenzyme and by measuring caspase-3 activity with a fluorogenic substrate. Furthermore, pretreatment of HL-60 cells with N-acetyl-Asp-Glu-Val-Asp-aldehyde, a specific inhibitor of caspase-3, but not N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a specific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process of peroxynitrite-induced apoptosis of HL-60 cells.