Protective Effects and Mechanisms of Melatonin on Stress Myocardial Injury in Rats.

ABSTRACT: Prolonged and intense stress can exceed the body's normal self-regulation and limited compensatory and repair capacity, resulting in pathological damage to the body. In this study, we established a rat stress myocardial injury (SMI) model to explore the protective effect of melatonin (MLT) on SMI and its possible mechanisms of action. Adult female Sprague Dawley (SD) rats were randomly divided into 5 groups: blank control group (NC), SMI group, MLT low-dose group, MLT medium-dose group, and MLT high-dose group, and 10 rats in each group were used to establish a SMI model by the water immersion restraint method. We observed the changes in body weight and tail vein glucose of each group. Serum levels of corticosterone (Cort), creatine kinase isoenzyme (CK-MB), and Troponin Ⅰ (Tn-Ⅰ) and activity of lactic acid dehydrogenase were measured by ELISA. Transcriptome sequencing was used to find differentially expressed genes in the control and model groups, and the results were verified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). HE staining was used to visualize the pathological changes in the heart tissue of each group, and Western blot was used to study the differences in protein expression in the cardiomyocytes of each group to further corroborate the results. The body weight growth rate of rats in the SMI group was significantly lower than that of the NC group ( P < 0.01), and the body weight growth rate of rats in the MLT high-dose group was significantly higher than that of the SMI group ( P < 0.05) with no significant difference compared with the NC group rats. The mean blood glucose of rats in the SMI group was significantly higher compared with the NC group ( P < 0.001), while the mean blood glucose of rats in the MLT administration groups was dose-dependently reduced compared with the SMI group. By RNA-seq and bioinformatics tools such as KEGG and Gene ontology, we found that the circadian clock-related genes Ciart , Arnt1 , Per1 , and Dbp were significantly downregulated in the SMI group during water immersion stress, and differentially expressed genes were enriched in the p38MAPK signaling pathway and p53 signaling pathway. Moreover, genes related to inflammation and apoptosis were differentially expressed. ELISA results showed that Cort, CK-MB, and Tn-Ⅰ levels were significantly higher in the SMI group compared with the NC group ( P < 0.01) and melatonin reduced the levels of Cort, CK-MB, and Tn-Ⅰ and decreased lactic acid dehydrogenase activity in rat serum. HE staining results showed that melatonin could attenuate stress-generated myocardial injury. Western blot showed that melatonin reduced the expression of p38MAPK, p53, Bax, and caspase-3 and increased the expression of Bcl-2 protein in rat heart. Melatonin can inhibit myocardial injury caused by water immersion, and its mechanism of action may be related to the regulation of the expression of circadian clock genes such as Ciart , Arnt1 , Per1 , and Dbp ; the inhibition of the expression of proapoptotic proteins such as p38MAPK, p53, Bax, and caspase-3; and the increase of the expression of Bcl-2 antiapoptotic protein.

Apigenin Inhibits Human SW620 Cell Growth by Targeting Polyamine Catabolism.

Apigenin is a nonmutagenic flavonoid that has antitumor properties. Polyamines are ubiquitous cellular polycations, which play an important role in the proliferation and differentiation of cancer cells. Highly regulated pathways control the biosynthesis and degradation of polyamines. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the metabolism, and spermidine/spermine-N1-Acetyl transferase (SSAT) is the rate-limiting enzyme in the catabolism of polyamines. In the current study, the effect of increasing concentrations of apigenin on polyamine levels, ODC and SSAT protein expression, mRNA expression, cell proliferation and apoptosis, and the production of reactive oxygen species (ROS) was investigated in SW620 colon cancer cells. The results showed that apigenin significantly reduced cell proliferation, decreased the levels of spermidine and spermine, and increased previously downregulated putrescine contents. Apigenin also enhanced SSAT protein and mRNA levels and the production of reactive oxygen species in SW620 cells, though it had no significant effect on the levels of ODC protein or mRNA. Apigenin appears to decrease the proliferation rate of human SW620 cells by facilitating SSAT expression to induce polyamine catabolism and increasing ROS levels to induce cell apoptosis.

Cytotoxic and Antitumor Effects of Curzerene from Curcuma longa.

Curzerene is a sesquiterpene and component used in oriental medicine. It was originally isolated from the traditional Chinese herbal medicine Curcuma rhizomes. In this study, anticancer activity of curzerene was examined in both in vitro and in vivo models. The result of the MTT assay showed that curzerene exhibited antiproliferative effects in SPC-A1 human lung adenocarcinoma cells in a time-dependent and dose-dependent manner. The anticancer IC50s were 403.8, 154.8, and 47.0 µM for 24, 48, and 72 hours, respectively. The flow cytometry analysis indicated curzerene arrested the cells in the G2/M cell cycle and promoted or induced apoptosis of SPC-A1 cells. The percentage of cells arrested in the G2/M phase increased from 9.26 % in the control group cells to 17.57 % in the cells treated with the highest dose (100 µM) of curzerene. Western blot and RT-PCR analysis demonstrated that curzerene induced the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Tumor growth was significantly inhibited in SPC-A1 cell-bearing nude mice by using curzerene (135 mg/kg daily), meanwhile, curzerene did not significantly affect body mass and the organs of the mice, which may indicate that curzerene has limited toxicity and side effects in vivo. In conclusion, curzerene could inhibit the proliferation of SPC-A1 human lung adenocarcinoma cells line in both in vitro and in vivo models. Focusing on its relationship with GSTA1, curzerene could induce the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Curzerene might be used as an anti-lung adenocarcinoma drug candidate compound for further development.

Simultaneous determination of thirteen flavonoids from Xiaobuxin-Tang extract using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry.

A simple and reliable high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis method was established to simultaneously determine thirteen flavonoids of Xiaobuxing-Tang in intestine perfusate, namely onpordin, 3'-O-methylorobol, glycitein, patuletin, genistein, luteolin, quercetin, nepitrin, quercimeritrin, daidzin, patulitrin, quercetagitrin and 3-glucosylisorhamnetin. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ionization mode. Negative ion ESI was used to form deprotonated molecules at m/z 315 for onpordin, m/z 299 for 3'-O-methylorobol, m/z 283 for glycitein, m/z 331 for patuletin, m/z 269 for genistein, m/z 285 for luteolin, m/z 301 for quercetin, m/z 477 for nepitrin, m/z 463 for quercimeritrin, m/z 461 for daidzin, m/z 493 for patulitrin, m/z 479 for quercetagitrin, m/z 477 for 3-glucosylisorhamnetin and m/z 609.2 for rutin. The linearity, sensitivity, selectivity, repeatability, accuracy, precision, recovery and matrix effect of the assay were evaluated. The proposed method was successfully applied to simultaneous determination of these thirteen flavonoids, and using this method, the intestinal absorption profiles of thirteen flavonoids were preliminarily predicted.

Curcumol induces apoptosis in SPC-A-1 human lung adenocarcinoma cells and displays anti-neoplastic effects in tumor bearing mice.

Curcumol is a sesquiterpene originally isolated from curcuma rhizomes, a component of herbal remedies commonly used in oriental medicine. Its beneficial pharmacological activities have attract significant interest recently. In this study, anti-cancer activity of curcumol was examined with both in vitro and in vivo models. It was found that curcumol exhibited time- and concentration-dependent anti-proliferative effects in SPC-A-1 human lung adenocarcinoma cells with cell cycle arrest in the G0/G1 phase while apoptosis-induction was also confirmed with flow cytometry and morphological analyses. Interestingly, curcumol did not display growth inhibition in MRC-5 human embryonic lung fibroblasts, suggesting the anti-proliferative effects of curcumol were specific to cancer cells. Anti-neoplastic effects of curcumol were also confirmed in tumor bearing mice. Curcumol (60 mg/kg daily) significantly reduced tumor size without causing notable toxicity. In conclusion, curcumol appears a favorable anti-cancer candidate for further development.

The effect of acori graminei rhizoma and extract fractions on spatial memory and hippocampal neurogenesis in amyloid beta 1-42 injected mice.

Acori graminei Rhizoma (AGR), the dry rhizoma of Acorus gramineus Soland (Araceae), has been used as an Asian traditional herbal medicine against senile dementia, stroke, and cardiovascular disease. Previous studies have revealed neuroprotective effects of AGR on neuronal damage and learning impairment, while mostly focused on the effect of volatile oil fraction of AGR. This study aimed to investigate the neuroprotective effects of different extract fractions from AGR against Alzheimer disease-like symptoms induced by Amyloid Beta (Aß) 1-42 intra-hippocampal injection. On day 7 after intra-hippocampal injection of saline or Aß1-42, spatial memory was assessed by the first Morris water maze, followed by 3-week intra-gastric administration of saline or water extract, volatile oil fraction, or defatted decoction fraction of AGR respectively. Mice were subsequently subjected to the second Morris water maze task. Levels of Aß1-42 and expressions of doublecortin and nestin in the hippocampus were examined using immunohistochemistry. Our results suggested that treatment with these different extract fractions from AGR could ameliorate cognitive impairment and down-regulate expressions of doublecortin and nestin in the hippocampus of Aß1-42 injected mice, in which water extract and volatile oil fractions were more effective in spatial memory than defatted decoction fraction.

[Absolute bioavailability of caffeic acid in rats and its intestinal absorption properties].

OBJECTIVE: To investigate the absolute bioavailability of caffeic acid in rats and its intestinal absorption properties. METHOD: The absolute bioavailability (Fabs) of caffeic acid was obtained after iv (2 mg x kg(-1)) or ig (10 mg x kg(-1)) administration to rats. The intestinal absorption of caffeic acid was explored by the recirculating vascularly perfused rat intestinal preparation. Caco-2 cell model was applied to measure the permeability of caffeic acid from apical to basolateral said (A-B) and from basolateral to apical said (B-A). RESULT: A two-compartment pharmacokinetic model was best to describe the pharmacokinetics of caffeic acid following iv or ig administration. The Fabs of caffeic acid was 14. 7% , and its intestinal absorption was 12.4%. The values of Papp A-->B and Papp B-->A of caffeic acid were retained stable while its concentration was changed. The efflux ratio values in this study surveyed were above 2.0, and suggesting caffeic acid was active transport. CONCLUSION: Caffeic acid was shown to have poor permeability across the Caco-2 cells, low intestinal absorption and low oral bioavailability in rats.

Antihyperlipidemic effect of protodioscin, an active ingredient isolated from the rhizomes of Dioscorea nipponica.

Developing drugs against metabolic-related disorders, including obesity and hyperlipidemia, is of importance for human health. Here, a bioactive phytochemical, protodioscin, isolated from the rhizomes of Dioscorea nipponica (Rhizoma Dioscoreae Nipponicae), was identified for its anti-hyperlipidemic effect. In hyperlipidemic rats, the post-administration of protodioscin significantly reduced the time of blood coagulation. In addition, the blood levels of triglyceride, cholesterol, low-density and high-density lipoproteins were also changed accordingly. Taken together, this was the first report of an antihyperlipidemic effect of protodioscin. Its great availability in various vegetables and medicinal plants will be useful in developing health food supplement(s) and/or drug(s) against hyperlipidemia.

Neuroprotective effects of a standardized flavonoid extract from Diospyros kaki leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Flavonoids, extracted from the leaves of Diospyros kaki, are the main therapeutic components of NaoXingQing (NXQ), a potent and patented Chinese herbal remedy widely used in China for the treatment of apoplexy syndrome. AIM OF THE STUDY: To investigate the neuroprotective effects of FLDK-P70, a standardized flavonoid extract, using in vivo rat models of both focal ischemia/reperfusion (I/R) injury induced by middle cerebral artery occlusion (MCAO) and on transient global brain ischemia induced by four-vessel occlusion (4-VO). We also aim to examine the effects of FLDK-P70 on glutamate-induced cell injury of hippocampal neurons as well as on hypoxia-induced injury of cortical neurons in primary cell culture. MATERIALS AND METHODS AND RESULTS: Administration of FLDK-P70 for 12 days (40, 80 mg/kg body weight, p.o., 5 days before and 7 days after 4-VO) increased the survival of hippocampal CA1 pyramidal neurons after transient global brain ischemia. Similarly, administration of FLDK-P70 for 7 days (40, 80 mg/kg body weight, p.o., 3 days before and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of rats. In primary rat hippocampal neuronal cultures, pretreatment with FLDK-P70 (5, 10 microg/ml) protected neurons from glutamate-induced excitotoxic neuronal death in a dose-dependent manner. In primary rat cerebral cortical neuronal culture, pretreatment with FLDK-P70 (25, 100 microg/ml) also reduced hypoxia-reoxygen induced neuronal death and apoptosis in a dose-dependent manner. CONCLUSIONS: These in vivo and in vitro results suggest that FLDK-P70 significantly protects rats from MCAO and 4-VO ischemic injury in vivo and protects hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia-induced injury in vitro. The mechanisms of these effects may be due to the antioxidative activity of the flavonoids. These results convincingly demonstrate that FLDK-P70 may be useful for the prevention and treatment of ischemia/reperfusion injury and other related neurodegenerative diseases.

Screening and identification of a peptide specifically targeted to NCI-H1299 from a phage display peptide library.

In this study, a NCI-H1299 (Non-Small Cell Lung Cancer, NSCLC) and a normal lung cell line (Small Airway Epithelial Cells, SAEC) were used for the subtractive screening in vitro with a phage display-12 peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the NCI-H1299 cells, and the output/input ratio of phages increased about 875-fold (from 0.4x10(4) to 3.5x10(6)). A group of peptides being capable of binding specifically to the NCI-H1299 cells were obtained, and the affinity of these peptides to bind to the targeted cells and tissues was studied. Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, a M13 phage isolated and identified from the above screenings, and a synthetic peptide ZS-1 (sequence EHMALTYPFRPP) corresponded to the sequence of the surface protein of the M13 phage were demonstrated to be capable of binding to the tumor cell surfaces of NCI-H1299 and A549 cell lines and biopsy specimens, but not to normal lungs tissue samples, other different cancer cells, or nontumor surrounding lung tissues. In conclusion, the peptide ZS-1 may be a potential candidate of biomarker ligands used for targeted drug delivery in therapy of lung cancer.

Neuroprotective effects of a standardized extract of Diospyros kaki leaves on MCAO transient focal cerebral ischemic rats and cultured neurons injured by glutamate or hypoxia.

Naoxinqing (NXQ, a standardized extract of Diospyros kaki leaves) is a patented and approved drug of Traditional Chinese Medicine (TCM) used for the treatment of apoplexy syndrome for years in China, but its underlying mechanism remains to be further elucidated. The present study investigates the effects of NXQ against focal ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats and against glutamate-induced cell injury of hippocampal neurons as well as against hypoxia injury of cortical neurons. Oral administrations of NXQ at 20, 40, 80 mg/kg/day for 7 days (3 days before MCAO and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of the rats. In primary rat hippocampal neuron cultures, treatment with NXQ at 5 - 20 microg mL concentration protects the neurons against glutamate-induced excitotoxic death in a dose-dependent manner. In primary rat cerebral cortical neuron cultures, pretreatment with 5 - 100 microg/mL NXQ also attenuates hypoxia-reoxygen induced neuron death and apoptosis in a dose-dependent manner. These results suggest that NXQ significantly protects the rats from MCAO ischemic injury in vivo and the hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia injury in vitro by synergistic mechanisms involving its antioxidative effects. NXQ:Naoxinqing CNS:central nervous system MCAO:middle cerebral artery occlusion I/R:ischemia and reperfusion.