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.

Effects of berberine on tumor growth and intestinal permeability in HCT116 tumor-bearing mice using polyamines as targets.

The prognosis of colorectal cancer (CRC) is seriously affected by high intestinal mucosal permeability accompanied by increasing tumor load. Berberine, a natural plant-derived product, can protect the intestinal mucosal barrier and suppress tumor growth, but its effects on the intestinal mucosal barrier dysfunction of CRC have not yet been evaluated. Herein, we assessed the effects of berberine on the intestinal mucosal permeability of HCT116 tumor-bearing mice and the underlying mechanism. Berberine (6.25, 12.5, 25 mg/kg) was administered to tumor-bearing mice for 3 weeks by intraperitoneal injection, and saline was given to controls and models. Compared with the control group, tumor-bearing mice had increased intestinal mucosal permeability in the third week. Meanwhile, the body weight decreased by 4%-7%, the concentration of D-lactic acid in plasma increased, and the expressions of ZO1 and Occludin were down-regulated. The intestinal mucosa was impaired. Compared with the model group, berberine inhibited tumor growth in a dose-dependent manner (6.25, 12.5, 25 mg/kg), reduced the permeability of intestinal mucosa, and alleviated intestinal mucosal damage. HPLC showed that berberine decreased the content of polyamines in tumor tissue, whereas increased that in intestinal mucosa tissue. Western blot showed that berberine inhibited the expressions of ODC, C-MYC and HIF-1α, but up-regulated those of OAZ1 and SSAT. In short, berberine may exert antitumor effects by suppressing tumor growth and elevating the intestinal mucosal permeability.

Pharmacokinetics of isochlorgenic acid C in rats by HPLC-MS: Absolute bioavailability and dose proportionality.

ETHNOPHARMACOLOGICAL RELEVANCE: Isochlorgenic acid C (IAC), one of the bioactive compounds of Lonicera japonica, exhibited diverse pharmacological effects. However, its pharmacokinetic properties and bioavailability remained unresolved. AIM OF THE STUDY: To determine the absolute bioavailability in rats and the dose proportionality on the pharmacokinetics of single oral dose of IAC. MATERIALS AND METHODS: A validated HPLC-MS method was developed for the determination of IAC in rat plasma. Plasma concentration versus time data were generated following oral and intravenous dosing. The pharmacokinetic analysis was performed using DAS 3.0 software analysis. Absolute bioavailability in rats was determined by comparing pharmacokinetic data after administration of single oral (5, 10 and 25mgkg(-1)) and intravenous (5mgkg(-1)) doses of IAC. The dose proportionality of AUC(0-∞) and Cmax were analyzed by linear regression. RESULTS: Experimental data showed that absolute oral bioavailability of IAC in rats across the doses ranged between 14.4% and 16.9%. The regression analysis of AUC(0-∞) and Cmax at the three doses (5, 10 and 25mgkg(-1)) indicated that the equations were y=35.23x+117.20 (r=0.998) and y=121.03x+255.74 (r=0.995), respectively. CONCLUSIONS: A new HPLC-MS method was developed to determine the bioavailability and the dose proportionality of IAC. Bioavailability of IAC in rats was poor and both Cmax and AUC(0-∞) of IAC had a positive correlation with dose. Evaluation of the pharmacokinetics of IAC will be useful in assessing concentration-effect relationships for the potential therapeutic applications of IAC.

Absorption mechanism of oxymatrine in cultured Madin-Darby canine kidney cell monolayers.

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin-Darby canine kidney (MDCK) cell monolayers. Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0 × 10(-5)-1.0 × 10(-3) M in 2 h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10(-4) M was studied. Moreover, Papp of OMT was determined in the absence/presence of cyclosporine and surfactants at 100 µM to further confirm the relative transport mechanism. Results The Papp AP→BL ranged from (3.040 ± 0.23) × 10(-6) to (3.697 ± 0.19) × 10(-6 )cm/s as the concentration varied from 10(-5) to 10(-3) M. OMT showed similar Papp at 4 and 37 °C (p > 0.05). Increasing the apical pH 7.4 and 8.0 resulted in Papp versus pH 5.0 (p < 0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, Papp was (0.318 ± 0.033) × 10(-5), (0.464 ± 0.048) × 10(-5), (0.897 ± 0.115) × 10(-5) and (1.341 ± 0.122) × 10(-5 )cm/s, respectively. In the presence of surfactants, Papp significantly increased up to 1.5-4.3-fold (p < 0.05). Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.

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.

Expression and function of GSTA1 in lung cancer cells.

Glutathione S-transferase A1 (GSTA1) appears to be primarily involved in detoxification processes, but possible roles in lung cancer remain unclear. The objective of this study was to investigate the expression and function of GSTA1 in lung cancer cells. Real-time PCR and Western blotting were performed to assess expression in cancer cell lines and the normal lung cells, then verify the A549 cells line with stable overexpression. Localization of GSTA1 proteins was assessed by cytoimmunofluorescence. Three double-strand DNA oligoRNAs (SiRNAs) were synthesized prior to being transfected into A549 cells with Lipofectamine 2000, and then the most efficient SiRNA was selected. Expression of the GSTA1 gene in the transfected cells was determined by real-time PCR and Western blotting. The viability of the transfected cells were assessed by MTT. Results showed that the mRNA and protein expression of A549 cancer cells was higher than in MRC-5 normal cells. Cytoimmunofluorescence demonstrated GSTA1 localization in the cell cytoplasm and/or membranes. Transfection into A549 cells demonstrated that down-regulated expression could inhibit cell viability. Our data indicated that GSTA1 expression may be a target molecule in early diagnosis and treatment of lung cancer.

[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.

[Apoptosis of cultured cortical neurons of rat's brain induced by heroin].

OBJECTIVE: To investigate whether heroin can directly induce apoptosis in primary cultured cortical neurons of rat's brain. METHODS: Cultured primary neurons cultures were obtained from cerebral cortex of embryo rats. After 7 days, the cells were incubated with different concentrations of heroin (purity-80%) for 24 hours. The neuronal survival was assessed by cell viability counting with fluorescent diacetate (FDA) staining. The morphological and biochemical changes were observed with Hoechst 33258 fluorescent staining and then analyzed by agarose gel electrophoresis, respectively. RESULTS: After treatment with different concentrations of heroin, the neurons showed a decreased survival rate in a dose dependent manner, and there was a significant difference in the survival rate between the heroin group and the control group (P < 0.05). When exposed to different concentrations of heroin, neurons exhibited the morphological and biochemical features of apoptosis, including cell shrinkage, neurite degeneration, network disappearance, condensation and aggregation of nuclear chromatin, and the formation of DNA ladders. With the increase of heroin concentration of rat's brain more apoptotic bodies were seen. CONCLUSION: Heroin can directly induce apoptosis in primary cultured cortical neurons in rat's brain.

[Bioinformatics analysis of YZ-2 and preparation of polyclonal antibody].

AIM: To analyze YZ-2, a novel protein associated with cerebral ischemia and prepares its polyclonal antibody. METHODS: According to the bioinformatics analysis and prediction of the possible high structure, hydrophilicity and antigenicity of YZ-2, a 22-amino acid residue partial peptide of YZ-2 was synthesized. The synthesized peptide was then used to immunize. And the properties of anti-YZ2 were analyzed by ELISA and Western blot. RESULTS: The hydrophilicity and antigenicity were predicted by methods of bioinformatics. The polyclonal antibody of YZ-2 was successfully obtained and its specificity and sensitivity were conformed by ELISA and Western blot. CONCLUSION: By the bioinformatics analysis and prediction, the hydrophilicity and antigenicity of YZ2 were analyzed. The antibody of YZ-2 was successfully obtained.