Effects of combined microplastic and cadmium pollution on sorghum growth, Cd accumulation, and rhizosphere microbial functions.

The interaction between microplastics (MPs) and cadmium (Cd) poses a threat to agricultural soil environments, and their effects on plant growth and rhizosphere microbial community functions are not yet clear. In this study, energy sorghum was used as a test plant to investigate the effects of two types of MPs, polystyrene (PS) and polyethylene (PE), at different particle sizes (13 µm, 550 µm) and concentrations (0.1%, 1% w/w), and Cd, as well as their interactions, on the growth of sorghum in a soil-cultivation pot experiment. The results showed that the combined effects of MP and Cd pollution on the dry weight and Cd accumulation rate in sorghum varied depending on the type, concentration, and particle size of the MPs, with an overall trend of increasing stress from combined pollution with increasing Cd content and accumulation. High-throughput sequencing analysis revealed that combined MP and Cd pollution increased bacterial diversity, and the most significant increase was observed in the abundance-based coverage estimator (ACE), Shannon, and Sobs indices in the 13 µm 1% PS+Cd treatment group. Metagenomic analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed that 19 groups of metabolic pathways, including microbial metabolism and methane metabolism, differed significantly under combined MP and Cd pollution. Hierarchical clustering results indicated that Cd treatment and combined MP and Cd treatment affected the abundances of sorghum rhizosphere soil nitrogen (N) and phosphorus (P) cycling genes and that the type of MP present was an important factor affecting N and P cycling genes. The results of this study provide a basis for exploring the toxic effects of combined MP and Cd pollution and for conducting soil environmental risk assessments.

Uncovering the key pharmacodynamic material basis and possible molecular mechanism of extract of Epimedium against liver cancer through a comprehensive investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver cancer is a worldwide malignant tumor, and currently lacks effective treatments. Clinical studies have shown that epimedium (YYH) has therapeutic effects on liver cancer, and some of its prenylflavonoids have demonstrated anti-liver cancer activity through multiple mechanisms. However, there is still a need for systematic research to uncover the key pharmacodynamic material basis and mechanism of YYH. AIM OF THE STUDY: This study aimed to screen the anti-cancer material basis of YYH via integrating spectrum-effect analysis with serum pharmacochemistry, and explore the multi-target mechanisms of YYH against liver cancer by combining network pharmacology with metabolomics. MATERIALS AND METHODS: The anti-cancer effect of the extract of YYH (E-YYH) was first evaluated in mice with xenotransplantation H22 tumor cells burden and cultured hepatic cells. Then, the interaction between E-YYH compounds and the cytotoxic effects was revealed through spectrum-effect relationship analysis. And the cytotoxic effects of screened compounds were verified in hepatic cells. Next, UHPLC-Q-TOF-MS/MS was employed to identify the absorbed components of E-YYH in rat plasma to distinguish anti-cancer components. Subsequently, network pharmacology based on anti-cancer materials and metabolomics were used to discover the potential anti-tumor mechanisms of YYH. Key targets and biomarkers were identified and pathway enrichment analysis was performed. RESULTS: The anti-cancer effect of E-YYH was verified through in vitro and in vivo experiments. Six anti-cancer compounds in plasma (icariin, baohuoside Ⅰ, epimedin C, 2″-O-rhamnosyl icariside Ⅱ, epimedin B and sagittatoside B) were screened out by spectrum-effect analysis. Forty-five liver-cancer-related targets were connected with these compounds. Among these targets, PTGS2, TNF, NOS3 and PPARG were considered to be the potential key targets preliminarily verified by molecular docking. Meanwhile, PI3K/AKT signaling pathway and arachidonic acid metabolism were found to be associated with E-YYH's efficacy in network pharmacology and metabolomics analysis. CONCLUSIONS: Our research revealed the characteristics of multi-component, multi-target and multi-pathway mechanism of E-YYH. This study also provided an experimental basis and scientific evidence for the clinical application and rational development of YYH.

Long noncoding RNA SPRY4-IT1 acts as a miR-101-5p sponge to promote gastrointestinal stromal tumor progression by inhibiting ZEB1.

OBJECTIVES: Research on long noncoding RNAs (lncRNAs) has been conducted in different areas of oncology. Currently, the biological significance of lncRNAs and their regulatory features in gastrointestinal stromal tumors (GIST) remain largely unknown. We have previously identified SPRY4-IT1 overexpression in GIST through lncRNA sequencing of GIST tissues. Coincidentally, SPRY4-IT1 is an intron of the SPRY4 gene, and SPRY4 is specifically highly expressed in GIST. Thus the aim of the present study was to investigate the role of lncRNA SPRY4-IT1 in GIST pathogenesis. METHODS: Herein, we screened for SPRY4-IT1 and analyzed its possible phenotypes using Gene set enrichment analysis (GSEA). The phenotypes of GIST were verified using CCK-8, colony formation, and wound-healing assays. The ceRNA mechanism was determined by the location of lncRNA SPRY4-IT1, and its relationship to the Ago2 protein. The SPRY4-IT1/miR-101-5p/ZEB1 axis was predicted using online software and sequencing. Luciferase and pull-down assays were performed for verification. Pathway-associated and phenotype-associated proteins were detected by western blotting. RESULTS: Sequencing analysis revealed 117 differentially expressed lncRNAs in GIST and normal gastric tissue samples. Accordingly, SPRY4-IT1 was screened out and its phenotype was predicted by GSEA. Mechanistically, SPRY4-IT1 was identified as a competing endogenous RNA (ceRNA) that downregulated miR-101-5p and upregulated ZEB1, which activated extracellular signal-regulated kinase (ERK) signaling to stimulate GIST proliferation, invasion, and epithelial-mesenchymal transition. Although this effect was regulated by a negative feedback loop through SPRY4, it was still controlled by SPRY4-IT1. CONCLUSIONS: In GIST, we revealed a ceRNA mechanism by which SPRY4-IT1 modulates ZEB1 by sponging miR-101-5p, eventually driving tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT).

Enhancing Mechanisms of the Plant Growth-Promoting Bacterial Strain Brevibacillus sp. SR-9 on Cadmium Enrichment in Sweet Sorghum by Metagenomic and Transcriptomic Analysis.

To explore the mechanism by which the plant growth-promoting bacterium Brevibacillus sp. SR-9 improves sweet sorghum tolerance and enriches soil cadmium (Cd) under pot conditions, the effect of strain SR-9 inoculation on the microbial community of sorghum rhizosphere soil was analyzed by metagenomics. Gene expression in sweet sorghum roots was analyzed using transcriptomics. The results showed that strain SR-9 promoted the growth of sweet sorghum and improved the absorption and enrichment of Cd in the plants. Compared with the uninoculated treatment, the aboveground part and root dry weight in strain SR-9 inoculated with sorghum increased by 21.09% and 17.37%, respectively, and the accumulation of Cd increased by 135% and 53.41%, respectively. High-throughput sequencing showed that strain SR-9 inoculation altered the rhizosphere bacterial community, significantly increasing the relative abundance of Actinobacteria and Firmicutes. Metagenomic analysis showed that after inoculation with strain SR-9, the abundance of genes involved in amino acid transport metabolism, energy generation and conversion, and carbohydrate transport metabolism increased. KEGG functional classification showed that inoculation with strain SR-9 increased the abundance of genes involved in soil microbial metabolic pathways in the rhizosphere soil of sweet sorghum and the activity of soil bacteria. Transcriptome analysis identified 198 upregulated differentially expressed genes in sweet sorghum inoculated with strain SR-9, including those involved in genetic information processing, biological system, metabolism, environmental information processing, cellular process, and human disease. Most of the annotated differentially expressed genes were enriched in the metabolic category and were related to pathways such as signal transduction, carbohydrate metabolism, amino acid metabolism, and biosynthesis of other secondary metabolites. This study showed that plant growth-promoting bacteria can alter the rhizosphere bacterial community composition, increasing the activity of soil bacteria and upregulating gene expression in sweet sorghum roots. The findings enhance our understanding of the microbiological and botanical mechanisms by which plant growth-promoting bacterial inoculation improves the remediation of heavy metals by sorghum.

[High-Throughput Sequencing Combined with Metabonomics to Analyze the Effect of Heavy Metal Contamination on Farmland Soil Microbial Community and Function].

Heavy metal contamination affects microbial composition and diversity. The interaction between heavy metal contamination and soil microorganisms has been a hot topic in ecological research. Battery manufacturing has been going on for over six decades in Xinxiang City, resulting in severe soil heavy metal contamination due to battery wastewater runoff. Few studies have investigated the effect of heavy metal contamination due to long-term battery wastewater runoff on microbial diversity and metabolomics in Xinxiang City. In this study, we collected samples from three heavy metal contaminated sites in Xinxiang City and found that Cd and Pb exceeded the recommended thresholds by 34-66 fold and 1.5-2.32 fold, respectively. High-throughput sequencing showed that Bacillus, Arthrobacter, Sphingomonas, and Streptomyces were the dominant bacteria genera, while Olpidium, Plectosphaerella, and Gibellulopsis were the dominant fungi genera, indicating that heavy metal contaminated soil in Xinxiang City was rich in heavy metal tolerant bacteria and fungi due to the long-term heavy metal stress. Correlation analysis showed that total Cu, DTPA extract Cu, and water soluble Pb were significant factors in bacterial diversity, while total Cd, total Ni, total Pb, total Zn, DTPA extract Cu, and water soluble Pb were significant factors in fungal diversity. To better understand the effect of heavy metal contamination on the metabolism of soil microorganisms, we conducted non-targeted metabolomic profiling, which showed significant differences in metabolites across the samples. Pathway enrichment analysis showed that these differential metabolites were involved in pathways such as metabolism, environmental information processing, and genetic Information Processing, which may play a role in heavy metal stress mitigation and environmental adaptation.

Accuracy of segmented measurement of axial length in ultra-high myopia filled with silicone oil using immersion B-scan ultrasonography.

AIM: To evaluate the accuracy of segmented measurement of axial length (AL) in high myopia filled with silicone oil by immersion B-scan ultrasonography (immersion B-scan). METHODS: From June 2016 to June 2020, a total of 67 ultra-high myopia inpatients (67 eyes) who underwent silicone oil removal combined with cataract extraction and intraocular lens (IOL) implantation were retrospectively enrolled. The preoperative axial length (AL) of 31 patients with severe cataract were segmented measured using immersion B-scan (B-scan group) and another 36 patients with mild or moderate cataract were measured using IOLMaster 500 (IOLMaster group). The post-operative ALs in two groups were both measured using IOLMaster 500. The IOL power was calculated with Haigis formula. The differences in ALs between pre- and post-surgery, as well as the postoperative refractive spherical equivalent, absolute refractive error, the prediction deviation of postoperative refraction and best corrected visual acuity (BCVA) were compared. RESULTS: The pre- and post-operative ALs were 30.46±1.63 mm (range 28.09-33.51 mm) and 30.42±1.70 mm (range 28.03-33.90 mm) in B-scan group (t=0.644, P=0.542) and 30.51±1.21 mm (range 28.03-33.90 mm) and 30.43±1.27mm (range 28.54-33.50 mm) in IOLMaster group (t=1.843, P=0.074), respectively. Three months after surgery, BCVA were 0.45±0.13 (range 0.3-0.9) and 0.44±0.20 (range 0.2-1.0) in B-scan and IOLMaster group respectively (t=0.086, P=0.932). There was no significant difference of the postoperative spherical equivalent (-3.11±0.65 D vs -3.21±0.51 D, t=0.671, P=0.505) and the absolute refractive error (0.589±0.340 vs 0.470±0.245 D, t=1.615, P=0.112) between two groups. In B-scan group, absolute refractive error within ±0.50 D was found in 18 eyes (58.1%), within ±1.00 D in 26 eyes (83.9%), and within ±1.50 D in 31 eyes (100%). In IOLMaster group, absolute refractive error within ±0.50 D was found in 23 eyes (63.9%), within ±1.00 D in 34 eyes (94.4%), and within ±1.50 D in 36 eyes (Z=0.757, P=0.449). CONCLUSION: The segmented measurement of ALs by immersion B-scan shows comparable measurement accuracy with that of IOLMaster 500 in ultra-high myopia patients with severe cataract secondary to silicone oil filling and can obtain an ideal postoperative refractive state.

Red Nucleus Interleukin-6 Evokes Tactile Allodynia in Male Rats Through Modulating Spinal Pro-inflammatory and Anti-inflammatory Cytokines.

Our previous studies have clarified that red nucleus (RN) interleukin (IL)-6 is involved in the maintenance of neuropathic pain and produces a facilitatory effect by activating JAK2/STAT3 and ERK pathways. In this study, we further explored the immune molecular mechanisms of rubral IL-6-mediated descending facilitation at the spinal cord level. IL-6-evoked tactile allodynia was established by injecting recombinant IL-6 into the unilateral RN of naive male rats. Following intrarubral administration of IL-6, obvious tactile allodynia was evoked in the contralateral hindpaw of rats. Meanwhile, the expressions of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1ß, and IL-6 were elevated in the contralateral spinal dorsal horn (L4-L6), blocking spinal TNF-α, IL-1ß, or IL-6 with neutralizing antibodies relieved IL-6-evoked tactile allodynia. Conversely, the levels of anti-inflammatory cytokines transforming growth factor-ß (TGF-ß) and IL-10 were reduced in the contralateral spinal dorsal horn (L4-L6), an intrathecal supplement of exogenous TGF-ß, or IL-10 attenuated IL-6-evoked tactile allodynia. Further studies demonstrated that intrarubral pretreatment with JAK2/STAT3 inhibitor AG490 suppressed the elevations of spinal TNF-α, IL-1ß, and IL-6 and promoted the expressions of TGF-ß and IL-10 in IL-6-evoked tactile allodynia rats. However, intrarubral pretreatment with ERK inhibitor PD98059 only restrained the increase in spinal TNF-α and enhanced the expression of spinal IL-10. These findings imply that rubral IL-6 plays descending facilitation and produces algesic effect through upregulating the expressions of spinal pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 and downregulating the expressions of spinal anti-inflammatory cytokines TGF-ß and IL-10 by activating JAK2/STAT3 and/or ERK pathways, which provides potential therapeutic targets for the treatment of pathological pain.

An unnatural tripeptide structure containing intramolecular double H-bonds mimics a turn hairpin conformation.

A series of unnatural tripeptides, each consisting of two aromatic γ-amino acid residues and an ϖ-amino acid residue, are designed to probe their folding into hairpin conformations. The ϖ-amino acid residues, with aliphatic or aromatic spacers of different sizes, serve as the loop of the hairpins. Studies based on one-dimensional (1D) 1H NMR performed at different concentrations, solvent polarity, and temperature, along with 2D-NMR studies, demonstrated that the doubly H-bonded aromatic γ-amino acid residues play important roles in driving these tripeptides into the hairpin conformation. The loop based on 5-aminovaleric acid, which offers a four-carbon (CH2)4 spacer, enhanced the stability of the corresponding hairpin, while loops having a shorter, a longer and a more rigid spacer disfavored the formation of the hairpins. Results from computational studies are in good agreement with the experimental observations. Furthermore, the crystal structure of peptide 1b revealed the expected hairpin conformation in the solid state. This turn motif, which contains H-bonded aromatic γ-amino acid residues as the core unit and an ϖ-amino acid residue serving as the loop, provides a new platform that can be used to obtain a variety of turn conformations by incorporating diverse amino acids into the loops.

Iron Nitride Nanopowders Prepared by High-Pressure Gas-Solid Reaction.

Fe4N nanopowders were prepared using specially-made high-pressure gas-solid reaction equipment, and their composition, morphology, and magnetic properties were analyzed by X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). An average particle size of 35 nm was obtained at 0.4 MPa, an ammonia-to-hydrogen ratio of 3:1, 623 K, in an ammonia solution for 2 h. The hysteresis loop displayed a thin and narrow shape at 673 K during VSM tests. The saturation magnetization (Ms) reached 169.80 emu/g.

[Determination of heavy metal contents in Gardeniae Fructus and fried Gardeniae Fructus and their health risk assessment].

In this study, the contents of Cu, As, Cd, Pb and Hg in 10 batches of Gardeniae Fructus and 10 batches of fried Gardeniae Fructus from Fuzhou in Jiangxi were determined by inductively coupled plasma mass spectrometry(ICP-MS), and the target hazard coefficient(THQ) for different drug users(adults and children) was calculated by using the international health risk assessment model. According to the ISO and green industry standard, the content of Hg in 4 batches of Gardeniae Fructus exceeded the standard with an over-standard rate of 40%. The THQ and total THQ of Hg in 2 batches of Gardeniae Fructus were higher than the international standard limit of Gardeniae Fructus. For 10 batches of fried Gardeniae Fructus, the content of every heavy metal and total amount of five heavy metals did not exceed the standard. However, the THQ and total THQ of Hg in 1 batch of fried Gardeniae Fructus were higher than the international standard limit of Gardeniae Fructus. As compared with Gardeniae Fructus, the contents of Cu, Pb and Hg in fried Gardeniae Fructus decreased by 34.0%, 77.6% and 23.1%; the THQ of Cu, Pb and Hg for adults decreased by 33.3%, 75.0% and 96.9%; and the THQ of Cu, Pb and Hg for children decreased by 37.5%, 75.0%, 90.7%. It showed that the contents of heavy metals in individual batches of Gardeniae Fructus in this experiment had a certain risk to human health, but the contents of these heavy metals in fried Gardeniae Fructus had no obvious effect on human health. This study provided experimental basis and research ideas for safety evaluation of Gardeniae Fructus and fried Gardeniae Fructus.

Network Analyses of the Differential Expression of Heat Shock Proteins in Glioma.

Heat shock protein (HSP) is a family of highly conserved protein, which exists widely in various organisms and has a variety of important physiological functions. Currently, there is no systematic analysis of HSPs in human glioma. The aim of this study was to investigate the characteristics of HSPs through constructing protein-protein interaction network (PPIN) considering the expression level of HSPs in glioma. After the identification of the differentially expressed HSPs in glioma tissues, a specific PPIN was constructed and found that there were many interactions between the differentially expressed HSPs in glioma. Subcellular localization analysis shows that HSPs and their interacting proteins distribute from the cell membrane to the nucleus in a multilayer structure. By functional enrichment analysis, gene ontology analysis, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, the potential function of HSPs and two meaningful enrichment pathways was revealed. In addition, nine HSPs (DNAJA4, DNAJC6, DNAJC12, HSPA6, HSP90B1, DNAJB1, DNAJB6, DNAJC10, and SERPINH1) are prognostic markers for human brain glioma. These analyses provide a full view of HSPs about their expression, biological process, as well as clinical significance in glioma.

[Differences in intestinal absorption of Gegen Qinlian Decoction between normal rats and rats with large intestinal damp-heat syndrome].

The study aimed to compare the difference in intestinal absorption of the components of Gegen Qinlian Decoction between normal rats and those with large intestinal damp-heat syndrome in the pathological state, in order to explore the rational application of Gegen Qinlian Decoction in the treatment of large intestinal damp-heat syndrome. Puerarin, daidzin, liquiritin, scutellarin, baicalin, wogonoside, coptisine, jatrorrhizine, berberine and palmatine were used as the detection indexes in the in vitro everted gut sacs absorption experiment. The cumulative absorption amount(Q/µg) and the absorption rate(K_a) of each component in each intestine segment were calculated and compared. It was found that the absorption of each component in different intestinal segments were linear absorption, with R~2 greater than 0.9, which conformed to the zero-order absorption rate. There were differences between normal rats and model rats in the absorption of the components in Gegen Qinlian Decoction with the same concentration. Intestinal absorption of most components of Gegen Qinlian Decoction in the model of large intestinal damp-heat syndrome increased to some extent. The components of Gegen Qinlian Decoction with the concentration of 200 g·L~(-1) had the highest absorption in the jejunum of the model rats, and the absorption in the ileum, duodenum and colon successively decreased except daidzin and baicalin. In terms of the absorption rate constant, the absorption in the duodenum and jejunum were significantly increased(P<0.01) compared with normal rats, and the absorption in the ileum was significantly decreased(P<0.01) compared with normal rats. In addition, the absorption of puerarin, daidzin, glycyrrhizin, coptisine and berberine increased selectivity in the colon. Therefore, pathological model animals were recommended in the study of the components relating to absorption effect, in order to really lay a research foundation for the symptomatic treatment of large intestinal damp-heat syndrome.

[Feasibility study of QAMS for quantitative analysis of multiple structural types of ingredients in Atractylodis Rhizome by GC].

In this study， quantitative analysis of multi-components with single marker(QAMS) was established and validated to simultaneously determine four sesquiterpenoids(ß-eudesmol， atractylon， atractylolideⅠ， atractylolide Ⅱ) in Atractylodis Rhizome based on the gas chromatographic method(GC). Using ß-eudesmol as the contrast， the relative correctionfactors(RCF) of the other three sesquiterpenoids were determined by GC. Within the line arranges，the values of RCF of ß-eudesmol to atractylon， atractylolideⅠand atractylolide Ⅱ were 0.823， 0.690 and 0.766， respectively. The RCF had a good reproducibility in various instruments， chromatographic columns. According to their RCF， we simultaneously determined four sesquiterpenoids in Atractylodis Rhizome only using one marker. The results of QAMS method were validated by comparing with that of internal standard method， and no obvious significant difference was found.

Near-infrared light-triggered theranostics for tumor-specific enhanced multimodal imaging and photothermal therapy.

The major challenge in current clinic contrast agents (CAs) and chemotherapy is the poor tumor selectivity and response. Based on the self-quench property of IR820 at high concentrations, and different contrast effect ability of Gd-DOTA between inner and outer of liposome, we developed "bomb-like" light-triggered CAs (LTCAs) for enhanced CT/MRI/FI multimodal imaging, which can improve the signal-to-noise ratio of tumor tissue specifically. IR820, Iohexol and Gd-chelates were firstly encapsulated into the thermal-sensitive nanocarrier with a high concentration. This will result in protection and fluorescence quenching. Then, the release of CAs was triggered by near-infrared (NIR) light laser irradiation, which will lead to fluorescence and MRI activation and enable imaging of inflammation. In vitro and in vivo experiments demonstrated that LTCAs with 808 nm laser irradiation have shorter T1 relaxation time in MRI and stronger intensity in FI compared to those without irradiation. Additionally, due to the high photothermal conversion efficiency of IR820, the injection of LTCAs was demonstrated to completely inhibit C6 tumor growth in nude mice up to 17 days after NIR laser irradiation. The results indicate that the LTCAs can serve as a promising platform for NIR-activated multimodal imaging and photothermal therapy.

MRI-guided tumor chemo-photodynamic therapy with Gd/Pt bifunctionalized porphyrin.

Porphyrin derivatives have been widely applied in MR imaging and photodynamic cancer therapy. We here report a novel Gd/Pt bifunctionalized porphyrin derivative (Gd/Pt-P1) for MRI-guided chemo-photodynamic cancer therapy. Gd/Pt-P1 was prepared from tetra(4-pyridyl) porphyrin (P1) via step by step coordination to cisplatin and gadolinium (Gd(iii)). Gd/Pt-P1 showed a particularly high synergetic chemo-photodynamic antitumor effect in vivo with a tumor inhibition rate (TIR) of 96.6% and excellent MR imaging performance.

Enhanced degradation of azo dye by a stacked microbial fuel cell-biofilm electrode reactor coupled system.

In this study, a microbial fuel cell (MFC)-biofilm electrode reactor (BER) coupled system was established for degradation of the azo dye Reactive Brilliant Red X-3B. In this system, electrical energy generated by the MFC degrades the azo dye in the BER without the need for an external power supply, and the effluent from the BER was used as the inflow for the MFC, with further degradation. The results indicated that the X-3B removal efficiency was 29.87% higher using this coupled system than in a control group. Moreover, a method was developed to prevent voltage reversal in stacked MFCs. Current was the key factor influencing removal efficiency in the BER. The X-3B degradation pathway and the types and transfer processes of intermediate products were further explored in our system coupled with gas chromatography-mass spectrometry.

Effects of caulophine on caffeine-induced cellular injury and calcium homeostasis in rat cardiomyocytes.

Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine-induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre-treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine-pre-treated cardiomyocytes were incubated with Fluo-3/AM, and then caffeine or KCl was used to induce Ca(2+) overload. The total intracellular Ca(2+) concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine-injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca(2+) concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl-induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine-induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.

[Oral acute toxicity of (+)-usnic acid in mice and its cytotoxicity in rat cardiac fibroblasts].

OBJECTIVE: To observe the oral acute toxicity of of (+)-usnic acid in mice and assess its cytotoxicity in rat cardiac fibroblasts. METHODS: The mice with acute poisoning of (+)-usnic acid at different doses by oral administration were observed for toxic manifestations, and the LD(50) was determined. The survival time and survival rate of the mice receiving different doses of (+)-usnic acid were observed. Cultured rat cardiac fibroblasts were inoculated with different concentrations of (+)-usnic acid, and the cell growth inhibition rate was estimated and the IC(50) determined using MTT assay. RESULTS: Higher dose of (+)-usnic acid resulted in more obvious symptoms of poisoning and shorter survival time of the mice. The LD(50) of (+)-usnic acid in mice by oral administration was 388 mg/kg. The manifestations of poisoning such as apathism, pilomotor, chill, dyspnea, torpidity and anorexia was observed. Rat cardiac fibroblasts incubated with (+)-usnic acid showed obvious growth inhibition, which was positively correlated to the dose of (+)-usnic acid, and high dose of (+)-usnic acid caused severe cell injuries. The IC(50) of (+)-usnic acid in rat cardiac fibroblasts was 322 microg/ml. CONCLUSION: (+)-usnic acid is a natural compound of low toxicity in mice, and low to medium dose of (+)-usnic acid dose not produce obvious cytotoxicity.

[In vitro effect of (+)-usnic acid on Toxoplasma gondii tachyzoites].

OBJECTIVE: To explore the effect of usnic acid on Toxoplasma gondii tachyzoites in vitro. METHODS: There are four groups named as (+)-usnic acid group, acetylspiramycin group, DMSO group and normal saline group. Groups of (+)-usnic acid and acetylspiramycin were further divided into 4 subgroups with final concentration of 5, 10, 25, 50 microg/ml respectively. Normal saline group and DMSO group were respectively given equal volume normal saline and 1% DMSO. Each group have 15 parallel tubes with 1 ml (1 x 10(6)/ml) T. gondii tachyzoites aqueous suspension. At 1 h, 2h and 4 h after drug treatment, tachyzoites were counted by light microscope with 0.4% Trypan blue staining. Tachyzoites in aqueous suspension was collected, and washed 3 times by PBS solution. Normal mice were inoculated intraperitoneally and observed for three generations. The cultivated rat cardiofibroblasts were then infected in vitro with T. gondii tachyzoites. At the same time, rat cardiomyocytes invasion by T. gondii tachyzoites was investigated. RESULTS: At 4 h treated by 10, 25 and 50 microg/ml (+)-usnic acid, 100% T. gondii tachyzoites were stained. Some tachyzoites were swelling, blunt or round in the two ends; and granules appeared in the cytoplasm, the nuclei were deep stained. The changes of tachyzoites in acetylspiramycin group were similar to (+)-usnic acid group, 100% T. gondii tachyzoites were stained in 50 microg/ml acetylspiramycin subgroup. In inoculation tests, mice died at 8th to 9th days in 5 microg/ml (+)-usnic acid subgroup and numerous tachyzoites were detected in ascites. However, most mice survived to be killed in the other (+)-usnic acid subgroups and the tachyzoites were not found in ascites. All mice in acetylespirmycin groups died at 6th to 8th days after inoculation and many tachyzoites or pseudocysts were observed in mice ascites. In infecting cell tests, the cultivated rat cardiofibroblasts were infected in vitro by the tachyzoites after treated with 5 microg/ml (+)-usnic acid for 4 h, and pseudocysts were formed in infected cells. It was negative in the other subgroups of (+)-usnic acid. But the cultivated rat cardiofibroblasts were infected to varying degree in acetylspiramycin groups, normal saline group and DMSO group. CONCLUSION: (+)-Usnic acid has a remarkable effect on T. gondii tachyzoites.