Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target.

Trichophyton rubrum causes ringworm worldwide. Citral (CIT), extracted from Pectis plants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial against T. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism against T. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p < 0.01 or 0.05), higher cellular leakage rates (p < 0.01), increased conidia with damaged membranes (p < 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3ß-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral against T. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis.

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10-2 and 3.7 ×10-2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria.

Neuroprotective Effects of Kinetin Against Glutamate-Induced Oxidative Cytotoxicity in HT22 Cells: Involvement of Nrf2 and Heme Oxygenase-1.

Oxidative stress is considered as one of key factors related to Alzheimer's disease (AD), while kinetin (KT) has been reported to exert anti-oxidative activities as well as neuroprotective effects both in vivo and in vitro. Thus, in this study, the neuroprotective effects of KT against glutamate-induced oxidative toxicity in HT22 cells were investigated. To evaluate the anti-oxidative capabilities of KT itself, several anti-oxidative assays in vitro were conducted. To evaluate the neuroprotective effects of KT, the levels of intracellular reactive oxygen species (ROS) and calcium influx, mitochondrial membrane potential (MMP), and cell death were measured by flow cytometry. Nuclear translocation of apoptosis inducing factor (AIF) and content of intracellular ATP were also determined. In addition, the phosphorylation levels of apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (p38) were evaluated as well. Besides, nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) were also examined to reveal underlying mechanisms. Results showed that KT rescued cell death, and suppressed the accumulation of intracellular ROS and the increase of intracellular calcium influx. In addition, KT maintained normal function of mitochondria and inhibited the phosphorylation of ASK-1, JNK, and p38. KT also promoted nuclear translocation of Nrf2 and enhanced the expression of HO-1 both at protein and mRNA level. Importantly, blockage of Nrf2 almost completely abolished the neuroprotective effects of KT, while blockage of HO-1 expression partly neutralized its neuroprotective effects. Our results indicated that KT can protect HT22 cells from glutamate-induced cell death by activating Nrf2 pathway and inducing expression of HO-1, suggesting KT might be a drug candidate for treatment of AD and other neurodegenerative disorders related to oxidative stress.

Protective effects of kinetin against aluminum chloride and D-galactose induced cognitive impairment and oxidative damage in mouse.

Increasing evidence indicates that aluminum exposure and oxidative stress play crucial roles in the initiation and development of Alzheimer's disease (AD). Aluminum chloride (AlCl3) and d-galactose (d-gal) combined treatment of mice is considered as an easy and cheap way to obtain an animal model of AD. Kinetin is a plant cytokinin, which is also reported to exert neuro-protective effects in vivo and in vitro. Thus, in this study, neuro-protective effects of kinetin were investigated in an AD model of mice induced by AlCl3 and d-gal. The Morris water maze (MWM) test was performed to directly evaluate neuro-protective effects of kinetin on the memory and spatial learning abilities, while the histopathological changes were examined by hematoxylin and eosin (H & E) staining method. To further investigate mechanisms involved, Al content in cortex and hippocampus was determined. In addition, related detection kits were used to determine acetylcholine (ACh) content and activity of acetylcholinesterase (AChE). Activities of anti-oxidative enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and the content of heme oxygenase-1 (HO-1) were also measured. Besides, the content of oxidative damage bio-markers including 8-iso-prostaglandin F (8-iso-PGF), advanced glycation end products (AGEs) and 8-hydroxy-2-deoxyguanosine (8-OHdG) were determined by ELISA kits. Finally, the distribution of beta-amyloid protein 1-42 (Aß1-42) was detected by immunohistochemistry (IHC), while the expression levels of amyloidogenic proteins including ß-amyloid precursor protein (APP), ß-secretase, γ-secretase and Aß1-42 were detected by western blotting (WB) method. Results showed that kinetin improved performance in MWM test, attenuated histopathological changes, reduced Al level in cortex and hippocampus, increased ACh content and decreased AChE activity. In addition, kinetin elevated activities of anti-oxidative enzymes and reduced the levels of oxidative damage biomarkers in AD model of mice. Furthermore, kinetin also increased the content of HO-1, and inhibited the distribution of Aß1-42 and the expressions of amyloidogenic proteins (APP, ß-secretase, γ-secretase and Aß1-42) in brain tissue of AD mice. Our results indicate that kinetin has neuro-protective effects on the AD model of mice induced by AlCl3 and d-gal, suggesting that kinetin may be a candidate drug for treatment of AD.

Effects of Carbopol® 934 proportion on nanoemulsion gel for topical and transdermal drug delivery: a skin permeation study.

Nanoemulsions (NEs) are used as transdermal drug delivery systems for systematic therapeutic purposes. We hypothesized that the skin permeation profile of an NE could be modulated by incorporating it into a hydrogel containing differing proportions of thickening agent. The objectives of this study were as follows: 1) to determine the stability and skin irritability of NE gels (NGs) containing 1%, 2%, and 3% (w/w) Carbopol® 934 (CP934) (termed NG1, NG2, and NG3, respectively); 2) to compare the skin permeation profiles and drug deposition patterns of the NGs; and 3) to visualize the drug delivery routes of the NGs. Terbinafine and citral were incorporated into the NGs as model drugs. Ex vivo skin permeation tests indicated that the percutaneous flux rates of terbinafine decreased in the order NE (215 µg/cm2) > NG1 (213 µg/cm2) > NG2 (123 µg/cm2) > NG3 (74.3 µg/cm2). The flux rates of citral decreased in the order NE (1,026 µg/cm2) > NG1 (1,021 µg/cm2) > NG2 (541 µg/cm2) > NG3 (353 µg/cm2). The NGs accumulated greater amounts of the drugs in the stratum corneum and less in the epidermis/dermis than did the NE (P<0.05) over a period of 12 h. Laser scanning confocal microscopy indicated that the NGs altered the main drug delivery routes from skin appendages to intercellular paths. Histological images suggested that perturbations to the skin structure, specifically the size of the epidermal intercellular spaces and the separation distance of dermal collagen bundles, could be significantly minimized by increasing the proportion of CP934. These results suggest that adjustments of the CP934 proportions can be used to modulate the skin permeation profiles of NGs for specific therapeutic purposes.

[Effect of kinetin on immunity and splenic lymphocyte proliferation in vitro in D-galactose-induced aging rats].

The purpose of this paper is to study the effect of kinetin (Kn) on immunity and splenic lymphocyte proliferation in vitro of aging rats induced by D-galactose (D-gal). Fifty SD rats were randomly divided into five groups: control group, aging model group, Kn low dose group, Kn middle dose group and Kn high dose group. The aging model group was proposed by napes subcutaneous injection of D-gal (125 mg/kg) for 45 d, and anti-aging groups were intragastrically administered with 5, 10, 20 mg/kg of Kn respectively from day 11. IgG, IgA, IgM contents of serum, the apoptosis percentage, stimulation index (SI) and proliferation index (PI) of splenic lymphocyte in vitro were evaluated. The results showed that the apoptosis percentage of splenic lymphocyte in aging model rats was higher, the serum IgG, IgA and IgM contents, SI and PI were lower than control group. Kn significantly decreased the apoptosis percentage of splenic lymphocyte, while increased the serum IgG, IgA and IgM contents, SI and PI in aging model group. These results suggest that Kn could inhibit the apoptosis, while promote the proliferation of splenic lymphocyte, and then effectively enhance the immune power of the aging rats and slow down the aging process.

Kinetin inhibits apoptosis of aging spleen cells induced by D-galactose in rats.

Kinetin (Kn) is a cytokinin growth factor that exerts several anti-aging and antioxidant effects on cells and organs. To investigate the mechanism underlying apoptotic events in aging cells induced by D-galactose (D-gal), we examined the effect of Kn delivered via nuchal subcutaneous injection on D-gal-induced aging and apoptosis in rats. Our results showed that interleukin (IL)-2 levels and mitochondrial membrane potential (ΔΨm) were decreased by Kn in aging rats while IL-6 production and apoptosis increased. In addition, the expression of anti-apoptotic Bcl-2 was low while that of Bax was high in the aging group. After treated with Kn, compared with aging group, there showed obvious difference in Kn group with elevated IL-2, proliferation index, Bcl-2, ΔΨm and decreased IL-6 and Bax in splenic lymphocyte. Based on these results, we concluded that Kn can effectively protect the rat spleen from aging, apoptosis, and atrophy.

[Effect of kinetin on ovary and uterus in D-galactose-induced female mouse model of aging].

The present study was to investigate the effect of kinetin on ovary and uterus of D-galactose-induced female mouse model of aging. Aging female mice model caused by D-galactose were used as model group, the aging model mice intragastrically administered with kinetin solution (daily 25 mg/kg or 50 mg/kg) were used as kinetin groups, and the mice with solvent as normal group (n = 20). To detect the effects of kinetin, estrous cycle, estradiol content, ovarian and uterine wet weight and organ index, SOD and GSH-Px activities, MDA and total protein contents, as well as the reserve function of ovaries were examined. The results showed that, kinetin-induced changes in two kinetin groups were observed, compared with the model group: (1) the estrous cycle was shortened; (2) serum estradiol content was significantly increased; (3) the wet weights of the ovary and uterus were increased significantly; (4) SOD and GSH-Px activities of ovary and uterus were significantly higher; (5) the MDA contents of the ovary and uterus were reduced significantly; (6) total protein contents of the ovary and uterus were increased significantly; (7) the numbers of mature oocytes in fallopian tubes were increased significantly. The results show that kinetin can protect ovary and uterus against oxidative damage, prevent low estrogen secretion caused by ovarian oxidative damage, shorten the estrous cycle in mice, and eventually maintain ovarian and uterine vitalities.

A combination of a microemulsion and a phospholipid complex for topical delivery of oxymatrine.

Oxymatrine (OMT), a water-soluble drug, has a very low oral bioavailability because of its low membrane permeability and its biotransformation in the gastrointestinal tract. Formulated as an oxymatrine-phospholipid complex (OMT-PLC) can improve the lipid solubility and effectiveness of OMT. The purpose of this study was to explore the utility of the combination of a microemulsion and an OMT-PLC as a topical delivery vehicle for enhancing the absorption and efficacy of OMT. The solubility of OMT-PLC was determined and phase diagrams of microemulsions were constructed. Various microemulsion formulations were developed and characterized by their physicochemical properties, and their in vitro and in vivo permeability through skin. An optimal microemulsion (ME4), which presented as spherical droplets and consisted of 10.0% OMT-PLC, 8.0% isopropyl myristate, 30.0% Cremophor RH40/polyethylene glycol 400 (1:1) and 52.0% water, was selected. It possessed an average droplet size of 32.4 nm, a low viscosity of 113.7 mPa · s, and a high cloud point of 88°C. Compared to the control solution, ME4 provided better skin permeability in vitro and a higher retention ratio of OMT in skin in vivo. Moreover, ME4 significantly enhanced the antiproliferative activity of OMT on scar fibroblasts. These results indicate that the combination of a microemulsion and a phospholipid complex represents an effective vehicle for topical delivery of OMT.

[Preparation of O/W ginseng saponins-based nanoemulsion and its amplified immune response].

OBJECTIVE: To prepare an O/W ginseng saponins-based nanoemulsion and investigate its amplified immune response. METHOD: The formulation of ginseng saponins-based nanoemulsion was optimized via the range of nanoemulsion zone in phase diagrams and the solubility of ginseng saponins. Its physicochemical properties were investigated, including morphology, particle size distribution, pH, viscosity and stability. Ginseng saponins-based nanoemulsion as adjuvant was co-administrated with a model antigen ovalbumin (OVA) in mice. Two weeks after the boosting, the serum levels of OVA-specific antibody and its isotypes were determined. RESULT: The optimized ginseng saponins-based nanoemulsion formulation consisted of ginseng saponins, IPM, Cremophor RH 40, glycerol and water (with the weight ratio of 2 : 4 : 17.8 : 17.8 : 58.4), which was a light yellow fluid. The shape of droplets was spherical under transmission electron microscopy with an average diameter of 72.20 nm and a polydispersity index of 0.052. The viscosity and pH value of it were 4.20 s and 6.02, respectively. And it showed good stability. When co-administered with OVA, no obvious side effects were observed in the mice immunized with ginseng saponin-based nanoemulsion. The serum levels of IgG, IgG1 and IgG2a antibody in the group of ginseng saponin-based nanoemulsion immunized mice was significantly increased compared to the groups of OVA and the saline solution of ginseng saponin. Compared with the adjuvant aluminium hydroxide, the serum levels of IgG and IgG1 antibodys in the groups of ginseng saponins-based nanoemulsion had no significant difference, but the level of IgG2a was obviously higher. CONCLUSION: ginseng saponin-based nanoemulsion could amplify the Th1 and Th2 immune responses, and can be used as the vaccine adjuvant.

[Effects of vinblastine nanoparticles on growth and apoptosis of glioma cell line C6].

OBJECTIVE: To compare effects of vinblastine (VLB) nanoparticles (NPS) and VLB physiologic saline solution on inhibiting glioma cell lines C6 growth and inducting its apoptosis. METHOD: Glioma cell lines C6 were respectively treated with 500 micro x L(-1) VLB NPS and VLB physiologic saline solution for 7 days. Amount of cells were counted by blood cell counting chamber. Glioma C6 growth curve was draw according to cells amount. Clone formation rate of glioma C6 was detected after 500 microg x L(-1) VLB NPS and VLB physiologic saline solution incubation for 2 weeks. In addition, the whole morphology of glioma C6 were observed by inverted microscope and inverted fluorescence microscope after 500 microg x L(-1) VLB NPS and VLB physiologic saline solution incubation for 48 h. RESULT: Entrapment of VLB in NPS may significantly inhibit glioma cells C6 growth from 2 to 7 days compared with VLB physiologic saline solution in the same dose (P < 0.05). Clone formation rate of glioma C6 in VLB physiologic saline solution group is 1. 3 times better than VLB NPS. The difference between VLB NPS and VLB physiologic saline solution is significant (P < 0.05). Results of morphology change indicated glioma cells C6 with the VLB NPS treatment were intermediate or end stage, missed structure integrality. Amount of cells was distinctly decreased, and apoptosis cells number was apparently increased compared with VLB physiologic saline solution group. CONCLUSION: VLB NPS have stronger cytotoxicity to glioma cells line C6 compared with VLB physiologic saline solution in the same dose. NPS may be effective as promising carrier for the transport of VLB into the glioma cells.

[Effect of vinblastine nanoparticles on antiproliferation in human glioma cell lines BT325].

OBJECTIVE: To compare antiproliferation effects of vinblastine nanopraticles and vinblastine water solution in human glioma cell lines BT325. METHOD: Vinblastine nanoparticles were prepared by emulsion polymerization process and using dextran as a stabilizing agent. It was characterized by means of morphology, size, drug entrapment efficiency and loading efficiency. Human glioma cell lines BT325 were treated with different concentrations of vinblastine nanoparticles and vinblastine water solution for 48 h, Antiproliferation effect was measured by MTT method. Morphological changes were observed by inverted microscope, transmission electron microscope and scanning electron microscope. RESULT: Mean diameter of VLB-PBCA-NP was about 74.4 nm, and drug entrapment efficiency and loading efficiency was 78.47% and 39.24%, respectively. Cell growth inhibition rate of vinblastine nanoparticles group and vinblastine water solution group in a concentration range (5-5 000 g x L(-1)) for 48 h was 41%, 49%, 73%, 83% and 28%, 33%, 54%, 60% respectively. Entrapment of VLB in NPS may distinctly degrade absorbency as compared to free drugs. Glioma cell BT325 which treated with VLB water solution were initial stage of apoptosis, and apoptosis body were forming. But VLB NPS-treated BT325 cells were intermediate or end stage, and missed structure integrality. CONCLUSION: VLB-PBCA-NP and VLB water solution could inhibit the growth of human glioma cell lines BT325, and VLB nanoparticles have stronger inhibition effect compared with VLB water solution in the same dose. PBCA may be effective as promising carrier for the transport of vinblastine into the glioma cells.

[Experimental study of cardiac muscle tissue engineering in bioreactor].

OBJECTIVE: This study investigates construction of cardiac muscle cell-porous collagen scaffold complex in a bioreactor so as to unveil the possibility of generating 3-dimensional cardiac muscle tissue under the environment that mimics microgravity in vitro. METHODS: 1-2-day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods, then seeded onto porous collagen scaffold. The cell-collagen complex was transferred into rotary cell culture system (RCCS) and incubated for 7 days. Cells cultured in 75 ml flasks and constructs cultures on plates served as control. Morphological changes of the cells were observed by light microscope and metabolic rate was recorded. Ultrastructure of the cells growing in porous collagen was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. H-E and anti-sarcomeric alpha-actin stains were performed in comparison with native cardiac muscle. RESULTS: The isolated cardiac muscle cells adhered to the bottom of the flasks 24 hours after plating and began to beat spontaneously. When incubated for 7 days in RCCS, cell-collagen constructs of form a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior of the construct was less in density than the outer part. Transmission electron microscopy demonstrated that subcellular elements characteristic of cardiac myocytes were in the outermost layer of constructs. A strongly positive stains of anti-sarcomeric alpha-actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle and approximately 40% of that in adult rat ventricles. Construsts in plates contained a few of cells which were less than those in RCCS. Metabolic activity of cells cultured in RCCS was higher than that in flasks and plates. CONCLUSIONS: Dissociated cardiac muscle cells cultured on 3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.