Neurotoxicity of ß-HgS differs from environmental mercury pollutants (MeHgCl and HgCl2) in Neuro-2a cell.

ß-HgS, differing from environmental mercury pollutants (MeHgCl and HgCl2) in chemical form, is used as traditional medicine in Asian countries for thousands of years. In this study, Neuro-2a cells were exposed to ß-HgS, MeHgCl and HgCl2 (5 µM) for 6-24 h. The cell viability of ß-HgS was higher than MeHgCl with 25.9% and 72.4% in 12 h and 24 h respectively. As the incubation time increased, MeHgCl had obvious damage to cell morphology, decreased the ratio of Bcl-2 and Bak and increased the expressions of TNF-α, IL-6 and IL-1ß significantly. Furthermore, the expressions of IL-1ß and IL-6 in HgCl2 group were increased significantly in 6 h and 24 h. The apoptotic rates in MeHgCl and HgCl2 group were respectively higher than ß-HgS with 32.2% and 7.30% in 24 h. Our findings indicate that ß-HgS is much less neurotoxicity than MeHgCl and HgCl2 in Neuro-2a cells.

Effects of ß-HgS on cell viability and intracellular oxidative stress in PC-12 cells.

Traditional Tibetan medicines containing ß-HgS have been used to treat chronic ailments for thousands of years. However, there has recently been speculation regarding the safety of these medicines due to their high mercury content. Although the toxic effect of ß-HgS has been previously investigated in vivo, the mechanism underlying the toxicity of this compound remains unclear. In this study, we investigate the mechanism of ß-HgS cytotoxicity via experiments performed on rat adrenal gland tumor cells (PC-12). Specifically, we analyze the viability and intracellular oxidative stress state of PC-12 cells treated with varying concentrations of ß-HgS. For comparison purposes, the effects of MeHgCl and HgCl2, two Hg-based compounds, on ROS generation and MDA, GSH/GSSG, Nrf2, NQO-1, and HO-1 levels are also determined. It should be noted that we used the small-molecule thiols of cell culture medium, such as cysteine, to increase the solubility of ß-HgS and prepare a ß-HgS solution to treat PC-12 cells. The obtained results show that ß-HgS inhibits cell viability at concentrations of 200-1000 ng Hg mL-1 (48 h treatment). In the concentration range of 200-600 ng Hg mL-1 (24 h treatment), the inhibitory effect of ß-HgS is stronger than that of MeHgCl; however, this trend is reversed at higher concentrations (800-1000 ng mL-1) and longer exposure times (48 h). Moreover, ß-HgS significantly promotes MDA, but has no appreciable influence on cell apoptosis and ROS generation in PC-12 cells, which suggests that its inhibitory effect on cell viability might be related to the stimulation of ROS-independent oxidative stress. Notably, ß-HgS and HgCl2 significantly increase the GSH content, GSH/GSSG ratio, NQO-1 mRNA expression, and HO-1 protein expression in PC-12 cells, indicating that the antioxidant protection against these compounds is triggered by Nrf2 activation. HPLC-AFS analysis shows that in ß-HgS and HgCl2 solutions, mercury exists in the same form of Hg2+, but the cytotoxicity of the former is greater. This is probably due to the additional oxidative damage induced by the S2- ion in ß-HgS. In conclusion, ß-HgS induces ROS-independent oxidative stress in PC-12 cells, and thus, is obviously cytotoxic. At the same time, it promotes the antioxidant capacity of cells by activating the Nrf2 pathway.

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium.

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 µm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (90:10 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 µg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.

Hormesis of methylmercury-human serum albumin conjugate on N9 microglia via ERK/MAPKs and STAT3 signaling pathways.

Methylmercury (MeHg+) is an extremely toxic organomercury cation that can induce severe neurological damage. Once it enters the body, methylmercury binds to amino acids or proteins containing free sulfhydryl groups. In particular, methylmercury is known to bind with human serum albumin (HSA) in human plasma; however, the effects of methylmercury-HSA conjugate (MeHg-HSA) on the central nervous system (CNS) are not fully understood. In the present study, we used the microglial cell line N9 as the target cells to evaluate the effect of MeHg-HSA on physiological function of the CNS preliminarily. The various factors in the cell culture were monitored by MTT assay, total lactate dehydrogenase assay, ELISA, qPCR, Western blot and flow cytometry techniques. The results showed that low-dose treatment with MeHg-HSA activated N9 cells, promoting cell proliferation and total cell number, enhancing NO and intracellular Ca2+ levels, and suppressing the release of TNFα and IL1ß without cytotoxic effects; while high-dose MeHg-HSA exhibited cytotoxic effects on N9 cells, including promoting cell death and increasing the secretion of TNFα and IL1ß. These results indicate that MeHg-HSA causes hormesis in microglia N9 cells. Furthermore, ERK/MAPKs and STAT3 signaling pathways related to the hormesis of MeHg-HSA on N9 cells. In addition, low dose of MeHg-HSA might be viewed as something very close to a lowest observed adverse effect level (LOAEL) for N9 cells. These findings will be useful for investigating the hormesis mechanism of MeHg+ and exploring the specific functions of MeHg-sulfhydryl conjugates on the central nervous system.

Development of a safety and efficacy nanoemulsion delivery system encapsulated gambogic acid for acute myeloid leukemia in vitro and in vivo.

This study aimed to improve the solubility, reduce the side effects and enhance the efficacy of gambogic acid against acute myeloid leukemia in vitro and in vivo. This oil-in-water nanoemulsion (average size 17.20 ±â€¯0.11 nm, zeta potential 4.17 ±â€¯0.82 mV) containing Tween-80, glycol, squalene and gambogic acid with improving 4000 times solubility was prepared by pseudoternary phase diagrams. We found that this nanoemulsion successfully encapsulated gambogic acid; it was stable and showed an obvious delayed release effect for the drug in three different phosphate-buffered saline (pH = 2.0, 5.8 and 7.4). The half inhibiting concentration (IC50) of this nanoemulsion (480.7 µg/mL and 408 µg/mL) were 1.67 times and 1.98 times higher than those of its water solution (287 µg/mL and 206 µg/mL) after acting on the toxicity standard cell line (L929 line) for 24 h and 48 h, respectively. Importantly, acute injection toxicity indicated that the half lethal dose (LD50) of this nanoemulsion (23.25 mg/kg, 95% LD50, 21.7-25.16 mg/kg) was 1.26 times higher than that of its water solution (18.59 mg/kg, 95% LD50, 16.84-20.53 mg/kg). Compared with its suspension, the bioavailability of this nanoemulsion was 318.2%. Furthermore, this nanoemulsion had a better efficacy against the acute myeloid leukemia in vitro and in vivo by improving the time and percent of survival (MV4-11 engrafts mice) and reducing half inhibiting concentration values in acute myeloid leukemia such as Jurket, HL-60 and MV4-11 cells. Our studies suggested that this nanoemulsion may be a promising therapeutic medicine for acute myeloid leukemia.

Hormesis of mercuric chloride-human serum albumin adduct on N9 microglial cells via the ERK/MAPKs and JAK/STAT3 signaling pathways.

Mercury chloride (HgCl2), a neurotoxicant that cannot penetrate the blood-brain barrier (BBB). Although when the BBB are got damaged by neurodegenerative disorders, the absorbed HgCl2, mainly in form of Hg (II)-serum albumin adduct (Hg-HSA) in human plasma, can penetrate BBB and affect central nervous system (CNS) cells. Current study planned to evaluate the effect of Hg-HSA on the physiological function of N9 microglial cells. At low dosage (15 ng/mL) of Hg-HAS, the observed outcomes was: promoted cell propagation, Nitric Oxide (NO) and intracellular Ca2+ levels enhancement, suppressed the release of TNF-α and IL-1ß and inhibited cell proliferation. At high dosage (15 µg/mL) we observed decline in NO and intracellular Ca2+ levels, and increment in the release of TNF-α and IL-1ß. These biphasic effects are similar to hormesis, and the hormesis, in this case, was executed through ERK/MAPKs and JAK/STAT3 signaling pathways. Study of quantum chemistry revealed that Hg2+ could form stable coordination structures in both Asp249 and Cys34 sites of HSA. Although five-coordination structure in Asp249 site is more stable than four-coordination structure in Cys34 site but four-coordination structure is formed easily in-vivo in consideration of binding-site position in spatial structure of HSA.

Effect of the Tibetan Medicine Zuotai on Degranulation and Inflammatory Mediator Release in RBL-2H3 Cells.

Zuotai is a drug containing mercury considered to be the king of Tibetan medicine. The biosafety of Zuotai led people's attention and so far little is known about the toxicity of Zuotai to mast cells. RBL-2H3 cells which used as an alternative model of mast cells were treated with Zuotai, ß-HgS and positive drug Compound 48/80 respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of drugs to RBL-2H3 cells. The degranulation of RBL-2H3 cells was studied from ß-hexosaminidase, histamine, interleukin (IL)-4 and tumor necrosis factor-α (TNF-α). The result showed that Zuotai can affect the cytotoxicity and degranulation of RBL-2H3 cells and the results can provide reference for the toxicity evaluations of Tibetan medicine Zuotai.

An antioxidant α-glucan from Cladina rangiferina (L.) Nyl. and its protective effect on alveolar epithelial cells from Pb2+-induced oxidative damage.

Air pollution is a serious global health problem nowadays. So, it is an emergency to pay sufficient attention to treat and prevent the diseases caused by air pollution, especially respiratory disease and lung damage. Cladina rangiferina (L.) Nyl. is an edible lichen that has been used in medicinal diets to treat respiratory and other diseases for over 500 years. In this study, a water-soluble polysaccharide, CRWP-P, was obtained from C. rangiferina by hot-water extraction, freeze-thawing separation, and Fehling reagent purification. Structural analysis showed that CRWP-P is a linear α-(1 → 3),(1 → 4)-d-glucan without branches. Its Mw was determined to be 1.05 × 105 Da. Its (1,3)-α-d-glucopyranosyl: (1,4)-α-d-glucopyranosyl ratio is approximately 1:2. Antioxidant activity assay showed that C. rangiferina polysaccharides, especially CRWP-P, had appreciable DPPH radical-scavenging activity and reducing power. Notably, they could effectively decrease cell breakdown and ROS generation, inhibit lipid peroxidation, increase key antioxidase activity, and promote glutathione redox cycling in Pb2+-oxidative injured A549 alveolar epithelium cells. Overall, the results of this study indicated that C. rangiferina polysaccharides, especially CRWP-P, have the potential to be natural antioxidants for the treatment of lung oxidative damage induced by lead of air pollutants.

pH-Dependent Effects of L-Cysteine on Mercury Dissolution of α-HgS and ß-HgS.

Mercury sulfide is an insoluble inorganic mercury compound, and it is the main chemical form in traditional oral mercury-containing medicines. Hg2+ has a high affinity for thiols, and small molecule thiols in the gastrointestinal tract may promote mercury dissolution of mercury sulfide by binding to Hg2+. L-cysteine is the only amino acid that possesses a reducing sulfhydryl group (-SH), out of the 20 amino acids. This study investigates the effect of L-cysteine on mercury dissolution of mercury sulfide at pHs ranging from 1.2 to 7.2. The results showed that L-cysteine had different pH-dependent effects on the mercury dissolution of α-HgS and ß-HgS. For α-HgS, the dissolved mercury concentration increased from 5.47 ± 0.97 ng/mL to 12.49 ± 0.54 ng/mL when the pH rose from 1.2 to 4.2, and decreased to 3.37 ± 0.70 ng/mL at pH 6.0 and then increased to 9.36 ± 0.79 ng/mL at pH 7.2. For ß-HgS, the dissolved mercury concentration increased from 151.09 ± 2.25 ng/mL to 2346.71 ± 62.62 ng/mL when the pH increased from 1.2 to 7.2. In conclusion, L-Cys was distinctly enhanced upon mercury dissolution of α-HgS and ß-HgS with increasing pH. These results may contribute to our understanding of the mercury absorption mechanism of traditional oral mercury-containing medicines.

Characterization and antitumor efficacy of poly(L-lactid acid)-based etoposide-loaded implants.

Etoposide is widely used in the chemotherapy of a variety of malignancies. But the strong lipophilicity, poor bioavailability, and severe side effects of etoposide limit its clinical application. The aim of this study was to develop sustained-release etoposide-loaded implants and evaluate antitumor activity of the implants after intratumoral implantation. We prepared the implants containing etoposide, poly(L-lactid acid) and polyethylene glycol 4000 by the direct compression method. The implants were characterized regarding drug-excipient compatibility, content uniformity, morphology, sterility, in vitro, and in vivo release profiles. Then the antitumor activity of the implants was tested in xenograft model of A549 human non-small cell lung cancer. SEM images displayed smooth surface of the implant and indicated that etoposide was homogeneously dispersed in the polymeric matrix. The results of content uniformity met the requirements of the Chinese Pharmacopoeia. Both in vitro and in vivo release profiles of the implants were characterized by high burst release followed by sustained release of etoposide. Intratumoral implantation of etoposide-loaded implants could efficiently delay the tumor growth. Furthermore, increasing the dose of implants led to higher tumor suppression rate without adding systemic toxicity. These results indicated that etoposide-loaded implants have significant antitumor efficacy in xenograft model without dose-limiting side effects and they possess a strong potential to be used as an intratumoral chemotherapy option for lung cancer treatment.

Mercury sulfides are much less nephrotoxic than mercury chloride and methylmercury in mice.

Mercury sulfides (α-HgS, ß-HgS) are frequently included in traditional medicines. Mercury is known for nephrotoxicity, their safety is of concern. To address this question, mice were orally administrated with Zuotai (54% ß-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHgCl (3.1mg/kg) for 7days, and nephrotoxicity was examined. Animal body weights were decreased by HgCl2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl2 and MeHg produced renal tubular vacuolation, interstitial inflammation and cell degeneration with protein cysts in the tubular lumen, while these pathological lesions were mild in Zuotai and HgS-treated mice. Electron microscopy showed that HgCl2 and MeHg produced spotted swelling endothelium reticulum, while these lesions were mild or absent in Zuotai and HgS-treated mice. Renal Hg contents reached 250-300ng/mg kidney in HgCl2 and MeHg groups as compared to 2-3ng/mg in Zuotai and HgS groups. The expression of kidney injury biomarkers, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), were increased after HgCl2 and MeHg, but unaltered after Zuotai and HgS. The expression of renal influx transporters Oat3 and Oatp4c1 was decreased, while the expression of renal efflux transporter such as Mrp2, Mrp4, and Mate2 was increased following HgCl2 and MeHg. These gene expressions were unchanged after Zuotai and HgS. In summary, both α-HgS and ß-HgS are less nephrotoxic than HgCl2 and MeHg, indicating that chemical forms of mercury are a major determinant of mercury disposition and toxicity.

[Metallic Elemental Analysis of Tibetan Herbal Medicines and Tibetan Medicine Preparations by Synchrotron Radiation X-Ray Fluorescence].

To discuss the relationship between metallic element and disease through determine the elementals in Tibetan Herbal Medicines and Tibetan Medicine Preparations that have obvious effect on hepatobiliary diseases by Synchrotron Radiation X-ray Source, then to reveal the substance foundation of pharmacological action. The results show that all the Tibetan Herbal Medicines used in the experiment have the 9 kinds of metallic elements of potassium(K), calcium(Ca), titanium(Ti), vanadium(V), chromium(Cr), manganese(Mn), ferrum(Fe), zinc(Zn) and lead(Pb), the content of the elements are in the ppb or ppm level though the element constitute and the content have obvious difference. Tibetan Medicine Preparations have another 6 kinds of metallic elements of nickel(Ni), copper(Cu), rubidium(Rb), mercury(Hg), cobalt(Co), gallium(Ga) and 1 kind of nonmetallic elements of arsenic(As) when compare with Herbal Medicines, and the element constitute and the content also have obvious difference. Take advantage of SR-XRF, the test gets the basic data of elements of Tibetan Herbal Medicines and Preparations, supply the scientific support to discuss the interaction of pharmacological mechanism and the metallic elements, and find the suitability of the technique for the metallic elements detection in Tibetan Medicines.

Anticancer and immunostimulating activities of a novel homogalacturonan from Hippophae rhamnoides L. berry.

Our previous study isolated an anti-fatigue polysaccharide (HRWP) from the Hippophae rhamnoides berry. In this study, using ion-exchange chromatography and gel filtration chromatography in turn, a water-soluble homogenous polysaccharide HRWP-A was isolated from HRWP. Structural analysis determined that HRWP-A was a polysaccharide with repeating units of (1→4)-ß-d-galactopyranosyluronic residues, of which 85.16% were esterified with methyl groups. An antitumor activity assay showed that HRWP-A could significantly inhibit the Lewis lung carcinoma (LLC) growth in tumor-bearing mice. Further experiments suggested that the antitumor effect of HRWP-A might be mediated through immunostimulating activity, as it enhances the lymphocyte proliferation, augments the macrophage activities, as well as promoting NK cell activity and CTL cytotoxicity in tumor-bearing mice. To our knowledge, this is the first report on a natural antitumor high-methoxyl homogalacturonan pectin from the H. rhamnoides berry-a compound that acts as a potential immunostimulant and anticancer adjuvant.