Regulatory Role of Chinese Herbal Medicine in Regulated Neuronal Death.

Ischemic neuronal injury results from a complex series of pathophysiological events, including oxidative, excitotoxicity, inflammation and nitrative stress. Consequently, many of these events can induce cell death, including necrosis (unregulated cell death) and apoptosis (a type of regulated cell death). These are long-established paradigms to which newly discovered regulated cell death processes have been added, such as necroptosis (a regulated form of necrosis) and autophagydependent cell death. Moreover, many researchers have targeted products associated with Chinese herbal medicine at regulated pathways for the treatment of ischemic neuronal injury. In East Asia, these drugs have been known for centuries to protect and improve the nervous system. Herbal extracts, especially those used in Chinese herbal medicine, have emerged as new pharmaceuticals for the treatment of ischemic neuronal injury. Here, we review the evidence from preclinical studies investigating the neuroprotective properties and therapeutic application of Chinese herbal medicines (Chinese herbal monomer, extract, and medicinal compounds) and highlight the potential mechanisms underlying their therapeutic effects via targeting differently regulated cell death pathways. Notably, many herbs have been shown to target multiple mechanisms of regulated cell death and, in combination, may exert synergistic effects on signaling pathways, thereby attenuating multiple aspects of ischemic pathology. In this review, we summarize a generally regulated pathway of cell death as a target for novel natural herbal regimens against ischemic neuronal injury.

Analysis of factors related to prognosis and death of fish bile poisoning in China: A retrospective study.

Fish bile has long been considered to have therapeutic benefits in folk medicine in some Asian countries. However, poisoning incidents and even death sporadically occurred when people consumed fish bile. Herein, we summarize the main characteristics of fish bile poisoning in China including clinical symptoms, treatment strategies and factors being associated with death and affecting prognosis, hoping to provide a reference for the diagnosis and treatment of fish bile poisoning, as well as forensic identification of death cases induced by fish bile poisoning. We suggest that the health authorities should make an effort to enhance people's awareness of the safety of traditional medicine like fish bile so as to reduce the incidence of adverse events.

Auto-degradable and biocompatible superparamagnetic iron oxide nanoparticles/polypeptides colloidal polyion complexes with high density of magnetic material.

HYPOTHESIS: Superparamagnetic iron oxide nanoparticles (SPIONs) are extensively used as building block of colloidal nanocomposites for biomedical applications. Strategies employed to embed them in a biodegradable and biocompatible polymer matrix often fail to achieve a high density of loading which would greatly benefit to applications such as imaging and hyperthermia. In this study, poly(acrylic acid) coated SPION (γ-Fe2O3-PAA) are self-assembled with hydrolysable poly(serine ester) by electrostatic complexation, leading to perfectly defined spherical particles with ultra-high density of magnetic material and an ability to auto-degrade into individual SPION and biocompatible byproducts. EXPERIMENTS: Self-assembly and auto-degradation of γ-Fe2O3-PAA/poly(serine ester) and γ-Fe2O3-PAA/poly(serine ester)-b-PEG colloidal particles are studied by light scattering and microscopy. Colloidal stability in bio-fluids, hyperthermia under alternating magnetic field, cellular uptake, cytotoxicity and degradation of γ-Fe2O3-PAA/poly(serine ester)-b-PEG in living cells are investigated. FINDINGS: A remarkably slow electrostatic complexation leads to dense superparamagnetic γ-Fe2O3-PAA/poly(serine ester)-b-PEG polyion complexes (PICs) with controlled sizes (150-500 nm) and times of degradation in aqueous solvents (700-5000 h). The material shows good sustainability during hyperthermia, is well taken up by MC3T3 cells and non-cytotoxic. TEM images reveal a mechanism of degradation by "peeling" and fragmentation. In cells, PICs are reduced into individual SPIONs within 72 h.

Pin1 Is Regulated by CaMKII Activation in Glutamate-Induced Retinal Neuronal Regulated Necrosis.

In our previous study, we reported that peptidyl-prolyl isomerase 1 (Pin1)-modulated regulated necrosis (RN) occurred in cultured retinal neurons after glutamate injury. In the current study, we investigated the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in Pin1-modulated RN in cultured rat retinal neurons, and in an animal in vivo model. We first demonstrated that glutamate might lead to calcium overloading mainly through ionotropic glutamate receptors activation. Furthermore, CaMKII activation induced by overloaded calcium leads to Pin1 activation and subsequent RN. Inactivation of CaMKII by KN-93 (KN, i.e., a specific CaMKII inhibitor) application can decrease the glutamate-induced retinal neuronal RN. Finally, by using an animal in vivo model, we also demonstrated the important role of CaMKII in glutamate-induced RN in rat retina. In addition, flash electroretinogram results provided evidence that the impaired visual function induced by glutamate can recover after CaMKII inhibition. In conclusion, CaMKII is an up-regulator of Pin1 and responsible for the RN induced by glutamate. This study provides further understanding of the regulatory pathway of RN and is a complementary mechanism for Pin1 activation mediated necrosis. This finding will provide a potential target to protect neurons from necrosis in neurodegenerative diseases, such as glaucoma, diabetic retinopathy, and even central nervous system diseases.

[Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala].

In this study， an endophytic bacteria strain BZJN1 was isolated from Atractylodes macrocephala， and identified as Bacillus subtilis by physiological and biochemical tests and molecular identification. Strain BZJN1 could inhibit the growth of mycelia of Ceratobasidium sp. significantly， and the inhibition rate was more than 70%. The mycelium growth deformity with bulge as spherical and partially exhaustible in apex or central with microscopic observation. The inhibitory rates under 3% and 6% concentrations of the cell free fermentation were 22.7% and 38.7% expectively. The field test proved that the control efficacy of treatment of 1×108 cfu·mL⁻¹ is 75.27% and 72.37% after 10 and 20 days. All the treatments of strain BZJN1 was able to promote the growth of A. macrocephala， the treatment of 1×108 cfu·mL⁻¹ could able to increase the yield to 14.1%.

The Toxic Effect of ALLN on Primary Rat Retinal Neurons.

N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 µM, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 µM but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 µM. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 µM after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 µM after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 µM after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 µM, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.

The effect and underlying mechanism of Timosaponin B-II on RGC-5 necroptosis induced by hydrogen peroxide.

BACKGROUND: Necroptosis is an important mode of cell death, which is due to oxidant stress accumulation. Our previous study indicated that oxidant stresses could be reduced by Timosaponin B-II (TBII), a kind of Chinese herb RhizomaAnemarrhenae monomer extraction. We wonder the possible effect of Timosaponin B-II, whether it can protect cells from necroptosis via reducing the oxidant stress, in RGC-5 following hydrogen peroxide (H2O2) insult. METHODS: RGC-5 cells were grown in DMEM, the model group was exposed in H2O2 with the concentration of 300 µM, and the experimental group was pre-treated with Timosaponin B-II at different concentrations (1 µM, 10 µM, 100 µM and 1000 µM) for 24 hrs. MTT assay was carried out to measure the cytotoxicity of H2O2, MDA concentration assay was executed to evaluate the degree of oxidative stress, TNF-α ELISA Assay was used to measure the concentration of TNF-α, finally, the degree of necrosis were analyzed using flow cytometry. RESULTS: We first constructed the cell injury model of necroptosis in RGC-5 upon H2O2 exposure. Morphological observation and MTT assay were used to evaluate the degree of RGC-5 death. MDA assay were carried out to describe the degree of oxidant stress. Annexin V/PI staining was used to detect necroptotic cells pre-treated with or without Timosaponin B-II following H2O2 injury. TNF-α ELISA was carried out to detect the TNF-α accumulation in RGC-5. Upon using Timosaponin B-II with concentration of 100 µM, the percentage of cell viability was increased from 50% to 75%, and the necrosis of cells was reduced from 35% to 20% comparing with H2O2 injury group. Oxidant stress and TNF-α was reduced upon injury which decreased the ratio of RGC-5 necroptosis. CONCLUSION: Our study found out that Timosaponin B-II might reduce necroptosis via inhibition of ROS and TNF-α accumulation in RGC-5 following H2O2 injury.

Timosaponin-BII inhibits the up-regulation of BACE1 induced by ferric chloride in rat retina.

BACKGROUND: Our previous studies indicated that oxidative stress up-regulated the expression of ß-amyloid precursor protein cleavage enzyme-1 (BACE1) in rat retina. Pharmacological reports have shown Timosaponin-BII, a purified extract originating from Chinese medical herb Rhizoma Anemarrhenae, is characterized as an antioxidant. Our present study aimed to determine whether Timosaponin-BII affected the expression of BACE1, ß-amyloid precursor protein cleavage production of Aß1-40 and ß-C-terminal fragment (ß-CTF) in rat retina, which were pre-treated with the oxidizing agent (solution of FeCl3). RESULTS: Few distinctions of BACE1 distribution were observed among all groups (normal control group, model group, Timosaponin-BII treated and vehicle control groups). Rat retinas in model group and vehicle control group manifested an apparent up-regulation of BACE1 expression. Meanwhile, the level of malonaldehyde (MDA), Aß1-40 and ß-CTF were increased. However, when comparing with the vehicle control group, the retinas in Timosaponin-BII treated group showed significantly less BACE1 (p<0.05) and accumulated less Aß1-40 or ß-CTF (p<0.05). It also showed significantly decreased level of MDA (p<0.05) and prolonged partial thromboplastin time (p<0.05). CONCLUSION: Our data suggested that Timosaponin-BII remarkably inhibited the up-regulation of BACE1 and reduced the over-production of ß-CTF and Aß in rat retina, which was induced by FeCl3. The mechanism of Timosaponin-BII on BACE1 expression may be related to its antioxidant property.

Mitochondrial respiratory inhibition and oxidative stress elevate beta-secretase (BACE1) proteins and activity in vivo in the rat retina.

Cerebral hypometabolism, oxidative stress and beta-amyloid peptide (Abeta) accumulation are key pathological events in Alzheimer's disease (AD). Beta-secretase (BACE, i.e., BACE1), a prerequisite for Abeta genesis, is elevated in sporadic AD. Recent studies show BACE upregulation in experimental conditions likely associated with energy insufficiency and/or oxidative stress. We investigated the effect of sublethal doses of mitochondrial respiratory inhibitors and potential endogenous oxidative substances on BACE expression in vivo using the retina as a model. Retinas were analyzed biochemically and anatomically 48 h following intraocular applications of mitochondrial complex I, II and IV inhibitors including rotenone, 3-nitropropionic acid and sodium azide, and plaque-containing oxidants including Fe(3+) and Abeta42 fibrils (Abeta42f). All agents caused elevations of BACE proteins and beta-site amyloid precursor protein (APP) cleavage product, beta-CTF, in retinal lysates in a dose-dependant manner. BACE activity and Abeta40 levels were also increased in agent-treated retinas relative to vehicle controls. BACE immunoreactivity in normal adult rat retina was present mostly in the plexiform layers, indicating a localization of the enzyme to synaptic terminals. No apparent change in laminar or cellular distribution of BACE labeling was detected in the experimental retinas. However, signs of neuronal stress including glial activation were observed in agent-treated retinas especially in high dosage groups. Our data suggest that mitochondrial respiratory inhibition and oxidative stress facilitate BACE expression in vivo. In addition, plaque constituents such as Fe(3+) and Abeta42f may participate in a self-enforcing cycle of amyloidogenesis via BACE upregulation.