Cong Yang

(-)-Epigallocatechin-3-gallate attenuates cognitive deterioration in Alzheimer's disease model mice by upregulating neprilysin expression.

Epigenetic changes are involved in learning and memory, and histone deacetylase (HDAC) inhibitors are considered potential therapeutic agents for Alzheimers disease (AD). We previously reported that (-)-epigallocatechin-3-gallate (EGCG) acts as an HDAC inhibitor. Here, we demonstrate that EGCG reduced β-amyloid (Aβ) accumulation in vitro and rescued cognitive deterioration in senescence-accelerated mice P8 (SAMP8) via intragastric administration of low- and high-dose EGCG (5 and 15 mg/kg, respectively) for 60 days. The AD brain has decreased levels of the rate-limiting degradation enzyme of Aβ, neprilysin (NEP). We found an association between EGCG-induced reduction in Aβ accumulation and elevated NEP expression. Further, NEP silencing prevented the EGCG-induced Aβ downregulation. Our findings suggest that EGCG might be effective for treating AD.

A novel assay for high-throughput screening of anti-Alzheimer's disease drugs to determine their efficacy by real-time monitoring of changes in PC12 cell proliferation

Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by the accumulation of senile plaque and neurofibrilary tangle formation in the brain, including the cerebral cortex and hippocampus. Nowadays, the first-line treatment for AD is the application of acetylcholinesterase inhibitors. However, acetylcholinesterase inhibitors are basically anti-symptomatic for a limited aspect of AD pathology and are associated with serious side-effects. With the advantage of multiple targets, pathways and systems, Chinese herbal compounds hold promising potential for the development of drugs for the treatment of AD. Over the past few years, with the development of Chinese herbal compounds and in vitro pharmacological studies, cell-based disease models are one of the main methods used to screen Chinese herbal compounds for potential efficacy. Testing the efficacy of possible anti-Alzheimer’s disease drugs and the development of new drugs are hindered by the lack of objective high-throughput screening methods. Currently, the assessment of the effects of drugs is usually made by MTT assays, involving laborious, subjective, low-throughput methods. Herein, we suggest a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess the effective composition of Chinese herbal compounds by assessing amyloid-β peptide Aβ1-42-induced apoptosis in PC12 cells. We detected the proliferation and motility of the cells using a fully automated high-throughput and real-time system. We quantitatively assessed cell motility and determined the real-time IC50 values of various anti-AD drugs that intervene in several developmental stages of Aβ1-42-induced apoptosis in PC12 cells, Then, we identified the optimal time phase by curative efficacy. Our data indicate that this technique may aid in the discovery and development of novel anti-Alzheimer’s disease drugs. It is possible to utilize a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance of 9–12. The impedance is displayed as a dimensionless parameter termed the cell index, which is directly proportional to the total area of tissue culture well that is covered by the cells. Hence, the cell index can be used to monitor cell adhesion, spreading, morphological variation and cell density.

Network pharmacology-based study on the mechanism of action for herbal medicines in Alzheimer treatment.

ETHNOPHARMACOLOGICAL RELEVANCE Alzheimers disease (AD), as the most common type of dementia, has brought a heavy economic burden to healthcare system around the world. However, currently there is still lack of effective treatment for AD patients. Herbal medicines, featured as multiple herbs, ingredients and targets, have accumulated a great deal of valuable experience in treating AD although the exact molecular mechanisms are still unclear. MATERIALS AND METHODS In this investigation, we proposed a network pharmacology-based method, which combined large-scale text-mining, drug-likeness filtering, target prediction and network analysis to decipher the mechanisms of action for the most widely studied medicinal herbs in AD treatment. RESULTS The text mining of PubMed resulted in 10 herbs exhibiting significant correlations with AD. Subsequently, after drug-likeness filtering, 1016 compounds were remaining for 10 herbs, followed by structure clustering to sum up chemical scaffolds of herb ingredients. Based on target prediction results performed by our in-house protocol named AlzhCPI, compound-target (C-T) and target-pathway (T-P) networks were constructed to decipher the mechanism of action for anti-AD herbs. CONCLUSIONS Overall, this approach provided a novel strategy to explore the mechanisms of herbal medicine from a holistic perspective.

Bioavailability enhancement of osthole after oral administration of Bushen Yizhi prescription extract to rats followed by Cnidium monnieri (L.) Cusson fruits extract in comparison to pure osthole at different doses.

ETHNOPHARMACOLOGICAL RELEVANCE Bushen Yizhi prescription (BSYZ) is a traditional Chinese compound prescription, which is commonly used in China for treating ShenXu and hypophrenia based on traditional Chinese medicine and Alzheimers Disease according to modern Chinese medicine. Cnidium monnieri (L.) Cusson fruits (CM) is treated as the main herb of BSYZ, and its main active ingredient Osthole (OST) is considered as one of the major active ingredients of BSYZ. Even though OST plays an important role in the BSYZ its bioavailability is poor. In order to investigate whether the bioavailability of OST was influenced by BSYZ and CM extract, the comparative evaluations on pharmacokinetics of OST after oral administration of pure OST at different doses, CM and BSYZ extract were studied. MATERIALS AND METHODS 30 rats were randomly assigned to five groups and orally administered with pure OST at different doses (15, 75 and 150 mg/kg), CM (15 mg/kg OST) and BSYZ (15 mg/kg OST) extract. At different predetermined time points after administration, the concentrations of OST in rat plasma were determined by using the HPLC-UV method, and main pharmacokinetic parameters were investigated. RESULTS The results showed that the pharmacokinetic parameters of OST were significantly different (p<0.05) among the groups. The AUC(0→t), AUC(0→∞) and Cmax of OST were significantly increased after oral administration of BSYZ extract, followed by CM extract, in comparison to pure osthole at different doses. CONCLUSIONS This present study indicated that the bioavailability of pure OST after oral administration was extremely low and it was dramatically enhanced because of the synergistic effect of the traditional Chinese Bushen Yizhi prescription.

Sodium Tanshinone IIA Sulfonate Attenuates Scopolamine-Induced Cognitive Dysfunctions via Improving Cholinergic System

Sodium Tanshinone IIA sulfonate (STS) is a derivative of Tanshinone IIA (Tan IIA). Tan IIA has been reported to possess neuroprotective effects against Alzheimers disease (AD). However, whether STS possesses effect on AD remains unclear. This study aims to estimate whether STS could protect against scopolamine- (SCOP-) induced learning and memory deficit in Kunming mice. Morris water maze results showed that oral administration of STS (10 mg/kg and 20 mg/kg) and Donepezil shortened escape latency, increased crossing times of the original position of the platform, and increased the time spent in the target quadrant. STS decreased the activity of acetylcholinesterase (AChE) and increased the activity of choline acetyltransferase (ChAT) in the hippocampus and cortex of SCOP-treated mice. Oxidative stress results showed that STS increased the activity of superoxide dismutase (SOD) and decreased the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in hippocampus and cortex. In addition, western blot was carried out to detect the expression of apoptosis related proteins (Bcl-2, Bax, and Caspase-3). STS upregulated the protein expression of Bcl-2 and downregulated the proteins expression of Bax and Caspase-3. These results indicated that STS might become a promising therapeutic candidate for attenuating AD-like pathological dysfunction.

Bushen‑Yizhi formula ameliorates cognition deficits and attenuates oxidative stress‑related neuronal apoptosis in scopolamine‑induced senescence in mice

Bushen-Yizhi formula (BSYZ), a traditional Chinese medicine formula consisting of six herbs has been reported to possess a neuroprotective effect. The present study aimed to investigate the effects of BSYZ on learning and memory abilities, as well as oxidative stress and neuronal apoptosis in the hippocampus of scopolamine (SCOP)-induced senescence in mice, in order to reveal whether BSYZ is a potential therapeutic agent for Alzheimer’s disease (AD). A high-performance liquid chromatography (HPLC) fingerprint was applied to provide a chemical profile of BSYZ. Extracts of BSYZ were orally administered to mice with SCOP-induced memory impairment for two weeks. The learning and memory abilities were determined by the Morris water maze test. The oxidant stress-related indices, such as activity of superoxide dismutase (SOD) and levels of glutathione (GSH) and malondialdehyde (MDA) were examined in hippocampus of SCOP-treated mice. The cell death ratio was assessed by TUNEL staining, while apoptotic-related proteins including Bcl-2 and Bax were determined by immunofluorescent staining and western blot analysis. Caspase-3 was determined by western blot analysis. Consequently, a chromatographic condition, which was conducted at 35°C with a flow rate of 0.8 ml/min on the Gemini C18 column with mobile phase of acetonitrile and water-phosphoric acid (100:0.1, v/v), was established to yield common fingerprint chromatography under 203 nm with a similarity index of 0.986 within 10 batches of BSYZ samples. BSYZ at a dose of 2.92 g/kg significantly improved the cognitive ability, restored the abnormal activity of SOD and increased the levels of MDA and GSH induced by SCOP. Moreover, the neural apoptosis in the hippocampus of SCOP-treated mice was reversed by BSYZ by regulating the expression of Bcl-2, Bax and caspase-3. The results demonstrated that BSYZ had neuroprotective effects in SCOP-induced senescence in mice by ameliorating oxidative stress and neuronal apoptosis in the brain, supporting its potential in AD treatment.

Alleviating Effects of Bushen-Yizhi Formula on Ibotenic Acid-Induced Cholinergic Impairments in Rat

This study explored the curative effect and underlying mechanisms of a traditional Chinese medicine compound prescription, Bushen-Yizhi formula (BSYZ), in ibotenic acid (IBO)-induced rats. Morris water maze and novel object recognition tests showed that BSYZ significantly improved spatial and object memory. Brain immunohistochemistry staining showed that BSYZ significantly up-regulated expression of choline acetyltransferase (ChAT) and nerve growth factor (NGF) in the hippocampus and cortex. The protein tyrosine kinase high-affinity receptor TrkA was slightly increased in the hippocampus and cortex, and significantly enhanced in the nucleus basalis of Meynert (NBM) after BSYZ intervention. The immunoreactivity of the p75 low-affinity receptor in BSYZ-treated rats was significantly strengthened in the cortex. Similar expression trends of nerve growth factor (NGF), TrkA, and p75 mRNA were observed in the hippocampus and cortex. Additionally, BSYZ reversed IBO-induced disorders of acetylcholine (ACh) levels, ChAT, and cholinesterase (ChE) in the cortex, which was consistent with the changes in mRNA levels of ChAT and acetylcholinesterase (AChE). Expression of ChAT and AChE proteins and mRNA in the hippocampus was up-regulated, whereas the apoptosis-relative protein cleaved caspase-3 was decreased after administration of BSYZ. Moreover, changes in cell death were confirmed by histological morphology. Thus, the results indicated that the BSYZ formula could ameliorate memory impairments in IBO-induced rats, and it exerted its therapeutic action probably by modulating cholinergic pathways, NGF signaling, and anti-apoptosis. Overall, it is suggested that the BSYZ formula might be a potential therapeutic approach for the treatment of Alzheimers disease (AD) and other cholinergic impairment-related diseases.

(−)-Epigallocatechin-3-gallate induces cancer cell apoptosis via acetylation of amyloid precursor protein

Abstract Epigenetic modifications are involved in cancer pathogenesis, and HDACis are considered potential therapeutic agents. We and others have shown the inhibitory activity of EGCG on HDAC1. But little is known about the effect of EGCG as on epigenetic regulation in cancer. Here, we try to demonstrate that EGCG acts as an HDACi downregulated APP expression, which was pathophysiologically upregulated in cancers and exerts a key role in cancer cell growth. We used PC-12 cells, SK-N-SH cells and primary tumor tissues for our analysis. Male 4-week-old athymic nude mice were used for heterotopic tumor growth assay. We employed Western blotting analysis to detect Bcl-2, Bax, APP, caspase-3, caspase-7, HDAC1 and H4Ac. We used AnnexinV-FITC and TUNEL staining for apoptosis detection. Tumor tissues were examined by immunohistochemical staining. We demonstrated that EGCG suppresses the growth of xenografted adrenal pheochromocytoma. Flow cytometry analysis and TUNEL staining showed that EGCG induced the apoptosis. Treatment with EGCG resulted in decrease in Bcl-2 but increase in Bax and activated caspase-3 and caspase-7. HDAC inhibitor EGCG leaded to hyperacetylated histone H4 by immunofluorescence. EGCG decreased APP levels by immunofluorescence staining and Western blot analysis. Silencing specific to HDAC1 leaded to caspase-3 and caspase-7 activation and cleavage. Our results are the first to demonstrate a functional interaction between EGCG and APP in suppression tumor growth, and provide a new epigenetic effects of EGCG on antitumor.

Neuroprotective effect of β-asarone against Alzheimer’s disease: regulation of synaptic plasticity by increased expression of SYP and GluR1

Aim β-asarone, an active component of Acori graminei rhizome, has been reported to have neuroprotective effects in Alzheimer’s disease. As the underlying mechanism is not known, we investigated the neuroprotective effects of β-asarone in an APP/PS1 double transgenic mouse model and in NG108 cells. Materials and methods APPswe/PS1dE9 double transgenic male mice were randomly assigned to a model group, β-asarone treatment groups (21.2, 42.4, or 84.8 mg/kg/d), or donepezil treatment group (2 mg/kg/d). Donepezil treatment was a positive control, and background- and age-matched wild-type B6 mice were an external control group. β-asarone (95.6% purity) was dissolved in 0.8% Tween 80 and administered by gavage once daily for 2.5 months. Control and model animals received an equal volume of vehicle. After 2.5 months of treatment, behavior of all animals was evaluated in a Morris water maze. Expression of synaptophysin (SYP) and glutamatergic receptor 1 (G1uR1) in the hippocampus and cortex of the double transgenic mice was assayed by Western blotting. The antagonistic effects of β-asarone against amyloid-β peptide (Aβ) were investigated in vitro in the NG108-15 cell line. After 24 hours of incubation, cells were treated with 10 μm Aβ with or without β-asarone at different concentrations (6.25, 12.5, or 25 μM) for an additional 36 hours. The cytotoxicity of β-asarone was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of cell viability, and cell morphology was evaluated by bright-field microscopy after 24 hours of treatment. The expression of SYP and GluR1 in cells was detected by Western blot assay in the hippocampus and brain cortex tissues of mice. Results β-asarone at a high dose reduced escape latency and upregulated SYP and GluR1 expression at both medium and high doses. Cell morphology evaluation showed that β-asarone treatment did not result in obvious cell surface spots and cytoplasmic granularity. β-asarone had a dose-dependent effect on cell proliferation. Conclusion β-asarone antagonized the Aβ neurotoxicity in vivo, improved the learning and memory ability of APP/PS1 mice, and increased the expression of SYP and GluR1 both in vivo and in vitro. Thus, β-asarone may be a potential drug for the treatment of Alzheimer’s disease.

β-Asarone Mitigates Amyloidosis and Downregulates RAGE in a Transgenic Mouse Model of Alzheimer’s Disease

Elevated β-amyloid (Aβ) is a hallmark of Alzheimer’s disease (AD). Recent evidence has suggested that the receptor of advanced glycation end products (RAGE) is a key target for Aβ-induced perturbation in AD, and blockade of RAGE significantly alleviates synaptic injury. Our previous study has suggested that β-asarone could reduce neuronal apoptosis and improve memory deficits in β-amyloid precursor protein and presenilin-1 (APP/PS1) double transgenic AD-model mice. In the present study, we evaluated the effects of β-asarone on amyloidosis in APP/PS1 mice. We found that the survival of neurons of APP/PS1 mice was improved by β-asarone, meanwhile, β-asarone decreased Aβ deposition and down-regulated Aβ1–42 levels in cortex and hippocampus of APP/PS1 mice brain. Interestingly, the level of RAGE was also significantly down-regulated by β-asarone. Our findings suggest that β-asarone might be effective for the treatment of AD, and the decreasing effects of β-asarone on Aβ might associate with its down-regulation of RAGE.