Clinical-grade human umbilical cord-derived mesenchymal stem cells improved skeletal muscle dysfunction in age-associated sarcopenia mice.

With the expansion of the aging population, age-associated sarcopenia (AAS) has become a severe clinical disease of the elderly and a key challenge for healthy aging. Regrettably, no approved therapies currently exist for treating AAS. In this study, clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were administrated to two classic mouse models (SAMP8 mice and D-galactose-induced aging mice), and their effects on skeletal muscle mass and function were investigated by behavioral tests, immunostaining, and western blotting. Core data results showed that hUC-MSCs significantly restored skeletal muscle strength and performance in both mouse models via mechanisms including raising the expression of crucial extracellular matrix proteins, activating satellite cells, enhancing autophagy, and impeding cellular aging. For the first time, the study comprehensively evaluates and demonstrates the preclinical efficacy of clinical-grade hUC-MSCs for AAS in two mouse models, which not only provides a novel model for AAS, but also highlights a promising strategy to improve and treat AAS and other age-associated muscle diseases. This study comprehensively evaluates the preclinical efficacy of clinical-grade hUC-MSCs in treating age-associated sarcopenia (AAS), and demonstrates that hUC-MSCs restore skeletal muscle strength and performance in two AAS mouse models via raising the expression of extracellular matrix proteins, activating satellite cells, enhancing autophagy, and impeding cellular aging, which highlights a promising strategy for AAS and other age-associated muscle diseases.

An optimized method for obtaining clinical-grade specific cell subpopulations from human umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are heterogeneous populations with broad application prospects in cell therapy, and using specific subpopulations of MSCs can enhance their particular capability under certain conditions and achieve better therapeutic effects. However, no studies have reported how to obtain high-quality specific MSC subpopulations in vitro culture. Here, for the first time, we established a general operation process for obtaining high-quality clinical-grade cell subpopulations from human umbilical cord MSCs (hUC-MSCs) based on particular markers. We used the MSC-CD106+ subpopulations, whose biological function has been well documented, as an example to explore and optimize the crucial links of primary preparation, pre-treatment, antibody incubation, flow sorting, quality and function test. After comprehensively evaluating the quality and function of the acquired MSC-CD106+ subpopulations, including in vitro cell viability, apoptosis, proliferation, marker stability, adhesion ability, migration ability, tubule formation ability, immunomodulatory function and in vivo wound healing ability and proangiogenic activity, we defined an important pre-treatment scheme which might effectively improve the therapeutic efficiency of MSC-CD106+ subpopulations in two critical clinical application scenarios-direct injection after cell sorting and post-culture injection into bodies. Based on the above, we tried to establish a general five-step operation procedure for acquiring high-quality clinical-grade MSC subpopulations based on specific markers, which cannot only improve their enrichment efficiency and the reliability of preclinical studies, but also provide valuable methodological guidance for the rapid clinical transformation of specific MSC subpopulations.

HGF Mediates Clinical-Grade Human Umbilical Cord-Derived Mesenchymal Stem Cells Improved Functional Recovery in a Senescence-Accelerated Mouse Model of Alzheimer's Disease.

Stem cells have emerged as a potential therapy for a range of neural insults, but their application in Alzheimer's disease (AD) is still limited and the mechanisms underlying the cognitive benefits of stem cells remain to be elucidated. Here, the effects of clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the recovery of cognitive ability in SAMP8 mice, a senescence-accelerated mouse model of AD is explored. A functional assay identifies that the core functional factor hepatocyte growth factor (HGF) secreted from hUC-MSCs plays critical roles in hUC-MSC-modulated recovery of damaged neural cells by down-regulating hyperphosphorylated tau, reversing spine loss, and promoting synaptic plasticity in an AD cell model. Mechanistically, structural and functional recovery, as well as cognitive enhancements elicited by exposure to hUC-MSCs, are at least partially mediated by HGF in the AD hippocampus through the activation of the cMet-AKT-GSK3ß signaling pathway. Taken together, these data strongly implicate HGF in mediating hUC-MSC-induced improvements in functional recovery in AD models.

Neuroprotective effect of ß-asarone against Alzheimer's disease: regulation of synaptic plasticity by increased expression of SYP and GluR1.

AIM: ß-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 ß-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, ß-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. ß-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 ß-asarone against amyloid-ß peptide (Aß) were investigated in vitro in the NG108-15 cell line. After 24 hours of incubation, cells were treated with 10 µm Aß with or without ß-asarone at different concentrations (6.25, 12.5, or 25 µM) for an additional 36 hours. The cytotoxicity of ß-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: ß-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 ß-asarone treatment did not result in obvious cell surface spots and cytoplasmic granularity. ß-asarone had a dose-dependent effect on cell proliferation. CONCLUSION: ß-asarone antagonized the Aß 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, ß-asarone may be a potential drug for the treatment of Alzheimer's disease.

[Thinking and Methods for Demonstrating the Effectiveness of Chinese Herbal Compounds from the Perspective of Pharmacokinetics].

To exert pharmacological effects, no matter therapeutic effect or toxic/side effect, it's necessary to achieve enough plasma concentration. Chinese medical compounds, which contain various ingredients, influence the metabolism of some active ingredients through the interaction of ingredients to improve curative effects or reduce toxic/side effects. Pharmacokinetics can be used to explore how Chinese medical compounds influence the in vivo metabolism of some active ingredients to achieve better curative effects.

(-)-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 Alzheimer's disease (AD). We previously reported that (-)-epigallocatechin-3-gallate (EGCG) acts as an HDAC inhibitor. Here, we demonstrate that EGCG reduced ß-amyloid (Aß) 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 Aß, neprilysin (NEP). We found an association between EGCG-induced reduction in Aß accumulation and elevated NEP expression. Further, NEP silencing prevented the EGCG-induced Aß downregulation. Our findings suggest that EGCG might be effective for treating AD.

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 Alzheimer's disease (AD) and other cholinergic impairment-related diseases.

Bushen­Yizhi formula ameliorates cognition deficits and attenuates oxidative stress­related neuronal apoptosis in scopolamine­induced senescence in mice.

Bushen­Yizhi 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 high­performance liquid chromatography (HPLC) fingerprint was applied to provide a chemical profile of BSYZ. Extracts of BSYZ were orally administered to mice with SCOP­induced memory impairment for two weeks. The learning and memory abilities were determined by the Morris water maze test. The oxidant stress­related indices, such as activity of superoxide dismutase (SOD) and levels of glutathione (GSH) and malondialdehyde (MDA) were examined in hippocampus of SCOP­treated mice. The cell death ratio was assessed by TUNEL staining, while apoptotic­related proteins including Bcl­2 and Bax were determined by immuno-fluorescent staining and western blot analysis. Caspase­3 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 water­phosphoric 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 SCOP­treated mice was reversed by BSYZ by regulating the expression of Bcl­2, Bax and caspase­3. The results demonstrated that BSYZ had neuroprotective effects in SCOP­induced senescence in mice by ameliorating oxidative stress and neuronal apoptosis in the brain, supporting its potential in AD treatment.

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-ß peptide Aß1-42-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 Aß1-42-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 para-meter 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.

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 Alzheimer's 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.