Identifying potential therapeutic targets of mulberry leaf extract for the treatment of type 2 diabetes: a TMT-based quantitative proteomic analysis.

BACKGROUND: Mulberry (Morus alba L.) leaf, as a medicinal and food homologous traditional Chinese medicine, has a clear therapeutic effect on type 2 diabetes mellitus (T2DM), yet its underlying mechanisms have not been totally clarified. The study aimed to explore the mechanism of mulberry leaf in the treatment of T2DM through tandem mass tag (TMT)-based quantitative proteomics analysis of skeletal muscle. METHODS: The anti-diabetic activity of mulberry leaf extract (MLE) was evaluated by using streptozotocin-induced diabetic rats at a dose of 4.0 g crude drug /kg p.o. daily for 8 weeks. Fasting blood glucose, body weight, food and water intake were monitored at specific intervals, and oral glucose tolerance test and insulin tolerance test were conducted at the 7th and 8th week respectively. At the end of the experiment, levels of glycated hemoglobin A1c, insulin, free fat acid, leptin, adiponectin, total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were assessed and the pathological changes of rat skeletal muscle were observed by HE staining. TMT-based quantitative proteomic analysis of skeletal muscle and bioinformatics analysis were performed and differentially expressed proteins (DEPs) were validated by western blot. The interactions between the components of MLE and DEPs were further assessed using molecular docking. RESULTS: After 8 weeks of MLE intervention, the clinical indications of T2DM such as body weight, food and water intake of rats were improved to a certain extent, while insulin sensitivity was increased and glycemic control was improved. Serum lipid profiles were significantly reduced, and the skeletal muscle fiber gap and atrophy were alleviated. Proteomic analysis of skeletal muscle showed that MLE treatment reversed 19 DEPs in T2DM rats, regulated cholesterol metabolism, fat digestion and absorption, vitamin digestion and absorption and ferroptosis signaling pathways. Key differential proteins Apolipoprotein A-1 (ApoA1) and ApoA4 were successfully validated by western blot and exhibited strong binding activity to the MLE's ingredients. CONCLUSIONS: This study first provided skeletal muscle proteomic changes in T2DM rats before and after MLE treatment, which may help us understand the molecular mechanisms, and provide a foundation for developing potential therapeutic targets of anti-T2DM of MLE.

Efficacy and safety of acupuncture for postpartum hypogalactia: protocol for a systematic review and meta-analysis.

INTRODUCTION: Breast milk is recognised as the best natural food for neonates, but many women experience postpartum hypogalactia (PH). Randomised trials have found that acupuncture exert therapeutic effect on women with PH. However, systematic reviews on the efficacy and safety of acupuncture are still lacking; therefore, this systematic review aims to evaluate the efficacy and safety of acupuncture for PH. METHODS AND ANALYSIS: Six English databases (PubMed, Cochrane Library, EMBASE, EBSCO, Scopus, and Web of Science) and four Chinese databases (China National Knowledge Infrastructure, Wan-Fang, Chinese Biomedical Literature and Chinese Scientific Journal) will be systematically searched from their establishment to 1 September 2022. Randomised controlled trials of the efficacy of acupuncture for PH will be reviewed. The study selection, data extraction and research quality evaluation will be conducted independently by two reviewers. The primary outcome is the change in serum prolactin level from baseline to the end of treatment. Secondary results include milk secretion volume, total effectiveness rate, degree of mammary fullness, rate of exclusive breast feeding, and adverse events. A meta-analysis will be performed using RevMan V.5.4 statistical software. Otherwise, a descriptive analysis will be conducted. The risk of bias will be assessed using the revised Cochrane risk-of-bias tool. ETHICS AND DISSEMINATION: This systematic review protocol does not require ethical approval because it does not include private information/data of the participants. This article will be published in peer-reviewed journals. PROSPERO REGISTRATION NUMBER: CRD42022351849.

16S rDNA analysis of osteoporotic rats treated with osteoking.

Introduction. Osteoporosis (OP) is characterized by microstructural degeneration of bone tissue, low bone mass, bone fragility and even brittle fracture (osteoporotic fracture, OPF). OP and OPF are common and there are many disadvantages to the current medications for OP/OPF. Osteoking is a traditional Chinese medicine (TCM) originating from the Yi nationality (Yunnan, China) that has been used to treat bone diseases for decades.Hypothesis/Gap Statement. This study will reveal the changes in the intestinal microbiota of OP rats after 70 days of osteoking treatment.Method. With duplication of sham and OP rats, eight groups were established, with six rats in each group. The intestinal microbiotas were analysed by 16S rDNA sequencing.Results. The results showed that osteoking changed the intestinal microbiota of sham rats and OP rats. The mechanism by which osteoking improves OP is related to the functions of the intestinal microbiota. After 70 days of treatment with osteoking, the contents of Pseudonocardia, Pedomicrobium, Variovorax, Niastella and Actinosynnema were decreased in OP rats. The functions of the above intestinal microbiota related to iron metabolism affected calcifediol and 25(OH)D, and measuring these bone metabolic indicators is required for further study.Conclusion. Osteoking changes the intestinal microbiota to improve OP, and further study which reveals these intestinal microbiota and mechanism is needed.

Symptoms Based on Deficiency Syndrome in Traditional Chinese Medicine Might Be Predictor of Frailty in Elderly Community Dwellers.

BACKGROUND: The most widely used frailty phenotype and frailty indexes are either time-consuming or complicated, thus restricting their generalization in clinical practice; and therefore, an easier and faster screening tool is needed to be developed. OBJECTIVE: To select sensitive symptoms in traditional Chinese medicine (TCM) and study whether they can improve the risk prediction of frailty. METHODS: This is a cross-sectional study enrolling 2249 Chinese elderly community dwellers. Data were collected via face-to-face inquiries, anthropometric measurements, laboratory tests, and community health files. Frailty was the main outcome measure, and it was evaluated by Fried's frailty phenotype (FP). The ordinal logistic regression model was used to identify the factors associated with frailty. The risk assessment plot was used to compare the discriminative ability for frailty among models with and without TCM symptoms. RESULTS: The identified sensitive influential factors for frailty included age, education level, dietary habits, chronic obstructive pulmonary disease, diabetes, cerebral infarction, osteoporosis, cold limbs, lethargy and laziness in speaking and moving, weakness of lower limbs, slow movement, dry mouth and throat, and glazed expression. The risk prediction for "frailty cumulative components ≥1" was not significantly increased, while for "frailty cumulative components ≥2", a new model developed with the above selected TCM symptoms had a higher AUC than the baseline model without it (0.79 VS 0.81, P=0.002). And the NRI and IDI for the new model were 41.4% (P=0.016) and 0.024% (P=0.041), respectively. CONCLUSION: This research might provide an easier and faster way for early identification and risk prediction of frailty in elderly community dwellers.

Ginsenoside Rg1 improves cognitive capability and affects the microbiota of large intestine of tree shrew model for Alzheimer's disease.

Ginsenoside Rg1 (Rg1) is traditional Chinese medicine with neuroprotective activity. Previous studies have demonstrated that Rg1 improves Alzheimer's disease (AD) and alters gut microbiology, but its mechanism remains to be elucidated, and thus far, its use in the treatment of AD has not been satisfactory. The present study investigated the improvement effects of Rg1 and its association with the microbiota of the large intestine. Following treatment with Rg1 in AD tree shrews, the treatment group demonstrated significantly shorter escape latency and crossed a platform more frequently in a water maze test. Western blotting demonstrated that Rg1 inhibited the expression of ß-secretase 1, while increasing microtubule-associated protein 2 and Fox-3 in the hippocampus. Immunohistochemical analysis revealed that Rg1 decreased the expression of amyloid ß, tau phosphorylated at serine 404 and pro-apoptotic factor Bax, while increasing the expression of Bcl-2 in the hippocampus and cortex. High throughput sequencing of 16S rRNA demonstrated that Rg1 altered the microbiota abundance of the large intestine. In conclusion, Rg1 affected the expression of apoptosis proteins, possessed a neuroprotective effect and may have a close association with the microbiota of large intestine by significantly reducing the abundance of Bacteroidetes and increasing the energy requirement of tree shrews.

Osteoking downregulates Mgp in an osteoporotic fracture rat model.

OBJECTIVE: To investigate the effectiveness of osteoking, a Traditional Chinese Medicine originating from Yi nationality, against osteoporosis (OP) and osteoporotic fracture (OPF), and to elucidate its mechanism of action. METHODS: An osteoporotic fracture rat model was established; animals were divided into three treatment groups: parathyroid hormone, osteoking and 0.9%NaCl. After 4, 8 and 12 weeks of treatment, serum and bone tissues were collected. Enzyme-linked immuno sorbent assay, x-ray, histopathological evaluation and proteomics were used. Proteomics and GO annotation were performed based on identified peptides. The relative network was obtained from the STRING database and verified by polymerase chain reaction and Western blotting. RESULTS: After osteoking treatment, the bone mineral density (BMD) increased with time in the osteoking group. At week 12, the BMD and bone mineral salt content of the osteoking group were 4.5% and 20.6% higher than those of the negative control group, respectively. Furthermore, the body weight followed the order of positive control group > osteoking group > negative control group, with significant differences among the groups (P < 0.05). Micro-CT analysis of femur sections revealed that the bone surface/volume ratio was significantly higher in the osteoking group than that in the negative control group. X-ray images demonstrated that the osteoking group showed clear callus. Moreover, high-voltage micro-CT demonstrated a massive cortical bone accumulation in the osteoking group. The gray values of callus in the osteoking group were higher than those in the negative group. From week 4 to 12, the serum bone alkaline phosphatase level increased by 49.6% in the osteoking group and the serum propeptide of type Ⅰprocollagen level decreased by 80.6%. Alizarin red staining demonstrated that the calcium deposition in the osteoking group was higher than that in the negative control group. Notably, the expression of Mgp, a key osteogenesis inhibitor, was lower in the osteoking group compared with the negative control group. Moreover, Sparc, bone morphogenetic protein-2 and Bglap expression was higher in the osteoking group through activation of the transforming growth factor-receptor activator of nuclear factor κB Ligand pathway. CONCLUSION: Osteoking treatment increased bone quality and promoted calcium deposition. The results suggest that osteoking inhibits Mgp through the TGF-ß/RANKL pathway to improve OP/OPF.

Osteoking improves OP rat by enhancing HSP90­ß expression.

Osteoporosis (OP) is a chronic bone disease that affects individuals worldwide. Osteoporosis is primarily asymptomatic, and patients with OP suffer from pain, inconvenience, economic pressure and osteoporotic fracture (OPF). Osteoking, a Traditional Chinese Medicine compound that originates from the Yi ethnic group, has been used for a number of years to treat fractures. In our previous study, osteoking exhibited therapeutic effects on rats with OPF by promoting calcium deposition. Based on bioinformatics and network pharmacology analyses of a component­target­disease database, heat shock protein HSP 90­ß (HSP90­ß), also known as HSP90­ß, was identified to be a key target of osteoking in OP. High HSP90­ß expression levels were observed in osteoporotic rats and rat bone mesenchymal stem cells (rBMSCs) following osteoking treatment. After 12 weeks of administration in vivo, there was increased bone mineral density (BMD) (P<0.05), increased bone alkaline phosphatase (P<0.05), and improved bone microstructure in the osteoking group compared with those of the negative control group. In vitro, increased calcium deposition in rBMSCs was observed after 4 weeks of osteoking treatment. These results suggest that the mechanisms of osteoking are closely associated with HSP90­ß and activate the bone morphogenetic protein (BMP) signalling pathway, primarily through BMP­2. Osteoking treatment improves OP in rats by enhancing HSP90­ß expression.

Mulberry leaf reduces inflammation and insulin resistance in type 2 diabetic mice by TLRs and insulin Signalling pathway.

BACKGROUND: It has been testified that Diabetes mellitus (DM) has a close association with chronic inflammation and Toll-like Receptors (TLRs), and DM could be prevented by mulberry leaf. Therefore, a hypothesis came into being that mulberry leaf could ameliorate proinflammation and insulin resistance (IR) through TLRs and insulin signalling pathways. METHODS: Water extracts of mulberry leaf (WEM) was given to diabetic mice by gavage for 10 weeks, and the diabetic mice was injected with low-dose streptozocin, fed with high-fat and high-sugar diet. Oral glucose tolerance tests (OGTTs) were conducted. At the same time, homeostasis model assessment of insulin (HOMA-IR) and the level of the inflammatory factor, tumour necrosis factor-α (TNF-α) was measured. The expressions of critical nodes of TLRs and insulin signalling pathway were also examined. RESULTS: WEM contributed to a significant decrease in fasting blood glucose, AUC from the investigation of OGTTs and HOMA-IR. The levels of the inflammatory factor, tumour necrosis factor-α (TNF-α) also declined. Moreover, WEM suppressed the expression of TLR2, myeloid differentiation primary-response protein 88 (MyD88), tumour-necrosis-factor receptor-associated factor 6 (TRAF6), nuclear factor kappa B (NF-κB) in the skeletal muscle. WEM could up-regulate the expression of insulin receptor (InsR) and insulin receptor substrate 1 (IRS1), and down-regulate the phosphorylation of IRS1 in adipose tissue. CONCLUSION: Through this study, a conclusion could be made that WEM mitigates hyperglycemia, IR, and inflammation through the interactions among TLR2 signalling pathway, insulin signalling pathway and TNF-α.

Anti-osteoporosis effects of osteoking via reducing reactive oxygen species.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoking is a Traditional Chinese Medicine consisting of seven types of medicinal herbs originated from Yi nationality and has been used in clinic to treat bone diseases for thousands of years in China. Osteoking shows excellent clinical therapeutic effects on osteoporosis, but it is not clear whether Osteoking could exhibit beneficial effects against osteoporosis via reducing reactive oxygen species (ROS). AIM OF THE STUDY: To explore whether the protective effects of Osteoking on osteoporosis related to ROS, we investigated the effects of Osteoking on osteogenesis differentiation under oxidative stress. MATERIALS AND METHODS: The ovariectomized (OVX) osteoporosis model was established by ovarian surgery, and Osteoking was orally administrated for 84 days. Then the pathogenesis changes of femur were analyzed by Hematoxylin and eosin (H&E) and Masson's trichrome staining. The levels of ROS, malondialdehyde (MDA)and superoxide dismutase (SOD) from rats' serum were further measured. In vitro, mouse pre-osteoblastic MC3T3-E1 cells pre-treated with or without 0.25 mM tert-butyl hydroperoxide (t-BHP) for 2 h were cultured and treated with different dilutions of Osteoking or 20 µM N-Acetyl-L-cysteine for another 24 h, respectively. The intracellular ROS production and markers of oxidative damage of the MC3T3-E1 cells were determined using corresponding kits, respectively. The expressions of alkaline phosphatase (ALP), collagen type I, osteoprotegerin (OPG), TGF-ß1, ß-catenin, receptor activator of nuclear factor-κB ligand (RANKL) and interleukin-6 (IL-6) were further analyzed by qRT-PCR and western blotting upon treatment. RESULTS: Our results showed that Osteoking significantly improving trabecular microstructure by promoting collagen fiber repair and new bone or cartilage regeneration was demonstrated in OVX osteoporosis rat models by micro-CT analysis and histological staining results. Osteoking supplementation reduced the levels of ROS and MDA in OVX rat serum and increased SOD activities. In addition, Osteoking could also up-regulate the proteins expression levels of Runx2, osteocalcin (BGP) and osteoprotegerin (OPG) but reducing the expression of tartrate-resistant acid phosphatase (TRAP). In vitro, Osteoking could effectively inhibit the t-BHP-induced intracellular excessive ROS production and protect cells from oxidative stress in mouse pre-osteoblastic MC3T3-E1 cells. Meanwhile, the mRNA expressions of ALP, collagen type I, OPG, TGF-ß1 and ß-catenin were also up-regulated whereas the RANKL and IL-6 were down-regulated in Osteoking-treated MC3T3-E1 cells. CONCLUSIONS: A novel therapeutic mechanism of Osteoking on osteoporosis reveals by present investigation. Clinic effects of Osteoking to treat osteoporosis are closely related to its ability to reduce oxidative stress.

High-Dose Electron Radiation and Unexpected Room-Temperature Self-Healing of Epitaxial SiC Schottky Barrier Diodes.

Silicon carbide (SiC) has been widely used for electronic radiation detectors and atomic battery sensors. However, the physical properties of SiC exposure to high-dose irradiation as well as its related electrical responses are not yet well understood. Meanwhile, the current research in this field are generally focused on electrical properties and defects formation, which are not suitable to explain the intrinsic response of irradiation effect since defect itself is not easy to characterize, and it is complex to determine whether it comes from the raw material or exists only upon irradiation. Therefore, a more straightforward quantification of irradiation effect is needed to establish the direct correlation between irradiation-induced current and the radiation fluence. This work reports the on-line electrical properties of 4H-SiC Schottky barrier diodes (SBDs) under high-dose electron irradiation and employs in situ noise diagnostic analysis to demonstrate the correlation of irradiation-induced defects and microscopic electronic properties. It is found that the electron beam has a strong radiation destructive effect on 4H-SiC SBDs. The on-line electron-induced current and noise information reveal a self-healing like procedure, in which the internal defects of the devices are likely to be annealed at room temperature and devices' performance is restored to some extent.

Effect of Osteoking on the osteogenic and adipogenic differentiation potential of rat bone marrow mesenchymal stem cells in vitro.

BACKGROUND: Bone damage is a condition that affects the quality of life of patients. Mesenchymal stem cells (MSCs) are important for bone repair. Osteoking is a natural compound in traditional Chinese Medicine used to treat bone diseases; however, the effect of Osteoking on the differentiation of MSCs has not been reported. In this study, we aimed to investigate the effect of Osteoking on the osteogenic and adipogenic differentiation potential of rat bone marrow mesenchymal stem cells (rbMSCs). METHODS: The effects of Osteoking on the proliferation and differentiation of rbMSCs were investigated. Different concentrations of Osteoking were prepared, and its cytotoxicity was evaluated by CCK-8 assay. The expression of osteogenic and adipogenic genes were determined, and several staining methods were used to reveal the osteogenic and adipogenic differentiation potential of rbMSCs. RESULTS: Our results show that appropriate concentrations of Osteoking can enhance osteogenic differentiation of rbMSCs and reduce adipogenic differentiation without any effect on proliferation. This may be related to the changes in related gene expression. CONCLUSION: Osteoking enhances osteogenic differentiation and inhibits adipogenic differentiation of rbMSCs. Therefore, Osteoking may have a therapeutic potential for treating bone disease caused by changes in differentiation function of MSCs.

[Effect of Osteoking on Bone Mineral Density and Dickkopf-1 Protein Levels in Rabbits with Osteoporotic Fracture].

OBJECTIVE: To explore the effect of Osteoking on bone mineral density (BMD) and serum Dickkopf-1 (DKK-1) protein levels in rabbits with osteoporotic fracture (OPF). METHODS: Totally 45 female Japanese big-ear rabbits were randomly divided into the treatment group, the model group, and the blank control group (as the control group), 15 in each group. Bilateral ovaries were ectomized for 24 weeks in the treatment group and the model group. Their left radial factures were induced after confirmed osteoporosis. Rabbits in the treatment group were administered with Osteoking by gastrogavage, once per two days. Equal volume of normal saline was given to rabbits in the model group. The general BMD and serum DKK-1 protein levels were detected before ovariectomy, at week 24 and 48 after ovariectomy. RESULTS: There was significant difference in the general BMD at week 24 after ovariectomy between the model group and the control group, and it was lower in the model group. Compared with the model group, the general BMD significantly increased and serum DKK-1 protein levels significantly decreased in the treatment group after intervention. Serum DKK-1 protein levels were significantly lower after intervention than before intervention in the treatment group. CONCLUSION: Osteoking could improve the BMD of OPF rabbits, and reduce their serum DKK-1 protein levels as well.

[Wnt/ß-catenin signal pathway mediated Salidroside induced directional differentiation from mouse mesenchymal stem cells to nerve cells].

OBJECTIVE: To explore the molecule mechanism of Salidroside inducing directional differentiation of mouse mesenchymal stem cells (MSCs) into neuronal cells. METHODS: The mouse multipotent mesenchymal precursor cell line (D1) was taken as the objective. Cultured MSCs were divided into the negative control group (complete culture solution), the positive control group (containing 1 mmol/L ß-mercaptoethanol), the Salidroside induced group (20 mg/L Salidroside), and the blocked group (20 ng/ ml DKK1, a special inhibitor of Wnt/ß-catenin signal pathway). All cells were inoculated in a 6-well plate (1 x 10(4) cells/cm2) and grouped for 24 h. The expression of p-catenin was detected by fluorescence Immunochemistry in the negative control group, the positive control group, and the Salidroside induced group. The expression of neuron-specific enolase (NSE), beta 3 class III tubulin (ß-tubulin III), nuclear receptor related factor 1 (Nurr1), glial fibrillary acidic protein (GFAP) mRNA, Wnt3a, ß-catenin, low-density lipoprotein receptor-related protein6 (LRP6), Axin mRNA were detected using reverse transcrip- tion PCR (RT-PCR). The expression of ß-catenin and NSE protein were analyzed by Western blot in the negative control group, the positive control group, and the Salidroside induced group. Ca2+ chelating agents (EGTA), L-type Ca2+ channel blocker (Nifedpine), and IP3Ks special inhibitor (LY294002) were used to block Ca2+ signal pathway respectively. The expression of Wnt3a, LRP-6, Axin, glycogen syn- thase kinase (GSK-3), and ß-catenin mRNA were detected by RT-PCR. The ß-catenin protein expression was analyzed using Western blot. RESULTS: Compared with the positive control group, ß-catenin protein was strong positively expressed; the expression of Wnt3a, ß-catenin, LRP6, Axin, NSE, ß-tubulin III, Nurr1 mRNA, and NSE protein were obviously up-regulated in the Salidroside induced group (P < 0.01). Compared with the positive control group and the Salidroside induced group, ß-catenin, NSE, Nurr1, and ß-tubulin III mRNA expression decreased; ß-catenin and NSE protein expression were also down-regulated in the blocked group (P < 0.01). Compared with the Salidroside induced group, the expression of Wnt3a, LRP-6, ß-catenin, and Axin mRNA were down-regulated in the Ca2+ signal blocked group and the salidroside induced group (P < 0.01, P < 0.05). CONCLUSION: Salidroside affected directional differentia- tion of MSCs into neuronal cells through Wnt/ß-catenin and Ca2+ signal pathway.

Effects of OsteoKing on osteoporotic rabbits.

Heng-Gu-Gu-Shang-Yu-He-Ji, also known as OsteoKing, is used as a herbal Traditional Chinese Medicine for the treatment of bone disease, including femoral head necrosis and osteoarthritis. However, whether OsteoKing has anti-osteoporotic properties has remained to be elucidated. The purpose of the present study was therefore to investigate the effects of OsteoKing on ovariectomy-induced osteoporosis in rabbits. Female New Zealand white rabbits were randomly divided into an ovariectomized (OVX) group and a sham-surgery group. The rabbits in the OVX group were subjected to an ovariectomy, while the rabbits in the sham group were subjected to the removal of an area of fat near the two ovaries. Bone mineral density, mechanical properties, serum biochemical parameters and micro-architecture were examined at 150 days post-OVX to characterize the experimental animal model. Once the osteoporotic rabbit model had been established, the rabbits in the OVX group were divided into the following groups: Model group, nilestriol group and 300 and 600 mg/kg OsteoKing groups, containing 16 rabbits in each group. OsteoKing and nilestriol were administered orally. The bone mineral density, mechanical properties, serum biochemical parameters, histology and micro-architecture were examined using dual-energy X-ray absorptiometric analysis, mechanical assessments, enzyme-linked immunosorbent assays, histopathological evaluation and micro-computerized tomography examination following 60 days and 120 days of treatment, respectively. Treatment with OsteoKing led to an elevation in the bone mineral density of the vertebra and serum phosphorus levels, reduced serum concentrations of osteocalcin, procollagen type I N-terminal peptide, tartrate-resistant acid phosphatase 5b and cross-linked N-telopeptide of type I collagen, improved mechanical properties (maximum load, stiffness and energy absorption capacity), and micro-architecture of the lumbar vertebra in the OVX osteoporotic rabbit model following treatment for 120 days. In conclusion, it was demonstrated that OsteoKing is effective in the prevention of estrogen deficiency-associated bone loss and may be a promising drug for the treatment of post-menopausal osteoporosis.

Salidroside induces neuronal differentiation of mouse mesenchymal stem cells through Notch and BMP signaling pathways.

Salidroside (p-hydroxyphenethyl-ß-D-glucoside, SAL), a phenylpropanoid glycoside isolated from a popular traditional Chinese medicinal plant Rhodiola rosea L., possesses multiple pharmacological actions. Previous study showed that SAL could induce rat mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons and induce mouse MSCs D1 to differentiate into neuronal cells. However, the mechanisms of SAL-induced neuronal differentiation of MSCs still need investigation. In this study, we observed the effects of SAL on neuronal differentiation of D1 cells and the possible involvement of Notch and BMP signaling pathways. SAL inhibited the proliferation, induced neuronal phenotypes, and upregulated the expressions of neuronal-specific marker molecules, such as neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (MAP2), and beta 3 class III tubulin (Tubb3/ß-tubulin III) in D1 cells. SAL not only downregulated the expressions of Notch1 and hairy enhancer of split 1 (Drosophila) (Hes1) but also upregulated the expression of Smad1/5/8 and its phosphorylation (p-Smad 1/5/8). The neuronal differentiation effects of SAL on D1 cells were promoted by a Notch signaling antagonist, DAPT, but attenuated by a BMP signaling pathway antagonist, Noggin. Our findings suggest that SAL might be promising in inducing neuronal differentiation of mouse MSCs mediated by both Notch signaling pathway and BMP signaling pathway.

[Salidroside via ERK1/2 and PI3K/AKT/mTOR signal pathway induces mouse bone marrow mesenchymal stem cells differentiation into neural cells].

To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/ mTOR signal pathway inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells, mouse bone marrow-derived mesenchymal stem cell lines D1 cells were used as research object. And they were divided into control groups and salidroside (SD) groups. Different concentrations (5, 25, 50, 100 and 200 microg x mL(-1) of SD were used and SD (100 microg x mL(-1)) was used to induce at different time (0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). The immunofluorescence staining chemical technology, real-time PCR and Western blotting were used to detect the positive rates of NSE, MAP2, beta-Tubulin III, NES, GFAP and the expression levels of beta-Tubulin III, NSE, ERK1/2, AKT. The expression of ERK1/2 and NSE was detected when the ERK1/2 and PI3K/AKT/ mTOR signal pathway was blocked by PD98059 and LY294002. It indicated that the positive rates of NSE, MAP2, beta-Tubulin III, NES and GFAP were gradually enhanced with time increased. The expression level of NSE and beta-Tubulin III protein were significantly higher than those in control groups (P < 0.01). The expression of ERK1/2, AKT mRNA and protein were higher with concentration and time increased. When the ERK1/2 and PI3K/AKT/mTOR signal pathway were blocked, the expression levels of NSE, NES and beta-Tubulin III mRNA and NSE protein were inhibited significantly. It points out that SD can stimulate the ERK1/2 and PI3K/AKT/mTOR signal pathway to promote BMSCs differentiation into neural cells.

[Chinese herbal medicine for patients with mild to moderate Alzheimer disease based on syndrome differentiation: a randomized controlled trial].

BACKGROUND: Alzheimer disease (AD) is a chronic neurodegenerative disease that is characterized by its gradual progression. At present, the cause and mechanism of AD are yet unclear, and there is no effective therapy for treating it. With development of global aging, the prevalence rate of AD is increasing. The life quality of elderly people is affected severely by AD that is ultimately life-threatening. Recently, study on treating AD with traditional Chinese medicine (TCM) has deepened. OBJECTIVE: To explore the therapeutic effects of a syndrome differentiation-based TCM regime in treating patients with mild to moderate AD for improving cognition, and to evaluate the changes in brain function of AD patients observed by resting-state functional magnetic resonance imaging (fMRI) technique. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Adopting the internationally recognized criteria developed by National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer's Disease and Related Disorders Association, the clinical trial was conducted on 131 patients with mild to moderate AD from 5 communities and 7 social welfare institutions. Participants were accepted after informed consent was received, and laboratory tests and a head imaging study were conducted. The patients were randomly divided into Chinese medicine group (CMG) (66 cases) or Western medicine group (WMG) (65 cases). Patients in the CMG were treated monthly with Chinese medicine according to syndrome differentiation. Patients in the WMG were treated with donepezil at a dose of 5 mg once daily. The therapeutic course lasted 48 weeks. MAIN OUTCOME MEASURES: The scores of Mini-Mental State Examination (MMSE), Fuld Object-Memory Evaluation (FOM), Block Design (BD) and Digit Span (DS) were used to evaluate the cognitive function; resting-state fMRI was used for observing brain function. The questionnaires and fMRI were performed before and after treatments. RESULTS: The cognitive functions of the patients in the CMG and WMG were improved after treatment. MMSE score was improved significantly in both groups (P<0.05 or P<0.001). After 48 weeks of treatment, 70.91% patients in the CMG had an improved MMSE score and 20% got worse, however, 55.77% patients in the WMG were improved in MMSE score and 34.62% got worse. Scores of FOM denominator and BD increased significantly in both groups; scores of FOM numerator and DS were also increased in the CMG (P<0.05 or P<0.01). The results of fMRI suggested that both Chinese medicine and donepezil treatment improved the connectivity between posterior cingulated gyrus and specific areas in the brain. The influence range of Chinese medicine primarily impacted on the left parietal lobe, being less than the influence range of donepezil, which primarily affected both sides of frontal lobes. CONCLUSION: TCM treatment based on syndrome differentiation is effective in improving cognitive function of patients with mild to moderate AD and increasing the brain function by increasing connectivity between posterior cingulated gyrus and specific areas in the brain.

[General and light microscope observation on histological changes of femoral heads between SANFH rabbit animal models and it were intervened by Osteoking].

OBJECTIVE: To evaluate the histological changes on the femoral heads of the SANFH rabbit animal models and after it were intervened by Osteoking (herbs of the Yi minority in Yunnan province) using general and light microscope observation. METHOD: A total of 150 New Zealand white rabbits were randomly divided into a non-treatment control group (A group, n = 24), normal rabbits with Osteoking treatment group (B group, n = 24), and the experimental group (n = 102). The experimental group was injected with escherichia coli endotoxin (10 microg x kg(-1)) into auricular vein twice by 24-hour intervals, and prednisolone (20 mg x kg(-1)) was injected into buttock three times by 24-hour intervals to make steroid-induced femoral head necrosis model. At the fifth week, 48 out of 53 rabbits were equally divided into model group (C group, n = 24, models with non-treatment Osteoking) and abnormal rabbits with Osteoking treatment group (D group, n = 24). B group and D group were intragastrically administrated with Osteoking, once every two days. A group and C group were intragastrically administrated with the equal volume of saline. At 8th, 12th and 16th week after model preparation, the femoral head specimens were observed under the general and a light microscope. RESULT: Macroscopic and light microscopic analysis showed that, clear bone necrosis of femoral head was observed in the C group, and a large number of fat cell proliferation was found in the bone marrow cavity. As compared with C group, the damage level of cells in D group was milder, however, the density of bone trabecula from Osteoking treatment was high, and the ratio of bone lacuna was very low. It is also demonstrated that the surface area of bone necrosis was decreased, and the number of cells from adiposities was reduced significantly. The phenomenon of bone necrosis repaired apparently. The morphology of femoral head from A group and B group is normal. CONCLUSION: It suggested that Osteoking could effectively help repair steroid-induced femoral head necrosis in the early stage.

[Effect of mechanical strain on differentiation of mesenchymal stem cells into osteoblasts].

This study sought to elucidate the effect of mechanical strain on the differentiation of mesenchymal stem cells into osteoblasts. Under the conditons of inducing osteoblasts, Immunohistochemical methods and RT-PCR technology were applied in osteogenic supplements medium to detect: (1) the expression of Alkaline phosphatase (ALP), Type I collagen (COL I ), Osterx (Osx) and Osteocalcin (OCN) mRNA, with cyclic strain (3%, 0.5 Hz) applied for 15 min, 30 min, 1 h, 2 h, 4 h, 3 d, 7 d, 14 d; (2) the expression of Osx mRNA and OCN mRNA with 3% strain for 1 h. The results showed: (1) ALP mRNA expression was higher at 7 days; COL I mRNA expression was greater obviously at 7 days and 14 days than that at 3 days and that of the unstrained cells; (2) the expression of Osx mRNA was up-regulated after 15min by strain stimulation,which was significantly increased at 30 min and 1 h in the unstrained cells. The expression of OCN mRNA was not affected in the unstrained cells at 15 min, whereas strain could promote the expression of OCN mRNA at this period. The expression of OCN mRNA was more obviously upregulated in the strained cells at 30 min and 1 h when compared with that in the unstrained cells; (3) the strain (1% and 3%) significantly promoted the expression of Osx mRNA; 10% strain had a little effect on Osx mRNA expression. The expression of OCN mRNA was up-regulated by 3% strain, whereas it had little effect at 1% and 10% strain. In summary, mechanical strain can promote the differentiation of mesenchymal stem cells into osteoblasts.

[Experimental study on osteoking in promoting gene expression of core binding factor alpha 1 in necrotic femoral head of rabbits].

OBJECTIVE: To study the effects of Osteoking in promoting gene expression of core binding factor alpha 1 (cbfalpha 1) in necrotic femoral head of rabbits. METHODS: Rabbit model of femoral head necrosis (FHN) was induced by intravenous injection of lipopolysaccharide (LPS) 10 microg/kg body weight twice with an interval of 24 h and intramuscular injection with methyl prednisone (MPS) 20 mg/kg body weight 3 times. The dynamic changes of cbfalpha 1 gene expression in the femoral head were observed with immunohistochemistry and real-time quantitative PCR. RESULTS: The protein expression of cbfa 1 gene was negative in both model and treatment groups at the 4th week, it turned to weakly positive in the treatment group at the 8th and 12th week but still negative in the model group. The mRNA expression of cbfalpha 1 in the treatment group was 2.87 times that in the model group at the 12th week. There was no significant difference in cbfalpha 1 expression between the normal rabbits with or without Osteoking treatment. CONCLUSION: Osteoking could promote the endogenous cbfalpha 1 expression in the FHN, the effect is better along with the prolonging of the time applied. But it showed no affection on cbfalpha 1 expression in the normal femoral head of rabbits.