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 induces rat mesenchymal stem cells to differentiate into dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron-like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/ß-tubulin III). It also increased expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine-beta-hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1-12 days. DA steadily increased after treatment with salidroside for 1-6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.

[Effects of polychlorinated biphenyl on the expression of c-fos and c-jun in mesenchymal cells of rats].

OBJECTIVE: To study the effects of polychlorinated biphenyl (PCB126) on the expression of c-fos and c-jun in mesenchymal cells (MSCs) of rats. METHODS: MSCs were separated by percoll cell separating medium (density 1.073 g/cm3). After being cultured and transferred, the cells were divided into a control group, a low-dose group (10(-7) mol/L PCB126) and a high-dose group (10(-6) mol/L PCB126). The cellular growth rate and the expression of c-fos and c-jun protein were analyzed by MTT, immunofluorescence chemical assay, RT-PCR and Western blot. RESULTS: The cellular growth rate in the low-and high-dose group were 13.8% and 19.1% at 12 h, 31.5% and 36.1% at 24 h, 42.5% and 43.6% at 48 h, respectively (P < 0.01). The positive rates of c-fos expression in the low-and high-dose groups were 54.6% and 51.3% at 12 h, and 83.2% and 73.0% at 24 h. The expression of c-fos mRNA detected by RT-PCR was upregulated at 30 min and 1h in PCB126 groups. The expression of c-jun mRNA were much higher in PCB126 groups than that in the control group. The expression of c-fos and c-jun protein was upregulated in the low-and high-dose groups at 12 h and 24 h. CONCLUSION: PCB126 could promote the proliferation of MSCs and upregulate the expression of cancer associated gene c-fos and c-jun. The effect of PCB126 on the function of MSCs might be associated with the abnormal expression of c-fos and c-jun gene.

[Effect of salidroside on rat bone marrow mesenchymal stem cells differentiation into cholinergic nerve cells].

OBJECTIVE: To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs) differentiation into the cholinergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cells for clinical therapy of nervous system diseases. METHODS: BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks,weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 microg/mL) and retinoic acid (5 micromol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular proliferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerve growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), beta-Tubulin III, glial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, beta-Tubulin III, GFAP,brain derived neurotrophic factor (BDNF),and gamma-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot. RESULTS: The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative,which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P < 0.05). RT-PCR results showed that the expression level of NSE,BDNF, beta-Tubulin III,GFAPmRNA were increased in group A at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of beta-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P < 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, beta-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P < 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P < 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P < 0.01), but no significant difference of BDNF was found between groups A and B (P > 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P < 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B. CONCLUSION: Salidroside could induce rat BMSCs differentiate into cholinergic nerve cells in vitro.

[Effects of polychlorinated biphenyl on the expressions of c-fos, c-Myc and beta-catenin in the rat testis].

OBJECTIVE: To study the effects of polychlorinated biphenyl (PCB) on the phenotype of the testis tissue and the testis tissue and the expression c-fos, c-Myc and beta-catenin in the rat testis. METHODS: Forty-five Wistar male rats were divided into a control and three perimental groups, the former fed normally, and the latter with PCB at 0.1, 1 and 10 mg/kg respectively for 90 days. Then the effects of PCB on the phenotype of the testis tissue and the expressions of c-fos, c-Myc and p-catenin were determined by histopathology and immunohistochemistry. RESULTS: Histopathological examinations revealed testis edema, damage of the mesenchymal phenotype, morphological changes of the contorted seminiferous tubules, absence of stromal cells, spermiocytes and prespermatids, and decreased number of sperm. The expressions of c-fos and c-Myc were significantly higher in the 1 and 10 mg/kg PCB groups than in the control and 0.1 mg/kg PCB groups (P < 0.01). The expression of beta-catenin was downregulated in the 0.1 mg/kg PCB group, with significant differences from the other groups (P < 0.01), but it was higher in the 1 mg/kg PCB than in the control and 10 mg/kg PCB groups (P < 0.01). CONCLUSION: PCB causes changes in the phenotype of the testis tissue, and the abnormal expressions of c-fos, c-Myc and beta-catenin are closely related to the PCB-induced testis injury.

[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.

[Effects of polychlorinated biphenyl on bcl-2 and TGFbeta1 expression in rat testes].

OBJECTIVE: To study the effects of polychlorinated biphenyl (PCB) on bcl-2 and TGFbeta1 expression in rat testes. METHODS: Forty male Wistar rats were divided into 4 groups at random: Group A (normal control), Group B (fed on 10(-8) mol/L PBC), Group C (feb on 10(-7) mol/L) and Group D (feb on 10(-6) mol/L). After three months, all the rats were killed, the animal model established, and observations made on the expression of bcl2 and TGFbeta1 in the rat testis using the optical microscope and immunohistochemical techniques. RESULTS: The damage to the structure of the testis was related to the dosage of PCB: the higher the dodage, the more serious the damage. PCB induced the expression of bcl-2 and TGFbeta1. The TGFbeta1 expression was significantly higher in the highest dosage group than in others (P < 0.01 ), and the bcl-2 expression was dramatically higher in Group C than in other groups (P < 0.01). CONCLUSION: PCB can cause injury in rat testes.

[Random amplified polymorphic DNA analysis of the genomes among 7 species of ticks].

OBJECTIVE: To study genomic polymorphic DNA and genetic distance of 7 species of ticks. METHODS: Ticks used in this study were Dermacentor nuttalli, D. silvarum, Haemaphysalis qinghaiensis, H. formosensis, H. punctata, Amblyomma testudinarium, and Ixodes ovatus. DNA extracts of the 7 species of ticks were amplified by random amplified polymorphic DNA (RAPD) and PCR technique using 5 primers with different arbitrary single chain polynucleotide sequences. DNA fingerprint maps were analyzed and the genetic distance among 7 species of ticks were counted. RESULTS: The amplified products of the 7 species of ticks by RAPD all showed their specific DNA band. The average genetic distance among them was 0.71. CONCLUSION: RAPD can differentiate the 7 species of ticks.