Targeting of the Sonic Hedgehog pathway by atractylenolides promotes chondrogenic differentiation of mesenchymal stem cells.

Molecules that enhance chondrogenic differentiation in mesenchymal stem cells (MSCs) were identified and isolated using an in vitro Gli reporter gene assay in MSCs incorporating a Sonic Hedgehog (Shh) target. Atractylenolide III, which promoted Gli1-mediated transcriptional activity, was isolated from an ethyl acetate extract of the Rhizoma, Atractylodis macrocephalae. After dehydration, atractylenolide III was transformed to atractylenolide I. Both atractylenolides were confirmed by MS, UV, IR, 1H- and 13C-NMR spectra. Atractylenolide III (which contains -OH at the 8-position) and atractylenolide I (which lacks -OH at the 8-position) were found to effectively promote the activity of the Gli promoter. While the hydroxyl group of atractylenolide III was not essential for the effect of atractylenolide, its effect was dependent on Shh signaling. Phenotypic cellular analysis indicated that atractylenolides induced MSCs to differentiate into chondrocytes, as shown by increased expression of specific chondrogenic markers including collagen II, aggrecan and the cartilage related transcription factor, Sox9. Atractylenolides significantly increased the expression of Shh and its target gene Gli-1, indicating that Shh signaling was activated by atractylenolides. Moreover, inhibition of Shh signaling reduced the effect of atractylenolides on the chondrogenic phenotype. The discovery that atractylenolides induce chondrocytes from MSCs is promising for bony disease therapy.

[Effects and mechanism of Plastrum testudinis extracts on PC12 apoptosis].

OBJECTIVE: To observe the inhibitive effects of Plastrum testudinis Extracts (PTE) on 6-Hydroxydopamine (6-OHDA) induced PC12 cells apoptosis and explore its mechanism. METHODS: PC12 apoptosis model was established by serum starvation and damaged for 24 hours. The cells were randomly divided into four groups:control group, 6-OHDA group, PTE 3, 30 microg/mL group. Cell optical density was determined by MTT; Ratio of cell apoptosis was examined by Annexin V/PI double stain flow cytometry (FCM), and Western blot was applied to detect the BCL-X/L expression. RESULTS: MTT and FCM analysis demonstrated that PTE can elevate PC12 cells viability and reduce their apoptotic ratio in a dose dependent manner. Western blot showed that PTE promoted the expression of BCL-X/L. CONCLUSION: PTE can inhibit the apoptosis of PC12 induced by 6-OHDA in a dose dependent manner, and its mechanism maybe associated partially with up-regulating BCL-X/L signaling pathway.

A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro and in vivo.

BACKGROUND: To date, it has repeatedly been demonstrated that infusing bone marrow-derived stem cells (BMSCs) into acellular nerve scaffolds can promote and support axon regeneration through a peripheral nerve defect. However, harvesting BMSCs is an invasive and painful process fraught with a low cellular yield. METHODS: In pursuit of alternative stem cell sources, we isolated stem cells from the inguinal subcutaneous adipose tissue of adult Sprague-Dawley rats (adipose-derived stem cells, ADSCs). We used a co-culture system that allows isolated adult mesenchymal stem cells (MSCs) and Schwann cells (SCs) to grow in the same culture medium but without direct cellular contact. We verified SC phenotype in vitro by cell marker analysis and used red fluorescent protein-tagged ADSCs to detect their fate after being injected into a chemically extracted acellular nerve allograft (CEANA). To compare the regenerative effects of CEANA containing either BMSCs or ADSCs with an autograft and CEANA only on the sciatic nerve defect in vivo, we performed histological and functional assessments up to 16 weeks after grafting. RESULTS: In vitro, we observed reciprocal beneficial effects of ADSCs and SCs in the ADSC-SC co-culture system. Moreover, ADSCs were able to survive in CEANA for 5 days after in vitro implantation. Sixteen weeks after grafting, all results consistently showed that CEANA infused with BMSCs or ADSCs enhanced injured sciatic nerve repair compared to the acellular CEANA-only treatment. Furthermore, their beneficial effects on sciatic injury regeneration were comparable as histological and functional parameters evaluated showed no statistically significant differences. However, the autograft group was roundly superior to both the BMSC- or ADSC-loaded CEANA groups. CONCLUSION: The results of the present study show that ADSCs are a viable alternative stem cell source for treating sciatic nerve injury in lieu of BMSCs.

[Effects of Niupo Zhibao Pellet on high-mobility group box-1 protein expression in lung tissues of endotoxin shock rats].

OBJECTIVE: To observe the effects of Niupo Zhibao Pellet, a compound traditional Chinese herbal medicine, on high-mobility group box-1 protein (HMGB1) expression in lung tissues of rats with endotoxin shock. METHODS: Thirty SPF Sprague-Dawley rats were randomly divided into control group, lipopolysaccharide (LPS) group and Niupo Zhibao Pellet group. Rats in Niupo Zhibao Pellet group were consecutively administered 7 days with 3 mL (1 g/L) Niupo Zhibao Pellet saline suspension every day by intragastric administration. Endotoxin shock was induced in rats of the LPS and Niupo Zhibao Pellet groups by intravenous injection of LPS (1.5 mg/kg) and intraperitoneal injection of D-galactosamine (100 mg/kg). Expression of HMGB1 in lung tissues was measured by immunohistochemical method with diaminobenzidine (DAB) coloration, fluorescein isothiocyanate (FITC) labeling, and by Western blotting. RESULTS: Expression of HMGB1 in lung tissues in the LPS group was increased and that in Niupo Zhibao Pellet group was higher than that in the LPS group and the control group. HMGB1 was presented in the cytoplasm of positive cells in the LPS group, but in the nucleus of positive cells in the Niupo Zhibao Pellet group. However, HMGB1 was little expressed in the lung tissues of normal rats. CONCLUSION: Niupo Zhibao Pellet can increase HMGB1 expression and locate HMGB1 in the nucleus but not the cytoplasm, which may be one of its mechanisms in reducing endotoxin shock.

(+)-Cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells by activating vitamin D receptor.

In our previous reports, it was revealed that steroids in traditional Chinese medicine (TCM) have the therapeutic potential to treat bone disease. In the present study, an in vitro model of a vitamin D receptor response element (VDRE) reporter gene assay in mesenchymal stem cells (MSCs) was used to identify steroids that enhanced osteogenic differentiation of MSCs. (+)-cholesten-3-one (CN), which possesses a ketone group that is modified in cholesterol and cholesterol myristate, effectively promoted the activity of the VDRE promoter. Phenotypic cellular analysis indicated that CN induced differentiation of MSCs into osteogenic cells and increased expression of specific osteogenesis markers, including alkaline phosphatase, collagen II and Runt-related transcription factor 2. Furthermore, CN significantly increased the expression of osteopontin, the target of the vitamin D receptor (VDR), which indicated that CN may activate vitamin D receptor signaling. Over-expression of VDR or knockdown studies with VDR-small interfering RNA revealed that the pro-differentiation effects induced by CN required VDR. Furthermore, the present study determined that the C-terminal region of the VDR is responsible for the action of CN. Taken together, the present findings demonstrated that CN induced osteogenic differentiation of MSCs by activating VDR. The present study explored the regulation of stem cells by using a series of similar steroids and provided evidence to support a potential strategy for the screening of novel drugs to treat bone disease in the future.

[Effect of Niupo Zhibao Pellet on expression of neuronal nitric oxide synthase in brain of endotoxin-induced shock rats].

OBJECTIVE: To investigate the effect of Niupo Zhibao Pellet (NPZBP) on the expression of neuronal nitric oxide synthase (nNOS) in the brain of endotoxin-induced shock rats. METHODS: SD rats were randomly divided into normal control group, endotoxin-induced shock model group and NPZBP-treated group. Lipopolysaccharide (LPS) (1.5 mg/kg i.v.) and tsD-galactosamine (D-GalN) (100 mg/kg i.p.) were administered to the rats in endotoxin-induced shock model group, as well as to the rats in NPZBP-treated group after seven-day treatment, to induce the shock. The expression of nNOS in the brain of the rats in each of the 3 groups was measured by immunohistochemical methods. RESULTS: In the 3 groups, nNOS immuno-positive cells distributed widely in layer II, III, IV of the cerebral cortex, the molecular layer of hippocampus, the polymorphic layer of the dentate gyrus, the reticular formation of brain stem, and the molecular, granular and Purkinje cell layer of the cerebellar cortex. The number of immuno-positive cells in the NPZBP-treated group was slightly higher than that of the normal control group, and significantly lower than that of the model group (P<0.05) in many regions of the brain, including cerebral cortex, hippocampus, brain stem and cerebellar cortex. CONCLUSION: NPZBP can inhibit the over-expression of nNOS in wide area of the brain in endotoxin-induced shock rats.

ß-Asarone inhibits neuronal apoptosis via the CaMKII/CREB/Bcl-2 signaling pathway in an in vitro model and AßPP/PS1 mice.

ß-Asarone, an active component of the Acori graminei rhizome that has been used as traditional Chinese herb, has been reported to be capable of inhibiting neuronal apoptosis. However, the signaling mechanism underlying the inhibitory effect of ß-asarone has remained elusive. This study was aimed to investigate whether the CaMKII signaling pathway is involved in the ß-asarone mediated neuroprotection. Using PC12 cells and primary cultures of cortical neurons treated with amyloid-ß (Aß)(1-40) or Aß(1-42) peptide, we demonstrated that ß-asarone can protect PC12 cells and cortical neurons and inhibit neuronal apoptosis by activating the CaMKII-α/p-CREB/Bcl-2 pathway. Moreover, CaMKII-α overexpression enhanced the ß-asarone-induced p-CREB-Bcl-2 expression and anti-apoptotic effects. Interestingly, suppression of CaMKII-α by siRNA or a specific inhibitor can significantly reduce the ß-asarone-induced p-CREB and Bcl-2 expression and Aß(1-40) induced neuronal apoptosis in PC12 cells. AßPP/PS1 mice at the age of 3 months and age-matched wild-type mice were intragastrically administered ß-asarone (7 mg/kg/day, 21 mg/kg/day) or a vehicle daily for 4 months. ß-asarone improved cognitive function of the AßPP/PS1 mice and reduced neuronal apoptosis in the cortex of the AßPP/PS1 mice. A significant increase in CaMKII/CREB/Bcl-2 expression was observed in the cortex of the AßPP/PS1 mice treated with ß-asarone. In summary, our observations demonstrated that ß-asarone can inhibit neuronal apoptosis via the CaMKII/CREB/Bcl-2 signaling pathway in in vitro models and in AßPP/PS1 mice. Therefore, ß-asarone can be used as a potential therapeutic agent in the long-term treatment of Alzheimer's disease.

Muscone exerts neuroprotection in an experimental model of stroke via inhibition of the fas pathway.

Identifying small molecules that are neuroprotective against stroke injury will be highly beneficial for treatment therapies. A cell viability assay and gas chromatography-mass spectrometry were used to identify active small molecules in XingNaoJing, which is a well known Chinese medicine prescribed for the effective treatment of stroke. Studies have found that muscone is the active compound that prevents PC12 cell and cortical neuron damage following various injuries. Analysis of apoptosis indicated that muscone inhibited glutamate-induced apoptotic cell death of PC12 cells and cortical neurons. Fas and caspase-8 expression were upregulated following glutamate treatment in cortical neurons, and was markedly attenuated in the presence of muscone. Furthermore, muscone significantly reduced cerebral infarct volume, neurological dysfunction and inhibited cortical neuron apoptosis in middle cerebral artery occluded (MCAO) rats in a dose-dependent manner. Moreover, a significant decrease in Fas and caspase-8 expression in the rat cortex was observed in MCAO rats treated with muscone. Our results demonstrate that muscone may be a small active molecule with neuroprotective properties, and that inhibition of apoptosis and Fas is an important mechanism of neuroprotection by muscone. These findings suggest a potential therapeutic role for muscone in the treatment of stroke.