Panax notoginseng saponins promote osteogenic differentiation of bone marrow stromal cells through the ERK and P38 MAPK signaling pathways.

The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. Here, we investigated whether PNS could promote osteogenesis of bone marrow stromal cells (BMSCs) through modulating the MAPK signaling pathways, which are implicated in BMSC osteogenesis. We found that PNS markedly increased the mineralization of BMSCs by alizarin red S assays and stimulate alkaline phosphatase activity of these cells. Additionally, PNS significantly increased the mRNA levels of alkaline phosphatase, core-binding factor a1, and bone sialoprotein while decreasing PPARγ2 mRNA levels. Furthermore, inhibitors of ERK, PD98059, and p38, SB203580 inhibited the osteogenesis-potentiating effects by PNS. PNS stimulated the activation of ERK and p38 as evidenced by increased phosphorylation of these proteins, which was inhibited by PD98059 and SB203580. Our findings indicate that PNS could promote BMSC osteogenesis by activating the ERK and p38 signaling pathways.

Panax notoginseng saponins promotes proliferation and osteogenic differentiation of rat bone marrow stromal cells.

AIM OF THE STUDY: Panax notoginseng saponins (PNS) is the main effective component of Panax notoginseng, have various pharmacologic activities such as antioxidant, anti-inflammatory, and estrogen-like bioactivities, have been shown to be an effective agent on anti-osteoporosis. Bone marrow stromal cells (BMSCs) play a crucial homeostatic role in skeletal modeling and remodeling due to their capability to differentiate into osteooblasts. Whether PNS has effect on osteogenic differentiation of BMSCs are unknown. This study was designed to investigate the effects of PNS on the proliferation and osteogenic differentiation of BMSCs in vitro. MATERIALS AND METHODS: When BMSCs cultivated in the basal medium or the osteogenic induction medium (OS with or without PNS), cell proliferation was analyzed using an MTT assay, the mineralization was assessed using Alizarin red S staining, the alkaline phosphatase activity was measured using a commercial kit, the mRNA level of osteogenic gene and PPARγ2 gene were determined using RT-PCR, the protein level of PPARγ2 was analyzed by Western blotting. RESULTS: BMSCs cultured in the basal medium with PNS caused a significant increase in proliferation. PNS treatment increased ALP activity, Alizarin red S staining and mRNA level of ALP, Cbfa 1, OC, and BSP, whereas decreased the mRNA level and protein expression of PPARγ2 during osteogenic induction. In addition, the effects of PNS treatment were dose-dependent relationship. CONCLUSION: PNS could stimulate BMSCs proliferation and promote their osteogenic differentiation by up-regulation expression of osteogenic marker gene and down-regulation expression of adipogenic marker gene in a dose-dependent manner. Thus, PNS may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Panax notoginseng saponins potentiate osteogenesis of bone marrow stromal cells by modulating gap junction intercellular communication activities.

AIMS: The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. We investigated the effects of PNS on gap junction intercellular communication (GJIC) and osteogenesis-associated genes in rat bone marrow stromal cells (BMSCs). METHODS AND RESULTS: Our MTT assays demonstrated that PNS enhanced BMSC proliferation under basal medium culture in vitro. Alkaline phosphatase (ALP) assays and alizarin Red staining showed that PNS stimulated ALP activity and calcium deposition by BMSCs. Measurement of the traversing of Lucifer yellow through intercellular junctions revealed that PNS significantly stimulated GJIC activities. RT-PCR assays further showed that PNS augmented the increase in the mRNA levels of ALP, core-binding factor a1, and bone sialoprotein while decreasing the mRNA level of PPARγ2. PNS also reduced RANKL levels and increased osteoprotegerin levels. Gap junction inhibitor, 18a-glycyrrhetinic acid, could partially reverse the actions of PNS on BMSCs. CONCLUSIONS: Our findings indicate that PNS could promote osteogenesis of BMSCs by targeting osteogenesis-associated genes, which could be mediated by their actions on GJIC.

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A metabolism in mice.

This study aimed to clarify the effects of single and repeated administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities or expression of some metabolic enzymes of retinoids and the influence of supplemental vitamin A on changed vitamin A homeostasis by TCDD. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight, with or without continuous administration of 2,500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, and 28. In Experiment II, the mice were daily given 0.1 microg TCDD/kg body weight, with or without supplemental 2,000 IU vitamin A/kg body weight, and were killed on day 14, 28, and 42. In both experiments, TCDD significantly decreased the hepatic all-trans-retinol level and increased the hepatic all-trans-retinoic acid (RA) content, increased the mRNA and enzymatic activities of retinal oxidase. In TCDD + vitamin A mice, the all-trans retinol content was significantly higher, and the retinal oxidase mRNA was significantly lower on day 3 or 7 in Experiment I and on day 14 in Experiment II, compared to TCDD-treated mice. The induction of the retinal oxidase may contribute to the decrease in hepatic all-trans-retinol level and the increase in hepatic all-trans-RA caused by TCDD. Supplemental vitamin A might decelerate the effect of TCDD on retinal oxidase mRNA.

Inhibitory effects of vitamin A on TCDD-induced cytochrome P-450 1A1 enzyme activity and expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potent environmental contaminant that produces a wide range of adverse biological effects, including the induction of cytochrome P450 1A1(CYP1A1) that may enhance the toxic effects of TCDD. Several studies indicated that concurrent supplementation of vitamin A could reduce the toxicity, and potentially inhibit CYP1A1 activity (measured as ethoxyresorufin-O-deethylase [EROD] activity). In the present study, we investigated the in vivo effects of vitamin A on EROD activities and the expression of CYP1A1 in the liver of TCDD-treated mice. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight with or without the continuous administration of 2500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, or 28. In Experiment II, the mice were given daily an oral dose of 0.1 mug TCDD/kg body weight with or without supplement of 2000 IU vitamin A/kg body weight, and were killed on day 14, 28, or 42. In both experiments, TCDD caused liver damage and increase in relative liver weights, augmented the EROD activities and CYP1A1 expression, and increased the aryl hydrocarbon receptor (AhR) mRNA expression, but did not alter the AhR nuclear translocator (ARNT) mRNA expression. CYP1A1 mRNA expression and AhR mRNA expression showed a similar time course. The liver damage in TCDD + vitamin A-treated mice was less severe than that in TCDD-treated mice. EROD activities, CYP1A1 expression, and AhR mRNA expression in vitamin A + TCDD-treated mice were lower than those in TCDD-treated mice, indicating that supplementation of vitamin A might attenuate the liver damage caused by TCDD.

[Cloning and analysis of NADP(H) -dependent retinol dehydrogenase/reductase gene: a novel member of short-chain dehydrogenase/reductase].

The total RNA was purified from bovine liver. According to the cDNA sequences of human, mouse and rabbit NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) gene, the gene-specific primers were designed and synthesized. In this study, we cloned the full-length cDNA of bovine liver NRDR by rapid amplification of cDNA ends (RACE) and RT-PCR methods. The bovine NRDR cDNA is 1 266 bp and the ORF, like other NRDR cDNAs, is 783 bp, encoding 260 amino acid residues. The bovine NRDR exhibited the identity in amino acid sequence to those of human, mouse and rabbit NRDR. It contains short-chain dehydrogenase/reductase (SDR) conservative motif and the peroxisomal targeting singal (PTS1) sequence at their C-terminal. In conclusion, the bovine NRDR cDNA was successfully cloned with RACE methods and submitted to GenBank(AF487454), whose nucleotide sequence and the deduced amino acid sequence were analyzed with Bioinformatics, that belongs to a novel peroxisomal SDR superfamily and plays an important role in the rate-limiting step of synthesizing retinoic acid. This is the first report suggesting the SDR participation of mammalian peroxisomers in retinoid metabolism and it provides a reliable foundation to further investigate the biological function of this protein and retinoic acid biosynthesis.

CDNA cloning of a short isoform of human liver NADP (H) -dependent retinol dehydrogenase/reductase and analysis of its characteristics.

In this report we found a new short PCR product when we amplified a 635 bp of NRDR fragment by RT-PCR. With 3'-Race and 5'-Race,we obtained two full-length cDNA sequences from human liver tissue,one 1 261 bp NRDR cDNA,another 1 003 bp NRDR isoform (NRDRiso,GenBank accession number:AY071856). The NRDR gene comprises eight exons and seven introns. The NRDRiso cDNA is produced by alternative splicing of NRDR cDNA, with the removal of 4, 5, 6 exons composed of consecutive 258 bp. The open reading frames of the NRDRiso cDNA predict a single polypeptide of 174 amino acids with the calculated minimum molecular mass of 18.6 kDa.