BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) have been widely accepted by medical investigators due to their advantages including easy obtaining, minimal invasion, with infinite proliferation and multi-differential potential, and without immunological rejection in the autologous transplantation. OBJECTIVE: The goal of this study is to isolate and purify rat bone marrow MSCs in vitro, so as to observe the effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow MSCs.DESIGN: A randomized controlled animal experiment.SETTING: Hip Center, First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.MATERIALS: Forty healthy male SD rats of SPF grade, weighing 170 to 180 g, were provided by the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine. The protocol was performed in accordance with ethics guidelines for the use and care of animals. The involved rats were divided into 4 groups by random digit table with 10 rats in each: normal control group, high-, middle-, and low-concentration groups. METHODS: This study was performed at the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine between January and March 2005. Rat bone marrow MSCs were isolated and purified by Percoll density gradient centrifugation, and cultured in vitro to establish rat bone marrow MSCs culture system. Rats in the high-, middle-, and low-concentration groups were intragastrically administrated with 4.4, 2.2 and 1.1 g/kg serum containing kidney-tonifying and blood-activating Chinese herbs, which equaled to 20, 10 and 5 times of adult dosage, respectively. Rats in the normal control group were intragastrically administrated with purified water for 1 week. One hour after the last administration, 6 mL blood was taken from abdominal aorta of each rat under the aseptic condition. Then, it was centrifuged at 2 000 r/min for 15 minutes, and meanwhile serum was collected. 10% rat serum containing kidney-tonifying and blood-activating Chinese herbs was added to the medium in the high-, middle-, and low-concentration groups, while 10% fetal bovine serum was added in the normal control group. MAIN OUTCOME MEASURES: ① MSCs growth status; ② MSCs morphology was observed by HE staining and Giemsa's staining; ③ MSCs antigen expression was detected by an immunocytochemical method; ④ Effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on MSCs growth.RESULTS: ①The primarily cultured bone marrow MSCs began to adhere to the wall 24 hours later and 80% of them reached the confluence 7 days later. ② MSCs took appearance in long shuttle shape or polygon. These cells were little. Nuclei were located in the middle part of cells or a little deviation. The ratio of nucleus to cytoplasm was a little high. ③CD44 expression was found in the cytoplasm of mononuclear cells, and colored blue. Partial MSCs expressed c-Kit. Their cytomorphology and phenotypic expression have the characteristics of MSCs. ④Three days after serum containing kidney-tonifying and blood-activating Chinese herbal medicine being added to high-, middle-, and low-concentration groups, the number of bone marrow MSCs was dose-dependently increased as compared with that in the normal control group. CONCLUSION: Serum containing kidney-tonifying and blood-activating Chinese herbs promotes the in vitro proliferation of bone marrow MSCs.

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) hold the potential of differentiating into osteoclasts. Polygonatum sibiricum polysaccharide (PSP) may inhibit the differentiation of BM-MNCs into osteoclasts and it is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE: To investigate the effect of PSP on the differentiation of mouse BM-MNCs into osteoclasts induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and bone resorption in vivo. METHODS: Mouse bone marrow-derived macrophages cultured in vitro, the effect of macrophage colony stimulating factor and PSP (5, 10, 20, 40, 80,160, 320, 640, 1280, 2560 mg/L) on the proliferation of mouse BM-MNCs was detected by cell counting kit-8 assay to determine the PSP concentration range; the mouse BMMs were cultured and induced in DMEM medium containing macrophage colony stimulating factor, RANKL and 5, 10, 20, 40, 80,160, 320, 640 mg/L PSP, respectively; those cultured without PSP served as control group. The morphological changes of cells were observed under an inverted microscope.; the number of osteoclasts was detected by tartrate-resistant acid phosphatase staining; the mRNA expression levels of osteoclast-related genes including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 were evaluated by quantitative real-time PCR. A mouse model of calvarial osteolysis induced by lipopolysaccharide was established to receive PSP intervention, and then micro CT scanning, three-dimensional reconstruction and relevants software were used for quantitative analysis of bone volume/volume percentage, trabecular number, trabecular bone spacing and thickness. The number of osteoclasts was identified by tartrate-resistant acid phosphatase staining and quantitative analysis of bone resorption area was conducted. RESULTS AND CONCLUSION: Compared with the control group, the concentration of PSP below 640 mg/L showed no significant effect on the proliferation of BMMs (P > 0.05). Different concentrations of PSP (40-640 mg/L) significantly reduced the number of osteoclasts, osteoclast differentiation and maturation, and the mRNA expression levels of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 TRAP, MMP-9, CtsK and NFATc1 (P < 0.05). Compared with lipopolysaccharide, PSP could effectively alleviate the lipopolysaccharide-induced calvarial osteolysis, and the bone volume/volume percentage, trabecular number, and trabecular bone spacing were significantly decreased (P < 0.05); additionally, the number of osteoclasts and the area of bone resorption were decreased significantly (P < 0.01). To conclude, PSP can inhibit the differentiation and maturation of mouse BMMs to osteoclasts and alleviate lipopolysaccharide-induced calvarial osteolysis.

BACKGROUND:Our previous studies have found that polygonatum sibiricum polysaccharide (PSP) promotes osteogenic differentiation of bone marrow mesenchymal stem cel s (BMSCs) by Wnt/β-catenin signaling pathway, but the molecular mechanism is unclear.OBJECTIVE:To investigate the effect of PSP promoting the osteogenic differentiation via Wnt signaling pathways in BMSCs after LRP5 silencing. METHODS:LRP5 interference vectors were constructed and then transfected into C57BL/6 mouse BMSCs cultured in vitro. The transfection efficiency of cel s was calculated under fluorescence inverted microscope and the expression of LRP5 protein was detected by western blot assay. The osteogenic potential of BMSCs after LRP5-siRNA transfection was analyzed by alkaline phosphatase staining, alizarin red staining and western blot assay. Effect of PSP on the osteogenic differentiation of LIRP5-silenced mouse BMSCs was detected by real-time PCR and dual luciferase assay. RESULTS AND CONCLUSION:Compared with the control group, the mineralization ability, the mRNA expressions of Runx2 and Osterix, and the protein expression of LRP5 were significantly decreased in the LRP5-siRNA group (P<0.05). PSP could promote LRP5-siRNA transfected mouse BMSCs differentiating into osteoblasts and significantly upregulated the expressions ofβ-catenin and Osterixin, and also induced the high expression of luciferase reporter gene (TOPFlash) containing wild type TCF binding sites (P<0.05). To conclude, LRP5 plays an important role in the process of mouse BMSCs differentiating into osteoblasts. PSP can promote the osteogenic differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway independent on LRP5.

Objective To investigate PSP on bone microstructures,Ca,P,OPG and RANKL of osteoporotic rat model.Methods Thirty female rats randomly divided into five groups:Sham,OVX,H-,M-,L-PSP.Sham and OVX were irrigated stomachsaline;PSP solution was gavaged to other groups.After 8-week,bone microstructures of tibial metaphyseal,Ca,P,OPG and RANKL were measured.Results Body weight,Ca,P,RANKL,Tb.Sp of OVX were significantly increased compared to Sham,OPG,BV/TV,Tb.Th,Tb.N decreased.Body weight of H-,M-PSP,Ca and Tb.Sp of PSP,P and RANKL in H-PSP were decreased compared to OVX,OPG in H-,M-PSP,BV/TV,Tb.Th,Tb.N of PSP group increased.The differences were statistically significant (P ＜ 0.05).Conclusion PSP prevents osteoporosis by improving the microstructure of trabecular bone,reducing bone turnover,increasing OPG and reducing RANKL expression.

Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.
Amino Acid Substitution ; Animals ; Arginine ; genetics ; Ganglia, Spinal ; drug effects ; Mutagenesis, Site-Directed ; Mutant Proteins ; pharmacology ; Neurons ; drug effects ; Patch-Clamp Techniques ; Peptides ; chemistry ; genetics ; pharmacology ; Rats ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; drug effects ; Spider Venoms ; chemistry ; genetics ; isolation & purification ; pharmacology ; Spiders ; Tetrodotoxin ; pharmacology

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

Objective To investigate the constitutive characteristics and the change trend of gynecologic malignant tumors in hospitalized patients in Guangxi Zhuang Autonomous Region over the recent 20 years.Methods Clinical data of 8009 in-patients who suffered from gynecologic malignant tumors in 23 hospitals from 1985 to 2004 in Guangxi Zhuang Autonomous Region were analyzed,with respect to the tumor types and change trend.Results(1)The leading 4 types of malignant tumors were cervical cancers, ovarian cancers,endometrial cancers,and malignant trophoblastic tumors according to the constitutive ratios of the tumors.The constitutive ratio of cervical cancer patients rose year by year,from 17.48% during the 1985-1989 period to 49.25% during the 2000-2004 period(P0.05).(2)The occurring age of cervical cancers became younger obviously,from≥60 years old dropped to