Rehmanniae Radix Preparata suppresses bone loss and increases bone strength through interfering with canonical Wnt/ß-catenin signaling pathway in OVX rats.

Rehmanniae Radix Preparata (RRP) improves bone quality in OVX rats through the regulation of bone homeostasis via increasing osteoblastogenesis and decreasing osteoclastogenesis, suggesting it has a potential for the development of new anti-osteoporotic drugs. INTRODUCTION: Determine the anti-osteoporotic effect of RRP in ovariectomized (OVX) rats and identify the signaling pathway involved in this process. METHODS: OVX rats were treated with RRP aqueous extract for 14 weeks. The serum levels of tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa-Β ligand (RANKL), alkaline phosphatase (ALP), and osteoprotegerin (OPG) were determined by ELISA. Bone histopathological alterations were evaluated by H&E, Alizarin red S, and Safranin O staining. Bone mineral density (BMD) and bone microstructure in rat femurs and lumbar bones were determined by dual-energy X-ray absorptiometry and micro-computed tomography. Femoral bone strength was detected by a three-point bending assay. The expression of Phospho-glycogen synthase kinase 3 beta (p-GSK-3ß), GSK-3ß, Dickkopf-related protein 1 (DKK1), cathepsin K, OPG, RANKL, IGF-1, Runx2, ß-catenin, and p-ß-catenin was determined by western blot and/or immunohistochemical staining. RESULTS: Treatment of OVX rats with RRP aqueous extract rebuilt bone homeostasis demonstrated by increasing the levels of OPG as well as decreasing the levels of TRAP, RANKL, and ALP in serum. Furthermore, RRP treatment preserved BMD and mechanical strength by increasing cortical bone thickness and epiphyseal thickness as well as improving trabecular distribution in the femurs of OVX rats. In addition, RRP downregulated the expression of DKK1, sclerostin, RANKL, cathepsin K, and the ratio of p-ß-catenin to ß-catenin, along with upregulating the expression of IGF-1, ß-catenin, and Runx2 and the ratio of p-GSK-3ß to GSK-3ß in the tibias and femurs of OVX rats. Echinacoside, jionoside A1/A2, acetoside, isoacetoside, jionoside B1, and jionoside B2 were identified in the RRP aqueous extract. CONCLUSION: RRP attenuates bone loss and improves bone quality in OVX rats partly through its regulation of the canonical Wnt/ß-catenin signaling pathway, suggesting that RRP has the potential to provide a new source of anti-osteoporotic drugs.

The effect of electro-acupuncture on pro-oxidant antioxidant balance values in overweight and obese subjects: a randomized controlled trial study.

Objective To undertake a randomized controlled trial in 196 obese subjects to examine the effect of electro-acupuncture on serum pro-oxidant antioxidant balance (PAB) values. Methods Subjects received authentic acupuncture (cases) or sham acupuncture (controls) for 6 weeks in combination with a low-calorie diet. In the following 6 weeks, they received the low-calorie diet alone. Serum PAB was measured at baseline, and 6 and 12 weeks later. Results We found that serum PAB values decreased significantly in the group receiving the authentic acupuncture compared to the sham treatment (p<0.001) at week 6, and whilst serum PAB increased significantly (p<0.05) in the second phase of the study, a significant difference between two groups remained at 12 weeks (p<0.05). Conclusions Electro-acupuncture in combination with a low-calorie diet was more effective at reducing serum PAB values in obese subjects compared to diet alone. Further work is required to determine the mechanism by which electro-acupuncture has this effect.

Experimental study on 1,25(OH)2 D3 amelioration of oral lichen planus through regulating NF-κB signaling pathway.

OBJECTIVE: To explore the protective function of vitamin D (VD)/vitamin D receptor (VDR) on the development of oral lichen planus (OLP) and elaborate the underling mechanism of it. METHODS: H&E staining, myeloid peroxidase (MPO) assays, quantitative PCR (qPCR), Western blotting, and Elisa were used to test the human biopsies and serum. QPCR, Western blotting, Elisa, and siRNA transfection were also performed in LPS-induced keratinocytes to observe the functions of vitamin D and VDR. RESULTS: The lack of VDR in the diseased biopsies from OLP patients was associated with activated helper T-cell type 1 (Th1)-driven inflammatory response. Importantly, the status of serum 25-hydroxyvitamin D of OLP patients was reduced consistently. In a cultured cell model, 1,25(OH)2 D3 could downregulate excessive production of pro-inflammatory factors induced by lipopolysaccharide (LPS) in keratinocyte HaCat cells. Mechanistically, even though LPS-induced cytokines in keratinocytes were inhibited both by nuclear factor-κB (NF-κB) inhibitor and by activator protein 1 (AP-1) inhibitor, VDR-dependent 1,25(OH)2 D3 blocked the activation of phosphorylated-NF-κB p65 rather than c-Jun/c-Fos in the presence of LPS stimulation. CONCLUSION: These results suggest that 1,25(OH)2 D3 plays an anti-inflammatory role in OLP by mediating NF-κB signaling pathway but not AP-1 signaling pathway with a VDR-dependent manner, predicting vitamin D supplement may be a potential strategy for the OLP management.

[The differentiation and vicissitude of variety of Aucklandiae Radix].

Aucklandiae Radix is a commonly used Chinese herbal medicine. Historically, the varieties of Aucklandiae Radix for its medicinal use are complicated. Early Radix Aucklandiae was produced domestically, and it began to be imported since the Northern and Southern Dynasties. The two families, four genera, including Saussure Aucklandiae Radix of Compositae, Inula Inulae Radix, Vladimiria Vladimiriae Radix, dali Aucklandiae Radix and Aristolochiaceae Aristolochic aristolochia debilis, are all applied for medicinal purposes. In modern time, there are 3 major classes of crude drugs of Aucklandiae Radix, namely, Aucklandiae Radix, Inulae Radix, root of common Vladimiriae Radix.

PM10 mass concentration and oxidative capacity of moxa smoke.

BACKGROUND: The burning of moxa floss in moxibustion releases moxa smoke containing a substantial amount of particulate matter (PM10) into the environment, which has generated safety concerns about its potential health impact. DESIGN: Plasmid scission assay was performed using PM10 collected from moxibustion clinics. METHODS: PM10 was collected in winter 2012 by burning three types of moxa floss samples in moxibustion simulation clinics, and the resulting PM10 mass concentration was calculated. Oxidative capacity of the PM10 samples was measured by plasmid scission assay and the percentage of DNA damage at dosage 500 µg ml(-1) (D500) was calculated by linear regression analysis. RESULTS: The average PM10 mass concentration of samples A (3 years and 3:1 ratio), B (3 years and 8:1 ratio) and C (10 years and 3:1 ratio) was 273.33, 172.22 and 168.89 µg/m(3), respectively. The D500 oxidative capacity of PM10 was on average 24.25%, 27.83% and 28.07% for samples A, B and C, respectively. No significant difference was found in the PM10-induced oxidative damage by moxa smoke produced from the three types of moxa floss. CONCLUSIONS: PM10 mass concentrations from the three types of moxa floss combustion exceeded internationally recommended levels. Despite so, PM10 mass concentration of moxa smoke was much lower than biomass and coal combustion and similar to that of gas combustion. The oxidative DNA damage induced by individual PM10 in moxibustion environment was lower than that reported in other environments, indicating that moxibustion-derived PM10 might not be as injurious to human health as generally assumed.

Moxibustion attenuates inflammatory response to chronic exhaustive exercise in rats.

Exercise is recognized as an activator to elicit an inflammatory response whilst moxibustion in traditional Chinese medicine has been previously found to modulate immune functioning. However, whether moxibustion can alleviate the inflammatory cytokines response to chronic exhaustive exercise remains unknown. In the present study, rats were randomly assigned to a sedentary control group (Sed), a sedentary moxibustion group (Sed + Moxa), and 2 trained groups- one submitted to a 3-week exhaustive swimming (Trained), and the other a trained moxibustion group (Trained + Moxa). We found that chronic exhaustive exercise significantly increased the serum levels of pro-inflammatory cytokines (IL-1ß, TNF-α, IFN-γ) and the IFN-γ/IL-4 ratio, and decreased the anti-inflammatory cytokines (IL-4, IL-10). Moxibustion treatment markedly reduced the serum levels of IL-1ß, IFN-γ and the IFN-γ/IL-4 ratio, while elevated the IL-4 and IL-10 productions in trained rats. However, TNF-α level was not significantly affected. Our results suggested that an excessive inflammatory response and a potential inflammatory damage may be involved during chronic exhaustive exercise. Moxibustion could attenuate the inflammatory impairment and have an anti-inflammatory effect. The beneficial effects of moxibustion might be mediated by reducing the pro-inflammatory cytokines, increasing the anti-inflammatory cytokines, and modulating the balance between pro- and anti-inflammatory cytokines.

Tat-modified leptin is more accessible to hypothalamus through brain-blood barrier with a significant inhibition of body-weight gain in high-fat-diet fed mice.

Obesity in human was found mainly due to the poor transportation of leptin through brain-blood barrier (BBB), called as leptin resistance. To produce a leptin capable of penetrating BBB, we have added Tat-PTD(9) to the C terminal of leptin to construct a fusion protein. The fusion Tat-leptin and native leptin genes were synthesized by single-step insertion of a polymerase chain reaction and expressed in Escherichia coli BL21 (Rosseta). The expressing products were purified and renatured by Ni-NTA affinity chromatography, and identified by the molecular size in SDS-PAGE gel and by its immunoreactivity to specific antibody with Western-blotting assay. To bio-functionally evaluate the fusion protein, Balb/c mice fed with high-fat diet (HFD) were given Tat-leptin, leptin or saline for 19 days. The immunohistochemical staining showed the increases in positive stains for the leptin in the region of hypothalamus of the HFD mice with either Tat-leptin or leptin as compared to saline group, but the staining intensity and frequency in the group with Tat-leptin were stronger and higher than those in the group with leptin. Furthermore, the most efficiency in preventing the body-weight gain caused by HFD was found in Tat-leptin group among these three groups. These results suggest that Tat-modified leptin may become a great potential candidate for the prevention or therapy of obese patients.

Assessment of genetic stability in tissue-cultured products and seedlings of Saussurea involucrata by RAPD and ISSR markers.

To control the genetic quality during the whole process of tissue culture of the traditional Chinese medicinal plant, Saussurea involucrate Kar. et Kir., DNA polymorphisms and genetic variations were investigated using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. The genetic stability/variation in tissue-cultured products, including three calli, three adventitious shoots, regenerated plantlets and 2 year-old regenerated plantlets cultivated in the planting base in Tianshan Mountain, were assessed compared with 1 year-old and 2 year-old seedlings cultivated in the same planting base using aseptic seedlings as reference. Apparent genetic variation was detected in the 11 type of plant materials. The percentages of polymorphic bands in the RAPD and ISSR analysis were, respectively, 35% and 33%. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 11 type of plant materials were respectively ranged from 0.823 to 0.995 with a mean of 0.878 and 0.825 to 0.974 with a mean of 0.885, which classified the samples into three groups. The similarity coefficient also revealed that differences among three calli were not remarkable by both RAPD and ISSR analysis, and only chemical components and growth properties needed consideration in the screening of callus used for the next redifferentiation studies. But there are remarkable differences among three adventitious shoots analyzed by ISSR markers. Therefore, RAPD and ISSR markers are efficient tools in genetic variation assessment and quality control in plant tissue culture process.

Hydrogenated castor oil nanoparticles as carriers for the subcutaneous administration of tilmicosin: in vitro and in vivo studies.

Tilmicosin-loaded solid lipid nanoparticles (SLN) were prepared with hydrogenated castor oil (HCO) by o/w emulsion-solvent evaporation technique. The nanoparticle diameters, surface charges, drug loadings and encapsulation efficiencies of different formulations were 90 approximately 230 nm, -6.5 approximately -12.5 mV, 40.3 approximately 59.2% and 5.7 approximately 11.7% (w/w), respectively. In vitro release studies of the tilmicosin-loaded nanoparticles showed a sustained release and the released tilmicosin had the same antibacterial activity as that of the free drug. Pharmacokinetics study after subcutaneous administration to Balb/c mice demonstrated that a single dose of tilmicosin-loaded nanoparticles resulted in sustained serum drug levels (>0.1 microg/mL) for 8 days, as compared with only 5 h for the same amount of tilmicosin phosphate solution. The time to maximum concentration (Tmax), half-life of absorption (T(1/2) ab) and half-life of elimination (T(1/2) el) of tilmicosin-loaded nanoparticles were much longer than those of tilmicosin phosphate solution. Tissue section showed that drug-loaded nanoparticles caused no inflammation at the injection site. Cytotoxicity study in cell culture and acute toxicity test in mice demonstrated that the nanoparticles had little or no toxicity. The results of this exploratory study suggest that the HCO-SLN could be a useful system for the delivery of tilmicosin by subcutaneous administration.

Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids.

Mammals cannot naturally produce omega-3 (n-3) fatty acids--beneficial nutrients found mainly in fish oil--from the more abundant omega-6 (n-6) fatty acids and so they must rely on a dietary supply. Here we show that mice engineered to carry a fat-1 gene from the roundworm Caenorhabditis elegans can add a double bond into an unsaturated fatty-acid hydrocarbon chain and convert n-6 to n-3 fatty acids. This results in an abundance of n-3 and a reduction in n-6 fatty acids in the organs and tissues of these mice, in the absence of dietary n-3. As well as presenting an opportunity to investigate the roles played by n-3 fatty acids in the body, our discovery indicates that this technology might be adapted to enrich n-3 fatty acids in animal products such as meat, milk and eggs.

Effects of adenoviral gene transfer of C. elegans n-3 fatty acid desaturase on the lipid profile and growth of human breast cancer cells.

BACKGROUND: Current evidence from both experimental and human studies indicates that omega-6 polyunsaturated fatty acids (n-6 PUFAs) promote breast tumor development, whereas long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) exert suppressive effects. The ratio of n-6 to n-3 fatty acids appears to be an important factor in controlling tumor development. Human cells usually have a very high n-6/n-3 fatty acid ratio because they cannot convert n-6 PUFAs to n-3 PUFAs due to lack of an n-3 desaturase found in C. elegans. MATERIALS AND METHODS: Adenoviral strategies were used to introduce the C. elegans fat-1 gene encoding an n-3 fatty acid desaturase into human breast cancer cells followed by examination of the n-6/n-3 fatty acid ratio and growth of the cells. RESULTS: Infection of MCF-7 cells with an adenovirus carrying the fat-1 gene resulted in a high expression of the n-3 fatty acid desaturase. Lipid analysis indicated a remarkable increase in the levels of n-3 PUFAs accompanied with a large decrease in the contents of n-6 PUFAs, leading to a change of the n-6/n-3 ratio from 12.0 to 0.8. Accordingly, production of the eicosanoids derived from n-6 PUFA was reduced significantly in cells expressing the fat-1 gene. Importantly, the gene transfer induced mass cell death and inhibited cell proliferation. CONCLUSION: The gene transfer of the n-3 fatty acid desaturase, as a novel approach, can effectively modify the n-6/n-3 fatty acid ratio of human tumor cells and provide an anticancer effect, without the need of exogenous n-3 PUFA supplementation. These data also increase the understanding of the effects of n-3 fatty acids and the n-6/n-3 ratio on cancer prevention and treatment.

Dietary isothiocyanates, glutathione S-transferase -M1, -T1 polymorphisms and lung cancer risk among Chinese women in Singapore.

Chinese populations consume a diet relatively high in isothiocyanates (ITCs), a derivative of cruciferous vegetables known to have cancer-protective effects. This class of compounds is metabolized by the glutathione S-transferase family of enzymes, which are also involved in the detoxification of tobacco-related carcinogens such as polycyclic aromatic hydrocarbons and alkyl halides. We evaluated the association between dietary isothiocyanate intake, GSTM1 and GSTT1 polymorphisms, and lung cancer risk in 420 Chinese women: 233 histologically confirmed lung cancer patients and 187 hospital controls. Among these, 58.8% of cases and 90.3% of controls were lifetime nonsmokers. An allele-specific PCR method was used to detect the presence or absence of the GSTM1 and GSTT1 genes in DNA isolated from peripheral blood. Higher weekly intake of ITCs (above the control median value of 53.0 micromol) reduced the risk of lung cancer to a greater extent in smokers [adjusted odds ratio (OR), 0.31; 95% confidence interval (CI), 0.10-0.98] than nonsmokers (OR, 0.70; 95% CI, 0.45-1.11). The inverse association was stronger among subjects with homozygous deletion of GSTM1 and/or GSTT1. Among nonsmokers with GSTM1-null genotype, higher intake of ITCs significantly reduced the risk of lung cancer (OR, 0.54; 95% CI, 0.30-0.95), an effect not seen among those with detectable GSTM1 (OR, 1.07; 95% CI, 0.50-2.29). Our results, in a Chinese female population, are consistent with the hypothesis that ITC is inversely related to the risk of lung cancer, and we show that among nonsmokers this effect may be primarily confined to GST-null individuals. Conjugation and elimination of ITCs is enhanced in GST-non-null relative to -null individuals, such that the GST metabolic genotype modifies the protective effect of ITCs on lung cancer development.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.

[Studies on chemical constituents in fruit of Lycium barbarum L].

OBJECTIVE: To study the chemical constituents in the fruit of Lycium barbarum. METHOD: The chemical constituents were isolated by column chromatography and identified by spectral data. RESULT: The compounds obtained were identified as scopoletin(I), beta-sitosterol(II), p-coumaric acid(III), glucose(IV), daucosterol(V) and betaine(VI). CONCLUSION: Compounds III, IV and V were isolated from Lycium barbarum for the first time.

[The analgesic and antispasmodic effects of guang tong xiao aerosol of TCM].

OBJECTIVE: To observe the analgesic and antispasmodic effects of Guang Tong Xiao Aerosol (GTXA). METHOD: Writhing test and tail-flick of physical stimulation were made to study the analgesic effect on mice and rats. RESULT AND CONCLUSION: GTXA given by gastrogavage in dose of 18.75 g.kg-1 or 12.50 g.kg-1 could markedly raise the pain threshold after chemical stimulation in mice and physical stimulation in rats, and had antispasmodic effects.

[A herbalogical study on traditional Mongolian medicine "lideri"].

By herbalogical study and investigation, "lideri" used by Mongolia doctors in different areas mainly contains 10 species from 4 genera of 4 families, but the quality materials only contains 3 species, Tinospora sinensis (Lour.) Merr., T. cordifolia Miers and T. capillipes Gagnep.