[Study on estrogenic effect of genistein and apigenin in vitro].

OBJECTIVE: To detect the estrogenic activity of genistein and apigenin with ER-positive cell line MCF-7 human breast cancer cells. METHOD: MTT method was adopted to study the impact of genistein and apigenin on MCF-7 proliferation in vitro. Real-time RT-PCR method was used to detect their impact on ERalpha, ERbeta, PR and PS2 mRNA expression levels. RESULT: Genistein and apigenin promoted the proliferation of MCF-7. Genistein 1 x 10(-10) mol x L(-1) group showed a significant increase in the expression of ERa mRNA levels or a 17. 76 times more than the control group and a 1.75 times more than the E2 group. Apigenin notably promoted the PR mRNA expression or a 4. 57 times more than the control group and a 1.11 times more than the E2 group. Both of them had different effect in promoting ERalpha, ERbeta, PR or PS2 mRNA. CONCLUSION: Both genistein and apigenin have a strong estrogen-like effect. Although they have different effect in promoting estrogenic response genes (such as ERa, ERbeta, PR and PS2 mRNA), genistein shows a stronger activity than apigenin. It also suggests that the signaling pathways of phytoestrogens showing estrogen-like effect are not completely identical with estrogen pathways. The B-cycle position of flavonoids is one of the key sites to estrogen-like activity, and isoflavones (cycle B on site 3) show stronger estrogen-like activity than flavones (B-cycle lies in site 2).

[Effect of sinusoidal electricity magnetic fields on the proliferation and differentiation of osteoblasts in vitro].

OBJECTIVE: To investigate the effect of sinusoidal electricity magnetic fields (SEMFs) on the proliferation and differentiation of osteoblasts in vitro. METHODS: Calvarial osteoblasts of newborn rats were isolated by enzyme digestion and randomly divided into 3 groups after subculture. Two groups of cells were exposed to 50 Hz 1.8 mT SEMFs for 30 min/d in parallel and vertical, respectively. Those without SEMFs exposure served as control. The cells were observed under the contrast phase microscope each day. After 48 h, cell proliferation was assayed by MTT method. The alkaline phasphatase (ALP) activities and calcium contents were measured after 3, 6, 9, and 12 days. The ALP positive colonies were histochemically stained after 10 days and the calcified nodules were stained by Alizarin Bordeaux after 12 days. Expressions of ALP, bone morphogenetic protein-2 (BMP-2) and Osterix (OSX) mRNA were measured at 0 h, 24 h, 48 h and 96 h. RESULTS: The cells exposed to the SEMFs were arranged in spiral appearance after 3 days. Compared with control, SEMFs inhibited cell proliferation (P < 0.01 or P < 0.05), but enhanced the maturation and mineralization of the osteoblasts. The results showed that SEMFs improved ALP activities, promoted calcium contents, increased calcified nodulues numbers, boosted expressions of ALP, BMP-2 and OSX mRNA. SEMFs with magnetic lines of force in parallel has stronger activities than those in vertical. CONCLUSION: The SEMFs at 1.8 mT and 50 Hz inhibit the proliferation of osteoblasts, but enhance the maturation and mineralization of osteoblasts.

[Study on mechanism of the anti hypoxia effect of genistein on osteoblasts in vitro].

OBJECTIVE: To study the protective effect of genistein on osteoblasts treated with hypoxia. METHODS: Rat osteoblasts were isolated from calvarias of newborn Sprague-Dawly rat by enzyme digestion and hypoxic environment was made by triple-gases incubator. Rat osteoblasts treated with hypoxia for 36 haurs. After 36 hours, cell viability, content of reactive oxygen species (ROS), analysis of cellular cycle and apoptosis, expression of proliferating cell nuclear antigen (PCNA), activity of iNOS and area of calcified nodules were detected. Total RNA was isolated and the gene expression of hypoxia inducible factor-1alpha (HIF-1alpha), BCL-2 and Caspase-3 was investigated by Real Time RT-PCR. RESULTS: Genistein could significantly improve cell viability, percentage of G1 phases, area of calcified nodules and decrease apoptosis rate, ROS content, expression of PCNA, activity of iNOS. Besides, mRNA levels of HIF-1alpha and BCL-2 were enhanced and that of Caspase-3 was inhibited. CONCLUSION: Genistein can protect osteoblasts from hypoxia and enhance osteogenic differentiation significantly.

[Protective effect of genistein on hypoxic injuries of osteoblasts cultivated in vitro].

OBJECTIVE: To investigate the effect of genistein on osteoblast proliferation, cellular cycle, apoptosis and differentiation of osteoblasts cultivated under hypoxia conditions. METHOD: Rat osteoblasts were isolated from calvarias by enzyme digestion and a hypoxic model was established by in a triple-gas incubator. Rat osteoblasts were grouped into the normoxic control group, the hypoxia control group and the hypoxia administration group which was subdivided into Ge-6 group, Ge-5 group and Ge-4 group, to which genistein was administered at doses of 1 x 10(-6), 1 x 10(-5), 1 x 10(-4) mol x L(-1). The cell survival rate, lactic dehydrogenase leakage rate, apoptosis and differentiation of osteoblasts were observed for each group at 3 h after hypoxia, and the gene expression of HIF-1alpha, Bcl-2, Caspase-3 was detected by Real time RT-PCR. Forty-eight hours after hypoxia, osteogenic differentiation markers including alkaline phosphatase activity and nodules were detected. RESULT: Compared with the hypoxia control group, the hypoxia administration group displays a significant increase in the survival rate and a decreased in LDH leakage rate, apoptosis rate and percentage of S + G2 phases. Besides, the mRNA level of HIF-1alpha and Bcl-2 were enhanced, the mRNA level of Caspase-3 was inhibited. CONCLUSION: Genistein has an effect on protecting osteoblasts from hypoxia.

[Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro].

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.

[Experimental studies on the early treatment of soft tissue explosion injury by vacuum-assisted closure].

OBJECTIVE: To investigate the effect on early treatment with vacuum-assisted closure(VAC) to wound healing of acute explosion injury in pigs, and provide a new way for early treatment of battle wounds. METHODS: Eight healthy 3-month Landrace pigs of both sexes with the body mass of (50 +/- 5) kg were selected in the study. Sixteen battle wounds were made by explosion of same type detonator (pattern number: 660929F48840-55, included DDNP 0.3 g, RDX 0.7 g) in hibateral skin of buttock of 8 pigs, which were divided into experimental group and control group (pair wounds of left and right). The raw sufaces were thorough debrided at 3 h after exposure, according to the characteristics of treatment on the battlefield, experimental group was treated with VAC under the pressure of (-50 +/- 5) Kpa after debridement and sterilization and control group was treated with routine dry sterile gauze draping. Results of bacteriology (bacterial counts and the proportion of G+ bacteria) and pathology (HE stain and Masson stain) were detected at every wound before and after treatment. RESULTS: At the 3 days after treatment,the bacterial number in the experimental group was [(7.82 +/- 0.55) x 10(4) ] CFU/g, in control group was [(1.07 +/- 0.14) x 10(6)] CFU/g. There was significant difference between two groups. The proportion of G+ bacteria in experimental group was significantly increased. The raw surface in experimental group was clean with affluent and neoformative granulation tissue, blood vessels and collagen, necrotic tissue decreased obviously by pathological observation. CONCLUSION: VAC could reduce the quantity of bacteria, improve the proportion of G+ bacteria, and promote the formation of granulation tissue and the healing of wound. The VAC for the treatment of battle wounds has a positive effect.

Sinusoidal electromagnetic field stimulates rat osteoblast differentiation and maturation via activation of NO-cGMP-PKG pathway.

Nitric oxide (NO) is an important intracellular and intercellular messenger, critically affecting bone metabolism. The purpose of this research is to investigate whether the effect of sinusoidal electromagnetic field (SEMF) on the differentiation and maturation of osteoblasts is mediated by the NO-cGMP-PKG signal pathway. We examined the impact of SEMF on nitric oxide synthase (NOS) activity, and found that L-NAME, nitric oxide synthase's inhibitor, prevents SEMF-mediated increase in NOS activity and NO levels. We showed that an inhibitor of soluble guanylyl cyclase (ODQ) blocks the increase in cGMP levels triggered by exposure to SEMF. The inhibitor PDE5, which hydrolyzes 3',5'-cyclic-GMP to 5'-GMP, prevents the SEMF's stimulation of PKG activity. We also blocked the NO-cGMP-PKG pathway to determine whether the maturation and mineralization of osteoblasts, stimulated by SEMF, would be inhibited. This was evaluated by measuring alkaline phosphatase (ALP) activity, osterix gene expression and mineralized bone modulus. After treatment with SEMF, the NOS activity increases in comparison with the control group (P<0.01), reaching the highest level after 0.5h. Osterix gene expression, ALP activity and mineralized bone nodules in the SEMF experimental group also increase significantly. However, these effects are partially blocked in the L-NAME treated cultures. Surprisingly, all the osteogenic markers in the SEMF+L-NAME group were slightly higher than in the control culture, but lower than in the cells exposed to SEMF only. We conclude that the NO-cGMP-PKG signal pathway is activated by SEMF treatment, the stimulatory effect of SEMF on the differentiation and mineralization of osteoblasts is attenuated when the pathway is blocked.