The protective effect of fluoxetine hydrochloride on brain tissues of rats with high altitude cerebral edema / 中国药理学通报

Aim To study the protective effect of fluoxetine hydrochloride on brain tissues of rats with simulated high altitude cerebral edema(HACE)and its mechanism.Methods The optimal dosage and time of fluoxetine hydrochloride were determined by the hypoxia tolerance test of mice under normal pressure.The rat model of brain edema at high altitude was established by large-scale low-pressure oxygen chamber.HE staining was used to observe the pathological changes of brain tissues in rats.Microplate reader was used to detect the corresponding indexes of oxidative stress such as malondialdehyde(MDA)level and superoxide dismutase(SOD)activity.The expressions of hypoxia-related proteins HIF-1α,VEGF,MMP-9,AQP4 and SERT were detected by Western blot.Results Compared with the hypoxia model group,after the intervention of fluoxetine hydrochloride,the survival time of mice was prolonged,and the middle dose of fluoxetine(14 mg·kg-1)had the best effect,with an extension rate of 17.78%.The pathological damage of brain was improved,the water content of brain decreased,and the permeability of blood-brain barrier decreased.MDA content in rat brain decreased and SOD activity increased.Western blot results showed that HIF-1α,VEGF,MMP-9,AQP4,SERT protein were significantly down-regulated.Conclusions Fluoxetine has protective effect on rats with brain edema at high altitude,and its mechanism may be related to improving oxidative stress,activating HIF-1α/VEGF/MMP-9 signaling pathway and affecting the expression of SERT protein.SERT may be a potential target for treating brain edema at high altitude.

Low-frequency pulsed electromagnetic fields promote osteoblast mineralization and maturation of rats through the PC2/sAC/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway.@*METHODS@#We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference.@*RESULTS@#The expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation.@*CONCLUSION@#PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.

Protective effect of fluoxetine against hypoxia induced injury on PC12 cells / 中国药理学通报

Aim To study the protective effect of fluoxetine against hypoxia induced injury on PC12 cells. Methods PC12 cells were randomly divided into control group, hypoxia group, and fluoxetine hydrochloride group. The last two groups were put into a hypoxic culture chamber for 18 hours, the cell state was observed under inverted microscope, and cell viability was detected using CCK-8 assay. Reactive oxygen species (ROS) level was evaluated by DCFH-DA. Lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) in cell culture supernatant were evaluated by enzyme labeling method. The expression levels of Bcl-2, Bax and caspase-3 were determined by Western blot. Results Compared with normal group, hypoxia caused obvious damage to PC12 cells. Fluoxetine hydrochloride at 10

Resveratrol and puerarin on peak bone mass in young rats / 中国骨伤

<p><b>OBJECTIVE</b>To compare effects of resveratrol, puerarin and the compounds on peak bone mass in rats.</p><p><b>METHODS</b>Forty SPF Wistar rats weighed 109.45 g to 119.44 g with an average of 115.87 g were selected. After 3 days' adaption, rats were divided into control group (the same volume of distilled water per day), puerarin group(15.4 mg/kg puerarin daily), resveratrol group (8.4 mg/kg resveratrol daily), compound drug group (daily dose of 8.4 resveratrol added 15.4 mg/kg of puerarin) and 10 in each group. The body weight of the rats was monitored at every 7 days and body bone density was measured at every month. All rats were sacrificed after 3 months. The bone mineral density of femur and vertebrae was detected by dual energy X-ray absorptiometry; bone biomechanics, VG staining was used to analyze bone histomorphometry;ELISA was used to detect serum bone metabolic index and microstructure of femur were scanned with Micro-CT scanner.</p><p><b>RESULTS</b>There were no significant differences in body weight among groups during exoeriment. Bone mineral density results showed BMD of femur and vertebrae in the other three groups were significantly increased, and R+P group was significantly higher than PR group and RES group(<0.05) by compared with CON group;three-point bending and compression test results showed compared with CON group, other three groups of femoral and vertebral maximum load values were significantly increased, and P+R group was higher than PR group and RES group, but elastic modulus was not statistically significant. Bone histomorphometry showed that number of trabecular bone in other three groups were significantly increased compared with CON group, separation of trabecular bone were significantly reduced, continuity was improved, and R+P group was significantly better than RES and PR group. The results of Micro-CT scan showed that separation of trabecular bone were significantly reduced, continuity were improved in other three groups, and R+P group was significantly better than RES and PR group. The numbers of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), volume of trabecular bone (BV/TV) in PR group, RES group and R+P group were significantly higher than CON group, but trabecular bone separation (Tb.Sp) was significantly reduced. Serum levels results showed, level of OC in the other three groups were higher than control group(<0.05), content of TRACP 5b decreased, and level of OC in P+R group was significantly higher than PR group and RES group, content of TRACP 5b was no significant change.</p><p><b>CONCLUSIONS</b>Compound of puerarin and resveratrol assigned in a 1:1 ratio could improve bone mineral density and bone mass in young rats, enhance biomechanical properties of bone, promote mineralization and maturation of osteoblasts, inhibit osteoblastic bone resorption, and is better than the role of their respective monomers. The paper showed that traditional Chinese medicine compound medicine will be used as a new way to prevent and treat osteoporosis.</p>

Effect of Compound Medicine of Tanshinone 2A and Resveratrol on Peak Bone Mass in Growing Rats / 中国医学科学院学报

Objective To study the effect of the compound medicine of tanshinone 2A and resveratrol on peak bone mass in growing rats and to explore its possible mechanism,so as to explore anti-osteoporosis mechanisms of new traditional Chinese medicine (TCM) drugs. Methods Totally 40 1-month-old female Wistar rats were randomly divided into tanshinone 2A group,resveratrol group,compound group (tanshinone 2A and resveratrol),and normal control group,with 10 rats in each group. Body weight was measured once every two weeks,and the whole body bone mineral density was measured with dual-energy X-ray monthly. When the whole-body bone mineral density became statistically significant between medication groups and control group,all animals were sacrificed to determine the bone mineral density of vertebrae and right femoral bone. The biomechanical properties of femur and vertebrae were measured by AGS-X series universal test,then the bone morphology was analyzed with Fuchsin picric acid staining. Finally,the levels of tartrate-resistant acid phosphatase 5b and osteocalcin were measured with enzyme-linked immunosorbent assay.Results The body weights were not statistically significant among all groups (P>0.05). The whole-body bone mineral density showed no significant difference (P>0.05) after feeding for 1 month;however,two months later,it was significantly different between medication groups and control group;in particular,the whole-body (P=0.016),femoral (P=0.001),and vertebral bone mineral density (P=0.034),bone trabecular number (P=0.024),thickness (P=0.040),and area (P=0.038) were significantly increased in the compound group,along with the significantly decreased trabecular separation degree (P=0.032). Compared with the control group,the compound group had significantly increased osteocalcin (P=0.033) and tartrate-resistant acid phosphatase 5b (P=0.028) levels in serum.Conclusion The compound of tanshinone 2 A and resveratrol can improve the bone density and bone quality in rats,and such effect is higher than either tanshinone 2 A monomer or resveratrolmonomer.

Effect of Xianling Gubao capsule on bone metabolism in ovariectomized rats / 中国中药杂志

To investigate the effect of Xianling Gubao capsule in preventing postmenopausal osteoporosis, forty-eight female Wistar rats were randomly divided into four groups: sham group (Sham), ovariectomized group (OVX), ethinylestradiol group (EE) and Xianling Gubao capsule group (XLGB). Rats in each group received ovariectomy, except for sham group. The XLGB group received Xianling Gubao capsule at the dose of 378 mg·kg⁻¹·d⁻¹. The dosage of EE group was 200 μg·kg⁻¹·d⁻¹, and OVX and Sham groups were only fed with equal volume of distilled water. All of the rats were put to death two months later. Bone mineral density, bone biomechanics, bone histomorphometry Micro-CT scanning and organ index of vital organs were calculated and pathologically observed. There was no significant difference in the body weight of rats and organ indexes of lung, kidney, heart and spleen in the experimental groups. There was also no significant change in their pathological observation, but the uterine index of OVX group and XLGB group was significantly lower than that of Sham group. According to the results of BMD test, compared with the OVX group, femurs and vertebrae BMD of the other three groups were increased, with statistically significant differences. On the basis of the results of bone biomechanical test, compared with OVX group, the maximum load values of femur and vertebrae of the other three groups were increased, with statistically significant differences, while the change of elastic modulus was not statistically significant. According to the bone histomorphometry results of VG staining, compared with Sham group, the number of trabecular bone was significantly lower than that in OVX group. Compared with OVX group, the number of trabecular bone in EE group and XLGB group was increased, but with no significant difference between EE and XLGB groups. The results of serum biochemical indexes showed that compared with Sham group, osteocalcin (OC) decreased, while tartrate resistant acid phosphatase 5b (TRACP 5b) increased in OVX group, with statistically significant differences. Compared with OVX group, the OC content of XLGB group and EE group increased, while the content of TRACP 5b decreased, with statistically significant differences. On the basis of the results of Micro-CT scanning, the change trends of femur volume BMD, number of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp), bone volume/tissue volume (BV/TV) in the groups were consistent with those of bone histomorphometry. There was no significant change in femoral cortical bone between the two groups. Xianling Gubao capsule can prevent osteoporosis in ovariectomized rats. The possible mechanism is the dual activity of inhibiting bone resorption and improving bone formation.

Optimization of Extraction and Enrichment Process and Anti-hypoxia Activity of Total Triterpenes from Sibiraea Angustata / 中国中医药信息杂志

Objective To optimize the extraction and enrichment process of total triterpenes from Sibiraea Angustata; To study its anti-hypoxia activity. Methods The extraction solvent of total triterpenoids of Sibiraea Angustata was screened by atmospheric pressure anti-hypoxia test. The content of total triterpenes and extraction rate were set as evaluation indexes. The effects of extraction time, extraction times and ratio of material to liquid on extraction were investigated by orthogonal test. The extraction process was optimized. Resin type, concentration of sample, eluent concentration and eluent volume were investigated to optimize the enrichment process of total triterpenes of Sibiraea Angustata. Finally, the anti-hypoxia activity was studied by atmospheric pressure test and acute decompression test. Results The optimum extraction process was as follows: 70% ethanol extraction, the ratio of material to liquid was 1:20, extrat 2 times with 1.5 h for each time, and the content of total triterpenes was 32.25%. The optimum enrichment process was: AB-8-type macroporous adsorption resin, the loading concentration of 2 mg/mL, 80% ethanol 5 BV elution, and total triterpenes content was up to 61.09%; Sibiraea Angustata total triterpenes anti-hypoxia activity test was strong, and had a certain dose dependency. Conclusion The extraction and enrichment process of total triterpenes from Sibiraea Angustata is stable, reproducible and has significant anti-hypoxia activity.

Protective Mechanism of Tempol on Hypobaric Hypoxia-Induced Heart Damage in Mice / 中国药学杂志

OBJECTIVE: To study the effect mechanism of tempol against hypobaric hypoxia-induced heart damage in mice. METHODS: One hundred and ten BALB/c mice were randomly divided into normal control group, hypoxia model group, acetazolamide group and tempol group. After single intraperitoneal injection for 30 min, the mice were exposed to a simulated high altitude of 8 000 m for 12 h. After hypoxic exposure, blood was collected from the eye sockets and separated into serum to measure the activities of lactic dehydrogenase (LDH)and creatine kinase (CK). Then the mice were sacrificed and the content of H2O2 and malondialdehyde (MDA) as well as ATPase and antioxidant enzyme activity in heart were determined. HIF-1, VEGF, Nrf2, and HO-1 were detected by immunohistochemistry. RESULTS: Compared with normal control group, the activities of plasma CK and LDH in hypoxia model group significantly increased. In addition, the content of H2O2 and MDA in hypoxia model group significantly increased while ATPase and antioxidant enzymes activity markedly decreased compared with the normal control group. Moreover, the expression of HIF-1α, VEGF, Nrf2 and HO-1 increased. Prior administration of tempol effectively decreased the activities of plasma CK and LDH as well as the content of H2O2 and MDA in heart tissue. Tempol could increase ATPase and antioxidant enzyme activities and decreased the expression of HIF-1α and VEGF compared with hypoxia model, while it could further increase the expression of Nrf2 and HO-1. CONCLUSION: Tempol has protective effect on heart injury induced by hypobaric hypoxia in mice. Its mechanism may be attributed to the amelioration of energy metabolism, scavenging free radical, improvement of antioxidant enzyme activity the activation of the Nrf2/HO-1 pathway as well as alleviation of oxidative stress.

Establishment of osteoblast primary cilia model removed by chloral hyrate / 中国骨伤

<p><b>OBJECTIVE</b>To establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.</p><p><b>METHODS</b>Three 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.</p><p><b>RESULTS</b>Compared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).</p><p><b>CONCLUSION</b>4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.</p>

Liquid chromatography tandem mass/mass spectrometry for the quantification of fudosteine in human serum without precolumn derivatization

A quantitative analysis method for fudosteine in human serum by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry [HPLC-ESI/MS/MS] was established, which shows high sensitivity and selectivity. The mobile phase composition was 75% 20 mM acetic acid and 25% acetonitril, which was pumped at a flow rate of 0.40 mL/ min. The overall chromatographic run time was approximately 7 min. The autosampler was set with an injection volume of 10 microL. The calibration curve was linear in the concentration range of 0.1-15.0 microg/mL. The coefficient of determination [r] was greater than 0.9998. This method has been fully validated and shown to be specific, accurate and precise. The method was simple, rapid and the sample preparation was minimal. It was successfully applied to the analysis of healthy volunteer

Comparative study on effect of osthole and genistein on peak bone mass in rats / 中国骨伤

<p><b>OBJECTIVE</b>To compare the ability of osthole (OST) and genistein (GEN) in enhancing bone peak bone mass of rats to prevent osteoporosis.</p><p><b>METHODS</b>Thirty-six female one-month-old SD rats of (125 +/- 3) g body weight were randomly divided into three groups, 12 rats in each group, one group was orally administered osthole at 9 mg x kg(-1) d(-1), one group was given genistein at 10 mg x kg(-1) d(-1) and another was given equal quantity of distilled water as the control. The body weight was monitored weekly and the bone mineral density (BMD) of total body was measured every month. All rats were sacrificed after three months, the femoral bone mineral density, the serum levels of osteocalcin (OC) and anti-tartaric acid phosphatase 5b (TRACP 5b) were measured by Elisa. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine.</p><p><b>RESULTS</b>No significant differences were observed between O-treated or GEN group and the control for the food-intake and body weight during three months. However, the rats treated with OST had significant higher BMD for both total body and femur than the control and GEN group. The O-treated rats also had higher level of serum OC and lower level of TRACP 5b. Besides, they owned bigger bone volume/tissue volume, trabecular thickness, trabecular number but smaller trabecular spacing. In the three point bending tests of femurs,they were found to have larger maximum load, the young's modulus and structural model index (SMI).</p><p><b>CONCLUSION</b>Orally administered osthole could efficiently increase the peak bone mass of rats,which provide new ideas for preventing osteoporosis.</p>

Protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats under constant pressure and closed conditions.</p><p><b>METHOD</b>The PESI dosage-dependent experiment for hypoxia rats was conducted under constant pressure and closed conditions by intraperitoneally injecting 125, 250, 500 mg x kg(-1) to finalize that the optimum dosage is the high dose of PESI. Afterwards, 90 Wistar rats were randomly divided into the hypoxic model group, the acetazolamide 250 mg x kg(-1) group and the PESI high dose group. Each group was further divided into three subgroups according to different hypoxia times, with 10 rats in each subgroup. Under the same hypoxia and administration conditions, the rats were sacrificed after 0, 3, 6 h respectively. Their brain samples were collected for common pathological observation and immunohistochemical staining of HIF-1alpha. Real-time RT-PCR was used to detect HIF-1alpha, EPO, HO-1 and Caspase-3 gene expressions. And the Western blot assay was adopted to detect HIF-1alpha protein expression.</p><p><b>RESULT</b>The brain tissues of the hypoxia model group were severely damaged with the increase in the hypoxia time. The acetazolamide group and the PESI high does group were damaged in a much lower degree. According to the gene expression and the Western blot assay, high dose of PESI could inhibit HIF-1alpha expression. According to the pure gene expression test, high dose of PESI could increase EPO and HO-1 mRNA expressions, but inhibit Caspase-3 mRNA expression.</p><p><b>CONCLUSION</b>PESI's protective mechanism for brain tissues of hypoxia rats under constant pressure and closed conditions may be related to its effects in inhibiting HIF-1alpha expression, increasing EPO expression and resisting cell apoptosis.</p>

Comparative study on effect of 8-prenlynaringenin and narigenin on activity of osteoclasts cultured in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the effects of 8-prenylnaringenin (PNG) and naringenin (NG) on the activity and apoptosis of osteoclasts cultured in vitro, in order to study physiological activity of 8-prenyl perssad.</p><p><b>METHOD</b>Osteoclasts were separated from long-limb bones of newly born rabbits, cultured in alpha-MEM containing 10% FBS, and then added with PNG and NG with the concentration of 1 x 10(-5) mol x L(-1). They were stained with TRAP and determined for enzymatic activity with TRAP after 4 d, and analyzed by toluidine blue staining after 7 d. The apoptotic osteoclasts were analyzed by Annexin V-FITC staining after 2, 4, 8, 12, 24, 36, and 48 hours, to observe their apoptosis. Their total RNAs were extracted, and analyzed for TRAP and Cathepsin K expressions by Real-time RT-PCR.</p><p><b>RESULT</b>Compared with the control group, both of the PNG group and the NG group showed much less osteoclasts (TRAP positive cells), lower TRAP activity and TRAP and Cathepin K (CTSK) expression, and smaller number of bone resorption pits and areas. The PNG group show lower indexes than the NG group. Additionally, the PNG group reached the apoptotic peak of osteoclasts at 12 h after drug administration, whereas the NG group reached after 24 h. And the former had more apoptotic cells than the latter.</p><p><b>CONCLUSION</b>8-PNG is much more active than NG in inhibiting the resorption of osteoclasts and inducing apoptosis of osteoclasts. Their only difference lies in 8-prenyl perssad, which is proved to be able to enhance the anti-bone resorption activity of 8-prenylnarigenin.</p>

Effects of icariin and genistein on peak bone mass in rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the effects of icariin (ICA) and genistein (GEN) on rats bone peak mass and thus screen for a drug that can more effectively prevent osteoporosis.</p><p><b>METHODS</b>Totally 36 one-month SD rats were randomly divided into three groups: ICA group [25 mg/(kg·d), intragastric administration], GEN group [10 mg/(kg·d), intragastric administration], and control group (fed with equal volume of distilled water). The body weight was monitored weekly and the bone mineral density of total body was measured monthly. All rats were sacrificed three months later. The femoral bone mineral density and the serum levels of osteocalcin and anti-tartaric acid phosphatase 5b, N-terminal propeptide of type 1 procollagen, and C-terminal propeptide of type 1collagen were measured. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine.</p><p><b>RESULTS</b>The body weight and organ index showed no significant difference among these three groups(P>0.05). No obvious pathological change was found. The bone mineral density was also not significantly different in the first and second months; however, in the third months, the ICA group had significant higher bone mineral density for both total body and femur than those in the control and GEN group (P<0.05). The same trends were found for both femur bone mineral density and whole-body bone mineral density (P<0.05). The ICA group also had significantly higher serum levels of osteocalcin (P<0.05) and lower level of anti-tartaric acid phosphatase 5b(P<0.05). Besides, rats in the ICA group had significantly larger bone volume/tissue volume, trabecular thickness, and trabecular number than the control group, whereas the trabecular spacing and model coefficients were signicantly lower(all P<0.05), which, however, were not significantly different between ICA group and GEN group (P>0.05). Femoral maximum load, Youg's modulus, and yield load were significantly higher in these two groups than in the control group (P<0.05), which, again, were not significantly different between ICA group and GEN group (P>0.05).</p><p><b>CONCLUSION</b>Orally administered ICA is more efficient than GEN in inhibiting resorption and promoting bone formation, and thus can dramatically improve the peak bone mineral density and bone quality.</p>

Comparison of effects of kaempferide and anhydroicaritin on biomineralization of cultured osteoblasts / 药学学报

This study is to compare the effects of kaempferide and anhydroicaritin on biomineralization of rat osteoblasts (ROB) in vitro. Calvarias were dissected aseptically from newborn SD rats, the osteoblasts were obtained by enzyme digestion and were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subculture was performed when cells covered with 90% of the dish. Kaempferide and anhydroicaritin were separately added with final concentrations of 1 x 10(-4), 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) mol x L(-1) under the conditions of osteogenic differentiation. The proliferation was measured by MTT, and the optimal concentration was detected by the ALP activity at the 9th day after osteogenic induction culture. The osteogenic indexes of kaempferide, anhydroicaritin and control group with the optimal concentration were compared. The result showed that the anhydroicaritin at concentration of 1 x 10(-5) mol x L(-1) had significantly promoted the activity of ALP, calcium content and osteocalcin content, increased the number of CFU-F(ALP) and mineralized nodules, enhanced the mRNA level of BMP-2, OSX and Runx-2, which are key genes of osteogenic differentiation, and raised the protein content of collagen-I. However, the kaempferide group had not significantly represented the ability that promoted osteogenic differentiation of ROB. The difference of osteogenic differentiation on ROB between kaempferide and anhydroicaritin was caused by the prenyl group on C-8 of icariin.

The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II / 药学学报

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

Effect of Osthol on the proliferation and differentiate of osteoblasts in vitro / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the effects of Osthol on the proliferation and differentiation of osteoblasts of rats (rat calvarial osteoblasts, ROB) cultured in vitro.</p><p><b>METHODS</b>The neonatal SD rat skull was segregated, and enzyme digestion was used to obtain bone cells which were cultured in MEM containing 10% FBS. The medium was changed every three days, and serial subcultivation was performed when cells covered with 90% of the culture dish. The Osthol was added to 96-well plates with final concentration of 1 x 10(-4) mol/L, 1 x 10(-5) mol/L, 1 x l0(-6) mol/L and 1 x10(-7) mol/L, and MTT method was used to evaluate the proliferation. Differentiation analysis: the alkaline phosphatase (ALP) activity was determined at the 3rd, 6th, 9th, 12th and 15th days separately after osteogenic induction culture. The synthesis of type I collagen was observed using immunohistochemical method at the 8th day. The ALP stain was performed at the 12th day. The alizarin red staining was done and calcified nodules was counted at the 14th day.</p><p><b>RESULTS</b>The Osthol with final concentration of 1 x 10(-4) mo/L inhibit the proliferation of ROB. The Osthol with final concentration of 1 x 10(-5) mol/L had no obvious influence on the proliferation of ROB, but it significantly promoted the activity of ALP, enhanced the synthesis of collagen type I and increased the number of calcified nodules.</p><p><b>CONCLUSION</b>The Osthol with final concentration of 1 x 10(-5) mol/L can promote differentiation and maturation of ROB, which may be active ingredients of Chinese drugs for the osteoporosis prophylaxis.</p>

Effects of rat serum containing total flavonoid extract of Epimedium sagittatum on development of osteoblasts / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of total flavonoid extract of Epimedium sagittatum (TFE) on the proliferation and differentiation of newborn rat calvarial osteoblasts (ROB).</p><p><b>METHOD</b>TFE was supplemented into the culture medium of ROB at 0. 1, 1, 10 and 100 microg x mL(-1) respectively. The serum of rats administered TFES (SRAT) was also added into the medium in a parallel treatment at 2.5%, 5% and 10% respectively. Their effects on cell proliferation and differentiation was studied by MTT and the analysis of osteogenic differentiation marks.</p><p><b>RESULT</b>TFE had no appreciable and on cell proliferation and differentiation at any concentration. However, 2.5% and 5% SRAT stimulated cell proliferation strongly and, 5% SRAT significantly promoted the maturation and function of osteoblast by improving the alkaline phosphatase activity, osteocalcin secretion, calcium deposition and the number of mineralized nodular structures.</p><p><b>CONCLUSION</b>The metabolites of TFE should be the anti-osteoporosis constitutes of Epimedium sagittatum.</p>