This paper, based on recent 10 years pharmacological experimental studies, reviews the effects of total flavones of epimedium on cardiovascular system, circulatory system, immune system and bone marrow system, etc.

Aim To investigate the effects of retinoic acid (RA) on induction osteroporosis model rats andpreventive effects of Danshengubao.Methods 4-month-old female Sprague-Dawley rats were given RA at 70 mg·kg~(-1)·d~(-1) and were given Danshengubao at different doses at the same time.All rats were treated by oral gavaged for 28 days.The static and dynamic parameters in cancellous bone of the fifth lumbar vertebrae (LV5) were examined and the dynamic changes of the tibial shaft (Tx) were observed with histomorphometrical analyses; the forth lumbar vertebrae (LV4) was used to perform the compression test.Results Compared with control group, biomechanical properties of LV4, the static parameters ( total tissue area, trabecular area, trabecular perimeter) and the dynamic parameters of LV5 were significantly decreased in RA group.Compared with control group, bone formation of Tx was decreased in periosteal surfaces but enhanced in endocortical surfaces in RA group.Compared with RA group, the biomechanical properties of LV4 were increased significantly in low and medium dose of Danshengubao groups.Conclusion sRA can decrease the size and the biomechanical properties of LV, but it can not change the percentage trabecular area. The mechanism may be related to the act that RA can inhibit cancellous bone formation, decrease the modeling of cortical bone in periosteal surfaces and enhance the remolding of cortical bone in endocortical surfaces. Danshengubao can improve biomechanics of LV induced by RA in rats.

BACKGROUND: Prevention of male osteoporosis is attracting more and more attention. OBJECTIVE: To study the effect of etidronate disodium on different skeletal sites by using bone histomorphometry through establishing a castrated rat model of osteoporosis. DESIGN, TIME AND SETTING: A completely randomized grouping controlled animal experiment was performed in the Animal Experimental Center and Department of Bone Biology Laboratory, Guangdong Medical College between October 2002 and September 2006. MATERIALS: Forty 3.5-month-old male Sprague Dawley rats, weighing (299+_22) g, were selected. Etidronate disodium was produced by Chengdu Chemical Pharmaceutical Factory with the batch number of 970101. Methyltestosterone was produced by Guangzhou Pharmaceutical Jacob with the batch number of 990701. METHODS: Rats were randomly divided into five groups: control group, sham operation group, castration group, castration with methyltestosterone group and castration with etidronate disodium group, eight rats in each group. Rats in the control group were sacrificed at the beginning of the study. Rats in the sham operation group underwent skin incision to expose the testis, but not removed. The remaining rats were treated to remove the testis by the method reported in the literature. Rats in the sham operation group and castration group were given normal saline, rats in the castration with methyltestosterone group were given methyltestosterone at 1.8mg/kg/d, rats in the castration with etidronate disodium group were given etidronate disodium at 36mg/kg/d. All of the rats were treated with intragastric administration at 5mL/kg for 90 days. MAIN OUTCOME MEASURES: Bone histomorphometric analysis of the proximal tibial metaphysis (PTM), tibial shaft (Tx) and the fifth lumbar vertebral body (LVB) were performed in undecalcified sections. RESULTS: Compared with the sham operation group, trabecular area percentage (%Tb.Ar), trabecular number (Tb.N) or trabecular thickness (Tb.Th) of PTM and LVB in the castration group were decreased (P < 0.05 and 0.01 ), while trabecular separation (Tb.Sp), percent labeled perimeter (%L.Pm), bone formation rate (BFR/BV) and osteoclast number per mm (Oc.N) were increased (P < 0.05 and 0.01 ). Tb.Ar of PTM and LVB were increased both in the etidronate disodium group and in the methyltestosterone group compared to those of the castration group, while bone formation indices and bone resorption perimeter were decreased. There was no significant difference between the etidronate disodium and methyltestosterone groups, and no significant change was in Tx in all groups. CONCLUSION: Etidronate disodium can prevent the cancellous bone loss of PTM and LVB in castrated rats, but has no effects on the cortical bone of Tx.

AIM To investigate the inhibitory effect of paeonol on hydrogen peroxide(H2O2)-induced apoptosis in PC12 cells. METHODS The injury model in PC12 cells was generated by H2O2 treatment. The cell viability was determined using methylthiazolyl tetrazolium reduction assay. Apoptotic cells and reactive oxygen species (ROS) were measured by flow cytometry. Lactate dehydrogenase (LDH) activity and malonyldialdehyde (MDA) content were measured by spectroscope respectively. RESULTS After PC12 cells were treated with H2O2 (100 μmol*L-1) for 10 h,its viability obviously decreased, and apoptotic cells, LDH release into the culture media, ROS and MDA contents in PC12 cells significantly increased. When the cells were pretreated with paeonol (12, 25 and 50 μmol*L-1)for 1 h prior to incubation with H2O2, its viability was greatly increased, and apoptotic cells, LDH release, ROS and MDA contents significantly decreased. CONCLUSION Paeonol protects PC12 cells from H2O2-induced apoptosis and this effect is probably achieved through its antioxidative action.

BACKGROUND: It has been reported that a combination of estrogen and progestin has a protective synergistic effect on osteoporosis with only little side effects.OBJECTIVE: This study was designed to investigate the effect of a combination of norethisterone and ethinyl estradiol (EE) on bone mass in ovariectomized rats.DESIGN: This study was a randomized controlled experiment.SETTING:It was conducted at the Department of Pharmacology of Guangdong Medical University.MATERIALS: Twenty-four specific pathogen free (SPF) unmated SD rats were selected, aging 4 and half months and weighing 230±15 g.METHODS: The experiment was conducted in the Department of Pharmacology of Guangdong Medical College from May to November 2002.These rats were randomly divided into 3 groups: pseudo-operation group, ovariectomy group and compound norethisterone group, each containing 8 rats. For the former two groups, ethanol solution (volume fraction=0.056), at a dose of 5 mL/(kg.d), was administered by gavage. While for compound norethisterone group, 60μg/(kg·d) norethisterone and 3.5μg/(kg·d) EE were given by gavage (according to the dosage for human, which was 20-35 μg EE combined with norethisterone). Duration of treatment was 90 days for all the animals. Then their tibias were removed. Employing a fullyautomatic imaging analysis system, osteoclasts and the relevant dynamic and static parameters reflecting secondary trabeculaes formation region in proximal tibias were measured. Respectively, the humeral samples were removed and employing the palsma emission spectrograph of full-spectrum direct reading, calcium content and hydroxyproline content in bone samples were measured. Meanwhile, urine calcium and hydroxyproline concentrations were examined as well.MAIN OUTCOME MEASURES: ①The trabecular area (Th. Ar), trabecular thickness (TbkTh), trabecular number (Tb.N) and trabecular separation (Tb. Sp) and the changes in static parareters of perimeters of osteoclasts were investigated. Variance in percent labeled perimeter (L. Pm ％), mineral apposition rate (MAR) and bone formation rate (BFR/BV) were also calculated. ②Changes in serum alkaline phosphatase (AKP), calcium and hydroxyproline contents in bone and urine were all measured.RESULTS: All the 24 rats entered the analysis procedure. Compared to pseudo-operation group, for the ovariectomy group, Tb. Ar and Tb.N decreased, Tb. Sp increased and osteoclast perimeter significantly increased (P＜0.01). Addtionally, the bone formation markers increased apparently with an increase in L. Pm ％ and MAR (P＜0.05) and a significant increase in BFR/BV (P＜0.01). Compared with the ovariectomy group, for the compound norethisterone group,the bone mass and the Tb.N increased, marked by an increase of 82％ in Tb. Ar and an increase of 83％ in Tb.N (P＜0.05), and the Tb.Sp decreased, marked by a decrease of 51％ (P＜0.05). Meanwhile, there was a decrease of 52.5％ in osteoblast perimeter (P＜0.01), an increase in organic bone matrix and a decrease in urine hydroxyproline (P＜0.05).CONCLUSION: A combination of estrogen and progestin has a protective synergistic effect on ovariectomized rats with osteoporosis, and it is capable of increasing the organic bone matrix without significant inhibitory effects on bone formation. The experimental dosage of the compound was calculated according to the clinical dosage, 20-35 μg estrogen combined with a progestin, which will yield optimal protective effects on bone sometimes.

BACKGROUND: Long-term large dose application of glucocorticoid can cause osseous loss and even femoral head necrosis,which is one of the reasons of pharmaceutical damages. Researches on its intervention have practical significance.OBJECTIVE: To study the osteoporosis induced by long-term large dose administration of glucocorticoid, and investigate the preventive effects of compound danshen(CD) in male rats.DESIGN: A randomized and controlled study by employing experimental animals as subjectsSETTING: An Experimental Animal Center, a Central Laboratory and an Institute of Pharmacology of a Medical CollegePARTICIPANTS: The study was conducted in the Experimental Animal Center, the Central Laboratory and the Institute of Pharmacology of Guangdong Medical College between 2002 and 2003. Totally 40 SD rats were employed.INTERVENTION: SD rats were treated with prednisone(2.7 mg/kg per day) by oral gavages and CD including danshen huangqi , baishu and yinyanghuo at dose of 2.5 g/kg per day,5.0 g/kg per day or 10.0 g/kg per day respectively,once a day for 12 weeks. At the experimental endpoint,the impacts of long term large dose (beyond physiologic dose) application of glucocorticoid on bone metabolism and the preventive effects of CD were observed through the measurement of the static and dynamic indicators for bone growth in un-decalcified superior tibia,the detection of Ca2 + and hydroxyproline contents in ulna,and the length and width of thighbone.MAIN OUTCOME MEASURES: Principal consequences: the impacts of CD on quantitative static and dynamic parameters of osseous morphology in rats with prednisone-induced osteoporosis; Secondary consequences: the comparison of the impacts of CD on bone biochemical indictors and femoral physical indicators in rats with prednisone-induced osteoporosis.RESULTS: In glucocorticoid control group (GC group),bone mass significantly decreased(P<0. 01); as indicated by bone morphological indicators,the number of bone trabecula[(1.98±0.20) / mm]and the percentage of bone trabecular size [(8.83 ±0.98)%] significantly reduced; the ratio of osteogenesis rate at bone surface (8.91±3.97) /neogenesis bone trabacular size to total bone trabecular size(332. 8±142.5)/neogenesis bone trabecular size to bone size(29.6±13.2) significantly decreased; bone absorption perimeter significantly increased(P<0. 01); osseous content in ulna reduced[ (0. 155±0. 01) g]; and femoral length[ (32.64±0.51) mn]significantly shortened (P<0. 05) . But in CD group,CD had certain preventive effects on bone injury induced by prednisone while there was no significant difference among each subgroup with different dose.CONCLUSION: Long-term application of prednisone can significantly inhibit bone growth and induce bone loss. CD has favorablepreventive effects on bone loss through its promotion of osteogenesis and inhibition of osteoclast bone resorption.

Objective To construct human apolipoprotein O (apolipoprotein O, ApoO) expression vector and obtain recombinant fusion protein thioredoxin (Trx)-ApoO by pET prokaryotic expression system. Methods The ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was cloned into pET-32a(+) vector and sequenced. The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21 (DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside (IPTG).The fusion protein was purified by Ni-NTA resin. Results The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid. ApoO cDNA gene fragment was induced by IPTG, and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide (SDS-PAGE). Conclusion Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.

AIM: To observe the changes of different parts of bones in rats 90 days after ovariectomized (OVX). METHODS: Twenty 4.5 month old virgin female Sprague Dawley rats were randomly divided into 2 groups: sham group and OVX group. Rats in sham group were sham operated, while rats in OVX group were bilaterally OVX. Rats in two groups were treated with 5 ml?kg -1 ?d -1 ethanol for 90 days. Parameters of the proximal tibial metaphysis (PTM), tibial shaft (Tx) and the fifth lumbar vertebral body (LVB) were analyzed by bone histomorphometry method. RESULTS: %Tb.Ar and Tb.N were decreased by 80.5 % and 76.1 %, Tb.Sp and Oc.N were increased by 468.0 % and 356.6 %, and MAR and BFR/BV were increased by 43.9 % and 95.9 %, respectively, in PTM. The changes in LVB were not remarkable as those in PTM. %Tb.Ar and Tb.Th were decreased by 35.0 % and 31.0 %, while Oc.N and BFR/BV were increased by 106.9 % and 126.8 %, respectively. The cortical bone and marrow areas of tibial shaft did not change in Tx, but bone formation parameters (%P LPm, %E LPm) were increased. CONCLUSION: After OVX for 90 days, high bone turnover osteopenia model is duplicated successfully in rats. Different parts of the bones have different reaction to ovariectomization in rats.

Aim To investigate the preventive effects of misoprostol on osteoporosis in aged ovariectomized(OVX)rats.Methods Female,10-month-old SD rats were ovariectomized(OVX)and,2 months later,were treated with misoprostol or controls for 2 months.The static and dynamic parameters in trabecular bone of the forth lumbar vertebrae(LV4)were examined with histomorphometrical analyses;the fifth lumbar vertebrae(LV5)was used to perform the compression test.Results Compared with the data from the sham-operated rats,the percent trabecular area and elastic modulus significantly decreased in OVX rats.Correspondingly,the bone break load and break stress decreased of post OVX was compared with those of sham-operated rats.Misoprostol increased the percent trabecular area by 21.6% compared with OVX rats,but it couldn't meet the statistical significance.Misoprostol enhanced the break load and elastic modulus compared with OVX rats.Conclusion Misoprostol can improve biomechanics of bone in ovariectomized osteoporosis rats.

AIM: To study the effects of epimedium pubescens flavonoids (EF) on the skeleton in ovariectomized rats. METHODS: Forty 4.5-month-old Sprague-Dawley female rats were randomly divided into 4 groups. Rats in sham group were sham-operated and treated by daily oral gavage with vehicle. Rats in other three groups were bilaterally ovariectomized (OVX) and treated by either daily oral gavage with vehicle, or diethylstilbestrol (DES) at 22.5 ?g?kg~ -1?d~ -1, or EF at 300 mg?kg~ -1?d~ -1 for 90 days. Bone histomorphometric analysis of the proximal tibial metaphysis (PTM), fifth lumbar vertebrae (LV5) and tibial shaft (Tx) was performed in undecalcified sections. The left femur was collected to determine bone weight, contents of calcium (Ca) , phosphorus (P ) and hydroxyproline. The uterine weight and the uterine luminal epithelial thickness (ULET) were determined. RESULTS: A significant increase in contents of Ca and P of femur was found in EF group. A tendency of increase was found in %Tb.Ar of PTM, but no significant change was found in bone histomorphometric parameters of LV5 and Tx in EF group. EF had no effect on uterine weight and ULET. CONCLUSION: EF can prevent OVX-induced bone mineral loss of femur, but does not prevent bone loss of PTM and LV5.