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.

OBJECTIVE To investigate the effect of berberine on differentiation of rat bone marrow mesenchymal stem cells (MSCs) to adipocytes and its mechanism. METHODS Rat MSCs were isolated and cultured, adipocytic differentiation was induced with adipogenesis-inducing medium (AIM). Cells were assigned into 6 groups:normal control, AIM group, AIM+berberine 0.1, 0.3, 1 and 3 μmol·L-1 groups, respectively. Morphology characteristics of mesenchymal stem cells were observed under an inverted microscope and adipocyte levels were analyzed by oil O staining. Alkaline phosphatase (ALP) activity was detected using p-nitrophenyl phosphate as a substrate. The cell survival was determined by MTT assay. Expressions of peroxisome proliferator activated receptor γ (PPARγ), fatty acid binding protein (aP2) and CCAAT enhancer-binding protein α (C/EBPα) mRNA were detected by semiquantitative RT-PCR. RESULTS Compared with normal control group, MSCs adipogenic differentiation, PPARγ, aP2 and C/EBPα mRNA expression significantly increased in AIM group (P<0.01), ALP activity in AIM group significantly decreased (P<0.01). Compared with AIM group, berberine inhibited MSCs adipogenic differentiation (P<0.01) and berberine 0.1, 0.3, 1 and 3 μmol·L-1 increased ALP activity by 26%, 54%, 81% and 122%, respectively. Berberine 3 μmol·L-1 significantly downregulated PPARγ expression (0.91±0.10 vs 1.34±0.06) (P<0.01), aP2 (1.05±0.10 vs 1.53±0.09) (P<0.01) and C/EBPα mRNA (1.24±0.06 vs 1.54±0.09) (P<0.01). Berberine had no effect on proliferation of MSCs. CONCLUSION Berberine inhibits differentiation of MSCs into adipocytes, which might be closely related to the downregulation of PPARγ, aP2 and C/EBPα mRNA.

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.

Objective To establish the high performance liquid chromatography(HPLC) fingerprint of wuzi yanzong pills. Methods HPLC was performed on Agilent Extend C18 column (250 mmí4. 6 mm,5 μm) with a gradient elution system using acetonitrile:methanol (101)-0. 4% phosphoric acid as the mobile phase. The column temperature was set at 30 ℃ and the flow rate was 1. 0 mL·min-1 . The eluate was detected at the wavelength of 254 nm. Chromatographic peaks were identified by LC-MS method. Results Nine common peaks in wuzi yanzong pill samples were identified by comparing their LC-MS data with those of reference compounds and related reference reports. The HPLC fingerprint of wuzi yanzong pills was finally developed based on the analysis of sixteen batches of samples and their similarities were above 0. 93. Conclusion This method has high precision,stability and repeatability. This study could be used for overall quality assessment of wuzi yanzong pills.

Objective To investigate the hypoglycemic and antioxidation effects of the red alga Porphyridium cruentum in diabetic mice induced by alloxan. Methods The diabetic models were established by intravenous administration of alloxan 70 mg?kg~(-1).Then the diabetic mice randomly were divided into four groups, Among them ,two groups were given ig the algal biomass 600,1200mg?kg~(-1) ,respectively. After 18 days, the contents of blood glucose, plasma lipids and the activities of SOD were detected. Results Compared with the diabetic model, the algal biomass 600,1200mg?kg~(-1) could significantly decrease the blood glucose.A trend towards lower plasma triglyceride level was observed in test groups. In addition the activity of plasma SOD was significantly enhanced in high dose group. Conclusion Porphyridium cruentum could reduce the level of blood glucose and effectively enhance the ability of antioxidation in the diabetic mice induced by alloxan.

AIM: To study the factors that affect bone volume in ovariectomized rats. METHODS: The bone volume and factors were analyzed by the grey system theory method in ovariectomized rats. RESULTS: Serum estradiol was the most important factor for the bone volume, followed by the bone contents of calcium, phosphorus and magnesium. Serum calcium, phosphorus and the bone contents of hydroxyproline were the less important factors for the bone volume. CONCLUSION: Serum estradiol and bone contents of calcium are the most important factors that affect bone volume in ovariectomized rats.KEY WOLDS grey correlative analysis; bone volume; factors; ovariectomized rats; osteoporosis

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.

AIM: To study the correlative changes of bone mineral contents and histomorphometry of lumbar vertebrae in ovariectomized rats by analysis of T-type correlation degree. METHODS: Forty 10.5-month-old virgin female Sprague-Dawley rats were randomly divided into five groups: (1) Basal: at 10.5 mon of age; (2) sham-1:sham-operated at 13 mon of age; (3) OVX-1: ovariectomized at 13 mon of age; (4) sham-2: sham-operated at 16 mon of age; (5) OVX-2:ovariectomized at 16 mon of age. After ovariectomy, all rats were treated orally with NS at 5 ml?kg -1?d -1. At the end-point of study, the undecalcified longitudinal fourth lumbar vertebra (LV4) sections were cut and stained with Goldner's Trichrome for bone histomorphometric analyses. The fifth lumbar vertebra (LV5) was dried with temperature and digested with acid for testing of bone mineral content. Then the effects of bone mineral contents on bone histomorphometry were assessed by analyzing the T-type correlation degree in the Grey system. RESULTS: All degrees of correlation between bone mineral contents and static histomorphometric parameters (trabecular bone volume) (BV/TV), trabecular thickness (Tb.Th) were positive, but for dynamic histomorphometric parameter (BFR/BV), the correlation degrees were negative. The effect of contents of calcium (Ca) and phosphorus (P) on histomorphometric parameters of lumbar vertebrae was much greater than that of the other bone mineral contents. CONCLUSIONS: Analysis of T-type correlation degree can evaluate the correlative changes of bone mineral contents and histomorphometry of lumbar vertebrae in ovariectomized objectively and fairly.

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 determine whether norethisterone in combination with ethinylestradiol can completely prevent bone loss in ovariectomized rats. METHODS Twenty-four Sprague-Dawley female rats at the age of 4.5 months were sham-operated and treated orally with vehicle, or ovariectomized (OVX) and treated orally with either vehicle or combined norethisterone (norethisterone at 60 ?g?kg -1?d -1 and ethinylestradiol at 3.5 ?g?kg -1?d -1) for 90 days. Double in vivo fluorochrome labeling was administrated. The undecalcified longitudinal proximal tibial metaphyseal sections were used for the bone histomorphometric analysis. The humerus and urine calcium contents were assayed by the method of atomic absorption spectrometry. The humerus and urine hydroxyproline contents were assayed by colorimetry. Blood serum alkaline phosphatase (ALP) were tested by BECKMAN auto bio-chemistry analyzer. RESULTS After 90 days post OVX the cancellous bone mass and showed high bone turnover indices. Bone hydroxyproline contents were lost markedly. The ALP activity increased. The urine hydroxyproline contents increased significantly. The combined norethisterone treated group prevented bone lost when compared with OVX. The combined norethisterone were shown to inhibit osteoclasts surface and decrese bone turnover rate. The combined norethisterone can increase hydroxyproline contents and reduce urine hydroxyproline contents significantly from OVX group. CONCLUSION Combining norethisterone can prevent OVX-induced cancellous bone loss in rats.