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试驾体验MG51.5T
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14-07-25 13:07来源：作者：Cherry Yang
2014年，MG全球车主俱乐部已成立84个年头。深入了解他们，你会发现MG的车主有一些共同的特征，比如热爱运动和冒险，再比如，享受一群志同道合的人在一起时的精神满足等等。
纵观中国，作为世界上规模最大的汽车市场，也慢慢开始出现一批批忠实的MG粉丝。虽然这跟欧洲很多国家相比要晚了很多，但随着众多全新车型的引入，这股力量似乎越来越强。近日有机会试驾搭载1.5T Hyperboost中冷涡轮增压发动机的MG5，让我对MG粉丝们的狂热多少有些认同和理解。
外观延续英式美学设计
与老款MG5相比，涡轮增压版本的外观和内饰变化不大，继续延续英式美学设计理念。但如果你认为这种&凌厉&的设计风格仅仅符合时尚的要求那就错了，凭借黄金两厢车身比例带来的超大后排乘坐空间和行李空间，让我在装运四个大尺寸全家福相框的时候得心应手，毫不费力。
动力升级 带来赛道般快感
虽然作为女性，但对于动力的要求可一点都不低。此次试驾多次往返京沪高速之间，享受几秒钟内提速到120公里/时的快感和推背感，将驾驶的乐趣发挥的淋漓尽致。全新发动机是以现款车型的1.5L自然吸气发动机为基础，配合一颗由上汽和通用联合研发的涡轮增压器升级而来，最大功率95kW/5500rpm，在rpm可爆发出210N&m的峰值扭矩。
除了1.5T发动机，值得一提的是智能6速手自一体变速箱。相比老款车型的4速自动变速箱，新车无论是在提速还是在燃油经济性方面都有了一定的提升。尽管为了试驾需要，在复杂的路况条件频繁加减速的情况下，百公里油耗始终维持在10个油左右，还是相当令人满意的。
inkaNet3.0 实现人车合一
客观来说，在试用inkaNet以前版本的过程中，经常会碰到语音识别错误等问题。但这次体验的升级版本让我对其完全改观。
首先，上汽inkaNet3.0配备了分辨率高达800*480的LCD显示屏，屏幕清晰，触摸体验和主流电容屏相近。另外，在试驾的过程中，我体验了从搜索导航、音乐播放到收发短信、拨打电话等一系列的功能，可以说通过语音就可以满足我在驾驶过程中所需的全部需求，而且充分保证了驾驶安全。新版本还提供wifi功能，车内和车外约5米范围内都能搜索到信号，每个月提供400M免费流量可供使用。
总结来说，MG51.5T除了可以充分满足日常驾驶所需之外，还能带来与赛车相似的速度激情和最前沿的智能互联体验，就12万元左右的价格区间来说，值得推荐。（文章来源：）
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闲置全新澳洲Lex S2000胎盘素绵羊油100g　含维E＆角鲨烯全天保湿
真实姓名::
姑姐系澳洲翻嚟，带咗劲多绵羊油，其中呢只牌子系绵羊油之中系最好级别咁制，而且含有胎盘素能修复细胞，促进生长，从而恢复柔嫩肌肤。
换物 换价值￥100以上物品
现金￥80& &淘宝澳洲代购要￥95
饼卷￥100&&
适合任何年龄使用，最适合送俾妈妈啦，一盒就搞掂。
共有￥18盒
我想要的换物：
茶葉有冇盒都得，酒類，能送人
餅卷 東海堂，美心的
牙膏，洗髮水，沐浴露，髮模，護髮素
記憶卡&&8G
零食，沖劑的，杯面，奶茶
笔记本音箱
BB用品，全新奶瓶，用品等
以下物品絕對不要：
衣物，飾品，書，雜誌，只要上海服飾
化妝品，護膚品，只要面膜
雜物不要，鑰匙扣，贈品出來的什麼什麼
電話線不要
产品介绍：
The Ultimate Skin Care – moisturizer with placenta and squalene. 24 hours time release.
Lex S2000 Placenta Cream Moisturiser
WITH 24 HOURS TIME RELEASE is formulated to high grade moisturizing anti-wrinkle nourishment cream. Contains enriched lanolin, placenta extracts, squalene, and Vitamin E specially formulated with Liposome. Its time – release for 24 hours, which beautifies by nourishing both the outer, under skin layers by moisture balance. With regular use skin will feel younger, soft and glowing.
Instructions
Your normal cleansing and toning routine – apply some cream to your damp skin and gently massage into face and neck, repeat this routine continuously. Avoid near eye area.
这款24小时级释保湿极品羊胎素绵羊油使用的是高品质的润肤防皱成分。富含羊胎素萃取，角鲨烯以及维他命E，特别是内含有脂质体成分--24小时缓释保湿，由内到外充分滋养您的皮肤各层，使水分平衡，经常使用本品会使您的皮肤年轻，柔嫩，充满光泽。
使用方法：洁面爽肤后，使用本品轻轻涂摸于面部及颈部皮肤，避免涂摸于眼部周围，眼部周围另使用眼霜。
闲置全新澳洲Lex S2000胎盘素绵羊油100g　含维E＆角鲨烯全天保湿
姑姐系澳洲翻嚟，带咗劲多绵羊油，其中呢只牌子系绵羊油之中系最好级别咁制，而且含有胎盘素能修复细胞，促进生长，从而恢复柔嫩肌肤。
换物 换价值￥100以上物品
现金￥80& &淘宝澳洲代购要￥95
饼卷￥100&&
适合任何年龄使用，最适合送俾妈妈啦，一盒就搞掂。
共有￥18盒
我想要的换物：
茶葉有冇盒都得，酒類，能送人
餅卷 東海堂，美心的
牙膏，洗髮水，沐浴露，髮模，護髮素
記憶卡&&8G
零食，沖劑的，杯面，奶茶
笔记本音箱
BB用品，全新奶瓶，用品等
以下物品絕對不要：
衣物，飾品，書，雜誌，只要上海服飾
化妝品，護膚品，只要面膜
雜物不要，鑰匙扣，贈品出來的什麼什麼
電話線不要
产品介绍：
The Ultimate Skin Care – moisturizer with placenta and squalene. 24 hours time release.
Lex S2000 Placenta Cream Moisturiser
WITH 24 HOURS TIME RELEASE is formulated to high grade moisturizing anti-wrinkle nourishment cream. Contains enriched lanolin, placenta extracts, squalene, and Vitamin E specially formulated with Liposome. Its time – release for 24 hours, which beautifies by nourishing both the outer, under skin layers by moisture balance. With regular use skin will feel younger, soft and glowing.
Instructions
Your normal cleansing and toning routine – apply some cream to your damp skin and gently massage into face and neck, repeat this routine continuously. Avoid near eye area.
这款24小时级释保湿极品羊胎素绵羊油使用的是高品质的润肤防皱成分。富含羊胎素萃取，角鲨烯以及维他命E，特别是内含有脂质体成分--24小时缓释保湿，由内到外充分滋养您的皮肤各层，使水分平衡，经常使用本品会使您的皮肤年轻，柔嫩，充满光泽。
使用方法：洁面爽肤后，使用本品轻轻涂摸于面部及颈部皮肤，避免涂摸于眼部周围，眼部周围另使用眼霜。
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您看到的内容均为会员发表,并不代表本站立场,转载时请注明作者和出处!Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Impact Factor 2.175
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Evidence-Based Complementary and Alternative MedicineVolume ), Article ID
pagesdoi:10.7698Research ArticleWnt-Signaling-Mediated Antiosteoporotic Activity of Porcine Placenta Hydrolysates in Ovariectomized Rats,1 ,2 ,2 ,2 ,1 and 21Korea Institute of Oriental Medicine, Daejeon 305811,
Republic of Korea2Department of Food and Nutrition, Institutes of Basic Sciences, Hoseo University, Asan 336795,
Republic of KoreaReceived 23 August 2012; Accepted 18 November 2012Academic Editor: Roja Rahimi Copyright (C) 2012 Byoung-Seob Ko et al. This is an open access article distributed under the , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Anti-osteoporotic effects of two types of porcine placenta hydrolysates (PPH) were evaluated in ovariectomized (OVX) rats orally administered PPH without (WPPH) or with (NPPH) ovarian hormones (1 g/kg bw/day). PPH groups were compared with OVX rats with estrogen replacement (0.1 mg/kg bw EST), or dextrose ( OVX-control) All rats received high-fat/calcium-deficient diets for 12 weeks. NPPH contained less estrogen and progesterone, but more essential amino acids, whereas the opposite was true for WPPH. NPPH decreased body weight and peri-uterine fat pads, and maintained uterus weight. NPPH rats had higher femur and lumbar spine bone mass density
but less than those of EST rats. Serum phosphorus and urinary calcium and phosphorus levels were reduced in NPPH rats compared to OVX-controls. Serum bone-specific alkaline phosphatase, osteocalcin, and bone turnover marker levels were reduced NPPH rats compared to OVX-controls. WPPH produced results similar to those of NPPH, but less significant. Both NPPH and estrogen upregulated low-density lipoprotein receptor-related protein 5 and β-catenin in OVX rats, while the expression of dickkopf-related protein 1 was suppressed. In conclusion, NPPH exerted anti-osteoporotic effects by activating osteogenesis and stimulating Wnt signaling, possibly mediated by the various amino acids and not ovarian hormones.1. IntroductionPostmenopausal osteoporosis is characterized by a net bone loss due to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation []. Osteoporosis increases susceptibility to bone fractures and reduces quality of life []. Bone mineral density (BMD), a major determinant of osteoporosis, peaks during the early 20 s, after which it decreases continuously at a slow rate and is significantly reduced after menopause [, ]. Hormone replacement therapy (HRT) has proven to be efficacious in preventing bone loss and reducing the incidence of skeletal fractures in postmenopausal women [] but has several side effects that reduce its popularity []. However, remedies other than HRT have been used to prevent and treat postmenopausal osteoporosis [–]. For example, administration of placenta extracts or hydrolysates from human or other animals by oral or subcutaneous injection [, ] has been used to treat osteoporosis.Treatment with 17β-estradiol achieves its positive bone effects through two key actions, facilitating vitamin D-related intestinal calcium absorption [] and suppressing bone resorption by decreasing the osteoprotegerin/receptor activator of nuclear factor kappa-B l (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL) system via the Wnt/β-catenin signaling pathways []. Furthermore, defective Wnt signaling plays a major role in osteoporosis []. Premenopausal women with amenorrhea have lower 17β-estradiol levels and reduced BMD []. In addition, combination treatment with estrogen and progesterone has a more positive effect on BMD than does estrogen alone []. Thus, BMD may be increased by HRT, and it is a suitable treatment for preventing bone loss. However, HRT has an adverse effect such as the induction of breast cancer. Researchers have investigated herbs that contain estrogen-like compounds for preventing bone loss [–]. Placenta products may be potential substitutes of HRT for preventing osteoporosis. Placental estrogen and progesterone may be effective components if these hormones are bioavailable in placenta hydrolysates and/or extracts. The placenta contains various nutrients and hormones that can improve the health of both fetuses and adults and can alleviate postmenopausal symptoms [, ]. Hominis placenta, dried human placenta, is used as one of several components of traditional remedies such as Yukmi-Jihangtang-Jahage and Honghwain-Jahage, which are used to treat inflammation, hyperlipemia, arteriosclerosis, and gynecological diseases such as osteoporosis and bone resorption [, , ]. The remedies contain placenta which has been prepared as a hot water extract for use in Chinese medicine. Since the availability of Hominis placenta is limited, porcine placenta has also been used. The ovarian hormones and amino acid profiles of porcine placenta are similar to those of Hominis placenta []. However, the effects and mechanisms underlying the placenta-associated health benefits remain poorly characterized and its active components have not been identified. Since the suppression of bone resorption is associated with Wnt/β-catenin signaling, this signaling was studied to determine the anti-osteoporosis mechanism of porcine placenta hydrolysates []. Among the major active components in placenta are ovarian hormones such as estrogen and progesterone, since estrogen plays a positive role in bone biology and osteoporosis prevention and treatment, primarily by decreasing bone resorption [, , ]. The active compounds in the placenta are likely water soluble (such as amino acids) since the remedies are prepared by boiling in water. However, estrogen and progesterone are not extractable by hot water. Thus, the positive effect of remedies such as Hominis placenta may not be due to estrogen and progesterone, but due to the profiles of amino acids and modified amino acids. It is therefore important to study the anti-osteoporotic activity of porcine placenta hydrolysates with and without removing ovarian hormones by modifying the filtering system to change the contents of lipids and amino acids in placenta hydrolysates. In the present study, the anti-osteoporotic effects of porcine placenta hydrolysates with and without ovarian hormones were examined in ovariectomized (OVX) rats fed calcium-deficient diets. 2. Materials and Methods2.1. Production and Composition of Porcine Placenta HydrolysatesPorcine placenta hydrolysates were prepared following the procedures of Codebio Inc. (Cheonan, Republic of Korea). Placentas were first thawed using a defroster and washed with saline at 16°C to remove blood and cords. They were then hydrolyzed using papain, bromelain, pronase, and Alcalase at °C for 48 h (pH 4.5&#x). Next, the protein hydrolysis enzymes were inactivated at °C for 30 min and the hydrolysates were filtered through activated carbon. The lipids were then removed by mixing with calcium and phosphate salts and by filtering through two types of absorbents. Finally, the hydrolysates were adjusted to pH
using calcium and phosphate salts. The 17β-estradiol and progesterone contents of the hydrolysates were measured using an ELISA kit, according to the manufacturer’s instructions (Abcam, Cambridge, MA, USA). Hydrolysates with both various ovarian hormone contents and with no hormones were prepared.
WPPH and NPPH were mixed with AccQ-Fluor buffer and Acc-Fluor reagent (Waters Corporation, Milford, MA, USA), and the mixture was heated at 80°C. An aliquot of the sample was passed through a 0.45 &#x3m filter and injected into the chromatographic system. Chromatographic separations were performed on an AccQ-Tag column (150 × 2.1 mm, 3 &#x3 Waters Corporation) in an HPLC instrument. A gradient mixture of AccQ-Tag Eluent (A) and acetonitrile (B) was used as the mobile phase at a flow rate of 1 mL/min at 30°C. The eluent composition started with 100% A and was linearly decreased to 67% over 33 min. It was switched to 100% B for 3 min and then switched back to 100% A for 26 min. Amino acids were detected using a fluorescence detector at excitation and emission wavelengths of 250 and 395 nm, respectively. A standard solution containing 22 amino acids at concentrations of 0.25–10 mg/L was prepared with 0.1 M HCl. Six-point calibration curves were generated by plotting the peak area versus concentration for each amino acid. 2.2. Animals Female Sprague-Dawley rats (weighing  g) were used. The animals were housed individually in stainless steel cages in a controlled environment (23°C and with a 12 h light/dark cycle). All surgical and experimental procedures were performed according to the guidelines of the Animal Care and Use Review Committee of the Hoseo University, Republic of Korea (). Since bone formation and resorption are slow processes, the experiment needed to proceed for at least 12 weeks and a calcium-deficient diet was provided to accelerate bone loss [–]. Experimental animals were provided water and calcium-deficient diets ad libitum during the 12-week experimental period. The calcium-deficient diet was prepared by a semipurified method with a modified AIN-93 formulation for experimental animals []. The diet consisted of 40 energy percent (En%) carbohydrates, 20 En% protein, 40 En% fats, and 1.7 g calcium/kg diet (one-third of the AIN-93 formulation). The major carbohydrate, protein, and fat sources were starch plus sugar, casein (milk protein), and lard (CJ Co, Seoul, Republic of Korea), respectively. 2.3. Experimental Design Rats underwent ovariectomy or a sham operation under anesthesia by intramuscular injection of a ketamine and xylazine mixture (100 and 10 mg, resp.). Forty OVX rats were randomly assigned to the following four groups: (1) porcine placenta hydrolytes with ovarian hormones (WPPH), (2) porcine placenta hydrolytes without ovarian hormones (NPPH), (3) estrogen replacement (EST), and (4) dextrose ( OVX-control). Ten sham-operated (Sham) rats were assigned to the placebo group (Sham-control). Our preliminary study found that the original placenta hydrolytes increased femur and spine BMD in a dose-dependent manner and that daily administration of 1,000 mg/kg bw/day original placenta hydrolysates improved the bone-mass density of OVX rats. In the present study, OVX rats in the appropriate groups were orally administered 1,000 mg/kg bw/day of porcine placenta hydrolysates with or without ovarian hormones or dextrose, while OVX rats in the EST group orally received 0.1 mg/kg bw conjugated estrogen (Dalim Biotech, Repulic of Korea). Sham rats were orally provided 1,000 mg/kg bw/day dextrose as a normal control. Food and water intake, as well as body weight, were measured every Tuesday at 10 AM. One day before being euthanized, urine samples were collected from the rats, which were then housed individually for 24 h in metabolic cages. At the end of the study, rats were anesthetized with ketamine and xylazine (100 and 10 mg/kg body weight, resp.). Peri-uterine and retroperitoneal fat mass and uteruses were then removed and weighed. Uterus index was calculated as uterus weight divided by body weight. Blood samples for serum isolation were collected by abdominal cardiac puncture. Urine and serum samples were then stored at −70°C for biochemical analysis. 2.4. BMD Measurement After anesthetization with ketamine and xylazine (100 and 10 mg/kg bw, resp.), BMDs of the right femur and lumbar spine were measured using a dual-energy X-ray absorptiometer (Norland pDEXA S Norland Medical Systems Inc., Fort Atkinson, WI, USA), which was equipped with the appropriate software for assessment of bone density in small animals. 2.5. Assay for Serum and Urine ChemistrySerum calcium (Ca), serum phosphorus (P), serum alkaline phosphatase (ALP), urine Ca, urine P, and urine creatinine levels were measured by standard colorimetric methods using commercial kits (Asan, Seoul, Republic of Korea). Serum osteocalcin (Osteocalc in-EIA, Biomedical Technology Inc., USA) and bone-specific alkaline phosphatase (BSALP) and urinary deoxypyridinoline (DPD; METRATM BAP EIA kit, Quidel Corp.) concentrations were determined by ELISA using the respective kits according to the manufacturer’s instructions.2.6. Isolation of Total Femur RNA and Real-Time PCRRat femurs were collected at the end of treatment. Femurs were individually powdered with a cold steel mortar and pestle and then mixed with a monophasic solution of phenol and guanidine isothiocyanate (TRIzol reagent, Gibco-BRL, Rockville, MD, USA) for total RNA extraction, following the manufacturer’s instructions. cDNA was synthesized from equal amounts of total RNA using superscript III reverse transcriptase, and polymerase chain reaction (PCR) was performed with high-fidelity Taq DNA polymerase. Equal amounts of cDNA were added to SYBR Green mix (Bio-Rad, Richmond, CA, USA) and amplified using a real-time PCR instrument (Bio-Rad). The expression levels of the genes of interest were normalized to that of the housekeeping gene, β-actin. To assess changes in the expression of Wnt pathway genes, the following primers were used: GGCTCGGATGAAGCTAACTG (forward) and CAGGATGATGCCAATGACAG (reverse) for low-density lipoprotein receptor-related protein 5 (LRP5), GCTGCATGAGGCACGCTAT (forward) and GGGCATGCATATTCCGTTT (reverse) for dickkopf-related protein 1 (DKK1), GGAAAGCAAGCTCATCATTCT (forward) and AGTGCCTGCATCCCACCA (reverse) for β-catenin, GCTCGAAAGTACAGGAACAGA (forward) and GCCGAGGAAGGGAGAGAACGAT (reverse) for RANKL, and AGCGTGGCTACAGCTTCACC (forward) and AAGTCTAGGGCAACATAGCACAGC (reverse) for β-actin. At least four PCR reactions per group were performed. 2.7. Statistical AnalysisStatistical analysis was performed using SAS, version 7.0. Results are expressed as means ± standard deviations. The anti-osteoporotic effects of NPPH, WPPH, and EST were compared by one-way analysis of variance. Significant differences between groups were identified by Tukey’s tests. The OVX and Sham groups were compared using two-sampled t-tests. A value of
was considered to indicate statistical significance.3. Results3.1. Composition of Porcine Placenta Hydrolysates Ovarian hormones and amino acid profiles were altered by removal of lipids by filtration. NPPH contained negligible levels of ovarian hormones, while WPPH contained substantial amounts of estrogen and progesterone due to incomplete lipid elimination (Table ). The amino acid profiles of NPPH and WPPH also differed (Table ). The total amino acid level of NPPH was lower than that of WPPH but contained a greater proportion of essential amino acids. Thus, some amino acids may have existed in modified forms, such as hydroxyproline and hydroxylysine.Table 1: Ovarian hormones and amino acid compositions of porcine placenta hydrolysates.3.2. Body and Organ Weights After 12 weeks, body weights and peri-uterine fat and retroperitoneal fat masses were higher in OVX rats than in Sham rats. NPPH inhibited the increment of OVX-induced weight gain, but the inhibition was less than that of estrogen. WPPH had no effect on body weight and fat mass increases (Table ). OVX caused significant atrophy of uterine tissue compared to the Sham group (), and NPPH significantly increased both absolute and relative uterine weights (Table ). EST had no significant effect. As expected, serum 17β-estradiol levels were significantly reduced in OVX rats but were unaffected by NPPH or WPPH. However, EST increased 17β-estradiol levels compared to those in Sham rats.Table 2: Body weights, peri-uterine and retroperitoneal fat weights, uterus weights, and serum 17β-estradiol levels at the end of the 12-week study period.3.3. Bone Mineral DensityRight femur and lumbar spine BMDs in OVX rats were decreased by 18.6 and 15.8%, respectively (), compared with Sham rats at the end of the 12-week experimental period (Figure ). The NPPH group exhibited significant 18.7 and 17.9% increases in the BMDs of the right femur and lumbar vertebra, respectively, compared to the OVX-control group (Figure ). However, these increases were lower than that elicited by estrogen treatment. Generally, WPPH elevated BMD, but the effects were not significant. Estrogen treatment ameliorated the OVX-mediated reduction of right femur and lumbar spine BMD, returning the levels to the baseline (Sham group). Thus, NPPH treatment prevented the reduction in BMD; however, estrogen did not play a role since serum estrogen levels of NPPH-treated rats were similar to those of the OVX-control rats.Figure 1: Total bone mineral densities (BMDs) in the femur and lumbar spine at the end of the study period. Ovariectomized (OVX) rats were divided into four groups, and for 12 weeks each group was orally administered 1 g/kg bw/day of porcine placenta hydrolysates with (WPPH) or without ovarian hormones (NPPH), 0.1 mg/kg bw/day conjugated estrogen (EST), or dextrose (OVX-control), while Sham rats were orally administered 1 g/kg/day dextrose as a normal control (Sham-control). At the end of the experimental period, BMDs of the femur and lumbar spine were measured by DXA. Values are expressed as means ± SD ().
different among the treatments at .
different among the OVX groups by Tukey’s test at .
different between the OVX-control and Sham-control groups at . 3.4. Serum and Urinary Ca and P Levels OVX rats exhibited significantly increased serum P levels and increased urinary Ca and P levels compared to Sham rats (Table ). NPPH administration reversed the OVX-induced changes in serum P levels and urinary Ca and P levels (), similar to EST-treated rats. However, serum Ca levels were not significantly altered by OVX, NPPH, or EST (Table ).Table 3: Serum and urinary Ca and P levels at the end of the 12-week treatment.3.5. Bone Turnover Markers The OVX operation significantly increased serum osteocalcin, ALP, and BSALP concentrations by 44.3, 38.7, and 24.5%, respectively, compared to the Sham-operated rats (Table ). There were no significant differences in the levels of WPPH-treated rats compared to the OVX-control rats. NPPH treatment of OVX rats significantly decreased serum osteocalcin, ALP, and BSALP concentrations in comparison to the OVX-control group (Table ). However, this decrease was not as significant as that induced by estrogen (Table ). Table 4: Bone turnover marker levels at the end of the 12-week treatment.3.6. Gene Expression Involved in Wnt Signaling in the FemurTo determine the expression levels of the Wnt pathway genes, LRP5, β-catenin, DKK1, and RANKL mRNA levels were monitored. β-catenin and DKK1 mRNA levels in the femur were upregulated in the OVX-control group compared to the Sham-control group (Figure ). However, LRP5 and RANKL mRNA levels in OVX rats were not significantly altered. LRP5 and β-catenin mRNA levels were greater in NPPH-treated OVX rats than that in the OVX-control rats and were further increased in OVX rats by estrogen treatment (Figure ). However, DKK1 expression was decreased by NPPH and estrogen, while RANKL expression was not affected by any treatment.Figure 2: Femur relative mRNA levels of the Wn LRP5, β-catenin, DKK1, and RANKL. Ovariectomized (OVX) rats were divided into four groups, and for 12 weeks each group was orally administered 1 g/kg bw/day of porcine placenta hydrolysates with (WPPH) or without ovarian hormones (NPPH), 0.1 mg/kg bw/day conjugated estrogen (EST), or dextrose (OVX-control), while Sham rats were orally administered 1 g/kg/day dextrose as a normal control (Sham-control). After the experimental period, mRNA levels in the femur were measured by real-time PCR. Values are expressed as means ± SD ().
different among the treatments at .
different among the OVX groups by Tukey’s test at .
different between the OVX-control and Sham-control groups at . 4. DiscussionThe regulation of bone mass is an active and dynamic process orchestrated by bone-forming osteoblasts and bone-resorbing osteoclasts [, ]. Osteoporosis, the most common bone remodeling disease, is caused mainly by an increase in bone resorption, which is not compensated for by a similar increase in bone formation [, ]. Postmenopausal osteoporosis is characterized by bone fragility due to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation []. Estrogen plays a crucial role in the regulation of bone mass by maintaining the balance between bone resorption and formation [, ]. Hormone replacement therapy can prevent or alleviate post-menopausal osteoporosis but has adverse side effects such as cancer []. The OVX rat model has many similarities to actual bone loss in humans [, ] and has been widely used to evaluate potential treatments for osteoporosis. Therefore, we investigated the anti-osteoporotic effect of PPH with and without ovarian hormones in OVX rats. The 12-week treatment with NPPH prevented reduction of the BMD of the femur and spine by reducing urinary excretion of Ca and P and bone turnover by decreasing Wnt signaling in OVX rats. WPPH exhib however, these were not significant. This suggests that the anti-osteoporotic effect of NPPH was not due to estrogen, since NPPH did not contain 17β-estradiol. Thus, the active compounds may include typical or modified amino acids.Serum Ca and P levels play important roles in the release of calcitonin and PTH from the thyroid and parathyroid glands, respectively []. These hormones regulate serum Ca levels within the normal range by modulating Ca accumulation in the bone, reabsorption from kidney tubules and absorption from the intestines [, ]. OVX rats exhibit higher serum P levels and urinary Ca and P levels due to increased bone resorption, but their serum Ca levels remain unclear [, ]. Cheng et al. [] demonstrated that Er-Zhi-Wan, a Chinese herbal remedy, reduces the OVX-induced increased rates of urinary Ca and P excretion. Our findings indicate that serum Ca levels were increased in OVX rats compared to Sham rats, but the difference was not significant. None of the treatments affected serum Ca levels in OVX rats. However, serum P levels and urinary Ca and P levels were higher in OVX rats than Sham rats and were significantly decreased after treatment with NPPH and EST. Thus, our data suggest that the bone turnover rate was downregulated by NPPH. Serum ALP, BALP, and osteocalcin, and urinary DPD, levels are widely accepted as bone turnover markers [–]. Osteocalcin is an abundant noncollagenous protein in the bone matrix, and the osteocalcin content of bone is an important bone growth marker []. BSALP is a glycoprotein ectoenzyme linked to the osteoblast membrane []. Osteocalcin and BSALP are released into the circulation during the bone remodeling process and during pathophysiological states. Bones are the main contributors to BALP and osteocalcin expression [, ]. In addition, DPD and pyridinoline are found in osteoclasts and are released and excreted into urine upon loss of bone cells [, ]. Therefore, serum BSALP and osteocalcin, as well as urinary DPD, are useful markers of bone turnover. The present study showed that 12-week treatment with NPPH in OVX rats significantly increased serum BALP and osteocalcin levels, and urinary DPD levels. These results are consistent with the report by Chae et al. [] that Hominis placenta extract reduced serum P, ALP, and thyroxine levels and decreased bone loss in OVX rats. In previous studies, after ovariectomy, rat BMD was markedly decreased compared to that of Sham-operated rats, due to an increase in bone turnover [–]. Estrogen is known to regulate bone remodeling before menopause in women [, ], and estrogen deficiency has been reported to accelerate bone loss []. Estrogen replacement therapy is used to alleviate post-menopausal osteoporosis via estrogen receptor activation to enhance osteogenic properties [, ]. However, the adverse effects of long-term estrogen usage (such as breast cancer and cardiovascular disease) reduce the popularity of this treatment [, ]. Our findings indicate that OVX rats had significantly decreased femur and lumbar spine BMDs, and after 12 weeks of NPPH treatment, BMD loss in the femur and lumbar vertebra was significantly inhibited in OVX rats with no increase in serum estrogen levels. This inhibition was less marked than that caused b however, the estrogen-mediated inhibition was not significant (). Since the experimental period was relatively short, this effect may be significant over longer periods of time. Considering the adverse effects of estrogen replacement, NPPH might be sufficient to prevent post-menopausal bone loss. Consistent with the present study, Hominis placenta inhibited the BMD decrease in OVX rats [].A Few studies have examined the placenta itself [, ]. A recent study by Takuma et al. [] has demonstrated that porcine placenta has not anti-osteoporotic activity in mice, but this study has some limitations for determining the prevention of osteoporosis due to a short-term treatment (5 weeks) and a diet containing sufficient Ca. However, some remedies that contain Hominis placenta, such as Yukmi-Jihangtang-Jahage, Saenghyuldan, and Honghwain-Jahage, are known to prevent bone resorption through their anti-inflammatory activities [, , ]. Cytokines in bone play a crucial role in the pathogenesis of osteoporosis. In estrogen-deprived bone cells, levels of cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and/or interleukin-6 (IL-6) are elevated and may be responsible for bone loss []. In addition, receptor activator of nuclear factor-&#x3B ligand (RANKL) has been identified as the major cytokine in osteoblast and osteoclast communication []. Yukmi-Jihangtang-Jahage inhibited COX-2 expression and suppressed IL-1β, TNF-α, and IL-6 levels in mouse calvarial osteoblasts, which may decrease bone resorption []. In addition, Yukmi-Jihangtang-Jahage decreased bone resorption in OVX rats. Similarly, Honghwain-Jahage inhibited IL-1β-induced bone resorption in cultured osteoblast cells derived from mouse calvarial bone explants []. Thus, placenta-containing remedies prevented bone loss by decreasing its resorption via suppression of inflammatory processes. However, RANKL expression was not significantly reduced by NPPH, WPPH, or estrogen replacement in the present study. Thus, the major pathway of NPPH-mediated bone loss prevention may not involve alleviation of inflammation by reducing cytokine concentrations.
Proteins in the Wnt signaling pathway are regulators of bone mass [, ]. Canonical Wnt signaling can regulate osteoblast function, contributing to bone formation [, ]. Wnt signaling is initiated by binding of a Wnt family member to two receptor molecules, namely, Frizzled proteins and LRP5/6. This stabilizes cytoplasmic β-catenin, after which the accumulated β-catenin is translocated into the nucleus where it stimulates Wnt target genes, including c-myc, Runx2, and cyclin D1 []. Activation of the Wnt/β-catenin signaling pathway promotes proliferation and differentiation of osteoblast precursor cells and increases osteoblast activity, which favors the deposition of new bone and increases BMD []. DKK1 negatively regulates Wnt/β-catenin signaling by binding to and antagonizing the Wnt coreceptors Lrp5/6 []. In the present study, the expression of proteins involved in Wnt signaling was elevated in OVX compared to S however, NPPH and estrogen treatment increased these to levels higher than those of OVX-control rats. Thus, OVX-induced osteogenesis can be used to overcome osteoclast action but may not in itself be sufficient. NPPH and estrogen treatments stimulate Wnt signaling, thus overcoming osteoclast activity. In addition, the expression of DKK1, a negative regulator of Wnt signaling, was markedly increased in OVX rats and inhibited the stimulation of Wnt signaling. Also, NPPH and estrogen treatment suppressed DKK1 expression and activated Wnt signaling. Thus, NPPH and estrogen stimulated Wnt signaling in OVX rats, resulting in increased osteogenesis.
The limitation of this study was that the active component in NPPH was not identified. Since NPPH does not include ovarian hormones, its anti-osteoporotic activity is not associated with ovarian hormones, such as estrogen. WHHP had a lesser effect on anti-osteoporotic activity, which may be related to the low levels of ovarian hormones it contains. Thus, improvement of the amino acid profiles or the presence of modified amino acids in NPPH should be considered. Nutrients and trace minerals, including essential amino acids, such as arginine, lysine, vitamin K, Mn, B, vitamin D, Zn, Cu, folate, and Si, are often used to improve bone structure []. Several studies suggest that dietary arginine and lysine may play an important role in the development, growth, and modeling of long bones [–]. Arginine is involved both in the synthesis of substrates (polyamine and L-Pro) implicated in collagen synthesis and in the production of growth hormone, insulin-like growth factor-I, and nitric oxide []. In addition, arginine is thought to alleviate metabolic disturbances in Ca absorption, growth, dentition and ossification defects, rachitism, osteomalacia, decalcification, and convalescence []. NPPH contained large amounts of arginine and essential amino acids (more than WPPH). Thus, arginine and other essential amino acids may be used to treat osteoporosis.5. ConclusionsNPPH prevented BMD loss in the femur and lumbar spine of OVX rats compared to the OVX-control, but the effect was less marked than that mediated by estrogen treatment. However, WPPH had less of an effect than NPPH. The NPPH-mediated improvement in BMD was associated with reduced bone resorption, as indicated by the decreased serum osteocalcin and BSALP levels, bone turnover markers, and lower serum P levels and urinary Ca and P excretion. The increase in BMD induced by EST was low but more marked than that induced by WPPH. NPPH and estrogen upregulated the expression of proteins (LRP5 and β-catenin) involved in Wnt signaling in OVX rats, while that of DKK1, an antagonist of Wnt signaling, was suppressed. Further studies are needed to confirm which component has anti-osteoporotic activity and to study its safety and efficacy for treating osteoporosis. In conclusion, NPPH had an antiosteoporotic effect, activating osteogenesis by stimulating Wnt signaling. This effect may have been mediated by amino acids but was not due to ovarian hormones. Thus, NPPH may represent an alternative treatment for postmenopausal osteoporosis.Conflict of InterestsThe authors declare that there is no conflict of interests.AcknowledgmentsThis work was supported by a grant from the Korea Institute of Oriental Medicine (Grant no. K12080) and by a Grant of Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2011.References
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