Osteogenic potential of punica granatum through matrix mineralization,cell cycle progression and runx2 gene expression in primary rat osteoblasts

Background

Osteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal rats.

Methods

The ethanolic extract of PG was subjected to evaluate cell proliferation, regeneration, mineralization and formation of collagen matrix using MTT, alkaline phosphatase, Alizarin Red-S staining and Sirius Red dye, respectively. Cell cycle progression and osteogenic gene Runx2 expression were carried out by flow cytometry and real time PCR, respectively.

Results

Exposure of different concentrations (10–100 μg/ml) of the extract on osteoblastic cells showed characteristic morphological changes and increment in cell number. A significant growth in cell proliferation, ALP activity, collagen contents and matrix mineralization of osteoblasts in a dose dependent manner (p < 0.05), suggested that PG has a stimulatory effect on osteoblastic bone formation or potential activity against osteoporosis. In addition, PG extract also enhanced DNA content in S phase of cell cycle and Runx2 gene expression level in osteoblasts.

Conclusion

The data clearly indicated that PG promoting bone cell proliferation and differentiation in primary osteoblasts might be due to elevating the osteogenic gene Runx2 expression. The present study provides an evidence for PG could be a promising herbal medicinal candidate that able to develop drugs for osteoporosis.     

巴戟天丸防治D-半乳糖损伤成骨细胞骨丢失的作用及机制研究

目的 研究巴戟天丸防治D-半乳糖（D-gal）损伤成骨细胞骨丢失的作用及机制。方法 采用新生24 h Wistar大鼠提取的原代成骨细胞,利用D-gal对细胞进行干预,并给予巴戟天丸提取物行药物治疗。分别采用MTT法和碱性磷酸酶试剂盒评价细胞的增殖和分化水平;采用DCFH-DA荧光探针对成骨细胞内活性氧（ROS）水平进行测定。采用Western blotting法对磷酸化蛋白激酶B(p-AKT)、蛋白激酶B(AKT)、血红素氧合酶1(HO-1)、醌NADPH脱氢酶1(NQO1)等氧化相关蛋白的表达进行检测,并采用免疫荧光法测定细胞核因子E2相关因子2(Nrf2)的核内表达水平。结果 巴戟天丸能显著提高D-gal干预细胞的增殖水平和ALP活性,并显著降低细胞内ROS水平。巴戟天丸能够显著促进细胞AKT蛋白的磷酸化,提高HO-1、NQO1的表达水平,进而激活PI3K/AKT信号通路。此外,巴戟天丸提取物还可显著促进成骨细胞核内Nrf2的表达,激活Nrf2信号通路,促进骨形成。结论 本研究首次明确了巴戟天丸可防治D-半乳糖损伤引起的成骨细胞骨丢失,其作用机制可能与调控PI3K/AKT和Nrf...     

Sirt1 is involved in decreased bone formation in aged apolipoprotein E-deficient mice

Aim:

Apolipoprotein E (ApoE) plays an important role in the transport and metabolism of lipids. Recent studies show that bone mass is increased in young apoE^−/− mice. In this study we investigated the bone phenotype and metabolism in aged apoE^−/− mice.

Methods:

Femurs and tibias were collected from 18- and 72-week-old apoE^−/− mice and their age-matched wild-type (WT) littermates, and examined using micro-CT and histological analysis. Serum levels of total cholesterol, oxidized low-density lipoprotein (ox-LDL) and bone turnover markers were measured. Cultured bone mesenchymal stem cells (BMSCs) from tibias and femurs of 18-week-old apoE^−/− mice were used in experiments in vitro. The expression levels of Sirt1 and Runx2 in bone tissue and BMSCs were measured using RT-PCR and Western blot analysis.

Results:

Compared with age-matched WT littermates, young apoE^−/− mice exhibited high bone mass with increased bone formation, accompanied by higher serum levels of bone turnover markers OCN and TRAP5b, and higher expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. In contrast, aged apoE^−/− mice showed reduced bone formation and lower bone mass relative to age-matched WT mice, accompanied by lower serum OCN levels, and markedly reduced expression levels of Sirt1, Runx2, ALP and OCN in bone tissue. After BMSCs were exposed to ox-LDL (20 μg/mL), the expression of Sirt1 and Runx2 proteins was significantly increased at 12 h, and then decreased at 72 h. Treatment with the Sirt1 inhibitor EX527 (10 μmol/L) suppressed the expression of Runx2, ALP and OCN in BMSCs.

Conclusion:

In contrast to young apoE^−/− mice, aged apoE^−/− mice showe lower bone mass than age-matched WT mice. Long-lasting exposure to ox-LDL decreases the expression of Sirt1 and Runx2 in BMSCs, which may explain the decreased bone formation in aged apoE^−/− mice.     

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein⁻¹ min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.     

Mthfr gene ablation enhances susceptibility to arsenic prenatal toxicity

Background

In utero exposure to arsenic is known to adversely affect reproductive outcomes. Evidence of arsenic teratogenicity varies widely and depends on individual genotypic differences in sensitivity to As. In this study, we investigated the potential interaction between 5,10-methylenetetrahydrofolate reductase (Mthfr) genotype and arsenic embryotoxicity using the Mthfr knockout mouse model.

Methods

Pregnant dams were treated with sodium arsenate, and reproductive outcomes including: implantation, resorption, congenital malformation and fetal birth weight were recorded at E18.5.

Results

When the dams in Mthfr^+/− × Mthfr^+/− matings were treated with 7.2 mg/kg As, the resorption rate increased to 43.4%, from a background frequency of 7.2%. The As treatment also induced external malformations (40.9%) and significantly lowered the average fetal birth weight among fetuses, without any obvious toxic effect on the dam. When comparing the pregnancy outcomes resulting from different mating scenarios (Mthfr^+/+ × Mthfr^+/−, Mthfr^+/− × Mthfr^+/− and Mthfr^−/− × ^Mthfr+/−) and arsenic exposure; the resorption rate showed a linear relationship with the number of null alleles (0, 1 or 2) in the Mthfr dams. Fetuses from nullizygous dams had the highest rate of external malformations (43%) and lowest average birth weight. When comparing the outcomes of reciprocal matings (nullizygote × wild-type versus wild-type × nullizygote) after As treatment, the null dams showed significantly higher rates of resorptions and malformations, along with lower fetal birth weights.

Conclusions

Maternal genotype contributes to the sensitivity of As embryotoxicity in the Mthfr mouse model. The fetal genotype, however, does not appear to affect the reproductive outcome after in utero As exposure.     

Small heterodimer partner overexpression partially protects against liver tumor development in farnesoid X receptor knockout mice

Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are nuclear receptors that are critical to liver homeostasis. Induction of SHP serves as a major mechanism of FXR in suppressing gene expression. Both FXR^−/− and SHP^−/− mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one of the most strongly induced genes by FXR in the liver and is a tumor suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC development in the livers of FXR^−/− mice and therefore, increased SHP expression in FXR^−/− mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR^−/− mice with overexpression of SHP in hepatocytes (FXR^−/−/SHP^Tg) and determined the contribution of SHP in HCC development in FXR^−/− mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR^−/− mice. However, SHP overexpression led to a lower grade of dysplasia, reduced indicator cell proliferation and increased apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6 levels, which was associated with activation of hepatic STAT3. In conclusion, SHP partially protects FXR^−/− mice from HCC formation by reducing tumor malignancy. However, disrupted bile acid homeostasis by FXR deficiency leads to inflammation and injury, which ultimately results in uncontrolled cell proliferation and tumorigenesis in the liver.     

An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice

It has been postulated that fetal vascular abnormalities in aryl hydrocarbon receptor null (ahr^−/−) mice may alter cardiovascular homeostasis in adulthood. We tested the hypothesis that blood pressure regulation in adult heterozygous mice (ahr^+/−) would be normal, compared to ahr^−/− mice, since no vascular abnormalities have been reported in the heterozygote animals. Mean arterial blood pressure (MAP) was measured using radiotelemetry prior to and during treatment with inhibitors of the autonomic nervous system, nitric oxide synthase (NOS), angiotensin converting enzyme (ACE), or endothelin-1 A receptor (ET_A). Also, indices of renin-angiotensin system (RAS) activation were measured. ahr^+/− and ahr^−/− mice were normotensive and hypotensive, respectively, compared to wild-type (ahr^+/+) littermates. Responses of all genotypes to autonomic nervous system inhibition were normal. ahr^+/− mice responded normally to NOS inhibition, while the responses of ahr^−/− mice were significantly blunted. In contrast, ahr^+/− mice were significantly more responsive to inhibition of ACE, an ET_A antagonist, or both, while ahr^−/− mice were significantly less responsive to ACE inhibition and more responsive to an ET_A antagonist. ahr^+/− mice also exhibited significant increases in plasma renin and ACE activity, plasma sodium, and urine osmolality, indicative of RAS activation. Thus, normotension in ahr^+/− mice appears to be maintained by increased RAS and ET-1 signaling, while hypotension in ahr^−/− mice may result from decreased RAS signaling. In conclusion, despite the lack of overt fetal vascular abnormalities in ahr^+/− mice, the loss of a single ahr allele has a significant effect on blood pressure regulation.     

The genetic epidemiology of melanocortin 4 receptor variants

While rare MC4R mutations are the commonest cause of monogenic forms of extreme, early-onset obesity, growing evidence shows that common MC4R variants contribute to obesity in the general population. Candidate gene studies have focussed on the V103I and I251L MC4R variants that both affect MC₄ receptor function in vitro. Individual association studies, which are typically small and underpowered, have found no association between V103I (frequency of 103I-allele: ~ 4%) or I251L (251 L-allele: ~ 2%) and the risk of obesity in the general population. However, large-scale meta-analyses have confirmed that both variants reduce the risk of obesity by − 21% in 103I-allele carriers (P < 10⁻⁴) and by − 50% in 251 L-allele carriers (P < 10⁻⁴). Recently, genome-wide association studies have identified a common variant (minor allele frequency: ~ 27%) at ~ 188 kb downstream of MC4R showing robust association (P < 5 × 10⁻⁸) with BMI and obesity in adults and children. Each additional minor allele increases BMI by 0.20 kg/m², body weight by 700–1000 g, and obesity risk by 14% in adults. Interestingly, this variant also showed association with increased height, consistent with the phenotype seen for rare MC4R mutations. Although MC4R is the nearest gene and phenotypic associations are consistent with those of MC4R mutations, it has not yet been established whether this variant indeed reflects MC₄ receptor function. Taken together, common MC4R variants contribute to variation in BMI and obesity risk in the general population. Of particular interest is the finding from genome-wide association studies that suggests that the region downstream of MC4R contributes to its regulation.     

Endothelial cell-specific aryl hydrocarbon receptor knockout mice exhibit hypotension mediated, in part, by an attenuated angiotensin II responsiveness

Hypotension in aryl hydrocarbon receptor knockout mice (ahr^−/−) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr^−/− (ECahr^−/−) mice would exhibit a similar phenotype. We generated ECahr^−/− mice by crossing AHR floxed mice (ahr^fx/fx) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr^−/− mice were hypotensive (ECahr^+/+: 116.1 ± 1.4; ECahr^−/−: 107.4 ± 2.0 mmHg, n = 11, p < 0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr^+/+, whereas the increase in ECahr^−/− mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr^−/− mice (ECahr^+/+ 1297 ± 223 mmHg/30 min; ECahr^−/−_AUC: 504 ± 138 mmHg/30 min, p < 0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr^−/− mice. ECahr^−/− mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr^−/− mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose.     

The PI3K/Akt/FOXO3a/p27 signaling contributes to anti-inflammatory drug-suppressed proliferation of human osteoblasts

Akt has been reported to suppress p27^Kip1 promoter activity through Forkhead box O (FOXO) in different kinds of cells. Previous studies indicated that anti-inflammatory drugs up-regulated p27^Kip1, and this effect might play an important role in anti-inflammatory drug-induced cell cycle arrest of human osteoblasts (hOBs). In this study, we hypothesized that these drugs might increase p27^Kip1 expression in hOBs by altering the Akt/FOXO signaling. We tested this hypothesis by examining the influences of three anti-inflammatory drugs on the levels and/or activities of Akt, FOXO and p27^Kip1 as well as the relationship between these factors and proliferation of hOBs. We tested the effects of indomethacin (10⁻⁵ and 10⁻⁴ M), celecoxib (10⁻⁶ and 10⁻⁵ M), and dexamethasone (10⁻⁷ and 10⁻⁶ M) using PI3K inhibitor, LY294002 (10⁻⁵ M) as the basis of comparison. The three drugs suppressed the canonical level of phosphorylated Akt in hOBs. This was accompanied by elevated FOXO3a level and increased promoter activity, mRNA expression and protein level of p27^Kip1. Furthermore, the anti-inflammatory drugs suppressed the EGF-induced increases in proliferation, phosphorylation, and nucleus translocation of Akt. Simultaneously, they suppressed EGF-induced decreases of FOXO3a nucleus accumulation and p27^Kip1 mRNA expression. On the other hand, FOXO silencing significantly attenuated the drug-induced up-regulation of p27^Kip1 and suppression of proliferation in hOBs. To the best of our knowledge, this study represents the first to demonstrate that Akt/FOXO3a/p27^Kip1 pathway contributes to suppression of hOB proliferation by anti-inflammatory drugs. We suggest that anti-inflammatory drugs suppress hOB proliferation, at least partly, through inactivating Akt, activating FOXO3a, and eventually up-regulating p27^Kip1 expression.     

青天葵甲醇提取物通过ERK信号通路诱导鼻咽癌CNE-2细胞凋亡

目的 研究青天葵甲醇提取物（Nervilia fordii methanol extracts,NFME）体外对鼻咽癌CNE-2细胞的凋亡作用及其作用机制。方法 MTT法检测不同浓度（0,0.25,0.5,1,2,3 mg·mL^-1）的NFME处理CNE-2细胞24,48 h对CNE-2细胞生长抑制率的影响;克隆原形成试验观察NFME对CNE-2细胞克隆形成率的影响;Hoechst凋亡染色观察NFME对CNE-2细胞凋亡的影响;Western blotting测定NFME作用下caspase-3、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果 MTT试验结果显示,与对照组相比,在给药24 h时0.5 mg·mL^-1的NFME就能抑制CNE-2细胞的增殖（P<0.05）,抑制率为12.64%。而在给药48 h时0.25 mg·mL^-1的NFME就能抑制CNE-2细胞增殖（P<0.05）,抑制率为22.43%;克隆原形成能力试验表明,0.25 mg·mL^-1的NFME能够抑制CNE-2细胞集落的形成（P<0.05）;Hoechst33258凋亡染色观察到0.25 mg·mL^-1的NFME作用24 h能够观察到CNE-2细胞发生凋亡（P<0.05）,凋亡率达到17.91%;Western blotting结果表明0.5 mg·mL^-1的NFME能使CNE-2细胞中caspase-3发生剪切,随着给药浓度增加其剪切作用越明显（P<0.01）,同时0.5 mg·mL^-1的NFME能够降低CNE-2细胞中ERK1/2和c-Raf蛋白的磷酸化水平（P<0.05）。结论 青天葵甲醇提取物能抑制鼻咽癌CNE-2细胞的增殖并诱导其发生凋亡,其作用机制可能与抑制ERK信号通路有关。     

杜仲中槲皮素、京尼平苷及桃叶珊瑚苷对小鼠成骨样细胞系MC3T3-E1增殖和分化的影响

目的 对杜仲抗骨质疏松的药效成分进行初步筛选。方法 采用小鼠成骨细胞MC3T3-E1体外培养模型, 通过MTT法测定细胞增殖, ELISA方法测定碱性磷酸酶(ALP)活性, 观察杜仲中槲皮素、京尼平苷及桃叶珊瑚苷对成骨细胞增殖和分化的影响。结果 槲皮素促MC3T3-E1细胞增殖的有效浓度为10^-6 mmol/L, 桃叶珊瑚苷则为10^-5 mmol/L;10^-4 μmol/L京尼平苷在干预后4 d, 才逐渐显现出促进MC3T3-E1增殖作用。槲皮素(10^-5、10^-3 mmol/L)、京尼平苷(10^-3 mmol/L)和桃叶珊瑚苷(10^-3 mmol/L)可增加MC3T3-E1 ALP活性。结论 槲皮素、京尼平苷和桃叶珊瑚苷可促进成骨细胞的增殖和分化, 且作用强度具有浓度相关性和时间相关性, 可能为杜仲抗骨质疏松的药效物质基础。     

Dermal penetration of propylene glycols: Measured absorption across human abdominal skin in vitro and comparison with a QSAR model

The dermal penetration of undiluted monopropylene glycol (MPG) and dipropylene glycol (DPG) has been measured in vitro using human abdominal skin under conditions of infinite dose application, and the results compared with predictions from the SKINPERM QSAR model (ten Berge, 2009). The measured steady-state penetration rates (J_ss) for MPG and DPG were 97.6 and 39.3 μg/cm²/h, respectively, and the permeability coefficients (K_p) were 9.48 × 10⁻⁵ cm/h for MPG and 3.85 × 10⁻⁵ cm/h for DPG. In comparison, the SKINPERM model slightly over-predicted J_ss and K_p for MPG and DPG by between 2.6- and 5.1-fold, respectively. The model predictions of 254 μg/cm²/h and 24.6 × 10⁻⁵ cm/h for MPG, and 202 μg/cm²/h and 19.8 × 10⁻⁵ cm/h for DPG were in fairly good agreement with the measured values. Further, the model predicted a J_ss of 101 μg/cm²/h and a K_p of 9.9 × 10⁻⁵ cm/h for the homologue tripropylene glycol. Assuming that the measured J_ss was the same under conditions of finite dose application (taken to be 10 μL/cm²) and was maintained over a 24-h period (both conservative assumptions), the relative dermal absorption of the applied dose was estimated to be 23% (0.96%/h) for MPG and 9% (0.39%/h) for DPG. However, the extrapolation for MPG may be further overestimated due to possible residence in the stratum corneum under infinite conditions of exposure that would not be applicable to a finite loading dose.     

Comparative effect of cadmium on osteoblastic cells and osteoclastic cells

Cadmium(Cd) has been thought to disturb the bone metabolism directly. The mechanism for the bone lesion is unknown, however. To examine the effects of cadmium on bone metabolism, we compared its effects on osteoblasts and osteoclasts in vitro. We used an established cell line, MC3T3-E₁, as osteoblasts and tartrate resistant acid phosphatase (TRACP)-positive multi-nucleated cells (MNC) formed by a bone marrow culture system as osteoclasts. Alkaline phosphatase (ALP) activity was decreased by 10^–7 M Cd and DNA content and hydroxyproline content of osteoblastic cells were decreased by 10^–5 M Cd. Cadmium at 10^–7 M inhibited the osteoclastic cell formation from mouse bone marrow in the presence of 10^–8 M 1,25(OH)₂ vitamin D₃. A 100-fold higher concentration of zinc(Zn) simultaneously added to the cadmium-containing medium prevented the toxicity of cadmium to osteoclastic cells as observed in the culture of osteoblastic cells. These results indicate that both bone formation and bone resorption are inhibited by cadmium. The responses of osteoclasts and osteoblasts to cadmium in this culture system were the same and the responses of cadmium-damaged osteoblasts and osteoclasts to zinc were also similar. These results suggest that another mechanism by which cadmium could cause bone damage should be considered in addition to the specific induction of osteoclastic cells by Cd.     

Hepatocyte growth factor-induced BMP-2 expression is mediated by c-Met receptor, FAK, JNK, Runx2, and p300 pathways in human osteoblasts

Hepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Bone morphogenetic protein-2 (BMP-2) is a crucial mediator in bone formation during fracture healing. However, the effects of HGF in BMP-2 expression in human osteoblasts are large unknown. Here we found that HGF induced BMP-2 expression in human osteoblasts dose-dependently. HGF-mediated BMP-2 production was attenuated by c-Met inhibitor or siRNA. Pretreatment with FAK inhibitor or JNK inhibitor (SP600125) also blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced FAK phosphorylation, JNK phosphorylation, and RunX2 translocation from cytosol to the nucleus. HGF-mediated Runx2 binding to BMP-2 promoter was inhibited by c-Met inhibitor, FAK inhibitor, and SP600125. The binding of Runx2 to the BMP-2 promoter, as well as the recruitment of p300 and the enhancement of histones H3 and H4 acetylation on the BMP-2 promoter was enhanced by HGF. Our results suggest that HGF increased BMP-2 production in human osteoblasts via the c-Met receptor/FAK/JNK/Runx2 and p300 signaling pathways.