1. Introduction

Bisphosphonates (e.g., pamidronate, etidronate, clodronate,alendronate, tiludronate, risedronate, and ibandronate) are established as effective treatments of osteoporosis for decades and shown to be effective in prevention of bone fracture[1]. Osteoporosis is a common type of bone disease characterised by reduced bone mass and increased in skeletal fragility[2]. Bisphosphonate acts by decreasing bone resorption, thereby slowing bone loss through reduce osteoclastic bone resorption[3,4]. It possesses unique pharmacological character in which they have the potential to continue to affect bone metabolism long after discontinuance of its intake[5,6]. Several research has reported possible side effects of bisphosphonate which can cause possible osteonecrosis of the jaw in oncology patients as well as atypical femoral fractures caused by over suppression of bone remodelling[7,8]. Meanwhile, orally administering bisphosphonates has a very low bioavailability and may induce gastrointestinal disturbances[1]. These agents have little ability to stimulate new bone synthesis. Therefore, investigations of alternative agents that can improve synthesis of new bone are fairly significant.

Bone is a dynamic tissue that undergoes constant remodelling through bone formation and resorption. The process of bone remodelling comprised of sequential events triggered initially from resorption of existing bone by osteoclast followed by bone formation by osteoblast[9]. Thus, osteoblast is the key component in bone multicellular unit. Osteoblasts are differentiated from multipotent mesenchymal cells that are regulated by several cytokines which include bone morphogenetic proteins-2 (BMP-2), transforming growth factor-β and so on[10,11]. The BMP-2 then faciliatates to differentiate mesenchymal cells with osteoblasts and induces bone formation by activating Smad signaling pathway and regulating transcription of osteoblast specific transcription factor such as Runtrelated transcription factor-2 (Runx2)/Core-binding factor-1, Osterix(Osx), alkaline phosphatase, type Ⅰ collagen, osteocalcin, and bone sialoprotein 2[12,13].

Runx2 and Osx are osteoblast-specific transcription factors essential for the development of osteoblastic cells and bone formation[14,15].The process of osteoblast differentiation is directed by Runx2 in which mesenchymal progenitor cells evolves to preosteoblast. Then,preosteoblastis directed to immature osteoblast by Runx2, Osx and β-catenin[16]. On the other hand, Osx is the second transcription factor that is essential for osteoblast differentiation whereby it is specifically expressed in all developing bones[16,17]. Given that these two transcription factors are essential for osteoblast differentiation from independent progenitor cells, the quantification of these factors could be used to determine the osteoblastogenic potential in discovery of new anabolic agent acting on bone[18-20]. Therefore,both Runx2 and Osx are important transcription factors for osteoblast differentiation and controlling the expressions of osteoblastogenic marker that are required for the terminal osteoblast differentiation and bone mineralisation[21].

Quercus infectoria (Q. infectoria) Olivier (Fagaceae) is a small tree or a shrub mainly presented in Greece, Asia Minor, Syria and Iran[22]. The galls of Q. infectoria have a great medicinal value and have pharmacologically been deciphered to be astringent,ant diabetic, antitremorine, local anaesthetic, antipyretic and antiparkinsonian[23]. The main components of Q. infectoria comprise of 50% to 70% polyphenols, which is made up from abundance amount of tannins, gallic acid, syringic acid, ellagic acid, amentoflavone,hexamethyl ether, isocryptomerin, methyl betulate, methyl oleanate and hexagalloyl glucose[24]. Throughout the years, various study reported the effects of polyphenols on promoting the formation of new bone[25,26]. Recently, polyphenols derived from dried plum have been reported to enhance osteoblast activity and function by upregulating Runx2, Osx and IGF-Ⅰ expression[27]. In addition, our preliminary study has found that the level of alkaline phosphates in hFOB 1.19 increases significantly after being treated with Q.infectoria galls extract,providing initial insight on the ability of Q.infectoria in modulating bone formation[28]. However, the effects of combining Q. infectoria with readily available osteoporotic agent such as biophosphonate have not been examined and understood.

In the present study, the paper aims to investigate and understand the effects of combining the readily available and commonly used osteoporotic drug (pamidronate) with Q. infectoria semi-purified fraction (QIsm-F) on human foetal osteoblast cell model (hFOB 1.19 cell line) by assessing the levels and expression of Osx and Runx2 important osteoblastogenic marker.

2. Materials and methods

2.1. Extraction of Q. infectoria extract

Q. infectoria galls were obtained from local market. The galls were identified based on its morphology parameters such as external colour, size, surface, texture, odour, taste and thickness[29]. Aqueous extraction of Q. infectoria was done by grinding the Q. infectoria galls to obtain powdered form followed by weighing 50 g of crude Q. infectoria extract in 100 mL of sterile distilled water and refluxing in water bath at 50 ℃ for 24 h.

每年高温季节都是鱼病的多发季节，特别是精养鱼池的病害发生率更高，轻则影响池鱼的健康生长，重则造成池鱼大批死亡，甚至发生翻塘死鱼事故。因此在高温季节，应有针对性地进行鱼病预防，采取生态防控措施，坚持以防为主、防治结合的原则，确保养殖生产安全。那么，在高温季节精养鱼池应如何进行生态防病呢？

产品外观设计的内容会直接影响到产品外观设计的质量，因此，应严格规划产品外观设计的具体内容。将传统图案应用在产品外观设计中是体现中华民族文化和弘扬传统文化精神的重要表现形式。在多元化的市场环境下，重视产品外观的文化特色并与传统图案中的民族元素进行有效融合，可使产品在激烈的市场竞争中继续保持竞争优势。

2.2. Isolation and fractionation of QIsm-F

Throughout the years, the process of drug discovery utilizes the information obtained from natural product researches which are then being developed as well as incorporated towards discovering new drugs by combining the chemical structure with existing drug[43].Hence, it tends to be authentic that the data obtained from this research could be used to develop and adapt for discovery of new improved osteoporotic drug with minimal risk of side effects to human.

2.3. Cell culture conditions

Immuno-fluorescence staining technique utilises the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell. Each marker; Runx2 (Alexa Fluor 488) and Osx (FITC) were tagged with different fluorescence dyes.Cells were cultured and treated in 4-chamber slide with seeding density of 5×103. The cells have then been prepared for immunefluorescence staining at day 3 and day 7 of incubation by fixing in 4% paraformaldehyde for 5 min at room temperature and then been permeabalized in 0.5% Triton X-100 (Thermo Fisher Scientific,USA) in DPBS for 15 min. After being blocked in 3% bovine serum albumin in DPBS for 60 min, the cells were then incubated overnight at 4 ℃ with primary antibody against Runx2 (1:200) directly conjugated with Alexa Fluor 488 (Santa Cruz, USA) and Osx (1:200);(Santa Cruz, USA). The secondary antibodies, conjugating with fluoresce in isothiocyanate were added at (1:500) dilution for cells incubated with Osx antibody and incubated for 1 h at 37 ℃ away from light. The cells were then mounted with Prolonged Diamond antifade mountant with DAPI (Thermo Fisher Scientific, USA).Images were viewed under fluoresence microscrope (Olympus,Japan) and captured by employing image analyser (Olympus, Japan).

2.4. Cell viability assay/MTT assay

This study employs pamidronate as osteoporotic control drug and agent for treatment combination with QIsm-F. Meanwhile,tamoxifen, a type of selective estrogen receptor, on the other hand, was established as negative control drug due to its known negative effect on osteoblast cell[31,32] and untreated control was used as general control.hFOB 1.19 cells were plated in 96-well plate at a density 5×103 cells per well[33]. Twenty four hour after plating, the cells were exposed with different concentration of each treatment groups (pamidronate,QIsm-F and tamoxifen), ranging from 0.01 μg/mL to 99 μg/mL for 72 h. Each treatment was carried out in triplicate. At the end of the treatment, 20 μL of freshly prepared MTT (5 mg/mL in PBS) (Nacalai Tescue, Japan) was added into each of the 96-wells plate followed by incubation for 4 h in a CO2 incubator at 37 ℃. Then, formazon salts were dissolved with 100 μL dimethyl-sulphoxide (Nacalai Tesque,Japan) and the absorbance was determined at 570 nm in ELISA micro plate reader. The graph of the percentage of viable cell versus log10 concentration (μg/mL) of each fraction was plotted employing GraphPad Prism 6.0. The half maximal effective concentration (EC50)was determined by the plotted graph.

2.5. Western blot

The total proteins were extracted at day 3 and day 7 of incubation with lysis buffer (Nacalai Tesque, Japan) for 20 min on ice. The protein samples were run on 10% SDS –PAGE gel electrophoresis at 100 V for 1 h for protein separation. The protein was then being transferred onto a polyvinylidene di fluoride membrane using semidry transblot (BioRad, USA) at 15 V for 2 h. After transfer, the polyvinylidene di fluoride membrane was blocked in 1% skim milk in tris-buffer saline with Tween-20 for 1 h followed by incubation with primary antibodies (against ß-actin 1:1000, Runx2 1:400 and Osx 1:400 (Santa Cruz, USA) overnight at 4 ℃. The membrane were then matched with secondary antibody IgG HRP (1:1000)(Santa Cruz, USA).

2.6. Immunocytochemistry

Human foetal osteoblast cell, hFOB 1.19 (CRL-11372) was purchased from American Type Cell Culture (Manassas, USA).The cells were revived in Dulbecco’s Modified Eagle Medium F-12(Gibco, USA) and supplemented with 10% fetal bovine serum;(Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). The cells were incubated in 5% CO2 incubator at 37 ℃. All cell culture work was maintained in a sterile condition by applying aseptic technique and being carried out in the Bio safety cabinet class 2(ESCO, Singapore).

一到冬天，要检查几次。不是怕别的，怕老鼠打了洞。葡萄窖里很暖和，老鼠爱往这里面钻。它倒是暖和了，咱们的葡萄可就受了冷啦！

2.7. Statistical analysis

Data were expressed as the mean±SEM of n=3 independent experiments. Statistical analysis was conducted by applying SPSS(version 22.0 for windows). Data were tested for normality and homogeneity of variance. Statistical comparisons of the results were established using Repeated Measure ANOVA analysis. Differences among groups were conducted by using multiple comparison post hoc tests. Values of P＜0.05 were considered to be statistically significant to the control.

Immuno-fluorescence staining against similar osteoblastogenic marker Runx2 and Osx supported the finding of both protein expressions. Through immuno-fluorescence staining, the research could then observe the proliferation rate, cell morphology together with the expression of both markers. The images show that the rate of cells proliferation increases from day 3 to day 7 in all treatment groups except tamoxifen and are at peak in cells treated with the combination of QIsm-F with pamidronate treatment. Moreover,the cells supplied with combination treatment were observed to channel higher intensity of fluorescence staining expressed in the nuclei of the cells and spread to the cytoplasm; along with well attached character and elongated shape as well as appear to be overlapping on each other due to rapid cell proliferation which evidently shows heightened expression of Runx2 and Osx.Meanwhile, the rate of proliferation and expression of Runx2 and Osx in hFOB 1.19 cells treated with individual treatment; QIsm-F or pamidronate respectively has modest effect on hFOB1.19. Based on the data collected, the interaction of both compounds (QIsm-F and pamidronate) acts synergistically with each other and is proven to be a more effective treatment choice than individual treatment.

3. Results

3.1. EC50 and effects of QIsm-F and combine treatment on cell viability

The process of bone remodelling has been illustrated and well documented to be directed by two classes of cells; bone removing cell (osteoclast) and bone forming cell (osteoblast). This study adapted osteoblast model so as to understand the mechanism of QIsm-F acting as well as combination treatment of readily available bisphosphonate with natural QIsm-F on bone forming cell for synthesis of new bone. As mentioned, osteoblast is the key component in bone multicellular unit whereby its optimal functions are influenced by many factors and pathway[41]. Important signalling pathway involves in osteoblast differentiation are the smad signalling pathway mediated by BMP-2 via differentiation mesenchymal cell to the osteoblast and the activation of osteoblastogenic transcripton factors which include Runx2, Osx, alkaline phosphatase, osteocalcin and many more[13,42]. The molecular actions of Q. infectoria on osteoblast cell has not been reported in any previous study. However,molecular action of polyphenols from other natural sources; as such by Bu et al., stated that dried plum polyphenols enhance osteoblast activity and function by up-regulating Runx2 and Osx marker[27];meanwhile Dai and colleagues, also reported up-regulation of the same marker in human bone marrow-derived mesenchymal stem cell model triggered by resveratrol; a type of naturally occuring phenols[41]. Our study, considers these two important osteoblastogenic marker (Runx2 and Osx) controlling osteoblast differentiation through smad signalling pathway which leads to formation of new bone forming cells. From the data indicated, the analysis of both Runx2 and Osx expression by western blot analysis shows elevated expression in all treatment groups except tamoxifen in a time dependent manner. However, the highest expression of Runx2 and Osx marker is expressed in cells treated with combination of QIsm-F and pamidronate; particularly at day 7 of treatments. This study implies that the combination of the studied drugs and natural anabolic agent is a more efficient treatment on stimulating the proliferation and differentiation of hFOB1.19 cells when compares to each individual treatment. In addition, this study also points out the biological effects of polyphenols presence in QIsm-F on the hFOB 1.19 cell model. Thereby, it might provide a new insight in osteoporotic treatment discovery.

3.2. Effects of QIsm-F and combination of QIsm-F with pamidronate on Runx2 and Osx expression

ARTICLE INFO

3.3. Up-regulated Runx2 and Osx during osteoblast differentiation of hFOB 1.19 cells

The image (Figure 3 & Figure 4) shows rapid proliferation and enhanced expression of Runx2 and Osx in hFOB 1.19 cells treated with combined QIsm-F with pamidronate at day 3 and day 7.

4. Discussion

As stated earlier, osteoporotic drug bisphosphonate mostly function as a resorptive inhibitor. The mechanism action of these drugs leads to decrease in important bone marker as well as exhibits negligible ability to enhance new synthesis of bone[34,35]. Hence,it is necessary to investigate other potential anabolic agent that can helps to repair the imbalance of the bone remodelling process.Natural anabolic agents from plant sources could be effective for use in the treatment of osteoporosis. Polyphenols, which is a major compound manifestation in Q. infectoria has been reported in the study’s preliminary investigation to contribute to increasing in bone marker that plays a role in initial bone metabolism process[28].This experiment demonstrated detailed effects of QIsm-F isolated from Q. infectoria as well as the synergistic effects of combining established QIsm-F with pamidronate acting on osteoblast model(hFOB1.19 cell) in comparison to individual treatments. The results shows a promising effect when combining QIsm-F with pamidronate whereby it enhanced osteoblast proliferation as well as expression of important bone osteoblastogenic marker that involves in osteoblast differentiation.

As mentioned earlier, there has been several reported research on the ability of different polyphenols from different types of natural plants products contributing to osteoblast differentiation and proliferation[25-27]. This study incorporates crucial solvents interaction from chromatographic technique in order to obtain desired QIsm-F. The presence of polyphenol in isolated QIsm-F was observed as black spot on thin layer chromatography plate as a results of Fecl3 staining and similar Rf values when compared to standard polyphenols (gallic acid and tannins).

(三)新会计制度中增加预算会计的概念。使得单位会计要素包括财务会计要素和预算会计要素，实行财务会计和预算会计平行记账法，单位对于纳入部门预算管理的现金收支业务，在采用财务会计核算的同时又进行预算会计核算，增加了预算收支、预算结余的账务处理，不仅清晰反映了财务主体的运行状况还有效反映和监督了会计主体预算收支执行情况。

Cell viability assay is an essential tool to measure the number of viable cultured cells in order to determine the cytotoxicity and cell viability effects produced by the given treatments to cells[36].the research utilises the usage of calorimetric MTT assay for determining EC50 of the treatments drugs and QIsm-F as well as measures the percentage of cell viability using the established EC50 concentration. Cell viability of hFOB1.19 treated with QIsm-F and combined QIsm-F with pamidronate is more than 80%; whereby,these exhibit the efficiency of QIsm-F and synergistic potential of combined treatment of QIsm-F with pamidronate as an anabolic agent that can contribute to the process of new bone formation.Combination treatment therapy between natural plants compounds with readily available drugs has been shown to exhibit synergistic effects in various diseases such as cancer; multiple myeloma[37] and prostate cancer[38]; viral infection[39], diabetic[40] and many more.

The EC50 values of each treatment were established by using MTT assay (Figure 1A) with pamidronate (15.270 μg/mL), QIsm-F(11.600 μg/mL) and tamoxifen (1.568 μg/mL), respectively. The concentration of QIsm-F (11.600 μg/mL) required to induce cell proliferation in hFOB1.19 cells was lower than required by pamidronate (15.270 μg/mL) which showed preliminary indicator of QIsm-F efficacy on osteoblast cell in comparison to pamidronate.Percentage of cell viability of hFOB 1.19 cells treated with respective established EC50 values of each treatment were investigated and shown in Figure 1B. After treatment with pamidronate, QIsm-F,combined QIsm-F with pamidronate and tamoxifen after 72 h, there is a significant difference in hFOB1.19 cell treated with pamidronate,QIsm-F and tamoxifen when compared to control (Figure 1B).

美国乔治亚大学老年病学中心副主任安娜·格拉斯曾针对中国老年医学的状况发表过论文，对于中国对阿尔茨海默病的更名行动，格拉斯有所关注，她对《中国新闻周刊》表示，“比起‘认知障碍症’‘失忆症’等各种名词，‘阿尔茨海默病’这个名称界限清晰，是一个更为严谨的医学名词。”格拉斯说，“阿尔茨海默病的防治在美国也面临着不少难题，然而我对中国民众对这种病症的缺乏了解，更加感到震惊。”

二是高度重视阅读与写作。美国高中生的阅读量远远大于中国学生，又厚又大的课本，要在课前阅读，并写出阅读笔记和提出问题；教师还会发阅读资料及学习任务单；每个单元结束时都有综合写作，不论人文课还是科学课，学生们常常一写就是一篇论文。

但现有的研究并未将橡胶材料受润滑剂相容性的影响与气缸工作性能的变化联系起来，对气缸的润滑优化帮助较小。因此，本文从相容性出发，对O型圈与润滑剂的相容性对于气缸工作性能的影响进行了试验研究，为日后气缸中的润滑优化提供指导。

Flash column chromatography was performed in glass column(width: 40 mm; length: 500 mm) with silica gel 60, 0.063-0.200 mm (Merck Milipore, USA) using solvent mixture (ethyl acetate:methanol: acetonitrile: H2O); ratio (1:1:7:1). Flash column was packed manually by using the slurry method. Aqueous Q. in fectoria extract sample was prepared by dissolving in 95% ethanol. The solvent mix was continuously added as the final layer simultaneously with elution process until collections of fraction were accomplished. Collection of sample fractions was done in test tubes, graduated with the fraction volume, and visualised by spotting on thin layer chromatography plates using established solvent system (ethyl acetate: methanol:acetonitrile: H2O); ratio (6:1.5:1.5:1). The plate was then visualized under UV lamp at wavelength 254 nm. The fraction with the same Rf values will be pooled together and each fraction will be subjected to second column chromatography by using different solvent mix ratio (ethyl acetate: methanol: acetonitrile: H2O); ratio (5.5:1:2.5:1).The fraction with the same Rf value will again be pooled together and considered as final semi-purified product[30]. The presence of polyphenols is confirmed by performing ferric chloride (Fecl3)staining on thin layer chromatography plate and comparing to the standard.

The effect of Q. infectoria as astringent, antidiabetic, antitremorine and local anaesthetic are well documented, nevertheless its effect on bone cells and associated disease are relatively still scarce. This research shows conclusive evidence that combination of these two agents acts synergistically by up-regulating both Runx2 and Osx marker via smad signalling pathway. Overall, the paper concluded that combination treatment between QIsm-F and pamidronate is effective treatment candidate for bone associated disease particularly osteoporosis than individual treatments; at the same time helps to improve the efficiency of osteoporotic drug pamidronate acting on bone forming cell.

Conflicts of interest statement

The authors declare that they have no conflict of interests.

Acknowledgements

The authors were grateful to the Ministry of Higher Education,Malaysia for funding this work under the Fundamental Research Grant Scheme grant number 203/PPSK/6171169, as well as School of Health Sciences and School of Dental Sciences, Universiti Sains Malaysia for providing the research facility.

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