Yonsei Med J.  2015 May;56(3):760-771. 10.3349/ymj.2015.56.3.760.

Stimulating Effect of a Novel Synthesized Sulfonamido-Based Gallate ZXHA-TC on Primary Osteoblasts

• Jin P
• Liao L
• Lin X
• Guo Q
• Lin C
• Wu H
• Zheng L
• Zhao J

• KMID: 2450351
• DOI: http://doi.org/10.3349/ymj.2015.56.3.760

Abstract

PURPOSE
This study is intended to investigate the effects of plants or plant-derived antioxidants on prevention of osteoporosis through the maintenance of reactive oxygen species (ROS) at a favorable level.
MATERIALS AND METHODS
In this study, a novel antioxidant, namely 3,4,5-Trihydroxy-N-[4-(5-hydroxy-6-methoxy-pyrimidin-4-ylsulfamoyl)-phenyl]-benzamide (ZXHA-TC) was synthesized from gallic acid and sulfadimoxine. Its effect on osteoblast metabolism was investigated via the detection of cell proliferation, cell viability, production of ROS, and expression of osteogenic-specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), alpha-1 type I collagen (COL1A1), and osteogenic-related proteins after treatment for 2, 4, and 6 days respectively.
RESULTS
The results showed that ZXHA-TC has a stimulating effect on the proliferation and osteogenic differentiation of primary osteoblasts by promoting cell proliferation, cell viability, and the expression of genes BSP and OCN. Productions of bone matrix and mineralization were also increased by ZXHA-TC treatment as a result of up-regulation of COL1A1 and alkaline phosphatase (ALP) at the early stage and down-regulation of both genes subsequently. A range of 6.25x10(-3) microg/mL to 6.25x10(-1) microg/mL is the recommended dose for ZXHA-TC, within which 6.25x10(-2) microg/mL showed the best performance.
CONCLUSION
This study may hold promise for the development of a novel agent for the treatment of osteoporosis.

Keyword

Gallic acid; sulfonamido; osteoporosis; osteogenesis; osteoblast

MeSH Terms

Alkaline Phosphatase/metabolism
Bone Morphogenetic Proteins/pharmacology
Cell Differentiation/*drug effects
Cell Proliferation/*drug effects
Collagen Type I/genetics
Core Binding Factor Alpha 1 Subunit
Down-Regulation
Gallic Acid
Osteoblasts/*drug effects
Osteocalcin/metabolism
Osteogenesis/drug effects
Osteoporosis/*prevention & control
Reactive Oxygen Species
Up-Regulation
Alkaline Phosphatase
Bone Morphogenetic Proteins
Collagen Type I
Core Binding Factor Alpha 1 Subunit
Gallic Acid
Osteocalcin
Reactive Oxygen Species

Figure

• 

  Fig. 1 The synthetic route of ZXHA-TC was presented. Reagents and conditions. (A) Acetyl oxide, oil bath, 120℃. (B) SOCl2, oil bath, 80℃. (C) Sulfadimoxine, THF, pyridine, ice bath. (D) HCl, THF, 60℃.

• 

  Fig. 2 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to analyze the cytotoxicity of primary osteoblasts treated with ZXHA-TC at different concentrations. (A) A range of 0-2.56×105 µg/mL of ZXHA-TC was chosen. The result shows that ZXHA-TC at the concentrations ranging from 6.25×10-5 to 2.5×102 µg/mL showed low cytotoxicity, among which 6.25×10-3 µg/mL to 6.25×10-1 µg/mL have a significantly positive effect on cell viability (p<0.05). (B) The effect of ZXHA-TC at the concentrations of 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL on primary osteoblasts was detected (n=9). Different letters indicate that the two groups are significantly different from each other (p<0.05), and similar letters indicate no significant difference. Treatment of ZXHA-TC promoted cell viability in a dose-dependent manner; particularly at the concentration of 6.25×10-2 µg/mL, ZXHA-TC enhanced cell viability the most. *Significant difference (p<0.05, n=3).

• 

  Fig. 3 Cell viability was determined by fluorescein diacetate-propidium iodide staining, in which viable cells were stained green and dead cells were stained red. (A-D) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 2 days. (E-H) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 4 days. (I-L) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 6 days. (M) Statistical analysis of the data from the staining pictures from A-L (n=3). As time elapsed, more and more dead cells were found in each group. Comparatively, more viable cells were found in the ZXHA-TC-treated groups, which indicated the positive effect on the primary osteoblasts. Scale bar=200 µm.

• 

  Fig. 4 Cell proliferation was detected with a BrdU cell proliferation detection kit by flow cytometry. (A-D) Represent the results of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL, respectively, at 4 days. The results showed that ZXHA-TC promoted cell growth markedly, especially at the concentration of 6.25×10-2 µg/mL.

• 

  Fig. 5 Actin filament was detected with rhodamine phalloidin-Hoechst 33258 staining, in which the actin filament and nuclei were stained red and blue respectively. (A-D) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 2 days. (E-H) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 4 days. (I-L) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 6 days. (M) Statistical analysis of the data from the staining pictures from A-L (n=3). Cells in ZXHA-TC-treated groups grew in clumps, with the density of the groups at the concentration of 6.25×10-2 µg/mL being most salient, which indicates that cells in these groups communicate more closely than those in the control. Scale bar=100 µm.

• 

  Fig. 6 A quantitative real-time polymerase chain reaction was used to analyze the progression of the expression of osteogenic genes RUNX2 (A), BSP (B), OCN (C), and COL1A1 (D) in primary newborn osteoblasts cultured in different groups for 2, 4, and 6 days. Compared with the control, expressions of the four genes were all up-regulated. However, the expressions of genes BSP and OCN were both up-regulated over time, and those of genes RUNX2 and COL1A1 were up-regulated from 2 to 4 days and down-regulated from 4 to 6 days. The minimum value was set to 1, and values are expressed as mean±2 SD. The bars with different letters at the same time are significantly different from each other (p<0.05; n=3), and those with similar letters show no significant difference. RUNX2, runt-related transcription factor 2; BSP, bone sialoprotein; OCN, osteocalcin; COL1A1, alpha-1 type I collagen.

• 

  Fig. 7 Time-course of ALP activity and ALP staining of primary osteoblasts at different concentrations (0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL) of ZXHA-TC are exhibited. (A) Relative ALP activity (units/100 mL) was expressed as mean±2 SD, and the activity in 6.25×10-2 µg/mL was significantly higher than other groups. ALP activity in the ZXHA-TC-treated groups increased from 2 to 4 days and decreased slightly from 4 to 6 days. However, the activity in the control increased over time. The bars with different letters at the same time are significantly different from each other (p<0.05; n=3), and those with similar letters indicate no significant difference. (B-E) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 2 days. (F-I) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 4 days. (J-M) Staining of primary osteoblasts treated with ZXHA-TC at concentrations of 0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL at 6 days. ALP staining in the ZXHA-TC-treated groups was strengthened from 2 to 4 days and was weakened slightly from 4 to 6 days, and the concentration of 6.25×10-2 µg/mL performed best, which was in accordance the ALP activity results. Scale bar=200 µm. ALP, alkaline phosphatase.

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  Fig. 8 Production of intracellular ROS was detected through the intensity of fluorescence indirectly with a microplate fluorescence reader. After being treated with different concentrations of ZXHA-TC (0 µg/mL, 6.25×10-3 µg/mL, 6.25×10-2 µg/mL, and 6.25×10-1 µg/mL) for 2, 4, and 6 days, primary osteoblasts were labeled with new synthesized DNA and assayed. The level of intracellular ROS was markedly reduced by ZXHA-TC, especially at the concentration of 6.25×10-2 µg/mL. ROS, reactive oxygen species.

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