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Drug Design, Development and Therapy Volume 10, 2016 - Issue
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Original Research

Celastrol inhibits prostaglandin E2-induced proliferation and osteogenic differentiation of fibroblasts isolated from ankylosing spondylitis hip tissues in vitro

Yu-Cong Zou1 School of Traditional Chinese Medicine, Southern Medical University, Guang Zhou, People’s Republic of China
,
Xian-Wen Yang2 The Third Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine, Guang Zhou, People’s Republic of China
,
Shi-Guo Yuan1 School of Traditional Chinese Medicine, Southern Medical University, Guang Zhou, People’s Republic of China
,
Pei Zhang1 School of Traditional Chinese Medicine, Southern Medical University, Guang Zhou, People’s Republic of China
&
Yi-Kai Li1 School of Traditional Chinese Medicine, Southern Medical University, Guang Zhou, People’s Republic of ChinaCorrespondence[email protected]
Pages 933-948 | Published online: 07 Mar 2016

Figures & data

Table 1 Basic clinical data for AS patients

Figure 1 Fibroblasts were successfully cultured and subcultured regularly by changing the culture medium.

Notes: (A) Fibroblasts were isolated from the hip synovium tissue. (B) third generation of cultured fibroblasts. Scale bar is 100 μm and magnification is 1010. (C) and (D) indicates CD90-FITC-positive cells and self control, respectively. The line is the gate that contains wanted CD90-FITC-positive cells. R2 is the classification of CD90-FITC-positive cells. (E) and (F) indicates CD 106-negative and CD90-positive cells, respectively.
Figure 1 Fibroblasts were successfully cultured and subcultured regularly by changing the culture medium.

Figure 2 Cell proliferation growth curve treated by various doses of Celastrol, 1.0 μM Indometacin as well as negative controls.

Note: This experiment was repeated six times.
Abbreviation: DMSO, dimethyl sulfoxide.
Figure 2 Cell proliferation growth curve treated by various doses of Celastrol, 1.0 μM Indometacin as well as negative controls.

Figure 3 Celastrol inhibits cell proliferation by an MTS assay.

Notes: The results of the MTS assay indicated that celastrol inhibits fibroblast proliferation in a time- and dose-dependent manner. This experiment was repeated six times. Values are shown as mean ± standard deviation. The error bars represent the standard deviation of the mean. *P<0.05, **P<0.01 versus normal control group. OD Value (%) = (mean OD value at different time + mean OD value at baseline) ×100% (the same sample).
Abbreviations: DMSO, dimethyl sulfoxide; OD, optical density.
Figure 3 Celastrol inhibits cell proliferation by an MTS assay.

Figure 4 Cell proliferation is measured by performing an EdU incorporation assay on day 14.

Notes: Positive DAPI staining was in blue in the nucleus, showing the numbers of cells as the control. Red fluorescence revealed the number of EdU-positive cells (all ×100). Histogram indicates the positive rates of EdU-positive cells (*P<0.05). *P<0.05, **P<0.01 versus normal control group. The scale bar is 50 μm.
Abbreviation: DMSO, dimethyl sulfoxide.
Figure 4 Cell proliferation is measured by performing an EdU incorporation assay on day 14.

Figure 5 Effect of different doses of celastrol on ALP activity in isolated ankylosing spondylitis fibroblasts compared with indometacin and negative control.

Notes: ALP activity was shown as a specific activity (unit/g protein). The concentration of ALP in each chamber was plotted as the mean ± standard deviation in six replicated experiments. *P<0.05, **P<0.01 versus normal control group. Data from indometacin (1.0 μM) and different doses of celastrol as well as negative controls were compared at the same time duration and evaluated by one-way analysis of variance. Multiple comparisons were performed by Scheffe’s method.
Abbreviations: DMSO, dimethyl sulfoxide; ALP, alkaline phosphatase.
Figure 5 Effect of different doses of celastrol on ALP activity in isolated ankylosing spondylitis fibroblasts compared with indometacin and negative control.

Figure 6 Effect of different doses of celastrol on Alizarin red staining in isolated ankylosing spondylitis fibroblasts compared with indometacin and negative control.

Notes: Dimethyl sulfoxide (DMSO), 1.0 μM of nonselective nonsteroidal anti-inflammatory drugs–indometacin, and the indicated concentrations of celastrol (0.5, 1.0, and 2.0 μM) were added on day 12. Representative images of Alizarin red staining were demonstrated on days 14, 21, and 28. The area of mineral deposit in each well was calculated by randomly choosing from four microscopic fields. The Alizarin red was eluted with 10% cetylpyridinium chloride, and the OD value was read at 510 nm for quantification. Each bar represents the mean ± standard deviation in six replicated experiments. Data from cultured fibroblasts treated with different drugs were compared at the same time duration and evaluated by one-way analysis of variance. *P<0.05, **P<0.01 versus control group. The scale bar is 100 μm.
Abbreviation: OD, optical density.
Figure 6 Effect of different doses of celastrol on Alizarin red staining in isolated ankylosing spondylitis fibroblasts compared with indometacin and negative control.

Figure 7 An amount of 1.0 μM celastrol inhibits mRNA expressions of osteogenic genes in prostaglandin E2-induced ankylosing spondylitis fibroblasts compared with untreated control.

Notes: The mRNA expressions of bone morphogenetic protein 2 (BMP-2), type I collagen, Runx-2, and osteocalcin were detected by real-time PCR on days 14, 21, and 28. The relative expression level was calculated from the threshold cycle (Ct) value of each PCR product and normalized with that of GAPDH by using a comparative Ct method. *P<0.05, **P<0.01.
Figure 7 An amount of 1.0 μM celastrol inhibits mRNA expressions of osteogenic genes in prostaglandin E2-induced ankylosing spondylitis fibroblasts compared with untreated control.

Figure 8 Celastrol (1.0 μM) impacts the expression of related proteins in isolated fibroblasts.

Notes: Cultured fibroblasts were pretreated with 1.0 μM celastrol. The protein levels were detected by Western blot analysis. The bands of Western blot analysis were digitally detected and normalized with that of β-actin. Each bar represents the mean ± standard deviation in six replicated experiments. Data were evaluated by one-way analysis of variance, and multiple comparisons were performed by Scheffe’s method. *P<0.05, **P<0.01 versus normal control group.
Abbreviation: PGE-2, prostaglandin E2.
Figure 8 Celastrol (1.0 μM) impacts the expression of related proteins in isolated fibroblasts.

Table S1 Raw Data Of Western Blot Analysis Results with 1.0 μM celastrol compared with untreated group

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