4-1.1 LDH Cytotoxicity Assay
The OD490 obtained from 2D cultured porcine chondrocytes which were treated with aucubin and betulin are summarized in Figure 22 and Figure 23, respectively. In the aucubin-treated experimental groups, the chondrocytes treated with the lowest concentration of aucubin showed a significantly higher OD value than the control group (0.725 ± 0.081 vs. 0.690 ± 0.073, P = 0.045). However, the chondrocytes treated with the middle and the highest concentrations of aucubin lowered the OD value compared with the control group.
0
Figure 22 LDH cytotoxicity assay of aucubin-treated chondrocytes. The absorbances at 490 nm were obtained from one-week chondrocytes which were treated with different concentrations of aucubin.
In the betulin-treated experimental groups, the higher the concentrations of betulin, the lower the OD values. However, these differences are not statistically significant.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
O.D. 490nm
■ Control
■ Betulin
Figure 23 LDH cytotoxicity assay of betulin-treated chondrocytes. The absorbances at 490 nm were obtained from one-week chondrocytes which were treated with different concentrations of betulin.
4-1.2 MTT Cell Proliferation Assay
The OD570 obtained from 2D cultured porcine chondrocytes which were treated with aucubin and betulin are summarized in Figure 24 and Figure 25, respectively. In the aucubin-treated experimental groups, the chondrocytes treated with the lowest concentration of aucubin showed very little and not significantly higher OD value than the control group, and the chondrocytes treated with the middle concentration of aucubin showed a little but not significant lower OD value than the control group. In contrast, the chondrocytes treated with the highest concentration of aucubin showed a significantly higher OD value than the control group (0.253 ± 0.03 vs. 0.220 ± 0.03, P
= 0.0004).
0 0.05 0.1 0.15 0.2 0.25 0.3
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
O.D. 570nm
* ■ Control
■ Acubin
Figure 24 MTT cell proliferation assay of aucubin-treated chondrocytes. The absorbances at 570 nm were obtained from one-week chondrocytes which were treated with different concentrations of aucubin.
In the betulin-treated experimental groups, the chondrocytes treated with the lowest and the highest concentrations of betulin both showed significantly lower OD values than the control group (0.168 ± 0.023 vs. 0.220 ± 0.03, P = 0.003, and 0.176 ± 0.028 vs. 0.220 ± 0.03, P = 0.046), and the chondrocytes treated with the middle concentration of betulin showed lower but not statistical significant OD values than the control group.
0 0.05 0.1 0.15 0.2 0.25 0.3
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
O.D. 570nm
*
■ Control
■ Betulin
*
Figure 25 MTT cell proliferation assay of betulin-treated chondrocytes. The absorbances at 570nm were obtained from one-week chondrocytes which were treated with different concentrations of betulin.
4-1.3 Total DNA for Cell Proliferation Quantification
After the genomic DNA was isolated from cultured cells, genomic DNA concentration was calculated by using the formula: DNA concentration = O.D. 260
﹣O.D. 320 × dilute factor × 50 ug/ml. Total genomic DNA content obtained from 2D cultured porcine chondrocytes which were treated with aucubin and betulin are summarized in Figure 26 and Figure 27, respectively. In the aucubin-treated experimental groups, the chondrocytes treated with the lowest and the highest concentrations of aucubin both increased the DNA content in a statistical insignificant level than the control group. However, the chondrocytes treated with the middle concentration of betulin decreased the DNA content in a statistical significant level compared with the control group (2.322 ± 0.005 μg/mL vs. 2.682 ± 0.135 μg/mL, p = 0.006).
Figure 26 Total DNA content of one-week chondrocytes treated with different concentrations of aucubin. DNA concentration was calculated by equation: O.D. 260 - O.D. 320 x dilute factor x 50 ug/ml.
In the betulin-treated experimental groups, betulin increased the total DNA content of chondrocytes in every experimental group. Total DNA content in chondrocytes treated with the lowest and the middle concentrations of betulin averaged 3.05 ± 0.240 μg/mL and 3.185 ± 0.318 μg/mL respectively, which showed significant increase than the control group (p = 0.041 and p = 0.033). Total DNA content in chondrocytes treated with the highest concentrations of betulin averaged 2.711 ± 0.118 μg/mL, but this increase of DNA content was not statistically significant. (p = 0.393).
0 0.5 1 1.5 2 2.5 3 3.5 4
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc. (ug/ml)
DNA conc. (ug/ml)
* * ■ Control
■ Betulin
Figure 27 Total DNA content of one-week chondrocytes treated with different concentrations of betulin. DNA concentration was calculated by equation: O.D. 260 - O.D. 320 x dilute factor x 50 ug/ml.
4-1.4 DMMB Assay for Sulfated Glycosaminoglycans Content
Before conducting DMMB assay, a linear standard curve was obtained from GAG standards: 0–100 mg/mL of chondroitin-6-sulfate (C-4384, Sigma), which were then used to estimate the GAG content in each experimental sample. The OD525
obtained from C6S standards are summarized in Figure 28. The linear regression trend line of C6S standards was: y = 0.0043x + 0.4292.
GAGs Standard Curve
Figure 28 Linear standard curve of glycosaminoglycans standards. The absorbance at 525 nm was measured. The linear regression trend line of glycosaminoglycans standards was: y = 0.0043x + 0.4292.
Sulfated glycosaminoglycans content obtained from 2D cultured porcine chondrocytes which were treated with aucubin and betulin are summarized in Figure 29 and Figure 30, respectively. Both in the aucubin-treated experimental groups and the betulin-treated experimental groups, sulfated GAG content didn’t show any significantly change. The average sulfated GAG content in the control group was 0.43128 ± 0.052 ug/ml, and the average sulfated GAG content in chondrocytes treated with the lowest, middle, and highest concentrations of aucubin were 0.43126
± 0.006 ug/ml, 0.43133 ± 0.019 ug/ml, and 0.43134 ± 0.001 ug/ml, respectively.
0 0.1 0.2 0.3 0.4 0.5 0.6
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
GAGs conc. (ug/ml)
■ Control
■ Acubin
Figure 29 Glycosaminoglycans content of one-week chondrocytes treated with different concentrations of aucubin.
In the betulin-treated experimental groups, the average sulfated GAG content in chondrocytes treated with the lowest, middle, and highest concentrations of betulin were 0.43126 ± 0.006 ug/ml, 0.43125 ± 0.001 ug/ml, and 0.43136 ± 0.002 ug/ml, respectively.
0 0.1 0.2 0.3 0.4 0.5 0.6
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
GAGs conc. (ug/ml)
■ Control
■ Betulin
Figure 30 Glycosaminoglycans content of one-week chondrocytes treated with different concentrations of betulin.
4-1.5 Real-time Reverse-Transcriptase Polymerase Chain Reaction for mRNA Expression Quantification
In real-time PCR, the -△Ct values of each target gene were normalized by the house-keeping gene, GAPDH, and relative expressions of each target gene between the experimental groups and the control group are shown by the comparative CT (△△CT) changes and summarized as follows.
4-1.5.1 Gene Expression of Type I collagen
In the aucubin-treated experimental groups, gene expression of type I collagen showed a 1.33-fold increase under the lowest concentration of aucubin treatment.
Also, there was a 1.36-fold increase under the middle concentration of aucubin treatment. In contrast, the chondrocytes treated with the highest concentration of aucubin lowered the gene expression of type I collagen to a 0.94-fold compared with the control group.
Figure 31 The mRNA expression of type I collagen of one-week chondrocytes under different concentrations of aucubin treatment.
In the betulin-treated experimental groups, gene expression of type I collagen showed a 0.91-fold decrease under the lowest concentration of aucubin treatment, while there was a 1.26-fold and 1.21-fold increase under the treatment of the middle and highest concentrations of aucubin, respectively.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
Figure 32 The mRNA expression of type I collagen of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of type I collagen protein.
4-1.5.2 Gene Expression of Type II collagen
In the aucubin-treated experimental groups, gene expression of type II collagen showed a 1.26-fold and a 1.14-fold increases under the treatment of the lowest and middle concentration of aucubin, respectively. However, there was a 0.95-fold decrease under the highest concentration of aucubin treatment.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
*
■ Control
■ Acubin
Figure 33 The mRNA expression of type II collagen of one-week chondrocytes under different concentrations of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of type II collagen protein.
In the betulin-treated experimental groups, gene expression of type II collagen increased in each experimental group. Moreover, the result showed that the higher the concentration of betulin added, the higher the gene expression of type II collagen in chondrocytes. The chondrocytes treated with the lowest concentration of betulin showed a 1.04-fold increase, and the chondrocytes treated with the middle and highest concentrations of betulin showed significant increases up to a 1.307-fold and 1.311-fold, respectively.
Figure 34 The mRNA expression of type II collagen of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of type II collagen protein.
4-1.5.3 Gene Expression of Aggrecan
In the aucubin-treated experimental groups, gene expression of aggrecan showed a 1.58-fold increase under the treatment of the lowest concentration of aucubin. Also, there was a 1.11-fold increase under the treatment of the middle concentration of aucubin. However, gene expression of aggrecan under the highest concentration of aucubin treatment showed a 0.96-fold decrease compared with the control group.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
* ■ Control
■ Acubin
Figure 35 The mRNA expression of aggrecan of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of aggrecan protein.
In the betulin-treated experimental groups, although gene expression of aggrecan showed a 0.79-fold decrease under the treatment of the lowest concentration of betulin, gene expression of aggrecan showed a 1.31-fold and a 1.54-fold under the treatment of the middle and highest concentration of betulin, respectively. The result showed that the higher the concentration of betulin added, the higher the gene expression of aggrecan in chondrocytes.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
*
Figure 36 The mRNA expression of aggrecan of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of aggrecan protein.
4-1.5.4 Gene Expression of Decorin
In the aucubin-treated experimental groups, gene expression of decorin increased in each experimental group, although the result showed that the higher the concentration of aucubin added, the lower the gene expression of decorin in chondrocytes. The chondrocytes treated with the lowest and middle concentrations of aucubin showed significant increases up to a 1.39-fold and 1.33-fold, respectively.
Also, the chondrocytes treated with the highest concentration of betulin showed a 1.06-fold increase.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
* * ■ Control
■ Acubin
Figure 37 The mRNA expression of decorin of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of decorin protein.
In the betulin-treated experimental groups, although gene expression of decorin decreased in each experimental group, the chondrocytes treated with the middle concentration of betulin still showed a 0.99-fold expression compared with the control group. The chondrocytes treated with the lowest and the highest concentrations of betulin showed a 0.82-fold and a 0.94-fold decrease, respectively.
0 0.2 0.4 0.6 0.8 1 1.2
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
Figure 38 The mRNA expression of decorin of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of decorin protein.
4-1.5.5 Gene Expression of Sox9
In the aucubin-treated experimental groups, gene expression of sox9 decreased lower than a 0.4-fold in each experimental group. The gene expression of sox9 in chondrocytes treated with the lowest, middle, and highest concentrations of aucubin showed 0.35-fold, 0.14-fold, and 0.23-fold decreases, respectively.
0 0.2 0.4 0.6 0.8 1 1.2
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
*
*
*
■ Control
■ Acubin
Figure 39 The mRNA expression of Sox9 of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of Sox9 protein.
Also, in the betulin-treated experimental groups, gene expression of sox9 decreased lower than a 0.35-fold in each experimental group. The gene expression of sox9 in chondrocytes treated with the lowest, middle, and highest concentrations of betulin showed 0.14-fold, 0.12-fold, and 0.33-fold decreases, respectively.
0 0.2 0.4 0.6 0.8 1 1.2
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
* *
*
Figure 40 The mRNA expression of Sox9 of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of Sox9 protein.
4-1.5.6 Gene Expression of MT1-MMP
In the aucubin-treated experimental groups, gene expression of MT1-MMP increased in each experimental group. However, real-rime PCR analysis demonstrated that MT1-MMP mRNA levels were slightly but not significantly increased in chondrocytes treated with the lowest concentration of aucubin compared with the control group. Furthermore, the gene expression of mt1-mmp in chondrocytes treated with the middle, and highest concentrations of aucubin showed 1.14-fold and 1.24-fold increases, respectively.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
* ■Control
■ Acubin
Figure 41 The mRNA expression of MT1-MMP of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of MT1-MMP protein.
In the betulin-treated experimental groups, although gene expression of mt1-mmp showed a 0.82-fold decrease under the treatment of the lowest concentration of betulin, gene expression of mt1-mmp showed a 1.16-fold and a 1.13-fold increases under the treatment of the middle and highest concentration of betulin, respectively.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
Figure 42 The mRNA expression of MT1-MMP of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of MT1-MMP protein.
4-1.5.7 Gene Expression of MMP-2
In the aucubin-treated experimental groups, gene expression of mmp-2 decreased in each experimental group. The chondrocytes treated with the lowest concentration of aucubin showed a 0.72-fold decrease. Moreover, the chondrocytes treated with the middle and highest concentrations of aucubin showed significant decreases to a 0.15-fold and 0.22-fold, respectively.
0 0.2 0.4 0.6 0.8 1 1.2
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
*
*
■ Control
■ Acubin
Figure 43 The mRNA expression of MMP-2 of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of MMP-2 protein.
Similarly, in the betulin-treated experimental groups, gene expression of mmp-2 decreased in each experimental group. The chondrocytes treated with the lowest concentration of betulin showed a 0.48-fold decrease. Moreover, the chondrocytes treated with the middle and highest concentrations of betulin showed significant decreases to a 0.13-fold and 0.21-fold, respectively.
0 0.2 0.4 0.6 0.8 1 1.2
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
*
*
Figure 44 The mRNA expression of MMP-2 of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of MMP-2 protein.
4-1.5.8 Gene Expression of T1MP-1
In the aucubin-treated experimental groups, although gene expression of timp-1 showed a 0.79-fold decrease under the treatment of the lowest concentration of aucubin, gene expression of timp-1 showed a 1.13-fold increase under the treatment of the middle concentration of aucubin. The chondrocytes treated with the highest concentration of aucubin showed a little decrease to a 0.98-fold.
0 0.2 0.4 0.6 0.8 1 1.2
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
■ Control
■ Acubin
Figure 45 The mRNA expression of TIMP-1 of one-week chondrocytes under different concentrations of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of TIMP-1 protein.
In the betulin-treated experimental groups, the chondrocytes treated with the lowest concentration of aucubin showed a 1.13-fold increase in mRNA expression of timp-1 compared with the control group. Although gene expression of timp-1 showed a 0.82-fold and a 0.92-fold decreases under the treatment of the middle and highest concentration of betulin respectively, the value were still kept above 0.8-fold.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
Figure 46 The mRNA expression of TIMP-1 of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of TIMP-1 protein.
4-1.5.9 Gene Expression of IL-1beta
In the aucubin-treated experimental groups, gene expression of IL-1 beta extremely increased higher than a 100-fold in each experimental group. The gene expression of IL-1 beta in chondrocytes treated with the lowest, middle, and highest concentrations of aucubin showed 103-fold, 170-fold, and 144-fold increases, respectively.
Figure 47 The mRNA expression of IL-1beta of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of IL-1beta protein.
On the other hand, gene expression of IL-1 beta also increased in each betulin-treated experimental group. Nevertheless, the increasing extent is not as high as in each aucubin-treated experimental group. The result showed that when chondrocytes were treated with betulin, there were about 20-fold to 50-fold increases in mRNA expression of IL-1 beta compared with the control group. The gene expression of IL-1 beta in chondrocytes treated with the lowest, middle, and highest concentrations of betulin showed 22-fold, 23-fold, and 47-fold increases, respectively.
Figure 48 The mRNA expression of IL-1beta of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of IL-1beta protein.
4-1.5.10 Gene Expression of TGF-beta1
In the aucubin-treated experimental groups, although gene expression of TGF-beta1 showed a 0.82-fold and 0.92-fold decreases under the treatment of the middle and highest concentration of betulin respectively, the TGF-beta1 mRNA levels were still kept above 0.85-fold. However, the chondrocytes treated with the highest concentration of aucubin showed a 0.60-fold decrease in TGF-beta1 mRNA levels compared with the control group.
0 0.2 0.4 0.6 0.8 1 1.2
control 5.0μg/L 80μg/L 1280μg/L
AUCUBIN conc.
Log10 (relative quantity)
*
■ Control
■ Acubin
Figure 49 The mRNA expression of TGF-beta1 of one-week chondrocytes under different concentration of aucubin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of TGF-beta1 protein.
In the betulin-treated experimental groups, gene expression of TGF-beta1 showed a 1.25-fold increase under the treatment of the lowest concentration of aucubin, and the chondrocytes treated with the highest concentration of betulin kept the gene expression of TGF-beta1 just as the control group (1.00-fold). In contrast, there was a 0.75-fold decrease in TGF-beta1 mRNA levels under the highest concentration of betulin treatment.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
control 0.02μg/ml 0.32μg/ml 5.12μg/ml
BETULIN conc.
Log10 (relative quantity)
■ Control
■ Betulin
Figure 50 The mRNA expression of TGF-beta1 of one-week chondrocytes under different concentration of betulin treatment. The data was shown by the comparative CT (△△CT) changes in gene expression of TGF-beta1 protein.
4-1.6 Chemiluminescence Method for Detecting Free Radicals Scavenging
Formation of reactive oxygen species in 2D cultured chondrocytes was demonstrated by using chemiluminescence (CL) technique with luminol and lucigenin chemical probes. Luminol was used to detect the existence of hydrogen peroxide in cells, while lucigenin was selective to the existence of superoxide anion in cells.
Because there was no significant detection of hydrogen peroxide existing in 2D cultured chondrocytes of the control groups and the experimental groups, the luminol-dependent chemiluminescence results were not shown here.
In the aucubin-treated experimental groups, lucigenin-dependent chemiluminescence demonstrated that lucigenin CL levels were slightly decreased in the group treated with the lowest concentration of aucubin compared with the control group (54.82±4.8 vs. 60.5±4.04 CL count/10sec, p = 0.165>0.01), but the lucigenin CL levels were slightly but not significantly increased in the group treated with the middle concentration of aucubin (88.35±6.63 vs. 60.5±4.04 CL count/10sec, p
=0.018>0.01). In the group treated with the highest concentration of aucubin, the lucigenin CL levels were dramatically and significantly decreased compared with the control group (-53.81±6.16 vs. 60.5±4.04 CL count/10sec, p =0.001<0.01).
-80
Figure 51 The free radicals scavenging effect of different concentrations of aucubin on lucigenin-dependent chemiluminescence of one-week chondrocytes.
In the betulin-treated experimental groups, lucigenin CL levels significantly
In the betulin-treated experimental groups, lucigenin CL levels significantly