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Anti-inflammatory and antioxidative effects of genistein in a model of spinal cord injury in rats

Ercan Bal
Ercan Bal
, 
Şahin Hanalioğlu
Şahin Hanalioğlu
, 
Aydın Sinan Apaydın
Aydın Sinan Apaydın
, 
Ceylan Bal
Ceylan Bal
, 
Almila Şenat
Almila Şenat
, 
Berrak Gümüşkaya Öcal
Berrak Gümüşkaya Öcal
, 
Burak Bahadır
Burak Bahadır
 und 
Ömer Faruk Türkoğlu
Ömer Faruk Türkoğlu
   | 29. Okt. 2021
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Article Category: Original article
Online veröffentlicht: 29. Okt. 2021
Seitenbereich: 233 - 243
DOI: https://doi.org/10.2478/abm-2021-0029
Schlüsselwörter
© 2021 Ercan Bal et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Timeline for the experiment. In group A (n = 7, control group, only T7-8-9 laminectomy), in group B (n = 7, trauma group, after T7-8-9 laminectomy and trauma was induced with a modified Allen weight-drop method, in group C (n = 7, genistein group, T7-8-9 laminectomy and spinal trauma was induced as described above. It was planned that group C would receive the first dose of genistein at 15 min immediately after trauma and would be given genistein subcutaneously at 2 mg/kg/day for 7 days). The rats were killed on the 7th day and the spinal cord was removed as a whole. No rats were lost, and no infection, or additional problems were observed during the experiment. cat, catalase; Cox-2, cyclooxygenase-2; IMA, ischemia-modified albumin; NT, native thiol; OSI, oxidative stress index; SS, disulfide; TT, total thiol; TAS, total antioxidant status; TOS, total oxidative status.
Timeline for the experiment. In group A (n = 7, control group, only T7-8-9 laminectomy), in group B (n = 7, trauma group, after T7-8-9 laminectomy and trauma was induced with a modified Allen weight-drop method, in group C (n = 7, genistein group, T7-8-9 laminectomy and spinal trauma was induced as described above. It was planned that group C would receive the first dose of genistein at 15 min immediately after trauma and would be given genistein subcutaneously at 2 mg/kg/day for 7 days). The rats were killed on the 7th day and the spinal cord was removed as a whole. No rats were lost, and no infection, or additional problems were observed during the experiment. cat, catalase; Cox-2, cyclooxygenase-2; IMA, ischemia-modified albumin; NT, native thiol; OSI, oxidative stress index; SS, disulfide; TT, total thiol; TAS, total antioxidant status; TOS, total oxidative status.

Figure 2

Catalase serum levels. Significant difference was observed on the 7th day (P = 0.001). Compared with the baseline, serum catalase values increased slightly in the control group (A white bars), decreased in the trauma group (B light gray bars), and increased significantly in the genistein group (C dark gray bars) on the 7th day. B, baseline; 0.5, 12th hour. Bars indicate means. Error bars (SD).
Catalase serum levels. Significant difference was observed on the 7th day (P = 0.001). Compared with the baseline, serum catalase values increased slightly in the control group (A white bars), decreased in the trauma group (B light gray bars), and increased significantly in the genistein group (C dark gray bars) on the 7th day. B, baseline; 0.5, 12th hour. Bars indicate means. Error bars (SD).

Figure 3

SS serum levels. Significant difference was observed on the 1st, 5th, and 7th day between the control group (A white bars) and trauma group (B light gray bars) and genistein group (C dark gray bars), and between groups B and C (P = 0.004, P = 0.014, P = 0.004, respectively). Compared with the levels at baseline, on the 7th day there was no significant difference in group A; an increase in group B; and a significant decrease in group C. Bars indicate means. Error bars (SD). SS, disulfide
SS serum levels. Significant difference was observed on the 1st, 5th, and 7th day between the control group (A white bars) and trauma group (B light gray bars) and genistein group (C dark gray bars), and between groups B and C (P = 0.004, P = 0.014, P = 0.004, respectively). Compared with the levels at baseline, on the 7th day there was no significant difference in group A; an increase in group B; and a significant decrease in group C. Bars indicate means. Error bars (SD). SS, disulfide

Figure 4

Immunohistochemically stained rat spinal cord sections with hematoxylin and eosin counterstain. A mouse monoclonal Cox-2 antibody (clone D-12: catalog No. sc-166475, Santa Cruz Biotechnology; RRID: AB_2276666) was used for primary detection, the monoclonal antibody was visualized using a diaminobenzidine chromogen system showing brown staining. Cox-2 positive cells were counted per 3 HPFs for each spinal cord (left panel, trauma group (B), right panel, genistein group (C). Left panel scale bar indicates 0.5 mm, inset scale bar indicates 50 μm. Right panel scale bar indicates 0.5 mm. Cox-2, cyclo-oxygenase-2; HPFs, 400× high-powered fields; RRID, research resource identifier.
Immunohistochemically stained rat spinal cord sections with hematoxylin and eosin counterstain. A mouse monoclonal Cox-2 antibody (clone D-12: catalog No. sc-166475, Santa Cruz Biotechnology; RRID: AB_2276666) was used for primary detection, the monoclonal antibody was visualized using a diaminobenzidine chromogen system showing brown staining. Cox-2 positive cells were counted per 3 HPFs for each spinal cord (left panel, trauma group (B), right panel, genistein group (C). Left panel scale bar indicates 0.5 mm, inset scale bar indicates 50 μm. Right panel scale bar indicates 0.5 mm. Cox-2, cyclo-oxygenase-2; HPFs, 400× high-powered fields; RRID, research resource identifier.

Figure 5

Cox-2 immunoreactivity scoring. Significant difference (P = 0.008) was found between the genistein (C) and other groups (A, B). The highest number of Cox-2 IR cells per 3 HPFs was observed in the control group (A white bar) and the trauma group (B light gray bar). The lowest number of Cox-2-IR cells was observed in the genistein group (C dark gray bar). Bars indicate means. Error bars (SD). Cox-2, cyclo-oxygenase-2; HPFs, 400× high-powered fields; IR, immunoreactive.
Cox-2 immunoreactivity scoring. Significant difference (P = 0.008) was found between the genistein (C) and other groups (A, B). The highest number of Cox-2 IR cells per 3 HPFs was observed in the control group (A white bar) and the trauma group (B light gray bar). The lowest number of Cox-2-IR cells was observed in the genistein group (C dark gray bar). Bars indicate means. Error bars (SD). Cox-2, cyclo-oxygenase-2; HPFs, 400× high-powered fields; IR, immunoreactive.

Time-dependent comparison of TAS, TOS, OSI, and Cox-2 immunoreactivity between the groups

Control group (A) Trauma group (B) Genistein group (C) P
TAS (nmol Trolox Eq/mg protein) 35.8 (30.7–40.8) 32.7 (28.6–36.8) 30.8 (29.8–35.3) 0.26
TOS (nmol H2O2 Eq/mg protein) 1.08 (0.59–1.12) 0.67 (0.58–0.85) 0.83 (0.60–1.11) 0.40
OSI (TOS/TAS) 0.03 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.56
Cox 2 IR cells/3 HPFs 19.1 ± 5.3 17.1 ± 5.9 7.71 ± 5.0 0.008 (y, z)

Time-dependent comparison of BBB scores in rats

Time point Control group (A) Trauma group (B) Genistien group (C) P
12th hour 7 (6–7) 5 (3–6) 5 (2–6) 0.006 (x, y)
1st day 13 (12–13) 11 (9–12) 11 (10–12) 0.002 (x, y)
3rd day 13 (13–13) 11 (9–12) 12 (11–13) 0.001 (x, y, z)
5th day 13 (13–13) 11 (9–12) 13 (12–13) <0.001 (x, z)
7th day 21 (21–21) 18 (14–19) 21 (21–21) <0.001 (x, z)
P <0.001 <0.001 <0.001

Time-dependent comparison of oxidative and antioxidative serum markers between groups

Parameter Group Time P
Baseline 12th hour 1st day 3rd day 5th day 7th day
Catalase (kU/L)
A 79.0 ± 18.0 74.2 ± 9.2 31.5 ± 13.8 84.3 ± 6.1 61.7 ± 12.0 86.0 ± 10.0 <0.001
B 48.1 ± 9.9 74.9 ± 7.9 42.6 ± 9.2 48.0 ± 4.6 54.1 ± 12.3 37.9 ± 21.9 0.02
C 51.4 ± 11.9 51.8 ± 30.0 76.9 ± 13.9 69.4 ± 13.3 75.0 ± 9.1 75.0 ± 9.1 0.002
P 0.11 0.11 0.001 (x, z) 0.15 (x, y, z) 0.15 0.001 (x, z)
Ischemia-modified albumin (AbsU)
A 0.84 ± 0.05 0.82 ± 0.05 0.99 ± 0.05 0.93 ± 0.04 0.91 ± 0.05 1.01 ± 0.03 <0.001
B 0.87 ± 0.04 0.93 ± 0.03 0.91 ± 0.03 0.78 ± 0.08 0.92 ± 0.04 0.92 ± 0.03 0.001
C 0.91 ± 0.03 0.92 ± 0.04 0.85 ± 0.04 1.00 ± 0.04 0.98 ± 0.05 1.07 ± 0.09 <0.001
P 0.10 0.001 (x, y) <0.001 (x, y, z) <0.001 (x, y, z) 0.10 0.004 (x, z)
NT (mmol/L)
A 183.1 ± 37.8 110.9 ± 36.0 116.0 ± 16.2 71.7 ± 13.3 157.3 ± 26.0 87.4 ± 20.4 <0.001
B 190.8 ± 51.0 222.2 ± 27.1 96.0 ± 31.4 127.8 ± 24.3 130.7 ± 29.8 166.3 ± 22.5 <0.001
C 194.7 ± 38.7 99.9 ± 37.6 167.3 ± 24.7 99.8 ± 32.4 108.1 ± 28.6 71.2 ± 22.9 <0.001
P 0.91 0.007 (x, y) 0.002 (y, z) 0.006 (x) 0.02 (y) 0.001 (x, z)
TT (mmol/L)
A 250.3 ± 53.0 189.4 ± 47.5 158.5 ± 18.2 142.4 ± 12.5 202.5 ± 33.0 152.3 ± 21.0 0.002
B 248.1 ± 63.7 281.7 ± 29.4 182.8 ± 54.5 207.4 ± 31.4 213.0 ± 42.6 238.4 ± 40.4 0.02
C 245.0 ± 55.7 170.8 ± 23.0 210.4 ± 28.2 160.1 ± 40.3 187.7 ± 42.9 106.0 ± 19.7 0.02
P 0.99 0.007 (x, z) 0.04 (y) 0.01 (x) 0.51 <0.001 (x, y, z)
SS (mmol/L)
A 33.6 ± 10.2 39.2 ± 9.5 21.2 ± 6.1 35.3 ± 5.2 22.6 ± 5.0 32.4 ± 8.4 0.007
B 28.6 ± 11.3 29.8 ± 8.8 43.4 ± 14.0 39.8 ± 12.5 41.2 ± 17.6 36.1 ± 11.7 0.33
C 25.2 ± 10.5 35.5 ± 18.4 21.6 ± 4.2 30.2 ± 8.7 39.8 ± 9.6 17.4 ± 8.5 0.005
P 0.297 0.286 0.004 (x, z) 0.193 0.014 (x, y) 0.004 (x, z)
NT/TT (%)
A 73.4 ± 5.0 57.9 ± 6.7 73.3 ± 6.9 50.2 ± 7.3 77.7 ± 3.0 57.4 ± 9.8 <0.001
B 77.1 ± 5.6 78.9 ± 5.7 52.5 ± 7.5 62.0 ± 9.8 62.1 ± 12.3 70.2 ± 5.7 <0.001
C 70.0 ± 4.9 58.6 ± 18.9 79.5 ± 3.1 61.6 ± 11.5 57.4 ± 6.5 66.9 ± 18.5 <0.001
P 0.12 0.003 (x, z) 0.001 (x, z) 0.04 (x, y) 0.005 (x, y) 0.049 (x)
SS/TT (%)
A 13.3 ± 2.5 21.0 ± 3.4 13.3 ± 3.5 24.9 ± 3.7 11.2 ± 1.5 21.3 ± 4.9 <0.001
B 11.5 ± 2.8 10.5 ± 2.8 23.8 ± 3.7 19.0 ± 4.9 18.9 ± 6.2 14.9 ± 2.9 <0.001
C 10.0 ± 2.5 20.7 ± 9.5 10.3 ± 1.5 19.2 ± 5.7 21.3 ± 3.2 16.6 ± 9.2 <0.001
P 0.11 0.003 (x, z) 0.001 (x, z) 0.04 (x, y) 0.005 (x, y) 0.049 (x)
SS/NT (%)
A 18.4 ± 4.7 37.3 ± 9.8 18.8 ± 7.0 51.7 ± 16.3 14.5 ± 2.5 39.7 ± 17.6 <0.001
B 15.2 ± 5.1 13.6 ± 4.6 46.9 ± 13.5 32.6 ± 13.9 33.5 ± 18.5 21.6 ± 5.6 <0.001
C 12.7 ± 3.7 45.2 ± 36.4 13.0 ± 2.5 34.6 ± 22.1 38.2 ± 10.4 31.1 ± 28.5 0.001
P 0.12 0.003 (x, z) 0.001 (x, z) 0.04 (x, y) 0.005 (x, y) 0.052 (x)
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