High-mobility group box 1 (HMGB1) is released after focal cerebral ischemia/reperfusion (I/R), and aggravates brain tissue damage. Ginsenoside Rb1 (Rb1), isolated from
Ischemic stroke accounts for 75% of all stroke patients; it is a long-term disability and a leading cause of death worldwide (
Traditional Chinese medicines are believed to be effective in treating patients with cerebral ischemia, and to have few clinical side-effects.
A total of 50 Wistar rats (male; body weight, 270–330 g, age: 10 weeks-12 weeks) were obtained from the Animal Center of Shandong University (Jinan, China). All rats were maintained at 25±1°C, with 12 h light/12 h dark cycle of housing, food and water available. All animal experiment protocols were approved by the Institutional Animal Care Committee of Shandong University (Jinan, China), and were performed in strict consistence with its guidelines.
After 1 week of accommodation, the rats were subjected to middle cerebral artery occlusion (MCAO) surgery as previously described (
Rb1 was dissolved in saline and intravenously injected following initiation of ischemia. The animals were randomized distributed into 5 groups according to the random number table. Firstly, the rats were numbered by body weight. Second, we chose the any row in the random number table and copy 50 random numbers. Sort random numbers from small to large. Specify the first 10 numbers as the first group from the sorted numbers, followed by analogy: i) sham control, the ECA was surgically prepared for insertion of the filament as described above, but the filament was not inserted and saline was received intravenously; ii) MCAO group, subjected to MCAO and saline was received intravenously and iii) Rb1 group, subjected to MCAO and 50, 100 or 200 mg/kg of Rb1 was received intravenously. We conducted a pre-experiment to investigate the effect of Rb1 100 mg/kg on the focal cerebral ischemic reperfusion rats. The results manifested that Rb1 100 mg/kg remarkably decreased the ischemic injury. Therefore, we chose Rb1 50, 100 and 200 mg/kg as the dose of Rb1.
Neurological examination was performed blindly 24 h after reperfusion, according to Zea Longa's method (
Coronal brain sections (2-mm thickness) were incubated with 2% TTC at 37°C for 30 min with gentle agitation, then fixed with 10% formalin in PBS. Pale unstained sections were considered to be indicative of infarct regions, whereas red-stained sections were indicative of normal tissue. The slices were photographed from each side, and the infarct regions and were detected both hemispheres using a morphological image-analysis system (Jie Da software, China). Infarct volume was calculated as a percentage of the contralateral hemisphere volume using an ‘indirect method’ (area of intact contralateral hemisphere-area of intact regions of the ipsilateral hemisphere) to compensate for edema formation in the ipsilateral hemisphere. The volume of infarction was obtained according to the following formula, and expressed as percentage of infarction in the ipsilateral hemisphere (
V, volume of fraction; Ai, infarct area of each slice, and h, slice thickness.
After being anesthetized with 10% chloral hydrate (350 mg/kg; injected intraperitoneally) the rats were sacrificed by cardiac perfusion, the brains were immediately removed and the bregma-3~3.8 mm areas were immobilized in 4% neutral buffered formalin and embedded in paraffin. No peritonitis was observed in the rats during the entire experimental protocol. The areas were mounted onto slides, deparaffinized with xylene, rehydrated using a graded alcohol series, stained with hematoxylin and eosin and analyzed under a light microscope at magnification, ×100. The brains were sliced into 10-µm thick coronal sections at the level of the bregma. TUNEL staining was performed using an
Protein samples were prepared as previously described (
Cortex samples were homogenized in 1 ml homogenization buffer and centrifuged at 14,000 × g for 10 min at 4°C. ELISA kits were used to verify the levels of high-mobility group box 1 (HMGB1) and NF-κB p65, TNF-α, iNOS, NO and IL-6, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
All data are expressed as the mean ± standard deviation and analyzed using one-way analysis of variance followed by the Least Significant Difference test. All the statistics analyses were performed using SPSS software (v.18; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Neurological scores were determined 24 h after I/R injury. No neurological deficitobserved in sham animals, whereas MCAO animals suffered from I/R injury, displayed all the characteristics of neuron damage and had relatively high neurological deficit scores (2.07±0.24;
Infarct area of brain tissues from the animals measured 24 h after I/R injury by TTC staining are presented in
Hematoxylin and eosin staining was applied to examine the histopathological abnormalities following focal cerebral I/R (
The level of HMGB1 in the peri-infarct zones of ischemic cortex samples from each group was measured. A significantly increased level of HMGB1 was identified in brain tissue subjected to focal cerebral ischemia reperfusion. Furthermore, an increased level of HMGB1 was observed in MCAO rats compared with the sham group (P<0.01;
High levels of TNF-α, iNOS, NO and IL-6 were also identified in MCAO rats (P<0.05, sham vs. MACO;
Neuronal cell apoptosis was measured by TUNEL staining in the rats of each group (
The expression levels of cleaved caspase-3 and caspase-9 was also investigated. High expression of apoptosis-related proteins was exhibited by brain tissues from the MCAO group compared with the sham group (P<0.05;
Natural products can be used to modulate cytokine-activity for treatment of diseases (
Numerous studies have demonstrated the beneficial effects of Rb1 in the treatment of ischemic stroke (
Furthermore, HMGB1 is a crucial proinflammatory factor in ischemic stroke and the signal is transduced via its putative receptors, such as toll-like receptors (TLRs), receptor for advanced glycation end products (RAGE) and matrix metalloproteinase (MMP) enzymes during ischemic stroke. The present study suggests that Rb1 administration could markedly reduce the elevated levels of HMGB1 and NF-κB in MCAO rats. However, the effects of Rb1 on HMGB1-associated receptors remain to be elucidated. HMGB1 may be a novel subject of brain-immune communication and post-stroke immunomodulation research. In acute ischemic stroke patients, the peripheral percentage of some subsets of T-lymphocytes was associated with the level of neurological deficit, and a predictive role of the peripheral percentage of CD28-null cells in stroke diagnosis and TOAST subtyping was suggested (
I/R injury-induced oxidative stress and inflammation also triggers multiple-cell apoptotic pathways responsible for cell death by necrosis or apoptosis (
In summary, the present study demonstrated that Rb1 has a protective effect on cerebral neurons in I/R injury. The mechanisms underlying these actions are not well established, however, our results suggest that the inhibition of inflammatory HMGB1 signaling may serve an important role in the process. Furthermore, Rb1 may be a promising neuroprotective candidate, and requires further laboratory and clinical investigation.
The authors would like to thank the Central Research Laboratory, The Second Hospital of Shandong University for technical assistance and the generous support.
The present study was supported by the Natural Science Foundation of China (grant no. 81402962).
All of the materials used in the present study are commercially available and all data included in the present study were obtained by the co-authors.
AL, GH, ZC and LZ designed the study. HL, XX and WJ performed the experiments. WZ, LS analyzed the data. HL and XX wrote the manuscript.
All animal experiment protocols were approved by the Institutional Animal Care Committee of Shandong University (Jinan, China), and were performed in strict consistence with its guidelines.
Not applicable.
The authors declare that they have no competing interests.
Effect of Rb1 on cerebral infarct area in MCAO rats. (A) Cerebral infarct area stained with TTC in different groups. The coronal sections were obtained after 24 h of reperfusion. (B) Evaluation of infarct area after 24 h of reperfusion, the bar indicates the percentage of infarct area. Data are expressed as the mean ± standard deviation (n=8). #P<0.05, ##P<0.01, ###P<0.001 vs. MCAO group. MCAO, middle cerebral artery occlusion; TTC, Triphenyltetrazolium chloride.
Histopathology of the cortical and hippocampal CA1 neurons of rats examined by Hematoxylin and eosin staining. Representative micrographs of the (A) cortex and (B) hippocampus of the sham, MCAO and Rb1 treated groups, respectively. Magnification, ×100. MCAO, middle cerebral artery occlusion.
Analysis of the levels of HMGB1 and inflammatory factors. (A) HMGB1 levels, measured by ELISA. (B) NF-κB expression levels. Levels of pro-inflammation factors, (C) TNF-α, (D) IL-6, (E) iNOS and (F) NO measured by ELISA. *P<0.05, **P<0.01, ***P<0.001 vs. sham group; #P<0.05, ##P<0.01, ###P<0.001 vs. MCAO group. HMGB1, high-mobility group box 1; MCAO, middle cerebral artery occlusion.
Rb1 protects neuronal cells from cerebral ischemic reperfusion-induced apoptosis in the cortex. (A) Cell apoptosis in cortex tissue measured by TUNEL staining. Magnification, ×200. (B) Analysis of the percentage of TUNEL-positive cells. ***P<0.001 vs. sham group; ##P<0.01 and ###P<0.001 vs. MCAO group. (C) Protein expression analyzed by western blotting.
Effects of Rb1 on neurological deficit scores in rats 24 h after reperfusion.
Groups | Rat no. (n) | Neurological scores |
---|---|---|
Sham | 8 | 0.00±0.00 |
MCAO | 8 | 2.07±0.24 |
Rb1 50 mg/kg | 8 | 1.71±0.43 |
Rb1 100 mg/kg | 8 | 1.25±0.72 |
Rb1 200 mg/kg | 8 | 1.05±0.36 |
Data are presented as the mean ± standard deviation. Animals received different doses of Rb1 or saline vehicle following the onset of ischemia. Neurological deficit scores were evaluated after 24 h of reperfusion according to the Longa's method.
P<0.05
P<0.01 vs. MCAO. RB1, Ginsenoside Rb1; MCAO, middle cerebral artery occlusion.