Protective Effects And Mechanism Of Epigallocatechin-3-Gallate On The Acute Phase In Spinal Cord Injury In Rats

Objective:To explore the effects and its possible mechanisms of Epigallocatechin-3-gallate (EGCG) on the acute phase in spinal cord injury (SCI) in rats, several aspects of the anti-oxidative, anti-inflammatory, anti-apoptotic effect were studied in the present study by using hemisected SCI rats at thoracic vertebra 8 (T8) as SCI rat model and hydrogen peroxide (H2O2)-induced PC 12 cells with oxidative-stress injury as the neuronal damaga cell model.Methods:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.1 Experiment in vivo:(1) Spinal cord injury procedure, grouping and treatingSpinal cord was hemisected at thoracic vertebra 8 (T8) in adult female Sprague-Dawley rats. Rats were randomly divided into six different groups as follows:(1) sham-treated rats (saline,5 ml/kg, ip), (2) SCI (saline,5 ml/kg, ip), (3) Methyllprednisolone sodium succinate (MPSS,100 mg/kg, ip), (4) EGCG (25 mg/kg, ip), (5) EGCG (50 mg/kg, ip) and (6) EGCG (100 mg/kg, ip) with a single dose immediately following trauma. MPSS and EGCG were diluted in saline.(2) Micro-morphological examination by pathohistology observation by HE staining and transmission electron microscopic in SCI ratsTwenty-four hours after SCI and administration on rats, the spinal cord with 2 cm was fixed in 4% buffered paraformaldehyde to observe pathohistology change. The spinal cord with 1 cm was obtained by abscising vertebral canal, and 1 mm3 cross-section of gray and white matter at the epicenter of each spinal cord lesion was fixed in 2.5% glutaraldehyde to observe micro-morphology.(3) Determination the concentration of serum inflammatory factor by ELISA assay in SCI rats Twenty-four hours after SCI and administration on rats, animals were=removed right eyeball and exanguinated 5 ml. The serum was collected. ELISA assay was performed to determine the concentration of serum Interleukin-1β(IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor necrosis factor-α(TNF-α) and Intercellular adhesion molecule-1 (ICAM-1) using commercial kit according to the description of the manufacturer.(4) Measurement of the levels of SOD, MDA, O.2-, NO and iNOS protein in SCI ratsTwenty-four hours after SCI and administration on rats, spinal cord was made 1% tissue homogenate to detect the levels of SOD, MDA, O.2- and NO according to the manufacturer's instruction. The spinal cords from each rat were made into gel electrophoresis samples to determine iNOS protein levels by Western blot assay.(5) Measusement of B-cell lymphoma/leukemia-2 gene B (Bcl-2) and Bcl 2-associated X protein (Bax) protein levels by Western blot in SCI ratsTwenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine Bcl-2 and Bax protein levels by Western blot assay.(6) Measusement of NT-3 and BDNF protein levels by Western blot in SCI rats Twenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine NT-3 and BDNF protein levels by Western blot assay.2 Experiment in vitro:(1) PC12 cells culture and injury procedurePC 12 cells were plated at a density of 2×105 cells/cm2 in a 50 ml culture flask and cultured routinely. The culture media were changed every 24 h. The cells were subcultured when attached upon reaching 80～90% confluence in 48 h. The cells were synchronized by serum starvation before EGCG and H2O2 treatment. Varying concentrations of H2O2 (final concentration 100～600μmol/L) were added to each well and co-incubated for 2 h. Damaged PC 12 cells model was established by MTT assay.(2) The protective effects of EGCG on damaged PC 12 cellsBeing treated with EGCG and H2O2, the culture media were collected and tested depending on LDH kits to measure of extracellular LDH levels. The cellular viability and mitochondria integrity were detected by MTT assay. Being treated with EGCG and H2O2, the cell cycle was monitor by cytometry. The cells were seeded onto poly-L-lysinated 18×18 cm rectangular coverslips placed in 6-well plates (2×105 cells/ml in 1ml medium), and cultured, synchronized and treated with EGCG and H2O2. The media were replaced with serum-free DMEM media containing BrdU at a final concentration of 10μmol/L for 24 h. DNA synthesis of S phase in cell cycle was determinated by BrdU-labeled immunofluorescence assay.(3) The mechanism of EGCG on oxidative-stress damaged PC 12 cellsBeing treated with EGCG and H2O2, PC 12 cells were collected. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) enzymes, hydroxy radical (·OH) and superoxide anion (O.2-), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in PC12 cells were measured according to the kits. iNOS protein and mRNA levels were determinated by Western blot and real-time RT-PCR.Results:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.1 Experiment in vivo:(1) The effect of EGCG on micro-morphology and pathohistology in acute SCI ratsThe neuronal and axonal pathohistology with HE staining was observed by the optical microscope. In sham group, the construction in grey matter and white matter was normal size and regular. In SCI group, some blood cells appeared in central gray of damaged area. A lot of monokaryotic or polycaryotic inflammatory cells infiltrated into grey matter and white matter, with capsular space change. In EGCG 25～50 mg/kg group, vacuolar degeneration was lightened, along with blood cells and inflammatory cells, and lower than the one in SCI group. In EGCG 100 mg/kg group and positive drug MPSS, myeloid tissue in damaged part arranged in regular way, inflammatory edema and apoptosis appeared occasionally.The nerve fiber in white matter arranged in regular way.The neuronal and axonal changes were observed by transmission electron microscopy. In sham group, the spinal cord motoneurons of the anterior grey horn and myeline sheaths in the white matter were normal size and regular. In SCI group, the spinal cord motoneurons of the grey horn shrinked back, and myeline sheaths in the white matter arranged in loose. In EGCG 25～50 mg/kg group, the spinal cord motoneurons of the anterior grey horn appeared frequently oncotic. Myeline sheaths in the white matter arranged in loose way, along with primary demyelination. In EGCG 100 mg/kg group and positive drug MPSS, motoneurons and axon were almost normal size and regular.(2) Effect of EGCG on the concentration of serum inflammatory factor in acute SCI ratsTwenty-four hours after SCI, the concentration of serum inflammatory factor IL-1β, IL-6, IL-8, TNF-αand ICAM-1 increased significantly in SCI model group (P < 0.01 vs sham group), while the concentrations were reduced by 25,50 and 100 mg/kg EGCG treatment except the treatment effect of 25 mg/kg EGCG on serum IL-8 (P< 0.05 vs SCI group).(3) Antioxidation of EGCG in acute SCI ratsAntioxidase system was measured according to the kits. The results showed that SOD levels was reduced and MDA levels increased in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased SOD levels, reduced MDA levels (P < 0.05 vs SCI group).Reactive oxygen species were measured according to the kits. The capability of suppressing O.2- activity was lowed severely, and NO levels were expressed highly in SCI model group (P< 0.01 vs sham group). The capability of suppressing O.2-activity was increased by 25,50 and 100 mg/kg EGCG treatment (P< 0.05 vs SCI group). NO levels were reduced by three dosages of EGCG treatment as compared with SCI model group, with no statistical significance in 25 100 mg/kg (P> 0:05 vs SCI group), and the statistical significance in 50 and 100 mg/kg (P< 0.01 vs SCI group). Western blot assays showed that the levels of iNOS protein were expressed excessively in SCI model group (P< 0.01 vs sham group), along with significantly decreased by EGCG treatment (P< 0.01 vs SCI group).(4) Anti-apoptosis effect of EGCG in acute SCI ratsWestern blot assays showed that B-cell lymphoma/leukemia-2 (Bcl-2) levels were depressed, and Bax levels and the ratio of Bax to Bcl-2 expression were expressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG reversed the changes, along with 25 mg/kg EGCG no statistical significance (P>0.05 vs SCI group),50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).(5) Effect of EGCG on the neurotrophic factor neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) in acute SCI ratsWestern blot assays showed that NT-3 and BDNF levels were depressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased the expression levels, along with 25 mg/kg EGCG no statistical significance,50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).2 Experiment in vitro:(1) H2O2-induced PC 12 cells damaged modelObserved by inverted microscope, the Blank group cells with full size attached solidly to the wall of culture flask, and stretched full. Cells body with good refractive index was bright and clear. The damaged cells body shrinked back, accompaniment with enation disappearance, cell spaces augmentation and sparsate distribution. The obviously damaged morphous were showed in the damaged cells. The degree of injury was the severest in the 600μmol/L H2O2 group. MTT asays showed that H2O2 decreased PC 12 cells viability as compared with the control group, with the statistical significance in 400～600μmol/L (P<0.01 vs Control group), and no statistical significance in 100～300μmol/L (P> 0.05 vs Control group).(2) The neuroprotective effect of EGCG on damaged PC 12 cellsAn increase in the amount of membrane damaged cells resulted in an increase of LDH enzyme activity and membrane permeability. Extracellular LDH levels were enhanced by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased membrane permeability by depressing the LDH activity (P< 0.05 vs H2O2 group).The cellular viability and mitochondria integrity were detected by MTT assay, and the results showed that 5～30μmol/L EGCG could antagonize the mitochondrial injury and the decreasing viability induced by H2O2 except the 2.5 umol/L EGCG group, significantly higher than the H2O2 group (P< 0.05 vs H2O2 group).Flow cytometry assay showed that the percentage of G0/G1-phase cells was increased, and the percentage of S-phase cells and Proliferative Index decreased by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased the percentage of G0/G1-phase cells, increased Proliferative Index (P< 0.05 vs H2O2 group). Three dosages of EGCG also increased the percentage of S-phase cells as compared with Control group, with the statistical significance in 10,20μmol/L (P<0.01 vs H2O2 group), and no statistical significance in 5μmol/L (P> 0.05 vs H2O2 group).To detect further proliferating cells in the S-phase, BrdU incorporated in the cells was measured. The number of BrdU-positive cells decreased significantly in cells treated with H2O2 (P< 0.01 vs Control group), while the number of BrdU-positive cells increased in cells with EGCG pretreatment (P< 0.01 vs H2O2 group).(3)The neuroprotective mechanism of EGCG on H2O2-induced damaged PC 12 cellsAntioxidase system was measured according to the kits. The results showed that SOD and GSH-Px levels were reduced and MDA levels increased in H2O2-induced PC 12 cells (P< 0.05 vs Control group). The levels of SOD and GSH-Px were increased and the contents of MDA reduced by EGCG pretreatment (P< 0.01 vs H2O2 group). Reactive oxygen species (ROS) were measured according to the kits. The results showed that the capability of suppressing O.2- activity was lowed, and the capability of producing·OH was enhanced in H2O2 group (P< 0.01 vs Control group). Three dosages of EGCG reversed the changes (P< 0.01 vs H2O2 group). Extracellular NO contents and intracellular iNOS activity were expressed highly in H2O2 group (P< 0.01 vs Control group), and reduced by EGCG pretreatment in damaged cells (P< 0.01 vs H2O2 group). Western blot andreal time RT-PCR assays showed that the levels of protein and mRNA of iNOS were expressed excessively in H2O2 group (P< 0.01 vs Control group), along with significantly decreased by EGCG pretreatment except the 5μmol/L group(P< 0.05 vs H2O2 group).Conclusion:In the present study we studied the effects of EGCG on acute SCI model from the aspects of biochemical and pathological indicator by using hemisected SCI rats as SCI model in vivo and H2O2-induced PC 12 cells oxidative-stress injury as the neuronal damaga model in vitro. The conclusion as follows:(1) EGCG played an important role in spinal cord injury at the early stages. The neuroprotective effect attributed to several aspects:anti-inflammatory effect by decreasing the concentration of serum inflammatory factor in acute SCI rats; antioxidation by enhancing antioxidase system activity in spinal cord, inhibiting ROS activity, and depressing the activation of iNOS-NO pathway; anti-apoptosis effect by downregulating Bax expression and upregulating Bcl-2 expression; promoting endogenous neurotrophic factor NT-3 and BDNF expression.(2) The present study demonstrated that EGCG was able to protect H2O2-induced PC 12 cells by enhancing antioxidase SOD, GSH-Px activity, decreasing ROS activity, and depressing the activation of iNOS-NO pathway, which could decrease membrane permeability, maintain membrane integrity, protect mitochondria, prolong the S phase in cell cycle and promote DNA synthesis of S phase.