| OBJECTIVESpinal cord injury (SCI) can result in serious neurological deficit. SCI is very common condition caused by fracture and dislocation of spine vertebral. The incidence of spinal cord injury in China is as high as 6. 7 persons in every one million.There is both a serious impact on SCI patient’s daily life and a severe economic burden on society. The pathophysiology process of SCI includes two important segments: primary injury and secondary injury. Direct trauma can result in an irreversible damage to spinal cord in primary injury segment.A series of pathological and physiological changes such as edema, hemorrhage, ischemia, electrolyte disturbance follows the primary injury. The second process is the secondary injury segment which has a broad and profound effect on the whole nature history of SCI. It is composed of a series of biochemical changes including oxidative stress and inflammation reaction.Both of them have a huge damage on cells in spinal cord. The primary injury caused by direct violence can’ t be avoided in advance. However, as for the secondary injury, we can make an effective intervention. In current, this is the hot area of spinal cord injury research. Plumbagin is a potent antioxidant and shows anti-carcinogenic, anti-inflammatory and analgesic activities. However,its exact molecular mechanism of action in spinal cord injury has notyetbeenexplored.In our test,we built a mouse spinal cord injury model in order to explore the exact neuroprotect ion mechanism and the impact of Plumbagin on BBB scores, Nissl body staining, ROS generation,LP, antioxidant status, cytokines and pro-inflammatory factor,and transcription factor in post-SCI.Materials and Methods1. AnimalMale Wistar rats weighing 180 - 210 g were used for the present experimental study.Food and water was made available ad libitum. Animals were maintained in the windowless animal quarter with automatictemperature(22±℃) and light (12h light/dark cycle).The humidity ranged from 60-70 %. Animals received human care, according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the NIH.The experimental procedures for the present study were approved by Department of Orthopedics, Qilu Hospital, Shandong University, China. The animals were acclimatized to their environment for 10 days.2. The induced spinal cord impact injury model, group and tissue sampleAfter the process of acclimatization, the rats were randomly divided into 3 groups. Group (Ⅰ) Sham - underwent sham surgery. Group (Ⅱ) SCI group - underwent SCI induction and injected with ipsaline.Group (Ⅲ) - SCI-induction and Plumbagin-SCI and intraperitoneal injection of Plumbagin(20 mg/kg) was given after 1 h of SCI on day 1 which was followed for 9 consecutive days. The BBB score and inclined angle were recorded in Day1,Day3, Day5 and Day10 respectively. In partⅡ and Ⅲ, the rats were killed for obtaining tissue samples at different time point.All the rats received preoperative fasting food for 12h and fasting water for 4h.Be sure to keep warm in operative process. All surgeries were performed under aseptic conditions.The rats were anesthetized with intramuscular 10% Chloral Hydrate (3. 5mg/kg) solution. After a dorsal skin incision and soft tissue dissection at the T8-T10 level, the T9 vertebra was stabilized in a frame with steel clamps inserted under the transverse processes.Laminectomy was performed without opening the dura. The spinal cord was contused using an impactor with 1. 5mm base diameter and 10g weight from the height of 2. 5mm.Local antibiotic injection was used in operative area. Muscles and skin were sutured. In the sham group, there were no other procedures except laminectomy.The successful standard :congestion and edema in spinal cord area; Tail swing and lower limbs flapping at the moment of impact; obvious soft paralysis;feeling and power dysfunction of lower limbs. All rats were given a subcutaneous injection ofgentamicin(12mg/kg) to prevent infection.Postoperative care included twice daily bladder expression and dry environment keeping.3. Nissl’ s stainingThe tissue sample was obtained at 10 day after SCI. The sections were treated with xylene for 5-10min three times and rehydrated with descending series of ethanol and distilled water.Stain in nissl staining solution for 5min. Then, rinse quickly in distilled water. The sections were treated with 95%ethyl alcohol for 5 sec and rinsed in 70% ethyl alcohol twice.Mount with the mounting medium and check microscopically for the results.4. Reactive oxygen species generationThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI. DCFH-DA was chosen as the probe of ROS activity test. If ROS exists, DCFH will be oxidated to DCF (a kind of Strong green fluorescence). ROS levels were measured spectrofluorimetrically at excitation (485 nm) and emission(528 nm) wavelength.5. TBARS activity testThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI.The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. TBARS activity assay kit was used to test TBARS activity. Absorbance measured at 532 nm and results expressed as nMTBARS/mg of protein.6. Glutathione (GSH) contentThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI. The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. GSH activity assay kit was used to test GSH activity.The absorbance was measured at 412 nm in a spectrophotometer.GSH content was calculated from the standard curve and the results were expressed as nmoles of GSH/mg of protein.7. Catalase (CAT) activityThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI. The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. CAT activity assay kit was used to test CAT activity. The measurements were performed spectrophotometrically at 240 nm.8. Total (Cu - Zn and Mn) superoxide dismutase activityThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI.The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. SOD activity assay kit was used to test SOD activity.Incubate for 30min at 37℃ and the absorbance was measured at 560 nm in a spectrophotometer. SOD activity was calculated according to formula.9. Glutathione peroxdiase (GSH-Px) activityThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI.The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. GSH-Px activity assay kit was used to test GSH-Pxactivity.The absorbance was measured at 340 nm in a spectrophotometer at 25℃.10. Glutathione-S-Transferase (GST) activityThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI. The injury spinal cord tissues were obtained and processed to tissue homogenate. Supernatant was used as sample. GST activity assay kit was used to test GST activity. The product formed was measured spectrophometrically at 340 nm.11. NAD (P)H:Quinone oxidoreductase (NQ01) activityThe tissue sample was obtained at 1 day, 3 days, 5 days,10 days after SCI. Endogenous NQ01 enzymatic activity was measured using menadione as a substrate.The standard assay system contains 50 mM Tris-HC1, pH 7.5, 0.08% Triton X-100,0. 5 mM NADH or NADPH, and 40 μM DCPIP or 10 μ Mmenadione. The absorbance was measured at 570 nm spectrophometrically. The results are expressed as change in U/mg protein.12. ELISA: TNF-α and IL-1β levelsThe serum was collected as sample in advance at 1 day, 3 days, 5 days, 10 days after SCI. The levels of TNF-α and IL-1β were detected using an ELISA kit. The process included antigen binding, Streptavidin-HR adding, chromogen adding and stop solution adding. Read the absorbance at 450 nm. Read the plate within 30 minutes after adding the stop solution. Use curve-fitting software to generate the standard curve and make a statistical analysis.13. Immunohistochemistry staining of Nrf-2Tissue sample was obtained and paraffinised sections were made at 5 days after SCI. SP kit was used to stain the secitons.We chose six random microscopic fields per-slide and used the Image Pro Plus 5. 0 software for analysis.14. Cytosolic and nuclear extract preparation and western blotCytosolic and nuclear extract Preparation: For protein expression studies cytosolic and nuclear extracts were isolated by using cytosolic and nuclear extract kitrespectively.The extracts were aliquoted and stored at-80℃ until further analysis. Sample proteins from cytosolic(50 μg) and nuclear extracts (50 μg) were loaded onto 12 %sodium dodecyl sulfate polyacrylamide gel electrophoresis.The proteins separated were transferred to a nitrocellulose membrane (Bio-Rad,Munich, Germany) and blocked with 3 %non-fat milk in Tris-buffered saline (pH 7. 4) containing 0. 1 %Tween (TBST) for 1 h. The membranes were washed thrice in TBST and incubated at RTwith appropriate primary antibody (anti-NF-κB p65 and anti-Nrf-2, Santa Cruz Biotechnology) for 1 h.The blots were washed with TBST thrice and incubated overnight at 4℃ with secondary antibody, horseradish peroxidase-linked anti-rabbit IgG (Santa Cruz Biotechnology).Followed by which the blots were enhanced with chemiluminescence reagent (ECL plus Western-blotting detection system, GE Healthcare). Loading control was maintained fusing β -actin for cytosolic extract and GAPDH for nuclear extracts. The intensity of the bands was calculated with image J software.Results1. The induced spinal cord impact injury model was set up successfullyThe preoperative BBB scale of all rats was 21. The lower limbs of SCI rats can’t move after waking. All BBB scales of Group Ⅱ and Group Ⅲ are 0. Moving slowly was often found in Group I,but this phenomenon would return to normal on the 4-5th day after operation.2. The results of BBB score and inclined plane test and Nissl’ s stainingThe results showed that BBB score of sham Group was essentially normal; BBB scores of SCI group were significantly lower than sham group (P < 0. 05) ; BBB scores of Plumbagin treatment group were significantly higher than SCI group(P< 0. 05), but BBB scores of Plumbagin treatment group were still lower than sham group. The results of inclined plane test: The inclination angle of SCI group were significantly lower than sham group ( P< 0. 05) ; The angle of Plumbagin treatment group were significantly higher than SCI group ( P< 0. 05), but the angle of Plumbagin treatment group were still lower than sham group. The results of Nissl’s staining: The nissl body was in the normal form in the sham group. In SCI group, nissl body was not clearly visible and neurons exhibited swelling,atrophy or necrosis at Day 10 after SCI. In plumbagin treatment group, nissl body decreased in number and the rest was similar to the sham group.3. The results of Oxidative stress parametersWe investigated whether Plumbagin would ameliorate spinal cord injury-induced ROS levels and associated damage of lipid peroxidation. The results show that the degree of ROS begun to increase significantly on the Day1 and reached the peak level on the day 5. The degree of ROS begun to decrease in the following days and was still higher than sham group on the Day 10 (P<0. 05). Compared with SCI group, the degree of ROS in Plumbagin treatment group decreased significantly in different time point (P< 0. 05). Results of TBA: The TBA in SCI group and Plumbagin group increased rapidly after SCI and reached the peak on the Dayl. Then,the TBA in SCI group and Plumbagin group begun to decreased in the following days and was still higher than sham group on the Day 10 (P < 0. 05).Compared with SCI group, the TBA of Plumbagin treatment group decreased significantly in different time point(P< 0.05).4. Results of antioxidant vitalityPhase Ⅱ antioxidant enzymes are battery of critical proteins regulated by Nrf-2/ARE pathway and show protective effects in conditions of oxidative stress. In order to determine the effect of Plumbagin on redox imbalance caused during spinal cord injury, we evaluated the activities of various antioxidant enzymes such as GSH, CAT, SOD, GSH-Px,GST and NQ01. In SCI group, the activity of antioxidant enzymes decreased significantly after SCI and increased gradually in the following days.But the activity of antioxidant enzymes in SCI group was still significantly lower than sham group (P<0.05) on the Day 10. Compared with SCI group, the activity of antioxidant enzymes in Plumbagin treatment group increased significantly in different time point (P< 0.05).5. Plumbagin reduces spinal cord injury induced pro-inflammatory cytokinesSpinal cord injury induced inflammation could upregulate cytokine levels. As Plumbagin showed to suppress NF- κ B expression, we further tested if cytokine production in SCI rat model could be ameliorated by Plumbagin treatment. The results showed a significant upregulation of pro-inflammatory cytokines (TNF- α , IL-1 β ) when compared to sham operated rats.In addition, Plumbagin treatment significantly reduced TNF- α and IL-1β levels when compared to rats with spinal cord injury. Results of TNF-α and IL-1β in different time point:In SCI group, pro-inflammation cytokines increased significantly after SCI and reached the peak level on the Dayl.Then, pro-inflammation cytokines begun to decrease slowly and were still higher than sham group on the Day 10. Compared with SCI group, the degree of pro-inflammation cytokines in Plumbagin treatment group decreased significantly in different time point (P< 0. 05).6. Results of Nrf-2 immunohistochemistryNrf-2 protein expression in SCI group was higher than sham group(P < 0.05). Compared with SCI group,the Nrf-2 protein expression in plumbagin treatment increased obviously (P <0.05).7. Plumbagin modulates nuclear Nrf-2 and NF- κ B expressionsNF- κ B mediates pro-inflammatory mechanisms while Nrf2 functions by activating cellular antioxidant machinery. Thus,it is important to determine the levels of these factors incytosolic and nuclear fractions and thus determines oxidative stress and inf lammation. The present investigation showed a significant nuclear upregulation of NF- κ B and downregulation of Nrf-2 expression during SCI-Induction. We could also observe that the cytosolic levels of NF-κB were significantly decreased and Nrf-2 levels were increased significantly. However, rats treated with Plumbagin post SCI-injury modulated the nuclear levels of NF-κ B and Nrf-2 when compared to SCI-rats.ConclusionsPlumbagin can supress the ROS and LP levels, inflammation reaction and NF- κB expression in SCI, meanwhile it can upregulate antioxidant status and Nrf-2. Taken together, the data suggests potential and novel role of Plumbagin in cytoprotection by modulating NF-κB and Nrf-2 levels against spinal cord injury. |
PDF Full Text Request