| Objective:Spinal cord injury (SCI) is one of the important fields in medical research currently.Its incidence is high, and the followed high disability rate and mortality become ahotspot for medical experts both at home and abroad. Acute SCI includes twopathological processes: primary injury and secondary injury. The secondary SCI canobviously aggravate the primary injury, thus causing more severe nerve dysfunction.Primary SCI has no symptom in occurrence, so how to suppress secondary SCI hasbecome the main focus in research of spinal cord injury. Pathological processes ofsecondary SCI include a series of molecular and cellular reaction pathways, which leadto the occurrence and development of secondary injury. Excessive production of freeradicals is the major reason for secondary injury. Both microcirculation disturbances ofspinal cord and reduction of local blood flow can cause the excessive production of freeradicals, and then leads to the lipid peroxidation of the cytomembrane. Researches showthat oxidation of the large amount of unsaturated fatty acid in the cell membrane willlead to cell structural changes. Excessive free radicals attack and lipid peroxidation are the main cause of this phenomenon, which even lead to cell degeneration and necrosis.Cell membranes of the neuron and myelin sub-cells have a serious of pathologicchanges in acute SCI, thus affecting the normal metabolism. Inflammation is one of themain factors for secondary spinal cord injury, which is also the core content inpathological study. NF-κB, a member of the family of transcription factor protein, isconsidered the key regulatory factor in inflammation gene expression. In the centralnervous system injury, NF-κB can regulate the expression of various cytokines. It alsohas regulation effect on inflammation. In general, NF-κB and IκB are present in the cellcytoplasm in the form of complex. IKKs are the main protein kinase for NF-κBactivation, with IKKβsubunit as the main catalytic subunit. It will prompt the NF-κB–IκB complex reactive and then active. And this activation can prompt thephosphorylation of IκB. When the NF-κB is activated, the nuclear localization of NF-κB was exposed. In this case, the free NF-κB can quickly enter the nucleus, and reactwith the specific κB sequence, which has a great influence on gene transcription of cells.It causes the mutation of the gene transcription, forms a huge amount of pro-inflammatory factors and free radicals, thus making the cells diseased.Currently, only methylprednisolone is used against inflammation of SCI in clinical.But the high-dose and long-term application of methylprednisolone often cause sideeffects such as suppressed immune system, liver function impairment, immunesuppression and osteonecrosis, etc.. With the development of science and technology,effective ingredients have been extracted from a variety of traditional Chinese medicinein recent years, which have been widely used in in-vivo and in-vitro SCI experimentsand achieved good effect. Salvia miltiorrhiza is a traditional Chinese herbal medicine,whose water-soluble active ingredients salvianolic acid B and tanshinone IIA have beenextracted. Salvianolic acid B has be approved to have several pharmacological effects(antioxidant, free radical scavenging, anti-inflammatory, and anti-tumor, etc.) inresearches of cardiac ischemia reperfusion, cerebral ischemia reperfusion and liverfibrosis. But its role in SCI is unclear.The promotion role of salvianolic acid B on rehabilitation for dyskinesia of hindlegs in rat acute SCI models is intended to be analyzed in this experiment throughbehavioral observation. Its regulation effects on IKK/NF-κB signaling pathways in SCI,and activation effects on NF-κB expression, IκB phosphorylation and IKKβactivity after SCI will also be analyzed. Through detection on the content of superoxide dismutase(SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and nitric oxide(NO), the roles of salvianolic acid B in antioxidation in SCI will be analyzed.Furthermore, the effects of salvianolic acid B on the expressions of anti-apoptosis geneBc1-2and pro-apoptosis gene Bax will also be analyzed. It is expected that through theexperiment, free radicals scavenging ability and anti-inflammation effect of salvianolicacid B in SCI, as well as the positive role of salvianolic acid B in inhibiting neuronalapoptosis can be confirmed, to provide a theoretical and practice foundation for clinicalapplication of salvianolic acid BMethods:1.7-week-old female adult SD rats were selected with the weight range from220gto250g. Spinal cord injury model was established in SD rats by advanced Allen method.The targeted injury site was T-12vertebra, and the damage energy was set to25g*cm.2. In the experiments, the rats were randomly divided into three groups, the controlgroup (only remove vertebral plate but not injury the spinal cord), salvianolic acid Btreatment group (intraperitoneal injection of salvianolic acid B (10mg/kg) half an hourafter operation), and spinal cord injury group (intraperitoneal injection of PBS).3. The promotion roles of salvianolic acid B in functional recovery of the hind legsof acute SCI rats were observed: BBB rating, slope test and foot print test were given atthe first day after operation and then every week during the experiment. After4weeksthe rats were given swimming test.4. The roles that salvianolic acid B played in eliminating free radicals and reducingperoxidation were observed: animals were executed24hours after spinal cord injury;1cm of myoloid tissue was collected from the damage area for homogenate detection,and blood samples were separated from the heart for sera diagnosis. The content ofSOD, GSH-Px, MAD and NO in the sera and the damage area were detected.5. The impact of salvianolic acid B on nerve cells apoptosis was observed: animalsare killed at24hours after spinal cord injury, then1cm long spinal cord sample wascollected; Western blot analysis and immunohistochemistry were used to test and analyze anti-apotosis gene Bc1-2and pro-apoptosis gene Bax after spinal cord injury toobtain their genetic expressions respectively.6. The relation between salvianolic acid B and kinase activity of subunit IKKβwasobserved; animals were executed at12hours after spinal cord injury and1cm spinalcord tissue was collected; the impact of salvianolic acid B on subunit kinase IKKβwasmeasured by means of kinase spectrometry of subunit IKKβ.7. The impact of salvianolic acid B on the expressions of NF-kB p65andphosphorylated I-kBα was observed, meanwhile the relation between salvianolic acid Band MPO activity was observed; the rats were killed at24hours after spinal cord injury;then1cm long spinal cord sample was collected and the expressions of phosphorylatedI-kBα and NF-kB p65after spinal cord injury were analyzed and measured by means ofimmunohistochemistry and western blot; MPO activity of the spinal cord sample wastested by using MPO activity detection kit.8. The impact of salvianolic acid B on the syringomyelia rate was observed; afterfour weeks’ experiment, the animals were killed and1cm spinal cord sample wascollected to analyze the syringomyelia rate of all groups by HE coloration.Results:1. After four weeks’ experiment, based on BBB rating, slope test, foot print testand swimming test results, it is confirmed that expect the first after operation, the rats’hind limb dyskinesia in salvianolic acid B group at other time points has been mitigatedsignificantly compared to other treatment groups (P<0.05). But the promotion effect ofsalvianolic acid B is found very limited on rat tail movement function in the swimmingtest (P>0.05).2.24hours after the operation, SOD and GSH-PX activity were significantlyreduced in serum and damage zone of rats in SCI group (P<0.05). Compared with theSCI group, salvianolic acid B can significantly improve the SOD and GSH-PX activityin spinal cord tissue of damage zone and plasma after SCI (P<0.05), thus the secondaryinjury is reduced by increasing antioxidant effect.24hours after SCI, spinal cord tissueand plasma in damage zone are observed and it is found that contents of NO and MDAin spinal cord tissue and plasma of damage zone are significantly improved (P<0.01).Salvianolic acid B can effectively reduce NO and MDA (P<0.05), reduce lipid peroxidation, protect blood-brain barrier, and reduce tissue edema effectively, thusprotecting nerve cells.3.12hours after SCI, IKKβactivity and protein activity in SCI are significantlyenhanced (P <0.05). Salvianolic acid B can significantly reduce the IKKβ activity in the12hours after injury (P<0.05).24hours after SCI, compared with the control group,MPO activity in injury group is obviously elevated (P<0.01), which can be significantlyinhibited by the salvianolic acid B (P<0.05). In the24hours after surgery,immunohistochemistry detection confirms there are hardly any phosphorylated I-kBαand NF-kB p65-positive cells expressed in the control group. The expression number ofphosphorylated I-kBα and NF-kB p65-positive cells is significantly increased after SCI(P<0.01), but salvianolic acid B can significantly reduce these expressions (P <0.05).The results of Western blot method further validate the conclusions ofimmunohistochemistry. After4successive weeks of experiment, rat syringomyelia rateof salvianolic acid B group has been significantly lowered compared with that of theSCI group (P<0.05).4.24hours after SCI, Western blot method and immunohistochemical methodshow that compared to the control group, Bax expressions in both of the other twogroups are increased significantly after SCI (P<0.01), and it is significantly reduced bysalvianolic acid B (P<0.05);24hours after operation, no Bc1-2expression is found inthe control group, but Bc1-2expressions are obviously increased in salvianolic acid Bgroup and SCI group (P<0.01), and salvianolic acid B has no effect on Bc1-2expression(P>0.05).Conclusion:1. After the BBB rating, slope test, foot print test and swimming test, it isconfirmed that salvianolic acid B plays a significantly promoting role in the recoveryfor dyskinesia of acute SCI rats’ hind limbs.2. Salvianolic acid B can inhibit the peroxidation brought from acute SCI, andscavenge free radicals and peroxidation product, thereby inhibiting the apoptosis ofneuronal cells and protecting nerve function.3. Salvianolic acid B plays a role in protecting nerve function by suppressing IKK/NF-kB signaling pathway in rat acute SCI model. |
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