| Purpose: Spinal cord injury(SCI)is a severe trauma of the Central nervous system(CNS).Currently,the treatment strategy of SCI is mainly to combat the cascades of secondary injuries.Rosmarinic acid(RA),a naturally occurring polyphenolic compound,has biological functions such as antioxidant,anti-inflammatory and anti-apoptotic,and has a strong therapeutic potential for neurological diseases.However,the neuroprotective effect and mechanism of RA on SCI are still unclear.Therefore,we established a rat acute SCI model to explore the neuroprotective effect and molecular mechanism of RA on SCI through proteomics.Methods: 1.A total of 110 clean adult female SD rats were randomly divided into five groups(22 rats per group): Sham group,model group(SCI group),10,20,and 40 mg/kg RA groups.Medical cerebral aneurysm clamp(calibration force of 30 g,clamp for 30 s)was used to prepare the SCI rat model.In the Sham group,laminectomy was performed only without spinal cord clipping,and spinal cord clipping was performed in the other groups.1 h after successful modeling,rats in the treatment group were intraperitoneally injected with RA solution,and rats in the Sham group and SCI group were intraperitoneally injected with the same volume of normal saline every day.The intervention time for each group was 28 days.At day 1,3,5,7,14,21,and 28 after intervention,motor function of rats in each group was evaluated by BBB score and inclined plate test.The rats were sacrificed on the 7th day and spinal cord tissue was collected.The histopathological changes of the anterior horn of the spinal cord were observed by HE staining.The status of motor neurons in the anterior horn of spinal cord was observed by Nissl staining.The expressions of glial fibrillary acidic protein(GFAP),neuronal marker protein(NEUN),and microglial marker protein(Iba-1)were detected by immunofluorescence assay.Neural cell apoptosis was detected by TUNEL assay.Biochemistry kit was used to detect the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)in spinal cord tissue.The contents of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),and interleukin-10(IL-10)in tissues were determined by enzyme-linked immunosorbent assay(ELISA).The protein expression levels of GFAP,Neun,NF-H,BDNF,Bax,Bcl-2,Caspase-9,Caspase-3,TNF-α,and IL-10 in tissues were detected by Western blot.2.Twelve female SD rats were randomly divided into two groups(6 rats in each group): SCI group and 20 mg/kg RA group.After 7 days of successful intervention in the model building,collect spinal cord tissue and extract of total protein.Two-dimensional gel electrophoresis(2-DE)was used to analyze the differential protein spots in the SCI group and the treatment group,and the differential protein spots were identified and analyzed by MALDI-TOF-MS mass spectrometry.Then,the functional pathways of the differential protein and the potential target of RA were predicted by bioinformatics,and verified by Western blot.3.A total of 110 SD rats were randomly divided into five groups(22 rats in each group): Sham group,SCI group,RA treatment group(20 mg/kg),RA + ML385(30 mg/kg)intervention group and ML385(30 mg/kg)intervention group.Treatment was started as soon as 1 h after successful modeling.The treatment group was intraperitoneally injected with 20mg/kg RA solution or 30 mg/kg ML385 solution before the injection of RA for 30 min.Both the Sham group and the SCI group were intraperitoneally injected with the same volume of normal saline,once a day.BBB score and inclined plate test were performed on the rats at 7 d and 14 d after surgery,respectively.Spinal cord tissue was collected from the rats at 7 d after surgery.HE staining was used to observe the pathological changes of spinal cord anterior horn,Nissl staining was used to observe the status of motor neurons in spinal cord anterior horn,TUNEL method was used to observe the apoptosis of nerve cells,biochemical kit was used to detect the content and activity of SOD,MDA,GSH-Px and CAT in spinal cord tissue,ELISA was used to detect the content of TNF-α,IL-6,IL-1β and IL-10 in spinal cord tissue.The expression levels of Bax,Bcl-2,Caspase-9,Caspase-3,TNF-α,IL-10,Nrf2,HO-1,TLR4,My D88,and NF-κB p65 in spinal cord tissues were detected by Western blot.Results: 1.(1)BBB score and inclined plate test results showed that the SCI group was lower than the sham group at different time points,and the hindlimb movement of rats in RA group improved significantly after 5-28 days;(2)The results of HE staining showed that compared with the sham group,the spinal cord tissue structure of the rats in the SCI group was severely damaged.After RA treatment,the changes in neuron morphology and tissue structure could be prevented;(3)Nissl staining results showed that the RA treatment group could significantly promote the formation of Nissl bodies,and the number of Nissl bodies in the middle and high-dose groups is more;(4)The TUNEL results show that there are obvious apoptotic nerve cells in the SCI group.Compared with the SCI group,the number of apoptotic nerve cells in the treatment group was significantly reduced;(5)ELISA results showed that compared with the sham group,the content of TNF-α,IL-6,IL-1β,and IL-10 was increased in the SCI group;and the content of TNF-α,IL-6 and IL-1β was decreased,and increase the content of IL-10;(6)After SCI,the activity of antioxidant enzymes GSH-Px,CAT,and SOD decreased significantly and the content of MDA increased;and the SCI group,the SOD,GSH-Px,and CAT antioxidant enzyme activities in the treatment group gradually increased,while the MDA content gradually decreased;(7)Immunofluorescence results showed that compared with the SCI group,the RA treatment group could reduce GFAP and promote the expression of Neu N;(8)Western blot results showed that,the expression levels of Bax,Caspase-9,Caspase-3,TNF-α,and IL-10 in the SCI group were increased;the expression levels of of Bax,Caspase-9,Caspase-3,and TNF-α proteins were significantly reduced,while the expression levels of IL-10 protein increased after RA treatment.2.(1)2-DE was used to characterize the tissue proteins of the SCI group and the RA treatment group(20mg/kg),and the 2D electrophoresis protein differential expression profile was successfully established.The rat spinal cord before and after RA treatment can be observed tissue protein profiles,which have obvious differences;(2)The total of 25 protein spots with obvious expression differences were identified by MALDI-TOF-MS,and 16 protein spots were successfully identified;(3)The successfully identified proteins were analyzed by databases,such as DAVID and STRING.Function mining revealed that some proteins are related to the functional recovery of SCI,such as oxidative stress proteins(HO-1 and HSP70)and inflammation-related proteins(HSP27,ANXA2,AKR1B1 and ARG1).Further analysis revealed that RA may regulate Nrf2 and NF-κB pathway;(4)Western blot results showed that compared with the sham operation group,the HO-1 protein in the spinal cord tissue and the Nrf2 protein in the nucleus of the model group increased,while the expression of Nrf2 protein in the cytoplasm decreased.After RA treatment,the expression of HO-1 and Nrf2 protein in the nucleus was higher than that of the SCI group,and the expression of Nrf2 protein in the cytoplasm was lower than that of the SCI group;(5)At the same time,TLR4,My D88,p-Ik Bα,p-IKKα/β and NF of the SCI group.The expression level of NF-κB p65 increased,but the expression level of these inflammatory proteins decreased after different doses of RA treatment.3.(1)BBB score and inclined plate experiment showed that the hind limb activity of rats in the RA treatment group was improved significantly at 7 and 14 days after the operation;compared with the RA treatment group,the hind limb activity function of the rats was weakened after the intervention of ML385;(2)HE staining showed that compared with the SCI group,the degree of spinal cord structural damage in the RA treatment group was improved.After ML385 intervention,the degree of neuronal morphology damage was further aggravated;(3)Nissl staining results showed The RA treatment group can significantly promote the formation of Nissl bodies,but the intervention of ML385 will reduce the number of Nissl bodies;(4)TUNEL results show that the number of apoptotic nerve cells significantly reduced in the RA treatment group compared with the SCI group,ML385 intervention increased the number of apoptotic nerve cells;(5)ELISA results showed that TNF-α,IL-6 and IL-1β decreased and the IL-10 content increased in the RA group;ML385intervention group increased the content of TNF-α,IL-6,and IL-1β,while the content of IL-10 decreased;(6)Compared with SCI group,the SOD content,GSH-Px and CAT antioxidant enzyme activity in the RA treatment group were increased,while MDA content was significantly reduced,and ML385 intervention would reverse the therapeutic effect of RA;(7)Western blot results showed that the expression levels of Bax,Caspase-9,Caspase-3,and TNF-α in the RA treatment group compared with the model group,and the level of IL-10 increased.After the intervention of ML395,the expression levels of Bax,Caspase-9,Caspase-3,and TNF-α protein increased again,while the expression level of IL-10 protein decreased;(8)At the same time,both Nrf2 and HO-1 proteins in the spinal cord tissue of RA treated rats were increased,and ML385 would reverse the changes of Nrf2 and HO-1 proteins.At the same time,ML385 would also reverse the effects of RA on TLR4,My D88,p-Ik Bα,p-IKKα/β,and NF-κB p65 protein expression levels.Conclusion: 1.RA can improve spinal cord tissue structure damage and reduce neuronal apoptosis in SCI rats,increase neuron survival,and promote the recovery of motor and nerve function in SCI rats.2.RA can inhibit the activation of microglia and astrocytes after SCI,reduce the secondary oxidative damage and inflammation of the spinal cord tissue,thereby exerting a neuroprotective effect.3.The neuroprotective effect of RA on SCI rats is partly achieved by activating Nrf2/HO-1 and inhibiting the TLR4/NF-κB signaling pathway.At the same time,RA can also regulate the Nrf2/NF-κB signaling axis. |
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