| Background:Spinal cord injury(SCI)is a neurological disease with high disability and mortality rates.After SCI,SCI produces different degrees of motor,sensory and autonomic dysfunction,which greatly affects the survival and quality of life of patients and brings a heavy burden to families and society.Currently,there is no definite and effective measure for neurological repair after SCI.For a long time,researchers have been focusing on the neurobiological changes in the spinal cord during SCI,as well as the repair and regulation of the nervous system after the injury,in order to break through this problem.Microglia,as the inherent immune cells of the nervous system,play an important role in the repair of neurological injury.Existing studies have found that microglia activation and polarization after SCI can significantly improve the recovery of transport function after SCI.In addition,Icariin(ICA),a flavonoid glycoside,is extracted and purified mainly from Epimedium,a plant of the family Berberaceae.Existing studies have shown that it has various pharmacological effects such as anti-inflammatory,antioxidant,anti-apoptotic,promoting bone metabolism,anti-osteoporosis,enhancing immune function,and improving cardiovascular and cerebrovascular functions.Moreover,it has been reported that Epimedium can reduce the inflammatory response after SCI and promote the recovery of motor function after SCI,but the exact mechanism has not been fully elucidated.Objective:The aim of this study was to explore whether ICA could induce microglia polarization to improve neurological recovery after SCI.Methods:(1)Seventy-two male SD rats were selected and randomly divided into three groups: sham-operated group(Sham group),model group(SCI group),and patchouli intervention group(ICA group),24 rats in each group.The SCI model was established according to the modified Allen’s method,and after successful modeling,ICA solution was given daily by gavage in the ICA intervention group,and equal amounts of saline were given in the sham-operated and model groups for 14 d in each group.(2)BBB scores and slant-board tests were performed on the motor functions of rats immediately,1 d,3 d,5 d,7 d,and 14 d after SCI to assess the recovery of hind limb movement in SD rats at different time points.The recovery of hind limb movement was assessed at different time points.(3)Paraffin sections were prepared by perfusion with 4%paraformaldehyde at 7d and 14 d after SCI,and HE staining was performed at the center of SCI to observe the morphological damage area of spinal cord injury tissue;Nissler staining was used to observe the retention of neurons in the injured spinal cord tissue.(4)At the seventh day after SCI,0.9% saline was used for perfusion sampling,and the expression of pro-inflammatory factors(TNF-α,IL-6),anti-inflammatory factors(IL-4,IL-10),microglia M1 type marker i NOS and M2 type marker Arg-1 protein were examined by Western Blot at the seventh day after SCI;(5)At the seventh day after SCI,0.9% saline was used for perfusion sampling.(5)The number of microglia M1 and M2 cells and their differentiation were detected by immunohistochemistry at 7 d after SCI using 0.9% saline perfusion.(6)The m RNA content of the M1 and M2 microglia markers was measured by RT-q PCR at 7 d after SCI using 0.9% saline perfusion.Results:(1)A traumatic spinal cord injury(TSCI)model in SD rats was successfully prepared by the spinal cord percussion device,which showed that the motor function of the rats in the Sham group was normal after surgery,while the rats in the SCI and ICA groups showed bilateral lower limb paralysis immediately after surgery.(2)The results of BBB scores after SCI showed that in the Sham group,the BBB scores were 21 before and after modeling;compared with the SCI group,the motor function BBB scores of rats after ICA intervention increased significantly at 5d,7d and 14 d and had higher maintenance angles at 7d and 14 d in the inclined plate experiment,and the differences were statistically significant(p < 0.05).(3)The results of HE staining at 7d and 14 d after SCI showed that the spinal cord tissue in the Sham group was morphologically intact,with a tight structure,clearly visible neuronal nuclei,contrasting nuclei and cytoplasm,no inflammatory cell infiltration,and few vacuoles;compared with the Sham group,the SCI group showed significant edema,congestion,inflammatory cell infiltration,and large vacuoles between the central gray matter and white matter As time progressed,the loss of normal tissue structure increased and the vacuolation phenomenon increased.Compared with the SCI group,the spinal cord tissue in the ICA group was less congested,edematous,and structurally destroyed,and the area of the vacuoles was smaller.The results of Nissl staining showed that the number of neurons at the spinal cord tissue injury was significantly higher in the ICA group than in the SCI group at the 7th and 14 th postoperative days,and the Nissl body was more completely preserved.(4)At the 7th day after SCI,Western Blot showed that the content of pro-inflammatory factors(TNF-α,IL-6)in the spinal cord injury tissues of the ICA group was significantly less than that of the SCI group,while the content of anti-inflammatory factors(IL-4,IL-10)was significantly higher than that of the SCI group,and the difference was statistically significant(p < 0.05);the M1 type marker of microglia in the SCI group was significantly higher than that of the SCI group.i NOS was significantly higher in the SCI group than in the ICA group,while the M2 marker Arg-1 was significantly higher in the ICA group than in the SCI group,and the difference was statistically significant(p < 0.01).The expression of M1-type marker i NOS was significantly higher in the SCI group than in the ICA group,whereas the expression of M2-type marker Arg-1 was significantly higher in the ICA group than in the SCI group,with statistically significant differences(p < 0.05).Similarly,it was observed that in the RT-q PCR results,the m RNA content of M1-type marker i NOS was significantly higher in the SCI group than in the ICA group,whereas the m RNA content of M2-type marker Arg-1 was significantly higher in the ICA group than in the SCI group,and the difference was statistically significant(p < 0.05).Conclusion:1.ICA significantly improved the recovery of motor function after SCI in rats;2.ICA inhibited the expression of pro-inflammatory factors and increased the expression of anti-inflammatory factors after SCI;it inhibited the polarization of microglia to M1 type and induced the polarization of microglia to M2 type microglia,thus reducing the harm caused by secondary injury after SCI.ICA is valuable to further investigate the protective effect of Epimedium on SCI by providing new ideas and targets. |
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