Preliminary Study On The Effect Of Shikonin In Spinal Cord Injury

Objective Shikonin is extracted and isolated from Lithospermum officinale,andhas a variety of pharmacological activities.Many studies have shown that shikonin has a biological role of anti-oxidation,anti-tumor,anti-inflammatory,anti-apoptotic,anti-viral and has low toxicity and side effects,it also has been shown to protect human organs from harm and a good potential for clinical application.However,the effect of shikonin on the recovery of spinal cord injury(SCI)and the protein expression levels and signaling pathways involved remains unknown.Previous report demonstrated that NF-κB signaling pathway could be suppressed by shikonin.However,the molecular mechanism of shikonin interaction with NF-κB pathway in the alleviation process of SCI remains unknown.In this experiment,we try to build a model of spinal cord injury in rats,after shikonin intervention treatment,HE staining,ELISA,TUNEL and Western blot were used to determine the effect of shikonin on spinal cord injury in rats.Spinal cord edema and inflammation before and after intervention,neurological deficits were evaluated to study the potential of shikonin in the treatment of spinal cord injury and to explore its mechanism.Methods Forty male Sprague-Dawley(SD)rats weighing 180-220 g were divided into sham group(n = 8)and operation group(n = 32).The rats were treated with a modified Allen’s weight reduction device(8 g weight,vertical height 40 mm,8 g x 40mm)on the spinal cord to induce moderate spinal contusion.Sham group: exposed thoracic vertebral body 10(T10)laminectomy,no damage to the spinal cord area;operation group after the establishment of SCI were given different treatment,namely spinal cord injury model group(SCI)8,strong methyl 8 rats in the group of MPSS,85 rats in the low dose shikonin intervention group(SHI10mg)and 8 rats in the high doseshikonin group(SHI100mg).Each rat was housed separately.Three rats were randomly selected from each group.Basso,Beattie,and Bresnahan(BBB)scores were assessed at 24 hours,48 hours and 72 hours after SCI.Spinal cord edema was assessed by the water content of the spinal cord tissue,HE staining was used to observe the tissue repair at 72 h after SCI,and IL-1β,IL-6 and TNF-α and other inflammatory cytokines Western blot was used to detect the related proteins.TUNEL was used to analyze the apoptosis of spinal cord tissue after SCI.IBA-1(1: 400,Abcam)and GFAP(1: 500,Sigma)antibodies were used to evaluate microglial activation and reactive gliosis in the injured spinal cord.Results 1.The BBB score in SCI group was remarkably lower than other groups at different time points.The BBB score in the shikonin treatment group at different doses was significantly higher than SCI treatment rats at selected time points(P<0.01).The recovery level of locomotor function after SCI injury between MPSS and 100 mg/kg of shikonin group had no significant difference.2.SCI group HE staining obvious spinal cord tissue was loose,multiple cavity formationand neutrophil infiltration.MPSS and different doses of shikonin can significantly reduce spinal cord injury.In addition,SCI injury enhanced IBA-1 and GFAP immunoreactivity compared with control group,while IBA-1 and GFAP intensity obviously attenuated by MPSS and shikonin with different doses treatment.3.The water content of SCI group was obvious increased compared with sham group.However,after treatment with different doses of shikonin,the mean value of water content was significantly reversed,which suggested that shikonin treatment could alleviate SCI-induced spinal corded ema in rats(P<0.001).The cytokine levels of SCI group were obviously increased compared with sham group.In addition,different doses of shikonin treatment could dramatically decrease IL-1β,IL-6 and TNF-α expression compared with SCI group.The results revealed that shikonin could inhibit inflammation levels after SCI injury.4.The apoptotic cells in SCI group were obviously increased compared with those in sham group.In the group of shikonin treatment following SCI injury,the apoptotic cells in the spinal cord were significantly decreased compared with SCI group.In addition,compared with 10mg/kg of shikonin group,100mg/kg of shikonin treatment induced less apoptotic cells after SCI injury.Conclusion The experiment successfully established a rat model of spinal cord injury,the model has the advantages of easy replication,high success rate,stable model,is an ideal model for the study of spinal cord injury.Shikonin can promote motor function recovery and tissue repair after SCI in rats,reduce spinal cord edema and inhibit inflammation after SCI injury.Further studies have concluded that shikonin improves motor function and tissue repair by inhibiting the levels of inflammatory cytokines and the expression of related proteins in spinal cord tissue.This newly mechanism provides further understanding of molecular signaling pathway in spinal cord injury during secondary injury and brings new insight into spinal cord injury.