Effects Of Salidroside On NF-κB And MAPK Signaling Pathway And Motor Function In Rats With Spinal Cord Injury

Spinal cord injury(SCI)referred to the damage of the spinal cord structure caused by various factors.SCI often leaded to disability,reduced the quality of life,caused great physical and mental dysfunction,lowered employment,brought heavy economic and psychological burden to the family and community.SCI was usually composed of "primary injury" and "secondary injury".The primary injury was directly caused by injury,which directly affected the spinal cord.Secondary injury referred to a series of damage caused by the primary injury,including edema,inflammation,ischemia,hypoxia,peroxidation and release of various chemicals.Secondary injury could cause ischemia and hypoxia,apoptosis,and a series of immune and biochemical reactions through a series of cascade amplification of molecular biochemistry and immune response,Secondary injury aggravated the damage of local tissue structure,expanded the scope of injury,be resulted in further damage to the spinal cord injury,could produce far greater harm than the primary damage.The mechanisms of secondary injury included: ischemia and hypoxia and oxygen free radical formation,the formation of inflammatory cytokines and immune response,excitotoxicity,calcium overload,apoptosis and glial scar formation.The generation of inflammatory cytokines and a series of immune cascade reactions played an important role in the pathogenesis of SCI,and had an important influence on the prognosis.It was one of the most important targets for SCI.At present,the SCI research in animals and human trials consumed huge amounts of resources did not produce revolutionary improvements in neurological function or the result of nerve regeneration,clinical practice found that some drugs had relatively large adverse reactions.These unsatisfactory results are largely due to the complexity of the primary injury and secondary injury mechanism was not entirely clear,the lack of strong interventions to control the primary injury and a series of vascular cells,and biochemical reactions in secondary injury.Accordingly,It was important that we needed to find a more effective and safe methods to promote the recovery of neural function of SCI.Salidroside was one of the active components of Rhodiola rosea.Salidroside could improve microcirculation,anti oxygen free radical,inhibit intracellular calcium overload,scavenge free radical,anti apoptosis and inflammation,so it had potential application value to SCI.In this study,we aimed to investigate the effect of salidroside on the recovery of neurological function in SCI rats,and to find a new way to treat SCI.In this study,a series of experiments in vitro was used to verify the inhibitory effect of salidroside on release of inflammatory cytokines from astrocyte stimulated by lipopolysaccharide(LPS),Including: the toxicity of salidroside to astrocyte cell,Real-time PCR method to detect the effect that salidroside inhibits the release of IL-1β,IL-6,TNF-α from astrocytes stimulated by LPS,ELISA method to detect that salidroside dercase the expression of IL-1β,IL-6 and TNF-α in astrocytes stimulated by LPS,Western Blot detection of the effect of salidroside on NF-κB and MAPK signal pathway which activated by LPS in astrocytes.We also carried out experiments in vivo to observe the effects of salidroside on motor function recovery and inhibitory effect of salidroside on release of inflammatory cytokines in rats with SCI.Experiments in vivo included: establishment of rat spinal cord injury model,evaluation of spinal cord injury repair function index and histological change,Real-time PCR to detect the expression of IL-1β,IL-6 and TNF-α in spinal cord,ELISA method was used to detect the releases of IL-1β,IL-6 and TNF-α in spinal cord tissues.This experiment consists of two parts: Part One: the Effect and Mechanism of Salidroside on Inflammatory Cytokines Released from Astrocytes Activated by LPSObjective: The aim of this study was to investigate the effects of salidroside on the release of IL-1β,IL-6,TNF-α from astrocytes activated by LPS,and investigate the regulatory mechanism of salidroside on the release of inflammatory cytokines from astrocytes activated by LPS.Methods: In vitro,We recover astrocytes,and examined the toxic effect of different concentrations of salidroside to astrocytes by MTS;RNA was extracted from the cells,and real-time PCR and ELISA were used to detect the effect of salidroside on the release of IL-1β,IL-6 and TNF-α in astrocytes activated by LPS,explored effects of salidroside on NF-κB and MAPK signaling pathway in astrocytes activated by LPS through Western blots.Results: Salidroside had no toxic effect on astrocytes.Real-time PCR and ELISA test showed that salidroside could decrease the expression of IL-1β,IL-6 and TNF-α in astrocytes activated by LPS.Western blot demonstrated that salidroside inhibited NF-κ B,ERK and p38 MAPK signaling pathway in astrocytes stimulated by LPS.Conclusion: Salidroside could decrease the release of IL-1β,IL-6 and TNF-α from astrocytes stimulated by LPS.The mechanism of anti-inflammatory on salidroside might be achieved by inhibiting NF-κB,ERK and p38 MAPK signaling pathway.Part Two: the Effect of Salidroside on Motor Function and Inflammatory Cytokines in Rats with SCIObjective: The aim of this study was to explore the effect of salidroside on the motor function and inflammatory cytokines of SCI model in rats.Methods: A total of 128 Sprague Dawley rats were selected to make the model with modified Allen’s method in the SPF Animal Center,the rats were randomly divided into Sham,SCI,methylprednisolone(MP)and salidroside(SAD)groups.The sham group did not damage the spinal cord.There was no neuroprotective therapy in the SCI group after injury.MP group and SAD group were treated with methylprednisolone and salidroside respectively.The function was evaluated at 1,6,24 hours and 7 days after modeling.The spinal cord specimens were taken for histological examination after being perfused with 4% paraformaldehyde.The spinal cord specimens taken for Real-time PCR and ELISA were obtained after being perfused with normal saline.Real-time PCR and ELISA were used to detect the effects of salidroside on the release of IL-1β,IL-6 and TNF-α after SCI.Results: After modeling,the BBB score of SCI group,methylprednisolone group and salidroside group were reduced.Both methylprednisolone and salidroside could improve the BBB score of hind limbs in rats,and the difference of the two drugs was not statistically significant.HE staining and Nissl staining showed that the tissue damage of SAD group and MP group was less than the SCI group.After 1,6,24 hours of SCI,the expression of IL-1β,IL-6 and TNF-α were increased,there was statistical significance in SCI,MP and SAD group contrast Sham group,there was no significant difference between SCI group and MP group and SAD group.After 7 days of injury,compared with SCI group,the expression of IL-1β,IL-6 and TNF-α in the MP group and SAD group were decreased,the difference was statistically significant,and there was no significant difference between MP group and SAD group.Conclusion: Salidroside could promote the recovery of lower limbs motor function in rat model of SCI,reduce the expression of IL-1β,IL-6 and TNF-α in injured spinal cord segments,retain more normal spinal cord gray matter and white matter,had application potential of neural protection on SCI.