The Neuroprotective Effects Of Luteolin Against Spinal Cord Injury In Mice Based On NF-κB/NLRP3 Signaling Pathway

Objectives: Spinal cord injury(SCI)is a common disease of central nerveous system(CNS),but there is still a lack of effective therapeutic measures.As functional neural tissue is subject to loss progressively after trauma,early neuroprotective intervention is important.Luteolin(LUT)is a flavonoid which has been reported with therapeutic efficacy against a variety of neurological diseases,but there are few studies in terms of SCI.Therefore,we prepared to investigate whether LUT had neuroprotective effects on SCI in mice,and tried to reveal the mechanistic relationship between these effects and NF-κB/NLRP3 signaling pathway.Methods: Seventy-two male C57BL/6 mice were randomly and equally divided into three groups: sham group,SCI group,and LUT group,and each group continued to be divided into three subgroups according to the injury time.The SCI model was prepared using the modified Allen’s method;in the sham group,only laminectomy was performed without spinal cord hit.Immediately after surgery,mice in the LUT group were intraperitoneally injected with luteolin solution at a dose of 30 mg/kg once daily;the sham group and SCI group was also intraperitoneally injected with an equal volume of vehicle solution with same frequency.Basso Mouse Scale(BMS)score and inclined-plate test were used to assess the hindlimb motor function of mice before and on the 1st,3rd,and 7th day after SCI.Hematoxylin-eosin(HE)staining was performed to evaluate the structural and morphological changes of spinal cord tissues.TUNEL fluorescence staining was used to detect cell apoptosis,while Nissl staining to observe the physiological status of neurons.The expressions of NF-κB/NLRP3 signaling pathway related proteins,including p-IκBα,NF-κBp65,NLRP3,caspase-1,and its downstream inflammatory factor IL-1β were detected by Western blot(WB).Results:(1)The BMS score of mice in all groups was 9 points preoperatively,and it was 9points in the sham group on the 1st,3rd,and 7th day after laminectomy.After SCI,no or slight joint activity was observed in the SCI group and the LUT group.On the 7th day,the BMS score of LUT group was significantly higher than that of SCI group(P(27)0.05).(2)In the inclined-plate test,the mean value of inclination angle in the sham group was measured to be above 63°.Compared with the sham group,the angle value of the SCI group and the LUT group was significantly decreased(P(27)0.01).The value of LUT group was greater than that of SCI group(P(27)0.05)on the 7th day.BMS score and inclined-plane test indicated that the administration of LUT could promote neurological recovery in mice.(3)HE staining showed that the inflammatory response was significantly alleviated in the LUT group compared with the SCI group.On the 7th day after SCI,the remaining area of spinal cord tissue in the LUT group was more than that in the SCI group(P(27)0.05),which indicated that LUT could alleviate structural disorder and reduce loss of spinal cord tissue following SCI.(4)Tunel fluorescence staining indicated that LUT could significantly reduce cell apoptosis in the acute phase of SCI(P(27)0.05)when the inflammatory response was gradually aggravated.(5)The Nissl bodies in the sham group were large patchy or granular,while they were disintegrated,or even disappeared in the SCI group.The results of cell count showed that the number of Nissl staining positive cells in LUT group was significantly more than that in SCI group(P(27)0.01),which indicated that LUT could reduce neurons loss and alleviated SCI.(6)After the administration of LUT,the expressions of p-IκBα,NF-κBp65,NLRP3, caspase-1,and IL-1β in the LUT group were significantly decreased compared with those in the SCI group(P(27)0.05).Conclusions:(1)LUT could improve hindlimb motor function in mice after SCI and promote neurological recovery.(2)LUT could reduce the neuron loss,ameliorate the tissue edema,and alleviate structural disorder of spinal cord following SCI.(3)That LUT exerting neuroprotective effects was associated with down-regulating NF-κB signaling pathway transduction,inhibiting activitation of NLRP3 inflammasome,and reducing the expression of IL-1β,to alleviate the inflammatory response after injury.