Alleviative Effects Of Echinacoside Against Spinal Cord Injury Via Inhibiting NF-?B/NLRP3 Signaling Pathway

Objectives: Spinal cord injury(SCI)always leads to serious inflammatory response,which in turn greatly aggravates the damage of spinal cord.Considerable literature has reported that nuclear factor-kappa B(NF-?B)and NLRP3 inflammasome signaling pathways play an essential role in the initiation of inflammatory response.Neuroinflammation in the central nervous system(CNS)is mainly regulated by microglia,which are the resident immune cells in the CNS.Echinacoside(ECH)is a phenylethanoid glycoside which has been reported to possess prominent anti-inflammatory and neuroprotective effects.However,few studies had investigated the effects of ECH on SCI.Therefore,this study was conducted with the following purposes: 1)looking into whether ECH had suppressive effects on the activation of BV-2 cells and the NF-?B signaling pathway in BV-2 cells;2)investigating whether ECH had suppressive effects on the activation of NLRP3 inflammasome in BV-2 cells and the underlying mechanism;(3)exploring whether ECH had therapeutic effects on SCI and the underlying mechanisms.Methods: 1)Cell Counting Kit-8(CCK-8)assays were used to estimate the cytotoxic effect of ECH on BV-2 cells with different concentrations,and harmless concentrations were selected for further experiments.The experiment included five groups: control group,model group,low-dose ECH group,middle-dose ECH group,and high-dose ECH group.To trigger the activation of BV-2 cells,1?g/ml lipopolysaccharide(LPS)was used to stimulate the BV-2 cells.The morphological change of BV-2 cells was observed by inverted phase contrast microscope.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of IL-1? and IL-18 in the supernatant of culture medium.Western blot(WB)was used to analyze the expressions of p-NF-?B,p-I?B?,and iNOS;2)The experiment included five groups: control group,model group,low-dose ECH group,middle-dose ECH group,and high-dose ECH group.BV-2 cells were stimulated with 500ng/ml LPS followed by 1mM ATP to establish the model of NLRP3 inflammasome activation.ELISA was used to detect the expressions of IL-1? and IL-18 in the supernatant of culture medium.To investigate the activation of NLRP3 inflammsome and the underlying molecular mechanism,WB was used to analyze the expressions of NLPR3 inflammasome-associated proteins including NEK7,NLRP3,ASC,and Caspase-1,as well as the NF-?B signaling pathway-associated proteins including p-NF-?B,NF-?B,p-I?B?,and I?B?.Immunofluorescence(IF)staining was also used to detect the expression of NLRP3 in BV-2 cells.Intracellular reactive oxygen species(ROS)were measured by flow cytometry using ROS assay kits,which marked the cells with DCFH-DA probe.To analyze the change of mitochondrial membrane potential(MMP),Mito-tracker Red probe was used to mark the mitochondria,followed by confocal scanning and image J analysis;3)Sprague-Dawley(SD)rats were randomly divided into three groups: Sham group,SCI group,and SCI+ECH group.The SCI model was established by the modified Allen's method.Neurological function was assessed using the Basso-Beattie-Bresnahan(BBB)open-field locomotor scale and inclined-plane test before and after SCI.The degree of edema was evaluated via determination of the precise spinal cord water content after SCI.Nissl staining and Hematoxylin-Eosin(HE)staining were performed respectively to evaluate neuronal survival and morphological changes of spinal cord.The expressions of IL-1? and IL-18 in spinal cord tissues were detected by ELISA.The expressions of NLPR3 inflammasome-associated proteins,including NEK7,NLRP3,ASC,Caspase-1,and NF-?B signaling pathway related proteins were detected by WB.In addition,myeloperoxidase(MPO)and ionized calcium binding adapter molecule 1(Iba-1),which represented the neutrophils infiltration and the activation of microglia,were also detected by WB.Results: 1)The results of CCK-8 indicated that administration of ?50?g/ml ECH had no significant cytotoxic effect on BV-2 cells.Therefore,10,20,and 50?g/ml ECH were selected for further experiments.Under the normal culture medium,BV-2 cells were in the resting state.After stimulation with LPS,BV-2 cells turned into activated condition with larger morphology and less processes.The administration of ECH could inhibit the morphological change of BV-2 cells.ELISA results showed the secretions of IL-1? and IL-18 were significantly increased after stimulation with LPS,and the administration of ECH significantly reduced the secretions of IL-1? and IL-18(P<0.01).The level of p-NF-?B,p-I?B?,and iNOS were significantly increased in response to LPS stimulation compared to the control.After the administration of ECH,p-NF-?B,p-I?B?,and iNOS decreased significantly in a dose-dependent manner(P<0.01);2)ELISA results showed the secretions of IL-1? and IL-18 were significantly increased after the stimulation with LPS+ATP,and the administration of ECH significantly reduced the secretions of IL-1? and IL-18(P<0.01).WB results showed that ECH inhibited the expressions of NLRP3,Caspase-1,ASC,and NEK7 in a dose-dependent manner(P<0.01),suggesting that ECH could inhibit the activation of NLRP3 inflammasome.The ratios of p-NF-?B/NF-?B and p-I?-B?/I?-B? increased significantly after stimulation with LPS+ATP,and the ratios were reduced after the administration of ECH(P<0.01).In addition,ECH reduced the production of ROS and prevented LPS+ATP-induced mitochondrial dysfunction in BV-2 cells;3)BBB scale and inclined-plane test indicated that the administration of ECH could significantly promote motor function recovery(P<0.05),reduce the neuron loss(P<0.05),ameliorate the tissue edema(P<0.05),and alleviate structural disorder of spinal cord following SCI.The results of ELISA suggested that ECH could significantly inhibit the expressions of IL-? and IL-18(P<0.01).Results of WB indicated that the levels of NLRP3 inflammasome and NF-?B signaling pathway related proteins were significantly increased after SCI,and the proteins obviously decreased after the administration of ECH(P<0.01).In addition,the proteins of MPO and Iba-1 were highly expressed after SCI and significantly decreased after treatment with ECH.Conclusions: 1)The findings of this study indicate that ECH significantly inhibits the activation of M1 microglia and NF-?B signaling pathway;2)ECH significantly reduces the generation of ROS,improves the MMP,and blocks the NF-?B signaling pathway,thus inhibiting the activation of NLRP3 inflammasome;3)ECH inhibits the activation of NLRP3 inflammasome via regulating the NF-?B/NLRP3 signaling pathway,thus alleviating the inflammatory response after SCI.In addition,ECH relieves the spinal cord edema,blocks the neutrophils infiltration,inhibits the activation of microglia,consequently ameliorates the neurological function after SCI.