| Objectives: Secondary neuroinflammation is an important cause of the aggravation of spinal cord injury(SCI).Neuronal damage after spinal cord injury is closely related to neuroinflammation.The NLRP3 inflammasome is mainly released by the immune cells of the central nervous system,microglia,and is a major contributor to neuroinflammation.Endoplasmic reticulum stress(ERS)plays an important role in spinal cord injury-induced neuroinflammation.NLRP3 inflammasome and endoplasmic reticulum stress play important roles in neuroinflammatory cascades.Urolithin A(UA)is a natural polyphenolic compound produced by intestinal microbial metabolism of pomegranate and nuts rich in ellagitannin and ellagic acid.Recently,in vivo and in vitro studies have found that UA has significant anti-inflammatory and neuroprotective effects,but few literatures report its role in SCI.The main purposes of this study are: 1)use BV-2 microglia to establish a neuroinflammation model,and explore the effect of UA on neuroinflammation and ERS pathway in vitro;2)use BV-2 microglia The NLRP3 inflammasome model was established,and the effect and mechanism of UA on the NLRP3 inflammasome pathway were further explored;3)The SCI model was established by the modified Allen’s method.exploring whether UA had therapeutic effects on SCI and the underlying mechanisms.Methods: 1)The cell proliferation and cytotoxicity detection kit(cell counting kit-8,CCK-8)was used to detect the effect of different concentrations of urolithin A and lipopolysaccharide(LPS)on the activity of BV2 cells to determine the safety of BV2 cells and the optimal concentration for subsequent studies.The experiment was divided into five groups: control group,model group,low-dose,medium-dose and high-dose urolithin A groups.BV-2 cells were treated with 1μg/ml LPS to prepare an inflammatory injury model.The contents of IL-1β and IL-18 in the supernatants of each group were measured using ELISA kits.At the same time,a nitric oxide detection kit was used to measure the release of NO in the supernatant of each group.The morphology of endoplasmic reticulum in BV-2 cells was observed using transmission electron microscopy(TEM).ER-associated proteins(GRP78,PERK,p-PERK,ATF-6,EIF-2α,p-EIF-2α,CHOP,ATF-4)and i NOS protein expression level.2)BV2 microglia were induced with 1μg/ml LPS to construct an NLRP3 inflammasome activation model.Group as above.Mitochondrial membrane potential detection kit(JC-1)was used to detect changes in mitochondrial membrane potential,and confocal microscopy was used to observe and collect pictures.The morphological changes of cell mitochondria were observed by TEM.The protein extracts of each group were detected by WB,and NF-κB(p-NF-κB,NF-κB,p-IκBα,IκBα)and NLRP3 inflammasomerelated proteins(NLRP3,ASC,Caspase-1,IL-1β,IL-18)expression levels.3)Healthy male SD rats were randomly divided into sham group,SCI group,SCI+L-UA group and SCI+HUA group.The SCI model was prepared by the modified Allen’s weight falling method.The motor function of hindlimbs of rats was assessed by Basso-Beanie-Bresnahan(BBB)open-field locomotor score and inclined plate test.Nissl staining and Hematoxylin-Eosin(HE)staining were performed respectively to evaluate neuronal survival and morphological changes of spinal cord.Spinal cord myelin was stained with LFB staining to assess spinal cord demyelination.The water content of spinal cord tissue was measured on the third day after operation.WB technology was used to detect the changes of ERS-related proteins,NLRP3 inflammasome-related proteins,NF-κB-related proteins,i NOS and Iba-1 protein levels.The morphological changes of endoplasmic reticulum and mitochondria in microglia of spinal cord tissue were observed by TEM.Results: 1)The results of CCK-8 showed that the concentration of ≤ 40 μM UA had no obvious toxic effect on BV-2 cells(P>0.05),and UA at concentrations of 2.5μM,5μM and 10μM were selected for subsequent experiments.ELISA results showed that the expression levels of IL-1β and IL-18 were significantly increased after LPS treatment of BV-2 cells compared with the control group,while the application of UA could reduce the expressions of IL-1β and IL-18(P<0.01).WB results showed that the expression levels of ERS-related proteins and i NOS proteins were significantly increased after the stimulation with LPS(P < 0.01),and different concentrations of UA could reduce the expression of these proteins(P<0.01),and there is a dose-dependent manner.The measurement results of NO concentration showed that the release of NO was significantly increased in the model group,and UA inhibited the release of NO.The TEM results show that the endoplasmic reticulum was significantly swollen and expanded after the stimulation with LPS,and ribosome shedding could be observed in the rough endoplasmic reticulum,and UA could significantly reduce the endoplasmic reticulum damage.2)According to WB results,it was found that in the model group,the expressions of NF-κB and NLRP3 inflammasome-related proteins were increased(P<0.01),while UA treatment could reduce the expression of these proteins(P<0.01).The results of the study on mitochondria showed that in the model group,the mitochondrial membrane potential was significantly decreased(P<0.01),and the mitochondrial membrane was ruptured and dissolved under TEM,and the mitochondrial cristae were unclearly fused,and even disappeared to form vacuoles,while uroliths were observed.UA could significantly improve the mitochondrial membrane potential and mitochondrial morphology of LPStreated cells.3)BBB scale and inclined-plane test indicated that the administration of UA could significantly promote motor function recovery(P<0.05),reduce the neuron loss,ameliorate the tissue edema(P < 0.01),and alleviate spinal cord demyelination and structural disorder after SCI.WB results showed that the expression levels of ERS-related proteins,p-NF-κB,p-IκBα and NLRP3 inflammasome-related proteins were significantly increased in SCI group(P<0.01),and UA treatment could reduce the expression levels.The expression of the above proteins(P<0.01).The TEM results showed that in the spinal cord microglia,endoplasmic reticulum swelling and degranulation,mitochondrial membrane dissolution and swelling,mitochondrial cristae disappearance and even vacuolization were observed.UA alleviated mitochondrial and endoplasmic reticulum damage.Conclusion: 1)The findings of this study indicate that UA significantly inhibits the activation of M1 microglia and ERS signaling pathway;2)UA can reduce mitochondrial damage,Inhibit the NF-κB signaling pathway,thus inhibiting the activation of the NLRP3 inflammasome;3)UA can inhibit the activation of the NLRP3 inflammasome by regulating the ERS/NLRP3 signaling pathway,thus alleviating the inflammatory response after SCI.In addition,UA can also improve spinal cord edema,inhibit the activation of microglia,and alleviate endoplasmic reticulum and mitochondrial damage,thus improving neuromotor function after SCI... |
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