| Arctigen(ARG)has been reported to possess potent anti-inflammatory properties.This study aims to evaluate the protective effects and underlying mechanisms of ARG on lipopolysaccharide(LPS)-induced neuroinflammati on and neurobiological alternations as well as cognitive impairment.Method Male C57BL/6J mice aged two months(n=50,weight 22±2g)were randomly assigned into the following groups: control group,model group(LPS,250μg/kg),ARG treatment group(LPS+ARG,100mg/kg)and single-use group(ARG,100mg/kg).The animal model of neuroinflammatio n was constructed by intraperitoneal injection of LPS(250μg/kg/d)for consecutive 7 days,and ARG dissolved in 0.5% carboxymethyl cellulose was treated by oral gavage for 3 weeks before intraperitoneal injection of LPS.The control group was intraperitoneally injected with 0.9% sterile saline and 0.5% sodium carboxymethyl cellulose.After the treatment,Morris water maze was employed to assess the cognitive abilities.Nissl staining,combined with the expressions of cleaved-caspese3 and cleaved-PARP1 in the hippocampus was used to examine the neuronal injury.Thioflavin-S staining and immunofluorescence staining were performed to evaluate the Aβ dimers and Aβ,and the generation of Aβprotein was further analyzed by western blot.Synapse density was detected by immunofluorescence SYP and PSD95 co-staining.The activation of neuroinflammatory microglia and astrocytes in the hippocampal CA1、CA3and DG subregions was conducted by immunohistochemistry.The expression of microglia markers IBA1 and astrocyte marker GFAP were detected by western blotting.The levels of TNF-α、IL-6 and IL-1β in brains were determined by ELISA.Western blotting was employed to assess the expressions of the TLR4/CD14/NF-кB signal pathway.In microglial BV-2cell lines,the CCK8 method was used to detect the cell viability,and the appropriate concentration of ARG was selected.ELISA method was used to identify the effects of different concentrations of ARG on the levels of inflammatory cytokines TNF-α、 IL-1β and IL-6 in LPS-treated BV-2 cells.Furthermore,TLR4/CD14/NF-кB signal activation was detected by immunofluorescence to study the anti-inflammatory and neuroprotective effects of ARG.Results The LPS-treated mice took a significantly longer time to find the hidden platform when compared to the control group during hidden platform tests,whereas the administration of ARG to LPS-treated mice exhibited a significantly lower escape latency(p<0.05)).In the probe trial,the duration in the target quadrant was remarkably reduced by 31.4% when compared to the control group,whereas the administration of ARG to LPS-treated mice spent a significantly longer time in the target quadrant.The nissl bodies were markedly decreased by 41.5% 、 56.5% and 58.4%,respectively,in the hippocampal CA1 、 CA3 and DG subregions of the LPS-treated mice when compared to the control group.Following ARG administration,the nissl bodies were increased by 34.1%、39.1% and 35.9%,respectively,in the hippocampal CA1、CA3 and DG subregion of LPS+ARG group.Compared with the control group,the expressions of cleaved-Caspase3 and cleaved-PARP1 were increased by 64% and 48%,respec tively,in the hippocampus of LPS-treated mice.Compared with the model group,the expressions of cleaved-Caspase3 and cleaved-PARP1 were decreased by 31.8% and 36.7%,respectively,in the hippocampus of the LPS+ARG mice.The content of Aβ dimers and Aβ protein were significantly increased in the hippocampus of the LPS group,whereas the ARG inhibited the deposition of Aβ dimers and Aβ protein in the hippocampus.The expressions of APP and BACE1 were increased by 74.5%and 58.6%,respectively,in the hippocampus of LPS mice when compared to the controls.Compared with the model group,the expressions of APP and BACE1 were decreased by 30.4% and 31.6%,respectively,in the hippocampus of LPS+ARG mice.The synaptic density was reduced by 43%in the hippocampus of LPS mice when compared to the controls.Compared with the model group,the synaptic density was increased by 36.9% in the hippocampus of LPS+ARG mice.The expressions of synaptic markers SYP and PSD95 were decreased by 39% and 41.9%,respectively,in the hippocampus of LPS mice.Compared with the model group,the expressions of SYP and PSD95 were increased by 28.5%、59.9%,respectively,in the hippocampus of LPS+ARG mice.The GFAP-positive astrocytes were significantly increased by 36.5%、48.2% and 51.5%,respectively,and the IBA-1-positive microglia were dramatically elevated by 46.4%、 51.9% and23.5%,respectively,in the hippocampal CA1、CA3 and DG subregions of LPS group when compared to the controls.Compared with LPS group,the GFAP-positive astrocytes were significantly decreased by 50.5% 、 46.3%and 46.1%,respectively,and the IBA-1-positive microglia were significantly reduced by 41.7% 、 55.1% and 39.6%,respectively,in the hippocampal CA1 、 CA3 and DG subregions of LPS+ARG mice.The expression of GFAP and IBA1 in the hippocampus of LPS mice were significantly increased when compared to the control group.Compared with the model group,the expression of GFAP and IBA1 in the hippocampus of LPS+ARG mice were significantly decreased.The levels of TNF-α 、 IL-6and IL-1β were significantly increased in the brains of LPS mice,whereas the administration of ARG to LPS-treated mice exhibited a significant reduction in the contents of TNF-α 、 IL-6 and IL-1β.The expressions The expressions TLR4 、 CD14 、 p-IкBα and p-NF-кB were significantly upregualted in the hippocampus of LPS mice when compared to the controls.Following ARG administration,the expressions TLR4、 CD14、 p-IкBα and p-NF-кB were significantly downregulated.In BV-2 cell lines,ARG(12.5,25 and 50 mol/L)did not affect cell viability.ARG inhibited the release of the LPS-induced inflammatory cytokines TNF-α 、 IL-1β and IL-6 in a dose-dependent manner.In LPS-treated BV-2 cell lines,ARG inhibited the translocation of NF-кB to the nucleus,which further indicated that ARG suppressed LPS-evoked neuroinflammation through the inhibition of NF-кB signaling pathway.Conclusions ARG inhibits the TLR4/NF-кB signaling pathway and neuroinflammation,reduces the formation and aggregation of Aβ,and thereby alleviates LPS-induced synaptic dysfunction and neuronal injury,and ultimately improves neuroinflammation-induced cognitive impairment.It is recommended that ARG can be used as a treatment option for neuroinflammation-related neurological diseases,such as Alzheimer’s disease. |
