Protective Effect And Mechanism Of Water Extract Of Eucommia Ulmoides Oliver On The Neuroinflammation Induced By Lipopolysaccharide In Mice

Multiple studies have shown that neuroinflammation is a key process involved in the degeneration of neurons and is also a factor in neurodegenerative diseases.In recent years,researchers focused their studies on suppressing neuroinflammation to find anti-inflammatory drugs to relieve or treat these diseases.Eucommia ulmoides Oliver,a single species of Eucommia genus belonging to the Eucommiaceae family,has effect of anti-hypotensive,anti-hypoglycemic,antioxidant,anti-inflammatory and neuroprotective effects.There are more and more reports about the therapeutic effect of Eucommia ulmoides on the CNS diseases,and Eucommia ulmoides has been an indispensable and important part of Chinese medicine formulations for the treatment of neurodegenerative diseases.However,there are few reports on whether the water extract of Eucommia ulmoides Oliver(WEE)could prevent and reduce neuroinflammation,improve the cognitive dysfunction,depressive-like behavior and intestinal flora caused by neuroinflammation.In this study,LPS was used to establish inflammation models.In vivo and in vitro experiments were combined to explore the alleviating effect of WEE on LPS-induced inflammation and the potential molecular mechanism,aiming to provide scientific basis for the comprehensive utilization of Eucommia ulmoides and the development of new natural anti-inflammatory drugs.The main investigations and results are as follows:(1)WEE increased the activity of SOD,GSH-Px and the content of MDA in the brain which decreased by LPS,and showed its antioxidant activity.Through water maze test and pole test,we found that WEE improved the cognitive dysfunction induced by LPS in mice.Through open field test and elevated cross maze test,we found that WEE improved LPS-induced depression-like behavior in mice.To investigate the causes of the behavioral changes,we further analyzed the proportion of different spines and synaptic relative proteins in hippocampus.As a result,WEE increased the proportion of mushroom spines and the expression of synaptic proteins such as synaptophysin(SYP),spinophilin(SPN),and postsynaptic dense protein(PSD95)in the hippocampus compared with LPS group,which was in accordance with the improvement of cognitive dysfunction and depressive behaviors in WEE-pretreated mice.(2)LPS significantly induced a decrease in the number of Brd U~+cells and DCX~+cells in subgranular zone(SGZ)and an increase in the number of Brd U~+cells in molecular layer(ML),while compared to LPS group,WEE increased the number of DCX~+cells.Compared with LPS group,WEE decreased the degree of newborn cells differentiating into glial cells and increased the percentage of progenitor cells asymmetric division.According to the morphological analysis of microglia by Sholl and Skeleton/Fractal methods,we found that WEE significantly affected the morphological changes and complexity of microglia induced by LPS in the hippocampus.(3)The densities of astrocytes and microglia in different regions of hippocampus were analyzed by immunofluorescence staining.It was found that WEE significantly decreased the density of GFAP~+cells in CA1,CA3,ML and hilus,and the protein levels of GFAP compared with LPS group.Similarly,WEE obviously decreased the densities of Iba1~+cells in CA1,CA3and hilus of hippocampus compared with LPS group.Moreover,WEE significantly inhibited the m RNA levels of IL-1β,IL-6,IL-1ra,i NOS and Arg-1,and reduced the m RNA levels of MMP9,MCP-1 and m PPARγinduced by LPS.Through western blot,WEE played a neuroprotective role by inhibiting the protein levels of NFκB,p38 MAPK pathway,apoptosis and endoplasmic reticulum stress related proteins activated by LPS.(4)Immunofluorescence staining and western blot were performed to analyze the number and protein levels of microglia and astrocytes in cortex,the results showed that density of GFAP~+and Iba1~+cells and levels of relative proteins were obviously decreased by WEE compared with LPS group.The expression of inflammatory factors in the cortex was detected by q PCR,and the results showed that the m RNA levels of TNF-α,IL-1β,IL-6,IL-10 and IL-1ra were significantly increased in LPS group,and markedly inhibited by WEE.Moreover,WEE played a neuroprotective role by inhibiting the expression of LPS-activated NFκB,p38MAPK pathways and apoptotic related proteins.(5)Through 16S rRNA sequencing analysis,WEE protected the intestinal tract of mice by regulating the structural composition of intestinal flora damaged by LPS.Compared with LPS group,WEE significantly inhibited m RNA levels of TNF-α,IL-1β,IL-6,IL-10,,IL-1ra and Occludin induced by LPS,and increased the expression of tight junction proteins(ZO-1and Occludin),thus alleviating intestinal inflammation.(6)It was found that WEE increased the survival rate of BV2 cells,decreased the degree of differentiation and intracellular ROS content compared with LPS group.With the increase of WEE concentration,the expression degree of inhibiting inflammation-related factors induced by LPS also increased,showing a certain concentration dependence.Western blot results showed that WEE inhibited the expression levels of NFκB,p38 MAPK pathway and apoptotic related proteins in a concentration dependent manner.In conclusion,WEE improved the cognitive dysfunction and depressive behaviors induced by LPS,showing a protective effect on LPS-induced adult neurogenesis in the hippocampus.And WEE played a neuroprotective role by inhibiting the expression of inflammation-related factors,TLR4,NFκB,p38MAPK signaling pathways,apoptosis and endoplasmic reticulum stress relative proteins activated by LPS.