Neuroprotective Effect Of Fucoidan On Alcohol-exposed Mice And Its Mechanism

Objective:Alcohol usedisorder(AUD)is a serious problem affecting public health today,characterized by the inability to control alcohol and serious impairment of social functions.Excessive drinking can lead to neuroinflammation,cognitive decline,irritability,depression and motor dysfunction,which is closely related to intestinal flora disorder and intestinal mucosal barrier damage caused by long-term alcohol exposure.Through the two-way transmission between the gut and the central nervous system,the microorganisms in the intestinal tract can extend its influence to the brain and then regulate the physiological and pathological processes,and finally form the intestinal-microbial-brain axis in the central nervous system.Fucoidan is a natural active substance extracted from seaweed,which has antioxidant,anti-inflammatory and intestinal microecological activities.In previous studies,it was found that fucoidan can improve intestinal mucosal injury by changing the composition of intestinal microbiota,thus improving alcoholic liver injury in mice.However,it is rarely reported whether fucoidan can improve the brain damage induced by alcohol exposure through the intestinal-microbial-brain axis.Therefore,this study will investigate whether fucoidan can reduce neuroinflammation and improve depression-like behavior in alcohol-dependent mice by regulating intestinal microecology,enhancing intestinal mucosal barrier function.Methods:1.Alcohol-dependent mouse model construction and intervention scheme: Forty-five8-week-old male C57BL/6J mice were fed in cage for 3 weeks,which made intestinal flora composition more uniform.Before the intervention,according to the results of sucrose preference experiment,the mice were randomly divided into three groups(fifteen mice per group),Control,Model and Fucoidan groups.In the whole intervention experiment,the mice in the model group and fucoidan intervention group drank tap water freely from 8 to 16:00 every Monday to Friday,and freely drank 15% ethanol solution(v/v)the rest of the time.All mice in the control group were free to drink tap water during this period.All mice were given up on Saturday and all mice were free to drink tap water on Sunday.During the intervention period,the mice in the control group and model group were given 0.2 m L normal saline at 12:00 everyday,and the mice in the fucoidan intervention group were given 300 mg/kg fucoidan solution.The intervention experiment lasted for 10 weeks,and all mice eat freely.2.Behavioral experiments were used to detect the depression-like behavior of mice in each group,including sucrose preference test,forced swim test,open field test and Y-maze test.3.The levels of 5-hydroxytryptamine(5-HT),brain derived neurotrophic factor(BDNF)and nerve growth factor(NGF)in serum and brain tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA).ELISA method was used to detect the levels of lipopolysaccharide(LPS)in serum and interleukin-1β(IL-1β),IL-6,IL-10,tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β)in brain tissues.4.Western blot was used to detect the expressions of Iba-1 and CD68,the markers of microglia cells and activated microglia cells respectively,and the related proteins of TLR4/MyD88/NF-κB p65 signal pathway in hippocampal tissues of mice.5.The expressions of Iba-1,CD68 and p-NF-κB p65 in the hippocampus of mice were observed by immunofluorescence assay.The expression levels of tight junction proteins Claudin-2 and ZO-1 in ileum were observed.6.16S rDNA gene sequencing technology was used to analyze the changes of intestinal microbiota in mice.7.Spearman correlation analysis was used to analyze the correlation between serological indicators and species.The correlation between encephalitis factors and hippocampal protein expression and species was analyzed by Pearson correlation analysis.Results:1.After 10 weeks of alcohol exposure,the decrease of food intake(1.71±0.70 g)and body weight(24.47±2.17 g)in model group were significantly lower than those in control group(3.15±0.52 g and 27.68±2.21 g,respectively)(P<0.01).The food intake of mice in the intervention group(2.65±0.93 g)was higher than that in the model group(P<0.05).2.Compared with the control group,after 10 weeks of alcohol exposure,the preference for sweet solution in the model group decreased by 24.18%(P<0.05).In the forced swim test,the resting time increased significantly by 78.01%(P<0.01),and in the Y maze test,the time to enter the new odd arm decreased by 66.98%(P<0.01).In the open field test,the times of entering the center and staying time were significantly reduced,and the total exercise distance was also significantly different(P<0.05).Compared with the model group,the depression-like behavior test results of mice treated with fucoidan were significantly improved(P<0.05).3.Compared with model group,the serum levels of IL-1β and TNF-α were decreased by 40.96% and 49.02%,respectively,and the levels of TGF-β and IL-10 were increased by 33.16% and 88.27%,respectively(P<0.05 or P<0.01).The levels of serum NGF,BDNF and 5-HT in the model group were significantly lower than those in the control group(P<0.05 or P<0.01).After fucoidan intervention,the levels of BDNF and5-HT increased by 40.85% and 48.29%(P<0.05),respectively.In addition,fucoidan significantly reduced the level of serum LPS in alcohol-exposed mice by 19.79%(P<0.05).Compared with the control group,the changes of NGF,BDNF and 5-HT levels in the brain tissue of mice in the model group were similar to those in the serum,and the levels of BDNF and 5-HT were decreased by 49.70% and 39.33%,respectively,after the intervention of fucoidan(P<0.05).4.Western blot showed that compared with the control group,the expression of Iba-1 in hippocampal tissue of model group had no significant change,the expression of CD68 was significantly increased(P<0.01),and the levels of TLR4,MyD88 and p-NF-κB p65 protein were significantly increased(P<0.05).The expressions of tight junction proteins Claudin-2 and ZO-1 in ileum were significantly decreased(P<0.01).After the intervention of fucoidan,the decrease of protein expression caused by alcohol exposure was significantly reversed.5.Immunofluorescence assay revealed that there was no significant change in the number of microglia in the hippocampus of the three groups(P<0.05).Compared with the control group,the expression of CD68 protein in the hippocampus of the model group was significantly increased,indicating that alcohol exposure promoted the activation of microglia,and the staining of NF-κB p65 protein was enhanced,indicating that alcohol exposure upregulated TLR4/MyD88/NF-κB p65 pathway.Fucoidan could reverse the effect of alcohol on microglia cell activation and downregulate signal pathway.In addition,the protein staining of Claudin-2 and ZO-1 in ileal tissue was weakened,and the results were significantly improved after the intervention of fucoidan(P<0.05).6.The results of 16S rDNA sequencing showed that there were differences in the diversity and structure of intestinal flora among the three groups.Compared with normal controls,alcohol exposure increased the abundances of Bacteroides and Akkermansiareduced,and the abundances of Prevotella,Alloprevotella,Barnesiella and Alistipes were reduced(P<0.05 or P<0.01).The composition of intestinal microflora was more similar to that of normal control mice,and the abundance of Prevotella and Alloprevotella increased(P<0.05).7.Spearman correlation analysis showed that the abundances of Alloprevotella,Prevotella,Alistipes and Mucispirillum were positively correlated with serum BDNF levels.The abundance of Clostridium ⅩVIII,Blautia,Anaerostipes and Akkermansia were negatively correlated with serum BDNF levels.Prevotella and Barnella abundance was positively correlated with serum 5-HT levels,while Brautella abundance was negatively correlated with serum 5-HT levels.Pearson correlation analysis showed that Prevotella abundance was negatively correlated with IL-1β levels.The abundance of Alistipes was negatively correlated with the expression levels of TNF-α,IL-6,IL-1β in brain and CD68 in hippocampus.Conclusions:1.The oral administration of fucoidan can reduce the level of LPS and down-regulate the TLR4/MyD88/NF-κB p65 pathway,inhibit alcohol-induced inflammatory response,and play a neuroprotective role in alcohol-exposed mice,thereby improving the depression-like behavior of mice.2.The neuroprotective mechanism of fucoidan on alcohol-exposed mice may be related to the improvement of intestinal mucosal barrier function,the restoration of intestinal microbial species balance and the regulation of intestinal-microbial-brain axis.