| BackgroundFo shou san(FSS)is a chinese herbal formula,which has been reported to exert effective protection on blood deficiency,blood stasis,stroke and ischemic cerebral vesscular disease.A growing body of evidence also indicates that the gut-liver-brain axis is closely related to neurological diseases,including Alzheimer’ s disease(AD).However,whether it have protective effects on Alzheimer’ s disease(AD)and its mechanism is unknown.ObjectiveTo study the effects of different doses of FSS in APP/PS1 Alzheimer’s disease(AD)mice,and to explore whether FSS can improve the learning and memory ability of AD mice by regulating gut-liver-brain axis to regulating gut bacteria,alkaline phosphatase(AP)and attenuating lipopolysaccharides(LPS)and lipid peroxidation(Malondialdehyde,MDA).Methods1.Animal part:In this study,we used the classical APP/PS1 double-transgenic AD mouse model to explore the neuroprotective effects and related mechanisms of ancient herbal formula(FSS).Six-month-old male APP/PS1 mice were randomly divided into model group(APP/PS1 mice),High-,medium-,and low-dose FSS groups,age matched C57BL/6J mice were as Wild type(WT)group.The dose of FSS in the high-,medium-,and low-dose groups were 6.4g/kg/d,3.2g/kg/d and 1.6g/kg/d,respectively.Positive control group was given 2mg/kg/d donepezil,WT group and AD group were given an equal volume of ultra pure water.All the treatments were administered intragastrically once a day for 8 weeks.After the administration,Morris water maze,Y maze and Novel object recognition test were used to detect the cognitive function.2.Biochemical part:Hematoxylin-eosin staining were used to assess neuronal damage in the Hippocampus,immunohistochemistry,western blotting,chromogenic end-point Tachypleus amebocyte lysate(TAL)assay kit,Malondialdehyde(MDA)assay kit,AP assay Kit were used to assess the level of LPS,MDA,AP in the gut,liver,serum,brain and real-time quantitative PCR(qPCR)were used to assess the gut bacteria.Results1.Effect of FoShouSan on learning and memory ability of APP/PS1 mice(1)Morris water maze test results:compared with the normal group,the escape latency of the model mice was significantly prolonged(P<0.05),while the medium-dose,high-dose FoShouSan group and donepezil treatment group significantly shortened the escape latency(P<0.05).During the space exploration period,there was no significant difference in the average speed of parade between the groups,but the number of mice crossing the platform in the model group was significantly lower than that in the normal group(P<0.05),and the time stay in the III target quadrant of the model group in the target quadrant was significantly lower than that in the normal group(P<0.05).P<0.05).After 8 weeks of FSS treatment,both the high-dose group and the positive control group significantly increased the number of crossing platforms,and also significantly prolonged the time stay in the III target quadrant(P<0.05 or P<0.01),and the medium-dose group significantly extended the time stay in the III target quadrant(P<0.01),the low-dose group also significantly increased the number of crossing the platform(P<0.05).(2)Y-maze results showed that after 8 weeks of administration of FSH,the percentage of three-armed alternation in the model group was significantly lower than that in the normal group(P<0.01),and the drug-administered group improved the alternation rate of the model group(P<0.05),especially in the high-dose group and the middle-dose group(P<0.01).(3)The novel object recognition test results suggest that after FSS treatment,the preferential index was significantly raised than in AD mice,but only in the high-dose group(FSS-H)have a significantly increased(P<0.05)·(4)In summary,we found that after 8 weeks of administration of FSS,the learning and memory function of the APP/PS1 model group(AD)was effectively improved,especially in the high-dose and medium-dose groups.2.Effects of FoShouSan on Biochemical Indexes of APP/PS1 AD Mice(1)The results of HE staining showed that most neurons were lost,shrinkage,dark-stained,peripheral no staining and with severe distortion of neuron fibers in the CA1 areas of the hippocampus in AD mice compared with control WT mice.After FSS treatment,apart from the positive control mice(Donepezil),and also the neuronal damage of the medium-dose and high-dose treated mice(FSS-H,FSS-M)were obviously attenuated,and there still have significant neuronal damage in the low-dose treated mice(FSS-L).(2)the immunohistochemistry result showed that the mean area covered by Aβ 1-42 plaques and LPS in the hippocampus of AD mice were both significantly higher than in the control WT mice(P<0.05).But the mean area covered by A β 1-42 plaques and LPS were significantly lower after High-FSS or medium-FSS treatment.In addition,the integrated optical density(IOD)of LPS were showed a significant positive correlation with the IOD of Aβ 1-42(r=0.36,P<0.0001)(3)The test of the quantitative Chromogenic End-point Tachypleus amebocyte lysate(TAL)assay kit(Xiamen Houshiji,China)showed that the levels of LPS were significantly higher in the fecal,liver and serum of AD mice compared to the control WT mice.But the levels of LPS in the liver,blood and feces were significantly lower after High-FSS or medium-FSS treatment.(4)Western blot tested the Alkaline phosphatase(AP)activity of the brain,and Alkaline Phosphatase Assay Kit were to test AP activity of gut,liver and serum.The result showed that AP activity is significantly increased in the brain and serum,but significantly reduced in the gut and liver in the AD mice.After FSS treatment,the AP activity in the brain and serum of FSS-H mice,and in the serum of FSS-M mice,were obviously reduced compared to AD mice.In the opposite,the AP activity in the gut and liver of FSS-H mice and FSS-M mice were increased compared to AD mice.(5)Malondialdehyde(MDA)assay kits result showed the levels of MDA were significantly higher in gut,liver,serum and brain in AD mice than in control WT mice.But decreased sharply after 8-week oral administration of FSS compared with the AD mice.Except in the FSS-M mice of the gut,the levels of MDA of FSS-H mice and FSS-M mice in Liver,serum and brain were all obviously decreased.But the levels of MDA of FSS-L mice were only obviously decreased in brain,in the other tissues were slightly decreased.(6)real-time PCR(q PCR)result showed the amount of Lactobacillus were decreased in AD mice compared to control WT mice,and rise after High-FSS or medium-FSS treatment,However,the amount of E.coli was sharply increased in AD mice compared to control WT mice,and fell after High-FSS or medium-FSS treatment.In addition,the Escherichia coli(E.coli)and Lactobacillus are interactive,they have a negative correlation in amount(r=0.3318,P<0.0001).(7)In summary,medium or high doses of FSS(FSS-H,6.4g/kg/d,FSS-M,3.2g/kg/d)could ameliorate learning and memory impairments,inhibit lipid peroxidation,decrease levels of LPS,regulate AP activity,and regulate gut bacteria populationsConclusionsWe found that gut bacteria,lipopolysaccharide(LPS)levels,lipid peroxidation levels,and alkaline phosphatase(AP)activity levels have changed in the APP/PS1 double transgenic AD mouse model;FSS could regulate gut-liver-brain axis to regulate AP and gut bacteria,and attenuate the LPS-related systemic inflammation and lipid peroxidation(MDA),and thereby AD-related pathology in APP/PS1 AD mice.Therefore,the mechanism of FSS cure AD mice may be related to the regulation of intestinal-liver-brain axis,and FSS is likely to be an effective clinical prescription for the treatment of Alzheimer’ s disease in the future. |
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