The Regulatory Effect Of Low-fiber Diet During Pregnancy On Neural Memory In Offspring And Its Possible Mechanism

PARTⅠ MODULATION OF LOW-FIBER DIET DURING PREGNANCY ON GUT MICROBIOTA AND OFFSPRING NEURAL MEMORYBackground: Studies have found that dietary fiber intake is closely related to the development of cognitive and memory.However,in recent years,the consumption of dietary fiber in the diet,including the consumption of dietary fiber during pregnancy,has decreased significantly,which has caused us obvious concerns about the development of intrauterine fetal cognitive memory.Objectives:(1)To explore the effects of low-fiber diet and normal fiber diet during pregnancy on synaptic plasticity and cognitive memory in offspring mice.(2)To analyze the effects of different fiber diets during pregnancy on the composition of gut microbiota.Methods:(1)According to AIN-93 G standard of American Academy of Nutrition,low-fiber diet feed and normal fiber diet feed were prepared.(2)Immediately after pregnancy,female mice were randomly divided into low-fiber diet and normal-fiber diet,and this feeding method continued throughout the pregnancy until the pregnant mice gave birth.After the calving of pregnant mice,the animals in the two groups were changed to normal fiber feed until the end of the experiment.(3)Collecting fecal samples from pregnant mice in the third trimester fed with different fiber diets,and analyzing the effect of different fiber diets on the diversity of gut microbes;collecting fecal samples from offspring mice when they are weaned,and analyzing the diversity of gut microbial from offspring mice.(4)Complete behavioral tests at the age of six weeks,including open field experiment,elevated plus maze experiment and Barnes experiment.(5)Long-term potentiation experiments and synaptic plasticity proteins(NR2A,NR2 B and GLUR2)were detected at the age of six weeks.Results: Compared with normal fiber diet during pregnancy,(1)low fiber diet during pregnancy did not affect the weight gain of mother and offspring mice.(2)The offspring of the low-fiber diet during pregnancy had less exercise time in the central area and total distance in the open field experiment;the open arm exercise time was less,but the number and proportion of entering the open arm had no different significant change in the elevated plus maze test;longer time to find target holes and more errors in finding non-target holes in the Barnes test.(3)The f EPSP potentials of offspring mice fed with low-fiber diet during pregnancy were lower after high-frequency stimulation,and the expressions of synaptic plasticity proteins NR2 B and GLUR2 were significantly reduced,but the changes of NR2 A were not obvious.(4)The detection of gut microbial diversity found that low-fiber diet during pregnancy significantly reduced the bacterial abundance of Bacteroidetes in the gut of female mice,and increased the bacterial abundance of Firmicutes,and this change trend also exists in the detection of the diversity of intestinal microbes in offspring mice(but there is no significant difference).Conclusion: Compared with the normal fiber diet during pregnancy,the low-fiber diet during pregnancy resulted in decreased expression of synaptic plasticity proteins in the hippocampus of offspring,impaired synaptic plasticity,and impaired spatial memory in offspring.In addition,different fiber diets during pregnancy resulted in significant changes in the abundance of Bacteroidetes and Firmicutes in the gut of female mice,suggesting that gut microbiota may be involved in the regulation of offspring memory function by fiber diets during pregnancy.PARTⅡ THE PENETRATION EFFECT OF PLACENTA ON SCFAS AND THE REGULATORY EFFECT OF SCFAS SUPPLEMENTATION DURING PREGNANCY ON THE MEMORY FUNCTION OF OFFSPRINGBackground: In the partⅠof the study,we found that a low-fiber diet during pregnancy led to significant memory impairment in offspring mice.And changes in the microorganisms associated with the production of SCFAs were found in the gut of female mice,suggesting that SCFAs may be involved in the regulation of cellulose in offspring memory during pregnancy.In addition,the growth and development of the fetus in utero requires the delivery of nutrients from the placenta,but whether the placenta allows SCFAs to permeation and the efficiency of permeation remain unclear.Objectives:(1)To study the permeation effect of placenta on different SCFAs.(2)To observe whether SCFAs supplementation during pregnancy can reverse the memory impairment of offspring mice caused by low-fiber diet during pregnancy.Methods:(1)A total of 31 normal participants in the third trimester were enrolled in the study.Maternal venous blood and matched umbilical cord venous blood were collected during delivery,and serum SCFAs concentrations were detected.The informed consent was obtained and signed;and this study was reviewed by the unit ethics committee before the start of the study.(2)The serum of maternal mice and fetal brain tissue in the third trimester of pregnancy with different fiber diets were collected to detect the concentration of SCFAs.(3)On the basis of low-fiber diet during pregnancy,behavioral tests of mice supplemented with propionate or butyrate,respectively,were completed at the age of six weeks,including open field experiment,elevated plus maze experiment,novel object recognition and Barnes experiment.(4)On the basis of normal-fiber diet during pregnancy,behavioral tests of mice supplemented with propionate or butyrate,respectively,were completed at the age of six weeks.(5)Long-term potentiation experiments and synaptic plasticity proteins(NR2A,NR2 B and GLUR2)were detected at the age of six weeks.Results:(1)By the analysis of population sample data,it was found that the concentrations of propionate,butyrate,caproate and valerate in cord blood serum were significantly linearly correlated with the concentrations in the maternal serum.The Pearson correlation coefficients of propionate,butyrate,caproate and valerate were 0.425(p =0.017),0.453(p = 0.011),0.502(p = 0.004),and 0.501(p = 0.004),respectively;however,the concentrations of acetate,isobutyrate,and isovalerate in cord blood were no significant correlation with the concentrations in the maternal serum,and the Pearson correlation coefficients were 0.182(p = 0.327),0.061(p = 0.745)and 0.294(p =0.108),respectively.(2)The analysis of animal serum SCFAs showed that the propionate and butyrate levels of maternal serum fed with low-fiber diet during pregnancy were significantly reduced,while the concentrations of acetate,valerate,caproate,isobutyrate and isovalerate were not significantly different between the two groups.In the detection of SCFAs in brain tissue of offspring mice at birth,the concentrations of propionate and butyrate in brain tissue of offspring mice fed with low-fiber diet during pregnancy were significantly lower,while the concentrations of acetate,valerate,caproate,isobutyrate and isovalerate were no significant change;however,in the detection of SCFAs in the brain tissue of offspring mice at the age of six weeks,there was no significant difference in the concentrations of all SCFAs between the two groups.(3)Compared with the offspring on a low-fiber diet during pregnancy,the total exercise distance and central area exercise time of the offspring with butyrate supplementation during pregnancy did not change significantly in open field experiment;the open arm entry times,total movement distance and movement time increased significantly in elevated plus maze test;the contact time with unfamiliar objects increased significantly in the new object recognition experiment;the time to find the target hole was significantly shortened,and number of errors in finding non-target holes was significantly reduced in the Barnes test.There was no significant change in the results of the above-mentioned tests in the mice supplemented with propionate.(4)Compared with the offspring on a normal fiber diet during pregnancy,the results of the offspring with propionate or butyrate supplementation during pregnancy did not change significantly in the above test.(5)Compared with the offspring on a low-fiber diet during pregnancy,the offspring with butyrate supplementation during pregnancy had higher f EPSP potential after high-frequency stimulation,and significantly increased the expressions of synaptic plasticity proteins NR2 B and GLUR2,but not significantly increased NR2A;However,the results of the above-mentioned tests were not significantly changed in the offspring mice with propionate supplementation during pregnancy.Conclusion: Serum SCFAs can pass through the maternal placental barrier.And propionate,butyrate,caproate and valerate in maternal serum can proportionally cross the placental barrier to reach the fetus,and the results are also mostly verified by the detection of animal specimens.In addition,using SCFAs supplementation experiments,it was found that only butyrate supplementation,but not propionate supplementation,could reverse the memory loss of offspring mice caused by low-fiber diet during pregnancy.PARTⅢ THE ROLE AND MECHANISM OF HDAC4 PATHWAY IN BUTYRATE REGULATING NEURONAL MEMORY IN OFFSPRINGBackground: Butyrate is a histone deacetylase(HDAC)inhibitor,which can promote gene transcription by inhibiting the activity of HDAC.Many studies reported that butyrate plays an important role in regulating nerve function repair through HDAC pathway.However,the regulatory effect and specific mechanism of butyrate on neural memory during development still need to be further studied.Objective: To observe the regulatory effect of butyrate on memory-related synaptic plasticity in neurons and the specific mechanism.Methods:(1)The hippocampal neurons of fetal mice were cultured,and the neurons were intervened with different concentrations of butyrate to observe the changes of synaptic plasticity proteins.(2)The protein expression changes of HDAC1,HDAC2,HDAC3,HDAC4 and FFAR3 in neurons were detected after butyrate intervention.(3)The m RNA expression changes of all HDAC subtypes(from HDAC1 to HDAC10) and FFAR2 and FFAR3 were detected after butyrate intervened in neurons.(4)The changes of HDAC1,HDAC2,HDAC3,HDAC4 and FFAR3 proteins were detected by animal brain tissue.(5)After using si RNA to interfere with nuclear HDAC4 protein,the expression changes of synaptic plasticity proteins(NR2A,NR2 B and GLUR2)were detected.(6)After si-HDAC4 and butyrate intervention,the changes of plasticity-related proteins were detected.Results:(1)With the increase of butyrate concentration from 0 to0.5m M,the expressions of synaptic plasticity proteins NR2 B and GLUR2 gradually increased,and the NR2 A protein also slightly increased.With the increase of butyrate concentration from 0.5 m M to 2 m M,the protein expressions of GLUR2 and NR2 B gradually decreased,and the expression of NR2 A protein also slightly decreased.(2)The expression of HDAC4 in neurons treated with butyrate was significantly decreased,while the expressions of HDAC1,HDAC2,HDAC3 and FFAR3 proteins did not change significantly.(3)At the m RNA level,the m RNA level of HDAC4 was significantly decreased after butyrate intervention,while the m RNA levels of FFAR3,FFAR2,HDAC1,HDAC2,HDAC3,HDAC5,HDAC6,HDAC7,HDAC8,HDAC9 and HDAC10 did not change significantly.(4)The detection of animal specimens also found that butyrate supplementation significantly reduced the expression of HDAC4 protein,but had no significant effect on the expression of FFAR3,HDAC1,HDAC2 and HDAC3 proteins.(5)Silencing of si-HDAC4 significantly increased the expressions of synaptic plastic proteins GLUR2 and NR2B;however,the expressions of synaptic plastic proteins NR2 B and GLUR2 did not change further after si-HDAC4 and butyrate intervention.Conclusion: From the cellular level,the use of si-HDAC4 to interfere with the expression of target genes can induce the effect of butyrate on neurons,and the interference of si-HDAC4 can block the regulatory effect of butyrate on neuronal synaptic plasticity,proving that butyrate can regulate the plasticity of neural synapses by regulating the expression of HDAC4 in the nucleus.