Effects Of Maternal Vitamin A Deficiency On The Development Of Enteric Nervous System In Offspring

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MATERNAL VITAMIN A DEFICIENCY DURING PREGNANCY IMPAIRS THE DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM AND GASTROINTESTINAL MOTILITY IN THE OFFSPRINGObjective: To investigate the effect of maternal vitamin A deficiency on the enteric nervous system(ENS)and gastrointestinal function of offspring and explore the possible signaling pathways involved.Methods: Three-week-old SD female rats were selected to construct animal models of normal VA(VAN,> 1.05 ?mol/L),VA deficiency(VAD,<0.7 ?mol/L),and VA supplementation(VAS,intragastric supplementation of VA for 3.5 days of pregnancy).Serum VA levels were detected by HPLC.Immunofluorescence staining was used to observe TUJ1 migration in the embryonic gut of rats on day E12.5 and day E14.5.The protein expression levels of retinoic acid nucleus receptors(RARs),PGP9.5,S100?,Ret and SOX10 were assessed by western blot.The fecal water content,total gastrointestinal transmission time and colonic motility were measured to evaluate gastrointestinal function when the offspring of the three groups were reached the age of 8 weeks.Western blotting was used to detect the expression levels of RARs,SOX10,cholinergic(ChAT)and nitrergic(nNOS)enteric neurons in colon tissues.The colonic myenteric plexus was isolated,and ChAT and nNOS were stained with immunofluorescence to observe the distribution of cholinergic and nitrergic enteric neurons.Results:(1)Immunofluorescence staining on day E12.5 and day E14.5 showed that the migration rate of early intestinal neuron TUJ1 in the fetal gut of the VAD group was slower than that of the VAN group,and the VAS group was faster than that of the VAD group,with no significant difference from the VAN group.(2)The results of RARs protein expression levels in the fetal intestines of the three groups showed that there was no difference in the expression level of RAR? between the three groups(P > 0.05).However,the expression levels of RAR? and RAR?protein in the VAD group were lower than those in the VAN group(P >0.05),while those in the VAS group were higher than those in the VAD group(P < 0.05),indicating that the VA level during maternal pregnancy affects the expression levels of RAR? and RAR? in the fetal intestine of the offspring.(3)The expression of PGP9.5 and S100? in the fetal gut in the VAD group was significantly lower than that in the VAN group(P<0.05),and the protein expression levels of SOX10 and Ret genes were also significantly lower than those in the VAN group(P <0.05).It is worth noting that the expression levels of PGP9.5,S100?,SOX10 and Ret in the VAS group were significantly higher than those in the VAD group(P<0.05),and there was no difference from the VAN group(P > 0.05),indicating that the VA level during pregnancy affected the expression levels of PGP9.5,S100?,SOX10 and Ret in the offspring.(4)The gastrointestinal function of three groups of 8-week-old mice was evaluated,and the results showed that compared with the VAN group,the fecal water content of rat offspring in the VAD group was increased(P < 0.001),the total gastrointestinal transmission time was prolonged(P < 0.01),and colon motility was slowed(P < 0.001).Compared with the VAD group,the VAS group had lower fecal water content(P <0.001),shorter gastrointestinal transmission time(P < 0.01),and faster colon motility(P< 0.001).The difference between the VAN and VAS groups was not statistically significant(P>0.05),suggesting that the VA level affected the gastrointestinal function of adult offspring rats,especially gastrointestinal motility.(5)In adult rats,the expression levels of ChAT and nNOS proteins in colon tissue of the VAD group were lower than those of the VAN group(P <0.05),while that of the VAS group was higher than that of the VAD group(P <0.05).There was no significant difference between the VAN group and VAS group(P > 0.05).Immunofluorescence staining of the myenteric plexus of the colon showed that ChAT and nNOS enteric neurons in the VAD group were significantly decreased compared with the VAN group(P < 0.05),and they were significantly increased in the VAS group compared with the VAD group(P < 0.05).There was no statistically significant difference between the VAN and VAS groups(P > 0.05),suggesting that VA levels during pregnancy affect the number of cholinergic and nitrogenous neurons in the colon of young rats.Conclusion: Maternal VAD slowed the migration of intestinal neurons in fetal rats,reduced the number of cholinergic and nitrogenousneurons in the colon,and damaged gastrointestinal function in offspring.Maternal VA supplementation during pregnancy can improve the adverse effects of pregnancy VAD on the development of ENS in offspring.PART ? RETINOIC ACID AFFECTS THE PROLIFERATION OF ENTERIC NEURAL STEM PROGENITOR CELLS AND THE DIFFERENTIATION OF SUBTYPE ENTERIC NEURONS THROUGH RAR?Objective: To investigate the effects of retinoic acid(RA),the active metabolite of VA,on the proliferation of enteric nerve stem progenitor cells and the differentiation of subtypes of enteric neurons and the possible molecular mechanisms involved.Methods: Primary enteric neurospheres were isolated from day E14.5 fetal intestines by mechanical separation and enzymatic digestion.The expression of stem/progenitor cells(Nestin/P75NGF),neurons(TUJ1)and glial cells(S100?)in enteric neurospheres was detected by double immunofluorescence staining.Enteric neurospheres were treated with five different concentrations(0 ?mol/L,0.5 ?mol/L,1.0 ?mol/L,2 ?mol/L,and4 ?mol/L)of RA.The m RNA and protein expression levels of RARs,SOX10,P75 NGF,Nestin,Ch AT and n NOS in the cells were detected by real-time PCR and western blotting after 3 days of proliferation culture and 7 days of differentiation culture after RA treatment.Immunofluorescence staining was used to detect the expression of P75 NGF and Nestin in enteric neurospheres under proliferative culture and the expression of Ch AT and n NOS under differentiation culture.After infection with Ad-RAR? and si RAR? adenovirus,real-time PCR and western blot analysis of the changes in RAR?,SOX10,P75 NGF,Nestin,Ch AT and n NOS in enteric neurospheres was performed to explore the effect of RAR? expression changes on enterosphere proliferation and differentiation and to determine whether RAR? affects SOX10 expression.Results:(1)Enteric neurospheres were successfully isolated and cultured from fetal intestinal tissue.(2)Double-labeled immunofluorescence staining revealed that enteric neurospheres simultaneously expressed Nestin,P75 NGF,TUJ1,and S100?,confirming that the cells obtained in the experiment were enteric neurospheres.(3)Under proliferation culture for 3 days,compared with the RA 0 ?mol/L group,the levels of RAR b and SOX10 m RNA expression were significantly upregulated,the levels of P75 NGF and Nestin m RNA expression were significantly downregulated following the other four RA treatments,and the RA concentration of 1 ?mol/L was significantly different(P(27)0.05).The changes in the expression of these proteins were completely consistent with the levels of their m RNA expression.However,RA treatment did not change the expression levels of RARa and RARg in the enteric neurospheres.Differentiation culture for 7 days,real-time PCR and western blot results showed that compared with the 0 ?mol/L group,the expression levels of RAR?,SOX10,Ch AT and n NOS were significantly increased,and the effect was most significant with 1 ?mol/L RA(P <0.05).Immunofluorescence staining results showed that RA treatment reduced the expression of P75 NGF and Nestin in enteric neurospheres under proliferative culture(P < 0.05)but increased the number of Ch AT and n NOS neurons under differentiation culture(P <0.05),and the effect was most significant with 1 ?mol/L RA(P < 0.001).(4)The recombinant si-RAR? adenovirus infection significantly decreased the levels of RAR? m RNA and protein expression in the enteric neurospheres compared to the control group(P(27)0.001),and the expression levels of SOX10 were also obviously decreased(P(27)0.01),and the protein expression of Ch AT(P < 0.01)and n NOS(P < 0.05)was also significantly downregulated after 7 days of differentiation culture.However,the protein expressions of P75 NGF and Nestin increased significantly(P < 0.01).Meanwhile,the levels of RAR? and SOX10 expression were significantly increased in the enteric neurospheres compared to the control group when RAR? expression was overactivated by the Ad-RAR? adenovirus(P(27)0.01),and the protein expression of Ch AT and n NOS was also significantly upregulated after 7 days of differentiation culture(P < 0.05).However,the protein expressions of P75 NGF and Nestin decreased significantly(P < 0.01).These data further indicate that the expression level of RAR? gene affects the expression of SOX10,P75 NGF,Nestin,Ch AT and n NOS in the enteric neurospheres.Conclusion: RAR? affects SOX10 expression,and RA affects the proliferation of stem cells and progenitor cells and the differentiation of cholinergic and nitrogen energy neurons in enteric neurospheres through RAR?.