The Effects Of Vitamin A Nutritional Level On The Autistic-like Behavior Of Rats Induced By Neonatal Maternal Separation

PART ? THE EFFECTS OF VAD ON THE AUTISM-LIKE BEHAVIOR OF NEONATAL MATERNAL SEPARATION RATSObjective: To investigate the effects of VAD beginning from pregnancy on the autism-like behavior of rats and the expressions levels of RAR? in the hypothalamus,autism associated proteins CD38 and OXT.Methods: Rat's models of pregnancy VAN or VAD were established.Their offsprings were randomly divided with or without neonatal maternal separation(NMS)to make the four groups of VAN,VAD,VAN-NMS and VAD-NMS.At 6-week-old,the offsprings rats were detected by open field test,social interaction test,elevated plus maze and forced swimming to determine their behavior.The serum retinol levels were tested by high performance liquid chromatography(HPLC).Western blot was used to detect the expression levels of RAR? and CD38 proteins,and Enzyme-linked immune sorbent assay(ELISA)to the serum concentrition of OXT.Results: ? The serum retinol concentrations of the VAN or VAN-NMS groups were significantly higher than those of the VAD or VAD-NMS groups(P<0.001,P<0.001),which were met the experimental requirements.? Compared with those of the VAN group,rats of the VAD group spent more time in self-grooming(P<0.001),more time in object chamber(P<0.01)while less time in rat chamber(P<0.001)and open arm(P<0.05),and much more defecation(P<0.01).What's more,the first time to immobility and the total time in struggle were also decreased,whereas the total time in immobility was increased(P<0.001).These results suggested that VAD could increase repeat stereotyped behavior,social intercourse disorder,anxirty and despair of rats.? Compared with those of the VAN group,rats of the VAN-NMS group spent more time in self-grooming(P<0.05),more time in object chamber(P<0.05)while less time in rat chamber(P<0.05)and open arm(P<0.05),and much more defecation(P<0.05).Moreover,the first time to immobility and the total time in struggle were also decreased(P<0.001),whereas the total time in immobility was increased(P<0.01).The above data indicated that the NMS induced the autism-like behavior of rats.? Compared with those of the VAN-NMS group,rats of the VAD-NMS group spent more time in self-grooming(P<0.001),more time in object chamber(P<0.001)while less time in rat chamber(P<0.001)and open arm(P<0.05),and much more defecation(P<0.05).Furthermore,the first time to immobility and the total time in struggle were also decreased(P<0.01,P<0.001),whereas the total time in immobility was increased(P<0.01).These data showed that the VAD aggravated the rats' autism-like behavior induced by NMS.? No matter with or without NMS,the levels of RAR? and CD38 protein expressions in the hypothalamus of the VA deficiency rats were significantly decreased compared with those of VA normal rats(P<0.001,P<0.001),but also the serum concentrations of OXT in the VA deficiency rats were statistically lower than those of the VA normal rats(P<0.05,P<0.01).Conclusions: ? Neonatal maternal separation induces rats to display autism-like behavior.? Vitamin A deficiency begaining from preganancy aggravates the autism-like behavior induced by NMS.? VA deficiency results in decreases of the RAR?,CD38 expression levels and OXT concentration.VAD combined with NMS further reduces the levels of CD38 and OXT expressions.PART ? VA SUPPLEMENTATION IMPROVE THE AUTISM-LIKE BEHAVIOR OF RATS WITH VITAMIN A DEFICIENCY AND NEONATAL MATERNAL SEPARATION RATSObjective: To explore the effects of VA supplementation on autism-like behavior of the VAD-NMS rats,and the expression level changes of RAR? and autism associated proteins of CD38 and OXT.Methods: The offsprings of the VAD-NMS were given by VA supplementation(VAS-NMS group),and the the offsprings without VA supplementation served as its control(VAD-NMS group).At 6-week-old,the rats were detected by open field test,social interaction test,elevated plus maze and forced swimming.The serum retinol levels were tested by HPLC.Western blotting was used to detect the expression levels of RAR? and CD38 proteins,and ELISA to detect the serum content of OXT.Results: ? The serum retinol concentrations of the VAS-NMS rats were significantly higher than that of the VAD-NMS rats(P<0.05).? Compared with those of the VAD-NMS group,rats in the VAS-NMS group spent less time in self-grooming(P<0.001)and object chamber(P<0.05)while more time in rat chamber(P<0.05)and open arm(P<0.001).The defecation frequency was decreased(P<0.01).Moreover,the first time to immobility and the total time in struggle were also increased,while the total time in immobility was decreased(P<0.001).These data suggested that VA supplementation in time relieved the autism-like behavior of VAD-NMS rats.? Both the expression levels of RAR? and CD38 proteins and the serum concentration of OXT in the hypothalamus were statisticallyincreased in the VAS-NMS rats compared with those of the VAD-NMS rats(P<0.05,P<0.05).Conclusions: ? The serum retinol level of the VAD-NMS rats can be elevated via postnatal VA supplementation.? VA supplementation improves the autism-like behavior of rats induced by the VAD-NMS.? VA intervention upregulates the expression levels of RAR? and CD38 protein in the hypothalamus and the serum OXT concentration of the VAD-NMS rats.These indicate that VA supplementation in time can be as an adjuvant therapy method to autism-like behavior rats induced by the VAD-NMS.PART ? THE MOLECULAR MECHANISM OF RAR BETA REGULATING CD38 SIGNALING PATHWAYObjective: To investigate the molecular mechanism of RA signaling mediated by RAR? regulating the autism associated protein CD38 expression level.Methods: Hypothalamus primary neurons were primary cultured from embryonic rats and identified with neuron-specific enolase(NSE).The advantage retinoic acid receptors were found in the primary neurons of hypothalamus using RA treatments in different concentrations.The levelsof RAR? and CD38 m RNA and protein expressions were detected by Real-time PCR and Western blot.Furthermore,the adenoviruses of RAR? overexpression or si RAR? were used to infected promary neurons to make the four groups of RFP,RFP+RA,Ad-RAR?+RA and si RAR?+RA.The levels of RAR? and CD38 protein expressions were tested in the neurons of hypothalamus by Western Blot.The binding of RAR? on the promotor of CD38 was tested by Ch IP-q PCR following RA treatment,and cytoplasmic Ca2+concentration of the neurons was detected by calcium image technique.Results: ? Hypothalamus primary neurons were identified by NSE to dsiplayed more than 95% purify,which can be used for futher study.? Treated with RA in a series of concentrations,the levels of RAR? expression were tendy to obviuosly increase,especially at the RA concentration of 1?mol/L(P<0.001).Meanwhile,the levels of CD38 m RNA and protein expressions were significantly higher than that of RA untreamtnet group(P<0.001).? With infection of adenoviruses of RAR? overexpression or si RAR?,the level of CD38 protein in the Ad-RAR?+RA group was significantly increased compared with that of the RFP+RA group(P<0.05),while statistically decreased in the si RAR?+RA group compared with in the RFP+RA group(P<0.05).? The result of Ch IP-q PCR showed that RA treatment induced RAR? enriching on the CD38 promotor(P<0.05).? KCL activated the cytoplasmic Ca2+ concentration in the neurons following RA treatment(P<0.001).Conclusions: RA up-regulates the RAR? signaling pathway in the hypothalamus neurons to enhance the expression level of CD38.The molecular mechanism may be that RAR? binds on the promotor of CD38,to promote calcium activity in the neurons induced by RA and regulate the OXT release.