The Effects Of Vitamin A On Colonic Epithelial Barrier Function In Vivo

Part 1 Normal VA nutritional level promotes the barrier function of colonic mucosal epitheliumObjective To investigate the effects of vitamin A on barrier function of colonic epithelium in vivo.Methods The pregnant rat models of vitamin A normal (VAN)、 vitamin A deficiency (VAD) were used, and the neonatal pups were supplemented with vitamin A (VAS) at postnatal day 1. The levels of serum retinol were detected by high performance liquid chromatography (HPLC).The transepithelial resistance (TER) of colonic mucosal epithelium tissue were tested by Ussing chamber system, and the ELISA kit was used to determine the concentration of serum diamine oxidase (DAO). The level changes of RARβ,TLR4 and ZO-2 expressions were detected by real-time PCR and western blot.Results The level of serum retinol was significantly decreased in the VAD group compared with that of the VAN group (P<0.001). The transepithelial resistance (TER) of rat colonic mucosal epithelium layer was statistally higher in the VAN group than that of the VAD rats (P<0.001). And the serum DAO level of VAN group was significantly lower than that of the VAD group (P<0.001). These results suggest that gestational VA difeciency may lead to decrease of colonic barrier function in pups. Following supplemented by VA to the VAD pups after birth 1 day, the level of serum retinol was significantly increased compared with that of the VAD group (P<0.001), but no statistical difference was found between the VAN and VAS groups. The level of colonic TER in the VAS rats was also significantly higher than that of the VAD group (P<0.01). And the serum DAO level was obviously decreased after VA intervention, but it was not reached to the normal level of VAN rats yet, demonstrating that VA supplementation can effectively increase level of serum retinol and improve intestinal barrier function. The PCR results showed that the levels of RARβ, TLR4 and ZO-2 mRNA expression in the mucosal layer of VAD colon tissues were significantly decreased compared with those of VAN rats (P<0.05, P<0.001), while expression levels of these three genes were significantly increased in the VAS rats (P<0.001, P<0.01). The protein expression levels of RARβ and TLR4 in the VAN group were higher than those of the VAD group, but their expression levels in the VAS group displayed a rising trend compared with those of the VAD group, but couldn’t be restored to the protein expression levels of VAN.Conclusion Gestational VA deficiency can result in decrease of colonic barrier finction of pups after birth, which closely associates with RARβ signaling pathway and the expression levels TLR4 and tight junctions ZO-2 in the colonic epithelial tissue.Part 2 Vitamin A deficiency from the beginning of gestation decreases colonic epithelial barrier function in the offspringObjective To investigate the effect of LPS infection on colonic epithelial barrier function in the offspring of gestational vitamin A deficiency.Methods Lipopolysaccharide (LPS) was intragastric administrated to the VAN and VAD pups of 6-week old inducing their intestinal infection. The levels of serum retinol were detected by high performance liquid chromatography (HPLC), and the ELISA kit was used to test concentration of diamine oxidase (DAO) in rat serum. The levels of RARβ, TLR4 and ZO-2 mRNA and protein expressions in the mucosal layer of colon tissues were analyzed by real-time PCR and western blot.Results 1. The level of serum retinol in the VAD group was significantly decreased compared with that of the VAN group. And the serum retinol level was obviously lower in the VAN group following the LPS treatment than that of the LPS untreatment, however, the status of VA nutrition was the main influence factor to the serum retinol level (P<0.0001).2. Bonferroni post-tests found that the LPS challenge was the major factor leading to increase of DAO concentration (P<0.0001), though both the serum retinol level and LPS treatment were the effect factors to the levels of serum DAO (P=0.0463, P<0.0001)。3. The PCR and western blot results showed that the levels of RARp\TLR4 and ZO-2 both mRNA and protein expressions in the mucosal layer of VAD colon tissues were significantly decreased compared with those of VAN rats (P<0.05). The LPS infection significantly down-regulated the level of RARβ mRNA expression in the VAN rats (P<0.05), which was affected by the interaction of both VA nutritional level and LPS infection. LPS challenge was the main effect factor to the TLR4 expression in rat colonic mucosal tissue, and VA nutrition level was the key effect factor to the ZO-2 expression through the Bonferroni post-tests analysis.Conclusion Gastational vitamin A deficiency decreases the level of TLR4 expression to reduce the ability of intestinal immune regulation in the pups, and downregulates the level of ZO-2 expression to decrease the tight junction of colonic epithelium. These results suggest that gastational vitamin A deficiency weakens intestinal barrier function to affect the capability on the anti-infection of intestine in the pups.