Effects Of Dietary Protein Levels On Intestinal Morphology And Tight Junction Protein Occludin In Weaned Piglets

Protein is an indispensable nutrient for the growth of piglets.Due to the incomplete intestinal development of weaned piglets,it is more sensitive to dietary protein levels.Excessive or low protein levels can cause diarrhea in piglets.The breakdown of intestinal mucosal barrier is the main cause of diarrhea in piglets.Tight junction(TJ)is an important component of intestinal mucosal barrier.It is particularly important for intestinal permeability.Once TJ is destroyed,diarrhea easily occurs.Occludin is an important protein to maintain the integrity of TJ,and its changes are closely related to TJ.There are few reports about the effect of dietary protein levels on Occludin protein.In order to explore the effect of dietary protein level on the intestinal morphology and Occludin of weaned piglets,96 weaned Du-Chang Three-way Hybrid piglets aged 25 days were randomly divided into four groups after 7 days of feeding.The piglets were fed diets with the same energy levels of 18%,20%,22% and 24% respectively,and slaughtered 6,24,48,72 and 96 hours after feeding.Three piglets in each group were randomly selected after anesthesia,and their jejunum,ileum tissues were collected respectively,and stained with HE and transmitted.The morphological changes of intestinal mucosa in piglets were observed by electron microscopy.The expression of Occludin protein in the small intestine of piglets was localized by immunohistochemistry.The changes of Occludin protein in the small intestine of piglets were detected by fluorescence quantitative RCR(q RT-PCR)and Western-Blot.The results showed that:(1)The results of small intestinal histopathology showed that the jejunum and ileum mucosa of piglets in 18% protein group were damaged more seriously than those in 20%protein group at 6-72 hours,showing necrosis and exfoliation of epithelial cells and infiltration of inflammatory cells.Normal structure of jejunum could not be observed at 24 hours,but the intestine and ileum mucosa were repaired at 72 hours.The jejunum and ileum mucosa of piglets were repaired except 24,72 hours.Except for 48 hours,the epithelial cells of 20% protein group were arranged in a tight and uniform length without inflammatory cell infiltration at the other time points,while the ileal mucosa of 22% protein group and jejunum and ileum mucosa of 24% protein group showed exfoliation and necrosis of epithelial cells during the whole experimental period(2)The results of transmission electron microscopy showed that the microvilli of ileum mucosa of piglets in 18% protein group were broken and shed irregularly arranged,the intercellular junction widened and the mitochondria swelled,except 48 and 96 hours,compared with that in 20% protein group,the microvilli of ileum mucosa of piglets in 20%protein group were uniform in length,clear tight junction and abundant in other organelles.At stage 22% and 24% protein groups,the ileal mucosal microvilli of piglets dropped off,tight junction disappeared,mitochondria swelled,some organelles dissolved,but recovered at 96 h.(3)Immunohistochemical results showed that during the whole feeding period,the positive expression area of Occludin protein in 20% protein group was larger,and distributed evenly at the top of the junction of small intestinal epithelial cells,with strong brown signal,while the positive expression area of Occludin protein in other proteins group was smaller,and the staining became light.The optical density of Occludin protein in jejunal tissue of piglets in 18% protein group was significantly lower than that in 20%protein group(P < 0.01)and significantly higher than that in other protein groups(P < 0.01)at 96 h.The optical density of Occludin protein in jejunal tissue of piglets in 20% protein group was the highest at 6-48 h and lower than that in 22% protein group at 72 h,but there was no significant difference between the two groups(P > 0.05).Except 48 hours,the optical density of Occludin protein in ileum tissue of piglets in 20% protein group was the highest,but there was no significant difference between 72 hours and 18% protein group(P > 0.05).At 48 hours,the optical density of Occludin protein in ileum tissue of piglets in18% protein group was significantly higher than that of other three protein groups(P <0.01)(4)During the whole experiment period,the relative expression of Occludin in jejunum of piglets in 18% protein group was lower than that of 20% protein group,and there was no significant difference at 48 hours(P>0.05).Except 24 hours,the relative expression of Occludin in jejunum of piglets in 20% protein group was significantly or extremely significantly higher than that of other protein groups(P<0.05,or P<0.01).At 48 and 96 hours,the relative expression of Occludin in the ileum of piglets in 18% protein group was higher than that in 20% protein group,but there was significant difference only at 96 hours(P<0.01).(5)At 6-48 hours,the expression of Occludin protein in jejunum of piglets in 20%protein group was significantly higher than that in other protein groups(P<0.05,or P<0.01).At 72 hours,the expression of Occludin protein in jejunum of piglets in 22% protein group was significantly higher than that in 20% protein group(P<0.01).At 96 hours,the expression of Occludin protein in jejunum of piglets in 18% protein group was significantly higher than that in 20% protein group(P<0.01).The expression of Occludin protein in ileum of piglets in 20% protein group was the highest at 6-24 and 72 hours(P<0.01),while in 48 and 96 hours,the expression of Occludin protein in ileum of piglets in 18% protein group was the highest at 20% protein group(P<0.01).The results showed that feeding 22% and 24% protein level diets to weaned piglets will destroy the intestinal mucosal barrier and its morphological structure after feeding,reduce the effective absorption area of the small intestine,and lead to a large number of undigested dietary protein entering the large intestine.The same 22% and 24% protein level diets will reduce the expression of Occludin protein and its m RNA in the small intestine,thus changing its permeability and causing diarrhea in piglets.Therefore,controlling dietary protein level at 20% is beneficial to up-regulate the expression of Occludin protein and its m RNA,reduce the intestinal permeability,maintain the intestinal mucosal barrier and its absorption function,and is more conducive to the growth and development of piglets.