食淀粉乳杆菌干预PRV GDNF和GSNO的ZO-1调控

为探究食淀粉乳杆菌在猪轮状病毒(PRV)感染乳鼠空肠组织中GSNO及GDNF对紧密连接蛋白ZO-1的影响,选择2日龄清洁级中国昆明鼠乳鼠作为试验动物,分为5组,正常对照组、轮状病毒组(RV组)、食淀粉乳杆菌组(L.a组)、预防性干预组(BI组)、治疗性干预组(AI组).空肠组织作为靶器官,分别应用实时荧光定量PCR检测GDNF和ZO-1 mRNA相对表达量,采用比色法检测抗氧化指标(NO、GSH、SOD、MDA、GSH-Px、CAT),通过ELISA定量检测GSNO和5-HT含量,Western blot检测ZO-1、GDNF、GFAP蛋白含量变化,免疫组化检测ZO-1蛋白分布表达变化.结果表明,食淀粉乳杆菌可显著上调ZO-1和GDNF mRNA相对表达量(P<0.05),RV组、BI组和AI组ZO-1和GDNF的mRNA相对表达量变化趋势相似,先升后降.攻毒后各时间点,L.a组5-HT含量均显著高于其他组(P<0.05),RV组5-HT含量高于对照组,低于BI组和AI组;L.a组GSNO含量均显著高于其他组(P<0.05),RV组、BI组和AI组GSNO含量整体变化趋势相似,均是先升后降;对于ZO-1、GFAP、GDNF蛋白表达,整体趋势相似,在攻毒后第2、3天表达量较显著,L.a组ZO-1和GFAP蛋白表达量比其他4组高,AI组GDNF蛋白表达量比其他4组高.免疫组化结果表明,L.a组ZO-1蛋白分布比对照组更连续均匀,BI组ZO-1蛋白分布较RV组更连续,AI组与RV组无明显差异.RV组NO含量和MDA含量显著高于其他组(P<0.05),L.a组NO和MDA含量保持不变,BI组和AI组NO和MDA含量高于L.a组,低于RV组;L.a组CAT、SOD、GSH、GSH-Px含量均高于其他组,BI组和AI组CAT、SOD、GSH、GSH-Px含量整体变化趋势一致,先降后升,且高于RV组.可知,食淀粉乳杆菌通过上调GDNF和GSNO表达和增强乳鼠空肠抗氧化能力,共同促进ZO-1表达,使其在乳鼠空肠上更均匀连续,显著提高乳鼠空肠细胞屏障强度,干预轮状病毒感染.

Lactobacillus amylovorus intervention of PRV on ZO-1 regulation of GDNF and GSNO

In order to explore the effect of GSNO and GDNF on tight junction protein ZO-1 in jejunum of suckling mice infected with porcine rotavirus(PRV), 2-day-old clean grade Kunming mice were selected as experimental animals and divided into five groups: normal control group, rotavirus group(RV group), Lactobacillus amylovorus group(L.a group), preventive intervention group(BI group)and therapeutic intervention group(AI group). Jejunum used as the target organ, Real time PCR(qPCR)was used to detect the relative expression of GDNF and ZO-1 mRNA. Colorimetry was used to detect antioxidant indexes(NO, GSH, SOD, MDA, GSH-PxP, CAT), ELISA was used to quantitatively detect the content of GSNO and 5-HT, Western blot was used to detect the protein content of ZO-1, GDNF and GFAP, and Immunohistochemistry was used to detect the distribution and expression of ZO-1 protein. The results showed that Lactobacillus amylovorus significantly increased the relative mRNA expression of ZO-1 and GDNF(P<0.05).The relative mRNA expression of ZO-1 and GDNF in RV group,BI group and AI group showed a similar trend, which increased first and then decreased. At each time point after rotavirus infection, the content of 5-HT in L.a group was significantly higher than that in other groups(P<0.05); the content of 5-HT in RV group was higher than that in control group and lower than that in BI group and AI group; the content of GSNO in L.a group was significantly higher than that in other groups(P<0.05), and the overall change trend of GSNO in RV group, BI group and AI group was similar, which first increased and then decreased, for the expression of ZO-1, GFAP and GDNF, the overall trend was similar, and the expression level was significant on the 2 nd and 3 rd day after rotavirus infection. The expressions of ZO-1 and GFAP in L.a group were higher than those in the other four groups, and the expressions of GDNF in AI group were higher than those in the other four groups.Immunohistochemical results showed that the distribution of ZO-1 protein in L.a group was more continuous and uniform than that in control group, and that in BI group was more continuous than that in RV group. There was no significant difference between AI group and RV group. The contents of NO and MDA in RV group were significantly higher than those in other groups(P<0.05), the contents of NO and MDA in L.a group remained basically unchanged; the contents of NO and MDA in BI group and AI group were higher than those in L.a group, but lower than those in RV group; the contents of CAT,SOD, GSH and GSH-Px in L.a group were higher than those in other groups, the overall change trend of CAT, SOD, GSH and GSH-Px in BI group and AI group was similar, basically decreased first and then increased, and higher than that in RV group. The results showed that Lactobacillus amylovorus could jointly promote the expression of ZO-1 by up regulating the expression of GDNF and GSNO and enhancing the antioxidant capacity of neonatal rat jejunum, make it more uniform and continuous in neonatal rat jejunum, significantly improve the strength of neonatal rat jejunal cell barrier and intervene in rotavirus infection.