Responses of growth performance,antioxidant function,small intestinal morphology and mRNA expression of jejunal tight junction protein to dietary iron in yellow-feathered broilers

This study aimed to investigate the dose-effect of iron on growth performance,antioxidant function.intestinal morphology,and mRNA expression of jejunal tight junction protein in 1-to21-d-old yellow-feathered broilers.A total of 7201-d-old yellow-feathered maleb roilers were allocated to 9 treatments with 8 replicate cages of 10 birds per cage.The dietary treatments were consisted of a basal diet(contained 79.6 mg Fe kg^(-1))supplemented with 0,20,40,60,80,160,320,640,and 1,280 mg Fe kg^(-1)in the form of FeSO_(4)·7H_(2)O.Compared with the birds in the control group,birds supplemented with 20mg Fe kg^(-1)had higher average daily gain(ADG)(P<0.0001).Adding 640 and 1,280 mg Fe kg^(-1)significantly decreased ADG(P<0.0001)and average daily feed intake(ADFI)(P<0.0001)compared with supplementation of 20mg Fe kg^(-1).Malondialdehyde(MDA)concentration in plasma and duodenum increased linearly(P<0.0001),but MDA concentration in liver and jejunum increased linearly(P<0.05)or quadratically(P<0.05)with increased dietary Fe concentration.The villus height(VH)in duodenum and jejunum,and the ratio of villus height to crypt depth(V/C)in duodenum decreased linearly(P?0.05)as dietary Feincreased.As dietary Fe increased,the jejunal relative mRNA abundance of claudin-1 decreased linearly(P=0.001),but the jejunal relative mRNA abundance of zona occludens-1(ZO-1)and occludin decreased linearly(P?0.05)or quadratically(P?0.05).Compared with the supplementation of 20 mg Fe kg^(-1),the supplementation of640 mg Fe kg^(-1)or higher increased(P?0.05)MDA concentrations in plasma,duodenum,and jejunum,decreased VH in the duodenum and jejunum,and the addition of 1,280 mg Fe kg^(-1)reduced(P?0.05)the jejunal tight junction protein(claudin-1,ZO-1,occludin)mRNA abundance.In summary,640 mg of supplemental Fe kg^(-1)or greater was associated with decreased growth performance,increased oxidative stress,disrupted intestinal morphology,and reduced mRNA expression of jejunal tight junction protein.

Dietary protocatechuic acid ameliorates inflammation and up-regulates intestinal tight junction proteins by modulating gut microbiota in LPS-challenged piglets

Background: Weaning is one of the major factors that cause stress and intestinal disease in piglets. Protocatechuic acid(PCA) is an active plant phenolic acid which exists in Chinese herb, Duzhong(Eucommia ulmoides Oliver), and is also considered as the main bioactive metabolite of polyphenol against oxidative stress and inflammation. This study aimed to investigate the effect of PCA on growth performance, intestinal barrier function, and gut microbiota in a weaned piglet model challenged with lipopolysaccharide(LPS).Methods: Thirty-six piglets(Pig Improvement Company line 337 × C48, 28 d of age, 8.87 kg ± 0.11 kg BW) were randomly allocated into 3 treatments and fed with a basal diet(CTL), a diet added 50 mg/kg of aureomycin(AUR), or a diet supplemented with 4000 mg/kg of PCA, respectively. The piglets were challenged with LPS(10 μg/kg BW) on d 14 and d 21 by intraperitoneal injection during the 21-d experiment. Animals(n = 6 from each group) were sacrificed after being anesthetized by sodium pentobarbital at 2 h after the last injection of LPS. The serum was collected for antioxidant indices and inflammatory cytokines analysis, the ileum was harvested for detecting mRNA and protein levels of tight junction proteins by PCR and immunohistochemical staining, and the cecum chyme was collected for intestinal flora analysis using 16 S rRNA gene sequencing.Results: Dietary supplementation of PCA or AUR significantly increased the expression of tight junction proteins including ZO-1 and claudin-1 in intestinal mucosa, and decreased the serum levels of thiobarbituric acid reactive substances(TBARS) and IL-6, as compared with CTL group. In addition, PCA also decreased the serum levels of IL-2 and TNF-α(P < 0.05). Analysis of gut microbiota indicated that PCA increased the Firmicutes/Bacteroidetes ratio(P < 0.05). Spearman's correlation analysis at the genus level revealed that PCA reduced the relative abundance of Prevotella 9, Prevotella 2, Holdemanella, and Ruminococcus torques group(P < 0.05), and increased the relative abundance of Roseburia and Desulfovibrio(P < 0.05), whereas AUR had no significant effect on these bacteria.Conclusions: These results demonstrated that both PCA and AUR had protective effect on oxidative stress, inflammation and intestinal barrier function in piglets challenged with LPS, and PCA potentially exerted the protective function by modulating intestinal flora in a way different from AUR.

New tight junction protein 2 variant causing progressive familial intrahepatic cholestasis type 4 in adults: A case report

BACKGROUND Progressive familial intrahepatic cholestasis(PFIC)encompasses a group of autosomal recessive disorders with high morbidity and mortality.Variants in the gene encoding tight junction protein-2(TJP2)have been linked to PFIC type 4(PFIC4),which predominantly presents in childhood.However,there are only limited data from adults with TJP2-related PFIC4.We report a family with an autosomal recessive disorder with a novel variant in the TJP2 gene in adults with very variable expression of PFIC4.CASE SUMMARY The index patient presented at 19 years old with liver cirrhosis and variceal bleeding and was treated with endoscopic banding and beta-blockers.In 2018,he developed primary liver cancer that was treated with radiofrequency ablation followed by liver transplantation in 2019.Genetic testing revealed a novel homozygous TJP2 variant causing PFIC4(TJP2([NM_004817.3]:c.[3334C>T];[3334C>T])).The consanguineous family consists of the father and mother(both heterozygous)and their 12 children,of which five carry the variant in a homozygous state;however,these five siblings have highly variable expression of PFIC4.Two homozygous brothers had cirrhosis and portal hypertension at diagnosis at the ages of 19 and 36.Two other homozygous brothers,age 23 and 19,and the homozygous sister,age 21,have elevated liver enzymes but presently no cirrhosis,which may suggest an age-dependent penetrance.In addition,five sisters had severe and mild intrahepatic cholestasis of pregnancy and carry the TJP2 variant in a homozygous and heterozygous state,respectively.CONCLUSION This novel TJP2 variant is associated with PFIC4 causing severe liver disease with cirrhosis and primary liver cancer in adolescents/adults.

Cinnamicaldehyde regulates the expression of tight junction proteins and amino acid transporters in intestinal porcine epithelial cells

Background： Cinnamicaldehyde（CA） is a key flavor compound in cinnamon essential oil possessing various bioactivities. Tight junction（TJ） proteins are vital for the maintenance of intestinal epithelial barrier function,transport, absorption and utilization of dietary amino acids and other nutrients. In this study, we tested the hypothesis that CA may regulate the expression of TJ proteins and amino acid transporters in intestinal porcine epithelial cells（IPEC-1） isolated from neonatal pigs.Results： Compared with the control, cells incubated with 25 μmol/L CA had increased transepithelial electrical resistance（TEER） and decreased paracellular intestinal permeability. The beneficial effect of CA on mucosal barrier function was associated with enhanced protein abundance for claudin-4, zonula occludens（ZO）-1, ZO-2, and ZO-3. Immunofluorescence staining showed that 25 μmol/L CA promoted the localization of claudin-1 and claudin-3 to the plasma membrane without affecting the localization of other TJ proteins, including claudin-4, occludin,ZO-1, ZO-2, and ZO-3, compared with the control cells. Moreover, protein abundances for rBAT, xCT and LAT2 in IPEC-1 cells were enhanced by 25 μmol/L CA, while that for EAAT3 was not affected.Conclusions： CA improves intestinal mucosal barrier function by regulating the distribution of claudin-1 and claudin-3 in enterocytes, as well as enhancing protein abundance for amino acid transporters rBAT, xCT and LAT2 in enterocytes. Supplementation with CA may provide an effective nutritional strategy to improve intestinal integrity and amino acid transport and absorption in piglets.

Protective Effect of Simvastatin on Impaired Intestine Tight Junction Protein ZO-1 in a Mouse Model of Parkinson's Disease

Summary： Recently, several studies showed that gastrointestinal tract may be associated with patho- physiology of Parkinson＇s disease （PD）. Intestine tight junction protein zonula occluden-1 （ZO-1） is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 （MMP-9）. In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine （MPTP）-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-I expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvas- tatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that＇pass across the intestinal barrier.

Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients

AIM:To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction(TJ) proteins.METHODS:Sixty patients diagnosed with mild to moderate Crohn’s disease(CD)were allocated into the herb-partitioned moxibustion combined with acupuncture(HMA)group(n=30)or the mesalazine(MESA)group(n=30)using a parallel control method.There were 2 sets of acupoints used alternately for HMA treatment.The following points were included in Set A:ST25(Tianshu),RN6(Qihai),and RN9(Shuifen)for herb-partitioned moxibustion and ST36(Zusanli),ST37(Shangjuxu),LI11(Quchi),and LI4(Hegu)for acupuncture.The points for Set B included BL23(Shenshu)and BL25(Dachangshu)for herb-partitioned moxibustion and EX-B2 of T6-T1(Jiajixue)fo r acupuncture.The patients received the same treatment6 times a week for 12 consecutive weeks.The MESA group received 1 g of mesalazine enteric coated tablets4 times daily for 12 consecutive weeks.Intestinaltissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session.Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1(ZO-1),occludin,and claudin-1.The m RNA levels were also evaluated.RESULTS:After the treatment,both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients;the patients treated with HMA showed better improvement.HMA significantly increased the expression of ZO-1(P=0.000),occludin(P=0.021),and claudin-1(P=0.016).MESA significantly increased the expression of ZO-1(P=0.016)and occludin(P=0.026).However,there was no significant increase in the expression of claudin-1(P=0.935).There was no statistically significant difference between the two groups for the expression of occludin and claudin-1(P>0.05).The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group(2333.34±352.51 vs 2160.38±307.08,P=0.047).HMA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.017),and claudin-1 m RNA(P=0.017).MESA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.042),and claudin-1 m RNA(P=0.041).There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 m RNA(P>0.05).However,the HMA group showed a significant improvement in ZO-1 m RNA expression compared with the MESA group(2378.17±308.77 vs 2200.56±281.88,P=0.023).CONCLUSION:HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their m RNAs.

Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

AIM:To investigate the effect of side-stream smoking on gut microflora composition,intestinal inflammation and expression of tight junction proteins.METHODS:C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks.Cecal contents were collected for microbial composition analysis.Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins.RESULTS:Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria,Clostridium but decreased Fermicutes(Lactoccoci and Ruminococcus),Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice.Meanwhile,side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα,accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6.The contents of tight junction proteins,claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking.In addition,side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling,while inhibiting AMPactivated protein kinase in the large intestine.CONCLUSION:Side-stream smoking altered gut microflora composition and reduced the inflammatory response,which was associated with increased expression of tight junction proteins.

Effects of Simulated Weightlessness on Tight Junction Protein Occludin and Zonula Occluden-1 Expression Levels in the Intestinal Mucosa of Rats

This study investigated the tight junction（TJ） protein expression of the intestinal mucosa in a rat tail-suspension model under simulated weightlessness.Twenty-four Wistar rats were randomly divided into three groups：CON group（n=8）,control; SUS-14 d group（n=8）,tail-suspension for 14 days; SUS-21 d group（n=8）,tail-suspension for 21 days.Occludin and Zonula Occluden-1（ZO-1） expression levels were determined by immunohistochemical analysis and mRNA fluorescent quantitative PCR.Plasma levels of diamine oxidase（DAO） and d-lactate were determined using enzymatic spectrophotometry.Immunohistochemical results for occludin and ZO-1 showed disruption of the TJs in the intestinal mucosa in SUS-14 d and SUS-21 d groups.The expression levels of occludin and ZO-1 in SUS-21 d group were lower than those in SUS-14 d group,and significantly lower than those in CON group（Occldin：0.86±0.02 vs 1.01±0.03 vs 1.63±0.03 and ZO-1：0.82±0.01 vs 1.00±0.02 vs 1.55±0.01,P〈0.01）.Moreover,the levels of plasma DAO and d-lactate in SUS-21 d group were higher than those in SUS-14 d group,and significantly higher than those in CON group（DAO：27.58±0.49 vs 20.74±0.49 vs 12.94±0.21 and d-lactate：37.86±0.74 vs 28.26±1.01 vs 17.76±0.91,P〈0.01）.There were significant negative correlations between occludin or ZO-1 expression levels and DAO（r2=0.9014,r2=0.9355,P〈0.01） or d-lactate levels（r2=0.8989,r2=0.9331,P〈0.01）.Occludin and Zo-1 were reduced in intestinal mucosa both in mRNA and protein levels in the rat tail-suspension model.The significant negative correlations between expression levels of TJs and plasma levels of DAO or d-lactate support the hypothesis that intestinal permeability is increased due to a decrease in TJ protein expression during tail-suspension from 14 days to 21 days.

Effect of tight junction protein of intestinal epithelium and permeability of colonic mucosa in pathogenesis of injured colonic barrier during chronic recovery stage of rats with inflammatory bowel disease

Objective: To discuss the changes in the tight junction protein of intestinal epithelium and permeability of colonic mucosa and its possible mechanism by building the rat mode of inflammatory bowel disease at the chronic recovery stage. Methods: A total of 36 SD rats were divided into the model group and control one according to the random number table, with 18 rats in each group. Rats in the model group were given the 3% dextran sulfate sodium solution by the way of drinking for 7 d to build the rat model of inflammatory bowel disease, while rats in the control group were given free drinking of water. Six rats were executed at day 7, 14 and 21 respectively. The colonic tissues were collected from rats to observe the pathological changes of colonic mucosa. The activity of myeloperoxidase was detected and the white blood count was performed for rats in each group. The Ussing chamber technique was employed to detect the transepithelial electrical resistance(TER) and short-circuit current(SC) of colonic mucosa of rats in different time intervals; the quantum dots labeling technique was employed to detect the expression level of claudin-1 and claudin-2 in the colonic tissues. Results: After the successful modeling, the weight of rats in the model group was significantly reduced, while the disease activity index score was increased. The weight was at the lowest level at day 14 and then it began to increase afterwards. The disease activity index score was at the highest level at day 12 and then it began to decrease gradually. The activity of myeloperoxidase and WBC for rats in the model group all reached the peak value at day 14 and then decreased gradually. There was no significant difference in the changes of TER and SC in different time intervals for rats in the control group(P>0.05). TER of model group was at the lowest level at day 14 and then increased gradually; SC was at the highest level at day 14 and then decreased gradually. TER of model group at day 7, 14 and 21 was significantly lower than that of control group, while SC of model group was significantly higher than that of control group(P<0.05). There was no significant difference in the change of mean fluorescence intensity of claudin-1 and claudin-2 in different time intervals for rats in the control group(P>0.05). The claudin-1 and claudin-2 for rats in the model group reached the highest level at day 14 and then decreased gradually. The claudin-1 and claudin-2 of model group at day 7, 14 and 21 was significantly higher than that of control group(P<0.05). Conclusions: After the acute stage, the inflammatory bowel disease is then in the chronic recovery stage; the increased permeability of colonic mucosa and increased expression of tight junction protein of intestinal epithelium are closely related to the pathogenesis and development of disease. The tight junction protein plays a key role in the pathogenesis of injured colonic barrier of inflammatory bowel disease.

Effect of salvianolate on intestinal epithelium tight junction protein zonula occludens protein 1 in cirrhotic rats

AIM:To study the effect of salvianolate on tight junctions(TJs) and zonula occludens protein 1(ZO-1) in small intestinal mucosa of cirrhotic rats.METHODS:Cirrhosis was induced using carbon tetrachloride.Rats were randomly divided into the untreated group,low-dose salvianolate(12 mg/kg) treatment group,medium-dose salvianolate(24 mg/kg) treatment group,and high-dose salvianolate(48 mg/kg) treatment group,and were treated for 2 wk.Another 10 healthy rats served as the normal control group.Histological changes in liver tissue samples were observed under a light microscope.We evaluated morphologic indices of ileal mucosa including intestinal villi width and thickness of mucosa and intestinal wall using a pathological image analysis system.Ultrastructural changes in small intestinal mucosa were investigated in the five groups using transmission electron microscopy.The changes in ZO-1 expression,a tight junction protein,were analyzed by immunocytochemistry.The staining index was calculated as the product of the staining intensity score and the proportion of positive cells.RESULTS:In the untreated group,hepatocytes showed a disordered arrangement,fatty degeneration was extensive,swelling was obvious,and disorganized lobules were divided by collagen fibers in hepatic tissue,which were partly improved in the salvianolate treated groups.In the untreated group,abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts,and collagen fibers gradually decreased and damaged hepatic lobules were partly repaired following salvianolate treatment.Compared with the untreated group,no differences in intestinal villi width between the five groups were observed.The villi height as well as mucosa and intestinal wall thickness gradually thickened with salvianolate treatment and were significantly shorter in the untreated group compared with those in the salvianolate treatment groups and normal group(P < 0.01).The number of microvilli decreased and showed irregular lengths and arrangements in the untreated group.The intercellular space between epithelial cells was wider.The TJs were discontinuous,which indicated disruption in TJ morphology in the untreated group.In the treated groups,the microvilli in the intestinal epithelium were regular and the TJs were gradually integrated and distinct.The expression of ZO-1 decreased in the small intestine of the untreated cirrhotic rats.The high expression rate of ZO-1 in ileal mucosa in the untreated group was significantly lower than that in the medium-dose salvianolate group(21.43% vs 64.29%,χ 2 = 5.25,P < 0.05),high-dose salvianolate group(21.43% vs 76.92%,χ 2 = 8.315,P < 0.01) and normal group(21.43% vs 90%,χ 2 = 10.98,P < 0.01).CONCLUSION:Salvianolate improves liver histopathological changes,repairs intestinal mucosa and TJ structure,and enhances ZO-1 expression in the small intestinal mucosa in cirrhotic rats.

Preliminary Investigation on the Effect of <i>Lactobacillus</i>and Epidermal Growth Factor on Tight Junction Proteins in Experimental Clostridium <i>difficile</i>Infection

Clostridium difficile associated disease (CDAD) is the most common hospital acquired infection, due to exposure to various drugs. C. difficile toxins influence barrier function in intestinal epithelium. Biotherapeutic approaches, employing probiotic and epidermal growth factor (EGF) could help in barrier protein protection and aid in CDAD management. A preliminary investigation on the effect of Lactobacillus acidophilus and EGF on tight junction proteins in experimentally induced C. difficile infection was done. BALB/mice were divided into 5 groups. Group 1 was comprised of healthy controls, whereas animals in Groups 2 - 5 were sub-divided into 3 subgroups (a, b and c) each. Animals in Groups 2 - 5 received C. difficile inoculum either on day 1 (Group 2) or after pretreatment with ampicillin (Group 3), cyclosporine (Group 4) or lansoprazole (Group 5). Additionally animals in subgroups “b” and “c” also received L. acidophilus and EGF inocula respectively after C. difficile challenge. All animals were investigated for the presence of tight junction proteins (occludin, α-actinin and zonula occludens) in their colonic segments. Data were analyzed using the SPSS version 10 software. These three proteins were present in significantly less (P < 0.05) number of animals in the drug receiving animals, whereas they were found in significantly more (P < 0.05) number of animals receiving L. acidophilus and EGF after challenge with ampicillin, cyclosporine and lansoprazole, suggesting their role in protecting intestinal barrier function.

人脐带间充质干细胞来源外泌体降低脊髓损伤后血脊髓屏障的通透性

背景:研究发现,内皮素参与了脊髓损伤后血脊髓屏障的破坏,干细胞来源外泌体可降低血脊髓屏障的通透性,修复脊髓损伤。目的:观察人脐带间充质干细胞来源外泌体是否可以通过抑制内皮素1表达降低血脊髓屏障的通透性,进而修复脊髓损伤。方法:用超速离心法从人脐带间充质干细胞培养上清液中提取外泌体,透射电子显微镜观察其形态,Western blot检测tsg101、CD63的表达。80只SD大鼠随机分成假手术组、模型组、外泌体组、内皮素1组(n=20),采用改良Allen’s法制备大鼠脊髓损伤模型,内皮素1组用微量注射器直接向损伤部位注射10μL(1μg/mL)内皮素1,术后即刻及1,2 d,分别给予假手术组、模型组尾静脉注射200μL PBS,外泌体组、内皮素1组尾静脉注射200μL外泌体(200μg/mL)。脊髓损伤后第1,3,7,14,21天进行后肢运功功能评分;损伤后第7天通过伊文思蓝染色观察血脊髓屏障通透性,Western blot检测脊髓组织中紧密连接蛋白ZO-1、β-Catenin、Occludin和内皮素1的表达。结果与结论:①外泌体组BBB评分在损伤后3-21 d显著高于模型组(P<0.05),苏木精-伊红染色显示外泌体组脊髓损伤较模型组明显减轻;内皮素1组BBB评分较外泌体组显著降低(P<0.05),内皮素1组脊髓损伤较外泌体组加重;②模型组内皮素1表达量较假手术组显著增加(P<0.05),外泌体组内皮素1表达量较模型组显著降低(P<0.05);③与模型组比较,外泌体组血脊髓屏障伊文思蓝渗出量显著减少(P<0.05),紧密连接蛋白β-Catenin、Occludin、ZO-1的表达增加(P<0.05);与外泌体组比较,内皮素1组伊文思蓝渗出量显著增加(P<0.05),上述紧密连接蛋白表达量显著减少(P<0.05);④结果表明,人脐带间充质细胞来源外泌体通过下调内皮素1表达来保护血脊髓屏障的通透性,起到了修复脊髓损伤的作用。

短小芽孢杆菌对羔羊肠道炎症和屏障功能的影响

【目的】本试验旨在研究短小芽孢杆菌对羔羊生长性能及腹泻、血清指标、结肠组织形态、炎性细胞因子和紧密连接蛋白含量的影响。【方法】选取36只初生重3 kg左右的1日龄萨能奶山羊公羔,随机分为4组(每组9个重复,每重复1只羔羊);每天分别饲喂0(CON组)、1 mL(BP1组)、5 mL(BP5组)、10 mL(BP10组)短小芽孢杆菌菌液,其活菌数为1×10^(8) CFU/mL,试验期14 d。【结果】饲喂短小芽孢杆菌对羔羊末重、平均日增重、平均日采食和料重比均无显著影响(P> 0.05),但均降低了羔羊的粪便评分和腹泻频率(P <0.05);BP1组血清中谷草转氨酶含量低于对照组(P <0.05),二胺氧化酶含量低于对照组和BP5组(P <0.05),与对照组相比,其它三组均降低了D-乳酸含量(P <0.05);BP1组增加了结肠肌层厚度(P <0.05);与对照组相比,BP1组、BP5组和BP10组均降低了IL-1β、IL-6、TNF-α含量(P <0.05),增加了IL-10、TGF-β、IFN-γ、PPAR-γ的含量以及MUC2、Claudin-1、Claudin-4、Occludin和ZO-1的含量(P <0.05),而且减少了结肠组织中性粒细胞细胞浸润。【结论】在初生羔羊代乳粉中添加短小芽孢杆菌可以降低羔羊腹泻频率,改善结肠组织形态,缓解炎症反应,增强黏膜屏障功能,但对生长性能无显著影响。

牛至香酚对LPS免疫应激小鼠血清细胞因子含量和肠道细胞因子、紧密连接蛋白表达水平的影响

【目的】旨在探究牛至香酚是否能有效改善脂多糖(LPS)免疫应激小鼠免疫功能的损伤。【方法】将80只小鼠随机分成空白对照组、模型组和牛至香酚低、中、高剂量组。牛至香酚低、中、高剂量组分别按25、50、100 mg·kg^(-1)的剂量灌胃牛至香酚,空白对照组和模型组按10 mL·kg^(-1)的剂量灌胃橄榄油,每日1次,连续14 d。第15天时,牛至香酚组和模型组小鼠腹腔注射8 mg·kg^(-1)LPS,空白对照组注射等量生理盐水,6 h后采集血液和肠道组织,检测小鼠血清细胞因子(IL-1β、IL-6、TNF-α、IL-10)含量和肠道细胞因子(IL-1β、IL-6、TNF-α、IL-10)、紧密连接蛋白(ZO-1、Occludin、Claudin-1)mRNA的表达水平。【结果】(1)与空白对照组相比,模型组小鼠血清TNF-α、IL-10、IL-1β含量上升(P>0.05),IL-6含量极显著上升(P<0.01);模型组回肠和结肠组织TNF-α、IL-6、IL-1βmRNA表达水平极显著上调(P<0.01),回肠和结肠组织ZO-1 mRNA表达水平下调(P>0.05),结肠组织Occludin、Claudin-1 mRNA表达水平极显著下调(P<0.01)。(2)与模型组相比,牛至香酚试验组小鼠血清IL-6含量极显著下降(P<0.01),IL-1β含量显著下降(P<0.05);牛至香酚高剂量组血清TNF-α含量极显著下降(P<0.01);牛至香酚中、高剂量组血清IL-10含量显著上升(P<0.05)。(3)同模型组相比,牛至香酚中、低剂量组回肠组织IL-1β、TNF-α、IL-6 mRNA表达水平极显著下调(P<0.01);牛至香酚中、高剂量组回肠组织ZO-1、Occludin mRNA表达水平极显著上调(P<0.01);牛至香酚高剂量组结肠组织IL-10 mRNA表达水平极显著上调(P<0.01),TNF-αmRNA表达水平极显著下调(P<0.01),Occludin、Claudin-1 mRNA表达水平极显著上调(P<0.01)。【结论】牛至香酚通过改变小鼠血清细胞因子含量和肠道细胞因子、紧密连接蛋白mRNA的表达水平,调节LPS免疫应激小鼠的免疫功能。

大麻二酚对创伤性脑损伤大鼠脑皮质Occludin、ZO-1表达水平及血脑屏障通透性的影响

目的观察创伤性脑损伤(TBI)大鼠血脑屏障(BBB)中紧密连接蛋白闭合蛋白(Occludin)、闭锁小带蛋白1(ZO-1)的表达及变化趋势,探讨大麻二酚(CBD)对BBB的干预作用。方法采用改良Feeney自由落体法制备大鼠TBI模型,将大鼠随机分为3组:假手术组(Sham组)、模型组(TBI+vehicle组)和CBD干预组(TBI+CBD组),每组24只;每个组又分为损伤后8 h、1、2、3、5和7 d共6个时间点,通过免疫组化和免疫荧光染色,Western blot检测与BBB通透性密切相关的Occludin和ZO-1在不同时间点的表达情况;通过荧光素钠实验检测BBB通透性。结果免疫组化实验结果表明,与假手术组相比,TBI后随着时间推移Occludin和ZO-1蛋白阳性表达减少(P<0.05),2 d达到最低;CBD干预1 d后Occludin和ZO-1蛋白表达水平均上调(P<0.05);免疫荧光染色实验与Western blot结果趋势近似,与假手术组相比,TBI后Occludin和ZO-1荧光表达强度及蛋白表达量减少(P<0.05),CBD干预2 d后Occludin和ZO-1表达水平上调(P<0.05);荧光素钠实验结果表明,TBI后脑组织BBB完整性遭到破坏,通透性升高(P<0.01),CBD干预后BBB通透性下降(P<0.05)。结论TBI后紧密连接蛋白Occludin和ZO-1表达下降,BBB通透性升高,CBD干预可逆转TBI对BBB的破坏。

饲粮锌缺乏对22~42日龄肉仔鸡生长性能、小肠形态、屏障功能及空肠中紧密连接蛋白mRNA表达的影响

本试验旨在研究饲粮锌缺乏对22~42日龄肉仔鸡生长性能、小肠形态、屏障功能及空肠中紧密连接蛋白mRNA表达的影响。试验选用140只1日龄爱拔益加肉仔鸡公雏,1~21日龄统一饲喂正常添加七水合硫酸锌(ZnSO 4·7H 2O)的试验饲粮(实测锌含量为84.40 mg/kg);22日龄时,从中选取体重接近的112只肉仔鸡,采用单因子完全随机试验设计,随机分为2组(每组7个重复,每个重复8只鸡),对照组饲喂满足肉仔鸡锌需要量的试验饲粮(基础饲粮中添加40 mg/kg锌,实测锌含量为64.36 mg/kg),锌缺乏组饲喂不添加锌的试验饲粮(基础饲粮中不添加锌,实测锌含量为22.23 mg/kg)。试验期42 d。结果表明,与对照组相比:1)锌缺乏组22~42日龄肉仔鸡料重比显著增加(P<0.05);2)锌缺乏组42日龄肉仔鸡十二指肠和空肠隐窝深度显著增加(P<0.05),42日龄肉仔鸡十二指肠、空肠和回肠绒毛高度/隐窝深度以及空肠绒毛高度和绒毛表面积显著降低(P<0.05);3)锌缺乏组42日龄肉仔鸡翅静脉和肝门静脉血清二胺氧化酶活性和异硫氰酸荧光素-葡聚糖含量显著增加(P<0.05);4)锌缺乏组42日龄肉仔鸡空肠中黏膜闭合蛋白-1(Claudin-1)的mRNA表达水平显著降低(P<0.05)。综上所述,饲粮锌缺乏可能通过降低紧密连接蛋白Claudin-1的mRNA表达而损伤肉仔鸡小肠黏膜完整性和屏障功能,进而降低其饲料转化效率。

丁酸钠通过调节肠道通透性改善酒精性肝病小鼠炎症的机制

目的探讨丁酸钠通过肠—肝循环改善肠道通透性来降低酒精性肝病(ALD)小鼠的炎性因子水平。方法将60只雌性C57BL/6J小鼠随机分为阴性对照组、ALD模型组、丁酸钠(NaB)干预对照组、NaB干预模型组,每组15只。对照组给予Lieber-DeCarli对照液体饲料,模型组给予等热量的Lieber-DeCarli酒精液体饲料,同时NaB干预对照组给予含NaB(0.6 g·kg^(-1))的液体饲料,喂养6周后,收集组织。采用HE染色检测肠道炎性细胞浸润及黏膜完整性;免疫荧光检测肠道紧密连接蛋白ZO-1、Occludin的表达水平;鲎试剂盒检测血浆、肝脏及肠道内毒素(LPS)的水平;ELISA法检测血浆、肝脏及肠道白细胞介素(IL)-6、IL-1β、IL-17A的水平;并分析紧密连接蛋白与肠道炎性因子表达水平的相关性。结果与阴性对照组相比,HE染色结果显示,ALD模型组小鼠的肠道绒毛结构紊乱;免疫荧光结果显示,紧密连接蛋白表达降低(P<0.05);血浆、肝脏及肠道LPS均升高(P均<0.05);血浆、肝脏及肠道炎性因子水平均升高(P均<0.05)。与ALD模型组相比,NaB干预模型组小鼠肠道绒毛结构好转,肠道紧密连接蛋白表达升高,血浆、肝脏及肠道LPS水平降低,血浆、肝脏及肠道的炎性因子水平降低(P均<0.05)。相关性分析结果显示,肠道紧密连接蛋白与肠道炎性因子表达水平呈负相关(P<0.05)。结论丁酸钠通过肠—肝循环改善肠道通透性来降低ALD小鼠血浆、肝脏及肠道的炎性因子水平,从而改善ALD小鼠的炎性反应。

紧密连接蛋白介导的十三味辛夷滴鼻剂对小鼠鼻腔黏膜修复机制探究

目的 探讨十三味辛夷滴鼻剂对鼻腔黏膜损伤小鼠的修复作用及其可能的干预机制。方法 制备小鼠鼻腔黏膜热损伤模型,随机分为空白对照组、模型对照组、滴通鼻炎水阳性对照组[1μl/(只·次·侧)]、十三味辛夷滴鼻剂高剂量[5μl/(只·次·侧)]、中剂量[2.5μl/(只·次·侧)]、低剂量组[1.25μl/(只·次·侧)],每组各10只,雌雄各半。给药7 d后,分级评价鼻黏膜红肿程度,苏木素-伊红(HE)染色观察鼻黏膜组织病理结构改变,免疫组化分析鼻黏膜上皮ZO-1、Claudin-1、Occludin表达水平。结果 给药7 d后,模型对照组红肿程度评分显著高于空白对照组(P <0.05),滴通鼻炎水阳性对照组、十三味辛夷滴鼻剂高、中、低剂量组均低于模型对照组;HE染色显示,十三味辛夷滴鼻剂各组随剂量增高,黏膜层逐渐变薄、黏膜结构趋于整齐;与模型对照组比较,十三味辛夷滴鼻剂高剂量组ZO-1、Claudin-1、Occludin蛋白表达量显著提高(P <0.05)。结论 十三味辛夷滴鼻剂可能通过上调鼻黏膜上皮紧密连接蛋白ZO-1、Claudin-1、Occludin的表达,修复小鼠鼻腔黏膜损伤。

短链脂肪酸对苯并[a]芘致小鼠认知功能障碍的影响

目的 观察短链脂肪酸(short-chain fatty acids, SCFAs)对苯并[a]芘(benzo[a]pyrene, B[a]P)慢性染毒小鼠的认知功能、大脑皮质和结肠的影响,并探讨其作用机制。方法 将30只8周ICR雄性小鼠随机分为对照组、B[a]P组和B[a]P+SCFAs组,每组10只。B[a]P组以10.0 mg/kg剂量隔天定时灌胃染毒,共45次,对照组灌胃等体积的花生油,两组均自由饮用纯净水。B[a]P+SCFAs组在10.0 mg/kg B[a]P染毒同时,其饮用水为SCFAs混合液(67.5 mmol/L乙酸钠、40 mmol/L丁酸钠和25.9 mmol/L丙酸钠)。染毒结束,采用T迷宫、新位置识别实验、洞板实验和爬杆实验检测小鼠认知功能改变。HE法观察皮质、结肠病理改变;ELISA法测定皮质和结肠中炎症因子IL-1β和TNF-α表达水平;RT-PCR法和Western blot法分别检测结肠紧密连接蛋白ZO-1和Occludin的mRNA和蛋白表达水平。结果 与对照组相比,B[a]P组小鼠空间记忆力、运动能力减弱,焦虑样反应增加(P<0.05),提示小鼠认知功能障碍发生;同时,小鼠大脑出现大量红色神经元,肠道细胞排列紊乱,肠绒毛断裂不全且间隔稀疏,杯状细胞明显减少且分布不均;皮质和结肠中IL-1β含量均显著增加(P<0.05),TNF-α表达呈上升趋势但无统计学意义(P>0.05);结肠ZO-1、Occludin mRNA和蛋白表达水平均显著降低(P<0.05)。与B[a]P组比较,B[a]P+SCFAs组小鼠空间记忆力、运动能力增强,焦虑样反应显著降低(P<0.05),提示SCFAs干预显著改善B[a]P所致的小鼠认知功能障碍。同时,SCFAs干预显著减少大脑红色神经元,结肠杯状细胞数量有所恢复,并显著降低小鼠皮质和结肠中IL-1β、皮质中TNF-α的含量(P<0.05),结肠ZO-1、Occludin mRNA和蛋白表达水平均显著升高(P<0.05)。结论 SCFAs干预能有效改善B[a]P诱导的小鼠认知功能障碍,可能与其增加结肠紧密连接蛋白mRNA和蛋白的表达,降低肠道通透性,促进肠道屏障修复进而减轻肠道和神经炎症有关。

电针对2型糖尿病模型大鼠肠黏膜屏障的影响

目的:观察电针对2型糖尿病(T2DM)模型大鼠全身炎症及肠黏膜屏障功能的影响。方法:将SD大鼠随机分为正常组、模型组与电针组,每组6只。采用高脂饲料结合单次小剂量链脲佐菌素(STZ)腹腔注射建立T2DM模型。电针组取双侧“足三里”“三阴交”“脾俞”“肾俞”,每周3次,给予8周治疗。治疗后检测各组大鼠空腹血糖(FBG);用ELISA法检测大鼠血清中空腹胰岛素(FINS)、肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、内毒素(LPS)、D-乳酸(D-LA)及二胺氧化酶(DAO)含量,并计算胰岛素抵抗指数(HOMA-IR);透射电镜观察大鼠小肠黏膜上皮超微结构;WB检测大鼠小肠上皮紧密连接蛋白ZO-1、Occludin和Claudin-1的表达。结果:与模型组比较,电针能降低T2DM大鼠FBG、FINS和HOMA-IR(P<0.01),下调血清中炎症因子TNF-α、IL-6含量(P<0.01),下调血清LPS、D-LA和DAO含量(P<0.01),上调小肠上皮ZO-1、Occludin及Claudin-1蛋白表达(P<0.01)。结论:电针“足三里”“三阴交”“脾俞”“肾俞”能够降低T2DM大鼠空腹血糖,降低全身炎症并改善胰岛素抵抗状态,其作用机制与上调小肠上皮紧密连接蛋白、改善肠黏膜通透性和修复大鼠肠黏膜屏障功能有关。