AIM:To investigate whether miRNA-155(miR-155)dysregulates apical junctional complex(AJC)protein expression in experimental severe acute pancreatitis(SAP).METHODS:Twenty-four male BALB/c mice were randomly assigned to ...AIM:To investigate whether miRNA-155(miR-155)dysregulates apical junctional complex(AJC)protein expression in experimental severe acute pancreatitis(SAP).METHODS:Twenty-four male BALB/c mice were randomly assigned to two groups:the SAP group(n=12)receiving sequential intraperitoneal injection of 50μg/kg caerulein and 10 mg/kg lipopolysaccharide over 6h,and the control group(n=12)receiving intraperitoneal injection of normal saline.Animals were sacrificed3 h following the last injection for collection of blood samples and pancreas and distal ileal segment specimens.Routine pancreas and intestine histology was used to assess SAP pathology and intestinal epithelial barrier damage.Levels of serum amylase,diamine oxidase(DAO),and tumor necrosis factor(TNF)-αwere determined using commercial kits.Total RNA samples were isolated from intestinal epithelial specimens and reversely transcribed into cDNA.miR-155 and RhoA mRNA expression profiles were determined using quantitative real-time polymerase chain reaction.Target genes for miR-155 were predicted using the miRTarBase database,RNA22 and PicTar computational methods.Western blotting was performed to quantitate the protein expression levels of the target gene RhoA,as well as zonula occludens(ZO)-1 and E-cadherin,two AJC component proteins.RESULTS:Intraperitoneal injection of caerulein and lipopolysaccharide successfully induced experimental acute pancreatic damage(SAP vs control,10.0±2.0vs 3.2±1.2,P<0.01)and intestinal epithelial barrier damage(3.2±0.7 vs 1.4±0.7,P<0.01).Levels of serum amylase(21.6±5.1 U/mL vs 14.3±4.2 U/mL,P<0.01),DAO(21.4±4.1 mg/mL vs 2.6±0.8 mg/mL,P<0.01),and TNF-α(61.0±15.1 ng/mL vs 42.9±13.9 ng/mL,P<0.01)increased significantly in SAP mice compared to those in control mice.miR-155 was significantly overexpressed in SAP intestinal epithelia(1.94±0.50 fold vs 1.03±0.23 fold,P<0.01),and RhoA gene containing three miR-155-specific binding sites in the three prime untranslated regions was one of the target genes for miR-155.RhoA(22.7±5.8 folds vs 59.6±11.6 folds,P<0.01),ZO-1(46±18 folds vs68±19 folds,P<0.01),and E-cadherin proteins(48±15 folds vs 77±18 folds,P<0.01)were underexpressed in SAP intestinal epithelia although RhoA mRNA expression was not significantly changed in SAP(0.97±0.18 folds vs 1.01±0.17 folds,P>0.05).CONCLUSION:TNF-α-regulated miR-155 overexpression inhibits AJC component protein syntheses of ZO-1,and E-cadherin by downregulating post-transcriptional RhoA expression,and disrupts intestinal epithelial barrier in experimental SAP.展开更多
Trichomonas vaginalis,a protist parasite of the urogenital tract in humans,is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development.In the present study w...Trichomonas vaginalis,a protist parasite of the urogenital tract in humans,is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development.In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T.vaginalis and the influence of the iron concentration present in the parasite’s culture medium on the interaction effects.Our results show that T.vaginalis adheres to the epithelial cell causing alterations in the junctional complex,such as:(a)a decrease in transepithelial electrical resistance;(b)alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin,occludin and ZO-1;and(c)enlargement of the spaces between epithelial cells.These effects were dependent on(a)the degree of the parasite virulence isolate,(b)the iron concentration in the culture medium,and(c)the expression of adhesin proteins on the parasite surface.展开更多
Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal c...Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.展开更多
D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-ma...D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine.All 1800 grass carp(16.20±0.01 g)were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose(0.52,1.75,3.02,4.28,5.50 and 6.78 g/kg diet)for 70 d.The study revealed that D-mannose increased feed intake(P 0.05).D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value(P<0.05).D-mannose supplementation at 4.28 g/kg increased intestinal length,intestinal weight and fold height of grass carp compared to the control group(P<0.05).This improvement may be attributed to the phosphomannose isomerase(PMI)-mediated enhancement of glycolysis.This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content(P<0.05)and improved cellular and intercellular structures for the first time.The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum(ER)stress through the inositol-requiring enzyme 1(IRE1),RNA-dependent protein kinase-like ER kinase(PERK),and activating transcription factor 6(ATF6)signaling pathways.The alleviation of ER stress may be linked to the phosphomannomutase(PMM)-mediated enhancement of protein glycosylation.In addition,ubiquitin-dependent[PTEN-induced putative kinase 1(PINK1)/Parkin]and ubiquitin-independent[BCL2-interacting protein 3-like(BNIP3L),BCL2-interacting protein 3(BNIP3),and FUN14 domain containing 1(FUNDC1)]mitophagy may play a role in maintaining cellular redox homeostasis.The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures,which may be closely associated with the small Rho GTPase protein(RhoA)/the Rho-associated protein kinase(ROCK)signaling pathway.In conclusion,D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways,and enhanced intercellular structures related to tight junctions and adherent junctions.Furthermore,quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg,respectively.展开更多
基金Supported by The research grants from Shanghai Municipal Science and Technology CommissionNo.114119b2900+1 种基金Shanghai Municipal Key Laboratory of Pancreatic DiseaseNo.P2012006
文摘AIM:To investigate whether miRNA-155(miR-155)dysregulates apical junctional complex(AJC)protein expression in experimental severe acute pancreatitis(SAP).METHODS:Twenty-four male BALB/c mice were randomly assigned to two groups:the SAP group(n=12)receiving sequential intraperitoneal injection of 50μg/kg caerulein and 10 mg/kg lipopolysaccharide over 6h,and the control group(n=12)receiving intraperitoneal injection of normal saline.Animals were sacrificed3 h following the last injection for collection of blood samples and pancreas and distal ileal segment specimens.Routine pancreas and intestine histology was used to assess SAP pathology and intestinal epithelial barrier damage.Levels of serum amylase,diamine oxidase(DAO),and tumor necrosis factor(TNF)-αwere determined using commercial kits.Total RNA samples were isolated from intestinal epithelial specimens and reversely transcribed into cDNA.miR-155 and RhoA mRNA expression profiles were determined using quantitative real-time polymerase chain reaction.Target genes for miR-155 were predicted using the miRTarBase database,RNA22 and PicTar computational methods.Western blotting was performed to quantitate the protein expression levels of the target gene RhoA,as well as zonula occludens(ZO)-1 and E-cadherin,two AJC component proteins.RESULTS:Intraperitoneal injection of caerulein and lipopolysaccharide successfully induced experimental acute pancreatic damage(SAP vs control,10.0±2.0vs 3.2±1.2,P<0.01)and intestinal epithelial barrier damage(3.2±0.7 vs 1.4±0.7,P<0.01).Levels of serum amylase(21.6±5.1 U/mL vs 14.3±4.2 U/mL,P<0.01),DAO(21.4±4.1 mg/mL vs 2.6±0.8 mg/mL,P<0.01),and TNF-α(61.0±15.1 ng/mL vs 42.9±13.9 ng/mL,P<0.01)increased significantly in SAP mice compared to those in control mice.miR-155 was significantly overexpressed in SAP intestinal epithelia(1.94±0.50 fold vs 1.03±0.23 fold,P<0.01),and RhoA gene containing three miR-155-specific binding sites in the three prime untranslated regions was one of the target genes for miR-155.RhoA(22.7±5.8 folds vs 59.6±11.6 folds,P<0.01),ZO-1(46±18 folds vs68±19 folds,P<0.01),and E-cadherin proteins(48±15 folds vs 77±18 folds,P<0.01)were underexpressed in SAP intestinal epithelia although RhoA mRNA expression was not significantly changed in SAP(0.97±0.18 folds vs 1.01±0.17 folds,P>0.05).CONCLUSION:TNF-α-regulated miR-155 overexpression inhibits AJC component protein syntheses of ZO-1,and E-cadherin by downregulating post-transcriptional RhoA expression,and disrupts intestinal epithelial barrier in experimental SAP.
基金This work was supported by Fundção Ary Frauzino para Pesquisa e Controle do Cfincer.Instituto Nacional de Cãncer.Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro(FAPERJ)Conselho Nacional de Desenvolvimento Cientfico e Tecnológico (CNPq)Associação Universitfiria Santa Ursula(AUSU).
文摘Trichomonas vaginalis,a protist parasite of the urogenital tract in humans,is the causative agent of trichomonosis,which in recent years have been associated with the cervical cancer development.In the present study we analyzed the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T.vaginalis and the influence of the iron concentration present in the parasite’s culture medium on the interaction effects.Our results show that T.vaginalis adheres to the epithelial cell causing alterations in the junctional complex,such as:(a)a decrease in transepithelial electrical resistance;(b)alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin,occludin and ZO-1;and(c)enlargement of the spaces between epithelial cells.These effects were dependent on(a)the degree of the parasite virulence isolate,(b)the iron concentration in the culture medium,and(c)the expression of adhesin proteins on the parasite surface.
文摘Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.
基金supported by the earmarked fund for CARS (CARS-45),and the National Key R&D Program of China (2019YFD0900200).
文摘D-mannose,essential for protein glycosylation,has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis.In addition to evaluating growth performance,we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine.All 1800 grass carp(16.20±0.01 g)were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose(0.52,1.75,3.02,4.28,5.50 and 6.78 g/kg diet)for 70 d.The study revealed that D-mannose increased feed intake(P 0.05).D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value(P<0.05).D-mannose supplementation at 4.28 g/kg increased intestinal length,intestinal weight and fold height of grass carp compared to the control group(P<0.05).This improvement may be attributed to the phosphomannose isomerase(PMI)-mediated enhancement of glycolysis.This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content(P<0.05)and improved cellular and intercellular structures for the first time.The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum(ER)stress through the inositol-requiring enzyme 1(IRE1),RNA-dependent protein kinase-like ER kinase(PERK),and activating transcription factor 6(ATF6)signaling pathways.The alleviation of ER stress may be linked to the phosphomannomutase(PMM)-mediated enhancement of protein glycosylation.In addition,ubiquitin-dependent[PTEN-induced putative kinase 1(PINK1)/Parkin]and ubiquitin-independent[BCL2-interacting protein 3-like(BNIP3L),BCL2-interacting protein 3(BNIP3),and FUN14 domain containing 1(FUNDC1)]mitophagy may play a role in maintaining cellular redox homeostasis.The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures,which may be closely associated with the small Rho GTPase protein(RhoA)/the Rho-associated protein kinase(ROCK)signaling pathway.In conclusion,D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways,and enhanced intercellular structures related to tight junctions and adherent junctions.Furthermore,quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg,respectively.
