Role of Glutamine in the Mediation of E-cadherin, p120-catenin and Inflammation in Ventilator-induced Lung Injury

Background： Ventilator-induced lung injury （VILI） is commonly associated with barrier dysfunction and inflammation reaction. Glutamine could ameliorate VILI, but its role has not been fully elucidated, This study examined the relationship between inflammatory cytokines （interleukin JILl-6, tumor necrosis factor [TNF]-α, and IL-10） and adherens junctions （E-cadherin, p 120-catenin）, which were ameliorated by glutamine in VILI, both in vitro and in vivo. Methods： For the in vivo study, 30 healthy C57BL/6 mice weighing 25-30 g were randomly divided into five groups with random number table （n = 6 in each group）： control （Group C）; low tidal volume （Group L）; low tidal volume ＋ glutamine （Group L ＋ G）; high tidal volume （Group H）; and high tidal volume ＋ glutamine （Group H ＋ G）. Mice in all groups, except Group C, underwent mechanical ventilation for 4 h. For the in vitro study, mouse lung epithelial 12 （MLE- 12） cells pretreated with glutamine underwent cyclic stretching at 20% for 4 h. Cell lysate and lung tissue were obtained to detect the junction proteins, inflammatory cytokines, and lung pathological changes by the Western blotting, cytokine assay, hematoxylin and eosin staining, and immunofluorescence. Results： In vivo, compared with Group C, total cell counts （t= -28.182, P 〈 0.01）, the percentage of neutrophils （t = -28.095, P 〈 0.01）, IL-6 （t = -28.296, P 〈 0.01 ）, and TNF-α（t = - 19.812, P 〈 0.01 ） in bronchoalveolar lavage （BAL） fluid, lung injury scores （t = -6.708, P 〈 0.01）, and the wet-to-dry ratio （t = - 15.595, P 〈 0.01 ） were increased in Group H; IL- 10 in BAL fluid （t = 9.093, P 〈 0.01 ） and the expression of E-cadherin （t= 10.044, P 〈 0.01） and p120-catenin （t = 13.218, P 〈 0.01） were decreased in Group H. Compared with Group H, total cell counts （t - 14.844, P 〈 0.01 ）, the percentage of neutrophils （t = 18.077, P 〈 0.0 l ）, IL-6 （t - 18.007, P 〈 0.01 ）, and TNF-α （t =1 0.171, P 〈 0.01 ） in BAL fluid were decreased in Group H ＋ G; IL-10 in BAL fluid （t - -7.531, P 〈 0.01 ） and the expression of E-cadherin （t = - 14.814, P 〈 0.01 ） and p 120-catenin （t = -9.114, P 〈 0.01 ） were increased in Group H ＋ G. In vitro, compared with the nonstretching group, the levels of IL-6 （t = 21.111, P 〈 0.01 ） and TNF-α （t - 15.270, P 〈 0.01 ） were increased in the 20% cyclic stretching group; the levels of IL- 10 （t = 5.450, P 〈 0.01 ） and the expression of E-cadherin （t = 17.736, P 〈 0.01 ） and p 120-catenin （t = 16.136, P 〈 0.01 ） were decreased in the 20% cyclic stretching group. Compared with the stretching group, the levels of IL-6 （t = 11.818, P 〈 0.01） and TNF-α （t = 8.631, P 〈 0.01 ） decreased in the glutamine group; the levels of IL- 10 （t = 3.203, P 〈 0.05） and the expression of E-cadherin （t= 13.567, P 〈 0.01） and p 120-catenin （t = -10.013, P 〈 0.01） were increased in the glutamine group. Conclusions： High tidal volume mechanical ventilation and 20% cyclic stretching could cause VIM. Glutamine regulates VIM by improving cytokines and increasing the adherens junctions, protein E-cadherin and p 120-catenin, to enhance the epithelial barrier function.

Preparation and properties of poly HTBN-based urethane urea/organo reactive montmorillonite nanocomposites

With ultrasonic assistant mixing way, an intercalated mixture of polyol/ organo reactive montmorillonite （ORMMT） was pretreated. The prepolymer composed MMT clay was prepared by reaction of polyol/ORMMT mixture with toluene diisocyanate （TDI）. The resultant prepolymer reacted with extender （DMTDA） and then the polyur- ethane-urealorgano reactive montmorillonite （PUUIORMMT） nanocomposites were obtained. The structure, morphology and properties of PUU/ORMMT nanocomposites were characterized by FT-IR, TEM, AFM, strain-stress machine, TGA, and dynamic mechanical analysis （DMA）. The results showed that when tl-m OMMT content is 3%, the PUU/ORMMT nanocomposities performed super mechanical properties. Because of the presence of ORMMT, both Tg of the soft segment and tan5 of the PUU increased, and the decomposition temperature for the first step and the second step increased respectively. TEM images showed that the organophilic MMT particles in the PUU composite exhibit a high degree of intercalation and exfoliation.

TNF /TNFR1 signaling pathway and endocytoplasmic reticulum stress (ERs) mediated apoptosis are involved in ofloxacin and marbofloxacin-induced apoptosis of juvenile dog joint chondrocytes in early stage in vitro.

Quinolones (QNs) are widely used for their broad antibacterial spectrum and good antibacterial activities,desirable pharmacokinetic characteristics and few cross reactions with other therapeutic agents. However,QNs have adverse effects including chondrotoxicity in juvenile animals,so the use of QNs is contraindicated in children and adolescents. In regarding to the chondrotoxicity of QNs,numerous studies have been done. The current hypothesis suggests that QNs compete with the β1 integrin receptors residing on chondrocyte surface for extracellular Mg ions,which leads to alternation in β1 integrin expression,or function and eventually results in chondrocyte death. Stupack et al (2001)demonstrated that caspase-8 could be recruited to unligated integrins in adherent cells and further initiate apoptosis in a death receptor-independent manner. Sheng et al (2008) found that ofloxacin induced rabbit's chondrocyte apoptosis by causing disturbance of β1 integrin functions and subsequently through caspase-8-dependent mitochondrial pathway.Apoptosis could be initiated through the stimulation of death receptors and through an intrinsic pathway from mitochondria. Santangelo and Bertone (2011) and Wu et al (2011) found that TNFα could be expressed in human primary condrocytes inducing by interleukin-1beta (IL-1) or lipopolysaccharide (LPS). However,to date there have not been sufficient results to support that signaling from the death receptors was involved in QNs-induced chondrocyte apoptosis.In addition,dilated cisternae of rough endoplasmic reticulum (ER) have been noticed in QNs-induced arthropathy.ER can participate in the initiation of apoptosis. Varieties of harmful cellular stimuli could lead to ERs (ER stress). Elevated ERs results in cellular apoptosis. But whether ERs mediates apoptosis in QNs-treated chondrocytes is not clear yet.In this study,we chose two QNs agents and chondrocytes were treated with ofloxacin and marbofloxacin at final concentrations of 20 μg / mL,50 μg / mL and 100 μg / mL respectively in vitro for 2 h,8 h and 24 h. Cell survival rate,cell apoptosis rate and death receptor pathway factors TNFα (intracellular tumor necrosis factor-alpha),TNFR1 (TNF receptor-1),TRADD (TNF receptor 1 associated via death domain),FADD (Fas-associated protein with death domain),caspase-8 and ERs mediated apoptosis factors caspase-12,GADD153 (CHOP or DDIT3),GRP78 (Bip),calpain,and anti-apoptosis factors Bcl-2 (B-cell lymphoma 2),NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) gene expression levels were measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis to determine the dose-response relationship. We further silenced the expression of TNFR1 successfully by transferring TNFR1-siRNA to chondrocytes to confirm whether TNFα / TNFR1 signaling pathways are involved ofloxacin and marbofloxacin-induced apoptosis. Furthermore,expression of death receptor pathway representative proteins TNFα / TNFR1 and endocytoplasmic reticulum (ER) pathway representative protein caspase-12 were confirmed using Western Blot.We have found that ofloxacin and marbofloxacin could induce apoptosis of chondrocytes in a time-and dose-dependent fashion within 24 h. mRNA of TNFα,TNFR1,TRADD,FADD and caspase-8 (caspase-8 of ofloxacin treated group were at 24 h) were highly expressed at 8 h,and GADD153,GRP78,calpain and caspase-12 at 8 h or 24 h,and antiapoptosis factors NF-κB and Bcl-2 were also raised after 2 h,all in a dose-dependent fashion. Expression of caspase-8was downregulated after silenced TNFR1. TNFα and TNFR1 proteins were expressed at 8 h and caspase-12 proteins were expressed at 24 h. In addition,ofloxacin showed a higher toxicity.Our results indicate that death receptor pathway TNF / TNFR1 and ERs mediated apoptosis factors are involved in ofloxacin and marbofloxacin-induced apoptosis of in vitro cultured juvenile dog joint chondrocytes within 24 h.