Effects of cyclosporin A pre-treatment combined with etomidate post-treatment on lung injury induced by limb ischemia-reperfusion in rats

Objectives To investigate the influence of cyclosporin A (CsA) pre-treatment and etomidate (ETO) post-treatment on lung injury induced by limb ischemia-reperfusion (I/R) in rats. Methods Rats were randomly divided into five groups: sham, I/R, I/R+CsA, I/R+ETO, and I/R+CsA+ETO. Limb I/R lung injury was established by bilateral clamping of the femoral arteries for 2 hours. Following reperfusion for 3 hours, blood gas analysis was performed. Pathological changes were assessed using immunohistochemistry. The apoptosis index (AI) and wet/dry weight ratio (W/D) were calculated. Levels of Fas protein and FasL mRNA were assessed by western blotting and RT-PCR, respectively. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β were detected by ELISA. Results I/R resulted in decreased PaO2 but increased AI, W/D, Fas, FasL mRNA, TNF-α and IL-1β. Scattered punctate apoptosis and necrosis were observed by immunohistochemistry. Compared with the I/R group, the I/R+ETO and I/R+CsA groups showed increased SpO2, decreased AI, W/D, Fas, FasL mRNA, TNF-α and IL-1β, and decreased numbers of apoptotic and necrotic cells. Combined treatment with CsA+ETO resulted in more dramatic changes in these parameters. Conclusions ETO post-treatment and CsA pretreatment reduced lung injury induced by limb I/R in rats. The mechanism may be related to synergistic inhibition of Fas/FasL signaling.

Insights into the Molecular Mechanism Accounting for Neutropenia in T-Large Granular Lymphocytes Leukemia

INTRODUCTION: Neutropenia represents the most frequent clinical manifestation in T-Large Granular Lymphocytes Leukemia (T-LGLL) patients. Literature data provided evidence of the involvement of soluble Fas Ligand (sFasL) in this process. Consistently, we confirmed that neutropenic T-LGLL patients were characterized by higher levels of sFasL than non neutropenic patients. We also demonstrated that FasL transcription was mediated by the Signal Transducer and Activator of Transcription (STAT)-3 and we showed that high STAT3 activation correlated with high levels of sFas. However, the mechanism through which STAT3 regulates FasL production still remains elusive. It is well known that FasL expression depends on post-transcriptional events involving ARE-binding proteins, such as Human antigen R (HuR). Important regulators of post-transcriptional modifications are microRNAs (miRNAs), that are small non-coding RNA molecules able to bind target mRNAs, promoting their degradation or blocking protein translation. Among them, miR-146b was identified to be induced by STAT3 in non-transformed cells. This work aims to determine whether miR-146b might regulate STAT3-mediated expression of FasL, thus playing a role in the pathogenesis of neutropenia in T-LGLL patients. METHODS: T-Large Granular Lymphocytes (T-LGLs) were purified by FACSAria cell sorter from PBMCs of untreated T-LGLL patients. High throughput and single miRNA analysis were carried out on purified LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. Transcriptional and protein expression levels were evaluated by Real Time-PCR and Western Blot (WB) assays. RESULTS: By assessing the expression of 756 mature miRNAs on purified patients' T-LGLs, we identified miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC); selected miRNAs were then analyzed for correlation with ANC. Among them, miR-146b expression was the only one correlated with ANC, being down-regulated in neutropenic patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control, indicating that miR-146b affected FasL expression at a post-transcriptional level. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients. In the end, we demonstrated the mechanism affecting miR-146b expression in the presence of STAT3 activation, pointing to a role of epigenetic modulation taking place, since the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) can restore STAT3-miR-146b axis. CONCLUSIONS: In this work we suggest a pathogenetic link between STAT3 activation, a defective miR-146b expression and neutropenia development in T-LGLL. Treatment with a demethylating agent may restore STAT3-dependent induction of miR-146b and may represent a new potential therapeutic strategy for the treatment of neutropenia in T-LGLL patients. Disclosures No relevant conflicts of interest to declare.

Effect of Puerarin on Expression of Fas/FasL mRNA in Pulmonary Injury induced by Ischemia-Reperfusion in Rabbits

The effect of puerarin (Pur) on expressions of Fas/FasL mRNAs in pulmonary ischemia and reperfusion injury (PIRI) in rabbit was investigated. The sole side lung ischemia and reperfusion model was used. Rabbits were randomly divided into three groups, a sham operated group (sham, n = 10), PIR group (IR, n =30) and PIR + Pur group (Pur, n =30). Changes of several parameters including apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured after 60, 180 and 300 minutes of reperfusion. Meanwhile, the location and expression of Fas/FasL mRNA were investigated. Lung tissue was prepared for light microscopic and electron microscopic observation after 60, 180 and 300 minutes of reperfusion. Compared with group IR, Fas/FasL mRNAs were slightly expressed in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia in group Pur. The values of AI, W/D and IQA were significantly lower than those in group IR after 60, 180, and 300 minutes of reperfusion in lung tissue ( P <0.01 or P <0.05). Meanwhile, the abnormal changes in lung tissue morphology were markedly less in group Pur. Puerarin notably protects lung from PIRI by inhibiting Fas/FasL mRNA expression and decreasing lung cell apoptosis in rabbits.

Η επίδραση της ερυθροποιητίνης στην οξεία νεφρική ισχαιμία

Η επίδραση της ερυθροποιητίνης στην οξεία νεφρική ισχαιμία. Πειραματική μελέτη σε επίμυες.Η ερυθροποιητίνη (ΕΡΟ) εμφανίζει πλειοτροπικές ιδιότητες. Σκοπός της μελέτης ήταν η διερεύνηση της επίδρασης της ΕΡΟ στην οξεία νεφρική ισχαιμία/επαναιμάτωση (ΝΙ/Ε). Άρρενες επίμυες Wistar υποβλήθηκαν σε αμφοτερόπλευρη NΙ/Ε 45 λεπτών. Χωρίστηκαν τυχαία στις ομάδες Ι/Ε (n=34) και EPΟ (χορήγηση 500 IU/kg, i.p, 20 λεπτά πριν την ισχαιμία, n=36). Σε κάθε ομάδα ορίστηκαν τρεις υποομάδες, στις οποίες η επαναιμάτωση ολοκληρώθηκε στις 6, 24 και 48 ώρες αντίστοιχα. Χειρουργηθέντες επίμυες που δεν υποβλήθηκαν σε ισχαιμία αποτέλεσαν τους μάρτυρες (n=10). Η νεφρική βλάβη εκτιμήθηκε με βιοχημικούς δείκτες (ουρία και κρεατινίνη) και διαβάθμιση της έκτασης της σωληναριακής βλάβης. Η έκφραση των Cyt c, nNOS, iNOS, eNOS και COX-2 μελετήθηκε ανοσοϊστοχημικά και των Fas/FasL, nNOS και Cyt c με RT-PCR. Η ΝΙ/Ε προκάλεσε επιδείνωση των βιοχημικών δεικτών και εγκατάσταση ιστολογικών αλλοιώσεων. Στην ομάδα EPΟ διαπιστώθηκαν χαμηλότερες τιμές βιοχημικών δεικτών και μικρότερης έκτασης σωληναριακή βλάβη (p<0,05). mRNA των Fas/FasL και Cyt c απομονώθηκαν από τους φυσιολογικούς νεφρούς. Το Fas/FasL mRNA δεν μεταβλήθηκε στις δύο ομάδες. Η ΝΙ/Ε προκάλεσε αύξηση της γονιδιακής και πρωτεϊνικής έκφρασης του Cyt c (κυρίως στις 48 και στις 6 ώρες αντίστοιχα). Η ΕΡΟ μείωσε την γονιδιακή έκφραση του Cyt c στις 48 ώρες, ενώ περιόρισε την πρωτεϊνική έκφρασή του κυρίως στις 6 ώρες. Η έκφραση της nNOS στους φυσιολογικούς νεφρούς μειώθηκε σημαντικά στις 6 ώρες επαναιμάτωσης, ενώ στη συνέχεια έτεινε να αποκατασταθεί. Η ΕΡΟ διατήρησε την έκφραση της nNOS στις 6 ώρες στα επίπεδα των μαρτύρων. Περιορισμένη έκφραση της iNOS παρατηρείται στους φυσιολογικούς νεφρούς. Η iNOS ανοσοαντίδραση αυξήθηκε σταδιακά από τις 6 στις 24 ώρες επαναιμάτωσης στα σωληνάρια και αγγεία. Η ΕΡΟ περιόρισε την ένταση της iNOS έκφρασης μεταϊσχαιμικά. Η eNOS εκφράζεται στα φυσιολογικά σωληνάρια και αγγεία. Επαγωγή της σημειώθηκε στις 6 και 24 ώρες στην ομάδα Ι/Ε, ενώ στην ΕΡΟ ήταν εμφανής μόνο στις 24 και στους ιστούς με μεγάλη βλάβη. Η COX-2 εκφράζεται φυσιολογικά σε πυκνές κηλίδες και σωληνάρια. Επαγωγή της σημειώθηκε στις 6 ώρες επαναιμάτωσης, η οποία στη συνέχεια υποχώρησε. Η ΕΡΟ περιόρισε αυτήν την επαγωγική αιχμή της COX-2 των 6 ωρών. Συμπερασματικά, η χορήγηση της ΕΡΟ ήταν νεφροπροστατευτική. Η ΝΙ/Ε και η ΕΡΟ δεν επηρέασαν την έκφραση των Fas/FasL. Η πρώιμη επαγωγή του Cyt c υποδηλώνει συμμετοχή του στην μεταγενέστερη εγκατάσταση βλάβης. Η ευεργετική δράση της ΕΡΟ εκδηλώνεται μέσω του ενδογενούς αποπτωτικού μονοπατιού. Η nNOS συμμετέχει στη φυσιολογική νεφρική λειτουργία. Η πρώιμη εξασθένιση της μετά την ισχαιμία, εκτός από επακόλουθο της βλάβης, είναι πιθανά άμεσα επιζήμια. Η επαγωγική δράση της ΕΡΟ στην nNOS συσχετίζεται με αυξημένο αριθμό βιώσιμων σωληναρίων. Η μεταϊσχαιμική επαγωγή της iNOS συνδέθηκε με την εγκατάσταση σωληναριακής βλάβης. Η εξασθένιση της iNOS από την ΕΡΟ είναι αποτέλεσμα των αντιφλεγμονωδών και αντιοξειδωτικών ιδιοτήτων της. Η αύξηση της eNOS στη ΝΙ/Ε υποχώρησε μετά την εγκατάσταση σοβαρής βλάβης. Η δράση της ΕΡΟ δεν συσχετίζεται άμεσα με την eNOS. Η COX-2 συμμετέχει στους ομοιοστατικούς νεφρικούς μηχανισμούς. Η επαγωγή της στη ΝΙ/Ε ερμηνεύεται ως περαιτέρω ενεργοποίηση αυτών των μηχανισμών ή/και ως πυροδότηση φλεγμονής. Η ευεργετική δράση της ΕΡΟ περιορίζει την κινητοποίηση των μηχανισμών ομοιόστασης ή/και τον επιβλαβή ρόλο της COX-2.

p,p′-DDE Induces Apoptosis of Rat Sertoli Cells via a FasL-Dependent Pathway

One,1-dichloro-2,2 bis(p-chlorophenyl) ethylene (p,p′-DDE), the major metabolite of 2,2-bis(4-Chlorophenyl)-1,1,1-trichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. It has antiandrogenic effect. However, the mechanism by which p,p′-DDE exposure causes male reproductive toxicity remains unknown. In the present study, rat Sertoli cells were used to investigate the molecular mechanism involved in p,p′-DDE-induced toxicity in male reproductive system. The results indicated that p,p′-DDE exposure at over 30 μM showed the induction of apoptotic cell death. p,p′-DDE could induce increases in FasL mRNA and protein, which could be blocked by an antioxidant agent, N-acetyl-l-cysteine (NAC). In addition, caspase-3 and -8 were activated by p,p′-DDE treatment in these cells. The activation of NF-κB was enhanced with the increase of p,p′-DDE dose. Taken together, these results suggested that exposure to p,p′-DDE might induce apoptosis of rat Sertoli cells through a FasL-dependent pathway.

Mediators of apoptosis Fas and FasL predict disability progression in multiple sclerosis over a period of 10 years

TNF-α, IL-12p35, IL-12p40, IL-4, IL-10, TGF-β1, CCR3, CXCR3, CCR5, Fas and FasL mRNA levels in PBMC of 25 multiple sclerosis (MS) patients were quantified at baseline by real-time PCR according to a post-hoc study design. The baseline values of the different markers were analysed with respect to their correlation with the increase in disability over a period of 10 years. High levels of Fas mRNA were associated with a favourable disease course in relapsing-remitting (RR) MS (R2 = 0.74, P = 0.0001, n = 13), as measured by the Expanded Disability Status Scale (EDSS); high levels of FasL mRNA were associated with relatively mild disease progression (R2 = 0.86, P = 0.0001, n = 12) in secondary progressive (SP) MS. These findings suggest that Fas-mediated apoptosis plays a major role in the mechanism underlying long-term disease progression in MS.

Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo

Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highfschigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and β-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo. (Blood. 2006;108:123-133)

THE HISTOLOGICAL LOCALIZATION OF FasL mRNA IN HUMAN LUMBAR DISC TISSUE

Lumbar disc has become a degenerative tissue since teenage. Apoptosis plays an important role in the processes of degeneration. FasL, a ligand of Fas receptor (a trans-membrane protein) promote signal transduction of apoptosis. In this study, fetal disc and degenerative disc from adults were examined for FasL mRNA expression. hPBMC was first treated by PHA-P, a fragment of FasL cDNA was then synthesized through one-step RT-PCR. Verified by sequencing and recombinant plasmids linearizing, the DIG-cRNA probe was synthesized by in vitro transcription. The distribution of FasL mRNA was observed on lumbar intervertebral disc specimens using in situ hybridization. FasL mRNA signals were expressed in both notochord cells and chondrocyte-like cells in the nucleus of fetal discs. In degenerative disc, the quantity of intervertebral disc cells reduced dramatically and the expression of FasL mRNA was not detected. Expression of FasL in fetal disc implied that Fas/FasL triggered cell death signal pathway involved in lumbar disk transition and degeneration.