Potential influences of expression levels of MFGE8 and HMGB1 on the intestinal mucosal barrier function and inflammatory response after blunt abdominal injury in rats

Purpose: To explore the influence of milk fat globule-EGF factor 8 protein (MFGE8) on blunt abdominal injury in Sprague Dawley (SD) rats through the RhoA/ROCK signaling pathway. Methods: The blunt abdominal injury model was generated in SD rats. A total of 44 rats was randomly assigned into three groups. Rat blunt abdominal injury was assessed by the abbreviated injury scale (AIS). The rats were sacrificed for observing the morphology of the abdominal cavity and intestines. Hematoxylin and eosin staining was performed to visualize the pathological changes of rat intestines. Positive expressions of MFGE8 and high mobility group box 1 (HMGB1) in rat intestines were examined by immunohistochemical staining. Protein levels were determined by Western blot. Serum levels of tumor necrosis factor α (TNF-α), IL-1ß, IL-6 and malondialdehyde (MDA) were measured by enzyme linked immunosorbent assay (ELISA). Results: Blunt abdominal injury resulted in inflammatory response of intestinal tissues, increased serum levels of TNF-α, IL-1ß, IL-6 and MDA, upregulation of HMGB1, RhoA and ROCK2, and downregulation of MFGE8 in rats, which were significantly alleviated by intervention of rhMFGE8. Conclusions: MFGE8 protects the intestinal mucosal barrier function after blunt abdominal injury in rats by downregulating HMGB1. Moreover, it alleviates inflammatory response and oxidative stress caused by blunt abdominal injury in rats through downregulating RhoA and ROCK.

HMGB1 and inflammatory cytokines in experimental acute lung injury induced in rabbits / [HMGB1 e citocinas inflamatórias em lesão pulmonar aguda experimental induzida em coelhos]

The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)

O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)

HMGB1 and inflammatory cytokines in experimental acute lung injury induced in rabbits / [HMGB1 e citocinas inflamatórias em lesão pulmonar aguda experimental induzida em coelhos]

The aim of this work was to measure HMGB1, TNF-alpha, and IL-8 in bronchoalveolar lavage (BAL), serum and TLR2 and TLR4mRNA expression in lung tissue of rabbits with two grades of acute lung injury (ALI). The animals were randomly assigned to groups with severe (S) and mild/moderate (MM) ALI, induced with warm saline, and a control group. HMGB1, TNF-alpha, IL-8, TLR2mRNA and TLR4mRNA were measured after ALI induction. The results showed increased levels of IL-8, TNF-alpha, HMGB1 and TLR4mRNA in the ALI groups. HMGB1, IL-8 and TNF-alpha concentrations in BAL were higher in S compared MM. Increased TLR4mRNA was observed in S and MM versus control. The results suggest an early participation of HMGB1 in ALI together with IL-8 and TNF-alpha and association with severity. TLR4 has early expression and role in ALI pathophysiology but is not associated with severity.(AU)

O objetivo deste trabalho é determinar os níveis de HMGB1, TNF-alfa e IL-8 no lavado broncoalveolar (BAL), bem como quantificar a expressão sérica de TLR2 e TLR4 mRNA em tecido pulmonar de coelhos com dois graus de lesão pulmonar aguda (LPA). Os animais foram distribuídos aleatoriamente em grupos com LPA grave (S) e leve / moderada (MM), induzidas com solução salina morna, e um grupo controle. HMGB1, TNF-alfa, IL-8, TLR2mRNA e TLR4mRNA foram medidos após a indução de LPA e quatro horas de ventilação mecânica. Os resultados mostraram níveis aumentados de IL-8, TNF-alfa, HMGB1 e TLR4mRNA nos grupos com LPA. As concentrações de HMGB1, IL-8 e TNF-alfa no LBA foram maiores no S comparado ao MM. Aumento de TLR4mRNA foi observado em S e MM versus controle. Os resultados sugerem uma participação precoce da HMGB1 na LPA em conjunto com IL-8 e TNF-alfa e associação com a gravidade da LPA. O TLR4 foi expresso na ALI e possivelmente possui papel precoce na fisiopatologia da LPA, mas sem associação com a gravidade.(AU)

Effect of ozone oxidative preconditioning on inflammation and oxidative stress injury in rat model of renal transplantation

Purpose: To investigate the effect of ozone oxidative preconditioning (OzoneOP) on inflammation and oxidative stress injury in rat model of renal transplantation. Methods: Thirty six male Sprague Dawley (SD) rats were randomly divided into three groups. Sham group: rats were treated with opening and closing abdomen. Kidney transplantation group (KT group): SD rat received the donors left kidney derived from another SD rat. Ozone oxidative preconditioning and kidney transplantation (OOP+KT group): donor SD rats received OzoneOP treatments by transrectal insufflations before kidney transplantation. After transplantation, parameters of renal function of recipients were determined. Morphology and pathological changes of renal allograft were examined. Expression of NF-kBp65, HMGB-1 were also determined by Western-blot. Results: Compared to KT group, the morphology and pathological damages of renal allograft were less serious in OOP+KT group. Meanwhile, levels of SOD and GSH-Px of renal allograft in OOP+KT group were higher than those in KT group respectively. Western-blot showed that the expressions of NF-kBp65 and HMGB-1 in OOP+KT group were obviously less than those in KT group. Conclusion: Ozone oxidative preconditioning could attenuate the inflammatory reaction and oxidative stress injury in renal allograft, which might be related with the enhancement of anti-oxidative system and suppression of inflammatory reaction.(AU)

Allopurinol preconditioning attenuates renal ischemia/reperfusion injury by inhibiting HMGB1 expression in a rat model

PURPOSE:To investigate the potential effects of pretreatment with allopurinol on renal ischemia/reperfusion injury (IRI) in a rat model.METHODS:Twenty four rats were subjected to right kidney uninephrectomy were randomly distributed into the following three groups (n=8): Group A (sham-operated group); Group B (ischemic group) with 30 min of renal ischemia after surgery; and Group C (allopurinol + ischemia group) pretreated with allopurinol at 50 mg/kg for 14 days. At 72 h after renal reperfusion, the kidney was harvested to assess inflammation and apoptosis.RESULTS:Pretreatment with allopurinol significantly improved renal functional and histological grade scores following I/R injury (p<0.05). Compared with Group B, the expression levels of caspase-3 and Bax were markedly reduced in Group C, meanwhile, whereas expression of bcl-2 was clearly increased (p<0.05). A newly described marker of inflammation, High Mobility Group Box 1(HMGB1), showed reduced expression in Group C (p<0.05).CONCLUSION:Pretreatment with allopurinol had a protective effect on kidney ischemia/reperfusion injury, which might be related to the inhibition of HMGB1 expression.(AU)

Dexmedetomidine preconditioning inhibits the long term inflammation induced by renal ischemia/reperfusion injury in rats

PURPOSE:To investigate the protective effects of dexmedetomidine (Dex) against renal ischemia/reperfusion injury (IRI).METHODS:Sprague-Dawley rats were randomly divided to sham group, IRI group and Dex group. The SD rats were subjected to 45 min of ischemia followed by eight weeks of reperfusion. Prior to ischemia, rats were either treated with Dex or not. Blood samples were collected for the detection of blood urea nitrogen (BUN) and creatinine (Cr) levels. Immunohistochemistry was performed for CD3 T-cell infiltrates. Real-time PCR and western blot were detected for the expression of TNF-α, IL-1β, ICAM-1, HMGB1 and TLR4.RESULTS:Compared with sham group, renal IRI significantly increased the serum levels of BUN and Cr. The H&E staining indicated that renal IRI resulted in obvious renal injury and immunohistochemistry found that there were more CD3 T-cell infiltrates in IRI group. Also, renal IRI upregulated the expression of TNF-α, IL-1β, ICAM-1, HMGB1 and TLR4. However, all these changes were alleviated by the treatment with Dex.CONCLUSIONS:Dexmedetomidine has beneficial effects on long term inflammation induced by renal ischemia/reperfusion injury. Its mechanisms may be achieved through inhibiting the HMGB1/TLR4 pathway to exert protective effects.(AU)

Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and peri-implantitis

High mobility group chromosomal protein B1 (HMGB1) and N2 (HMGN2), two members of High mobility group (HMG) family, play important role in inflammation. The purposes of this study were to investigate the expression of HMGB1 and HMGN2 in periodontistis. The expression of HMGB1 and HMGN2 mRNA in gingival tissues and gingival crevicular fluid (GCF) in chronic periodontitis (CP), generalized aggressive periodontitis (G-AgP) patients and healthy subjects was detected by real-time PCR. The protein level of HMGB1 and HMGN2 in peri-implant crevicular fluid (PICF), peri-implant crevicular fluid of peri-implantitis (PI-PICF) and normal patients was determined by Western blotting. Furthermore, IL-1, IL-6, IL-8, TNF- and HMGB1 levels in GCF, PI-PICF and healthy-PICF samples from different groups were determined by ELISA. HMGN2 expression was increased in inflamed gingival tissues and GCF from CP and G-ApG groups compared to control group. HMGB1 expression was the highest in the gingival tissues and GCF from CP patients and was accompanied by increased concentrations of IL-1, IL-6, IL-8 proinflammaory cytokines. To our knowledge, this is the first study reporting that the expression of HMGB1 and HMGN2 was increased in the gingival tissues and GCF in CP and G-AgP and the PICF in PICF. Our data suggest that HMGB1 may be a potential target for the therapy of periodontitis and PI.

Expressão gênica de TLR-2, TLR-4, HMGB1 e VEGF em úlcera abomasal em bovinos de corte

As úlceras abomasais atingem bovinos de todas as idades e raças em todos os sistemas de produção, gerando perdas econômicas. A úlcera resulta da isquemia, atraindo leucócitos e macrófagos, estimulando fibroblastos, células endoteliais e epiteliais. A proteína do grupo de alta mobilidade 1 (HMGB1) liga -se a diferentes receptores de superfície celular, incluindo Toll-like-2 (TLR-2) e -4 (TLR-4), produzindo citocinas. A presença da HMGB1 causa aumento dos níveis do fator de crescimento endotelial vascular (VEGF), um regulador fundamental da angiogênese. Assim, investigou -se a participação da HMGB1, TLR-2, TLR-4 e VEGF em úlceras abomasais em bovinos de corte. Um total de 150 abomasos de bovinos de corte foi examinado em um abatedouro; 17 amostras da região cárdica foram colhidas. Os tecidos extraídos foram classificados em grupo normal (sem ulceração de mucosa); ulceração de grau 1 (erosões não perfuradas com lesões mínimas da mucosa) e ulceração de grau 2 (erosões não perfuradas combinadas com sangramento moderado da mucosa) e confirmado pela histopatologia. A expressão dos genes nas amostras normais ou ulceradas no abomaso foi avaliada pela RT qPCR. Os dados foram submetidos à ANOVA seguido por teste de Bonferroni ao nível de p<0,05. Não houve diferença de expressão de HMGB1, de TLR-4 e de VEGF entre os dois tipos de úlceras em relação aos abomasos normais. Úlceras de grau 2 tiveram expressão de TLR-2 superior a úlceras de grau 1. O aumento da expressão de TLR-2 pode estar associado à manutenção da cicatrização, promovendo a resposta inflamatória, evidenciado pela presença de infiltrado inflamatório mononuclear e neutrófilos.

Abomasal ulcers affect cattle of all ages and breeds in all production systems, leading to economic losses. The ulcer resulting from tissue ischemia, attracting leukocytes and macrophages, stimulates fibroblasts, endothelial and epithelial cells. The protein of high mobility group 1 (HMGB1) binds to different cell surface receptors, including Toll-like-2 (TLR-2) and 4 (TLR-4) resulting in cytokine production. The presence of HMGB1 causes increased levels of vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. Thus, it was investigated whether HMGB1, TLR-2, TLR-4 and VEGF play a role in abomasal ulcers in beef cattle. A total of 150 abomasums from beef cattle were examined in a slaughterhouse; 17 samples were collected from the cardiac region. The extracted tissues were divided into normal group (without ulceration of the mucosa); type 1 ulcers (unperforated erosions with minimal mucosal injury) and type 2 ulcers (unperforated erosions combined with moderate bleeding of the mucosa) and confirmed by histopathology. Gene expression was evaluated by RT qPCR in samples of normal or ulcerated abomasums. Data were analyzed by ANOVA followed by Bonferroni test at p <0.05. No difference in expression of HMGB1, TLR-4 and VEGF was detected between the two types of ulcers when compared to normal abomasums. TLR-2 expression was higher in type 2 ulcers than in type 1 ulcers. Increased TLR-2 expression might be associated with the maintenance of abomasal healing, promoting the inflammatory response, as evidenced by the presence of mononuclear cell infiltration and neutrophils.

Edição do gene TFAM pela engenharia CRISPR Cas9 em modelo bovino

O fator de transcrição A mitocondrial (TFAM) é um membro da subfamília HMGB que se liga a promotores do DNA mitocondrial (mtDNA). É um gene extremamente importante para a manutenção do mtDNA, pois regula o número de cópias e é essencial para inicialização da replicação e transcrição do mtDNA. Recentemente técnicas de edição genica vêm sendo utilizada como uma ferramenta bastante eficaz na manipulação genômica. A nova tecnologia chamada de CRISPR/ Cas9 (Regulary interspaced clustered short palindromic repeats) utiliza um RNA guia (gRNA) curto que contém 20 nucleotídeos complementares a sequência de DNA. Seu mecanismo é simples, quando o RNA guia se liga ao local alvo, a proteína Cas9 é recrutada para se ligar no local alvo e induzir a dupla quebra na cadeia de DNA. Neste contexto, este estudo propôs editar o gene TFAM pela tecnologia CRISPR Cas9, com o objetivo de gerar células ROS zero através do knock-out em fibroblastos bovinos. Os fibroblastos bovinos utilizados neste estudo foram derivados de uma biopsia de pele coletada de animais adultos. A sequência do gene foi obtida a partir do banco de dados GenBank (www.ncbi.nlm.nih.gov) e esta foi inserida no site CRISPR direct(crispr.dbcls.jp) e no site rgenome (rgenome.net) a fim de desenhar o gRNA. O gRNA foi desenhado no exon 1 do gene TFAM bovino e depois foi realizado todo o procedimento de clonagem. Os fibroblastos foram cultivados e após as células atingirem 80% de confluência, estas foram eletrotransfectados com Cas9 (Addgene 48668), GFP e plasmídeo controle. Foi utilizado o kit Primary Mammalian Fibroblasts (VPI-1002) e a transfecção foi realizada no equipamento AMAXA Nucleofector 2B. Após a transfecção foi realizada a citometria de fluxo para avaliar a taxa de transfecção, e as células pós transfectas foram plaqueadas em placas de 96 poços, pela técnica de sorting. O sorting separarou uma célula por poço de 96. Após dias em cultura essas células foram tripsinizadas em placas de 6 poços e o DNA genômico foi extraído, utilizando o kit Qiamp DNA microkit-Qiagen. Para avaliar a frequência de mutações, foi realizado a digestão com a enzima T7 endonuclease, e após confirmado mutações, os clones foram enviados para analise de sequenciamento. Observamos uma taxa de transfecção eficiente de 51,3%. Obtivemos 40 clones com DNA extraído para analise, no qual 6 destes possuiam mutações no local de inserção da CRISPR Cas 9. Concluímos até o momento que o desenho da CRISPR foi eficiente e que obtivemos uma deleção do gene TFAM.

The mitochondrial transcription factor A (TFAM) is a member of HMGB subfamily, that bind to promoters of mtDNA. It is a very important gene that maintains mtDNA, regulates the number of copies and is essential for the initiation of transcription mtDNA. Recently, gene edition techniques have been used as a very effective tool in genomic manipulation. The new technology called CRISPR/Cas9 (Regulary interspaced clustered short palindromic repeats) uses a short gRNA containing 20 nucleotides complementary to the DNA sequence. When gRNA binds to the target site, the Cas9 protein is recruited to bind in the chosen location and induce double strands breaks in DNA. In this context, this study proposed to edit the TFAM gene by CRISPR Cas9 technology aiming to generate ROS zero cell through the knock-out in bovine fibroblasts. Bovine fibroblasts used in this study were derived from a skin biopsy collected from an adult. The sequence obtained from the database GenBank (www.ncbi.nlm.nih.gov) was inserted in the CRISPR direct site (crispr.dbcls.jp) and in the rgenome site (rgenome.net) to design the RNA guide. The gRNA was designed in the CRISPR direct site (crispr.dbcls.jp) for the Exon 1 of the gene TFAM bovine and after was performed the CRISPR cloning. The fibroblast were cultured and after reaching 80% of confluence, were electro-transfected with Cas9 (Addgene 48668) and control plasmids using the Nucleofector TM Kit for Primary Mammalian Fibroblasts (VPI-1002) and transfected with Cas 9 (Addgene 48668), GFP and control plasmid. Were used the Primary Mammalian Fibroblasts (VPI-1002) and the transfection was performed on the AMAXA Nucleofector 2B. Post transfected cells were analyzed by flow cytometry to evaluate the rate of transfection. The cells post transfected were further split into 1 cell/well (96- well plates for cell cloning). After days in culture these cells were trypsinized in 6-well plates and the genomic DNA was extracted using the Qiamp DNA microkit- Qiagen. To assess the mutation frequency, T7 endonuclease assay were performed and after confirmed the mutations, the clones were sent for sequencing analysis. We observed that the cells were efficiently transfected since they have a rate of 51,3% transfection. We obtained 40 clones with extracted for analysis, in which 6 of these had mutations at the insertion site of CRISPR/Cas 9. We concluded that until this moment the CRISPR design was efficient and that we obtained a deletion of the TFAM gene.

Resposta de células trofoblásticas bovinas à infecção por Brucella abortus: uma abordagem proteômica

Brucella abortus é considerada o principal agente etiológico da brucelose bovina, uma zoonose que provoca grandes perdas econômicas em todo o mundo. O objetivo desse estudo foi determinar o perfil diferencial de expressão proteica de células trofoblásticas bovinas nas fases iniciais da infecção por B. abortus. Foi usado o modelo de explantes confeccionados a partir de membrana cório-alantóidea de fetos no último trimestre de gestação infectados por suspensão de B. abortus 2308 (1.0 x 108 bactérias - MOI 1:1000). No estudo da cinética da infecção foram avaliados os tempos de 0,5, 2, 4 e 8 h pós-infecção no qual foi obtida reprodutibilidade alta entre triplicatas dos géis comparados (Match > 84%; IC > 0,77). O perfil de expressão de proteínas foi similar entre os tempos avaliados (Match > 75%). Os tempos de 0,5 e 4 h pósinfecção apresentaram as maiores diferenças qualitativas entre os géis e foram escolhidos para análise diferencial em gel (DIGE). Nessa análise não foram identificados spots com diferenças quantitativas significativas entre os géis comparados. Por outro lado, 103 spots presentes apenas em um grupo experimental foram selecionados para identificação por espectrometria de massa. As proteínas BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 e TPM4 foram identificadas apenas nos extratos proteicos de células trofoblásticas infectadas. As proteínas identificadas nesse estudo servirão como alvos para novas pesquisas relacionadas à interação patógeno-hospedeiro na infecção por B. abortus

Brucella abortus is considered the main etiological agent of bovine brucellosis, a zoonotic disease that causes great economic losses worldwide. The aim of this study was to determine the differential profile of proteins expression of bovine trophoblast cells in the early stages of infection by B. abortus. Was used the explants model prepared from chorio-alantóidea of fetuses in the last trimester of pregnancy infected by suspending the B. abortus 2308 containing 1.0 x 108 bacteria (MOI 1:1000). In the study of the kinetics of infection was evaluated the times of 0.5, 2, 4 and 8 h post infection with B. abortus. Was obtained high reproducibility between triplicate of gels compared (Match> 84%; CI > 0.77) and the proteins expression profile was similar among the times evaluated (Match > 75%). Times of 0.5 and 4 h post infection showed the highest qualitative differences between the gels and were used for the differential in gel electrophoresis (DIGE). In this analysis have not been identified spots with significant quantitative differences between the compared gels. Moreover, 103 spots are present only in an experimental group were selected for identification by mass spectrometry. The proteins BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 and TPM4 were identified only in protein extracts of trophoblastic cells infected by B. abortus. The proteins identified in this study will serve as targets for further research related to host-pathogen interaction in infection by B. abortus