Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism / 中华烧伤杂志

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on pulmonary vascular endothelial cells (PMVECs) injury in septic mice and its mechanism. Methods: The experimental research method was adopted. The primary ADSCs were isolated and cultured from the discarded fresh adipose tissue of 3 patients (female, 10-25 years old), who were admitted to the First Affiliated Hospital of Air Force Medical University undergoing abdominal surgery, and the cell morphology was observed by inverted phase contrast microscope on the 5th day. The expressions of CD29, CD34, CD44, CD45, CD73, and CD90 of ADSCs in the third passage were detected by flow cytometry. The third to the fifth passage of ADSCs were collected, and their exosomes from the cell supernatant were obtained by differential ultracentrifugation, and the shape, particle size, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin of exosomes were detected, respectively, by transmission electron microscopy, nano-particle tracking analysis and Western blotting. Twenty-four adult male BALB/c mice were adopted and were divided into normal control group, caecal ligation perforation (CLP) alone group, and CLP+ADSC-exosome group with each group of 8 according to random number table (the same grouping method below) and were treated accordingly. At 24 h after operation, tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) levels of mice serum were detected by enzyme-linked immunosorbent assay, and lung tissue morphology of mice was detected by hematoxylin-eosin and myeloperoxidase staining, and the expression of 8-hydroxy-deoxyguanosine (8-OHdG) of mouse lung cells was detected by immunofluorescence method. Primary PMVECs were obtained from 1-month-old C57 mice regardless gender by tissue block method. The expression of CD31 of PMVECs was detected by immunofluorescence and flow cytometry. The third passage of PMVECs was co-cultured with ADSCs derived exosomes for 12 h, and the phagocytosis of exosomes by PMVECs was detected by PKH26 kit. The third passage of PMVECs were adopted and were divided into blank control group, macrophage supernatant alone group, and macrophage supernatant+ADSC-exosome group, with 3 wells in each group, which were treated accordingly. After 24 h, the content of reactive oxygen species in cells was detected by flow cytometry, the expression of 8-OHdG in cells was detected by immunofluorescence, and Transwell assay was used to determine the permeability of cell monolayer. The number of samples in above were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: The primary ADSCs were isolated and cultured to day 5, growing densely in a spindle shape with a typical swirl-like. The percentages of CD29, CD44, CD73 and CD90 positive cells of ADSCs in the third passage were all >90%, and the percentages of CD34 and CD45 positive cells were <5%. Exosomes derived from ADSCs of the third to fifth passages showed a typical double-cavity disc-like structure with an average particle size of 103 nm, and the protein expressions of CD9, CD63 and TSG101 of exosomes were positive, while the protein expression of β-actin of exosomes was negative. At 24 h after operation, compared with those in normal control group, both the levels of TNF-α and IL-1β of mice serum in CLP alone group were significantly increased (with t values of 28.76 and 29.69, respectively, P<0.01); compared with those in CLP alone group, both the content of TNF-α and IL-1β of mice serum in CLP+ADSC-exosome group was significantly decreased (with t values of 9.90 and 4.76, respectively, P<0.05 or P<0.01). At 24 h after surgery, the pulmonary tissue structure of mice in normal control group was clear and complete without inflammatory cell infiltration; compared with those in normal control group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP alone group were more obvious; compared with those in CLP alone group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP+ADSC-exosome group were significantly reduced. At 24 h after operation, endothelial cells in lung tissues of mice in 3 groups showed positive expression of CD31; compared with that in normal control group, the fluorescence intensity of 8-OHdG positive cells of the lung tissues of mice in CLP alone group was significantly increased, and compared with that in CLP alone group, the fluorescence intensity of 8-OHdG positive cells in the lung tissues of mice in CLP+ADSC-exosome group was significantly decreased. The PMVECs in the 3rd passage showed CD31 positive expression by immunofluorescence, and the result of flow cytometry showed that CD31 positive cells accounted for 99.5%. At 12 h after co-culture, ADSC-derived exosomes were successfully phagocytose by PMVECs and entered its cytoplasm. At 12 h after culture of the third passage of PMVECs, compared with that in blank control group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant alone group was significantly increased (t=15.73, P<0.01); compared with that in macrophage supernatant alone group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant+ADSC-exosome group was significantly decreased (t=4.72, P<0.01). At 12 h after culture of the third passage of PMVECs, and the 8-OHdG positive fluorescence intensity of PMVECs in macrophage supernatant alone group was significantly increased; and compared with that in blank control group, the 8-OHdG positive fluorescence intensity of PMVECs in macrophage+ADSC-exosome supernatant group was between blank control group and macrophage supernatant alone group. At 12 h after culture of the third passage PMVECs, compared with that in blank control group, the permeability of PMVECs monolayer in macrophage supernatant alone group was significantly increased (t=6.34, P<0.01); compared with that in macrophage supernatant alone group, the permeability of PMVECs monolayer cells in macrophage supernatant+ADSC-exosome group was significantly decreased (t=2.93, P<0.05). Conclusions: Exosomes derived from ADSCs can ameliorate oxidative damage in mouse lung tissue, decrease the level of reactive oxygen species, 8-OHdG expression, and permeability of PMVECs induced by macrophage supernatant.

Anti-placental growth factor antibody ameliorates hyperoxia-mediated impairment of lung development in neonatal rats

This study investigates the effect of the overexpression of the placental growth factor (PGF) and hyperoxia on lung development and determines whether anti-PGF antibody ameliorates hyperoxia-mediated impairment of lung development in newborn rats. After exposure to normoxic conditions for seven days, newborn rats subjected to normoxia were intraperitoneally or intratracheally injected with physiological saline, adenovirus-negative control (Ad-NC), or adenovirus-PGF (Ad-PGF) to create the Normoxia, Normoxia+Ad-NC, and Normoxia+Ad-PGF groups, respectively. Newborn rats subjected to hyperoxia were intraperitoneally injected with physiological saline or anti-PGF antibodies to create the Hyperoxia and Hyperoxia+anti-PGF groups, respectively. Our results revealed significant augmentation in the levels of PGF and its receptor Flt-1 in the lung tissues of newborn rats belonging to the Normoxia+Ad-PGF or Hyperoxia groups. PGF overexpression in these groups caused lung injury in newborn rats, while anti-PGF antibody treatment significantly cured the hyperoxia-induced lung injury. Moreover, PGF overexpression significantly increased TNF-α and Il-6 levels in the bronchoalveolar lavage (BAL) fluid of the Normoxia+Ad-PGF and Hyperoxia groups. However, their levels were significantly reduced in the BAL fluid of the Hyperoxia+anti-PGF group. Immunohistochemical analysis revealed that PGF overexpression and hyperoxia treatment significantly increased the expression of the angiogenesis marker, CD34. However, its expression was significantly decreased upon administration of anti-PGF antibodies (compared to the control group under hyperoxia). In conclusion, PGF overexpression impairs lung development in newborn rats while its inhibition using an anti-PGF antibody ameliorates the same. These results provided new insights for the clinical management of bronchopulmonary dysplasia in premature infants.

Angiotensin- (1-7) inhibits inflammation and oxidative stress to relieve lung injury induced by chronic intermittent hypoxia in rats

Obstructive sleep apnea is associated with inflammation and oxidative stress in lung tissues and can lead to metabolic abnormalities. We investigated the effects of angiotensin1–7 [Ang-(1–7)] on lung injury in rats induced by chronic intermittent hypoxia (CIH). We randomly assigned 32 male Sprague-Dawley rats (180–200 g) to normoxia control (NC), CIH-untreated (uCIH), Ang-(1–7)-treated normoxia control (N-A), and Ang-(1–7)-treated CIH (CIH-A) groups. Oxidative stress biomarkers were measured in lung tissues, and expression of NADPH oxidase 4 (Nox4) and Nox subunits (p22phox, and p47phox) was determined by Western blot and reverse transcription-polymerase chain reaction. Pulmonary pathological changes were more evident in the uCIH group than in the other groups. Enzyme-linked immunosorbent assays and immunohistochemical staining showed that inflammatory factor concentrations in serum and lung tissues in the uCIH group were significantly higher than those in the NC and N-A groups. Expression of inflammatory factors was significantly higher in the CIH-A group than in the NC and N-A groups, but was lower than in the uCIH group (P<0.01). Oxidative stress was markedly higher in the uCIH group than in the NC and N-A groups. Expression of Nox4 and its subunits was also increased in the uCIH group. These changes were attenuated upon Ang-(1–7) treatment. In summary, treatment with Ang-(1-7) reversed signs of CIH-induced lung injury via inhibition of inflammation and oxidative stress.

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats / Efeitos da eritromicina sobre a γ-glutamil-cisteína-sintetase e a interleucina-1β no tecido pulmonar exposto à hiperóxia de ratos recém-nascidos prematuros

Objective: To explore the effect of erythromycin on hyperoxia-induced lung injury. Methods: One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Conclusions: Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. .

Objetivo: Explorar o efeito da eritromicina sobre lesões pulmonares induzidas por hiperóxia. Métodos: Uma prole de ratos Sprague-Dawley (SD) prematuros com um dia de vida foi dividida aleatoriamente em quatro grupos: grupo 1 ar + cloreto de sódio, grupo 2 ar + eritromicina, grupo 3 hiperóxia + cloreto de sódio e grupo 4 hiperóxia + eritromicina. Com um, sete e 14 dias de exposição, foram detectadas Glutationa (GSH) e Interleucina-1 beta (IL-1 beta) pelo ensaio imunossorvente ligado à enzima (ELISA), e o ácido bicinconinico (BCA) foi utilizado para detectar a proteína GSH. O mRNA da γ-glutamil-cisteina-sintetase (γ-GCS) foi detectado por reação em cadeia da polimerase via transcriptase reversa (RT-PCR). Resultados: Comparadas ao grupo 1, as expressões do mRNA da GSH e da γ-GCS no grupo 3 aumentaram significativamente com um e sete dias de exposição (p < 0,05), porém a expressão de mRNA da γ-GCS diminuiu significativamente aos 14 dias; a expressão de IL-1 beta no grupo 3 aumentou significativamente aos 7 dias de exposição (p < 0,05) e diminuiu significativamente aos 14 dias. Comparadas ao grupo 3, as expressões do mRNA da GSH e da γ-GCS no grupo 4 aumentaram significativamente com um, sete e 14 dias de exposição (p < 0,05), porém as expressões de GSH mostraram uma tendência de queda aos 14 dias; a expressão de IL-1 beta no grupo 4 foi reduzida significativamente com um e sete dias de exposição (p < 0,05). Conclusões: As variações de IL-1 beta e GSH mediadas por oxidantes estão envolvidas no desenvolvimento de lesão pulmonar induzida por hiperóxia. A eritromicina poderá regular positivamente a atividade da γ-GCS, aumentando a expressão de GSH, inibindo os níveis de interleucina-1beta mediada por ...

Changes of aquaporins 1 expression in the contused lung of rats / 法医学杂志

OBJECTIVE@#To investigate the expression changes of aquaporins 1 (AQP1) in contused lung tissue of rats and its relationship with pulmonary edema.@*METHODS@#SD rats were randomly divided into experimental and control groups. The pulmonary contusion models were then prepared. The expression and distribution of AQP1 in lung tissue of the rats were detected by immunohistochemistry.@*RESULTS@#The lung tissue showed edema, hemorrhage, inflammatory cell infiltration 1 h, 3 h after pulmonary contusion, and the inflammatory response aggravated after 5 h. AQP1 expression at 1 h, 3 h and 5 h in the contusion group were significantly higher than that of the control group (P < 0.01). The expression of AQP1 continued to increase with time and aggravation of edema compared to the control group. AQP1 was mainly distributed in the capillary endothelial cells and interstitial cells of the bronchial and alveolar walls. Although there were no observed changes in AQP1 expression location in contused lung tissue, the intergrated optical density(IOD) showed significant statistical difference (P < 0.01).@*CONCLUSION@#There might exist an dysregulation of AQPs gene expression in contused lung tissue, leading to a large number of abnormal transmembrane water transportation and abnormal water accumulation, which may be one of the reasons for pulmonary edema in contused lung tissue.