OBJECTIVE: To investigate the association between the glucose-to-lymphocyte ratio (GLR) and prognosis of patients with sepsis-associated acute kidney injury (SA-AKI). METHODS: Based on the Medical Information Mart for Intensive Care-IV (MIMIC-IV), SA-AKI patients aged ≥ 18 years were selected. According to the tertiles of GLR, the patients were divided into GLR1 group (GLR ≤ 4.97×10^-9 mmol), GLR2 group (4.97×10^-9 mmol < GLR < 9.75×10^-9 mmol) and GLR3 group (GLR ≥ 9.75×10^-9 mmol). Patients with SA-AKI were divided into survival group and death group according to whether they survived 28 days after admission. The patient's gender, age, vital signs, laboratory test results, comorbidities, sequential organ failure assessment (SOFA), acute physiology score III (APS III) score and treatment measures were extracted from the database. Kaplan-Meier survival analysis was used to make the survival curves of patients with SA-AKI at 28 days, 90 days, 180 days and 1 year. Multivariate Logistic regression analysis model was used to explore the independent risk factors of 28-day mortality in patients with SA-AKI. Receiver operator characteristic curve (ROC curve) was used to analyze the predictive efficacy of GLR for the prognosis of patients with SA-AKI. RESULTS: A total of 1 524 patients with SA-AKI were included, with a median age of 68.28 (58.96, 77.24) years old, including 612 females (40.16%) and 912 males (59.84%). There were 507 patients in the GLR1 group, 509 patients in the GLR2 group and 508 patients in the GLR3 group. There were 1 181 patients in the 28-day survival group and 343 patients in the death group. Grouping according to GLR tertiles showed that with the increase of GLR, the 28-day, 90-day, 180-day and 1-year mortality of SA-AKI patients gradually increased (28-day mortality were 11.64%, 22.00%, 33.86%, respectively; 90-day mortality were 15.98%, 26.72%, 40.55%, respectively; 180-day mortality were 17.16%, 28.29% and 41.73%, and the 1-year mortality were 17.95%, 29.27% and 42.72%, respectively, all P < 0.01). According to 28-day survival status, the GLR of the death group was significantly higher than that of the survival group [×10^-9 mmol: 9.81 (5.75, 20.01) vs. 6.44 (3.64, 10.78), P < 0.01]. Multivariate Logistic regression analysis showed that GLR was an independent risk factor for 28-day mortality in patients with SA-AKI [when GLR was used as a continuous variable: odds ratio (OR) = 1.065, 95% confidence interval (95%CI) was 1.045-1.085, P < 0.001; when GLR was used as a categorical variable, compared with GLR1 group: GLR2 group OR = 1.782, 95%CI was 1.200-2.647, P = 0.004; GLR3 group OR = 2.727, 95%CI was 1.857-4.005, P < 0.001]. ROC curve analysis showed that the area under the ROC curve (AUC) of GLR for predicting 28-day mortality in patients with SA-AKI was 0.674, when the optimal cut-off value was 8.769×10^-9 mmol, the sensitivity was 57.1% and the specificity was 67.1%. The predictive performance was improved when GLR was combined with APS III score and SOFA score, and the AUC was 0.806, the sensitivity was 74.6% and the specificity was 71.4%. CONCLUSIONS GLR is an independent risk factor of 28-day mortality in patients with SA-AKI, and high GLR is associated with poor prognosis in patients with SA-AKI.
Male ; Female ; Humans ; Blood Glucose ; Glucose ; ROC Curve ; Prognosis ; Sepsis/diagnosis* ; Acute Kidney Injury ; Retrospective Studies ; Intensive Care Units

Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.

Objective:To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis.Methods:The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 μg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1β and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. Results:The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1β (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1β (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/β-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/β-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/β-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. Conclusion:iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.

Objective: To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Methods: Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCA^TM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. Results: The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2^-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2^-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2^-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2^-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. Conclusion The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

Objective: To analyze the specificity and sensitivity of the modified microbubble test in identifying the peripherally inserted central venous catheters (PICC) tip based on the chest X-ray location as the "gold standard", and to find out an accurate and noninvasive PICC tip positioning method that can save time and cost. Methods: Convenient sampling method was conducted. The patients under PICC guided by ultrasound in intensive care unit (ICU) or PICC clinic of the First Affiliated Hospital of Nanchang University from August 2017 to February 2018 were enrolled. All patients were followed up by ultrasound guided PICC catheter placement, modified microbubble test and chest X-ray localization. The relationship between the density of microbubbles in modified microbubble test and the location of PICC tip in chest X-ray localization was analyzed. Using chest X-ray localization as the "gold standard", the diagnostic evaluation indexes such as specificity and sensitivity of PICC tip identification by modified microbubble test were calculated. Results: A total of 120 patients were enrolled during the study period, excluding those who refused to participate in the study, unclear right atrial ultrasound, conscious intolerance, unclear chest X-ray, and finally 108 patients completed the modified microbubble test and chest X-ray tip localization. According to the chest X-ray localization results of 108 patients, 69 patients (63.9%) were in ideal locations, 33 (30.6%) were in dissatisfactory position, and 6 (5.5%) were in malposition. There was no significant difference in gender, age, tube placement, depth of catheterization, placement of catheterization room, and catheterization among the three groups. In the modified microbubble test, there were 74 patients (68.5%) with gradeⅠmicrobubble, 25 (23.2%) with gradeⅡ microbubble, and 9 (8.3%) with grade Ⅲ microbubble. There was a correlation between microbubble density and the tip position of the catheter, showing a moderate intensity correlation, and the contingency coefficient was 0.662. The sensitivity of the modified microbubble test for PICC tip positioning was 95.7% (66/69), the specificity was 89.7% (35/39), the rate of missed diagnosis was 4.4% (3/69), the misdiagnosis rate was 10.3% (4/39), the positive predictive value was 94.3% (66/70), the negative predictive value was 92.1% (35/38), and the Youden index was 0.85. The consistency between the two methods was good, and the Kappa value was 0.86. Conclusions Compared with the chest X-ray localization method, the modified microbubble test method has high sensitivity and specificity in identifying PICC in the position, and the operation is simple, noninvasive, with less time and low cost. The modified microbubble test can be used as a screening test for PICC tip position, especially in ICU. When there are technical limitations or suspicious patient, further chest X-ray is necessary.

OBJECTIVE: To analyze the specificity and sensitivity of the modified microbubble test in identifying the peripherally inserted central venous catheters (PICC) tip based on the chest X-ray location as the "gold standard", and to find out an accurate and noninvasive PICC tip positioning method that can save time and cost. METHODS: Convenient sampling method was conducted. The patients under PICC guided by ultrasound in intensive care unit (ICU) or PICC clinic of the First Affiliated Hospital of Nanchang University from August 2017 to February 2018 were enrolled. All patients were followed up by ultrasound guided PICC catheter placement, modified microbubble test and chest X-ray localization. The relationship between the density of microbubbles in modified microbubble test and the location of PICC tip in chest X-ray localization was analyzed. Using chest X-ray localization as the "gold standard", the diagnostic evaluation indexes such as specificity and sensitivity of PICC tip identification by modified microbubble test were calculated. RESULTS: A total of 120 patients were enrolled during the study period, excluding those who refused to participate in the study, unclear right atrial ultrasound, conscious intolerance, unclear chest X-ray, and finally 108 patients completed the modified microbubble test and chest X-ray tip localization. According to the chest X-ray localization results of 108 patients, 69 patients (63.9%) were in ideal locations, 33 (30.6%) were in dissatisfactory position, and 6 (5.5%) were in malposition. There was no significant difference in gender, age, tube placement, depth of catheterization, placement of catheterization room, and catheterization among the three groups. In the modified microbubble test, there were 74 patients (68.5%) with grade I microbubble, 25 (23.2%) with grade II microbubble, and 9 (8.3%) with grade III microbubble. There was a correlation between microbubble density and the tip position of the catheter, showing a moderate intensity correlation, and the contingency coefficient was 0.662. The sensitivity of the modified microbubble test for PICC tip positioning was 95.7% (66/69), the specificity was 89.7% (35/39), the rate of missed diagnosis was 4.4% (3/69), the misdiagnosis rate was 10.3% (4/39), the positive predictive value was 94.3% (66/70), the negative predictive value was 92.1% (35/38), and the Youden index was 0.85. The consistency between the two methods was good, and the Kappa value was 0.86. CONCLUSIONS Compared with the chest X-ray localization method, the modified microbubble test method has high sensitivity and specificity in identifying PICC in the position, and the operation is simple, noninvasive, with less time and low cost. The modified microbubble test can be used as a screening test for PICC tip position, especially in ICU. When there are technical limitations or suspicious patient, further chest X-ray is necessary.
Catheterization, Central Venous ; Catheterization, Peripheral ; Central Venous Catheters ; Humans ; Microbubbles ; Ultrasonography

OBJECTIVE: To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. METHODS: Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCA^TM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. RESULTS: The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2^-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2^-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2^-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2^-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. CONCLUSIONS The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
Animals ; Brain Diseases ; Cells, Cultured ; Fibroblasts ; Humans ; Kruppel-Like Factor 4 ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Sepsis

Objective To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). Methods The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. ① The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. ② Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. Results ① After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. ② Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. Conclusion In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

Objective To observe the amplification effect of mitochondrial DNA (mtNDA) on the inflammatory response of rat alveolar macrophage induced by lipopolysaccharide (LPS), and to preliminarily explore its mechanism. Methods mtDNA of Sprague-Dawley (SD) rat liver tissue was harvested, ultra-micro spectrophotometer and 1% agarose gel electrophoresis were used to detect the concentration and quality of mtDNA. The alveolar macrophages of SD rat were isolated and cultured, the macrophages in logarithmic growth phase were divided into phosphatic buffer solution (PBS) group, LPS group, mtDNA group and LPS+mtDNA group. The first three groups were added equal volumes of PBS, LPS 1 mg/L or mtDNA 10 mg/L to the alveolar macrophages medium for 6 hours and 12 hours, respectively; the alveolar macrophage medium of LPS+mtDNA group was stimulated with 1 mg/L LPS for 6 hours and then stimulated with 10 mg/L mtDNA for 6 hours. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA);the expression of the key protein of Pyroptosis-Gasdermin D (GSDMD) was detected by Western Blot. Results ① The mtDNA A260/280 ratios were between 1.8-2.0, and agarose gel electrophoresis showed a single band, with a size of about 16 kb. ② After alveolar macrophages stimulated by LPS or mtDNA for 6 hours or 12 hours, respectively, the levels of IL-1β and TNF-α were higher than those in PBS group. When cells were treated with mtDNA for 6 hours after LPS stimulation 6 hours, the levels of IL-1β were higher than those in LPS 12 hours group (ng/L: 366.27±23.35 vs. 154.70±23.32, 1 < 0.01), but the levels of TNF-α had no significant difference compared with LPS 12 hours group (ng/L: 836.13±25.01 vs. 802.67±30.48, 1 > 0.05). ③ The protein expressions of GSDMD in LPS group, mtDNA group and LPS+mtDNA group were significantly higher than those in PBS group (GSDMD/β-actin: 1.77±0.05, 1.65±0.04,2.40±0.05, 1.00±0.02, all 1 < 0.01), and the protein expression of GSDMD in LPS+mtDNA group was higher than that in LPS group (1 < 0.01). Conclusion mtDNA amplifies LPS-induced alveolar macrophage inflammatory responses,which mechanism may be related to the increase in pyroptosis mediated by mtDNA.

Objective To explore the inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide (LPS) on the alveolar macrophages (NR8383). Methods The alveolar epithelial cells disposed with different treatments were co-cultured with alveolar macrophages by using a Transwell system separately. Alveolar epithelial cells (RLE-6TN) were randomly divided into 4 groups: normal group, LPS-stimulated group, exosome inhibitor group, and exosome inhibitor pretreatment + LPS stimulation group. NR8383 cultured alone was considered as a blank control. After the 12-h co-culture, the real-time PCR (qPCR) was performed to examine the mRNA relative expression of IL-6, TNF-α, and IL-1β in NR8383 cells. To further explore the role of exosomes derived from RLE-6TN on alveolar macrophages mediated inflammationary response, the experimental exosomes (exosomes derived from LPS-induced RLE-6TN) and control exosomes exosomes derived from normal RLE-6TN were extracted by gradient ultracentrifugation. Transmission electron microscopy and Western blotting analyses was performed to identify the exosomes, and qNano particle diameter analyzer was conducted to measure the particle diameter of exosomes. In vitro, NR8383 cells were divided into 3 groups which were cultured with exosomes derived from LPS-stimulated RLE-6TN at a concentration of 10 μg/mL (experimental group), exosomes derived from untreated RLE-6TN at the same concentration of 10 μg/mL (control group), and the PBS at the same volume with experimental group (PBS group), respectively for 12 h. After the treatment, the phagocytosis of NR8383 cells was observed by laser confocal microscope and the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in supernatants of NR8383 was detected by enzyme-linked immunosorbent assay ELISA Results (1)In the co-culture experiment, the mRNA relative expression of pro-inflammatory cytokine in the LPS group was significantly increased compared with the blank control group (P<0.01), however comparing the exosome inhibitor pretreatment+LPS group with the LPS group, the expression of pro-inflammatory cytokine was decreased (P<0.01). (2) The extracted exosomes were observed as circular or elliptical vesicles with a diameter of 40-100 nm under the transmission electron microscopy. Western blotting analyses showed that the extracted exosomes express the protein marker, such as CD63 and CD9; After incubation with NR8383 cells for 5 h, laser scanning confocal microscope showed that the exosomes labeled with red fluorescent were uptaken by NR8383 cells. (3)After the exosomes derived from the LPS-disposed RLE-6TN and the normal RLE-6TN cells were incubated with NR8383 cells respectively. The ELISA test showed that treated the alveolar macrophages with LPS induced alveolar epithelial secreted exosomes led to a robustly increased release of pro-inflammatory cytokine (P<0.01), but there was no significant difference between the control group and PBS group (P>0.05). Conclusions Exosomes derived from LPS-disposed alveolar epithelial cells activate the alveolar macrophage-mediated inflammatory response.