| Objective: Through exploring the effect of C-X-C motif chemokine 10 (CXCL10) in lipopolysaccharide (LPS) induced ARDS in rats, our study aimed to provide theoretical basis and therapeutic target for ARDS treatment.Methods: We first established ARDS model in rats. Male Wistar rats weighing 180-220 g were randomly divided into two groups, LPS group and saline group. Each group had 24 rats. After the rats were anesthetized by intraperitoneal injection of pentobarbital, LPS was intratracheal instilled slowly at a dose of 2 mg/kg to induce ARDS, while the rats in control group received an equal volume of normal saline. At 2 h, 6 h and 12 h after exposure to LPS, rats were anesthetized by pentobarbital again and then blood was collected from abdominal aorta to perform arterial blood gas analysis. The rats were sacrificed immediately after blood collection and lung tissues were received. Pulmonary histopathological examination and lung injury score were performed to further establish whether ARDS model was built. At 2 h, 6 h and 12 h,serum and BALF concentrations of CXCL9, CXCL10 and CXCL11 were measured with ELISA. The gene and protein expression of CXCL10 and CXCR3 were further evaluated by quantitative PCR and western blotting. To explore the possible mechanism of CXCL10 in ARDS, anti-CXCL10 antibody was used to neutralize CXCL10. Lung wet/dry weight ratio, inflammatory mediators expression,inflammatory cells in BALF and CXCR3 expression in neutrophils and macrophages were further investigated.Results:1. Establishment of ARDS model by LPS induction1.1 General observations of rats After intratracheal instillation of normal saline or LPS, rats in the control group developed tachypnea that disappeared rapidly without any other abnormalities.Whereas the rats in LPS group exhibited increasing respiratory rate and heart rate,cyanotic limbs and lips, slow reactions and inaction. At 12 h, half of the rats in LPS group had mouth and nasal hemorrhage.1.2 Pulmonary histopathological manifestationLung tissues obtained from saline control group appeared normal structure with no significant signs of inflammatory infiltration or interstitial edema. In contrast, lung tissues in LPS groups showed extensive lung injury including pulmonary interstitial edema, alveolar wall thickening, hemorrhage and inflammatory cells infiltration that worsened over time. A significantly higher lung injury score was found in LPS group than the saline group (p<0.01).1.3 Arterial blood gasPaO2 and PaO2/FiO2 in LPS group were significantly lower over time. At the time point of 12 h, PaO2 was less than 60 mmHg and PaO2/FiO2 was less than 300 mmHg in LPS group. PaO2 and PaO2/FiO2 in LPS groups were significantly decreased than in control groups (p<0.01).2. CXCL10 and CXCR3 expressions in LPS-induced ARDS2.1. CXCL9, CXCL10, and CXCL11 Levels in LPS-induced ARDSTo investigate whether CXCR3 ligands were expressed in LPS-induced ARDS, we investigated the expression of CXCR3 ligands in serum and BALF by ELISA.LPS-induced ARDS in rats led to substantial increase in the expression of CXCL9,CXCL10 and CXCL11 levels in serum and BALF compared to saline control rats (p<0.05) and they further increased with longer exposure to LPS. Furthermore, the levels of CXCL10 were increased far more than CXCL9 and CXCL11.Therefore,compared with CXCL9 and CXCL11, CXCL10 may exert major function in the progression of ARDS induced by LPS.2.2. CXCL10 and its receptor CXCR3 in LPS-induced ARDS in lung tissueWe investigated CXCL10 and CXCR3 expression in lung tissues by both q-PCR and western blotting. After 12 h of LPS injection, the relative mRNA expression of CXCL10 and CXCR3 were significantly increased compared with saline control group (p<0.01). The protein expression of CXCL10 and CXCR3 were coincidently with the mRNA expression (p<0.01).3. Neutralization of CXCL10 decreased lung injury in ARDS model3.1. Neutralization of CXCL10 decreased pulmonary edema in ARDS model After LPS administration,the lung W/D ratio and the lung edema score were significantly increased compared with the saline group (p<0.01), while administration of anti-CXCL10 antibody was significantly decreased the lung W/D ratio and the lung edema score compared with the LPS+isotype control group (p<0.01), and there was no difference between the LPS groups and LPS+isotype control group (p>0.05).3.2. Neutralization of CXCL10 decreased the gene expression of inflammatory mediators in LPS-induced ARDS LPS induction significantly upregulated mRNA expression of IFN-y, IL-6, IL-10,and ICAM-1 (p<0.01). Furthermore, the rats pretreated with monoclonal anti-CXCL10 antibody showed significantly lower levels of ICAM-1,IFN-γ, and IL-6 compared with the LPS group or LPS+isotype control group (p<0.05 ). In contrast,CXCL10 neutralization did not suppress IL-10 mRNA expression.3.3. Neutralization of CXCL10 decreased inflammatory cell recruitment in LPS-induced ARDSWe utilized flow cytometry to investigate total cells, neutrophils, macrophages and T lymphocytes changes in BALF. After LPS was given, the total cell numbers, the percent of neutrophils, macrophages and CD3+CD8+ lymphocytes were significantly increased (p< 0.05), while there was no difference in CD3+ T lymphocytes and CD3+CD4+T lymphocytes between the two groups (p>0.05). After CXCL10 was blocked,total cells,neutrophils and macrophages (p<0.05) but not CD3+CD8+ T cells decreased compared with the isotype control group (p>0.05).3.4. Neutralization of CXCL10 decreased CXCR3 expression in neutrophils and macrophagesAs the majority of leukocytes in BALF after LPS induction were neutrophils and macrophages, we next investigated the proportion of CXCR3 positivity in neutrophils and macrophages. The percent of CXCR3+ neutrophils and CXCR3+ macrophages were significantly elevated in the LPS group compared with the saline group (p<0.01),moreover, treatment with anti-CXCL10 reduced the proportion of CXCR3+ cells among neutrophils and macrophages compared with the isotype control group( p<0.05).Conclusion:1. The ARDS model can be eatablished by intratracheal instillation of LPS (2mg/kg)in rats.2. Chemokine CXCL10 can promote the development of ARDS by binding to its receptor CXCR3.3. CXCL10 neutralization could ameliorate LPS-induced ARDS through the following possible mechanisms:(1) Decreasing the pulmonary edema;(2) Reducing the induction of important inflammatory factors that involved in the pathology of ARDS;(3) Suppressing the recruitment of inflammatory cells into lungs;(4) Decreasing the CXCR3 expression in both neutrophils and macrophages. |
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