Diabetes increases inflammation and lung injury associated with protective ventilation strategy in mice

International Immunopharmacology 13 (2012) 280–283

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Diabetes increases inflammation and lung injury associated with protective ventilation strategy in mice☆ Xiang-qing Xiong a, 1, Wan-tie Wang b, 1, Liang-rong Wang a, Li-da Jin a, Li-na Lin a,⁎ a b

Department of Anesthesiology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China Department of Pathophysiology, Wenzhou Medical College, Wenzhou, China

a r t i c l e

i n f o

Article history: Received 24 February 2012 Received in revised form 24 April 2012 Accepted 26 April 2012 Available online 9 May 2012 Keywords: Mechanical ventilation Low tidal volume Inflammation Diabetes Lung injury

a b s t r a c t Background: Mechanical ventilation may paradoxically cause lung injury. Protective mechanical ventilation strategy utilizing low tidal volume and high frequency has been shown to attenuate inflammation and reduce mortality in non-diabetic patients. The purpose of this present study was to observe the effects of diabetes on inflammation and lung injury in mice with protective ventilation strategy. Methods: Forty mice were included in our study. The mice in Group Dia-MV and Con-MV were subjected to 4 hour-ventilation. And the mice in Group Dia-SB and Con-SB were exposed to room air breathing spontaneously for 4 h. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), superoxide dismutase (SOD) and malondialdehyde (MDA) levels in serum were detected and the expression of inflammatory cytokine mRNA was also determined in lung tissue. Lung damage was assessed using a modified lung injury score. Results: The serum levels of TNF-α, IL-6, and IL-10 in Group Dia-MV were significantly higher than those in Group Dia-SB or Group Con-MV or Group Con-SB (P b 0.05). Quantitative RT-PCR analysis of pro-inflammatory cytokines in lung homogenates presented similar results. The mice in Group Dia-MV suffered obvious lung histological changes, whose lung injury scores were significantly higher in Group Dia-SB as compared to Group Con-SB , Group Con-MV or Group Dia-SB (P b 0.05). Conclusions: Diabetes increased the inflammation reaction and associated lung injury in mice in spite of the protective mechanical ventilation strategy based on low tidal volumes and high frequency. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Mechanical ventilation is the essential breath-support approach used in general anesthesia and also serves as a lifesaving intervention for acute respiratory failure. However, improper ventilation methods, especially with high tidal volumes, can lead to lung injury characterized by increased vascular permeability, alterations in lung mechanics, and increased production of inflammatory mediators [1]. Compared to the traditional high tidal volume method, protective ventilation strategy with low tidal volume and plateau pressure was shown to reduce mortality and decrease the length of ventilation duration in patients with acute respiratory distress syndrome (ARDS) [2]. A large body of evidence has revealed physiological and structural abnormalities in the lung tissue of both type 1 and type 2 diabetic animals; additionally, decreased capacity of the antioxidant defense ☆ This work was supported by the Scientific Research Foundation of Wenzhou, Zhejiang Province, China (No. Y20100030). ⁎ Corresponding author at: Department of Anesthesiology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, Zhejiang Province, China. E-mail address: [email protected] (L. Lin). 1 Co-first authors and contributed equally to this study. 1567-5769/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2012.04.020

system and increased oxidative stress were also observed [3]. However, the studies on the effects of the protective mechanical ventilation strategy on a diabetic lung remain unavailable. The purpose of the present study was to investigate the effects of diabetes on inflammation and lung injury in the streptozotocininduced diabetic mouse model under a low tidal volume and high frequency ventilation strategy. The production and expression of inflammatory cytokines, TNF-α, IL-6 and IL-10, in serum and lung tissue were measured to indicate inflammation. The oxidative stress was evaluated by detecting serum SOD and MDA levels. 2. Materials and methods 2.1. Animal preparation All experimental designs were approved by the Animal Ethics Committee of Wenzhou Medical College and strictly abided by the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male C57BL/6N mice weighing from 50 to 60 g were obtained from the Animal Experimental Center. Experimental diabetes was induced by using a single intraperitoneal administration of streptozotocin (65 mg/kg, Sigma-Aldrich Co. Ltd,

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USA), which was dissolved in 1 mL sodium citrate buffer (pH 4.5, 0.1 M) [4]. Blood samples were collected via the tail vein 2 days after the streptozotocin administration, and blood glucose levels were determined using a glucometer; mice with blood glucose levels >250 mg/dL were defined as diabetic and twenty of those mice were included in further studies. All mice were anesthetized using intraperitoneal administration of 90 mg kg ketamine and 1 mg/kg acepromazine, and placed in the supine position. Muscular relaxation was achieved by applying 1.5 mg/kg vecuronium intraperitoneally. Then, a tracheotomy was established using a 18-gage catheter and a polyethylene catheter was inserted into the left carotid artery for direct blood pressure monitoring and sampling. Anesthesia was maintained using continuous intraperitoneal administration of ketamine (45 mg/kg/h), acepromazine (0.5 mg/kg/h) and vecuronium (0.75 mg/kg/h). 2.2. Experimental design These twenty streptozotocin‐treated diabetics were randomized into two groups: Group Dia-MV and Group Dia-SB; another twenty control normal mice, which were not pretreated with streptozotocin, were randomly divided into Group Con-MV and Con-SB, n = 10 in each group. The mice in Group Dia-MV and Con-MV were subjected to 4 h of mechanical ventilation using an animal ventilator whose parameters were set as follows: tidal volume 8 mL/kg, frequency 150 breaths/min, and a fraction of inspired oxygen 0.35. The mice in Group Dia-SB and Con-SB were exposed to room air breathing spontaneously for 4 h. During the experimental procedure, oxygen saturation, blood pressure and heart rate were monitored continuously using multichannel physiologic recorder, and Ringer's solution was infused (8–10 mL/h) to resupply fluid losses and keep blood pressure within normal ranges. Blood samples were obtained from the left carotid artery every hour during ventilation for analysis of serum parameters and blood gasses including arterial oxygen and carbon dioxide partial pressures; the peak and plateau pressures were monitored and recorded every 30 min throughout the study. 2.3. Sample harvesting The blood sample was centrifuged at 8000 rpm for 15 min, and the serum was collected and stored at −80 °C for further testing. Immediately after 4 h of ventilation, the mice were euthanized and the lungs isolated. The right lung was inflated at standard pressure, fixed in 4% buffered formalin solution overnight at room temperature, dehydrated, and embedded in paraplast. The left lung was homogenized for the measurement of relative mRNA levels.

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the Sangon Biological Engineering Technology & Services (Shanghai, China) according to sequences previously described: Cytokines Forward primer

Reverse primer

TNF-α

5′-ACA TTC GAG GCT CCA GTG AAT TCG G-3′ 5′-ATG CTT AGG CAT AAC GCA CTA GGT T-3′ 5′- GCA ACC CAG GTA ACC CTT AAA GTC-3′

IL-6 IL-10

5′-GGC AGG TCT ACT TTG GAG TCA TTG C-3′ 5′-CTG GTG ACA ACC ACG GCC TTC CCT A-3′ 5′- TTT CTT TCA AAC GAA GGA CCA GAT G-3′

2.6. Lung injury score The right middle lung lobe was fixed in 4% buffered formalin solution overnight at room temperature, dehydrated, and embedded in paraplast. Four‐micrometer sections were used. The degree of lung damage was determined using a modified VILI score [5] described as follows: 1) thickness of the alveolar walls; 2) infiltration or aggregation of inflammatory cells; 3) hemorrhage. Each item was graded according to the following five-point scale: 0, minimal damage; 1, mild damage; 2, moderate damage; 3, severe damage; 4, maximal damage. The degree of lung damage was assessed with a sum of scores ranging from 0 to 12. The average of the sum of each field score was compared between groups. 2.7. Statistical analyses Statistical analyses were performed with SPSS software (version 15.0, SPSS, Chicago, IL). Continuous, normally distributed data were expressed as mean ± SD (standard deviation). Data were further analyzed for normality before a one-way ANOVA followed by the Student–Newman–Keuls post hoc test. For two group comparisons, the unpaired Student's t test was used. P b 0.05 was considered statistically significant. 3. Results 3.1. Comparison of parameters indicating inflammation and oxidative stress

The concentrations of TNF-α, IL-6 and IL-10 in serum were detected using an enzyme-linked immunosorbent assay (ELISA) based on the manufacturer's instructions. The detection limits for these kits were 3.5 pg/mL for TNF-α, 80 pg/mL for IL-6, and 15 pg/mL for IL-10. Serum SOD and MDA level were determined by the thiobarbituric acid reaction (Jiancheng Bio-engineering Research Institute, Nanjing, China).

The serum levels of TNF-α, IL-6, and IL-10 in Group Dia-MV and Group Dia-SB were higher than Group Con-MV and Con-SB at baseline, and those levels in Group Dia-MV were significantly higher than those in Group Dia-SB or Con-MV or Con-SB from first to fourth hour of ventilation (P b 0.05). Compared to baseline values, the serum levels of these cytokines were gradually increased over ventilation duration in Group Dia-MV, and no significant changes were found in Group Con-SB throughout the study (Figs. 1A–C). Quantitative RT-PCR analysis of pro-inflammatory cytokines in lung homogenates presented similar results (P b 0.05) (Fig. 2). At baseline, the activities of serum SOD in Group Dia-MV and Dia-SB were lower than Group Con-MV and Con-SB, while the levels of MDA were in reverse trend. The activities of serum SOD decreased and MDA levels were conversely increased along with ventilation duration in Group Dia-MV (P b 0.05) (Figs. 1D–E). No significant changes in MDA levels and SOD activities were found in Group Con-SB throughout the study

2.5. RT-PCR analysis of cytokines in lung tissue

3.2. Lung injury scores

The left lung tissue was homogenized and total RNA was extracted using TRIzol reagent (Sangon Biological Engineering Technology & Services, Shanghai, China) according to the manufacturer's instructions. For RT-PCR, 1 μg of total RNA from each sample was resuspended in a 25 μL final volume of reaction buffer. GAPDH was used as an internal control. The following primers were synthesized by

Compared with Group Con-SB, the lung injury scores were significantly higher in Group Dia-MV (3.83 ± 0.34, P b 0.05), Group Dia-SB (2.11 ± 0.20, P b 0.05) or Group Con-MV (1.98 ± 0.18, P b 0.05) (Fig. 3). The lung tissues in Group Dia-MV suffered serious lesions, which were demonstrated as lung mesenchymal dropsy, inflammatory cell infiltration, pulmonary alveolus bursts and hemorrhage,

2.4. Analysis of inflammatory cytokines and SOD/MDA levels in serum

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Fig. 1. Effect of diabetes on inflammatory cytokine levels in serum and oxidative stress in mice with protective ventilation strategy. The mice in Group Dia‐MV and Group Con‐MV were ventilated for 4 h with low tidal volume (8 mL/kg) and high frequency (150 breaths/min). The mice in Group Dia‐SB and Con‐SB were exposed to room air for 4 h. Then the levels of TNF‐α, IL‐6, IL‐10, SOD and MDA in the serum were analyzed. Graphs represent mean ± SD, n = 10 in each group. #P b 0.05, versus Group Con‐SB; *P b 0.05, versus Con‐MV; P b 0.05, versus Dia‐SB.

while mild lung microstructural changes were found in Group Dia-SB or Group Con-MV. 4. Discussion The main finding in the present study was that diabetes increases the inflammatory response and lung injury in mice ventilated by a protective strategy utilizing low tidal volumes and high frequency; this was indicated by increased levels of TNF-α, IL-6 and IL-10 in serum, lower serum SOD activity and increased serum MDA levels. Additionally, the diabetic lung suffered obvious histological changes resulting in high lung injury scores. A protective mechanical ventilation strategy consisting of low tidal volume (6 mL/Kg) and low plateau pressure was shown to decrease the length of ventilation duration and reduce mortality in

Fig. 2. Effect of diabetes on TNF-α, IL-6 and IL-10 mRNA of lung tissue homogenates in mice receiving protective ventilation strategy. The mice in Group Dia‐MV and Group Con‐MV were ventilated for 4 h with low tidal volume (8 mL/kg) and high frequency (150 breaths/min). The mice in Group Dia‐SB and Con‐SB were exposed to room air for 4 h. Then the left lung was homogenized and RNA was extracted, followed by RT‐ PCR for TNF‐α, IL‐6 and IL‐10 mRNA. Graphs represent mean ± SD, n = 10 in each group. #P b 0.05, versus Group Con‐SB; *P b 0.05, versus Group Con‐MV; P b 0.05, versus Group Dia‐SB.

patients with acute respiratory distress syndrome (ARDS) [6,7]. In order to avoid inadequate ventilation, our study utilized a mechanical ventilation strategy with relatively low tidal volumes (8 mL/kg) and high-frequency (150 breaths/min). As shown in our study, the levels of inflammatory cytokines and MDA significantly increased and SOD activities decreased along with ventilation duration. Quantitative RT-PCR analysis of lung homogenates revealed similar results. Additionally, interstitial edema, inflammatory cell infiltration, alveolar rupture or bleeding was obvious with high lung injury scores in diabetic mice, while the protective mechanical ventilation was well tolerated in non-diabetic mice. Increasing evidence has revealed physiological and structural abnormalities in the lung tissue of both type 1 and type 2 diabetic animals, thus highlighting the lung as a target organ [8]. Diabetes is associated with impaired pulmonary function in a restrictive pattern and reported basal lamina thickening of capillaries and epithelia in lungs, where nonenzymatic glycosylation-induced alteration of lung connective tissue is the most likely pathogenic mechanism underlying pulmonary microangiopathy. [9–11]. In addition, diabetes damages respiratory muscle endurance, causing a decline in lung elastic recoil [1]. Diabetic autonomic neuropathy and diastolic-stress disorder lead to dysfunction of ventilation–perfusion ratio also would contribute to lung injury [12]. Besides the potential structural pathogenic mechanism, decreased capacity of the antioxidative defense system and increased oxidative stress were reported to aggravate the diabetic lung injury [3]. An increase in the level of MDA, a lipid peroxidation marker, accompanied by the depressed activity of SOD, the most important endogenous antioxidase, was assessed to imply oxidant damage. Oxygen radicals are implicated in lung injury by causing lipid peroxidation of the pulmonary capillary membranes that are rich sources of polyunsaturated fatty acids as well as impairing alveolar– capillary barrier permeability. It is acknowledged that there are multiple sources of oxidative stress in the situations of hyperglycemia or diabetes: glucose can undergo autoxidation and generate oxygen radicals, in addition to the augmented generation of reactive species in diabetes including NOS, NAD(P)H oxidase and xanthine oxidase

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sensitive gene expression via transcription factor NF- B, which has been recognized to result in the release of inflammatory cytokines. Elevated TNF-α and IL-6 levels served as markers indicating a systemic inflammatory response in our study, where an increase in IL-10 level may shift the balance towards a compensatory antiinflammatory response. In conclusion, diabetes increased the inflammation reaction and associated lung injury in mice in spite of the protective mechanical ventilation strategy based on low tidal volumes and high frequency. However the potential mechanisms involved were not covered in our study, and the clinical significance also needs to be further elucidated. It is also remains unknown whether the lung injury would be aggravated in mice or patients that have suffered longer periods of diabetes or hyperglycemia. References

Fig. 3. Effect of diabetes on lung injury scores in mice with protective ventilation strategy. The mice in Group Dia‐MV and Group Con‐MV were ventilated for 4 h with low tidal volume (8 mL/kg) and high frequency (150 breaths/min). The mice in Group Dia‐SB and Con‐SB were exposed to room air for 4 h. The lungs had severe lesions in Group Dia‐MV, which was evidenced by lung mesenchymal dropsy, inflammatory cell infiltration, pulmonary alveolus bursts or hemorrhage. In Group Dia‐SB and Con‐MV, there were varying degrees of lung damage, but less than those in Group Dia‐MV. Lung injury scores were coordinated to microstructural changes. Graphs represent mean ± SD, n = 10 in each group. #P b 0.05, versus Group Con‐SB; *P b 0.05, versus Group Con‐MV; P b 0.05, versus Group Dia‐SB.

by their enzymatic sources [13–15]. In addition, the mitochondrial respiratory chain has been considered as another source of nonenzymatic generation of reactive species [16]. Diabetes also depresses the endogenous antioxidant enzymes, including GSH, SOD and catalase, leading to both ROS formation and activation of stress-

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