J. Cell. Mol. Med. Vol 20, No 9, 2016 pp. 1706-1717
Bone-marrow-derived mesenchymal stem cells inhibit gastric aspiration lung injury and inflammation in rats Jing Zhou
a, b, #
, Liyan Jiang c, #, Xuan Long a, #, Cuiping Fu a, Xiangdong Wang a, Xiaodan Wu a, Zilong Liu a, Fen Zhu a, Jindong Shi d, *, Shanqun Li a, *
a
Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China b Department of General Practice, Zhongshan Hospital, Fudan University, Shanghai, China c Department of Respiratory Medicine, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China d Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China Received: October 13, 2015; Accepted: March 3, 2016
Abstract Gastric aspiration lung injury is one of the most common clinical events. This study investigated the effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on combined acid plus small non-acidified particle (CASP)-induced aspiration lung injury. Enhanced green fluorescent protein (EGFP+) or EGFP BMSCs or 15d-PGJ2 were injected via the tail vein into rats immediately after CASP-induced aspiration lung injury. Pathological changes in lung tissues, blood gas analysis, the wet/dry weight ratio (W/D) of the lung, levels of total proteins and number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) were determined. The cytokine levels were measured using ELISA. Protein expression was determined by Western blot. Bone-marrow-derived mesenchymal stem cells treatment significantly reduced alveolar oedema, exudation and lung inflammation; increased the arterial partial pressure of oxygen; and decreased the W/D of the lung, the levels of total proteins and the number of total cells and neutrophils in BALF in the rats with CASP-induced lung injury. Bone-marrow-derived mesenchymal stem cells treatment decreased the levels of tumour necrosis factor-a and Cytokine-induced neutrophil chemoattractant (CINC)-1 and the expression of pp65 and increased the levels of interleukin-10 and 15d-PGJ2 and the expression of peroxisome proliferator-activated receptor (PPAR)-c in the lung tissue in CASP-induced rats. Tumour necrosis factor-a stimulated BMSCs to secrete 15d-PGJ2. A tracking experiment showed that EGFP+ BMSCs were able to migrate to local lung tissues. Treatment with 15d-PGJ2 also significantly inhibited CASP-induced lung inflammation and the production of pro-inflammatory cytokines. Our results show that BMSCs can protect lung tissues from gastric aspiration injury and inhibit lung inflammation in rats. A beneficial effect might be achieved through BMSC-derived 15d-PGJ2 activation of the PPAR-c receptor, reducing the production of proinflammatory cytokines.
Keywords: bone marrow mesenchymal stem cell gastric aspiration acute respiratory distress syndrome 15-deoxy-D12 14 prostaglandin J2 peroxisome proliferator-activated receptor-c
Introduction Gastric aspiration is a common clinical event. The content of the aspirate includes gastric contents, blood, bacteria, liquids and secretions. Gastric aspiration is a major direct cause of acute lung injury (ALI) and the more severe acute respiratory distress syndrome [1, 2]. Gastric aspiration may result in a variety of clinical lung injuries, ranging from mild, subclinical pneumonitis to a more severe, progressive disease with associated high mortality. Acute respiratory distress syndrome from gastric aspiration frequently occurs in an unconscious patient in one of every 2000–4000 anaesthetic cases [3, 4]. Acute res-
piratory distress syndrome associated with gastric aspiration accounts for approximately 30% of the mortality and 20% of all deaths attributable to anaesthesia [5]. Acid and food particles are the main components of gastric aspiration that induces lung injury. Food particles of gastric aspirates are not only responsible for airway obstruction but also directly contribute to inflammatory damage [6]. The aspiration of combined acid and small gastric particles (CASP) can lead to more severe lung injury in contrast to lung injury induced by either acid or gastric particles alone
# These authors contributed equally to this work. *Correspondence to: Shanqun LI E-mail: [email protected]
Jindong SHI E-mail: [email protected]
doi: 10.1111/jcmm.12866
ª 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
J. Cell. Mol. Med. Vol 20, No 9, 2016 and can generate more severe lung pathological changes than the sum of the individual components [4, 6]. Inflammatory injury is an important characteristic in gastric aspiration lung injury. Acid-induced lung injury is characterized by the stimulation of capsaicin-sensitive neurons and a direct chemical acid burn on the airway epithelium followed by an acute neutrophilic inflammatory response over 4–6 hrs [1]. Therefore, reducing or blocking the inflammatory cascade in gastric aspiration lung injury is an important treatment strategy [7]. 15-Deoxy-D-12,14-prostaglandin J2 (15d-PGJ2) has anti-inflammatory effects [8]. 15d-PGJ2 is formed by spontaneous PGD2 dehydration into cyclopentane (PGJ2 is forms first, then further dehydration leads to D-12-PGJ2 and finally to 15d-PGJ2). In contrast to other prostaglandins, 15d-PGJ2 may exert anti-inflammatory effects mainly through interacting with intracellular targets [9]. In particular, 15d-PGJ2 is an endogenous ligand for the intranuclear receptor PPARc. PPARc is a member of the nuclear receptor superfamily and a ligand-activated transcription factor, with pleiotropic effects on lipid metabolism, inflammation and cell proliferation. PPARc is expressed in both alveolar macrophages and neutrophils and plays a critical role in inflammatory responses. Many studies have demonstrated that the activation of PPARc by 15d-PGJ2 or other agonists has anti-inflammatory effects and leads to a substantial reduction in experimental ALI [9–11]. However, the roles of 15d-PGJ2 and PPARc in gastric aspiration-induced lung injury are unknown. Recently, bone-marrow-derived mesenchymal stem cells (BMSCs) have attracted a great deal of attention because they have a potent in vivo immunosuppressive effect in humans, as reported in 2004 [12]. Bone-marrow-derived mesenchymal stem cells exhibit anti-inflammatory effects in several diseases, including myocardial infarction, diabetes, sepsis, hepatic failure and acute renal failure. The compelling benefits of BMSCs for lung injury have also generated a great deal of interest [13]. Bone-marrow-derived mesenchymal stem cells can increase the release of prostaglandin E2, which acts on the EP2 and EP4 receptors of macrophages and stimulates the production and release of interleukin (IL)-10 [14]. However, the effects and underlying mechanisms by which BMSCs exert anti-inflammatory effects on CASP-induced aspiration lung injury are unknown. In this study, we investigated the effects of BMSCs on CASP-induced aspiration lung injury in a rat model.
Materials and methods BMSC isolation and culture Bone-marrow-derived mesenchymal stem cells were isolated from rat bone marrow. Briefly, three specific-pathogen-free (SPF) male Sprague– Dawley rats weighing 100–150 g were anaesthetized by an intraperitoneal injection of 10% chloral hydrate (4 ml/kg) and then executed by breaking the neck; the bone marrow tissue was harvested from bilateral femurs under sterile conditions. Cells were plated in 25 cm2 plastic flasks with DMEM-LG (Gibco, New York, NY, USA) containing 10% foetal bovine serum (FBS; Gibco), 10 ng/ml epidermal growth factor (Sigma-Aldrich, Saint Louis, MO, USA), 100 U/ml penicillin (Gibco) and
100 U/ml streptomycin (Gibco) at 37°C in humid air with 5% CO2 (Precision Scientific, MA, USA) for culture. After a 48-hr incubation, BMSCs adhered to the bottom of the culture plates and haematopoietic cells remained suspended in the medium; non-adherent cells were discarded. The medium was changed every 3–4 days thereafter. When the adherent cells were confluent to more than 80%, they were digested with 0.25% trypsin-EDTA (Gibco) and re-plated at 1:2 dilutions under the same culture conditions. The cells were further purified by passaging. The growth curves of BMSCs at passages 2, 4 and 6 were determined by a CCK-8 kit (Dojindo Chemical Co., Kyushu, Japan) in accordance with the manufacturer’s instructions.
Immunophenotypic analysis of BMSCs The BMSCs at passage 5 were trypsinized into a single cell suspension and re-cultured in multi-well tissue culture plates (4 wells) containing Poly-L-Lysine (Sigma-Aldrich, Munich, Germany)-pre-coated cover slides at a density of 1 9 103 cells/well in DMEM medium supplemented with 10% FBS. The cultures were maintained in a CO2 incubator under the same culture conditions until they reached confluence. Cell BMSCs were harvested by digestion with trypsin, washed with PBS, and stained with PE- or fluorescein isothiocyanate (FITC)-labelled antibodies to CD14, CD19, CD34, CD45, CD73, CD90 or CD105 (BioLegend, Shanghai, China). The BMSCs at passages 5 were used for flow cytometry (Beckman Coulter, Brea, CA, USA) using Expo32 ADC analysis software, concurrent with the time that they were used during the whole experiment. Bone-marrow-derived mesenchymal stem cells were positive for CD73, CD93 and CD105 and negative for the haematopoietic markers CD14, CD19, CD34 and CD45 [15, 16].
Animal models Specific-pathogen-free male Sprague–Dawley rats weighing 200–250 g (Shanghai Slac laboratory animal Co., Ltd., Shanghai, China) were divided into three groups: (i) Normal saline (NS) + PBS group: 12 rats were injected with PBS via the tail vein immediately after being instilled with NS intratracheally; (ii) Injury + PBS group: 12 rats were injected via the tail vein with PBS immediately after being instilled with CASP intratracheally; and (iii) Injury + BMSC group: 12 rats were injected via the tail vein with BMSCs immediately after being instilled with CASP intratracheally. All the animals were maintained at the Experimental Animal Center of Zhongshan Hospital, Fudan University. All the animal protocols were reviewed and approved by the ethical committee for Animal Care and Use at Zhongshan Hospital, Fudan University. All the animals were housed in high-efficiency particulate air-filter-topped cages in a sterile laminar flow environment and had free access to food and water before and after the operation.
CASP-induced aspiration lung injury and BMSC transplantation Gastric particulates were prepared from the stomach contents of SPF male Sprague–Dawley rats as described by Knight [17]. Briefly, five SPF male Sprague–Dawley rats weighing 100–150 g were anaesthetized by an intraperitoneal injection of 10% chloral hydrate (4 ml/kg) and were then killed by breaking the neck; the stomach contents were collected, washed
ª 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
1707
several times in NS, filtered through gauze and sterilized by autoclaving (final particle sizes were
Bone-marrow-derived mesenchymal stem cells inhibit gastric aspiration lung injury and inflammation in rats Jing Zhou
a, b, #
, Liyan Jiang c, #, Xuan Long a, #, Cuiping Fu a, Xiangdong Wang a, Xiaodan Wu a, Zilong Liu a, Fen Zhu a, Jindong Shi d, *, Shanqun Li a, *
a
Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China b Department of General Practice, Zhongshan Hospital, Fudan University, Shanghai, China c Department of Respiratory Medicine, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China d Department of Respiratory Medicine, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China Received: October 13, 2015; Accepted: March 3, 2016
Abstract Gastric aspiration lung injury is one of the most common clinical events. This study investigated the effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on combined acid plus small non-acidified particle (CASP)-induced aspiration lung injury. Enhanced green fluorescent protein (EGFP+) or EGFP BMSCs or 15d-PGJ2 were injected via the tail vein into rats immediately after CASP-induced aspiration lung injury. Pathological changes in lung tissues, blood gas analysis, the wet/dry weight ratio (W/D) of the lung, levels of total proteins and number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) were determined. The cytokine levels were measured using ELISA. Protein expression was determined by Western blot. Bone-marrow-derived mesenchymal stem cells treatment significantly reduced alveolar oedema, exudation and lung inflammation; increased the arterial partial pressure of oxygen; and decreased the W/D of the lung, the levels of total proteins and the number of total cells and neutrophils in BALF in the rats with CASP-induced lung injury. Bone-marrow-derived mesenchymal stem cells treatment decreased the levels of tumour necrosis factor-a and Cytokine-induced neutrophil chemoattractant (CINC)-1 and the expression of pp65 and increased the levels of interleukin-10 and 15d-PGJ2 and the expression of peroxisome proliferator-activated receptor (PPAR)-c in the lung tissue in CASP-induced rats. Tumour necrosis factor-a stimulated BMSCs to secrete 15d-PGJ2. A tracking experiment showed that EGFP+ BMSCs were able to migrate to local lung tissues. Treatment with 15d-PGJ2 also significantly inhibited CASP-induced lung inflammation and the production of pro-inflammatory cytokines. Our results show that BMSCs can protect lung tissues from gastric aspiration injury and inhibit lung inflammation in rats. A beneficial effect might be achieved through BMSC-derived 15d-PGJ2 activation of the PPAR-c receptor, reducing the production of proinflammatory cytokines.
Keywords: bone marrow mesenchymal stem cell gastric aspiration acute respiratory distress syndrome 15-deoxy-D12 14 prostaglandin J2 peroxisome proliferator-activated receptor-c
Introduction Gastric aspiration is a common clinical event. The content of the aspirate includes gastric contents, blood, bacteria, liquids and secretions. Gastric aspiration is a major direct cause of acute lung injury (ALI) and the more severe acute respiratory distress syndrome [1, 2]. Gastric aspiration may result in a variety of clinical lung injuries, ranging from mild, subclinical pneumonitis to a more severe, progressive disease with associated high mortality. Acute respiratory distress syndrome from gastric aspiration frequently occurs in an unconscious patient in one of every 2000–4000 anaesthetic cases [3, 4]. Acute res-
piratory distress syndrome associated with gastric aspiration accounts for approximately 30% of the mortality and 20% of all deaths attributable to anaesthesia [5]. Acid and food particles are the main components of gastric aspiration that induces lung injury. Food particles of gastric aspirates are not only responsible for airway obstruction but also directly contribute to inflammatory damage [6]. The aspiration of combined acid and small gastric particles (CASP) can lead to more severe lung injury in contrast to lung injury induced by either acid or gastric particles alone
# These authors contributed equally to this work. *Correspondence to: Shanqun LI E-mail: [email protected]
Jindong SHI E-mail: [email protected]
doi: 10.1111/jcmm.12866
ª 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
J. Cell. Mol. Med. Vol 20, No 9, 2016 and can generate more severe lung pathological changes than the sum of the individual components [4, 6]. Inflammatory injury is an important characteristic in gastric aspiration lung injury. Acid-induced lung injury is characterized by the stimulation of capsaicin-sensitive neurons and a direct chemical acid burn on the airway epithelium followed by an acute neutrophilic inflammatory response over 4–6 hrs [1]. Therefore, reducing or blocking the inflammatory cascade in gastric aspiration lung injury is an important treatment strategy [7]. 15-Deoxy-D-12,14-prostaglandin J2 (15d-PGJ2) has anti-inflammatory effects [8]. 15d-PGJ2 is formed by spontaneous PGD2 dehydration into cyclopentane (PGJ2 is forms first, then further dehydration leads to D-12-PGJ2 and finally to 15d-PGJ2). In contrast to other prostaglandins, 15d-PGJ2 may exert anti-inflammatory effects mainly through interacting with intracellular targets [9]. In particular, 15d-PGJ2 is an endogenous ligand for the intranuclear receptor PPARc. PPARc is a member of the nuclear receptor superfamily and a ligand-activated transcription factor, with pleiotropic effects on lipid metabolism, inflammation and cell proliferation. PPARc is expressed in both alveolar macrophages and neutrophils and plays a critical role in inflammatory responses. Many studies have demonstrated that the activation of PPARc by 15d-PGJ2 or other agonists has anti-inflammatory effects and leads to a substantial reduction in experimental ALI [9–11]. However, the roles of 15d-PGJ2 and PPARc in gastric aspiration-induced lung injury are unknown. Recently, bone-marrow-derived mesenchymal stem cells (BMSCs) have attracted a great deal of attention because they have a potent in vivo immunosuppressive effect in humans, as reported in 2004 [12]. Bone-marrow-derived mesenchymal stem cells exhibit anti-inflammatory effects in several diseases, including myocardial infarction, diabetes, sepsis, hepatic failure and acute renal failure. The compelling benefits of BMSCs for lung injury have also generated a great deal of interest [13]. Bone-marrow-derived mesenchymal stem cells can increase the release of prostaglandin E2, which acts on the EP2 and EP4 receptors of macrophages and stimulates the production and release of interleukin (IL)-10 [14]. However, the effects and underlying mechanisms by which BMSCs exert anti-inflammatory effects on CASP-induced aspiration lung injury are unknown. In this study, we investigated the effects of BMSCs on CASP-induced aspiration lung injury in a rat model.
Materials and methods BMSC isolation and culture Bone-marrow-derived mesenchymal stem cells were isolated from rat bone marrow. Briefly, three specific-pathogen-free (SPF) male Sprague– Dawley rats weighing 100–150 g were anaesthetized by an intraperitoneal injection of 10% chloral hydrate (4 ml/kg) and then executed by breaking the neck; the bone marrow tissue was harvested from bilateral femurs under sterile conditions. Cells were plated in 25 cm2 plastic flasks with DMEM-LG (Gibco, New York, NY, USA) containing 10% foetal bovine serum (FBS; Gibco), 10 ng/ml epidermal growth factor (Sigma-Aldrich, Saint Louis, MO, USA), 100 U/ml penicillin (Gibco) and
100 U/ml streptomycin (Gibco) at 37°C in humid air with 5% CO2 (Precision Scientific, MA, USA) for culture. After a 48-hr incubation, BMSCs adhered to the bottom of the culture plates and haematopoietic cells remained suspended in the medium; non-adherent cells were discarded. The medium was changed every 3–4 days thereafter. When the adherent cells were confluent to more than 80%, they were digested with 0.25% trypsin-EDTA (Gibco) and re-plated at 1:2 dilutions under the same culture conditions. The cells were further purified by passaging. The growth curves of BMSCs at passages 2, 4 and 6 were determined by a CCK-8 kit (Dojindo Chemical Co., Kyushu, Japan) in accordance with the manufacturer’s instructions.
Immunophenotypic analysis of BMSCs The BMSCs at passage 5 were trypsinized into a single cell suspension and re-cultured in multi-well tissue culture plates (4 wells) containing Poly-L-Lysine (Sigma-Aldrich, Munich, Germany)-pre-coated cover slides at a density of 1 9 103 cells/well in DMEM medium supplemented with 10% FBS. The cultures were maintained in a CO2 incubator under the same culture conditions until they reached confluence. Cell BMSCs were harvested by digestion with trypsin, washed with PBS, and stained with PE- or fluorescein isothiocyanate (FITC)-labelled antibodies to CD14, CD19, CD34, CD45, CD73, CD90 or CD105 (BioLegend, Shanghai, China). The BMSCs at passages 5 were used for flow cytometry (Beckman Coulter, Brea, CA, USA) using Expo32 ADC analysis software, concurrent with the time that they were used during the whole experiment. Bone-marrow-derived mesenchymal stem cells were positive for CD73, CD93 and CD105 and negative for the haematopoietic markers CD14, CD19, CD34 and CD45 [15, 16].
Animal models Specific-pathogen-free male Sprague–Dawley rats weighing 200–250 g (Shanghai Slac laboratory animal Co., Ltd., Shanghai, China) were divided into three groups: (i) Normal saline (NS) + PBS group: 12 rats were injected with PBS via the tail vein immediately after being instilled with NS intratracheally; (ii) Injury + PBS group: 12 rats were injected via the tail vein with PBS immediately after being instilled with CASP intratracheally; and (iii) Injury + BMSC group: 12 rats were injected via the tail vein with BMSCs immediately after being instilled with CASP intratracheally. All the animals were maintained at the Experimental Animal Center of Zhongshan Hospital, Fudan University. All the animal protocols were reviewed and approved by the ethical committee for Animal Care and Use at Zhongshan Hospital, Fudan University. All the animals were housed in high-efficiency particulate air-filter-topped cages in a sterile laminar flow environment and had free access to food and water before and after the operation.
CASP-induced aspiration lung injury and BMSC transplantation Gastric particulates were prepared from the stomach contents of SPF male Sprague–Dawley rats as described by Knight [17]. Briefly, five SPF male Sprague–Dawley rats weighing 100–150 g were anaesthetized by an intraperitoneal injection of 10% chloral hydrate (4 ml/kg) and were then killed by breaking the neck; the stomach contents were collected, washed
ª 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
1707
several times in NS, filtered through gauze and sterilized by autoclaving (final particle sizes were
