The Regulatory Role Of Costimulatory Molecule B7-H3 In LPS-induced Acute Lung Injury And Its Mechanisms

Acute lung injury(ALI), as an inflammatory injury in the lung, is a common severe condition in clinical practice, characterized by high morbidity and mortality. How to effectively prevent and treat ALI is one of the clinical challenges. B7-H3, as a costimulator, plays an important role in innate immunity. Currently, however, there is no literature on the role of B7-H3 in ALI. Based on the mouse ALI model, this study explores the effect of B7-H3 in the occurrence and development of ALI, clarifies the mechanism of the regulatory effect of B7-H3 in ALI, in an effort to provide a new target for the immunotherapy of ALI.Part I The establishment of a mouse ALI modelObjective: To explore and establish a mouse model of ALI with a high success and survival.Methods: Twenty male BALB/c mice were randomly divided into the LPS group and the control group treated with saline. Nasal dripping was performed to establish the mouse LPS-induced ALI model. The rate of success in the establishment of the model and the survival rate of the mice were recorded. After 24 hrs, the mouse lung tissue and bronchoalveolar lavage fluid(BALF) were collected. Pathological changes of lung tissues after HE staining were observed under the optical microscope. Cell counting and Wright’sstaining were done to determine the total count of white cells and the number of neutrophils in BALF. The total protein content in BALF was determined by the BCA method. Enzyme-linked immunosorbent assay(ELISA) was employed to analyze the changes in the protein level of the inflammatory cytokines(TNF-α, IL-1β and IL-6) and chemokines(CXCL2). Realtime PCR and ELISA were employed to respectively analyze the changes in the m RNA and protein levels of the inflammatory cytokines and chemokines in BALF and lung tissue. Pathological changes of lung tissues after H&E staining were observed under the optical microscope.Results: HE staining revealed lung congestion, microvascular endothelial cell bleeding, inflammatory exudation, neutrophil infiltration, thickened alveolar walls and hyalinosis in the LPS group while no such pathological changes were found in the control group. Compared with the control group, the total number of cells and neutrophils in BALF was significantly increased in the LPS group(p<0.01). Compared with the control group, the total protein content, inflammatory cytokines(TNF-α, IL-1β and IL-6) and chemokines(CXCL2) in BALF were also significantly increased(p<0.05). Compared with the control group, the m RNA and protein levels of the inflammatory cytokines and chemokines in BALF and lung tissue were significantly increased(p<0.01).Conclusions: Nasal dripping of LPS is applied to establish a mouse ALI model, and it is proved that the model has a high success and survival rate.Part II The regulatory effect of costimulatory molecule B7-H3 in ALI through regulating the functions of neutrophilsObjective: To evaluate the regulatory effect of costimulatory molecule B7-H3 in LPS-induced acute lung injury based on the mouse model of ALI.Methods: Forty male BALB/c mice were randomly divided into the control group treated with saline, the B7-H3 group, the LPS group and the B7-H3+LPS group. Nasal dripping of LPS was performed to establish the LPS-induced ALI model. 24 hrs after establishing the model, the mouse lung tissue and BALF of all groups were collected. Pathological changes of lung tissues after H&E staining were observed under the optical microscope. The total protein content in BALF was determined by the BCA method. Cell counting and Wright’s-staining were done to determine the total count of white cells and the number of neutrophils in BALF. The total number of cells and Wright’s-stained polymorphonuclear cells(PMNs) in BALF were counted. Myeloperoxidase(MPO) activity was evaluated by the MPO kit. Realtime PCR and ELISAwere employed to respectively analyze the changes in the m RNA and protein levels of the inflammatory cytokines and chemokines in BALF and lung tissue. ELISA was employed to analyze the changes in the protein level of the inflammatory cytokines and chemokines.Results: HE staining showed that B7-H3 can ameliorate the lung tissue injury of the LPS-induced ALI model. B7-H3 can significantly decrease the white cell count, the number of neutrophils and the total protein content in the BALF of the LPS-induced ALI model. B7-H3 can significantly decrease MPO activity in lung tissue(P<0.05) of the LPSinduced ALI model. B7-H3 can reduce the expression of chemokines(CXCL2) in BALF. These differences from the LPS group are statistically significant. But the changes in the expression of inflammtory cytokines TNF-α, IL-1β and IL-6 in BALF are not affected. Compared with the LPS group, B7-H3 can downregulate the expression of chemokines(CXCL2) in lung tissue without affecting the expression of inflammtory cytokines TNF-α、IL-1β and IL-6 in lung tissue, which was a significant difference.Conclusions: Research on the ALI model suggests that B7-H3 can ameliorate the lung tissue injury of the LPS-induced ALI model, lower pulmonary vascular permeability, depress MPO activity, lower the expression of inflammatory cytokines, decreasing the LPS-induced ALI in mice, and exerting a protective effect.Part III The mechanism of the protective effect of costimulatory molecule B7-H3 in ALI through downregulating the chemotaxis of neutrophils and macrophagesObjective: To study the regulatory effect of costimulatory molecule B7-H3 on neutrophils and alveolar macrophages and analyze the mechanism of B7-H3 in ALI.Methods: Forty male BALB/c mice were randomly divided into the control group treated with saline, the B7-H3 group, the LPS group and the B7-H3+LPS group. 24 hrs after establishing the mouse LPS-induced ALI model, BALF from all groups was collected. The apoptosis of neutrophils was analyzed by flow cytometry. Collagenase digestion, cell adhesion and MACS were used to isolate mouse lung endothelial cells,alveolar macrophages and marrow-origin neutrophils, which were analyzed for cell apoptosis, chemotaxis and transendothelial migration. FACS analysis was also employed to analyze the expression of CXCR2 and Mac-1. Chemiluminescence was used for Reactive Oxygen Spices(ROS). Realtime PCR and ELISA were used for detecting the chemokine m RNA and protein levels in alveolar macrophages. The phosphorylation of NF-k B p65 and MAPK p38 on alveolar macrophages was analyzed by FACS.Results: The recombinant protein B7-H3 does not affect PMN apoptosis both in vitro and in vivo. B7-H3 attenuates LPS-stimulated PMN chemotaxis, and the expression of CXCR2 on PMN is markedly downregulated. B7-H3 also attenuates LPS-stimulated PMN migration across pulmonary vascular endothelial cell monolayer, and the expression of Mac-1 on PMN is markedly downregulated. B7-H3 inhibits LPS-stimulated reactive oxygen species(ROS) release. B7-H3 attenuates LPS-stimulated CXCR2 expression in alveolar macrophages and leads to the downregulation of LPS-induced NF-k B p65 phosphorylation, but it does no affect LPS-induced MAPK p38 phosphorylation.Conclusions: B7-H3 affects neutrophil chemotaxis, tranendothelial migration, ROS generation, expression of chemokine CXCL2 on avelolar macrophages and the phosphorylation of signal pathway p65. Therefore, B7-H3 exerts a protective effect in ALI mainly through downregulating the chemotaxis of neutrophils and avelolar macrophages towards the affected area and reducing lung injury.