Study On The Role Of Gas6/Mer Signaling Pathway In Silica-induced Lung Inflammation And Fibrosis In Mice

Long-term inhalation of silica particles is the cause of silicosis,while the pathogenic mechanism of which is still unclear.Inhaled silica can cause lung inflammation,and lead to diffuse fibrosis in the lung tissue with time.Our previous research found that growth arrest specific protein 6（Gas6）and its tyrosine kinase receptor TAM family（Mer,Axl and Tyro3）were involved in the regulation of silica-induced lung inflammation and fibrosis,suggesting that the Gas6/TAM pathway might play a key role in the course of silicosis.In this study,we first explored the changes of Gas6 levels in the lung and peripheral blood in mice after silica exposure.Then,we established silica exposure models using Gas6 or Mer deficient mice by gene knockout technology,to explore the effect and the downstream mechanisms of Gas6/Mer pathway in silica-induced lung inflammation and fibrosis,aiming to find a new target for prevention and treatment of silicosis.PartⅠ.Effect of silica exposure on Gas6 level in lung and peripheral blood of mice.Objective:To establish a silica exposure model in wild type（WT）mice and observe the expression of growth arrest specific protein 6（Gas6）in bronchoalveolar lavage fluid（BALF）and peripheral blood.Methods:We generated a silica exposure model by intratracheal administration of silica suspension in mice.A total of 48 male mice,weighing 18-22 g,were randomly divided into two groups including control group and silica group（24 mice per group）.Mice in control group were subjected to sterilized normal saline（NS）instillation,while mice in silica group received an equivoluminal silica suspension.At days 7,28 and 84after exposure,8 mice in each group were randomly sacrificed to collect BALF and peripheral blood.The Gas6 concentrations in BALF and serum were determined by enzyme-linked immunosorbent assay（ELISA）.Result:Compared with respective control group,the protein level of Gas6 in BALF was significantly elevated on day 7 and maintained the stably high level on days28 and 84 in silica-exposed groups（P<0.05）.At day 7 after silica exposure,the Gas6concentration in serum was significantly increased and then had a slight decrease on days 28 and 84,which remained to be higher when compared with control group at the same time point（P<0.05）.Conclusions:Silica exposure induced a sustained elevation of Gas6 in mice lung,suggesting that Gas6 might play an important role in silicosis.PartⅡ.The role of Gas6 gene knockout in silica-induced lung inflammation and fibrosis in mice.Objective:To explore the potential role and mechanisms of Gas6 gene knockout in silica-induced lung inflammation and fibrosis in mice,and its effect on the expressions of TAM receptors.Methods:Mouse was subjected to intratracheal instillation of silica suspension to generate the silica exposure model.We identified Gas6-deficient mice(Gas6^-/-)and its WT littermates reproduced by Gas6 gene heterozygote for experiment,which were randomly divided into control groups and silica groups（24 mice per group）.There were four groups including WT control group,WT silica group,Gas6^-/-control group and Gas6^-/-silica group,which subjected to an equivoluminal of NS for control groups and silica suspension for silica groups.At days 7,28 and 84 after silica exposure,8 mice of each group were sacrificed,and the following indicators were measured:Ⅰ.The lung injury was revealed by levels of total protein（TP）,lactate dehydrogenase（LDH）,alkaline phosphatase（AKP）and acid phosphatase（ACP）in BALF.Ⅱ.To evaluate the lung inflammatory response,the concentrations of IL-1β,TNF-αand MCP-1 in BALF were determined by ELISA and the lung tissues were stained by hematoxylin-eosin（H&E）.To observe macrophages recruitment in lung tissues,the expression of F4/80 was detected by immunohistochemistry analysis（IHC）,quantitative real-time PCR（RT-PCR）and Western blot.Ⅲ.We investigated lung fibrosis after silica exposure revealed by Masson staining,the m RNA and protein expressions of collagen-related indictor（Fibronectin,Collagen?）.To explore the potential mechanism of Gas6 in regulating silica-induced fibrosis,RT-PCR and WB were used to detect the m RNA and protein levels of epithelial-mesenchymal transition（EMT）-related factors（TGF-β,E-cadherin,α-SMA）.IV.The influence of silica exposure and Gas6 gene knockout on expressions of TAM tyrosine kinase receptors including Mer,Axl and Tyro3 was also assessed at the m RNA and protein levels.Result:Ⅰ.Effect of Gas6 gene kockout on silica-induced lung injury:The expressions of TP,LDH,AKP and ACP in BALF elevated at days 7,28 and 84 after silica exposure in WT mice,which were largely prevented and maintained a low level in Gas6^-/-silica group（P<0.05）.Ⅱ.Effect of Gas6 gene kockout on silica-induced lung inflammation:（1）In silica-treated WT mice,H&E-stained sections showed varying degrees of inflammatory cell infiltration and granuloma at days 7,28 and 84 after silica exposure.Pathology inflammation scores of Gas6^-/-silica groups were significantly lower those of the corresponding WT silica groups at all post-exposure time points（P<0.05）.(（2）In WT silica group,the contents of IL-1β,TNF-αand MCP-1 in BALF increased at all post-exposure time points（P<0.05）.Compared with WT silica group,the levels of IL-1βand TNF-αin Gas6^-/-silica group decreased significantly on days 28 and 84,while MCP-1 concentration had a significant decrease on days 7 and 28（P<0.05）.（3）The IHC result showed that F4/80 expression in the lung tissues of WT silica group,a macrophage marker,was significantly higher than that of the respective control group at all post-exposure time points,which was partly prevented in Gas6^-/-mice（P<0.05）.Further,we quantified the severity of macrophage infiltration at both m RNA and protein levels,which confirmed the above results.Ⅲ.Effect of Gas6 gene kockout on silica-induced lung fibrosis:（1）In silica-treated group,Masson staining showed an obvious collagen deposition on days 28and 84 in WT mice（P<0.05）,which were significantly attenuated in Gas6-deficient mice.（2）In parallel to histopathological change,the expressions of Fibronectin and Collagen?had a significant increase on days 28 and 84 in both WT and Gas6^-/-mice after silica exposure（P<0.05）.However,their expressions were much lower in Gas6^-/-silica group than those in WT silica group at the same time point（P<0.05）.（3）In WT silica group,we detected EMT reflected by increased m RNA and protein levels of TGF-βandα-SMA,and decreased m RNA and protein levels of E-cadherin in mice lung tissues on days 28 and 84（P<0.05）.Genetic loss of Gas6 could partly reverse the silica-induced EMT（P<0.05）.Ⅳ.Effect of Gas6 gene knockout on the expressions of TAM receptor in silica-exposed mice lung:The m RNA and protein expressions of Mer receptor in silica-treated WT mice was significantly higher than that in respective control group at all post-exposure time points,while it was inhibited in Gas6^-/-silica group（P<0.05）.In addition,there was no significant difference in the m RNA and protein levels of Axl and Tyro3 in the lung tissues of each group.Conclusions:Gas6 deficiency alleviated silica-induced ling injury,pulmonary inflammation and fibrosis,possibly through inhibition of silica-induced macrophages recruitment and EMT process.Gas6 gene knockout significantly decreased the high expression of Mer receptor in mice lung after silica exposure.Gas6 has a certain role in regulating the expression of Mer receptor,so we continue to explore the role of Mer in silica-induced pulmonary inflammation and fibrosis in mice.PartⅢ.The role of Mer gene knockout in silica-induced lung inflammation and fibrosis in mice.Objective:To investigate the potential function and downstream mechanisims of Mer receptor gene knockout in silica-induced lung injury,inflammation and fibrosis.Methods:Mer gene heterozygote were bred to reproduce Mer-deficient mice(Mer^-/-)and its WT littermates for experiment for experiment,which were randomly divided into control groups and silica groups（24 mice per group）.There were four groups including WT control group,WT silica group,Mer^-/-control group and Mer^-/-silica group,which subjected to an equivoluminal of NS for control groups and silica suspension for silica groups.At days 7,28 and 84 after silica exposure,8 mice of each group were sacrificed,and the following indicators were measured:We detected the levels of TP,LDH,AKP and ACP in BALF to evaluate the effect of Mer gene knockout on silica-induced lung injury.IL-1β,TNF-αand MCP-1 concentrations in BALF,the m RNA and protein levels of F4/80 were measured for evaluation of silica-induced inflammation.Masson staining was used to observe collagen deposition in lung tissue,RT-PCR and Western blot were used to determine the m RNA and protein levels of fibrosis-related（Fibronectin,Collagen?）and EMT-related indicators（TGF-β,E-cadherin,α-SMA）.Result:Ⅰ.Effect of Mer gene kockout on silica-induced lung injury:Our data showed that Mer gene knockout could also markedly attenuate silica-induced increase of TP,LDH,AKP and ACP in BALF at all post-exposure time points（P<0.05）.Ⅱ.Effect of Mer gene kockout on silica-induced lung inflammation:（1）Mer gene knockout significantly alleviated silica-induced inflammatory lesion at all post-exposure time points（P<0.05）.（2）Results from ELISA showed that the concentrations of IL-1β,TNF-αand MCP-1 in WT silica group were significantly higher than those in respective control group at all post-exposure time points（P<0.05）.Compared with WT silica group,IL-1βconcentration in Mer^-/-silica-treated mice declined significantly at all post-exposure time points,while the content of TNF-αsignificantly decreased on days 28 and 84,MCP-1 concentration decreased on days 7and 28 in Mer^-/-silica group（all P<0.05）.（3）Expression of macrophage marker F4/80was elevated in WT silica group and peaked on day 28,which was largely prevented in Mer-deficient mice after silica exposure（P<0.05）.Ⅲ.Effect of Mer gene kockout on silica-induced lung fibrosis:（1）Compared with the control group,WT mice lung showed an obvious collagen deposition and increased expressions protein levels of Fibronectin and Collagen?on days 28 and 84after silica exposure,which were alleviated in Mer^-/-silica group at the same time point.（2）Mer deficiency significantly ameliorated silica-induced EMT in mice lung on days28 and 84,mainly reflected by decreased levels of TGF-βandα-SMA,increased E-cadherin expressions.Conclusions:Genetic loss of Mer obviously ameliorated silica-induced lung injury,inflammation,and fibrosis.Mer receptor and its ligand Gas6 have similar effects in silica-induced pulmonary inflammation and fibrosis in mice.Gas6 or Mer knockout may play its role in inhibiting inflammatory response and fibrosis by prevention of macrophages accumulation and downregulating EMT.