Regulation And Mechanism Of ASICla On TNF-α Expression In LPS Activated Macrophages In Vitro And In Vivo

Acute lung injury（ALI）is an inflammatory disease of the body caused by a variety of intrinsic and extrinsic factors.This inflammatory reaction causes an increase in alveolar-capillary-intimal damage in the lungs,resulting in increased permeability of alveolar-capillary vessels,which is a common clinically high incidence disease,and its pathogenesis has not been fully reported.Alveolar macrophages,as alveolar resident macrophages,are the first line of defense for the immune defense of the lungs.By secreting inflammatory factors such as TNF-α,they directly activate polymorphonuclear leukocytes（PMNs）,leading to lungs.An internal uncontrolled inflammatory burst that triggers the onset of ALI.Tissue acidification is a fundamental feature of the inflammatory response,with a slight decrease in the pH of the local tissue.Whether or not there is a protein sensitive to pH drop on the surface of macrophages,which mediates the occurrence of various inflammatory reactions,is worthy of further discussion.Acid-sensing ion channels 1a（ASIC1a）are a class of proton H⁺gated acid-sensitive channels.When a variety of inflammatory diseases occur,the common feature is tissue acidification,mild pH decline,and ASIC1a channel will sense this pH drop,aggravating the body damage caused by inflammation.Current studies indicate that extracellular acidification induces ASIC1a activation and affects the formation and function of bone macrophages.LPS stimulates spinal macrophages,activates the ASIC1a channel,and regulates the expression of inflammatory factors in cells.The endoplasmic reticulum is a Ca²⁺reservoir.When extracellular Ca²⁺flows into the cell,it causes intracellular Ca²⁺overload,which in turn causes the endoplasmic reticulum protein synthesis and folding functions to be deregulated,thereby activating endoplasmic reticulum Stress,ERS).Numerous studies have shown that ERS participates in the regulation of inflammation by activating NF-κB,MARK and other inflammatory signaling pathways.The previous study of the research team found that ASIC1a is elevated in the inflammatory environment caused by ALI,ERS plays an important role in the development of ALI.Activation of ASIC1a induces extracellular Ca²⁺influx,and changes in intracellular Ca²⁺homeostasis induce ERS.To this end,based on the previous research of the research group,this study investigated the effect of ASIC1a on the expression of TNF-αsecreted by LPS-activated macrophages and its relationship with ERS in an in vitro and in vivo inflammatory model.The main research contents are summarized as follows:1.Expression changes of ASIC1a and TNF-αin alveolar macrophages of ALI mouse modelIn this study,a mouse ALI model（LPS）was established,and bronchoalveolar lavage fluid was extracted by bronchial lavage technique.Mouse primary alveolar macrophages（AM）were isolated and extracted from bronchoalveolar lavage fluid.Lung tissue sections of the mouse ALI model were prepared and pathological changes were examined.The lung tissue was subjected to wet-to-dry weight ratio.AM was identified by Wright-Gissom staining.Compared with the normal group,the expression of ASIC1a,TNF-αmRNA and protein in the LPS group was significantly increased.The results showed that the mouse ALI model was successfully established,and the primary alveolar macrophages were successfully isolated and cultured.The high expression of ASIC1a was found in the ALI mouse model.2.Expression of ASIC1a,ERS-related proteins GRP78,CHOP,C/EBPα,TNF-αin alveolar macrophages of ALI mouse model after Amiloride blocked ASIC1aThe expression of ASIC1a,ERS-related proteins GRP78,CHOP,C/EBPα,TNF-αwere detected by Western Blot,q-PCR and ELISA experimental analysis methods.The results showed that the expressions of ASIC1a,GRP78,CHOP and TNF-αin AM in the LPS group were significantly up-regulated,and the expression of C/EBPαwas significantly down-regulated.It suggested that ASIC1a activated ERS to regulate the expression of inflammatory factor TNF-αsecreted by alveolar macrophages.3.Screening of optimal stimulation concentration and optimal action time of cell model LPSWe used NO kit to detect the secretion of NO in LPS at different concentrations and different time conditions.The results showed that the optimal concentration of LPS was0.25μg/mL,and the NO secretion was the highest.The optimal duration of LPS was 24h,and the NO secretion was the highest.The optimal stimulation concentration and optimal time of LPS activation of RAW264.7 cells was 0.25μg/mL,24 h.4.Effects of LPS-induced activation of RAW264.7 cells on the expression of ASIC1a and TNF-αRAW264.7 cells were activated by LPS（0.25μg/mL）,and the expression changes of ASIC1a and TNF-αin the cell inflammation model were detected by Western Blot,q-PCR and ELISA.The results showed that the expression of ASIC1a and TNF-αin the LPS group was significantly higher than that in the normal group（P<0.01）.It suggested that ASIC1a may play an important role in the inflammatory response of LPS-activated RAW264.7 cells.5.Effects of PcTx1 on the change of Ca²⁺concentration induced by LPS in RAW264.7 cellsThe laser confocal technique was used to dynamically detect the change of Ca²⁺concentration in RAW264.7 cells.From the results of confocal fluorescence images and relative concentration trajectories,the fluorescence intensity of Ca²⁺was significantly enhanced in RAW264.7 cells of LPS group,while the fluorescence intensity of Ca²⁺was weakened after PcTx1.It suggested that the ASIC1a channel is open to promote Ca²⁺influx.6.PcTx1/ASIC1a-ShRNA blocked ASIC1a,LPS induced activation of RAW264.7expression of ASIC1a,TNF-α,ERS-related proteinRAW264.7 cells were stimulated with LPS（0.25μg/mL）for 24 h to establish an in vitro inflammatory cell model.After blocking ASIC1a with PcTx1 and ASIC1a-ShRNA,ASIC1a gene expression was down-regulated.The protein expression changes of ERS marker proteins GRP78,CHOP and C/EBPαwere detected by Western Blot and q-PCR analysis methods.It was found that after PcTx1/ShRNA-ASIC1a treatment,the expression of ERS marker proteins GRP78 and CHOP was down-regulated,and C/EBPαexpression was up-regulated.The ELISA assay method was used to detect the expression of TNF-α,and the results showed that the expression of TNF-αwas significantly decreased.The q-PCR results were consistent with the ELISA results.It suggested that ASIC1a activated ERS to participate in the regulation of the expression of the inflammatory factor TNF-αsecreted by LPS activated RAW264.7 cells in vitro.7.Interaction between CHOP and C/EBPαproteinWe used the Co-IP kit to detect the results.The results showed that CHOP and C/EBPαwere expressed in the mixed protein solution.When the CHOP protein was precipitated,C/EBPαwas precipitated.Similarly,when the C/EBPαprotein was precipitated,the CHOP protein was also precipitated.The results indicated that CHOP interacts with C/EBPαin the cell and the two bind to each other.8.Effect of C/EBPαon the activity of TNF-αpromoterWe used the luciferase assay kit to detect the effect of C/EBPαon the activity of TNF-αpromoter and detected the activity of luciferase.The results showed that the plasmid was transfected with mC/EBPα,and the luciferase activity was unchanged compared with the empty vector.Transfection of TNF-αplasmid showed no change in luciferase compared with empty vector.When C/EBPαwas co-transfected with TNF-αplasmid,luciferase activity was significantly enhanced.It suggested that C/EBPαand TNF-α.The promoter region is combined.9.Overexpression of C/EBPαto detect the expression of TNF-αTo further observe the relationship between C/EBPαand TNF-αpromoter,we transfected RAW264.7 cells with overexpressed C/EBPαplasmid for 24 h.Both q-PCR and ELISA showed that overexpression of C/EBPαsignificantly decreased the expression of TNF-α,suggesting that C/EBPαnegatively regulates TNF-αexpression in LPS-activated RAW264.7 cells.