| Background Carbon nanotubes(CNTs),as the main nanomaterials,have superior physical and chemical properties.In recent years,they have been widely used in all walks of life,CNT nanoparticles are generated in various processes such as production,transportation,and storage,increasing the potential exposure of occupational populations and posing potential hazards to their health.In the occupational environment,CNT mainly enters the human body through the respiratory tract,and inhaled particles can be deposited in the lungs,causing damage to the respiratory system,including reducing lung function,inducing lung inflammation and pulmonary fibrosis.Single-walled carbon nanotubes(SWCNTs)in CNTs are more widely used than multi-walled carbon nanotubes(MWCNTs),and the previous research of our group found that SWCNTs have stronger ability to promote diseases than MWCNTs.Therefore,it is one of the important tasks of public health research to identify,evaluate,predict and control the health hazards of SWCNTs to occupational population and consumers.Objective1.To clarify that the relationship between neutrophil cluster and pulmonary inflammation and fibrosis,lung injury induced by SWCNT through single cell RNA sequencing of lung tissue from mice.2.To reveal the mechanism of enhancing or desensitizing the sensitivity of GPCR to regulate neutrophil cluster and mediate the lung inflammation and fibrosis induced by SWCNT in mice by adjusting the sensitivity of GPCR in vivo and in vitro.3.To investigate the molecular mechanism of GPCR regulating SWCNT-induced neutrophil clustering via the enrichment and functional analysis of single cell RNA sequencing results,GPCR desensitizing regulator and Si RNA interference.Materials and Methods1.The relationship between inflammation and fibrosis of lung tissue induced by SWCNT and neutrophil cluster in mice.C57BL/6J mice aged 8-10 weeks were divided into control group and SWCNT group.PBS and 40 μg/ SWCNT were given by tracheal instillation respectively.After treatment for 1 day,3 days,7 days,28 days and 56 days,the lung function was tested.Neutrophils were extracted from BALF,and the morphology,quantity,function and GPCR sensitivity were evaluated.Then the mice were killed to take lung tissue for detection.To analyze the relationship between lung function,inflammation and fibrosis of lung tissue and neutrophil cluster in mice,and analyze the cell subsets,intercellular interaction,neutrophil subsets and differential gene expression of single cell RNA sequencing results.2.Animal experiment of neutrophil colony regulating SWCNT-induced lung inflammation and fibrosis.In order to explore the mechanism of neutrophil clustering in regulating inflammation and fibrosis of lung tissue induced by SWCNT,the effects of neutrophil clustering on inflammation and fibrosis of lung tissue in mice were analyzed by using GPCR desensitization inhibitor in the early stage of SWCNT exposure through animal experiments.Firstly,C57BL/6J mice aged 8-10 weeks were randomly divided into control group,SWCNT group and GPCR desensitization inhibitor groups with different doses(12.5 μg/ml,25 μg/ml and 50 μg/ml).The control group and SWCNT group were given PBS and 40 μg/ SWCNT respectively by tracheal instillation,and the GPCR desensitization inhibitor group was injected intraperitoneally with 0.2m L on the 0 th day after SWCNT exposure.Lung function was tested on the 1st,3rd and7 th day after SWCNT exposure,and lung tissue was taken to detect inflammation and fibrosis,so as to determine the best inhibitor dosage.After determining that 25 μg/ml is the best dosage of GPCR desensitization inhibitor,C57BL/6J mice aged 8-10 weeks were randomly divided into control group,SWCNT group and 25 μg/ml GPCR desensitization inhibitor group at different time points in the early stage of SWCNT exposure.The control group and SWCNT group were given PBS or 40 μg/ SWCNT by intratracheal instillation,and the GPCR desensitization inhibitor group was injected with 0.2m L of 25 intraperitoneally on the first and second days after SWCNT exposure.After 3,7 and 28 days of SWCNT exposure,the lung function was tested,the morphology,quantity,function and sensitivity of GPCR of neutrophils in BALF were evaluated,and lung tissue was taken to detect inflammation and fibrosis.To explore the mechanism of neutrophil cluster inducing inflammation and fibrosis in mouse lung tissue through regulating the expression of chemokines and EMT and FMT mediating SWCNT.3.GPCR regulates neutrophil cluster,mediates SWCNT-induced chemokine secretion,EMT and FMT in vitro.In order to further validate the mechanism of inducing GPCR desensitization to limit self clustering and regulating EMT and FMT induced by SWCNT exposure,we conducted in vitro studies.Firstly,the neutrophils cultured in vitro were treated with SWCNT,and the sensitivity of neutrophils to GPCR,the expression of chemokines,chemotaxis and phagocytosis were detected with or without GPCR desensitizing inducer or inhibitor.Then,neutrophils treated with SWCNT were co-cultured with lung bronchial epithelial cells or lung fibroblasts with or without GPCR desensitization inducer or inhibitor,respectively,and the expressions of EMT markers E-cad and Vim in lung bronchial epithelial cells and FMT markers α-SMA and COL I in lung fibroblasts were detected,so as to verify in vitro the mechanism of inducing GPCR desensitization of neutrophils,limiting their clustering and regulating EMT and FMT in the middle and late period of SWCNT exposure.4.Molecular mechanism of GPCR regulating neutrophil cluster.4.1 Functional enrichment and pathway analysis of major differential genes of neutrophil subsets in lung tissue.In order to further explore the molecular mechanism of GPCR regulating neutrophil clustering in SWCNT-induced lung toxicity,the KEGG enrichment analysis and pathway analysis of the main differential genes of C1 and C2 subgroups with the largest number of neutrophils in mouse lung tissue were carried out to find possible molecules of GPCR regulating neutrophil clustering.4.2 K-ras regulates chemokine expression and promotes neutrophil clusteringThe enrichment of single cell RNA sequencing results and pathway analysis in early lung tissues of mice exposed to SWCNT showed that K-ras was related to neutrophil clustering in early stage of SWCNT exposure.In order to explore whether K-ras promoted neutrophil clustering and its possible mechanism,the expression of K-ras in BALF of mice with lung injury induced by SWCNT exposure was detected,and the relationship between K-ras and neutrophil chemokine expression,GPCR sensitivity and cell chemotaxis function was analyzed.In vitro,the expression of K-ras in neutrophils was up-regulated and down-regulated by overexpression and Si RNA interference,respectively,to explore the regulatory effect of K-ras on the sensitivity of neutrophils to GPCR,the expression of chemokines and the directional chemotaxis of cells.4.3 GRK2/β-arrestin regulates GPCR desensitization to limit neutrophil clustering.Enrichment of single cell RNA sequencing results and pathway analysis in lung tissue of mice exposed to SWCNT showed that GRK2 and β-arrestin were related to neutrophil colony self-limitation in the middle and late stage of SWCNT exposure.In order to explore whether GRK2 and β-arrestin mediate neutrophil colony self-limitation and its possible mechanism,The expressions of GRK2 and β-arrestin in neutrophils of BALF mice with lung injury induced by SWCNT exposure were detected,and the relationship between GRK2 and β-arrestin and neutrophil chemokine expression,GPCR sensitivity and cell-oriented chemotaxis function was analyzed.Si RNA vitro experiments were conducted to investigate the regulatory effects of GRK2 and β-arrestin on the sensitivity of neutrophils to GPCR,the expression of chemokines and the directional chemotactic function of cells.Results1.The relationship between inflammation and fibrosis of lung tissue induced by SWCNT and neutrophil cluster in mice.1.1 The lung injury caused by SWCNT exposure in mice is mainly inflammation in the early stage and fibrosis in the middle and late stage.The lung function of mice exposed to SWCNT began to decrease at the early stage of exposure,and it became more and more serious with the increase of time(P<0.05).At the early stage of SWCNT exposure,the infiltration of inflammatory cells in lung tissue of mice increased significantly,and the contents of pro-inflammatory cytokines IL-1β and TNF-α in BALF increased significantly(P<0.05),but pulmonary fibrosis was not obvious.In the middle and late stage of SWCNT exposure,the inflammation in the lung tissue of mice was relieved,but there was obvious collagen deposition.The content of HYP in lung tissue was significantly higher than that in PBS group,and the expressions of COL1,Vimentin and α-SMA in lung tissue were significantly higher than that in PBS group,while the expression of E-cad was significantly lower than that in PBS group(P <0.05).1.2 Neutrophils may rely on GPCR to mediate lung inflammation and fibrosis through chemokines and intracellular signaling pathways.Analysis of single cell RNA sequencing results shows,in the early and middle stages of SWCNT exposure,29 cell clusters were copolymerized,and 9 cell types were identified,including neutrophils,macrophages,fibroblasts,epithelial cells,T cells,B cells,NK cells,plasma cells and erythrocytes,among which the number of neutrophils was the most significant and the number of differential genes was the most.In the analysis of cell-cell interaction,at the early stage of SWCNT exposure,the signal intensity of neutrophils was much higher than that of other cells except fibroblasts;In the middle stage of SWCNT exposure,it was slightly higher than other cells except fibroblasts.These results indicate that neutrophils play an important role in the process of lung injury induced by SWCNT exposure in mice.This study identified six subpopulations of neutrophils(C1-C6),with C1 and C2 having the highest number,and there was a significant difference in the proportion in the early and middle stages of SWCNT exposure(P<0.001).The number of differential genes in C1 and C2 cell subgroups was the largest among the 6 subgroups.Twenty-eight of the common differential genes belong to inflammation and pulmonary fibrosis,including Cxcr4,Grk2,K-ras,Cxcl2,Smad3,etc.Among these28 differential genes,GO enrichment analysis showed that 6 genes were enriched to GPCR binding,15 genes were enriched to GO items related to chemokine response,and KEGG enrichment analysis showed that 10 genes were enriched to chemokine signal pathway.On the main chemokine CXCL pathway signal,neutrophils are the main senders and receivers.In the early stage of exposure,neutrophils mainly regulate macrophages and cooperate with macrophages.In the middle stage of exposure,it mainly regulates fibroblasts and cooperates with fibroblasts to play a role.KEGG enrichment analysis also found that the total differential genes of C1 and C2 cell subsets were mainly highly enriched in Endocytosis,MAPKand other signaling pathways,especially Endocytosis signaling pathway,which may be related to phagocytosis of neutrophils.There were 28 common differential genes in C1 and C2 cell subsets.Sh3kbp1,Cxcr4,Grk2,etc.are significantly up-regulated.Cxcr4 and Grk2 have an upstream and downstream regulatory relationship and also play an important role in chemokine signaling pathway,while Cxcr4 is a representative GPCR, and Grk2 is mainly involved in the desensitization of GPCR.1.3 SWCNT exposure can enhance the sensitivity and directional chemotaxis of neutrophils in the early stage,and promote the lung clustering of neutrophils;In the middle and late stage of exposure,the neutrophil GPCR is desensitized and the directional chemotaxis function is weakened,which limits the lung colony of neutrophils.In mice exposed to SWCNT,there were many granular protrusions on the surface of neutrophils in lung tissue at the early stage of exposure,and some cells ruptured,and the abnormality became more serious with the increase of time.Compared with PBS group,at the early stage of exposure,the proportion of neutrophils in BALF increased by more than 10 times,the expression of neutrophils’ chemokines BLT1 and CXCR2 and their phagocytic function increased significantly,and the calcium concentration and instantaneous influx level of neutrophils,a sensitive indicator of GPCR,increased significantly(P<0.05),but the expression of GRK2,a key regulator of GPCR desensitization,did not change significantly(P<0.05).In the middle and late stage of exposure,compared with the early stage of exposure,the proportion of neutrophils in BALF,the expression of chemokines BLT1 and CXCR2,and the phagocytic function were significantly decreased,and the concentration of neutrophils’ calcium ions and the instantaneous influx level were significantly decreased.However,the expression of GRK2 increased significantly(P<0.05).2.Animal experiment of neutrophil colony regulating SWCNT-induced lung inflammation and fibrosis.The results of dose screening experiment showed that the best dose of neutrophil GPCR desensitization inhibitor was 25μg/ml,then choose the early stage after SWCNT exposure,that is,the first and second days after exposure to inhibit neutrophil GPCR desensitization.2.1 Inhibition of neutrophil GPCR desensitization upregulates the expression of neutrophil chemokines in the early stage of SWCNT exposure,enhances the directional chemotaxis and phagocytic function of neutrophils,and promotes effective clustering of neutrophils.Mice that inhibited neutrophil GPCR desensitization on day 1 after exposure also experienced rupture and abnormalities of lung tissue neutrophils in the early phase of exposure compared with the SWCNT group,with significantly higher proportions of neutrophils,BLT1 and CXCR2 protein expression,and significantly enhanced phagocytosis in the BALF(P<0.05),but with no significant changes in directional chemotaxis,with significantly elevated calcium current concentrations and transient endocytosis levels were significantly elevated,and there was no significant change in GRK2 m RNA expression(P>0.05);In the middle and late stages of exposure,lung tissue neutrophil abnormalities improved,with significantly higher proportions of neutrophils,BLT1 and CXCR2 protein expression in the BALF,significantly enhanced directional chemotaxis and phagocytosis,and significantly higher calcium ion flow concentrations and transient endocannabinoid flow levels,but significantly lower GRK2 m RNA expression(P<0.05).Mice that inhibit neutrophil GPCR desensitization on the second day after exposure,compared with the SWCNT group,rupture and abnormality of lung tissue neutrophils occurred in the early stage of exposure,the proportion of neutrophils in the BALF,the expression of BLT1 and CXCR2 proteins were significantly elevated,phagocytosis and targeted chemotactic function were significantly enhanced,the concentration of calcium ion flow and the level of transient endocytosis were were significantly elevated(P<0.05),and there was no significant change in GRK2 m RNA expression(P>0.05);In the middle and late phases of exposure,lung tissue neutrophil abnormalities were significantly better,with a significantly lower proportion of neutrophils in BALF,significantly higher BLT1 and CXCR2 protein expression,significantly enhanced directional chemotaxis and phagocytosis(and higher than that of the day 1-treated group),and significantly higher calcium ion flow concentration and transient endocytosis levels(but lower than that of the day 1-treated group),whereas GRK2 m RNA expression was significantly reduced(P<0.05).2.2 Inhibiting neutrophil GPCR desensitization reduces lung inflammation induced by SWCNT exposure,alleviates mid to late stage pulmonary fibrosis induced by SWCNT exposure,and enhances lung function.Mice that inhibit neutrophil GPCR desensitization on the first day after exposure,compared with the SWCNT group,there was a significant inflammatory infiltrate in the lung tissue at the early stage of exposure,the levels of IL-1β and TNF-α in the BALF were significantly elevated(P<0.05),no significant collagen deposition was seen in the lung tissue,the levels of HYP in the lung tissue and the COL1,E-cad,Vimentin and α-SMA protein expression were not significantly changed(P>0.05),and Penh and EF50,the main indicators of lung function,were decreased and increased,respectively(P<0.05);In the middle stage of exposure,the inflammatory infiltration of lung tissue was obvious,the contents of IL-1β and TNF-α in BALF were significantly elevated,collagen deposition in lung tissue was significantly relieved,the contents of HYP and protein expression of COL1,Vimentin and α-SMA in lung tissue were significantly decreased,protein expression of E-cad was significantly elevated,and the main indexes of lung function,Penh and EF50,were respectively decreased and increased(P<0.05);In the late stage of exposure,the inflammatory infiltration of lung tissue was obvious,but the contents of IL-1β and TNF-α in BALF were significantly decreased,collagen deposition in lung tissue was significantly improved,the contents of HYP and protein expression of COL1,Vimentin andα-SMA in lung tissue were significantly decreased,the protein expression of E-cad was significantly increased,the main indexes of lung function Penh and EF50 decreased and increased respectively(P<0.05).Mice that inhibit neutrophil GPCR desensitization on the second day after exposure,compared with the SWCNT group,there was a significant inflammatory infiltration in lung tissue in the early stage of exposure,and the levels of IL-1β and TNF-α in BALF were significantly elevated(P<0.05),but lower than those in the day1 treatment group,and there was no significant collagen deposition in the lung tissue,and the levels of HYP in the lung tissue and the levels of COL1 and E-cad,Vimentin and α-SMA protein expression were not significantly changed,and Penh and EF50,the main indexes of lung function,were decreased and increased,respectively(P<0.05);In the middle and late stages of exposure,the inflammatory infiltrate of lung tissue was significantly relieved,the content of IL-1β and TNF-α in BALF was significantly decreased,collagen deposition in lung tissue was significantly better,the content of HYP in lung tissue and the protein expression of COL1,Vimentin,andα-SMA were significantly decreased,and the protein expression of E-cad was significantly increased,and the main indexes of lung function Penh significantly decreased and EF50 significantly increased(P<0.05).3.GPCR regulates neutrophil cluster and mediates SWCNT-induced chemokine secretion,EMT and FMT in vitro.3.1 GPCR regulates SWCNT induced neutrophil clustering and its cellular functionCompared with PBS,the morphology of neutrophils treated with SWCNT is quite abnormal,with more granular protrusions on the cell surface and some cells breaking.The level of calcium ion decreased significantly,the expression of GRK2 m RNA and protein increased significantly,and the chemotaxis and phagocytosis decreased significantly(P<0.05).Compared with the SWCNT group,the morphological abnormality of neutrophils in the SWCNT+GPCR desensitization inducer group was more obvious,the calcium ion level decreased significantly,the m RNA and protein expression of GRK2 increased significantly,and the chemotaxis and phagocytosis decreased significantly(P<0.05).However,the morphological abnormality of neutrophils in SWCNT+GPCR desensitization inhibitor group was improved,the calcium ion level was increased,the m RNA and protein expression of GRK2 were significantly decreased,and the chemotaxis and phagocytosis were significantly enhanced(P<0.05),which was close to that in PBS group.3.2 GPCR regulates neutrophil clustering and its cellular function,mediating SWCNT induced EMT and FMTCompared with PBS,the expression level of TGF-β1,the fluorescence intensity of ROS and the contents of MPO and NE in neutrophils treated with SWCNT were significantly increased(P<0.05).Neutrophils treated with SWCNT can up-regulate the expression of Vimentin and down-regulate the expression of E-cad in co-cultured BEAS-2B cells,and up-regulate the expression of COL1 and α-SMA in primary lung fibroblasts of mice(P<0.05).Compared with the SWCNT group,the expression level of TGF-β1,the fluorescence intensity of ROS and the contents of MPO and NE of neutrophils in the SWCNT+GPCR desensitization inducer group were significantly increased,and the expressions of Vimentin and E-cad in co-cultured BEAS-2B cells were up-regulated and the expressions of COL1 and α-SMA in primary mouse lung fibroblasts were up-regulated significantly(P<0.05).In the SWCNT+GPCR desensitization inhibitor group,the expression level of TGF-β1 protein in neutrophils,the fluorescence intensity of ROS and the contents of MPO and NE were significantly decreased,which could down-regulate the expression of Vimentin protein in BEAS-2B,up-regulate the expression of E-cad protein,and down-regulate the expression of COL1 and α-SMA protein in primary mouse lung fibroblasts(P<0.05).4.Molecular mechanism of GPCR regulating neutrophil cluster.4.1 GPCR may regulate the neutrophil colony in the lung tissue of mice exposed by SWCNT through K-ras,GRK2 and β-arrestin.The results of RNA sequencing of single cell in lung tissue of mice exposed to SWCNT showed that K-ras,β-arrestin,Smad3,etc.were co-expressed with the main differentially expressed genes Cxcr4 and Grk2 in neutrophil C1 and C2 subgroups.Compared with PBS group,the expression level of K-ras in lung neutrophils of mice in SWCNT group was significantly up-regulated on the 1st and 3rd day after exposure,but down-regulated on the 7th day after exposure.The expression levels of Grk2 andβ-arrestin did not change significantly after 1 and 3 days of exposure,but increased significantly after 7 days of exposure.4.2 K-ras regulates the expression and clustering of neutrophil chemokines in the early stage of SWCNT exposure.Compared with PBS group,the expression level of K-ras protein in BALF neutrophils in SWCNT group and GPCR desensitization inhibitor group increased significantly after 3 days of exposure(P<0.05),but decreased significantly after 7days and 28 days of exposure(P<0.05).Moreover,the GPCR desensitization inhibitor group was significantly higher than the SWCNT group after 7 days and 28 days of exposure(P<0.05).Compared with PBS,the expression level of K-ras protein in neutrophils cultured in vitro treated by SWCNT was significantly decreased(P<0.05).Compared with the neutrophils cultured in vitro treated with SWCNT,the expression level of K-ras protein in SWCNT+GPCR desensitization inducer group decreased significantly,while the expression level of K-ras protein in SWCNT+GPCR desensitization inhibitor group increased significantly(P<0.05).Overexpression of neutrophil K-ras can significantly up-regulate the expression of autologous chemokines BLT1 and CXCR2,and enhance the sensitivity and directional chemotaxis of neutrophils induced by SWCNT(P<0.05).Inhibition of neutrophil K-ras expression significantly decreased the expression of autologous chemokines BLT1 and CXCR2,promoted the desensitization of neutrophil GPCR induced by SWCNT(P<0.05),and decreased the directional chemotactic function of cells.4.3 GPCR/β-arrestin regulates the desensitization of neutrophils in the middle and late stage of SWCNT exposure,which limits their clustering.Compared with the PBS group,the expression levels of GRK2 and β-arrestin protein in BALF neutrophils of mice did not change significantly after 3 days of exposure(P>0.05),but increased significantly after 7 days and 28 days of exposure(P<0.05).Compared with SWCNT group,GPCR desensitization inhibitor group decreased significantly after 7 days and 28 days of exposure(P<0.05).Compared with PBS,the expression levels of GRK2 and β-arrestin protein of neutrophils cultured in vitro treated by SWCNT were significantly increased(P<0.05).Compared with SWCNT-treated neutrophils cultured in vitro,the expression levels of GRK2 andβ-arrestin in SWCNT+GPCR desensitization inducer group increased significantly,while the expression levels of GRK2 and β-arrestin in SWCNT+GPCR desensitization inhibitor group decreased significantly(P<0.05).Compared with PBS group,the level of neutrophil calcium in SWCNT group was significantly decreased,and the expression levels of autologous chemokines BLT1 and CXCR2 were significantly decreased,which indicated that neutrophil GPCR was desensitized,neutrophil colony was self-limited,and the directional chemotaxis function decreased.After overexpression of neutrophil GRK2,the protein expression levels of GRK2 and β-arrestin were further increased,while the calcium level was significantly decreased,and the protein expression levels of BLT1 and CXCR2 were further decreased(P<0.05).At this time,the neutrophil GPCR was further desensitized(P<0.05),and the directional chemotaxis function continued to decline.However,after the neutrophil β-arrestin was knocked out,the protein expression level of β-arrestin decreased significantly(P<0.05),the protein expression level of GRK2 did not change significantly(P>0.05),the calcium ion level increased significantly,and the protein expression levels of BLT1 and CXCR2 increased significantly(P<0.05).At this time,the sensitivity of neutrophil GPCR increased.Over-expressing GRK2 plasmid and knockout β-arrestin plasmid were added at the same time.Compared with adding knockout β-arrestin plasmid alone,the protein expression level of neutrophil β-arrestin was significantly increased,while the calcium ion level was significantly decreased,and the protein expression levels of BLT1 and CXCR2 were significantly decreased(P<0.05).These results indicate that GRK2/β-arrestin regulates GPCR desensitization and mediates neutrophil colony self-limitation.Conclusions1.The sensitivity of neutrophils to GPCR is enhanced in the early stage of SWCNT exposure,which promotes lung clustering and causes lung inflammation;The desensitization of neutrophils in the middle and late stage of exposure limits their clustering in the lung,and the acute inflammation of lung tissue gradually turns into low-grade chronic inflammation and forms fibrosis.2.Early activation of K-ras by SWCNT exposure can up-regulate the secretion of neutrophil chemokines,enhance the sensitivity of GPCR,promote neutrophil lung tissue clustering,and enhance its directional chemotaxis and phagocytosis.3.In the middle and late stage of SWCNT exposure,neutrophils aggregated in lung tissue can desensitize GPCR by activating their own GRK2-β-arrestin pathway,limit neutrophil aggregation,and damage the directional chemotaxis and phagocytosis. |
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