| Objective: Fungi widely existed in the air,soil and other environments,which human beings depend for survival.Inhalation of organic dust contaminated by fungi might lead to hypersensitivity pneumonitis.Repeated exposure might cause chronic inflammation,granuloma and even irreversible lung fibrosis.And it brought a huge burden to social economy and medical care.However,the specific pathogenesis was still unclear.As an important component of cell wall,1,3-?-glucan was a recognized biological marker of fungi.It was used to set up lung inflammation model to further study the mechanism of fungi induced inflammation.Epithelial cells were the first barrier in the lungs to defense fungal invasion,and their injury and death could cause the destruction of the immune barrier.Autophagy could regulate homeostasis to maintain cell survival,and restrict inflammation.HMGB1 was a traditional multifunctional factor,which could affect autophagy at multiple levels However,whether HMGB1 could regulate epithelial cells autophagy in 1,3-?-glucan induced lung inflammation? And its regulatory mechanism remained unclear.So,in this study,1,3-?-glucan was used to set up lung inflammation model,type 6 adeno-associated virus carried short-hairpin-HMGB1 was used to silence the expression of HMGB1 in the lung,to explore the regulatory mechanism of HMGB1 on 1,3-?-glucan induced lung inflammation.Methods: In this study,the male C57BL/6 mice were randomly divided into six groups:(1)Control group: saline,(2)Experimental group: 1,3-?-glucan,(3)Control empty virus group: saline+AAV-NC,(4)Experimental empty virus group: 1,3-?-glucan+AAV-NC,(5)Control silence group: saline+ AAV-sh-HMGB1,(6)Experimental silence group: 1,3-?-glucan+ AAV-sh-HMGB1.ELISA,Western Blot and Realtime-PCR analyses were used to detect the silencing effect of HMGB1.The protein expression of HMGB1 in mice was observed by immunofluorescence.The inflammatory responses of each group mice lung were observed by H&E staining.Luminex and Realtime-PCR analyses were used to detect the secretion of cytokines in the alveolar lavage fluid(BALF)and the transcriptional levels of the cytokines in the lung tissues.Collagen deposition was detected by measuring hydroxyproline content in lung tissues of mice and Masson staining.The above experiments were used to demonstrate the effect of silencing HMGB1 on 1,3-?-glucan induced lung inflammation.Human lung epithelial cell line A549 cells were treated with 1,3-?-glucan to establish an epithelial cell inflammation model in vitro,and HMGB1 silencing model in vitro was set by transfection of lentivirus carried sh-HMGB1.The expression and the secretion of cytokines in epithelial cells treated with 1,3-?-glucan were checked by Realtime-PCR and ELISA assays.Immunofluorescence assay was used to demonstrate the expression level of HMGB1 on epithelial cells.The expressions of autophagy-related proteins after silencing HMGB1 were detected by Realtime-PCR and Western Blot assays in 1,3-?-glucan induced lung inflammation.Transmission electron microscopy was used to observe the production of autophagosomes in epithelial cells of mice lung.The change of autophagy level in A549 cells after silencing HMGB1 was massured by immunofluorescence assay.The above experiments were used to demonstrate the effect of silenting HMGB1 on 1,3-?-glucan induced autophagy in epithelial cells.The effect of autophagy flow induced by silencing HMGB1 was measured by immunofluorescence,Western Blot and Realtime-PCR.The Beclin1-BCL2 binding was studied by immunoprecipitation assay after silencing HMGB1 in 1,3-?-glucan induced lung inflammation and immunoprecipitation assay was used to measure the effect of silencing HMGB1 on Beclin1 post-modification.The expression of apoptosis-related protein in each group mice lung was detected by Western Blot and Realtime-PCR.TUNEL staining was used to further demonstrate the effect of HMGB1 on apoptosis CCK8 method was used to measure A549 cell viability after autophagy activator and apoptosis inhibitor treatment.The above experiments were used to illustrate the specific regulatory sites after silencing HMGB1 in 1,3-?-glucan induced autophagy.Result: 1.Silencing HMGB1 could aggravate 1,3-?-glucan induced lung inflammation.Inflammatory cell infiltration and epithelial cells thicken,exfoliation was observed by H&E staining in the early and late stages of 1,3-?-glucan instillation.Silencing HMGB1 further aggravated the inflammatory injury by 1,3-?-glucan injection.Luminex assay was used to measure the secretion levels of related inflammatory factors and chemokines in the alveolar lavage fluid.Silencing HMGB1 increased the expression of CCL5 after 1,3-?-glucan instillation(P < 0.01),rather than TNF-? and ICAM-1.Realtime-PCR results showed that the expression of factors in lung tissue was consistent with Luminex results(P < 0.05).2.Silencing HMGB1 could aggravate 1,3-?-glucan induced collagen deposition.Masson staining didn't show significant collagen deposition of mice lung in the early and late stages of 1,3-?-glucan exposure.And relatively significant collagen expression was observed by Masson staining after silencing HMGB1 in the late stage of 1,3-?-glucan exposure.Silencing HMGB1 significantly increased hydroxyproline content in mice lung tissue of late stages of 1,3-?-glucan instillation(P < 0.05).3.Silencing HMGB1 aggravated 1,3-?-glucan induced epithelial cells' injury.Immunofluorescence assay was used to check the expression of HMGB1 and epithelial marker EPCAM in lung tissue,the results showed that HMGB1 was highly expressed on lung epithelial cells.The expression levels of cytokines in the supernatant of A549 cells were detected by ELISA and Realtime-PCR.The secretion and the expression of CCL5,TNF-? and ICAM-1 was increased by 1,3-?-glucan treatment.Silencing HMGB1 further increased the expression of CCL5 instead of TNF-? and ICAM-1(P < 0.01).4.Silencing HMGB1 could restrict 1,3-?-glucan induced autophagy.A large number of autophagosomes in epithelial cells was observed with a transmission electron microscopy in the early and late stages of 1,3-?-glucan exposure.The number of autophagosomes was significantly reduced after silencing HMGB1.Western Blot and Realtime-PCR assays were used to measure the protein levels of LC3 and P62 in mice lung.LC3 and P62 expressions were significant increased by 1,3-?-glucan instillation(P < 0.05),while silencing HMGB1 decreased the expression of LC3 and P62(P < 0.05).Immunofluorescence was used to detect the expression of LC3 in A549 cells.Results showed that autophagy was significantly increased after 1,3-?-glucan treatment in A549 cells(P < 0.05).Silencing HMGB1 significantly decreased the level of autophagy after 1,3-?-glucan treatment in A549 cells(P < 0.05).5.Silencing HMGB1 could affect autophagy flow by regulating Beclin1.The expression of LC3 B and LAMP1 in mice lung was detected by immunofluorescence assay.Results showed that the combination of autophagosomes and lysosomes was weakened in the early and late stages of 1,3-?-glucan exposure,and silencing HMGB1 could not restore the 1,3-?-glucan induced rupture of autophagy flow.Western Blot and Realtime-PCR assays showed that the expression of LAMP1 was significantly reduced by 1,3-?-glucan instillation(P < 0.05).And compared with the experimental group,the expression level of LAMP1 after HMGB1 silencing was not significantly changed.Western Blot and Realtime-PCR assay were used to measure the expression of m TOR,p-m TOR,and Beclin1.Results indicated that compared with the control group,p-m TOR/m TOR was significantly reduced and the expression of Beclin1 was significantly increased in experimental group mice(P < 0.05).Compared with the experimental group,there was no significant difference in p-m TOR/m TOR expression level in the experimental silence group,and the expression of Beclin1 was increased.6.Silencing HMGB1 could increase the binding of Beclin1 and BCL2 in 1,3-?-glucan induced autophagy.The location of Beclin1 and BCL2 in lung of mice in the early stage of 1,3-?-glucan exposure was detected by immunofluorescence assay,and results indicated that the positions of Beclin1 and BCL2 were significantly overlapped after silencing HMGB1.Significantly increased binding of Beclin1 and BCL2 in mice lung after silencing HMGB1 was observed by immunocoprecipitation assay in 1,3-?-glucan induced lung inflammation.7.Silencing HMGB1 could inhibit the post-modification of Beclin1 in 1,3-?-glucan induced autophagy.Lung tissues of the early stage of 1,3-?-glucan exposure mice were subjected to immunoprecipitation using Beclin1 antibody to detect ubiquitin expression.The result showed that silencing HMGB1 significantly reduced the ubiquitination level of Beclin1 in mice lung after 1,3-?-glucan exposure.8.Silencing HMGB1 increased the level of apoptosis induced by 1,3-?-glucan.Tunel staining was used to detect the level of apoptosis in mice lung at the early and late stages of 1,3-?-glucan exposure.The results indicated the apoptotic cells were significantly increased in 1,3-?-glucan group,compared with the control group.And HMGB1 silenced mice generated more apoptotic cells after 1,3-?-glucan exposure.Results of Western Blot and Realtime-PCR assay showed a higher level of BAX and a lower level of BCL2 in 1,3-?-glucan group at the early and late stages of 1,3-?-glucan exposure(P < 0.05).Silencing HMGB1 further increased the translation and transcription of BAX,and reduced the gene expression of Bcl2(P < 0.05).9.Silencing HMGB1 could affect the levels of epithelial cells' autophagy and apoptosis.CCK8 assay was used to detect the A549 cell viability.Results showed that A549 cell vitality was significantly decreased in HMGB1 silenced cells after 1,3-?-glucan treatment(P < 0.05).Autophagy activator rapamycin and apoptosis inhibitor Z-VAD-FMK could restore the reduced cell viability caused by silencing HMGB1 after 1,3-?-glucan treatment(P < 0.05).Conclusion: HMGB1 could enhance the nucleation stage of 1,3-?-glucan induced lung epithelial cell autophagy by regulating Beclin1 ubiquitination,affecting the binding of Beclin1 and Bcl2,thus promote the epithelial cell autophagy,restrict 1,3-?-glucan induced lung inflammation. |
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