| Objective:As a typical nanomaterial,MWCNTs has unique structure and superior physical properties.It is widely used in industrial productions and medical fields,therefore,its toxic effects have become a public health concern.Although epidemiological evidence of its respiratory toxicity is insufficient,animal and in vitro studies have confirmed its respiratory toxicity,which can induce pulmonary fibrosis and tumors.Autophagy is an important process for cells to maintain homeostasis,which has been shown to be closely related to the occurrence and development of a variety of respiratory diseases.MWCNTs have been proved in a number of studies to have effects on autophagy,which can induce the overactivation of autophagy or block autophagic flux,and then lead to cell dysfunction.However,its molecular mechanism is still unclear.Circ HECTD1 has multiple functions in the immune system and can influence autophagy.Meanwhile,it has been proved to promote the activation of alveolar macrophages and participated in the process of pulmonary fibrosis.In this study,bronchial epithelial BEAS-2B cells were used to study the effect of MWCNTs on autophagy of BEAS-2B cells and the role of circHECTD1 in this process.This study will provide a scientific basis for a better understanding of the toxicological effects of MWCNTs.Methods:CCK-8 assay was performed to examine the viability of BEAS-2B cells which were treated with different doses of MWCNTs(0,0.25,1,4 and 16μg/cm~2).BEAS-2B cells were treated with different doses of MWCNTs(0,0.25,1,4μg/cm~2)for24 h,or treated with 4μg/cm~2MWCNTs for different times(0,12,24 and 48 h),and the protein levels of LC3II and p62 were measured by Western blot.After transfected with m RFP-GFP-LC3 adenovirus for 2 h,BEAS-2B cells were treated with 4μg/cm~2of MWCNTs for 24 h to be observed the fusion of autophagosomes and lysosomes.BEAS-2B cells were pre-treated with 20μM chloroquine for 3 h and then were treated with 4μg/cm~2of MWCNTs for 24 h,and the protein levels of LC3II and p62 were measured by Western blot.After treated with 4μg/cm~2of MWCNTs for 24 h,BEAS-2B cells were stained by Lysotracker red for 2 h,then observed the staining under a fluorescent microscope.The random primer and oligo(dt)primer were used for reverse transcription to verify the circularization of circHECTD1 by q RT-PCR.q RT-PCR was used to detect the expression level of circHECTD1 in BEAS-2B cells,which were treated with different doses of MWCNTs(0,0.25,1,4 and 16μg/cm~2)for 24 h or treated with MWCNTs at 4μg/cm~2for different times(0,12,24 and 48 h).BEAS-2B cells were transfected with adv-circHECTD1 for 6 h,and subsequently treated with 4μg/cm~2of MWCNTs for 24 h,the protein levels of LC3II and p62 were measured by Western blot.BEAS-2B cells was transfected with m RFP-GFP-LC3 adenovirus for 2 h,and then transfected with adv-circHECTD1 for 6 h,and subsequently treated with 4μg/cm~2of MWCNTs for 24 h,then autophagic flux was observed by confocal microscope.BEAS-2B cells was transfected with adv-circHECTD1 for 6 h,and subsequently treated with 4μg/cm~2of MWCNTs for 24 h,then treated with Lysotracker red for 2 h to detect lysosomal p H by fluorescent microscope.Results:1.The effect of MWCNTs on the viability of BEAS-2B cells:compared with untreated cells,the viability of BEAS-2B cells treated with MWCNTs(1,4 and 16μg/cm~2)for 24 h were significantly decreased(P<0.01).2.Effects of MWCNTs on autophagic flux in BEAS-2B cells:(1)Effects of MWCNTs on the expression of LC3Ⅱand p62 proteins:the proteins level of LC3Ⅱand p62 in BEAS-2B cells treated with 4μg/cm~2of MWCNTs for 24 h were significantly increased(P<0.01).The proteins level of LC3Ⅱand p62 were significantly increased in the cells treated with 4μg/cm~2of MWCNTs at 24 h and 48 h(P<0.01).(2)The effect of MWCNTs on autophagic flux observed by transfection with m RFP-GFP-LC3:compared with the control group,the yellow fluorescent spots were increased in the MWCNTs treated group.(3)Chloroquine was used to study the effect of MWCNTs on autophagic flux in BEAS-2B cells:LC3II was significantly increased after pre-treatment with chloroquine(P<0.01),but MWCNTs treatment didn’t further increase the level of LC3Ⅱprotein in the cells pre-treated with chloroquine.These results suggested that MWCNTs induced autophagic flux blockage in BEAS-2B cells.3.The effect of MWCNTs on lysosomal p H:lysosomes were stained with Lysotracker red dye,and the red fluorescence was significantly reduced after treatment with 4μg/cm~2of MWCNTs,which indicated that MWCNTs increase the lysosomal p H in BEAS-2B cells.4.Verification of circularization of circHECTD1 in BEAS-2B cells:the expression of circHECTD1 was not affected by RNase R digestion;compared with random primer,the expression of circHECTD1 was significantly decreased by using oligo(dt)primer(P<0.01).5.The effect of MWCNTs on circHECTD1 expression in BEAS-2B cells:The expression of circHECTD1 was significantly decreased after treatment with 4μg/cm~2of MWCNTs for 24,48 and 72 h(P<0.01).6.Effects of circHECTD1 on autophagic flux blockage induced by MWCNTs in BEAS-2B cells:Compared with the negative control,circHECTD1 overexpression significantly decreased the expression of p62 and LC3II proteins level after the treatment with 4μg/cm~2of MWCNTs for 24 h,meanwhile,yellow fluorescence spots were reduced after circHECTD1 overexpression.These results suggested that the overexpression of circHECTD1 alleviated autophagic flux blockage in BEAS-2B cells induced by MWCNTs.7.The effect of circHECTD1 overexpression on lysosomal p H of BEAS-2B cells treated with MWCNTs:The red fluorescence intensity was recovered after circHECTD1overexpression in BEAS-2B cells treated with MWCNTs,indicating a decrease in lysosomal p H.Conclusions:MWCNTs can block autophagic flux in BEAS-2B cells;The autophagic flux blockage induced by MWCNTs was related to the increase of lysosomal p H.MWCNTs can decrease the expression of circHECTD1 in BEAS-2B cells,which leads to the increase of lysosomal p H.This may be one of the mechanisms of the autophagic flux blockage in BEAS-2B cells treated with MWCNTs. |
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