The Potential Effects Of Inorganic Arsenic On Macroautophagy And Chaperon-mediated Autophagy In J774A.1 Macrophages

Objective:Arsenic and its compounds are widely found in nature as metal-like poisons.The damage to human health caused by inorganic arsenic exposure has become a global environmental health problem.In recent years,more and more studies have shown that inorganic arsenic can affect the functions of macrophages in many aspects,including the disorder of innate immune function of macrophages,oxidative stress,etc.These functions are related to the lysosomal autophagy pathway,suggesting that the effect of arsenic on the function of macrophages may be related to the autosomal pathway.In this study,sodium arsenite（NaAsO₂）was used as the exposure factor to observe the changes of macrophage related pathways and key regulatory factor TFEB in J774A.1 macrophages under the influence of NaAsO₂,as well as to explore the changes of molecular chaperon-mediated autophagy function in macrophages.Methods:1.Cell culture and grouping:mouse macrophage line J774A.1 was cultured and grouped as follows:control group and 1μM NaAsO₂ treated group（6 h,9 h,12 h,15 h,18 h,24 h,36 h）.After NaAsO₂ treatment,the cells were extracted for subsequent treatment.2.Detection indexes and methods:（1）The protein expression levels of LC3,p62,Beclin1,ATG5,ATG12,TFEB,CTS B/D/L/S,HSC70,LAMP2A and PHLPP1 after NaAsO₂ treatment for 0-36 h were determined by Western blot.（2）Immunofluorescence was used to detect the immunofluorescence intensity of LC3 and p62 at 0-36 h,as well as to observe the nucleation of TFEB over time and observe the co-localization of HSC70 and LAMP2A to detect CMA activity.Results:1.Effects of inorganic arsenic on macrophage-related proteins.Compared with the control group,the protein expression levels of Beclin1,ATG5 and ATG12 in the 12 h group were all increased,and the difference was statistically significant（P<0.05）;The protein expression levels of Beclin1 and ATG12 in the 24 h treatment group were decreased compared with those in the 12 h group,and the differences were statistically significant（P<0.05）;The expression levels of the three proteins in the 36 h treatment group decreased compared with the 12 h and 24 h treatment groups,and the differences in Beclin1 and ATG12 protein expression levels were statistically significant（P<0.05）.2.Effect of inorganic arsenic on p62 protein.Compared with the control group,the protein expression of p62 after NaAsO₂treatment was increased,and the protein expression was the highest after 12 h treatment,and the difference was statistically significant（P<0.05）,suggesting the activation of macrophage;The expression level of p62 in the 24 h and 36 h groups was significantly decreased compared with the 12 h group,indicating the decrease of macrophage activity.In addition,the same conclusion could be obtained for the immunofluorescence of p62.The quantitative results of fluorescence intensity showed that the expression of p62 protein was the highest at 12 h,and the difference was statistically significant（P<0.05）,and the fluorescence intensity of P62 in 24 h and 36h groups was significantly lower than that in 12 h group,the difference was statistically significant（P<0.05）.3.Effect of inorganic arsenic on LC3 protein.Compared with the control group,LC3-II expression level was significantly increased after NaAsO₂ treatment for 12-36 h,and the difference was statistically significant（P<0.05）.In addition,the quantitative results of LC3 immunofluorescence intensity showed that the immunofluorescence intensity of LC3 protein in the 12 h group was higher than that of the control group,and the difference was statistically significant（P<0.05）.4.Protein expression and nuclear translocation of TFEB.Compared with the control group,the protein expression level of TFEB began to increase in the 6 h group,and significantly increased in the 9 h group and the subsequent treatment groups,the differences were statistically significant（P<0.05）.In addition,the degree of nuclear localization was significantly increased in the 12 h group and the subsequent time group.It was consistent with the results of protein level,suggesting that the activity of macrophage increased at 12 h.5.Effect of inorganic arsenic on lysosomal tissue protein expression.Compared with the control group,the protein expression level of CTSB decreased slightly with time,and the difference between the 24 h and 36 h groups was statistically significant（P<0.05）.The expression level of CTSD protein decreased significantly with time,and the difference was statistically significant.The expression level of CTSS protein did not change significantly over time,and the difference was not statistically significant compared with the control group（P<0.05）.CTSL protein expression level increased first and then decreased with time,and increased significantly in the 12 h and 24 h groups,and was statistically significant compared with the control group（P<0.05）.In addition,the expression level of CTSL protein in the 36 h group was significantly lower than that in the 24 h group,and the difference was statistically significant（P<0.05）.6.Effect of inorganic arsenic on the expression of molecular chaperone autophagy（CMA）related proteins.Compared with the control group,the protein expression levels of LAMP2A,HSC70and PHLPP1 in 12 h,24 h and 36 h groups were significantly increased,and the differences were statistically significant（P<0.05）.In addition,the protein expression levels of LAMP2A and PHLPP1 in the 36 h group were significantly higher than those in the 24 h group,and the differences were statistically significant（P<0.05）.7.Effect of inorganic arsenic on autophagy activity of molecular chaperones.Compared with the control group,the immunofluorescence intensity of LAMP2A,HSC70 and LAMP2A in 12 h,24 h and 36 h groups were significantly increased,and the degree of binding was significantly increased,and the binding phenomenon in 36h group was the most obvious.These results suggested that CMA activity increased with time and reached the peak value at 36 h.Conclusion:1.Sodium arsenite can activate macroautophagy in macrophages;2.Sodium arsenite can activate TFEB,a key transcription factor of autophagosomal pathway in macrophages;3.Sodium arsenite can activate molecular chaperon-mediated autophagy（CMA）in macrophages.