Inorganic Arsenic Inhibits The Adipogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells By Interfering The Level Of Cellular Reactive Oxygen Species

Object:Arsenic is a common metalloid,which enters into human body mainly through drinking water,coal burning and occupational exposure.Long-term arsenic exposure leads to multiple organs and multiple system demage increasimg the risk of metabolic diseases such as type 2 diabetes and malignant tumors.Chronic exposure to arsenic has been shown to increase ROS level and updet the balance of cellular redox homeostasis.Mesenchymal stem cells have the characteristics of self-renewal and multi-directional differentiation.MSCs,which the vital target cells of arsenic toxicity,are used as a cell model to figure out the mechanisms underlying the impaired adipogenesis caused by arsenite.This study would provide a new perspective for the mechanism analysis of metabolic diseases caused by arsenic exposure.Methods:The primary m BM-MSCs were obtained by the adherent separation method.The cell viability experiment was used to detect the effect of NaAsO₂ exposure on the cytotoxicity of m BM-MSCs.Adipogenic differentiation of m BM-MSCs was performed by DMIRI differentiation strategy;after exposured to 1μM,2μM,and 3μM arsenic to m BM-MSCs,oil red O staining was used to determine the adipogenic differentiation ability of m BM-MSCs.RT-q PCR technology was used to detect the m RNA levels of the key genes for adipogenesis such as Cebpα,Pparg 1,Pparg 2,Nrf1,Nrf2,Gclc,Gclm,Nqo1 and Hmox1.Flow cytometry was used to detect the effect of 1μM NaAsO₂exposure on the level of ROS in m BM-MSCs at the early stage of adipogenic differentiation.Western-blot technology was used to detect the protein expression levels of antioxidant proteins NRF2,GCLC,GCLM and NQO1.Results:1.CCK8 cell viability test results showed that at the concentration of NaAsO₂lower than 7μM,the cell viability did not change significantly;when the concentration was higher than 10μM,the cell viability decreased significantly（P<0.05）;2.NaAsO₂ caused m BM-MSCs increased expression of internal antioxidant-related transcription factor Nrf2 and its downstream genes:Compared with control group,the antioxidant-related genes Nrf1,Nrf2,Gclc and Nqo1 showed an upward trend.The expressions of Hmox1 were significantly elevated with increasing dose（P<0.05）;3.NaAsO₂ exposure can inhibit the adipogenic differentiation ability of m BM-MSCs:the Oil red results showed that compared with the DMIRI adipogenic differentiation group,cell lipid droplets in the 1,2 and 3μM NaAsO₂ exposure groups decreased,and Oil red O staining gradually increased with the arsenic dose;4.The inhibitory effect of NaAsO₂ played in the early stage of adipogenic differentiation of m BM-MSCs:the group exposed to 1μM NaAsO₂ at the early stage of differentiation had smaller lipid droplets and a significant difference in the quantitative Oil red O（P<0.05）.RT-q PCR results showed that the expression of adipogenic differentiation-related genes Cebpα,Pparg 1 and Pparg 2 in the DMIRI induced differentiation group increased significantly after 36 h（P<0.05）.Compared with the control group,the expression levels of Cebpα,Pparg 1 and Pparg 2 at 48 h after NaAsO₂ exposure significantly decreased（P<0.05）;5.NaAsO₂ exposure interfered with the level of ROS and thus affected the adipogenic differentiation of m BM-MSCs:the level of ROS in 1μM NaAsO₂ exposure group showed an overall downward trend,and down to 50%within 24 h（P<0.05）.The expression of ROS in the cytoplasm of the DMIRI induced differentiation group increased,reached a peak at 1 h then dwon to 1.3 times of the cont group at 24 h.The 1μM NaAsO₂ exposured group showed the same trend of ROS in the cytoplasm with control group,but the accumulation of ROS at 0-6 h was lower（P<0.05）;6.NaAsO₂ exposure activates the expression of antioxidant gene Nrf2 and its downstream genes:the m RNA level of Nrf2 in the differentiation group induced by DMIRI reached a peak at 2 h and then decreased（P<0.05）.The m RNA levels of genes Gclc,Gclm,Nqo1 and Hmox1 did not change significantly.Compared with the control DMIRI induced differentiation group,the expression of Nrf2 in the NaAsO₂ exposure group was higher,but the difference was not statistically.Compared with the control group,the m RNA expression levels of Gclc,Gclm,Nqo1 and Hmox1 in the NaAsO₂exposure group were higher（P<0.05）,and the overall expression increased first and then decreased,and reached a peak at 12 h（P<0.05）.After adipogenic differentiation induction,the expression of cellular antioxidant proteins NRF2 and GCLC increased first and then decreased,and reached a peak at 12 h（P<0.05）.The expression of GCLM protein continued to decline after 24 h（P<0.05）.The expression of NQO1 protein showed a trend of increasing first then decreasing,and reached a peak at 24 h（P<0.05）.Compared with the control DMIRI induced differentiation group,the expression level of NRF2 in the NaAsO₂ exposure group was higher（P<0.05）.The expression of NQO1protein increased first and then decreased in the early stage of adipogenic differentiation,and reached a peak at 24 h（P<0.05）.Compared with the DMIRI induced differentiation group,the expression of NRF2,GCLC and NQO1 protein was higher in the NaAsO₂exposure group（P<0.05）.The overall expression of GCLM protein was higher than that in the induced differentiation group,showing a trend of first increasing and then decreasing,and reached a peak at 36 h（P<0.05）.Conclusion:1.Exposure to environmentally relevant doses of NaAsO₂ can inhibit the adipogenic differentiation ability of m BM-MSCs.2.Environmentally relevant doses of NaAsO₂exposed in the early stage of adipogenicdifferentiation can inhibit the accumulation of ROS by activating the expression of the antioxidant gene Nrf2 and its downstream genes,inhibiting the committed differentiation function of m BM-MSCs to preadipocytes.