| Objectives: Formaldehyde is a kind of organic chemical raw material,which is widely used to synthesize decoration materials,preservatives,clothing anti-wrinkle agents and other products.Because the free formaldehyde in these products is volatile,people will often be exposed to formaldehyde in production and life.Studies have shown that formaldehyde may be associated with various diseases such as nasopharyngeal cancer,asthma,lung cancer,Alzheimer’s disease,and leukemia,and is a potential carcinogen.However,due to the high reactivity of formaldehyde,there is a great controversy about the distal tissue damage caused by formaldehyde.Our previous study found that the plasma levels of malondialdehyde in lung cancer patients and mice with lung cancer induced by benzo[a]pyrene increased,the antioxidant level decreased,and the mitochondrial function of peripheral blood mononuclear cells was significantly decreased,indicating that the immune function of tumor patients was significantly reduced.It has been reported in the literature that formaldehyde exposure can affect the number and function of immune cells,but the mechanism remains unclear.Therefore,we speculate that formaldehyde may lead to the decline of mitochondrial function of immune cells,thereby promoting the occurrence and development of diseases caused by other harmful factors.We carried out formaldehyde exposure experiments in mice,and found that formaldehyde did not cause a decrease in mitochondrial function of T and B lymphocytes,but led to a decrease in the level of mitochondrial reactive oxygen species mROS in T lymphocytes.mROS is an essential second signal in the process of T lymphocyte activation.For SPF mice,T lymphocytes are mostly inactive,and the level of energy supply metabolism of nonactivated T lymphocytes relying on fatty acid oxidation and oxidative phosphorylation is low,while the level of energy supply metabolism of activated T lymphocytes relying on aerobic glycolysis is high,which is prone to apoptosis.They have different responses to external stimuli.Therefore,we speculate that due to the different energy supply modes of nonactivated T lymphocytes and activated T lymphocytes,the effects of formaldehyde on nonactivated T lymphocytes and activated T lymphocytes may be different.Therefore,this study explored the effects and related mechanisms of formaldehyde on nonactivated T lymphocytes and activated Tlymphocytes,which may provide new ideas for the prevention and treatment of diseases caused by formaldehyde.Methods: In this experiment,the exposure model of gaseous formaldehyde in mice was established by dynamic inhalation to clarify the effect of formaldehyde on the function of T and B lymphocytes in mice.Blood routine,flow cytometry and chemiluminescence were used to detect the count of peripheral blood cells,the level of MMP in peripheral blood and spleen T and B lymphocytes and the level of ATP in peripheral blood mononuclear cells,so as to explore the effects of formaldehyde on the number of peripheral blood cells and the mitochondrial function of T and B lymphocytes;mito SOXTM staining was used to detect the effect of formaldehyde on the mROS content of T and B lymphocytes in peripheral blood and spleen of mice;The effect of formaldehyde on the production of plasma cytokines in mice was detected by electrochemiluminescence.The animal model and in vitro cell culture model of activated T lymphocytes were established by using formaldehyde exposed mice and primary spleen lymphocytes treated with ConA to clarify the effect of formaldehyde on T lymphocyte activation.The levels of plasma cytokines and T lymphocyte mROS in mice were detected to explore the effect of formaldehyde on T lymphocyte activation.The formaldehyde exposure of primary spleen lymphocytes was used to determine the mechanism of the effect of formaldehyde on the activation of nonactivated T lymphocytes.The expression of mitochondrial motility related protein drp1 and its receptor binding in primary spleen lymphocytes were detected by Western blot and co-immunoprecipitation,indicating the role of mitochondrial division in the injury caused by formaldehyde exposure.The primary spleen lymphocytes exposed to formaldehyde were administered ConA to activate T lymphocytes,and the mROS level and cytokine production of T lymphocytes were detected to further clarify the effect of formaldehyde on T lymphocyte activation.Using the T lymphocyte line Jurkat E6-1 and the activated T lymphocytes obtained by ConA activation of mouse primary spleen lymphocytes,a cell culture model of the effect of formaldehyde exposure on activated T lymphocytes was established to clarify the effect of formaldehyde exposure on activated T lymphocytes and its related mechanism.The mROS,mitochondrial function and apoptosis of activated T lymphocytes were detected to explore the damage of formaldehyde to activated T lymphocytes.The active oxygen scavenger NAC and formaldehyde were exposed at the same time to detect the oxidation and antioxidant level,mitochondrial function,and apoptosis level of activated T lymphocytes,and to clarify the role of oxidative damage in mitochondrial dysfunction and apoptosis of activated T lymphocytes induced by formaldehyde.Oxidative damage is an important factor leading to mitochondrial damage.We exposed Mdivi-1,a selective inhibitor of mitochondrial motility related protein DRP1,to formaldehyde at the same time to clarify the role of mitochondrial damage division in mitochondrial dysfunction and apoptosis of activated T lymphocytes induced by formaldehyde.The expression of DRP1 in activated T lymphocytes and its phosphorylation level at Ser616 site and the binding with its receptors MFF and FIS1 were detected to determine the expression changes of mitochondrial injury and division related proteins;The morphological and structural changes of activated T lymphocyte mitochondria were observed by transmission electron microscope;The levels of oxidation and antioxidation,mitochondrial dysfunction and apoptosis of activated T lymphocytes were detected to further illustrate the role of mitochondrial injury and division in mitochondrial dysfunction and apoptosis of activated T lymphocytes induced by formaldehyde.Results:1.Formaldehyde exposure significantly decreased the level of mROS in nonactivated T lymphocytes of miceIn order to explore the effect of formaldehyde on T and B lymphocytes of mice,we measured the levels of MMP in T and B lymphocytes of mice spleen,ATP in peripheral blood mononuclear cells,mROS and plasma cytokines.the results showed that compared with the control group,there were no significant changes in MMP of T and B lymphocytes,ATP content of peripheral blood mononuclear cells and plasma cytokine level in each formaldehyde exposure group;compared with the control group,the level of T lymphocyte mROS in 3 mg/m3 and 5 mg/m3 formaldehyde exposed mice decreased significantly(P < 0.05).mROS is a necessary stimulation signal for T lymphocyte activation,which also reflects the level of mitochondrial metabolism,indicating that formaldehyde may hinder T lymphocyte activation by reducing the production of T lymphocyte stimulation signal mROS.2.Formaldehyde blocked the activation of nonactivated T lymphocytes by reducing the level of mROSIn order to explore whether formaldehyde inhibits the activation of nonactivated T lymphocytes by reducing the production of mROS,we activated T lymphocytes of formaldehyde exposed mice with ConA,and detected the levels of plasma cytokines and T lymphocyte mROS in mice.The results showed that after administration of ConA,the levels of plasma cytokines and T lymphocyte mROS in formaldehyde exposed mice were significantly lower than those in control mice,the difference was statistically significant(P < 0.05).The normal proliferation and division of mitochondria is the basis for maintaining the physiological balance of mROS.Formaldehyde reduces the level of mROS and plasma cytokines produced by T lymphocyte activation,indicating that formaldehyde may hinder the activation of nonactivated T lymphocytes by inhibiting the proliferation and division of mitochondria.3.Formaldehyde blocks the activation of nonactivated T lymphocytes by inhibiting the binding of DRP1 to its receptor MFFIn order to verify whether formaldehyde hinders the activation of nonactivated T lymphocytes by inhibiting mitochondrial proliferation and division,we measured the mROS level,mitochondrial function,and apoptosis of nonactivated lymphocytes cultured in vitro.The results showed that compared with the control group,the levels of mROS and ATP of nonactivated T lymphocytes exposed to formaldehyde decreased significantly(P < 0.05),and the levels of MMP and apoptosis did not change significantly.These results showed that formaldehyde inhibited mitochondrial productivity.The expression results of mitochondrial proliferation and division related proteins showed that formaldehyde significantly decreased the expression of DRP1 in nonactivated lymphocytes,reduced its phosphorylation at Ser616 site,and significantly reduced the binding between DRP1 and receptor MFF(P < 0.05).It shows that formaldehyde inhibits the proliferation and division of mitochondria,resulting in the decrease of mROS level.After ConA administration activated the primary spleen lymphocytes exposed to formaldehyde,the cytokine level and mROS level of T lymphocytes in formaldehyde group decreased significantly(P < 0.05). In conclusion,formaldehyde inhibits the binding of DRP1 and its proliferation and division receptor MFF,hinders the proliferation and division of mitochondria,and then reduces the production of energy and mROS,thus hindering the activation of T lymphocytes.4.Formaldehyde causes mitochondrial dysfunction and apoptosis of activated T lymphocytes by producing a large amount of mROSIn order to explore the effect of formaldehyde exposure on the mitochondrial function of activated T lymphocytes,we measured the mROS level,mitochondrial function,and apoptosis of activated T lymphocytes in vitro.The results showed that compared with the control group,the level of mROS and the proportion of apoptosis in T lymphocytes increased significantly,and the content of ATP and MMP decreased significantly(P < 0.05).It suggests that formaldehyde exposure may lead to mitochondrial dysfunction and apoptosis of activated T lymphocytes through oxidative damage.In order to verify the role of formaldehyde exposure in mitochondrial dysfunction and apoptosis of activated T lymphocytes through oxidative damage,we used reactive oxygen species scavenger NAC and formaldehyde to detect the oxidation and antioxidant level,mROS level,mitochondrial function,and apoptosis of activated T lymphocytes.The results showed that compared with the control group,the levels of SOD,GSH-Px and GSH/GSSG in formaldehyde group decreased significantly,and the level of MDA increased significantly.After the administration of NAC,the antioxidant level recovered significantly,the levels of MDA,mROS and apoptosis decreased significantly,and the levels of ATP and MMP recovered significantly(P < 0.05).It shows that formaldehyde does lead to mitochondrial dysfunction and apoptosis of activated T lymphocytes through oxidative damage.5.Formaldehyde leads to mitochondrial dysfunction and apoptosis of activated T lymphocytes by increasing the binding of DRP1 to its receptor FIS1Oxidative damage is an important factor leading to mitochondrial damage and division.In order to explore the role of formaldehyde in mitochondrial dysfunction and apoptosis of activated T lymphocytes through mitochondrial damage and division,we used Mdivi-1,a selective inhibitor of DRP1,to act simultaneously with formaldehyde,The expression of mitochondrial injury and division related proteins in activated T lymphocytes and the recovery of cell phenotype by Mdivi-1 were detected.The results showed that compared with the control group,the change of DRP1 protein in T lymphocytes of formaldehyde group was not obvious,the protein expression level of p-DRP1(Ser616)increased significantly,the binding between DRP1 and receptor FIS1 increased significantly,and the protein expression level and binding with receptor of drp1 and p-DRP1(Ser616)decreased significantly after Mdivi-1 administration(P < 0.05);compared with the control group,the mitochondrial structure of cells in the formaldehyde group was unclear and the number was reduced,and the use of Mdivi-1 can alleviate the mitochondrial damage caused by formaldehyde(P < 0.05);compared with the formaldehyde group,the antioxidant level of Mdivi-1 was significantly restored,the levels of MDA,mROS and apoptosis were significantly decreased,and the levels of ATP and MMP were also significantly restored in the formaldehyde group(P < 0.05).It shows that formaldehyde does cause mitochondrial damage and division through oxidative damage,resulting in mitochondrial dysfunction and apoptosis of activated T lymphocytes.Conclusion:1.Formaldehyde reduces mitochondrial proliferation and division by inhibiting the binding of DRP1 to mitochondrial proliferation and division receptor MFF,and reduces the stimulation signal mROS required for T lymphocyte activation,resulting in the inhibition of nonactivated T lymphocyte activation.2.Formaldehyde increases mitochondrial injury and division by promoting the binding of DRP1 to mitochondrial injury and division receptor FIS1,resulting in mitochondrial dysfunction and apoptosis of activated T lymphocytes. |
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