| BackgroundAtherosclerosis is an inadaptive inflammatory reaction caused by the retention of cholesterol and apolipoprotein B-containing lipoproteins in the susceptible areas of the arterial system.Macrophages play an important role in the pathogenesis of atherosclerosis.Mitochondrial aldehyde dehydrogenase 2(ALDH2)is known for its strong role in ethanol metabolism.It is a key enzyme involved in the degradation of acetaldehyde,4-HNE and malondialdehyde(MDA)to acetic acid.In recent years,a number of studies have reported the anti-atherosclerotic effect of activated ALDH2 activity.ALDH2 can directly regulate the function of plaque cells,improve metabolism and mediate inflammatory response to affect the development of atherosclerotic lesions.Recent studies have found that in the ApoE-/-mouse model of spontaneous atherosclerosis,plaque area,stability and systemic pro-inflammatory response are related to the level of oxidative stress of macrophages and DNA damage.cGAS-STING signal pathway is an important signal pathway involved in DNA injury response in macrophages.Kim et al found that ethanol-mediated DNA damage was significantly aggravated in the liver tissues of mice with ALDH2 gene deletion.Although many studies have been carried out to evaluate the various mechanisms of ALDH2 on atherosclerotic diseases,the mechanism by which ALDH2 regulates the inflammatory response of macrophages in the process of atherosclerosis is unclear.To sum up,we propose the following hypothesis:ALDH2 inhibits the activation of cGAS-STING signal pathway and reduces macrophage inflammation by reducing DNA oxidative damage caused by oxidative stress in macrophages.Objectives1.To study the effect of ALDH2 on DNA damage of macrophages.2.To explore the effect of ALDH2 on inflammatory response of macrophages and its mechanism.Methods1.Effect of ALDH2 on cGAS-STING signal pathway of macrophages1.1 Effect of ALDH2 activity change on cGAS-STING signal pathway mediated by cisplatin or oxidized low density lipoprotein(ox-LDL)in RAW 264.7 cellsRAW 264.7 cells were preincubated with ALDH2 agonist Alda-1(20?M),inhibitor Daidzin(60 ?M)and isovolumic DMSO for 30 minutes,then cisplatin(16?M)was added and ox-LDL(80 ?g/ml)was used to detect the expression of cGAS-STING signaling pathway proteins in macrophages.1.2 Effect of ALDH2 gene expression on cGAS-STING signaling pathway mediated by cisplatin or ox-LDL in primary peritoneal macrophagesThe primary peritoneal macrophages of ALDH2 knockout and overexpression mice and wild type mice were extracted and treated with cisplatin(16 ?M)and ox-LDL(80?g/mL)for 24 hours.The expression of cGAS-STING signal pathway proteins in macrophages was detected.2.Effect of ALDH2 on the release of inflammatory cytokines from RAW 264.7 cellsRAW 264.7 cells were preincubated with ALDH2 agonist Alda-1(20 ?M),inhibitor Daidzin(60 ?M)and isovolumic DMSO for 30 minutes,followed by cisplatin(16 ?M)and ox-LDL(80 ?g/mL)for 24 hours.3.Effect of STING on ALDH2 regulating the release of inflammatory cytokines from macrophagesThe peritoneal macrophages of STING knockout mice and wild type mice were preincubated with Daidzin(60?M)for 30 minutes,and then treated with cisplatin(16?M)or ox-LDL(80 ?g/mL)for 24 hours.The expression of inflammatory factors in the cells was detected.4.Effect of ALDH2 on DNA damage of macrophages mediated by cisplatin or ox-LDL4.1 Effect of ALDH2 activity change on DNA damage in RAW 264.7 cells mediated by cisplatin or ox-LDLRAW 264.7 cells were preincubated with ALDH2 agonist Alda-1(20 ?M),inhibitor Daidzin(60 ?M)and isovolumic DMSO for 30 minutes,and then treated with cisplatin(16?M)and ox-LDL(80 ?g/mL)for 24 hours.The expression of?-H2A.X protein was detected.4.2 Effect of ALDH2 gene expression on DNA damage in primary peritoneal macrophages mediated by cisplatin or ox-LDLThe primary peritoneal macrophages of ALDH2 knockout and overexpression mice and wild type mice were extracted and treated with cisplatin(16?M)and ox-LDL(80 ?g/mL)for 24 hours.The expression of ?-H2A.X protein in macrophages was detected.4.3 Effect of ALDH2 on DNA oxidative adduct 8-OHG in RAW 264.7 cells mediated by cisplatin or ox-LDLRAW 264.7 cells were preincubated with ALDH2 agonist Alda-1(20 ?M),inhibitor Daidzin(60?M)and isovolumic DMSO for 30 minutes,then cisplatin(16?M)was added and ox-LDL(80 ?g/mL)was treated for 24 hours.after DNA purification,the content of 8-OHG in DNA was detected by 8-OHG detection kit.5.The effect of CD36 inhibition in foam cell lipid depositionEffects of changes in ALDH2 activity on oxidative stress levels in RAW 264.7 cells mediated by cisplatin or ox-LDL.6.Effect of ROS on inflammatory response of macrophages regulated by ALDH26.1 Effect of ALDH2 on H2O2-mediated cGAS-STING signal pathway in RAW 264.7 cellsThe cells were preincubated with Alda-1(20 ?M)or Daidzin(60 ?M)for 30 minutes,and then treated with H2O2(20 ?M)for 24 hours.The expression of cGAS-STING signal pathway proteins and the transcription of inflammatory factors were detected.6.2 Effect of ROS on ALDH2 regulating inflammatory response of RAW 264.7 cells.RAW 264.7 cells were preincubated with N-acetylcysteine(NAC,20?M)or Daidzin(60 ?M)or both for 30 minutes,then treated with cisplatin(16 ?M)and ox-LDL(80 ?g/mL)for 24 hours.The changes of cGAS-STING signal pathway protein expression,inflammatory factor transcription and DNA oxidative damage marker 8-OHG were detected respectively.Results1.Effect of ALDH2 on cGAS-STING signal pathway of macrophages1.1 Enhanced ALDH2 activity can inhibit the activation of cGAS-STING signal pathway mediated by cisplatin or ox-LDL in RAW 264.7 cells.In RAW 264.7 cell line,we found that increased ALDH2 activity could inhibit the increase of cGAS protein expression and STING,TBK1 phosphorylation in cisplatin or ox-LDL-mediated cGAS-STING signaling pathway.However,the inhibition of ALDH2 activity was the opposite.1.2 Overexpression of ALDH2 can inhibit the activation of cGAS-STING signal pathway mediated by cisplatin or ox-LDL in primary mouse peritoneal macrophagesIn the extracted primary mouse peritoneal macrophages,we found that ALDH2 overexpression could inhibit the increase of cGAS protein expression and STING,TBK1 phosphorylation in cisplatin or ox-LDL-mediated cGAS-STING signaling pathway.The result of ALDH2 gene knockout is the opposite.2.ALDH2 reduces the release of inflammatory cytokines from RAW 264.7 cells Alda-1,an active agonist of ALDH2,could reduce the increase of mRNA transcription of IFN-?,IL-1?,IL-6,TNF-? and Arg molecules in RAW 264.7 cells mediated by cisplatin or ox-LDL,while the mRNA transcription level of iNOS decreased and the release of IL-1? and IFN-? increased,while ALDH2 activity inhibitor Daidzin was the opposite.3.The regulation of ALDH2 on the release of inflammatory cytokines from macrophages is partially dependent on STINGAfter the peritoneal macrophages of STING knockout mice and wild type mice were preincubated with Daidzin(60 ?M)for 30 minutes and treated with cisplatin(16?M)or ox-LDL(80 ?g/mL)for 24 hours,we found that cGAS protein expression and TBK1 phosphorylation increased after ALDH2 activity inhibition,and the transcription levels of downstream inflammatory factors IFN-?,IL-1? and IL-6,TNF-? increased.In the primary peritoneal macrophages of STING knockout mice,these changes were partially inhibited except for cGAS protein.4.ALDH2 attenuates DNA damage induced by cisplatin or ox-LDL in macrophages4.1 Enhanced ALDH2 activity attenuates DNA damage in RAW 264.7 cells mediated by cisplatin or ox-LDLALDH2 agonist Alda-1 decreased the expression of ?-H2A.X protein in RAW 264.7 cells mediated by cisplatin or ox-LDL,while the inhibitor of ALDH increased the expression of y-H2A.X protein.4.2 Effect of ALDH2 gene expression on DNA damage in primary peritoneal macrophages mediated by cisplatin or ox-LDLOverexpression of ALDH2 gene decreased the expression of y-H2A.X protein in primary mouse peritoneal macrophages mediated by cisplatin or ox-LDL,while ALDH gene knockout increased y-H2A.X protein expression.4.3 Effect of ALDH2 on DNA oxidative adduct 8-OHG in RAW 264.7 cells mediated by cisplatin or ox-LDL.ALDH2 agonist Alda-1 reduced the content of 8-OHG in DNA of RAW 264.7 cells mediated by cisplatin or ox-LDL,while the inhibitor of ALDH increased the content of 8-OHG.5.Effects of changes in ALDH2 activity on oxidative stress levels in RAW 264.7 cells mediated by cisplatin or ox-LDLALDH2 agonist Alda-1 reduced the fluorescence intensity of ROS and enhanced the activity of SOD in RAW 264.7 cells mediated by cisplatin or ox-LDL,while Daidzin,an inhibitor of ALDH2,enhanced the fluorescence intensity of ROS and decreased the activity of SOD.6.Effect of ROS on inflammatory response of macrophages regulated by ALDH26.1 Effect of ALDH2 on H2O2-mediated cGAS-STING signal pathway in RAW 264.7 cellsAlda-1,an active agonist of ALDH2,could reduce the increase of cGAS protein expression and STING,TBK1 phosphorylation in RAW 264.7 cells mediated by H2O2,increase the transcription level of downstream inflammatory cytokines IFN-?,IL-1?and IL-6,TNF-? mRNA,and increase the release of IL-1? and IFN-?,while ALDH2 inhibitor Daidzin was the opposite.6.2 Effect of ROS on ALDH2 regulating inflammatory response of RAW 264.7 cellsALDH2 inhibitor Daidzin increased cGAS protein expression,STING,TBK1 phosphorylation,inflammatory cytokines,IFN-?,IL-1?,IL-6,TNF-? molecular transcription,IL-1?,IFN-? release and DNA oxidative adduct 8-OHG in RAW 264.7 cells mediated by cisplatin or ox-LDL.The use of ROS scavenger N acetylcysteine could inhibit the above results.Conclusions and Significances1.This study firstly discover that the increased activity of ALDH2 can inhibit the cGAS-STING signal pathway and reduce the inflammatory response of macrophages.2.It is suggested that ALDH2 can reduce the oxidative damage of DNA in macrophages by inhibiting oxidative stress and play a role in anti-cGAS-STING activation.3.It is suggested that the regulation of cGAS-STING signal pathway is an important target to reduce the inflammatory response of macrophages in patients with decreased ALDH2 activity. |
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