HSP22 Inhibites Mitochondrial Reactive Oxygen Species Formation And Suppresses Hyperglycemia-induced Endothelial Injury

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IntroductionType2 diabetes(T2D)is a chronic inflammatory and progressive disease that induces multiple microvascular and macrovascular complications,such as atherosclerosis,diabetic nephropathy,diabetic retinopathy,and cardiovascular disease.Endothelial cell injury and inflammatory response play critical roles in the development and progression of diabetic-induced complications.However,the molecular mechanisms underlying T2DM-induced endothelial dysfunction remain incompletely elucidated.Endothelial cell activation is the initiation of vascular injury.It has been found that the expression of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)induced by endothelial cell activation may promote monocyte adhesion and the release of inflammatory factors,destroying the integrity of endothelial cells,followed by smooth muscle cell proliferation,migration,eventually leading to vascular dysfunction.In short,endothelial cell activation would induce various vascular-related diseases,such as atherosclerosis.In addition,recent studies have confirmed that high glucose promotes vascular injury by inducing endothelial cell activation and releasing various inflammatory factors.Mitochondria are highly dynamic organelles that are involved in multiple cellular processes,including energy metabolism,reactive oxygen species(ROS)production,inflammatory response and cell death.To date,accumulating evidence suggests that increased mitochondrial reactive oxygen species(mtROS)represent the major intracellular source of superoxide anions in diabetes and that mtROS disrupts mitochondrial homeostasis and ultimately lead to cell damage.In addition,excessive mtROS and mitochondrial damage are critical for diabetes-induced endothelial dysfunction and complications.Interestingly,mtROS are signaling mediators that drive the activation of proinflammatory transcription factors,such as activator protein-1(AP-1)and nuclear factor-?B(NF-?B),and promote inflammatory cytokine production,ultimately leading to aggravating the injury of cells.For example,Yang and his colleague found that mtROS could promote the activation of endothelial cells.Therefore,inhibition of mtROS production in endothelial cells stimulated by hyperglycemia is of great significance for the prevention and treatment of diabetes and its complications.Heat shock protein 22(HSP22)is a member of the small heat shock proteins(sHSPs)family wihich is induced upon stress and pathological processes and protect cells against various stress stimuli and inflammation.Although HSP22 has also been reported to be upregulated in the diabetic retina and heart,HSP22 plays a role in diabetes-induced vascular endothelial injury remains largely unknown.Therefore,we focused on the molecular mechanism by which mtROS mediate endothelial injury to further understand the pathologic process of diabetes-induced vascular injury and to identify a potential therapeutic target for T2 DM.Hence,we propose the following hypothesis: HSP22 reduces hyperglycemiainduced endothelial cell activation and inflammatory response by inhibiting mtROS formation,ultimately reducing vascular injury.In the current study,we used a mouse model of T2 DM that was developed in three types of mice,HSP22 transgenic(TG)mice,HSP22 knockout(KO)mice and matched wild-type(WT)littermates,and human umbilical vein endothelial cells(HUVECs)treated with high glucose(HG)to investigate whether HSP22 is involved in hyperglycemia-stimulated endothelial injury.Section ? Hyperglycemia-Induced Vascular Endothelial Injury and Increased Expression of Heat Shock Protein 22(HSP22)Objective:To investigate whether HSP22 is up-regulated during hyperglycemia-stimulated vascular endothelial cells activation andinjury.Methods:1)We used a high-fat diet and streptozotocin injection model to induce type 2 diabetes(T2D),which is Diabetes group.Then the normal chow diet mice and high-fat diet mice were used as control group(Control group)and high fat control group(HF-control).The fasting blood glucose(FBG)level and body weight were measured once per 4 weeks.Mice with blood glucose levels >200 mg/dl were considered diabetic.2)The mouse aortic pathological morphology was observed using hematoxylin and eosin staining(H&E).And immunohistochemical staining of the aortas was used to detect the expression of HSP22,intracellular adhesion molecule 1(ICAM-1),and vascular cell adhesion molecule 1(VCAM-1).3)A mouse cytokine array kit was used to detect changes of inflammatory factors in serum in serum.4)RT-PCR was used to detect the expression of cytokines and HSP22 in mice aorta.5)HUVECs were treated with normal glucose(NG,5 mM D-Glucose),osmotic control(OC,30 mM D-mannitol)or HG(30 mM D-Glucose).Then,cell counting kit-8(CCK-8)was used to determine endothelial cell activity and lactic dehydrogenase-based toxicology assay Kit(LDH)was used to measure cytotoxicity.Both the two experiments were used to determine whether the hyperglycemia damage model was successfully constructed.6)Adhesion Assays was used to examine the adhesion ability of three groups.RT-PCR was used to detect the expression of adhesion molecule(ICAM-1 and VCAM-1)and cytokines in mice aorta.7)Immunofluorescence,RT-PCR and Western blot were used to examine the expression level of HSP22 in hyperglycemia-stimulated HUVECs.Results:1)The body weights and FBG levels were monitored once per 4 weeks.The diabetic mice exhibited significantly higher blood glucose levels and lower body weights than Control group and HF-control group(P<0.05).All these suggested that T2 D mouse model was successfully constructed.2)The results of aortic HE staining indicated that cells in aorta were well arranged,whose nuclei were uniform and cytoplasm were evenly stained in Control group;cells in aorta in HF-control group arrangement were in slightly disorder,whose nuclei were irregular and a small part of the cytoplasm were deeply stained;However,cells in dibetes group arranged in disorder,containing irregular nucleus and deeply stained cytoplasm.We further detected the levels of ICAM-1 and VCAM-1 proteins in the aortas by immunohistochemistry and found that the expression levels of ICAM-1 and VCAM-1 in the aortic endothelium from the diabetic group were higher than those in the nondiabetic control groups(P<0.05).3)Using a cytokine antibody array containing 18 cytokines,we found that 5 serum cytokines—IL5,IL6,IL13,Mip3 a and TGF?1— were clearly elevated in diabetic group.RT-PCR was further used to detect the expression of IL5,IL6,IL13,Mip3? and TGF?1 in the aortic endothelium.And the results showed that the levels of cytokines in diabetes group were higher than Control group and HF-control group(P<0.05).4)We examined the expression of HSP22 in the aortas using immunohistochemistry and RT-PCR and found that the upregulation of HSP22 from diabetes group was more obvious than that in control group and HF-control group(P<0.05).5)Compared with NG and OC,HG significantly decreased cell viability and increased cytotoxicity(P<0.05).6)Compared with NG and OC,the HG-treated HUVECs exhibited increased adhesion to untreated monocytes(P<0.05);the levels of adhesion molecule(ICAM-1 and VCAM-1)and cytokines were also increased in HG group than that in NG and OC group(P<0.05).7)Compared with the NG and OC treatment groups,a significant increase in HSP22 expression was found in the HG group by immunofluorescence,RT-PCR and Western blotting,but the vascular endothelial specific marker CD31 was significantly decreased(P<0.05).Conclusions:1)We found that hyperglycemia induces endothelial cell activation and injury in vivo and in vitro.2)Hyperglycemia induces HSP22 upregulation in endothelial cells.Section ? HSP22 Alleviates Hyperglycemia-Induced Endothelial Cell Activation and InjuryObjective: To investigate whether HSP22 can inhibit endothelial cell activation,inflammatory response and vascular injury.Methods: 1)The cells were transfected with plasmids containing the HSP22 gene or a dominant-negative form and HSP22-specific si RNA oligos or dominant-negative si RNA oligos.Then the cells were treated with 30 m M glucose,and the role of HSP22 was evaluated under the condition.Overexpression of HSP22 experimental groups: Normal glucose+ none control group(NG+Non),Normal glucose+ negative control group(NG+NC),Normal glucose+ HSP22 overexpression group(NG+HSP22),High glucose+ none control group(HG+Non),High glucose+ negative control group(HG+NC),High glucose+ HSP22 overexpression group(HG+HSP22).Silencing of HSP22 experimental groups: Normal glucose+ none control group(NG+Non),Normal glucose+ negative control RNA group(NG+NC si),Normal glucose+ HSP22 si RNA group(NG+HSP22 si),High glucose+ none control group(HG+Non),High glucose+ negative control RNA group(HG+NC si),High glucose+ HSP22 si RNA group(HG+ HSP22 si).2)Western blot was used to detect the expression of HSP22 in each group,and to determine whether the HSP22 cell model was successfully constructed by overexpression and silencing.3)CCK-8 and LDH were used to determine endothelial cell activity and cytotoxicity in each group.Then adhesion assays was used to examine the adhesion ability in each groups.RT-PCR was used to detect the expression of adhesion molecule(ICAM-1 and VCAM-1)and cytokines(IL5,IL6,IL13,Mip3 a and TGF?1).4)We used a mouse model of T2 DM that was developed in three types of mice,HSP22 transgenic(TG)mice,HSP22 knockout(KO)mice and matched wild-type(WT)littermates to investigate whether HSP22 is involved in hyperglycemia-stimulated endothelial injury.HSP22 overexpression transgenic mice groups: Wild type-control group(WT-control),Wild type-diabetes group(WT-diabetes),HSP22 transgene-control group(TG-control),HSP22 transgene-diabetes group(TG-diabetes);HSP22 knockout mice groups: Wild type-control group(WT-control),Wild type-diabetes group(WT-diabetes),HSP22 knockout-control group(KO-control),HSP22 knockout-diabetes group(KO-diabetes).5)The fasting blood glucose(FBG)level and body weight were measured once per 4 weeks.Mice with blood glucose levels >200 mg/dl were considered diabetic.Then immunohistochemical staining and RT-PCR was used to detect the expression of HSP22 inmice aorta.6)The mouse aortic pathological morphology was observed using H&E.And immunohistochemical staining of the aortas was used to detect the expression of ICAM-1)and VCAM-1.7)RT-PCR was used to detect the expression of cytokines(IL5,IL6,IL13,Mip3 a and TGF?1)in mice aorta.Results: 1)Compared with NG+NC group,the expression of HSP22 in endothelial cells of NG+HSP22 group was significantly higher(P<0.05);Compared with HG + NC group,the expression of HSP22 in endothelial cells of HG + HSP22 group was further increased,suggesting that the overexpression of HSP22 cell model was successfully constructed.Compared with NG+NC si group,the expression of HSP22 in endothelial cells of NG + HSP22 Si group was significantly lower(P<0.05);Compared with HG + NC si group,the expression of HSP22 in endothelial cells of HG + HSP22 si group was also significantly decreased(P<0.05),suggesting that silencing of HSP22 cell model was successfully constructed.2)Compared with NG+NC group,cell viability in HG + NC group was significantly reduced,and the cytotoxicity was significantly increased(P<0.05).However,a clear increase in cell viability and a decrease in cytotoxicity were observed in the HG-treated endothelial cells after overexpression of HSP22(P<0.05).After the silence of HSP22,the opposite result was obtained.Compared with NG+NC si group,the cell viability in HG+NC si group was significantly reduced,and the cytotoxicity was significantly increased(P<0.05).However,a decreased cell viability and higher cytotoxicity were observed following the silencing of HSP22(P<0.05).3)Results from monocyte adhesion assay showed that,compared with NG+NC group,the monocyte adhesion of endothelial cells were increased in HG + NC group(P<0.05).However,compared with HG+NC group,the degree of adhesion of endothelial cells in HG+HSP22 group was lower(P<0.05).After the silence of HSP22,the opposite result was obtained.Compared with NG+NC si group,the monocyte adhesion of endothelial cells were increased in HG + NC group(P<0.05).Furthermore,monocyte adhesion of HG-treated endothelial cells was significantly increased following the silencing of HSP22 compared with that in the NC group(P<0.05).4)RT-PCR was used to detect the expression of adhesion molecules(ICAM-1 and VCAM-1)and cytokines(IL5,IL6,IL13,Mip3 a and TGF?1)respectively.Compared with NG+NC group,HG + NC group had significantly higher levels of adhesion molecules and cytokines(P<0.05).However,compared with HG + NC group,the levels of adhesion molecules and cytokines were decreased in HG+HSP22 group(P<0.05).Compared with NG+NC si group,HG+NC si group had significantly higher levels of adhesion molecules and cytokines(P<0.05).Furthermore,the expression of adhesion molecules and cytokines were significantly increased following the silencing of HSP22 compared with that in the NC group(P<0.05).5)The body weights and FBG levels were monitored once per 4 weeks.Compared with the WT-Diabetic mice,HSP22 overexpression had no effect on the FBG level in the TG-diabetic mice,but the FBG level both of them was higher than that in TG-control group and WT-control group.Compared with WT-control group,the body weight of WT-diabetes group decreased significantly(P<0.05),but the body weight of TG-diabetes group were not significantly lower than those of the WT-diabetic mice(P<0.05).Compared with KO-control group and WT-control group,KO-diabetes group mice and WT-diabetes group mice had higher FBG level and lower body weight(P<0.05).However,there was no difference between the KO-diabetic mice and the WT mice in the FBG level or body weight.6)HSP22 expression in the aorta was tested by immunohistochemistry and RT-PCR.Compared with WT-control group,WT–diabeties group had higher expression of HSP22(P<0.05).Compared with TG-control group,TG–diabeties group also had higher expression of HSP22(P<0.05).Compared with WT-diabetes group,the expression of HSP22 in KO-diabetes group significantly decreased(P<0.05).7)The results of aortic HE staining indicated that,compared with WT-diabetes group,the aortic morphology of TG-diabetes group was more regular,it was characterized by more tidy cell arrangement,more uniform nuclei,more evenly cytoplasmic staining,and less fiber breakage.Contrarily,cells in KO-diabetes group were more disorder arranging,containing much more irregular nucleus and deeply stained cytoplasm than WT-diabetes group,as well as much more fiber breakage.We further detected the levels of ICAM-1 and VCAM-1 proteins in the aortas by immunohistochemistry and found that the expression of ICAM-1 and VCAM-1 from WT-diabetes group were higher than WT-control group(P<0.05).The expression of the ICAM-1 and VCAM-1 of TG-diabetes group were found to be lower than in WT-diabetes group(P<0.05).However,KO-diabetes group had much higher levels of ICAM-1 and VCAM-1 than WT-diabetes group(P<0.05).8)RT-PCR was further used to detecte the expression of IL-5,IL-6,IL-13,Mip3 a and TGF?1 in the aorta of mice.Compared with WT-control group,the expressions of cytokines in WT-diabetes group increased significantly(P<0.05).However,the levels of cytokines were decreased in the aortas from TG-diabetes group compared with WT-diabetes group(P<0.05),while the levels of cytokines in KO-diabetes group increased significantly(P<0.05).Conclusions: 1)HSP22 alleviates hyperglycemia-induced endothelial cell activation and inflammatory response,resulting in reducing vascular injury.2)HSP22 deficiency aggravates hyperglycemia-induced endothelial cell activation and inflammatory response,ultimately increasing vascular injury.Section ? Hyperglycemia Induces Oxidative Stress,Mt ROS Production and Mitochondrial Impairmentin in Vascular Endothelial CellsObjective: Confirmation of hyperglycemia-induced mt ROS synthesis and mitochondrial dysfunction in vascular endothelial cells.Methods: 1)Animal experiments were grouped as in Section 2,they were Control group,HF-control group and Diabetes group respectively.2)The intracellular ROS in the freshly isolated aortas were measured using dihydroethidium(DHE)staining.3)The levels of 8-hydroxy-desoxyguanosine(8-OHd G)were assessed by ELISA in each group.4)Cell experiments were grouped as in Section 2,they were NG,OC and HG,respectively.5)The cells were incubated with the Mt ROS-specific probe Mito SOX.Then,the level of mt ROS was analyzed using flow cytometry.6)The cells were incubated with mitotracker and JC-1.Then fluorescence was detected by confocal laser scanning microscopy.Results: 1)Compared with Control group and HF-control group,Diabetes group showed higher levels of ROS in the aortas(P<0.05).2)8-OHd G accumulation in Diabetes group was substantially greater than that in Control group and HF-control group(P<0.05).3)Mito SOX Red was used to examine the mitochondrial superoxide anion production in HUVECs.The results showed that the production of mt ROS in HG group was higher than that in NG and OC group(P<0.05).4)Mitochondrial morphology was detected by mitotracker and the results suggested that mitochondrial fluorescence density in HG group were significantly weaker than those in NG and OC groups.Mitochondrial membrane potential was observed by JC-1 staining and the results showed that,compared with NG and OC groups,HG group had lower membrane potential(P<0.05).Conclusions: 1)Oxidative stress levels increased significantly in aorta of diabetes mice.2)Hyperglycemia induces mt ROS production and mitochondrial impairment in vascular endothelial cells.Section ? HSP22 Inhibits Hyperglycemia-Induced Oxidative Stress,Mt ROS Production and Mitochondrial ImpairmentObjective: To investigate whether HSP22 can inhibit hyperglycemia-induced mt ROS formation and mitochondrial dysfunction.Methods: 1)Cell experiments were grouped as in Section 3,they were overexpression of HSP22 experimental groups(NG+Non,NG+NC,NG+HSP22,HG+Non,HG+NC,HG+HSP22)and silencing of HSP22 experimental groups(NG+Non,NG+NC si,NG+HSP22 si,HG+Non,HG+NC si,HG+ HSP22 si)respectively.2)The cells were incubated with the Mt ROS-specific probe Mito SOX.Then,the level of mt ROS was analyzed using flow cytometry.3)The cells were incubated with mitotracker and JC-1.Then fluorescence was detected by confocal laser scanning microscopy.4)Animal experiments were grouped as in Section 3,they were HSP22 overexpression transgenic mice groups(WT-control,WT-diabetes,TG-control,TG-diabetes)and HSP22 knockout mice groups(WT-control,WT-diabetes,KO-control,KO-diabetes)respectively.5)The intracellular ROS in the freshly isolated aortas were measured using DHE staining.6)The levels of 8-OHd G were assessed by ELISA in each group.Results: 1)Compared with NG+NC group,HG + NC group showed higher levels of mt ROS(P<0.05).However,compared with HG+NC group,HG+HSP22 group had lower levels of mt ROS(P<0.05).After the silence of HSP22,the opposite result was obtained.Compared with NG+NC si group,the levels of mt ROS in HG+NC si group was higher(P<0.05).Furthermore,mt ROS was significantly increased following the silencing of HSP22 compared with that in HG+NC si group(P<0.05).After the silence of HSP22,the opposite result was obtained.2)Mitochondrial morphology was detected by mitotracker and the results suggested that,compared with NG+NC group,mitochondrial fluorescence density in HG + NC group were significantly weaker(P<0.05).However,compared with HG+NC group,HG+HSP22 group had higher fluorescence density(P<0.05).After the silence of HSP22,the opposite result was obtained.Compared with NG+NC si group,fluorescence density in HG+NC si group was weaker(P<0.05).Furthermore,mitochondrial fluorescence density in HG+HSP22 si group was much weaker than that in HG+NC si group(P<0.05).3)Mitochondrial membrane potential was observed by JC-1 staining and the results showed that,compared with NG+NC group,HG + NC group had lower membrane potential(P<0.05).However,membrane potential in HG+HSP22 group significantly increased than HG+NC group(P<0.05).Compared with NG+NC si group,mitochondrial membrane potential in HG+NC si group was lower(P<0.05).Furthermore,membrane potential in HG+HSP22 si group was much lower than HG+NC si group(P<0.05).4)Compared with WT-control group,WT-diabetes group showed higher levels of ROS in the aortas(P<0.05).The levels o f ROS in TG-diabetes group obviously decreased than WT-diabetes group(P<0.05).However,the levels of ROS in KO-diabetes group significantly increased than that in WT-diabetes group(P<0.05).5)Compared with WT-control group,WT-diabetes group had more 8-OHd G accumulation(P<0.05).Compared with WT-diabetes group,8-OHd G accumulation was lower in TG-diabetes group(P<0.05).However,8-OHd G accumulation in KO-diabetes group was substantially greater than in WT-diabetes group(P<0.05).Conclusions: 1)Overexpression of HSP22 inhibits hyperglycemia-induced mt ROS production and mitochondrial dysfunction in endothelial cells.2)Silencing HSP22 promotes hyperglycemia-induced mt ROS production and mitochondrial dysfunction in endothelial cells.Section ? HSP22 Suppresses Hyperglycemia-Induced Endothelial Cell Activation and Injury by Inhibiting Mt ROS FormationObjective: To investigate whether HSP22 can reduce hyperglycemia-induced endothelial cell activation and injury by inhibiting mt ROS production.Methods: 1)After HSP22 si RNA was transfected into HUVECs,Mito TEMPO and high glucose were used to intervene cells to identify that HSP22 could reduce endothelial cells activation and injury by inhibiting t mt ROS production.The experimental groups: Normal glucose + negative control group(NG + NC),High glucose + negative control group(HG + NC),High glucose + negative control + Mito TEMPO group(HG + NC + Mito TEMPO),High glucose + HSP22 si RNA group(HG + si RNA-HSP22),High glucose + HSP22 si RNA + Mito TEMPO group(HG + si RNA-HSP22 + Mito TEMPO).2)CCK-8 and LDH were used to determine endothelial cell activity and cytotoxicity in each group.Then adhesion assays was used to examine the adhesion ability in each groups.RT-PCR was used to detect the expression of adhesion molecule(ICAM-1 and VCAM-1)and cytokines(IL5,IL6,IL13,Mip3 a and TGF?1).3)The cells were incubated with the Mt ROS-specific probe Mito SOX.Then,the level of mt ROS was analyzed using flow cytometry.4)The cells were incubated with mitotracker and JC-1.Then fluorescence was detected by confocal laser scanning microscopy.5)An ATP measurement kit was used to measure the levels of ATP in the cell homogenates.Western blot was used to detect the expression of DRP1 and p-DRP1.Results: 1)Compared with NG+NC group,cell viability in HG + NC group was significantly reduced,and the cytotoxicity was significantly increased(P<0.05).A clear increase in cell viability and a decrease in cytotoxicity were observed in HG+NC+Mito TEMPO group(P<0.05).However,compared with HG+NC+ Mito TEMPO group,the cell viability in HG+ si RNA-HSP22+Mito TEMPO group group was significantly reduced,and the cytotoxicity was significantly increased(P<0.05).Compared with HG+si RNA-HSP22 group,HG+ si RNA-HSP22+ Mito TEMPO group had higher cell viability and lower cytotoxicity(P<0.05).2)Compared with NG+NC group,the monocyte adhesion of endothelial cells and the expression of adhesion molecules(ICAM-1 and VCAM-1)were increased in HG + NC group(P<0.05).However,compared with HG+NC group,the degree of adhesion of endothelial cells and adhesion molecules levels in HG+NC+Mito TEMPO group were lower(P<0.05).Compared with HG+si RNA-HSP22,the monocyte adhesion of endothelial cells and adhesion molecules levels were significantly decreased in HG+si RNA-HSP22+Mito TEMPO(P<0.05).However,monocyte adhesion and adhesion molecules levels in HG+ si RNA-HSP22+Mito TEMPO group were significantly increased than HG+NC+Mito TEMPO group(P<0.05).3)RT-PCR was used to detect the expression of cytokines(IL5,IL6,IL13,Mip3 a and TGF?1).Compared with NG+NC group,HG + NC group had significantly higher levels of cytokines(P<0.05).However,compared with HG + NC group,the levels of cytokines were decreased in HG+NC+Mito TEMPO(P<0.05).Compared with HG+NC+Mito TEMPO group,HG+ si RNA-HSP22+Mito TEMPO group had significantly higher levels of cytokines(P<0.05).But,the levels of cytokines in HG+ si RNA-HSP22+Mito TEMPO group were obviously lower than HG+si RNA-HSP22 group(P<0.05)4)Compared with NG+NC group,HG + NC group showed higher levels of mt ROS(P<0.05).However,compared with HG+NC group,HG+NC+Mito TEMPO group had lower levels of mt ROS(P<0.05).Compared with HG+NC+Mito TEMPO group,the levels of mt ROS in HG+ si RNA-HSP22+Mito TEMPO group was higher(P<0.05).But mt ROS was further increased in HG+si RNA-HSP22 group than HG+ si RNA-HSP22+Mito TEMPO group(P<0.05).5)Mitochondrial morphology was detected by mitotracker and the results suggested that,compared with NG+NC group,mitochondrial fluorescence density in HG + NC group were significantly weaker(P<0.05).However,compared with HG+NC group,HG+NC+Mito TEMPO group had higher fluorescence density(P<0.05).Compared with HG+NC+Mito TEMPO group,fluorescence density in HG+ si RNA-HSP22+Mito TEMPO group was weaker(P<0.05).Furthermore,mitochondrial fluorescence density in HG+si RNA-HSP22 group was much weaker than that in HG+ si RNA-HSP22+Mito TEMPO(P<0.05).6)Mitochondrial membrane potential was observed by JC-1 staining and the results showed that,compared with NG+NC group,HG + NC group had lower membrane potential(P<0.05).Membrane potential in HG+NC+Mito TEMPO group significantly increased than HG+NC group(P<0.05).Compared with HG+NC+ Mito TEMPO group,mitochondrial membrane potential in HG+ si RNA-HSP22+ Mito TEMPO group was lower(P<0.05).Furthermore,membrane potential in HG+si RNA-HSP22 group was much lower than HG+ si RNA-HSP22+Mito TEMPO(P<0.05).7)Compared with NG+NC group,HG + NC group had lower levels of ATP(P<0.05).= ATP levels in HG+NC+Mito TEMPO group significantly increased than HG+NC group(P<0.05).Compared with HG+NC+Mito TEMPO group,ATP levels in HG+ si RNA-HSP22+Mito TEMPO group was lower(P<0.05).But,ATP levels in HG+si RNA-HSP22 group were much lower than HG+ si RNA-HSP22+Mito TEMPO(P<0.05).8)Compared with NG+NC group,HG + NC group had higher expression of p-DRP1/DRP1(P<0.05).The expression of p-DRP1/DRP1 in HG+NC+Mito TEMPO group significantly decreased than HG+NC group(P<0.05).Compared with HG+NC+Mito TEMPO group,the level of p-DRP1/DRP1 in HG+ si RNA-HSP22+ Mito TEMPO group was higher(P<0.05).But,the expression of p-DRP1/DRP1 in HG+si RNA-HSP22 group were much higher than HG+ si RNA-HSP22+Mito TEMPO (P<0.05).Conclusions: 1)HSP22 reduces endothelial cell activation and inflammatory response by inhibiting hyperglycemia-mediatedmt ROS.2)Mito TEMPO offsets the effect of HSP22 deficiency in endothelial cells which promoted mt ROS formation and mitochondrial dysfunction during hyperglycemia condition.Section ? SummaryTo summarize,in this study,we demonstrated that HSP22 reduced endothelial cell activation and inflammatory response by inhibiting endothelial-monocyte adhesion and inflammatory cytokine release during hyperglycemia condition,and ultimately reducing vascular injury.In addition,HSP22 inhibited hyperglycemiainduced oxidative stress in endothelial cells,result in reducing mt ROS production and mitochondrial dysfunction.We further explored the molecular mechanism of HSP22 inhibiting endothelial cell injury,and found that HSP22 reduced endothelial cell activation and inflammatory response by inhibiting hyperglycemia-mediated mt ROS,ultimately decreasing endothelial cell injury.In short,we explored the role of HSP22 in diabetic vascular injury and its molecular mechanism,which may provide new ideas and targets for the prevention and treatment of diabetic complications.