| Backgrounde:Atherosclerosis（AS）is an important pathophysiological mechanism leading to cardiovascular diseases.Currently,it is believed that lipid deposition and endothelial dysfunction are the initial stages of the development of AS.During the process of AS,mitochondrial dysfunction causes changes in cell metabolism and respiration,leading to excessive production of reactive oxygen,inducing oxidative stress.Although low levels of reactive oxygen play an important signaling role,excessive reactive oxygen can induce structural damage to cells,leading to further progression of AS and instability of plaques.Heat Shock Protein 22（HSP22）is a small heat shock protein that was initially discovered to be involved in tumorigenesis in various tumors by regulating cell growth and apoptosis.Recent studies have shown that HSP22 has a protective effect in myocardial injury,cerebral ischemia,and aging by regulating mitochondrial function,antioxidant and anti-apoptotic properties.However,its role and regulatory mechanism in AS disease is still unclear.Our team’s previous research found that tamoxifen can inhibit the development of AS by inducing the expression of HSP22and reducing oxidative low-density lipoprotein damage to vascular endothelial cells.Based on the previous research results,we speculate that HSP22 may be involved in the process of AS by regulating mitochondrial function.Part 1 The Role of HSP22 in Atherosclerosis in MiceObjective:To explore the role and mechanism of Heat Shock Protein 22（HSP22）in atherosclerosis（AS）in mice.Methods:1.Atherosclerotic mouse models were constructed by feeding C57BL/6 wild-type（WT）,Apo E-/-,Apo E-/-HSP22+/-（Tg）,and Apo E-/-HSP22-/-（KO）mice a high-fat diet for 12 weeks,and the body weights of the mice were measured one day and every 4 weeks after high-fat chow feeding.2.After 12 weeks of high-fat feeding,mouse serum was collected and lipid-related indicators,including Triglyceride（TG）,Total cholesterol（TC）,High-density lipoprotein cholesterol（HDL-C）,and Low-density lipoprotein cholesterol（LDL-C）levels,were detected using an automatic blood biochemistry analyzer.3.The aortic arch was stained with oil red O and the aortic arch frozen sections were stained with oil red O to detect the condition of the atherosclerotic plaque.4.Aortic slices were stained with hematoxylin and eosin（HE）to observe pathological change in the aorta.5.The expression levels of NO and oxidative stress molecules MDA,SOD,GSH-PX,and ROS levels were detected in serum using reagent kits,and ROS levels were detected in aortic slices stained with DHE.6.Western bolt was used to detect the protein expression levels of vascular endothelial injury indicators p-e NOS,e NOS,ET-1,mitochondrial outer membrane fusion indicator mitochondrial fusion protein 2（Mitofusion 2,Mfn2）,mitochondrial inner membrane fusion indicator Optic Atrophy 1（OPA1）and transcription factor Nrf2 in aortic tissues.Results:1.Body weight measurement:The body weight of mice in each group was measured and recorded one day before and every 4 weeks after high-fat feeding.The results showed that there was no significant difference in baseline body weight among the four groups of mice（p>0.05）.After 1-8 weeks of high-fat feeding,the body weight of mice in all groups increased,with a similar growth trend and no significant differences was observed（p>0.05）.From weeks 9-12s of high-fat feeding,the body weight of mice in each group showed a decreasing trend.Among them,the KO group showed the greatest decrease in body weight,but there was no significant difference in body weight among the groups（p>0.05）.2.Blood lipid levels:（1）Baseline levels:No significant difference in the baseline levels of lipids（TG,TC,HDL-C,LDL-C）in the mice of each group（p>0.05）.（2）After 12 weeks of high-fat feeding,the results showed that compared to the WT group,the levels of TG,TC,HDL-C,and LDL-C in Apo E-/-,Tg,and KO groups were significantly increased（p<0.05）.Compared to the Apo E-/-group,there were no significant changes in the levels of TG,TC,HDL-C,and LDL-C in Tg and KO groups of mice（p>0.05）.3.Aortic macroscopic and frozen section Oil Red O staining results analysis showed that compared to the WT group,the plaque area of Apo E-/-group significantly increased（p<0.05）.Compared to the Apo E-/-group,the plaque area of Tg group decreased（p<0.05）,but the plaque area of KO group further increased（p<0.05）.4.HE staining of the aortic arch revealed that there was no obvious plaque in the aorta of WT group mice,and the morphology was more regular,mainly showing neat cell arrangement,uniform nuclear size and uniform cytoplasmic staining;the Apo E-/-group,Tg group and KO group all had atheromatous plaque formation,and a fibrous cap was visible,with a large number of neutrophils and foam cells gathering under the fibrous cap;the fibrous caps of the plaques in the Apo E-/-and KO groups were uneven in thickness,and a large number of cholesterol crystals and necrotic materials were visible under the fibrous caps;the fibrous caps of the Tg group were relatively smooth,and there were fewer cholesterol voids and necrotic materials under the caps.5.The measurement of NO levels in mouse serum showed that compared to the WT group,the Apo E-/-group had a significant decrease in NO content（p<0.05）.Compared to the Apo E-/-group,the Tg group had a significant increase in NO content（p<0.05）,while the KO group had a further decrease in NO serum content and a significant difference（p<0.05）.6.The measurement of oxidative stress molecules MDA and antioxidant stress molecules SOD and GSH-PX in mouse serum showed that compared to the WT group,the Apo E-/-group had a significant increase in MDA serum content and a decrease in SOD and GSH-PX serum content（p<0.05）.Compared to the Apo E-/-group,the Tg group had a significant decrease in MDA serum content and an increase in SOD and GSH-PX serum content（p<0.05）,while the KO group had a further increase in MDA serum content and a further decrease in SOD and GSH-PX serum content（p<0.05）.7.The protein expression levels of vascular endothelial injury markers p-e NOS,e NOS,ET-1,mitochondrial dynamic markers OPA1,Mfn2,and transcription factor Nrf2 were further detected by Western Blot in mouse aortic tissues.The analysis results showed that compared with the WT group,the levels of p-e NOS,Mfn2,and OPA1 proteins were decreased in the Apo E-/-group（p<0.05）,while the expression levels of ET-1 and Nrf2 proteins were increased（p<0.05）.Compared with the Apo E-/-group,the levels of p-e NOS,Mfn2,OPA1,and Nrf2 proteins were increased in the Tg group（p<0.05）,while the expression level of ET-1 protein was decreased（p<0.05）.The protein expression levels of p-e NOS,Mfn2,OPA1,and Nrf2 proteins were decreased in the KO group（p<0.05）,and the expression level of ET-1 protein was increased（p<0.05）.Conclusion:1.Overexpression of HSP22 can significantly reduce oxidative stress levels in mice,induce mitochondrial fusion and promotion of Nrf2 protein expression,improve endothelial cell damage,and inhibit the formation of atherosclerotic plaques.2.Silencing HSP22 further induces an increase in oxidative stress levels in mice,inhibits of mitochondrial fusion and Nrf2 protein expression,leads to endothelial cell damage,and accelerates the formation of atherosclerotic plaques.Part 2 Ox-LDL induced endothelial cell jury and mitochondrial dysfunctionObjective:To investigate whether oxidized low-density lipoprotein（Ox-LDL）can induce endothelial cell injury and changes in mitochondrial biological function.Methods:1.Human umbilical vein endothelial cell（HUVECs）were divided into two groups:the control group,which received no treatment,and the Ox-LDL group,which was treated with 100μg/ml Ox-LDL for 24 hours to establish an endothelial cell injury model.2.Cell counting kit-8（CCK-8）assay was used to detect endothelial cell viability,and lactate dehydrogenase cytotoxicity assay kit（LDH assay kit）was used to detect cell toxicity level.3.Western blot was used to detect the expression levels of endothelial cell injury markers p-e NOS,e NOS,and ET-1.4.Flow cytometry was used to analyze the changes in mitochondrial membrane potential in the two groups of endothelial cells.5.Mito SOX Red probe and Mito Tracker fluorescent probe were used to detect the levels of reactive oxygen species（ROS）and mitochondrial morphology changes in different treatment groups.6.Western blot was used to detect the protein levels of Mfn2 and OPA1 in different treatment groups of endothelial cells.Results:1.The results of the CCK-8 and LDH assay show that compared to the control group,the cell viability of the Ox-LDL group was significantly decreased（p<0.05）,and the cell toxicity was significantly increased（p<0.05）.2.Western blot analysis showed that compared to the control group,the p-e NOS protein expression level was reduced in the Ox-LDL group（p<0.05）,while the ET-1protein expression level was increased（p<0.05）.3.Flow cytometry analysis revealed that compared to the control group,the level of mitochondrial JC-1 aggregates in endothelial cells of the Ox-LDL group was decreased and JC-1 monomers were increased,indicating a decrease in mitochondrial membrane potential and increased depolarization（p<0.05）.4.Confocal mt ROS staining analysis suggested that compared to the control group,the level of ROS in endothelial cells of the Ox-LDL group was significantly increased（p<0.05）.5.Confocal mitotracker analysis showed that compared to the control group,the number of mitochondrial monomers and networks in endothelial cells of the Ox-LDL group increased,but the average length,median length,average branch length,median branch length,and mitochondrial footprint perμm2 all decreased（p<0.05）,indicating that the mitochondrial morphology of the Ox-LDL group was damaged.6.Western blot analysis showed that compared to the control group,the protein levels of the mitochondrial fusion proteins Mfn2 and OPA1 were decreased（p<0.05）.Conclusion:1.Ox-LDL leads to reduced endothelial cell activity and increased LDH secretion,inducing endothelial cell damage.2.Ox-LDL leads to a decrease in both endothelial cell mitochondrial membrane potential,increased ROS production,and altered mitochondrial morphology,resulting in an imbalance in mitochondrial biological function.Part 3 The regulatory role of HSP22 in Ox-LDL induced endothelial cell mitochondrial dysfunction and its impact on Nrf2Objective:To explore whether HSP22 improves Ox-LDL-induced endothelial cell damage through the regulation of mitochondrial biological function.Methods:1.HUVECs were transfected with HSP22 overexpression plasmids and HSP22interference fragments.The effects of plasmids and interference fragments were determined by RT-q PCR and Western Blot.2.HSP22 overexpression plasmid and HSP22 interference fragment were transfected with HUVECs separately and then treated with Ox-DL.HUVECs were divided into 10 groups.These groups were Control,Ox-LDL,HSP22 overexpression negative control group（NC）,HSP22 overexpression group（HSP22）,HSP22overexpression negative control+Ox-LDL treatment group（NC+Ox-LDL）,HSP22overexpression+Ox-LDL treatment group（HSP22+Ox-LDL）,HSP22 interference negative control group（si-NC）,HSP22 interference group（si-HSP22）,HSP22interference negative control+Ox-LDL treatment group（si-NC+Ox-LDL）,and HSP22 interference+Ox-LDL treatment group（si-HSP22+Ox-LDL）.3.The endothelial cell activity level was detected by the CCK-8 kit.4.Flow cytometry was used to analyze the changes in mitochondrial membrane potential in endothelial cells of different treatment groups.5.The ATP kit was used to analyze the changes in ATP levels in endothelial cells of different treatment groups.6.Detection of intracellular ROS levels by Mito SOX Red fluorescent probe.7.Western Blot was used to detect the protein levels of the endothelial cell damage index p-e NOS,e NOS,ET-1,and mitochondrial biological function-related markers Mfn2,OPA1 in different treatment groups.8.Immunofluorescence staining was used to detect the expression levels of Nrf2in different treatment groups.9.Western Blot was used to detect the expression levels of Nrf2 protein in the cytoplasm and nucleus of different treatment groups.Results:1.RT-q PCR and Western Blot were used to detect the expression levels of HSP22 after transfection of HSP22 plasmid and interference fragment.The results indicated that there was no significant difference in the m RNA and protein levels of HSP22 between the si-NC and NC groups compared to the Control group（p>0.05）.Compared with the si-NC,the HSP22 expression in the si-HSP22 group m RNA and protein were significantly reduced in the si-HSP22 group（p<0.05）;HSP22 m RNA and protein expression were significantly increased in the HSP22 group compared with the NC group（p<0.05）.2.HUVECs were transfected with HSP22 plasmid or interference fragment,and then treated with Ox-LDL for 24 hours.Cell viability was measured by CCK-8 assay.The results showed that compared to the control group,cell viability was significantly decreased in the Ox-LDL group（p<0.05）.In the NC vs HSP22 group,cell viability increased in the HSP22 group（p<0.05）.In the si-NC vs si-HSP22 group,cell viability decreased in the si-HSP22 group（p<0.05）.There was no significant difference in cell viability between the NC+Ox-LDL group and si-NC+Ox-LDL group compared to the Ox-LDL group（p>0.05）,but cell viability was significantly increased in the HSP22+Ox-LDL group（p<0.05）and further decreased in the si-HSP22+Ox-LDL group（p<0.05）.3.Flow cytometry analysis revealed that compared to the Control group,the level of JC-1 polymer in endothelial cell mitochondria decreased,while the level of JC-1monomer increased,mitochondrial membrane potential decreased,and depolarization level significantly increased in the Ox-LDL group（p<0.05）.There was no significant difference in mitochondrial depolarization level between the NC and HSP22 groups（p>0.05）.In the si-NC vs si-HSP22 group,the mitochondrial depolarization level was significantly increased in the si-HSP22 group（p<0.05）.There was no significant difference in mitochondrial depolarization level between the NC+Ox-LDL group and si-NC+Ox-LDL group compared to the Ox-LDL group（p>0.05）,but mitochondrial depolarization level was significantly decreased in the HSP22+Ox-LDL group（p<0.05）and further increased in the si-HSP22+Ox-LDL group（p<0.05）.4.The ATP assay kit was used to analyze the changes in ATP levels in different treatment groups of endothelial cells.Compared with the Control group,the ATP content of endothelial cells in the Ox-LDL group was significantly decreased（p<0.05）;there was no significant difference in ATP levels between the NC group and the HSP22 group（p>0.05）;the si-HSP22 group had a lower level of ATP in cells compared with the si-NC group（p<0.05）;there was no significant difference in ATP levels in NC+Ox-LDL group,HSP22+Ox-LDL group and si-NC+Ox-LDL group compared with the Ox-LDL group（p>0.05）,while the ATP level in the si-HSP22+Ox-LDL group was further decreased（p<0.05）.5.The results of the co-localization of mt ROS staining indicate that compared to the control group,the Ox-LDL group had an increased production of ROS in endothelial cells（p<0.05）.There was no significant difference in ROS levels between the NC group and the HSP22 group（p>0.05）.However,the si-HSP22group had a significant increase in intracellular ROS production compared to the si-NC group（p<0.05）.When comparing the groups treated with Ox-LDL,the NC+Ox-LDL group and the si-NC+Ox-LDL group showed no significant difference in ROS levels compared to the Ox-LDL group（p>0.05）.In contrast,the HSP22+Ox-LDL group had a decreased intracellular ROS level（p<0.05）,while the si-HSP22+Ox-LDL group had a further increase in ROS levels（p<0.05）.6.Western Blotting detected the following:Compared with the Control group,p-e NOS,Mfn2 and OPA1 protein levels decreased and ET-1 protein expression levels increased in the Ox-LDL group,with significant differences in the results（p<0.05）;NC group vs HSP22 group,no significant differences in ET-1 protein levels in the HSP22 group（p>0.05）,p-e NOS,Mfn2 and OPA1 protein In the si-NC group vs.si-HSP22 group,the p-e NOS,Mfn2 and OPA1 levels were decreased（p<0.05）and ET-1 expression levels were increased（p<0.05）in the si-HSP22 group;compared with the Ox-LDL group,the p-e NOS,ET-1,Mfn2 and OPA1 expression levels in the NC+Ox-LDL and si-NC+Ox-LDL groups were decreased（p<0.05）.ET-1,Mfn2 and OPA1 protein expression levels were not significantly different between NC+Ox-LDL and si-NC+Ox-LDL groups（p>0.05）,while p-e NOS,Mfn2 and OPA1 levels increased in HSP22+Ox-LDL group（p<0.05）and ET-1 protein levels decreased in si-HSP22+Ox-LDL group（p<0.05）;while p-e NOS,Mfn2 and OPA1 levels further decreased in si-HSP22+Ox-LDL group（p<0.05）.levels were further reduced（p<0.05）and ET-1 protein expression levels were further increased（p<0.05）.7.Immunofluorescence staining observed the distribution of Nrf2 in different groups.In the group transfected with HSP22 plasmid,compared with the Control group,Nrf2 was mainly distributed in the cytoplasm,while in the Ox-LDL group,Nrf2 was expressed in both the cytoplasm and nucleus,and most of it was distributed in the nucleus（p<0.05）.Compared with the Control group,the NC group also showed Nrf2 mainly distributed in the cytoplasm,with no significant difference（p>0.05）,but the HSP22 group had significantly increased expression of Nrf2 in the nucleus（p<0.05）.Compared with the Ox-LDL group,the NC+Ox-LDL group mainly distributed in the nucleus（p>0.05）,while in the HSP22+Ox-LDL group,almost all Nrf2 was concentrated in the nucleus（p<0.05）.In the group transfected with HSP22 interference fragment,compared with the Control group,Nrf2 in the cytoplasm was mainly distributed in the Ox-LDL group,and Nrf2 was expressed in both the cytoplasm and nucleus,with most of it distributed in the nucleus（p<0.05）.Compared with the Control group,Nrf2 in the si-NC group was also mainly distributed in the cytoplasm,with no significant difference from the Control group（p>0.05）,but the expression of Nrf2 in the cytoplasm was further increased in the si-HSP22 group（p<0.05）.Compared with the Ox-LDL group,the si-NC+Ox-LDL group was mainly distributed in the nucleus,with no significant difference from the Ox-LDL group（p>0.05）,while only a small part of Nrf2 was distributed in the nucleus and most of it was distributed in the cytoplasm in the si-HSP22+Ox-LDL group（p<0.05）.8.In different treatment groups,Western blot was used to detect Nrf2 protein expression in the cytoplasm and nucleus.Compared with the Control group,the Ox-LDL group had elevated nuclear Nrf2 protein levels（p<0.05）and decreased cytoplasmic Nrf2 protein levels（p<0.05）.NC group vs.HSP22 group,the nuclear Nrf2 protein level increased（p<0.05）and the cytoplasmic Nrf2 protein level decreased（p<0.05）in the HSP22 group.Si-NC group vs.si-HSP22 group,nuclear Nrf2 protein level was decreased（p<0.05）and cytoplasmic Nrf2 protein level was increased（p<0.05）in si-HSP22 group.Compared with the Ox-LDL group,there was no significant difference in the cytoplasmic and nuclear Nrf2 protein levels in the NC+Ox-LDL group and the si-NC+Ox-LDL group（p>0.05）.The nuclear Nrf2protein level increased（p<0.05）and the cytoplasmic Nrf2 protein level decreased（p<0.05）in the HSP22+Ox-LDL group,while the nuclear Nrf2 protein level decreased（p<0.05）and the cytoplasmic Nrf2 protein level increased（p<0.05）in the si-HSP22+Ox-LDL group.Conclusion:1.Overexpression of HSP22 can inhibit the production of ROS,regulate mitochondrial biology,improve endothelial cell damage induced by high fat,and induce the nuclear translocation of Nrf2.2.Interference with the expression of HSP22 can induce a large amount of ROS production,lead to mitochondrial dysfunction,further aggravate endothelial cell damage induced by high fat,and inhibit the nuclear translocation of Nrf2.Part 4 HSP22 regulates mitochondrial function by promoting the nuclear translocation of Nrf2 to reduce endothelial cell damageObjective:To investigate whether HSP22 regulates mitochondrial biology and reduces endothelial cell damage induced by Ox-LDL by promoting the nuclear translocation of Nrf2.Methods:1.The HUVECs were treated with different concentrations of ML385（Nrf2inhibitor）for 24 hours（0Μm,5μM,10μM,100μM,and 200μM）,and the expression levels of Nrf2 protein were determined by Western blot（p<0.05）to determine the optimal treatment concentration of ML385,which was found to be 5μM.2.After transfection with HSP22 overexpression plasmids,the cells were divided into HSP22 group,HSP22+Ox-LDL group,HSP22+Nrf2 inhibitor group（HSP22+ML385）,and HSP22+Nrf2 inhibitor+Ox-LDL group（HSP22+ML385+Ox-LDL）,and were subjected to corresponding treatments.3.The CCK-8 assay was used to measure endothelial cell viability in different groups.4.The protein levels of endothelial cell injury markers p-e NOS,e NOS,ET-1,and mitochondrial biology-related markers Mfn2,and OPA1 were detected by Western blot in different treatment groups.5.The Mito SOX Red fluorescent probe was used to detect the levels of intracellular ROS in cells treated with different methods.Results:1.The optimal treatment concentration of Nrf2 inhibitor ML385 was found to be5μM.2.CCK-8 results showed that compared to the HSP22 group,cell viability was significantly decreased in the HSP22+Ox-LDL group（p<0.05）and in the HSP22+ML385 group（p<0.05）.Compared to the HSP22+Ox-LDL group,cell viability was further decreased in the HSP22+ML385+Ox-LDL group（p<0.05）.3.Western Blot results showed that compared with the HSP22 group,the p-e NOS,Mfn2,and OPA1 protein expression levels were decreased and ET-1 protein levels were increased in the HSP22+Ox-LDL group（p<0.05）,and similarly,the p-e NOS,Mfn2 and OPA1 protein expression levels were decreased and ET-1 protein levels were increased in the HSP22+ML385 group（p<0.05）.However,compared with the HSP22+Ox-LDL group,the p-e NOS,Mfn2,and OPA1 protein expression levels were further decreased and ET-1 protein expression levels were further increased in the HSP22+ML385+Ox-LDL group（p<0.05）.4.Analysis of the staining results of co-localized confocal mt ROS suggests that,compared to the HSP22 group,the HSP22+Ox-LDL group and the HSP22+ML385group showed a significant increase in ROS levels（p<0.05）.Furthermore,compared to the HSP22+Ox-LDL group,the HSP22+ML385+Ox-LDL group showed a further increase in ROS levels（p<0.05）.Conclusion:HSP22 can regulate mitochondrial biology by inducing Nrf2 nuclear translocation,reducing cellular reactive oxygen species levels,and thereby alleviating high-fat-induced vascular endothelial cell damage. |
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