| Objective:The aim of this study is to evaluate the function and the mechnaism of Geranylgeranylacetone（GGA）in the process ofatherosclerosis（AS）and to explore the mechanism of associating with heat shock protein 22（HSP22）.Methods:Cell:1.Explored the concentration of GGA,which was proper for Human Coronary Artery Endothelial Cells（HCAECs）;2.Divided the HCAECs,which was in good condition,into four groups,including the control group,the GGA group,the HSP22-siRNA group and the HSP22-si RNA+GGA group,then transfected each cell groups with HSP22-siRNA or negative segment of siRNA,after 24hours,treated GGA group and HSP22-siRNA+GGA group with GGA,which was in proper concentration,treated control group and HSP22-si RNA group with equal amount of ethanol as control,after 12hours,treated all groups with the media that containing80μ/ml ox-LDL one day;3.Detecting the apoptosis and the level of ROS by flow cytometer,detecting the mRNA of HSP22 and NF-κB with RT-qPCR and detecting the expression of HSP22、NF-κB、eNOS and ICAM-1 using Western Blot.Animal:1.We choice two groups of mice for experiment,all of these groups were 8-9weeks-old male one,which was divided by their genotype:HSP22-/-//ApoE-/-and HSP22+/+//ApoE-/-,16 each.After one week of adaptive feeding,divided the two groups into four subgroups,including the control group,the GGA treated group（GGA group）,the HSP22 knockout group（the KO group）and the HSP22knockout+GGA treated group（the KO+GGA group）.Keep all groups with high fat diet,12 weeks,treated the GGA group and the KO+GGA group with GGA（400mg/（kg·d））from the fifth week,and treated the others with equalamount of normal saline as control,8 weeks;2.The weight of all mice was recorded every week.The lipid levels were measured at baseline and the end of intervention;3.Measured the atherosclerotic lesion burdens of the wall from mice’s aortas by The Oil Red O staining and HE staining;4.Detecting the concentrations of serum HSP22 by ELISA and detecting the expression of HSP22、NF-κB、eNOS and ICAM-1 using Western Blot.Results:1,The optimum concentration of GGA was 1μmol/L.2,Flow cytometry（1）apoptosis:compared with the control group,the apoptosis level of the GGA group was decreased（31.78±3.27 vs 23.99±2.03,P<0.05）,and there was no significant change in the HSP22-siRNA group（31.78±3.27 vs 35.92±4.47,P>0.05）.Compared with the GGA group,the apoptosis level was increased in the HSP22-siRNA+GGA group（23.99±2.03 vs 33.24±4.85,P<0.05）,but there was no significant difference between the HSP22-siRNA group and the HSP22-siRNA+GGA group（35.92±4.47 vs 33.24±4.85,P>0.05）.（2）ROS level:compared with the control group,the level of ROS in the GGA group decreased significantly（308.0±23.52 vs 218.0±26.21,P<0.05）,the ROS level in the HSP22-si RNA group increased significantly（308.0±23.52 vs 422.7±41.26P<0.05）.Compared with the GGA group,the ROS level of the HSP22-siRNA+GGA group was significantly increased（218.0±26.21 vs 381.7±56.22,P<0.05）.There was no significant difference between the HSP22-si RNA group and the HSP22-si RNA+GGA group（422.7±41.26 vs 381.7±56.22,P>0.05）.3,RT-qPCR（1）The relative expression of HSP22 mRNA:compared with the control group,the relative expression of HSP22 mRNA in the GGA group was 1.85±0.189（P<0.05）,increased significantly,while the relative expression of HSP22 mRNA was very low in the HSP22-si RNA group and the HSP22-siRNA+GGA group,and there was no significant difference between those two groups（0.30±0.281 vs 0.25±0.215,P>0.05）.（2）The relative expression of NF-κB mRNA:compared with the control group,the relative expression of NF-κB mRNA in the GGA group was 0.68±0.052（P<0.05）,decreased gently,while the relative expression of NF-κB mRNA in the HSP22-siRNA group was 1.43±0.160（P<0.05）,increased significantly.In addition,compared with the GGA group,the relative expression of NF-κB mRNA in the HSP22-siRNA+GGA group increased significantly（0.68±0.052 vs 1.31±0.327,P<0.05）.There was no significant difference between the HSP22-siRNA group and the HSP22-siRNA+GGA group in the level of NF-κB mRNA（1.43±0.160 vs1.31±0.327,P>0.05）.4,Detected the expression of HSP22,NF-κB,ICAM-1 and eNOS using Western Blot:（1）Expression of HSP22:compared with the control group,the expression of HSP22 in the GGA group increased significantly（0.231±0.025 vs 0.364±0.058,P<0.05）,while the expression of HSP22 decreased significantly in the HSP22-siRNA group and the HSP22-siRNA+GGA group,and there was no statistical difference between those two groups（0.090±0.015 vs 0.089±0.016,P>0.05）.（2）Expression of NF-κB:compared with the control group,the expression of NF-κB in the GGA group was decreased significantly（0.781±0.092 vs 0.595±0.068,P<0.05）,whereas,the expression of NF-κB in the HSP22-siRNA group was increased（0.781±0.092 vs 1.042±0.102,P<0.05）.Compared with the GGA group,the expression of NF-κB in the HSP22-siRNA+GGA group,was significantly increased（0.595±0.068 vs 0.805±0.090,P<0.05）.Compared with the HSP22-siRNA group,the expression of NF-κB in the HSP22-siRNA+GGA group decreased slightly（1.042±0.102 vs 0.805±0.090,P<0.05）.（3）Expression of ICAM-1:compared with the control group,the expression of ICAM-1 in the GGA group was decreased（0.447±0.061 vs 0.305±0.064,P<0.05）,while the expression of ICAM-1 in the HSP22-siRNA group was increased（0.447±0.061 vs 0.589±0.045,P<0.05）.Compared with the GGA group,the expression of ICAM-1 was increased significantly in the HSP22-siRNA+GGA group（0.305±0.064 vs 0.571±0.127,P<0.05）.There was no significant difference between the HSP22-siRNA group and the HSP22-siRNA+GGA group（0.589±0.045 vs0.571±0.127,P>0.05）.（4）eNOS expression:compared with the control group,there was no obvious change in the expression of eNOS in the GGA group（0.438±0.053 vs 0.556±0.065,P>0.05）,and there was no obvious change in the HSP22-siRNA group,as well（0.438±0.053 vs 0.381±0.092,P>0.05）.Compared with the GGA group,the expression of eNOS was decreased gently in the HSP22-siRNA+GGA group（0.556±0.065 vs 0.406±0.048,P<0.05）.There was no significant difference between the HSP22-siRNA group and the HSP22-siRNA+GGA group（0.381±0.092 vs0.406±0.048,P>0.05）.5,Blood lipid levels:（1）Baseline level:compared with all groups of lipid level at baseline,there was no significant difference between each other（P>0.05）.（2）After intervention:there was no statistical difference in the level of TC,TG,LDL-C and HDL-C between all groups（P>0.05）.6,The concentration of serum HSP22 detected by ELISA:（1）Before intervention:compared with the WT group,there was no significant difference in the level of serum HSP22 between the WT+GGA group（68.30±7.92 vs70.77±7.78,P>0.05）.However,the level of serum HSP22 was extremely low,almost can not be detected,in the KO group and the KO+GGA group（17.53±3.01 vs20.03±2.19,P>0.05）.（2）After intervention:compared with the WT group,the level of serum HSP22was increased significantly in the WT+GGA group（192.3±25.69 vs 273.1±24.13,P<0.05）,and the level of serum HSP22 was still very low in the KO group and the KO+GGA group,and there was no significant deference between those two groups（34.27±5.78 vs 36.53±6.11,P>0.05）.7.Histopathological observation:（1）Aortic Oil Red O staining:compared with the WT group,the proportion of atherosclerotic plaque in aorta was decreased significantly in the WT+GGA group（12.42±1.49 vs 6.82±0.83,P<0.01）,however,there was no obvious change in the KO group（12.42±1.49 vs 13.70±0.86,P>0.05）.Compared with the WT+GGA group and the KO+GGA group,there was no significant difference in the proportion of atherosclerotic plaque in aorta between each other（6.82±0.83 vs 7.16±2.29,P>0.05）.Compared with the KO group,the proportion of atherosclerotic plaque in aorta was decreased obviously in the KO+GGA group（13.70±0.86 vs 7.16±2.29,P<0.01）.（2）HE staining in the root of the aorta:compared with the WT group,the proportion was decreased in the WT+GGA group（41.80±3.05vs 29.21±5.46,P<0.05）,while,the relative area of plaque in the KO group was increased gently（41.81±3.05vs 48.24±2.40,P<0.05）.Compared with the group of WT+GGA and KO+GGA,there was no significant difference between each other（29.21±5.46vs 35.82±3.67,P>0.05）,and compared with the KO group,the relative area of plaque in the KO+GGA group was significantly decreased（48.24±2.40 vs 35.82±3.67,P<0.05）.8,Detected the expression of HSP22,NF-κB,ICAM-1 and eNOS in the aorta by Western Blot:（1）The expression of HSP22:compared with the WT group,the expression of HSP22 in the aorta was increased in WT+GGA group（0.297±0.090 vs 0.593±0.080,P<0.05）,but there was no visible band of HSP22 in the KO group and the KO+GGA group（0.057±0.021 vs 0.050±0.010,P>0.05）.（2）The expression of NF-κB:compared with the WT group,the expression of NF-κB was decreased in the WT+GGA group（0.630±0.070 vs 0.357±0.090,P<0.05）,while,the KO group was increased（0.630±0.070 vs 1.117±0.175,P<0.05）.Compared with the WT+GGA group,the KO+GGA group was increased（0.357±0.090 vs0.703±0.133,P<0.05）,Compared with the KO group,the level of NF-κB was decreased gently in KO+GGA group 1.117±0.175 vs 0.703±0.133,P<0.05).（3）The expression of ICAM-1:compared with the WT group,the expression of ICAM-1 was decreased in the WT+GGA group（0.183±0.031 vs 0.113±0.031,P<0.05）,but the KO group was increased significantly（0.183±0.031 vs 0.327±0.080,P<0.05）.Compared with the WT+GGA group,the expression of ICAM-1 was significantly increased in the KO+GGA group（0.113±0.031 vs 0.297±0.090,P<0.05）.Compared with the KO group and the KO+GGA group,there was no significant difference between each other（0.327±0.080 vs 0.297±0.090,P>0.05）.（4）The expression of eNOS:compared with the WT group,the expression of eNOS in the WT+GGA group was not obvious change（0.263±0.040 vs 0.277±0.040,P>0.05）,while,the expression of eNOS in the KO group was decreased gently（0.263±0.04 vs 0.160±0.046,P<0.05）,compared with the WT+GGA group,the expression of eNOS in the KO+GGA group was decreased slightly（0.277±0.040 vs0.173±0.031,P<0.05）;there was no significant difference in the expression of ICAM-1 between the KO group and the KO+GGA group（0.160±0.046 vs0.173±0.031,P>0.05）.Conclusion:1,GGA can induced HCAECs over-express HSP22 in a dose dependent manner.2,The proper concentration of GGA can reduced the apoptosis of HCAECs induced by ox-LDL,and the primary machanism of this effect was to reduce the expression of NF-κB、ICAM-1 by induced HSP22 over-express.3,GGA can mitigated the response of inflammation in AS by induce the expression of HSP22,down-regulated NF-κB、ICAM-1,meanwhile,it can also alleviated the formation of atherosclerotic plaque in aorta,which play a protective role in both formation and progress of AS. |
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