SHP2抑制剂PHPS1对ApoE基因敲除小鼠动脉粥样硬化斑块及巨噬细胞的影响

Effects of SHP2 inhibitor PHPS1 on atherosclerotic plaques and macrophages in ApoE gene knockout mice

目的研究SHP2抑制剂PHPS1对ApoE基因敲除(ApoE-/-)小鼠动脉粥样硬化斑块及巨噬细胞的影响。方法体内实验:将ApoE-/-小鼠随机分成模型组和PHPS1组,每组10只,并给予高脂饲料喂养,模型组腹腔注射0.9%氯化钠溶液3 mg·kg-1·d-1,PHPS1组腹腔注射PHPS1剂量3 mg·kg-1·d-1,注射1次/d,注射20 d。通过Movat染色分析斑块面积;通过天狼星红染色分析胶原所占比例;通过免疫组化分别分析巨噬细胞与平滑肌细胞的表达量;通过Western Blotting检测p-SHP2、p-Syk、CD36蛋白磷酸化水平。体外实验:将小鼠巨噬细胞RAW246.7细胞培养于DMEM培养基中,将其分为RAW246.7组(未做其他任何处理的DEMD培养基)、RAW246.7+ox-LDL组(给予ox-LDL处理的DEMD培养基),RAW246.7+ox-LDL+PHPS1组(同时给予ox-LDL+PHPS1处理的DEMD培养基)。体外实验:将小鼠巨噬细胞RAW246.7细胞培养于DMEM培养基中,将其分为RAW246.7组(未做其他任何处理的DEMD培养基)、RAW246.7+ox-LDL组(给予ox-LDL处理的DEMD培养基),RAW246.7+ox-LDL+PHPS1组(同时给予ox-LDL+PHPS1处理的DEMD培养基)。通过免疫荧光检测细胞内CD36的表达量;通过荧光微球吞噬实验检测巨噬细胞的内吞能力;通过Western Blotting检测各组细胞中p-SHP2、p-Syk、CD36蛋白磷酸化水平。结果体内实验:Movat染色显示PHPS1组动脉粥样硬化斑块面积明显小于模型组,差异有统计学意义(P<0.05);天狼星红染色显示,PHPS1组动脉粥样硬化斑块内胶原纤维的含量较模型组明显减少,差异有统计学意义(P<0.05);免疫组化结果显示PHPS1组平滑肌细胞与巨噬细胞较模型组明显减少,差异有统计学意义(P<0.05);Western Blotting检测结果显示,PHPS1组p-SHP2、p-Syk、CD36蛋白的磷酸化水平较模型组均明显降低,差异有统计学意义(P<0.05)。体外实验:PHPS1组巨噬细胞内CD36的荧光强度明显低于ox-LDL组,差异有统计学意义(P<0.05);荧光微球吞噬实验结果显示PHPS1组巨噬细胞荧光强度明显低于ox-LDL组,差异有统计学意义(P<0.05);Western Blotting检测结果显示,RAW246.7+ox-LDL+PHPS1组p-SHP、p-Syk、CD36蛋白磷酸化水平较RAW246.7组明显降低,差异有统计学意义(P<0.05)。结论SHP2抑制剂PHPS1通过抑制SHP2、Syk蛋白磷酸化水平,从而减少巨噬细胞内吞脂质脂质的作用,最终阻止动脉粥样硬化斑块的形成。

Objective To investigate the effects of SHP2 inhibitor PHPS1 on atherosclerotic plaques and macrophages in ApoE-/-mice.Methods In vivo experiment:ApoE-/-mice were randomly divided into model group and PHPS1 group,with 10 mice in each group,the mice in PHPS1 group were given a high-fat diet,however,the mice in model group were given normal saline by intraperitoneal injection at the dose of 3mg·kg-1·d-1,then the mice in PHPS1 group were given PHPS1 at a dose of 3mg·kg-1·d-1 by intraperitoneal injection,once a day for 20 days.The plaque area was analyzed by Movat staining.and the proportion of collagen was analyzed by sirian red staining,and the expression levels of macrophages and smooth muscle cells were detected by immunohistochemistry,and phosphorylation levels of P-SHP2,P-SYk and CD36 proteins were detected by Western Blot.In vitro experiment,the mouse macrophage RAW246.7 cells were cultured in DMEM medium,which were divided into RAW246.7 group(DEMD medium without any other treatment),RAW246.7+Ox-LDL group(DEMD medium treated with ox-LDL),and RAW246.7+OX-LDL+PHPS1 group(DEMD medium treated with ox-LDL+PHPS1).The expression levels of CD36 were detected by immunofluorescence,the endocytosis of macrophages was detected by fluorescence microsphere phagocytosis experiment,and the phosphorylation levels of P-SHP2,P-SYk and CD36 proteins in each group were detected by Western Blot.Results in vivo experiment:Movat staining showed that the area of atherosclerotic plaque in PHPS1 group was significantly smaller than that in model group(P<0.05),and the sirius red staining showed that the content of collagen fiber in atherosclerotic plaque in PHPS1 group was significantly lower than that in model group(P<0.05).Immunohistochemical results showed that the numbers of smooth muscle cells and macrophages in PHPS1 group were significantly less than those in model group(P<0.05).In vitro experiment,the fluorescence intensity of CD36 in macrophages in PHPS1 group was significantly lower than that in OX-LDL group(P<0.05),and the fluorescence microspheres phagocytosis experiment results showed that the fluorescence intensity of macrophages in PHPS1 group was significantly lower than that in OX-LDL group(P<0.05).Western Blot showed that phosphorylation levels of P-SHP,P-SYk and CD36 proteins in RAW246.7+Ox-LDL+PHPS1 group were significantly lower than those in RAW246.7 group(P<0.05).Conclusion The SHP2 inhibitor-PHPS1 can inhibit the expression levels of SHP2 and Syk protein phosphorylation,so as to decrease the effects of macrophages in endocytosis of lipids,ultimately to prevent the formation of atherosclerotic plaques.