PHPS1促进ApoE基因敲除小鼠动脉粥样硬化斑块的不稳定性研究

The study of PHPS1 promoted the instability of atherosclerotic plaque in ApoE knockout mice

目的探讨蛋白酪氨酸磷酸酶SHP-2特异性抑制剂苯肼基吡唑啉酮磺酸盐1(PHPS1)对ApoE基因敲除小鼠动脉粥样硬化斑块的影响。方法2019年1—12月于河北省人民医院临床研究中心进行实验。6~8周龄雄性ApoE基因敲除小鼠18只,随机数字表法分为模型组(生理盐水灌胃,0.25 mg·kg^(-1)·d^(-1))和PHPS1组(PHPS1灌胃,0.25 mg·kg^(-1)·d^(-1)),各9只。4周后处死小鼠取血清,检测总胆固醇、三酰甘油、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇及IL-1β、TNF-α水平;主动脉根部病理切片HE染色评估斑块大小;免疫组化评估斑块内巨噬细胞、平滑肌含量;天狼星红染色评估斑块内胶原表达情况;TUNEL染色评估斑块内细胞凋亡情况;Movat染色评估斑块内坏死核心面积;Western-blot检测内质网应激凋亡相关通路蛋白表达量。结果与模型组比较,PHPS1组血脂水平差异无统计学意义(P>0.05),炎性因子IL-1β和TNF-α水平明显升高(t/P=2.934/0.010,2.240/0.040);斑块面积、斑块内巨噬细胞含量显著增加(t/P=4.869/0.001,4.241/0.001);斑块内平滑肌和胶原含量差异无统计学意义(P>0.05);TUNEL和Movat染色显示凋亡阳性区域和斑块内坏死核心面积显著增大(t/P=7.636/0.000,17.212/0.000);Western-blot结果显示内质网应激蛋白p-PERK、ATF4表达升高,凋亡相关蛋白CHOP、cleaved-caspase3、bax表达显著上升,bcl-2蛋白表达显著下降(P均<0.01)。结论蛋白酪氨酸磷酸酶SHP-2可通过抑制斑块细胞的内质网应激从而抑制凋亡进而稳定动脉粥样硬化斑块。

Objective To investigate the effect of protein tyrosine phosphatase SHP-2 inhibitor PHPS1 on atherosclerotic plaques in ApoE geneknockout mice.Methods The experiment was conducted in the Research Center of Hebei General Hospital from January to December 2019.Eighteen male ApoE gene knockout mice aged six to eight weeks were randomly divided into the model group,whice were given intragastric administration of normal saline(0.25 mg·kg^(-1)·d^(-1)),and the PHPS1 group,whice were given intragastric administration of PHPS1(0.25 mg·kg^(-1)·d^(-1)).After four weeks,the levels of TG、TC、LDL、HDL、IL-1βand TNF-αwere measured;HE staining was used to evaluate the plaque size of aortic roots;Immunohistochemical was used to evaluate the content of macrophages and smooth muscle in the plaques;Sirius red staining wasused to evaluate the contents of collagen in the plaques;TUNEL staining was used to assess apoptosis in plaques;Movat staining was used to evaluate the area of necrotic core in plaques;Western blot was used to evaluate the protein expression levels of apoptosis-related pathways induced by Endoplasmic Reticulum Stress.Results Compared with the model group,there was no significant difference in blood lipid levels in PHPS1 group(P>0.05),but the level of inflammatory factors((IL-1βand TNF-α))were significantly increased(t/P=2.934/0.010,2.240/0.040);The plaque area and the content of macrophages in the plaque increased significantly(t/P=4.869/0.001,4.241/0.001));The content of smooth muscle and collagen inthe plaques werenot statistically significant(P>0.05);TUNEL and Movat staining showed a significant increase in apoptotic positive areas and the necrotic core of the plaque(t/P=7.636/0.000,17.212/0.000);Western blot results suggested that the endoplasmic reticulum stress proteins p-PERK and ATF4 increased,the apoptosis-related proteins CHOP,cleaved-caspase3 and bax increased significantly,and the bcl-2 protein decreased significantly(P<0.01).Conclusion Protein tyrosine phosphatase SHP-2 can inhibit endoplasmic reticulum stressin plaque cells,so as toinhibit apoptosis,ultimately stabilizes atherosclerotic plaque.