CXCL14对糖尿病动脉粥样硬化小鼠脂肪组织焦亡及主动脉斑块的影响

Effects of CXCL14 on adipose tissue pyroptosis and aortic plaque of diabetic atherosclerotic mice

目的:探究CXC趋化因子配体14(CXCL14)对糖尿病动脉粥样硬化(atherosclerosis,AS)小鼠脂肪组织焦亡及AS斑块的影响。方法:①腹腔注射链脲佐菌素诱导糖尿病ApoE-/-小鼠,对糖尿病小鼠高脂喂养20周,构建糖尿病AS模型(模型组),对照小鼠仅高脂饮食(AS组);②在糖尿病小鼠后肢内侧皮下注射抗CXCL14短肽(抗CXCL14组),对照糖尿病小鼠仅皮下注射生理盐水;③在糖尿病小鼠后腹股沟皮下脂肪组织原位注射腺相关病毒(AAV)以抑制消化道皮肤素(GSDMD)介导的细胞焦亡,分为:AAV-shscramble组、AAV-shGSDMD组、抗CXCL14+AAV-shscramble组、抗CXCL14+AAV-shGSDMD组。各组小鼠高脂饮食饲养20周后,麻醉下收集小鼠血清,应用生化试剂盒检测小鼠血脂水平(总胆固醇、三酰甘油、LDL-C、HDL-C)。处死小鼠,取附睾脂肪组织进行电镜扫描及HE染色观察脂肪细胞变化;利用qRT-PCR技术分析附睾脂肪组织焦亡相关因子GSDMD、天冬氨酸蛋白水解酶-1(Caspase-1)、NLR家族吡啶结构域蛋白3(NLRP3)炎症小体、白介素-1β(IL-1β)和炎症因子白介素-6(IL-6)的变化。分离提取小鼠主动脉,通过HE染色和血管大体油红O染色观察主动脉AS斑块的相对面积。结果:与AS组相比,模型组小鼠附睾脂肪组织中的脂肪细胞肥大并数量减少(P<0.01),主动脉AS斑块面积显著增加,总胆固醇、三酰甘油、LDL-C水平明显升高,HDL-C明显降低;附睾脂肪组织的焦亡相关因子和IL-6水平明显升高(P<0.05),表明糖尿病促进小鼠AS斑块与脂肪组织焦亡。注射抗CXCL14短肽可以减少主动脉斑块面积,改善血脂水平,并抑制脂肪组织焦亡,脂肪细胞数量增多。GSDMD敲减后,附睾脂肪组织中脂肪细胞数量增多,主动脉AS斑块面积减少;然而注射抗CXCL14免疫肽后,AAV-shGSDMD组AS斑块面积变化不显著。结论:抗CXCL14可以减轻脂肪组织焦亡并缓解糖尿病AS的发展。

Objective:To explore the effect of CXCL14 on pyroptosis of adipocytes and the formation of atherosclerosis in diabetic microenvironment.Methods:①ApoE-/-mice were intraperitoneally injected with streptozotocin to construct diabetes mellitus,and diabetic ApoE-/-mice were fed with high fat diet for 20 weeks,and the diabetic atherosclerosis model(model group)was constructed,and control mice were only fed on a high-fat diet(AS group);②Anti-CXCL14 short peptide was injected subcutaneously on the medial hind limb of diabetic mice(anti-CXCL14 group),and control diabetic mice were injected subcutaneously with normal saline only;③Adeno-associated virus(AAV)was injected in situ into posterior inguinal subcutaneous adipose tissue in diabetic mice to inhibit gastrointestinal dermatin(GSDMD)-mediated pyroptosis,divided into:AAV-shscramble group,AAV-shGSDMD group,anti-CXCL14+AAV-shscramble group,anti-CXCL14+AAV-shGSDMDgroup.After 20 weeks of high-fat diet of the mice,the serum of the mice was collected under anesthesia,and the blood lipid levels(total cholesterol,triglycerides,LDL-C,and HDL-C)of the mice were analyzed by biochemical detection kit.The mice were sacrificed,and the epididymal adipose tissue was scanned by electron microscopy to observe the changes of fat cells.qRT-PCR was used to analyze the changes of pyroptosis-related factors GSDMD,aspartate proteolytic enzyme-1(Caspase-1),NLR family pyridine domain protein 3(NLRP3)inflammasome,interleukin-1β(IL-1β)and inflammatory factor interleukin-6(IL-6).The mouse aorta was isolated and extracted,and the relative area of atherosclerotic plaques was observed by HE staining and oil red O staining.Results:Compared with the AS group,the hypertrophy and number of adipocytes of adipose tissue in the epididymis in the model group decreased(P<0.01),the size of aorticplaque increased,the levels of total cholesterol,triacylglycerol and LDL-C were significantly increased,and HDL-C was significantly reduced.The levels of pyroptosis-related factors and IL-6 in adipose tissue of epididymis were significantly increased(P<0.05),indicating that diabetes promoted pyroptosis of AS plaque and adipose tissue in mice.Injection of anti-CXCL14 short peptide could reduce the size of aortic plaque,improve blood lipid levels,and inhibit adipose tissue pyroptosis,which increases the number of fat cells.After GSDMD knockdown,the number of adipocytes increased and the area of aortic plaques decreased.However,after the injection of anti-CXCL14 immune peptide,there was no significant change in atherosclerosis in the AAV-shGSDMDgroup.Conclusion:Anti-CXCL14 can attenuate adipose tissue pyroptosis and alleviate the development of diabetic atherosclerosis.