25-羟基维生素D_(3)聚乳酸微球通过NLRP3/Caspase-1/GSDMD信号轴对妊娠期糖尿病大鼠心肌损伤的影响

Effect of 25-hydroxyvitamin D_(3)-loaded polylactic acid microspheres on myocardial injury in rats with gestational diabetes mellitus through NLRP3/Caspase-1/GSDMD signaling axis

目的探讨25-羟基维生素D3聚乳酸微球对妊娠期糖尿病大鼠心肌损伤的影响以及对核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)/含半胱氨酸的天冬氨酸蛋白水解酶-1(Caspase-1)和消皮素D(GSDMD)信号轴的调节作用。方法采用W/O型乳化-溶剂挥发法制备25-羟基维生素D3聚乳酸微球, 扫描电镜下观察所得微球的形态, 激光粒度分析仪检测微球的直径, 并计算其累积释放率。妊娠大鼠通过腹腔注射链脲佐菌素建立妊娠期糖尿病大鼠模型, 随机分为模型组、对照组、微球组, 每组10只, 另取10只孕鼠作为假手术组。微球组大鼠沿左心室边缘取4点共注射微球悬液100 μl, 对照组用同样方法注射等量空白微球, 假手术组和模型组同样方法注射等量生理盐水。孕21 d取材进行后续实验。检测大鼠空腹血糖(FBG)、空腹胰岛素(FINS), 并计算胰岛素抵抗指数(HOMA-IR);全自动生化分析仪检测血清肌钙蛋白Ⅰ(cTnI)和肌酸激酶同工酶(CK-MB)水平;酶联免疫吸附试验(ELISA)检测心肌组织中白细胞介素(IL)-1β和肿瘤坏死因子-α(TNF-α)水平, 超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性以及丙二醛水平, 蛋白印迹法检测NLRP3、Caspase-1和GSDMD蛋白表达量。结果扫描电镜下微球呈比较规则的球形, 大小较为一致, 表面较为致密, 无裂缝。微球直径为(13.48±2.04)μm, 且粒径分布集中均一。微球前5天释放缓慢, 然后累积释药率大幅度上升, 第28天累积释药率达80%以上, 后趋于稳定。与假手术组比较, 模型组大鼠FBG、HOMA-IR、cTnI、CK-MB、IL-1β、TNF-α、丙二醛水平以及NLRP3、Caspase-1和GSDMD蛋白表达均升高, FINS、SOD和GSH-Px活性均降低(均P<0.05);与模型组和对照组比较, 微球组大鼠FBG、HOMA-IR、cTnI、CK-MB、IL-1β、TNF-α、丙二醛水平以及NLRP3、Caspase-1和GSDMD蛋白表达均降低, FINS、SOD和GSH-Px活性均升高(均P<0.05)。结论 25-羟基维生素D3聚乳酸微球可降低妊娠期糖尿病大鼠血糖水平以及胰岛素抵抗, 降低心肌组织炎症反应和氧化应激损伤, 减轻心肌损伤, 可能通过抑制NLRP3/Caspase-1/GSDMD信号轴发挥作用。

Objective To investigate the effects of 25-hydroxyvitamin D3-loaded polylactic acid microspheres on myocardial injury in rats with gestational diabetes mellitus as well as its regulatory effects on the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3)-cysteinyl aspartate specific proteinase-1(Caspase-1)-gasdermin D(GSDMD)signaling axis.Methods 25-Hydroxyvitamin D3-loaded polylactic acid microspheres were prepared by W/O emulsification and solvent volatilization method.The morphology of the microspheres was observed by scanning electron microscope,the diameters of the microspheres were detected by laser particle size analyzer,and the cumulative release rate was calculated.Pregnant rats were injected intraperitoneally with streptozotocin to establish a rat model of gestational diabetes mellitus and were randomly divided into the model group,the control group,and the microsphere group,with 10 rats in each group.Another 10 pregnant rats were taken as the sham operation group.In the microsphere group,a total of 100μl of microsphere suspension was injected at 4 points along the edge of the left ventricle,while the control group was injected with an equal amount of blank microspheres in the same way,and the sham operation group and the model group were injected with an equal amount of physiological saline in the same way.Samples were taken at 21 d of gestation for subsequent experiments.Fasting blood glucose(FBG),fasting insulin(FINS),and homeostasis model assessment of insulin resistance(HOMA-IR)of the rat model were detected.Cardiac troponin I(cTnI)and serum creatine kinase isoenzyme(CK-MB)levels were detected by a fully automated biochemical analyzer.Interleukin(IL)-1βand tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)activity,and malondialdehyde levels in myocardial tissues were detected by enzyme-linked immunosorbent assay(ELISA).Protein expression of NLRP3,Caspase-1 and GSDMD was detected by Western Blot.Results The scanning electron microscope observation showed that the microspheres were regular spherical with uniform size,compact surface and no cracks.The diameter of the microspheres was(13.48±2.04)μm,and the particle size distribution was concentrated and homogeneous.The release of 25-hydroxyvitamin D3-loaded polylactic acid microspheres was slow in the first 5 days,and then the cumulative release rate increased significantly,and the cumulative release rate reached more than 80%on the 28th day,and then stabilized.Compared with the sham operation group,the levels of FBG,HOMA-IR,cTnI,CK-MB,IL-1β,TNF-α,malondialdehyde as well as the protein expression of NLRP3,Caspase-1 and GSDMD were increased in the model group,and the activities of FINS,SOD and GSH-Px were decreased(all P<0.05).Compared with the model and control groups,the levels of FBG,HOMA-IR,cTnI,CK-MB,IL-1β,TNF-α,malondialdehyde,and protein expression of NLRP3,Caspase-1,and GSDMD were decreased in the microsphere group,and the activities of FINS,SOD,and GSH-Px were increased(all P<0.05).Conclusions The 25-hydroxyvitamin D3-loaded polylactic acid microspheres can reduce blood glucose level and insulin resistance,reduce myocardial inflammation and oxidative stress injury,and alleviate myocardial injury in rats with gestational diabetes mellitus,possibly by inhibiting the NLRP3/Caspase-1/GSDMD signaling axis.