人脐带间充质干细胞对2型糖尿病小鼠胰岛功能的影响及其对NLRP3炎性体的调节作用

The impact of human umbilical cord-derived mesenchymal stem cells on the pancreatic function of type 2 diabetic mice and their regulatory role on NLRP3 inflammasomes

目的观察人脐带间充质干细胞(UC-MSCs)对2型糖尿病(T2DM)小鼠胰岛功能的影响及其对NOD样受体家族核苷酸结合寡聚化结构域样受体3(NLRP3)炎性体和自噬过程的调节作用。方法实验研究。选取20只8周龄雄性C57BL/6J小鼠,分为正常对照组(n=5)和高脂喂养建模组(n=15);T2DM建模成功的小鼠进一步分为糖尿病组(n=7)和UC-MSCs治疗组(n=7)。UC-MSCs治疗组每周给予1次UC-MSCs(1×106/0.2 ml磷酸盐缓冲液)尾静脉输注治疗,共治疗4周;糖尿病组注射等量生理盐水,正常对照组不予处理。治疗结束后1周各组小鼠行腹腔糖耐量和胰岛素耐量试验检测糖耐量和胰岛素敏感性变化;采用免疫荧光染色法观察胰岛结构改变并检测胰岛中白细胞介素1β(IL-1β)和胰十二指肠同源框因子1(PDX-1)的表达。体外用脂多糖和三磷酸腺苷对小鼠骨髓诱导的原代巨噬细胞进行刺激(处理组)后,与UC-MSCs进行Transwell共培养实验24 h(治疗组),采用酶联免疫吸附法检测各组上清液中IL-1β的分泌水平,采用免疫荧光染色检测NLRP3炎性体和自噬相关蛋白的表达。统计学分析采用单因素方差分析及重复测量方差分析。结果体内实验表明,与糖尿病组相比,UC-MSCs治疗组小鼠胰岛结构部分修复,糖耐量和胰岛素敏感性均改善(均P<0.05),共聚焦显微镜下观察胰岛中PDX-1表达增高,IL-1β的表达水平降低。体外实验表明,相较于处理组,治疗组中巨噬细胞分泌的IL-1β水平降低[(85.9±74.6)比(883.4±446.2)pg/ml,P=0.001],共聚焦显微镜下观察NLRP3炎性体的表达降低,自噬底物蛋白P62表达降低,微管相关蛋白轻链β3(LC3B)、自噬效应蛋白Beclin-1表达增高。结论UC-MSCs可降低T2DM小鼠胰岛炎症水平,保护胰岛功能,可能与UC-MSCs抑制巨噬细胞NLRP3炎性体的活性,增强自噬水平有关。

Objective To investigate the effect and regulation of umbilical cord-derived mesenchymal stem cells(UC-MSCs)on islets function and NOD-like receptor family,pyrin domain containing 3(NLRP3)and autophagy in type 2 diabetic mellitus(T2DM)mice.Methods Experimental study.Twenty,8-week-old,male C57BL/6J mice were selected and divided into a normal control group(n=5)and a high-fat feeding modeling group(n=15).The model of T2DM was established by high-fat feeding combined with intraperitoneal injection of low-dose streptozotocin.After successful modeling,those mice were divided into a diabetes group(n=7)and a UC-MSCs treatment group(n=7).The UC-MSCs treatment group was given UC-MSCs(1×106/0.2 ml phosphate buffer solution)by tail vein infusion once a week for a total of 4 weeks;the diabetes group was injected with the same amount of normal saline,and the normal control group was not treated.One week after the treatment,mice underwent intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests,and then the mice were sacrificed to obtain pancreatic tissue to detect the expressions of interleukin-1β(IL-1β)and pancreatic and duodenal homeobox 1(PDX-1)by immunofluorescence.The bone marrow-derived macrophages were stimulated with lipopolysaccharide and adenosine triphosphate(experimental group)in vitro,then co-cultured with UC-MSCs for 24 h(treatment group).After the culture,enzyme-linked immunosorbent assay was used to detect the secretion level of IL-1βin the supernatant,and immunofluorescence staining was used to detect the expression of NLRP3 inflammasome,and related autophagy proteins.Statistical analysis was performed using unpaired one-way analysis of variance,repeated measure analysis of variance.Results In vivo experiments showed that compared with the diabetes group,the UC-MSCs treatment group partially repaired islet structure,improved glucose tolerance and insulin sensitivity(all P<0.05),and the expression of PDX-1 increased and IL-1βdecreased in islets under confocal microscopy.In vitro experiments showed that compared with the experimental group,the level of IL-1βsecreted by macrophages in the treatment group was decreased[(85.9±74.6)pg/ml vs.(883.4±446.2)pg/ml,P=0.001],the expression of NLRP3 inflammasome and autophagy-related protein P62 was decreased,and the expressions of microtubule-associated protein 1 light chain 3β(LC3)and autophagy effector Beclin-1 were increased under confocal microscopy.Conclusions UC-MSCs can reduce the level of pancreatic inflammation in T2DM mice,preserving pancreatic function.This might be associated with the ability of UC-MSCs to inhibit the activity of NLRP3 inflammasomes in macrophages and enhance autophagy levels.