羊膜间充质干细胞移植可抑制大鼠创伤性脑损伤模型中的炎性小体活性

Amniotic mesenchymal stem cells transplantation can inhibit inflammasomes activity in rat traumatic brain injury model

目的探讨羊膜间充质干细胞(AMSC)移植对创伤性脑损伤(TBI)大鼠炎性小体活性的影响。方法实验采用SD大鼠,分为4组,TBI模型组、生理盐水组、AMSC移植组、假手术组,通过立体定向的方法,将AMSC移植到TBI大鼠损伤周围区域,移植1周后通过实时定量聚合酶链反应(Real-time PCR)的方法检测TBI大鼠损伤周围区域炎性小体蛋白复合物组分:NLRP1、NLRP3、凋亡相关斑点样蛋白(ASC)、活性半胱氨酰天冬氨酸特异性蛋白酶1(Caspase-1)的表达,以及下游促炎因子:白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)的表达;同时采用免疫组织荧光法检测各组大鼠损伤局部的髓过氧化物酶(MPO)和单核/巨噬细胞抗原1(ED-1)的表达,测定各组损伤局部的炎性反应强度。通过改良的神经功能缺失评分(mNSS),分析AMSC移植对大鼠TBI的治疗作用。对各组炎性小体组分及促炎因子表达水平的组间比较采用采用单向方差分析,移植术后各组间神经功能评分采取重复测量方差分析,进一步的组间比较采用LSD的方法;NS组与AMSC移植组炎性反应强度比较采用两独立样本t检验。结果与假手术组比较,TBI模型组损伤周围区域NLRP1和NLRP3炎性小体复合物各组分及下游促炎因子表达均明显升高:NLRP1(0.98±0.06比12.50±2.00,F=53.547,P<0.05)、NLRP3(0.99±0.08比12.56±1.93,F=61.324,P<0.05)、ASC(1.00±0.07比11.07±1.95,F=52.320,P<0.05)、Caspase-1(1.01±0.09比11.59±1.89F=52.306,P<0.05)、IL-1β(1.00±0.04比6.77±1.28,F=35.365,P<0.05)、IL-18(0.98±0.06比9.64±1.74,F=44.510,P<0.05)、TNF-α(0.98±0.06比7.26±1.57,F=38.755,P<0.05)。而与生理盐水组比较,AMSC移植可降低炎性小体复合物各组分及下游促炎因子表达:NLRP1(12.55±1.80比8.06±1.97,F=53.547,P<0.05)、NLRP3(12.87±2.18比8.76±1.22,F=61.324,P<0.05)、ASC(12.22±2.25比5.88±1.15,F=52.320,P<0.05)、Caspase-1(12.43±2.28比6.73±1.33,F=52.306,P<0.05)、IL-1β(6.98±1.26比3.87±1.12,F=35.365,P<0.05)、IL-18(9.25±1.82比4.06±1.26,F=44.510,P<0.05)、TNF-α(8.35±1.42比4.09±1.12,F=38.755,P<0.05)。与生理盐水组比较,AMSC组可降低损伤周围区域的炎性反应强度。与生理盐水组比较,AMSC移植可促进TBI大鼠神经功能恢复。结论AMSC移植对NLRP1和NLRP3炎性小体的抑制作用是其抗炎作用的机制之一,从而促进TBI大鼠的神经功能恢复。

Objective To investigate the effect of amniotic mesenchymal stem cell transplantation on the activity of inflammasomes in rats with traumatic brain injury.Methods SD rats were divided into 4 groups,traumatic brain injury(TBI)model group,normal saline group,amniotic mesenchymal stem cells(AMSC)transplantation group,and sham operation group.AMSC was transplanted into the area around the injury of TBI rats.One week after transplantation,real-time quantitative polymerase chain reaction(Real-time PCR)were used to detect the expression of inflammatory body protein complex components and the pro-inflammatory factors:NLRP1,NLRP3,ASC,cysteinyl aspartate-specific protease(Caspase)-1,interleukin(IL)-1β,IL-18,and tumor necrosis factor-α(TNF-α);immunotissue fluorescence method was used to detect the myeloperoxidation the expression of MPO and ED-1.The modified neurological deficit score was used to analyze the therapeutic effect of AMSC transplantation on TBI in rats.Results Compared with the sham operation group,the expression of NLRP1 and NLRP3 inflammasome complexes and downstream pro-inflammatory factors in the area around the injury in the TBI model group were significantly increased:NLRP1(0.98±0.06 vs.12.50±2.00,F=53.547,P<0.05),NLRP3(0.99±0.08 vs.12.56±1.93,F=61.324,P<0.05),ASC(1.00±0.07 vs.11.07±1.95,F=52.320,P<0.05),Caspase-1(1.01±0.09 vs.11.59±1.89,F=52.306,P<0.05),IL-1β(1.00±0.04 vs.6.77±1.28,F=35.365,P<0.05),IL-18(0.98±0.06 vs.9.64±1.74,F=44.510,P<0.05),TNF-α(0.98±0.06 vs.7.26±1.57,F=38.755,P<0.05).Compared with the normal saline group,AMSC transplantation can reduce the expression of each component of the inflammatory body complex and the downstream pro-inflammatory factors:NLRP1(12.55±1.80 vs.8.06±1.97,F=53.547,P<0.05),NLRP3(12.87±2.18 vs.8.76±1.22,F=61.324,P<0.05),ASC(12.22±2.25 vs.5.88±1.15,F=52.320,P<0.05),Caspase-1(12.43±2.28 vs.6.73±1.33,F=52.306,P<0.05),IL-1β(6.98±1.26 vs.3.87±1.12,F=35.365,P<0.05),IL-18(9.25±1.82 vs.4.06±1.26,F=44.510,P<0.05),TNF-α(8.35±1.42 vs.4.09±1.12,F=38.755,P<0.05).Compared with the normal saline group,the AMSC group can reduce the intensity of inflammation in the area around the injury.Compared with the saline group and TBI model group,AMSC transplantation can promote the recovery of nerve function in TBI rats.Conclusion We conclude that inflammasome inhibition may be one of the mechanisms for the anti-inflammatory effect of AMSC in the TBI,thereby promoting the recovery of nerve function in TBI rats.