肝组织中NLRP3炎症小体活化与日本血吸虫感染小鼠肝纤维化程度的相关性

NLRP3 inflammasome activation in liver is associated with the extent of hepatic fibrosis in mice infected with Schistosoma japonicum

目的探讨BALB/c小鼠肝组织中NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体活化与日本血吸虫(Schistosoma japonicum)感染所致肝纤维化程度的关系。方法 6~8周龄BALB/c雌性小鼠48只,分为未感染组(n=8)、感染后第5、6、8和12周组(n=10)。感染组每鼠经腹部皮肤贴片法感染日本血吸虫尾蚴(20±3)条。取小鼠肝脏组织,用天狼星红染色法检测肝组织切片中胶原的含量。抽提肝组织RNA,逆转录成cDNA,实时荧光定量PCR(qPCR)检测NLRP3、凋亡相关微粒样蛋白(apoptosis associated speck-like protein containing a CARD domain,ASC)、半胱天冬酶原1(pro-caspase-1)、白细胞介素-1β前体(proIL-1β)和IL-18前体(pro-IL-18)的RNA表达水平。用免疫组化染色结合H-score评分法检验小鼠肝脏半胱天冬酶1(caspase-1)p10和IL-1β的蛋白质表达水平。结果天狼星红染色结果显示,日本血吸虫感染后第5、6、8、12周小鼠肝脏切片的胶原面积百分比在第8周最高(29.66±1.07)%,其后依次为第6周(21.69±1.24)%、第12周(11.98±0.95)%和第5周(1.76±0.34)%。qPCR结果显示,与未感染组相比,感染后第5、6、8、12周小鼠肝脏NLPR3、ASC、pro-caspase-1、pro-IL-1β以及pro-IL-18的相对表达量为:NLRP3分别为8.12±0.66(P<0.01)、24.13±2.81(P<0.01)、14.86±0.35(P<0.01)、6.74±0.67(P<0.01),ASC为0.82±0.14、7.12±0.90、3.08±0.87、4.13±0.93(P<0.01),pro-caspase-1为1.14±0.72、2.53±0.46(P<0.05)、3.16±0.80(P<0.01)、2.19±0.87,pro-IL-1β为9.95±1.04、117.76±10.01(P<0.01)、36.98±11.73(P<0.01)、7.74±2.27,pro-IL-18为2.42±0.36、1.85±0.11、1.74±0.10、1.69±0.15。免疫组化结合H-score评分的结果显示,感染后第5、6、8和12周小鼠肝脏的caspase-1 p10和IL-1β的H-score值分别为2.80±0.12、2.10±0.06、8.57±1.00(P<0.01)、4.55±0.50(P<0.01)和0.13±0.13、0.20±0.00、3.30±0.59(P<0.01)、1.80±0.43。结论日本血吸虫感染小鼠的肝纤维化程度在第8周最明显,继而是第6周、第12周和第5周;NLRP3炎症小体在感染第6周、第8周显著活化;NLRP3炎症小体的活化与日本血吸虫感染所致的肝纤维化程度呈正相关。

Objective To investigate the association of NOD-like receptor protein 3 （NLRP3） infiammasome activation with the extent of liver fibrosis in mice with Schistosoma japonicum infection. Methods Forty-eight SPF-grade female BALB/c mice at six to eight weeks of age were grouped as non-infection （n = g）, S. japonicum infection for 5, 6, 8 and 12 weeks （n = 10 in each）. Each mouse in the infection group was infected with 20 ± 3 cercariae through an abdominal transdermal patch. The liver tissues were collected for Sirius red staining to detect collagen deposition or liver fibrosis. SYBR Green quantitative PCR （qPCR） was performed to detect the relative mRNA expression of NOD-like receptor protein 3 （NLRP3）, apoptosis associated speck-like protein containing a CARD domain （ASC）, pro-easpase-1, pro-IL-1β and pro-IL-18. Immunohistochemical staining （IHC） and H-score were used to evaluate the protein levels of caspase-1 active fragment p10 and IL-1β. Results Sirius red staining revealed that in the S. japonicum infection group the collagen deposition area was the largest at 8 weeks post infection （29.66 ± 1.07）%, followed by 6 weeks （21.69 ± 1.24）%, 12 weeks （11.98± 0.95）%, and 5 weeks （1.76 ± 0.34）%. Compared with the non-infection group, qPCR results showed that at 5, 6, 8 and 12 weeks after infection, the relative mRNA expression of NLPR3 was 8.12 ± 0.66 （P 〈 0.01）, 24.13 ± 2.81 （P 〈 0.01）, 14.86 ± 0.35 （P 〈 0.01）, and 6.74 ± 0.67 （P 〈 0.01）, respectively; the relative mRNA expression of ASC was 0.82 ± 0.14, 7.12 ± 0.90, 3.08± 0.87, and 4.13 ± 0.93 （P 〈 0.01）; the relative mRNA expression of pro-caspase-1 was 1.14 ± 0.72, 2.53 ± 0.46 （P 〈 0.05）, 3.16 ± 0.80 （P 〈 0.01）, and 2.19 ± 0.87; and the relative mRNA expression of pro-IL-1β was 9.95 ± 1.04, 117.76± 10.01 （P〈 0.01）, 36.98 ± 11.73 （P〈 0.01）, and 7.74± 2.27. No significant changes were found for pro-IL- 18 （2.42 ± 0.36, 1.85 ± 0.11, 1.74 ± 0.10, and 1.69 ± 0.15, respectively）. The IHC H-scores of caspase-1 p10 at 5, 6, 8, and 12 weeks after infection were 2.80 ± 0.12, 2.10 ± 0.06, 8.57 ± 1.00 （P 〈 0.01）, and 4.55 ± 0.50 （P〈 0.01）, while those of IL-1β were 0.13 ± 0.13, 0.20 ± 0.00, 3.30 ± 0.59 （P 〈 0.01）, and 1.80 ± 0.43, respectively. Conclusion S. japonicum infection for 8 weeks shows the most serious liver fibrosis. NLRP3 inflammasomes were significantly activated at weeks 6 and 8 post infection. NLRP3 inflammasome activation in the liver is positively correlated with the extent of hepatic fibrosis in infected mice.