消退素D1治疗系统性红斑狼疮模型小鼠的分子机制

Molecular mechanism of resolvin D1 in the treatment of systemic lupus erythematosus model mice

目的探讨消退素D1(RvD1)治疗系统性红斑狼疮(SLE)模型小鼠的分子机制。方法选取8周龄雌性MRL/lpr小鼠(SLE小鼠模型,12只)和ICR小鼠(健康对照小鼠,6只),将MRL/lpr小鼠按照随机数字表法随机分为磷酸缓冲溶液(PBS)组和RvD1组(每组各6只),尾静脉分别注射0.2 ml PBS和5μg/kg RvD1,8周后检测PBS组和RvD1组小鼠血清抗双链DNA(dsDNA)抗体、尿蛋白和滤泡辅助T细胞(Tfh)比例。处死小鼠后分离PBS组小鼠脾脏CD4^(+)T细胞,按随机数字表法将细胞分为CD4^(+)T细胞+二甲基亚砜(DMSO)(A组)、CD4^(+)T细胞+RvD1(B组)、初始CD4^(+)T细胞+DMSO(C组)、初始CD4^(+)T细胞+RvD1(D组),检测各组细胞E26转录因子1(ETS1)mRNA的表达水平和Tfh比例和分化情况。将miR-338-3p抑制剂与PBS组脾脏CD4^(+)T细胞或初始CD4^(+)T细胞共培养,检测细胞ETS1 mRNA的表达水平和Tfh的分化情况。用shRNA-circ_0006779慢病毒或空载慢病毒感染ICR组小鼠的脾脏CD4^(+)T细胞,检测各组细胞miR-338-3p mRNA、ETS1 mRNA的水平和Tfh的分化情况(细胞实验的筛孔数均为5)。结果RvD1组小鼠脾脏CD4^(+)T细胞ETS1 mRNA表达量高于PBS组(1.00±0.33比0.34±0.13,P=0.014);RvD1组小鼠脾重、血清抗dsDNA抗体水平、尿蛋白水平、Tfh细胞比例均低于PBS组(均P<0.05)。RvD1与CD4^(+)T细胞体外共培养结果发现:B组CD4^(+)T细胞ETS1 mRNA表达量高于A组(1.00±0.08比0.25±0.05,P<0.001)、Tfh比例低于A组(16.06%±3.06%比21.76%±3.42%,P=0.020);RvD1与初始CD4^(+)T细胞体外共培养结果发现:D组初始CD4^(+)T ETS1 mRNA表达量高于C组(1.00±0.25比0.21±0.16,P<0.001)、Tfh分化程度低于C组(8.81%±1.01%比12.60%±1.86%,P=0.011)。与不加miR-338-3p抑制剂相比,miR-338-3p抑制剂可以增加PBS组CD4^(+)T细胞ETS1 mRNA表达量(1.00±0.24比0.10±0.01,P<0.001),降低Tfh细胞分化程度(9.56%±1.53%比13.60%±1.32%,P<0.001);与空载慢病毒组相比,shRNA-circ_0006779慢病毒可以增加ICR组CD4^(+)T细胞miR-338-3p水平(1.00±0.22比1.38±0.21,P=0.002),降低ETS1 mRNA水平(0.76±0.13比1.00±0.14,P=0.005),提高Tfh细胞分化程度(4.00%±0.65%比1.68%±0.60%,P<0.001)。结论RvD1通过circ_0006779/miRNA-3384-3p/ETS1轴抑制Tfh细胞分化,治疗小鼠的SLE。

Objective To investigate the molecular mechanism of resolvin D1(RvD1)in the treatment of systemic lupus erythematosus(SLE)model mice.Methods Eight-week-old female MRL/lpr mice(Lupus mice model,a total of 12)and ICR mice(healthy control mice,a total of 6)were selected.MRL/lpr mice were randomly divided into phosphate buffered solution(PBS)group and RvD1 group(6 in each group)according to the random number table method.PBS(0.2 ml)and RvD1(5μg/kg)were injected into the tail vein respectively,serum anti double stranded DNA(dsDNA)antibody levels,urine protein levels and T follicular helper cell(Tfh)ratio were detected in PBS and RvD1 groups of mice after 8 weeks.After the mice were killed,spleen CD4^(+)T cells of PBS group were isolated.CD4^(+)T cells were divided into CD4^(+)T cells+dimethyl sulfoxide(DMSO)(Group A),CD4^(+)T cells+RvD1(Group B),naive CD4^(+)T cells+DMSO(Group C),and naive CD4^(+)T cells+RvD1(Group D)according to random number table method.E26 transcription factor 1(ETS1)mRNA expression and Tfh ratio and differentiation were detected in each group.CD4^(+)T cells or naive CD4^(+)T cells were co-cultured with miR-338-3p inhibitors,to detect ETS1 mRNA expression and Tfh differentiation.Splenic CD4^(+)T cells of ICR mice were infected with shRNA-circ_0006779 lentivirus or empty lentivirus.miR-338-3p level,ETS1 mRNA expression and Tfh differentiation were detected in each group(The number of sieve holes in cell experiments all is 5).Results The expression level of EST1 mRNA on CD4^(+)T cells in the spleen of mice in the RvD1 group was higher than that in the PBS group(1.00±0.33 vs 0.34±0.13,P=0.014);The spleen weight,serum anti-dsDNA antibody level,urine protein level,and Tfh cell ratio of RvD1 group were all lower than those of PBS group(all P<0.05).The results of co-culture of RvD1 and CD4^(+)T cells in vitro showed that the ETS1 mRNA expression in CD4^(+)T cells in Group B was higher than that in Group A(1.00±0.08 vs 0.25±0.05,P<0.001),and the Tfh ratio was lower than that in Group A(16.06%±3.06%vs 21.76%±3.42%,P=0.020);The co-culture results of RvD1 and naive CD4^(+)T cells showed that the naive CD4^(+)T ETS1 mRNA expression in Group D was higher than that in Group C(1.00±0.25 vs 0.21±0.16,P<0.001),and Tfh differentiation was lower than that in Group C(8.81%±1.01%vs 12.60%±1.86%,P=0.011).Compared with the absence of miR-338-3p inhibitor,miR-338-3p inhibitor can increase the expression of ETS1 mRNA in CD4^(+)T cells in PBS group(1.00±0.24 vs 0.10±0.01,P<0.001),and reduce Tfh cell differentiation(9.56%±1.53%vs 13.60%±1.32%,P<0.001);Compared with the empty lentivirus group,shRNA-circ_0006779 lentivirus can increase the miR-338-3p level of CD4^(+)T cells(1.00±0.22 vs 1.38±0.21,P=0.002),reduce ETS1 mRNA level(0.76±0.13 vs 1.00±0.14,P=0.005),and improve the differentiation of Tfh cells in ICR group(4.00%±0.65%比1.68%±0.60%,P<0.001).Conclusion RvD1 can inhibit Tfh cell differentiation through circ_0006779/miRNA-338-3p/ETS1 axis and treats SLE in mice.