粪菌移植对肠道菌群失调小鼠SLE发生发展的影响及机制

Effect and mechanism of fecal bacteria transplantation on the occurrence and development of SLE in mice with intestinal dysbacteriosis

目的探讨粪菌移植对肠道菌群失调小鼠系统性红斑狼疮(SLE)发生发展的影响及机制。方法50只雌性6~8周龄ICR小鼠随机分成为正常对照组(NC组)、SLE模型对照组(MC组)、肠道菌群失调SLE模型组(LM组)、肠道菌群失调SLE模型小鼠泼尼松治疗组(LSP组)、肠道菌群失调SLE模型小鼠粪菌移植治疗组(LSF组),每组10只。MC组小鼠采用紫外线急性照射后腹腔注射脂多糖的方法诱导建立SLE模型;LM组、LSP组和LSF组先用盐酸林可霉素灌胃诱导肠道菌群失调模型,在菌群失调基础上进行SLE的诱导;然后,LSP组采用醋酸泼尼松给药治疗15 d;LSF组采用粪菌移植液回灌治疗15 d。实验结束后,进行小鼠肠道活菌选择性培养计数、血清自身抗体的检测、器官指数检测、苏木精-伊红染色观察肾脏病理改变、16S rRNA分析肠道菌群的变化以及实时荧光定量PCR检测脾脏组织中细胞因子白细胞介素-6(IL-6)、干扰素-γ(IFN-γ)、Toll样受体2(TLR2)和核转录因子-κB(NF-κB)p65 mRNA的表达水平。结果与NC组比较,MC组和LM组小鼠肠道大肠杆菌和粪肠球菌数量增加,双歧杆菌和乳酸杆菌数量减少,抗核抗体和抗双链DNA抗体水平升高,器官指数升高,肾脏病理损伤严重,IL-6、IFN-γ、TLR2和NF-κB p65的mRNA表达升高,差异均有统计学意义(P均<0.05);与MC组比较,LM组小鼠大肠杆菌和粪肠球菌增加更明显,双歧杆菌和乳酸杆菌减少更明显,抗核抗体[(8.14±0.08)pg/mL vs.(6.07±0.12)pg/mL]和抗双链DNA抗体[(18.10±0.21)ng/mL vs.(17.33±0.12)ng/mL]升高更明显,肾脏病理损伤更严重,IL-6[(1.01±0.10)vs.(0.67±0.07)]、IFN-γ[(1.00±0.09)vs.(0.70±0.07)]、TLR2[(1.01±0.12)vs.(0.62±0.13)]和NF-κB p65[(1.00±0.08)vs.(0.65±0.18)]的mRNA表达更高,差异均有统计学意义(P均<0.05);与LM组比较,LSP组和LSF组小鼠肠道大肠杆菌和粪肠球菌数量明显减少,双歧杆菌和乳酸杆菌数量明显增加,抗核抗体[(5.12±0.23)、(5.02±0.15)pg/mL]和抗双链DNA抗体[(14.69±0.16)、(14.25±0.16)ng/mL]水平均降低,肾脏组织病理损伤减轻,IL-6[(0.62±0.08)、(0.60±0.10)]、IFN-γ[(0.52±0.11)、(0.50±0.10)]、TLR2[(0.54±0.07)、(0.51±0.07)]和NF-κB p65[(0.53±0.03)、(0.52±0.03)]的mRNA表达呈下降趋势,以上差异均有统计学意义(P均<0.05)。基因测序结果显示,LSP组和LSF组菌群多样性和丰富度均有提高,并能增加小鼠肠道菌群中厚壁菌门、乳杆菌科、毛螺菌科和普氏菌科的比例,减少拟杆菌门和变形菌门的比例。相关性分析结果表明,葡萄球菌、克雷伯菌均与TLR2、NF-κB p65表达成正相关(P<0.05),艾森伯格氏菌与NF-κB p65和IL-6水平成正相关(P<0.05),嗜酸菌与IFN-γ和IL-6水平成负相关(P<0.05),乳酸球菌与IFN-γ水平成负相关(P<0.05),瘤胃球菌与TLR2和NF-κB p65表达成负相关(P<0.05)。结论粪菌移植可能通过TLR2/NF-κB信号通路关键因子表达的抑制和增加肠道有益菌来缓解肠道菌群失调SLE小鼠的发展。

Objective To investigate the effect and mechanism of fecal bacteria transplantation on the development of systemic lupus erythematosus(SLE)in mice with intestinal dysbacteriosis.Methods Fifty female 6-8 week old ICR mice were randomly divided into normal control group(NC group),SLE model control group(MC group),intestinal dysbacteriosis SLE model group(LM group),intestinal dysbacteriosis SLE model mice treated with prednisone(LSP group),and intestinal dysbacteriosis SLE model mice treated with fecal bacteria transplantation(LSF group).The mice in MC group were induced to establish SLE model by intraperitoneal injection of lipopolysaccharide after acute ultraviolet irradiation;LM group,LSP group and LSF group were treated with lincomycin hydrochloride by gavage to induce intestinal flora imbalance model,and SLE was induced on the basis of flora imbalance;LSP group was then treated with prednisone acetate for 15 days;LSF group was treated with fecal bacteria transplantation solution for 15 days.After the experiment,the mouse intestinal viable bacteria were selectively cultured and counted,the serum autoantibody was detected,the organ index was detected,the renal pathological changes were observed by hematoxylin-eosin staining,the intestinal flora was analyzed by 16S rRNA and the expression level of interleukin-6(IL-6),interferon-γ(IFN-γ),Toll-like receptor 2(TLR2),nuclear transcription factor-κB(NF-κB)p65 mRNA in spleen tissues was detected by real-time PCR.Results Compared with NC group,the number of intestinal Escherichia coli and Enterococcus faecalis in MC group and LM group increased,the number of Bifidobacterium and Lactobacillus decreased,the levels of antinuclear antibody and anti-double stranded DNA antibody increased,the organ index increased,the renal pathological injury was serious,and the expressions of IL-6,IFN-γ,TLR2 and NF-κB p65 mRNA increased,with statistical significance(all P<0.05).Compared with MC group,Escherichia coli and Enterococcus faecalis in LM group increased more obviously,while Bifidobacterium and Lactobacillus decreased more obviously,the antinuclear antibody[(8.14±0.08)pg/mL vs.(6.07±0.12)pg/mL]and anti-double stranded DNA antibody[(18.10±0.21)ng/mL vs.(17.33±0.17)ng/mL]increased more obviously,the renal pathological injury was serious more obviously,IL-6[(1.01±0.10)vs.(0.67±0.07)],IFN-γ[(1.00±0.09)vs.(0.70±0.07)],TLR2[(1.01±0.12)vs.(0.62±0.13)],NF-κB p65[(1.00±0.08)vs.(0.65±0.18)]mRNA increased more obviously,with statistical significance(all P<0.05).Compared with LM group,the number of intestinal Escherichia coli and Enterococcus faecalis in LSP group and LSF group decreased significantly,while the number of Bifidobacterium and Lactobacillus increased significantly,the anti-nuclear antibodies[(5.12±0.23),(5.02±0.15)pg/mL]and anti-double stranded DNA antibodies[(14.69±0.16),(14.25±0.16)ng/mL]were decreased;pathological damage of renal tissue was relieved,and IL-6[(0.62±0.08),(0.60±0.10)],IFN-γ[(0.52±0.11),(0.50±0.10)],TLR2[(0.54±0.07),(0.51±0.07)],NF-κB p65[(0.53±0.03),(0.52±0.03)]mRNA were decreased,with statistical significance(all P<0.05).The results of gene sequencing showed that the diversity and richness of bacterial flora in LSP group and LSF group were improved,and the proportion of Firmicutes,Lactobacteriaceae,Chaetomidae and Purpliae in intestinal flora of mice was increased,while the proportion of Bacteroides and Proteobacterium was decreased.The results of correlation analysis showed that Staphylococcus and Klebsiella were positively correlated with the expressions of TLR2 and NF-κB p65(P<0.05),Eisenbergiella was positively correlated with the expressions of NF-κB p65 and IL-6(P<0.05),Acidibacter were negatively correlated with IFN-γand IL-6 levels(P<0.05),Lactococcus bacteria were negatively correlated with IFN-γlevels(P<0.05),and Ruminococcaceae were negatively correlated with the expressions of TLR2 and NF-κB p65(P<0.05).Conclusion Fecal microbiota transplantation might alleviate the development of intestinal flora dysbiosis in SLE mice by inhibiting the expression of key factors in the TLR2/NF-κB signaling pathway and increasing intestinal beneficial bacteria.