TLR2mAb、TLR4mAb对DSS诱导的小鼠溃疡性结肠炎的干预作用及对肠道菌群的影响
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摘要
目的观察TLR2及TLR4单克隆抗体对葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的小鼠急性期溃疡性结肠炎的干预作用及对肠道菌群的影响。
方法50只25g左右的雄性BALB／c小鼠分为A-E五组：正常对照组(A)、模型组(B)、TLR2mAb干预组(C)、TLR4mAb干预组(D)、TLR2mAb和TLR4mAb共同干预组(E),每组10只。A组小鼠饮用蒸馏水,B-E组小鼠饮用水代以5%DSS水溶液以产生急性期溃疡性结肠炎,造模开始的同时,C-E组小鼠每隔48h分别予以TLR2mAb(10μg)、TLR4mAb(10μg)、TLR2mAb(10μg)+TLR4mAb(10μg)腹腔内注射,A、B两组小鼠予生理盐水腹腔内注射作为对照,期间观察小鼠的疾病活动指数(disease activity index, DAI)。造模及干预7d后处死所有小鼠,HE染色观察各组小鼠的结肠炎症情况,并以盲法进行结肠组织病理学评分(histopathological score, HS)。取结肠标本,使用Realtime-PCR法检测各组结肠粘膜中TLR2mRNA、TLR4mRNA的表达。留取小鼠盲肠内粪便对大肠杆菌、双歧杆菌及乳酸杆菌进行培养并比较各菌群菌落计数(Colony forming units, CFU)的变化。
结果(1)与A组相比,B组的DAI及HS显著增高(DAI.9.70±2.406 vs 0.50±0.707,P<0.01；HS：16.50±2.991 vs 6.40±3.273,P<0.01)；C、D组与B组相比DAI及HS差异均无统计学意义(P>0.05)；E组的DAI及HS明显低于B组(DAI：5.70±3.498vs 9.70±2.406,P<0.05；HS：9.50±3.308 vs 16.50±2.991,P<0.01)。(2)小鼠结肠粘膜TLR2mRNA表达：B组TLR2mRNA表达明显高于A组(P<0.01)；C、D、E干预组TLR2mRNA表达明显低于B组(P<0.05),与A组无明显差异(P>0.05)。小鼠结肠粘膜TLR4mRNA表达：B组TLR4mRNA表达明显高于A组(P<0.05)；C、D、E干预组TLR4mRNA表达明显低于B组(P<0.01,P<0.05,P<0.01),与A组无明显差异(P>0.05)。(3)肠道菌群：A组大肠杆菌数量较少,B-E四组大肠杆菌均明显生长(与A组相比P<0.05),C-E组大肠杆菌与B组相比数值有所下降,但无统计学差异(P>0.05)；B组双歧杆菌及乳酸杆菌数量均明显少于A组及C-E组(P<0.05),使用TLR2mAb、TLR4mAb干预后,C-E组双歧杆菌及乳酸杆菌数量上升,与A组相比无明显统计学差异(P>0.05)。
结论TLR2mAb及TLR4mAb联合干预可以减轻葡聚糖硫酸钠诱导的小鼠急性期溃疡性结肠炎的疾病活动指数及病理炎症改变,具有预防作用。使用两种单抗单独或联合干预均可以显著降低结肠粘膜TLR2及TLR4的表达。两种单克隆抗体均能明显提高小鼠肠道内双歧杆菌及乳酸杆菌的菌群数量,但对大肠杆菌数量无明显影响,且两抗体同时干预时对肠道菌群结构的改善未见显著协同作用。
Objective To evaluate the prevention effects of TLR2mAb and TLR4mAb on DSS-induced colitis in mice.
Methods Fifty healthy male BALB/c mice (SPF level) purchased from an experimental animal center (Shanghai Medical College, Fudan University, China), all weighing about 25 g, were randomly assigned into five groups:the normal control group (group A), the UC model group (group B), TLR2mAb intervention group (group C), TLR4mAb intervention group (group D) and TLR2mAb+TLR4mAb intervention group (group E). Group B to group E were given 5.0% DSS solution as drinking water for 7 days to induce acute intestinal inflammation while the normal control group drank distilled water freely. Groups C, D and E respectively received TLR2mAb (10μg), TLR4mAb(10μg) and TLR2mAb (10μg)+TLR4mAb(10μg) injection intraperitoneally once on days 1,3,5 and 7. Group A and B received a normal sodium injection intraperitoneally as control. All mice were killed on the eighth day. The daily disease activity index (DAI) assessment was carried out in the process of modeling. The tissues were fixed with 10% neutral formalin, embedded in paraffin for blinded histological analysis. The mucosal mRNA expressions of TLR2 and TLR4 were analyzed by Realtime PCR. Fecal samples were obtained directly from the cecum and were serially diluted in Ringer diluent for incubation under aerobic or anaerobic conditions. After incubation, colonies were identified according to standard procedures.
Results (1) The DAI score was the lowest in group A. In group B-E, DAI scores were higher than group A (P<0.01). Group B was the most different from group A. Groups C and D had relatively lower scores than group B, but there were no significant differences between each group (C/D vs B, P>0.05). Group E, with TLR2mAb+TLR4mAb intervention, showed a markedly lower score than group B (P<0.05). Compared with group A, group B showed a significant difference in HS(P<0.01). The inflammation was improved in groups C-E. Although HS seemed to be lower in group C and D than group B, there were no significant differences between each group (P>0.05). Notably, the inflammation was significantly improved in group E with a lower HS than group B (P<0.01). (2) In mice with DSS-induced colitis, expressions of both TLR2 and TLR4 in colonic epithelial cells were much higher than normal mice:group B was notably different from group A (TLR2:P<0.01; TLR4:P<0.05). Intervention with TLR2mAb or/and TLR4mAb could down-regulated their expressions to nearly the normal level (P>0.05). (3) Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp. Comparing with group A, group B showed a conspicuous increase of Escherichia coli and decrease of Lactobacillus spp(P<0.05) and Bifidobacterium spp(P<0.05). After being treated with TLR2mAb and TLR4mAb, Lactobacillus spp and Bifidobacterium spp in groups C, D and E increased noticeably to the normal level, and there was no statistical significance between every intervention groups and the normal control group (P>0.05). While counts of E. coli in group C-E were still nearly the same as that in group B (C/D/E vs B, P>0.05).
Conclusion Intervention of TLR2mAb combined with TLR4mAb is effective in suppressing DSS-induced colitis in mice. Both TLR2mAb and TLR4mAb can increase counts of Lactobacillus spp and Bifidobacterium spp in DSS-induced colitis. While these interventions have less influence in reducing the count of E. coli.
方法50只25g左右的雄性BALB／c小鼠分为A-E五组：正常对照组(A)、模型组(B)、TLR2mAb干预组(C)、TLR4mAb干预组(D)、TLR2mAb和TLR4mAb共同干预组(E),每组10只。A组小鼠饮用蒸馏水,B-E组小鼠饮用水代以5%DSS水溶液以产生急性期溃疡性结肠炎,造模开始的同时,C-E组小鼠每隔48h分别予以TLR2mAb(10μg)、TLR4mAb(10μg)、TLR2mAb(10μg)+TLR4mAb(10μg)腹腔内注射,A、B两组小鼠予生理盐水腹腔内注射作为对照,期间观察小鼠的疾病活动指数(disease activity index, DAI)。造模及干预7d后处死所有小鼠,HE染色观察各组小鼠的结肠炎症情况,并以盲法进行结肠组织病理学评分(histopathological score, HS)。取结肠标本,使用Realtime-PCR法检测各组结肠粘膜中TLR2mRNA、TLR4mRNA的表达。留取小鼠盲肠内粪便对大肠杆菌、双歧杆菌及乳酸杆菌进行培养并比较各菌群菌落计数(Colony forming units, CFU)的变化。
结果(1)与A组相比,B组的DAI及HS显著增高(DAI.9.70±2.406 vs 0.50±0.707,P<0.01；HS：16.50±2.991 vs 6.40±3.273,P<0.01)；C、D组与B组相比DAI及HS差异均无统计学意义(P>0.05)；E组的DAI及HS明显低于B组(DAI：5.70±3.498vs 9.70±2.406,P<0.05；HS：9.50±3.308 vs 16.50±2.991,P<0.01)。(2)小鼠结肠粘膜TLR2mRNA表达：B组TLR2mRNA表达明显高于A组(P<0.01)；C、D、E干预组TLR2mRNA表达明显低于B组(P<0.05),与A组无明显差异(P>0.05)。小鼠结肠粘膜TLR4mRNA表达：B组TLR4mRNA表达明显高于A组(P<0.05)；C、D、E干预组TLR4mRNA表达明显低于B组(P<0.01,P<0.05,P<0.01),与A组无明显差异(P>0.05)。(3)肠道菌群：A组大肠杆菌数量较少,B-E四组大肠杆菌均明显生长(与A组相比P<0.05),C-E组大肠杆菌与B组相比数值有所下降,但无统计学差异(P>0.05)；B组双歧杆菌及乳酸杆菌数量均明显少于A组及C-E组(P<0.05),使用TLR2mAb、TLR4mAb干预后,C-E组双歧杆菌及乳酸杆菌数量上升,与A组相比无明显统计学差异(P>0.05)。
结论TLR2mAb及TLR4mAb联合干预可以减轻葡聚糖硫酸钠诱导的小鼠急性期溃疡性结肠炎的疾病活动指数及病理炎症改变,具有预防作用。使用两种单抗单独或联合干预均可以显著降低结肠粘膜TLR2及TLR4的表达。两种单克隆抗体均能明显提高小鼠肠道内双歧杆菌及乳酸杆菌的菌群数量,但对大肠杆菌数量无明显影响,且两抗体同时干预时对肠道菌群结构的改善未见显著协同作用。
Objective To evaluate the prevention effects of TLR2mAb and TLR4mAb on DSS-induced colitis in mice.
Methods Fifty healthy male BALB/c mice (SPF level) purchased from an experimental animal center (Shanghai Medical College, Fudan University, China), all weighing about 25 g, were randomly assigned into five groups:the normal control group (group A), the UC model group (group B), TLR2mAb intervention group (group C), TLR4mAb intervention group (group D) and TLR2mAb+TLR4mAb intervention group (group E). Group B to group E were given 5.0% DSS solution as drinking water for 7 days to induce acute intestinal inflammation while the normal control group drank distilled water freely. Groups C, D and E respectively received TLR2mAb (10μg), TLR4mAb(10μg) and TLR2mAb (10μg)+TLR4mAb(10μg) injection intraperitoneally once on days 1,3,5 and 7. Group A and B received a normal sodium injection intraperitoneally as control. All mice were killed on the eighth day. The daily disease activity index (DAI) assessment was carried out in the process of modeling. The tissues were fixed with 10% neutral formalin, embedded in paraffin for blinded histological analysis. The mucosal mRNA expressions of TLR2 and TLR4 were analyzed by Realtime PCR. Fecal samples were obtained directly from the cecum and were serially diluted in Ringer diluent for incubation under aerobic or anaerobic conditions. After incubation, colonies were identified according to standard procedures.
Results (1) The DAI score was the lowest in group A. In group B-E, DAI scores were higher than group A (P<0.01). Group B was the most different from group A. Groups C and D had relatively lower scores than group B, but there were no significant differences between each group (C/D vs B, P>0.05). Group E, with TLR2mAb+TLR4mAb intervention, showed a markedly lower score than group B (P<0.05). Compared with group A, group B showed a significant difference in HS(P<0.01). The inflammation was improved in groups C-E. Although HS seemed to be lower in group C and D than group B, there were no significant differences between each group (P>0.05). Notably, the inflammation was significantly improved in group E with a lower HS than group B (P<0.01). (2) In mice with DSS-induced colitis, expressions of both TLR2 and TLR4 in colonic epithelial cells were much higher than normal mice:group B was notably different from group A (TLR2:P<0.01; TLR4:P<0.05). Intervention with TLR2mAb or/and TLR4mAb could down-regulated their expressions to nearly the normal level (P>0.05). (3) Group A showed a considerable predominance of Lactobacillus spp and Bifidobacterium spp. Comparing with group A, group B showed a conspicuous increase of Escherichia coli and decrease of Lactobacillus spp(P<0.05) and Bifidobacterium spp(P<0.05). After being treated with TLR2mAb and TLR4mAb, Lactobacillus spp and Bifidobacterium spp in groups C, D and E increased noticeably to the normal level, and there was no statistical significance between every intervention groups and the normal control group (P>0.05). While counts of E. coli in group C-E were still nearly the same as that in group B (C/D/E vs B, P>0.05).
Conclusion Intervention of TLR2mAb combined with TLR4mAb is effective in suppressing DSS-induced colitis in mice. Both TLR2mAb and TLR4mAb can increase counts of Lactobacillus spp and Bifidobacterium spp in DSS-induced colitis. While these interventions have less influence in reducing the count of E. coli.
引文
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