脊柱术后细菌感染过程中脂多糖通过TLR-4/NF-кB途径诱导降钙素原的机制

Mechanism of lipopolysaccharide inducing procalcitonin through TLR-4/NF-кB pathway during bacterial infection after spinal surgery

目的探究脊柱术后细菌感染过程中脂多糖通过TOLL样受体4(TLR-4)/核因子κB(NF-κB)途径诱导降钙素原(PCT)的机制。方法60只雌性C57BL/6小鼠(9周龄,18~22g)按照随机数字表法分为对照组、革兰阴性(G-)感染组和革兰阳性(G+)感染组,每组20只。通过体内生物发光测量小鼠细菌感染数量。通过蛋白印迹法分析小鼠组织脂多糖、TLR-4、NF-κB和PCT的蛋白表达。通过酶联免疫试剂盒检测小鼠血清炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)的浓度。通过蛋白印迹法分析Bax和B淋巴细胞瘤-2基因(Bcl-2)的蛋白表达。通过定量逆转录PCR(RT-qPCR)分析核因子KappaB抑制剂的激酶(IKK)α/β、IκB和p65的mRNA表达。结果术后第1天,各组小鼠细菌感染数量比较差异无统计学意义(P>0.05),第2天和3天,G-感染组和G+感染组的细菌数量较对照组升高,差异均有统计学意义(P<0.05);G-感染组与G+感染组细菌感染量比较差异无统计学意义(P>0.05)。G-感染组和G+感染组脂多糖、TLR-4、NF-κB和PCT的蛋白表达升高,差异均有统计学意义(P<0.05),G-感染组与G+感染组比较差异无统计学意义(P>0.05)。G-感染组和G+感染组血清TNF-α、IL-6和IL-1β浓度较对照组升高,差异均有统计学意义(P<0.05),G-感染组与G+感染组比较差异无统计学意义(P>0.05)。G-感染组和G+感染组Bax蛋白表达较对照组升高,Bcl-2蛋白表较对照组降低,差异均有统计学意义(P<0.05)。G-感染组和G+感染组IKKα/β、IκB和p65的mRNA表达较对照组升高,差异均有统计学意义(P<0.05),G-感染组与G+感染组比较差异无统计学意义(P>0.05)。结论脊柱术后G-/G+菌感染过程中,通过激活脂多糖、TLR-4、NF-κB、PCT途径,诱导促炎和细胞凋亡。TLR-4和PCT途径可能在未来成为治疗脊柱术后感染的潜在疗法。

Objective To explore the mechanism of lipopolysaccharide(LPS)inducing procalcitonin(PCT)through the Toll-like receptor 4 polypeptide(TLR-4)/nuclear factor-κB(NF-кB)pathway during bacterial infection after spinal surgery.Methods Sixty female C57BL/6 mice(9 weeks old,18-22 g)were divided into control group,Gram negative(G-)infected group and Gram positive(G+)infected group according to the random number table method,with 20 mice in each group.The number of bacterial infections in mice was measured by in vivo bioluminescence.The protein expression of LPS,TLR-4,NF-κB and PCT in mouse tissues was analyzed by Western blotting.The concentration of tumor necrosis factorα(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in serum of mice was detected by enzyme-linked immunoassay kit.The protein expression of Bax and B lymphocyte tumor-2 gene(Bcl-2)was analyzed by Western blot.The mRNA expression of the kinase of nuclear fac-tor-kappa B inhibitor(IKK)α/β,IκB and p65 was analyzed by RT-qPCR.Results On the first day after surgery,there was no difference in the number of bacterial infections in each group of mice(P>0.05).On the second and third days,the number of bacteria in the G-infected group and G+infected group was higher than that in the control group,the differences were statistically significant(P<0.05).Cell apoptosis was detected by load flow cytometry.The apoptosis rate of LPS group was higher than that of NC group(P<0.05).There was no difference in bacterial infection between G-infection group and G+infection group(P>0.05).The protein expressions of LPS,TLR-4,NF-κB and PCT increased in the G-infected group and G+infected group,the differences were statistically significant(P<0.05),and there was no difference between the G-infected group and the G+infected group(P>0.05).Serum levels of TNF-α,IL-6 and IL-1βin the G-infection group and G+infection group were higher than those in the control group,the differences were statistically significant(P<0.05),and there was no difference between the G-infection group and the G+infection group(P>0.05).The expression of Bax protein in the G-infection group and G+infection group was higher than that in the control group,nd the Bcl-2 protein expression was lower than that in the control group,the differences were statistically significant(P<0.05).The mRNA expressions of IKKα/β,IκB and p65 in the G-infection group and G+infection group were higher than those in the control group,the differences were statistically significant(P<0.05),and there was no difference between the G-infection group and the G+infection group(P>0.05).Conclusion In the process of G-/G+bacterial infection after spinal surgery,it induces pro-inflammatory and apoptosis by activating the pathways of LPS,TLR-4,NF-κB,and PCT.The TLR-4 and PCT pathways may become potential therapies for the treatment of postoperative spinal infections in the future.