南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (05): 647-653.doi: 10.12122/j.issn.1673-4254.2020.05.06

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胆碱能抗炎通路对脓毒症大鼠早期炎症与免疫反应具有负向调控作用

李红兵，李 媛，王武石，彭宗爽，吴 芳   

  • 出版日期:2020-05-20 发布日期:2020-05-20
  • 基金资助:

Cholinergic anti-inflammatory pathway plays negative regulatory role in early inflammatory and immune responses in septic rats

  

  • Online:2020-05-20 Published:2020-05-20

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摘要： 目的 通过对特异性胆碱能受体α7nAChR进行干预，探讨胆碱能抗炎通路（CAP）在脓毒症早期对炎症与免疫的调控。方法 采用SPF级SD大鼠64只，用随机数字表法分组，对照组8只：正常饲养，不做任何处理；假手术组8只：大鼠剖腹但不进行盲肠结扎穿刺法（CLP）制备脓毒症模型，并予哌拉西林（50 mg/kg，腹腔注射，3次/d，连续3 d）；脓毒症组共48只，采用CLP脓毒症模型制备，制备成功后随机分为模型组16只：予哌拉西林（50 mg/kg，腹腔注射，3次/d，连续3 d）及生理盐水（1 mL/100 g，腹腔注射，3次/d，连续3 d）；GTS-21组16只，哌拉西林使用同脓毒症组，并以GTS-21（4 mg/kg，腹腔注射，1次/d，连续3 d）进行干预；甲基牛扁亭（MLA）组16只：哌拉西林使用同脓毒症组，并以MLA（4.8 mg/kg，腹腔注射，1次/d，连续3 d）进行干预。记录各组大鼠MSS评分并进行短程HRV分析，3 d后处死大鼠采集血液，采用ELISA测定血清TNF-α、IL-1α、IL-10、IL-6、HMGB1、sCD14等细胞因子、采用细胞流式方法测定淋巴细胞CD4+CD25+ Treg与TH17阳性细胞比例。结果 与对照组比较，脓毒症大鼠MSS 评分明显升高，HRV指标（SDNN, RMSSD, HF, SD1, SD2）明显下降（P<0.05）；GTS-21明显改善了脓毒症MSS评分（P<0.05），MLA较GTS-21明显增加MSS评分（P<0.05），二者对HRV指标无显著影响（P>0.05）；脓毒症大鼠血清TNF-α、IL-1α、IL-10、IL-6、HMGB1、sCD14浓度、淋巴细胞CD4+CD25+ Treg与TH17淋巴细胞明显升高（P<0.05），Treg/TH17较对照组明显降低（P<0.05），GTS-21能明显降低脓毒症血清TNF-α、IL-1α、IL-6、HMGB1、sCD14浓度及TH17淋巴细胞百分比（P<0.05）；MLA增加脓毒症 大鼠血清TNF-α、IL-1α、IL-10、IL-6、HMGB1、sCD14浓度与CD4+CD25+ Treg，TH17淋巴细胞百分比、降低模型组大鼠Treg/TH17，与GTS-21组相比有显著差异（P<0.05）。结论 CAP对脓毒症早期炎症与免疫反应具有明显的负向调控作用，HRV部分指标能较好的反应CAP对炎症与免疫的调控效能。

Abstract: Objective To investigate the role of cholinergic anti-inflammatory pathway (CAP) in neuro-regulation of inflammatory and immune response in the early stage of sepsis. Methods Sixty-four SD rats were randomly divided into control Group (n=8) with normal feeding without any treatment; sham operation group (n=8) with laparotomy but without cecal ligation and puncture (CLP), followed by intraperitoneal injection 50 mg/kg piperacillin 3 times a day for 3 consecutive days; and sepsis group (n=48) with CLP-induced sepsis. The rat models of sepsis were randomized into model groups (n=16) with intraperitoneal injection of piperacillin (50 mg/kg) and normal saline (1 mL/100 g) for 3 times a day for 3 days; GTS-21 group (n=16) with additional intraperitoneal injection of 4 mg/kg GTS-21 (once a day for 3 days); and methyllycaconitine (MLA) group (n=16) with intraperitoneal injection of MLA (4.8 mg/kg) in addition to piperacillin (once a day for 3 days). Murine Sepsis Score (MSS) of the rats and short-range HRV analysis were recorded. Three days later, the rats were sacrificed and serum levels of TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 were measured with ELISA. The percentages of CD4+CD25+ Treg and TH17 lymphocytes and their ratios were measured using flow cytometry. Results Compared with the control rats, the septic rats had significantly increased MSS scores and lowered HRV indexes (SDNN, RMSSD, HF, SD1, and SD2; P<0.05); treatment with GTS-21 significantly decreased while MLA increased MSS scores (P<0.05), but neither of them obviously affected HRV of the rats. Serum levels TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 and the percentages of CD4+CD25+ Treg and TH17-positive lymphocytes were significantly higher and Treg/TH17 ratio was significantly lower in the septic rats compared with those in the control group (P<0.05); treatment with GTS-21 significantly decreased the levels of serum levels of TNF-α, IL-1α, IL-6, HMGB1, and sCD14 and TH17 lymphocyte percentage (P<0.05), whereas MLA treatment significantly increased serum levels of TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 and the percentages of CD4 +CD25 + Treg and TH17- positive lymphocytes and decreased Treg/TH17 ratio in the septic rats (P<0.05). Conclusion CAP plays negative regulatory role in early inflammatory and immune response to sepsis, and some of the HRV indicators can well reflect the regulatory effect of CAP on inflammation and immunity in the septic rats.