中药药理与临床

目的:探讨热毒宁注射液联合美罗培南治疗耐碳青霉烯克雷伯菌(carbapenem-resistant Klebsiella pneumoniae,CRKP)致大鼠肺部感染的作用机制。方法:采用气管滴注法建立CRKP致大鼠肺部感染模型，将其分为正常对照组、模型对照组、热毒宁2.7、5.4、10.7 g/kg组、美罗培南50 mg/kg组、热毒宁5.4 g/kg联合美罗培南50 mg/kg组，造模12 h后腹腔注射给药，连续给药3 d。观察大鼠一般状况，分析血常规、肺指数、肺组织载菌量、肺组织病理损伤情况；基于转录组学挖掘热毒宁与美罗培南联用增效的潜在机制，并检测炎症因子变化情况及肺组织Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子-κB(NF-κB)和补体成分5a(C5a)/补体成分5a受体1(C5aR1)信号通路相关靶点的蛋白和mRNA表达。结果:与正常对照组比较，模型对照组大鼠一般状况差，肺指数、肺组织载菌量、肺组织病理损伤评分显著升高(P<0.01),血清IL-6、IL-8、TNF-α含量升高，肺组织NF-κB、MyD88、C5a含量升高，肺组织TLR4、C5aR1、TIRAP蛋白阳性表达水平升高，肺组织Tlr4、Nfkb p65、Myd88、Tirap、C5a、C5ar1 mRNA表达上调(P<0.01);与模型对照组相比，单用美罗培南、热毒宁组大鼠体质量、肺组织载菌量改善不明显，联用组体质量升高(P<0.01),血中白细胞计数、中性粒细胞计数、单核细胞计数、肺指数、肺组织载菌量显著降低(P<0.01),血清IL-6、IL-8、TNF-α含量降低，肺组织NF-κB p65、MyD88、C5a含量降低，肺组织TLR4、C5aR1、TIRAP蛋白阳性表达水平降低，肺组织Tlr4、Nfkb p65、Myd88、Tirap、C5a、C5ar1 mRNA表达下调(P<0.05或P<0.01);与美罗培南50 mg/kg组比较，联合组WBC、NEU、MONO数降低，肺组织载菌量减少，肺指数降低，肺组织病理评分降低，血清IL-6、IL-1β、TNF-α含量降低，肺组织NF-κB p65、MyD88、C5a含量降低，肺组织TLR4、C5aR1、TIRAP蛋白阳性表达水平降低，肺组织Tlr4、Nfkb p65、Myd88、Tirap、C5a、C5ar1 mRNA表达下调(P<0.05或P<0.01)。HE染色结果表明单用美罗培南、热毒宁可以在一定程度上改善CRKP肺部感染大鼠肺组织的病理损伤，联用后改善效果更明显。转录组学提示热毒宁可能通过影响补体凝血级联系统和趋化因子通路协同美罗培南发挥抑菌抗炎作用。结论:热毒宁可以协同美罗培南发挥抗CRKP感染的作用，其机制可能与热毒宁和美罗培南联用调控补体C5a/C5aR1和TLR4/MyD88/NF-κB信号通路，促进机体对病原菌的清除作用，减轻CRKP致肺部感染大鼠的炎症反应有关。该研究结果为热毒宁联合美罗培南治疗临床CRKP感染提供实验数据支撑。

Objective:To investigate the mechanism of Reduning Injection(热毒宁注射液,RDNI) combined with Meropenem(MEM) in the treatment of pulmonary infection induced by carbapenem-resistant Klebsiella pneumoniae(CRKP) in rats. Methods:A rat model of CRKP-induced pulmonary infection was established by tracheal instillation. The rats were divided into normal control, model control, RDNI(2.7、5.4、10.7 g/kg),MEM(50 mg/kg),and RDNI(5.4 g/kg) + MEM(50 mg/kg) groups. Twelve hours after modeling, drugs were administered intraperitoneally for three consecutive days. The general condition of rats was observed, and blood routine, lung index, bacterial load in lung tissue, and pathological damage of lung tissue were analyzed. Transcriptomics was employed to explore the potential synergistic mechanisms of RDNI and MEM,while changes in inflammatory factors were analyzed. In addition, the protein and mRNA expression of signaling pathway targets related to Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB) and complement component 5a(C5a)/complement component 5a receptor 1(C5aR1) in lung tissues were detected. Results:Compared with the normal control group, the model control group showed poor general condition, with significantly increased lung index, bacterial load in lung tissues, and pathological injury scores(P<0.01). The serum levels of interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α) were elevated, along with increased levels of NF-κB, MyD88, and C5a in lung tissue. The protein expression of TLR4, CSaR1, and Toll/IL-1 receptor domain-containing adaptor protein(TIRAP) in lung tissue was upregulated, and mRNA expression of Tlr4, Nfkb p65, Myd88, Tirap, C5a, and C5ar1 was significantly increased(P<0.01).Compared with the model control group, body weight and bacterial load in lung tissues were not significantly improved in either the MEM group or the RDNI group alone. However, the combination of RDNI and MEM significantly ameliorated weight loss(P<0.01),and significantly reduced leukocyte count, neutrophil count, monocyte count, lung index, and bacterial load in lung tissues(P<0.01). The serum levels of IL-6, IL-8, and TNF-α decreased, the contents of NF-kB p65, MyD88, and C5a in lung tissue decreased, the positive expression levels of TLR4, C5aR1, and TIRAP proteins in lung tissue decreased, and the mRNA expressions of Tlr4, Nfkb p65, Myd88, Tirap, C5a, and C5ar1 in lung tissue were downregulated(P<0.05 or P<0.01).Compared with MEM 50 mg/kg group, the combination group showed decreased WBC, NEU, and MONO counts, reduced bacterial load in lung tissue, lower lung index, and improved pathological scores in lung tissue. The serum levels of IL-6,IL-1β,and TNF-α were significantly reduced, and the lung tissue levels of NF-κB p65,MyD88, and C5a were reduced, and the positive expressions of TLR4, CSaR1, and TIRAP proteins in lung tissue decreased, and the mRNA expressions of Tlr4, Nfkb p65, Myd88, Tirap, C5a, and C5ar1 in lung tissue were downregulated(P<0.05 or P<0.01) in the combination group. H&E staining results showed that MEM or RDNI alone could partially improve pathological lung injury in CRKP-infected rats, while the combined treatment had a more pronounced effect. Transcriptomic analysis suggested that RDNI may synergize with MEM by regulating the complement-coagulation cascade system and chemokine pathways to exert antibacterial and anti-inflammatory effects. Conclusion:This study demonstrates that RDNI can synergize with MEM to exert anti-CRKP effects. The mechanism may be associated with regulation of the C5a/C5aR1 and TLR4/MyD88/NF-κB signaling pathways by the combination, thereby enhancing pathogen clearance and alleviating inflammatory responses in CRKP-infected rats. These findings provide experimental support for the clinical application of RDNI combined with MEM in the treatment of CRKP infection.