南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (9): 2035-2045.doi: 10.12122/j.issn.1673-4254.2026.09.04
• • 上一篇
徐璐1(
), 潘缘瑾1, 汪雨辰2, 陈振宇1, 蒋伟2, 於江泉1,2(
), 郑瑞强1,2(
)
收稿日期:2026-02-04
出版日期:2026-09-20
发布日期:2026-09-30
通讯作者:
於江泉,郑瑞强
E-mail:sxmuxl@163.com;yujiangquan2021@163.com;zhengruiqiang2021@163.com
作者简介:徐 璐,在读硕士研究生,E-mail: sxmuxl@163.com
基金资助:
Lu XU1(
), Yuanjin PAN1, Yuchen WANG2, Zhenyu CHEN1, Wei JIANG2, Jiangquan YU1,2(
), Ruiqiang ZHENG1,2(
)
Received:2026-02-04
Online:2026-09-20
Published:2026-09-30
Contact:
Jiangquan YU, Ruiqiang ZHENG
E-mail:sxmuxl@163.com;yujiangquan2021@163.com;zhengruiqiang2021@163.com
Supported by:摘要:
目的 探讨血必净对脂多糖(LPS)诱导的脓毒症相关急性呼吸窘迫综合征(ARDS)小鼠模型的保护作用及其分子机制。 方法 体内实验:C57BL/6J小鼠随机分为对照组、LPS模型组、LPS+低剂量血必净注射液组(LPS+LXBJ)和LPS+高剂量血必净注射液组(LPS+HXBJ)(n=15)。检测生存率、肺组织病理(HE染色、肺损伤评分、湿/干质量比)、炎症因子[肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)]水平、磷脂转运蛋白(PLTP)表达(转录组测序、实时荧光定量PCR、Western blotting、酶联免疫吸附试验、免疫荧光)。体外实验:RAW264.7巨噬细胞给予LPS与不同浓度血必净处理,检测细胞活力、形态、炎症因子及PLTP表达,并利用过表达PLTP验证其功能。信号通路:检测JNK/c-JUN和NF-κB通路蛋白表达。 结果 与LPS组相比,血必净治疗(尤其是高剂量组)提高脓毒症ARDS小鼠的4 d生存率(P<0.05),降低肺组织病理学损伤评分(P<0.001)和湿/干质量比(P<0.001)。血必净降低了血清及肺泡灌洗液中TNF-α、IL-6和IL-1β的水平:在血清中,低剂量血必净即可降低上述指标(P<0.05),高剂量血必净作用更为显著;在肺泡灌洗液中,低、高剂量血必净均可降低IL-1β水平(P<0.05),高剂量血必净降低TNF-α、IL-6(P<0.05)水平,而低剂量血必净的作用未达到统计学意义(P>0.05)。转录组测序及实验验证显示,LPS下调肺组织PLTP表达(P<0.001),而血必净干预可逆转该趋势(P<0.05)。体外实验显示,血必净呈剂量依赖性地抑制LPS诱导的RAW264.7细胞炎症因子释放(P<0.05),并上调PLTP表达。机制研究显示,血必净通过上调PLTP,抑制了JNK/c-JUN和NF-κB信号通路的磷酸化激活(P<0.05),而过表达PLTP则增强其效果(P<0.05)。 结论 血必净通过上调PLTP表达,抑制JNK/c-JUN和NF-κB信号通路,减轻脓毒症相关ARDS的炎症反应与组织损伤,提示PLTP可能为其作用的关键靶点。
徐璐, 潘缘瑾, 汪雨辰, 陈振宇, 蒋伟, 於江泉, 郑瑞强. 血必净注射液通过上调磷脂转运蛋白调控巨噬细胞JNK/c-JUN通路改善脓毒症相关急性呼吸窘迫综合征[J]. 南方医科大学学报, 2026, 46(9): 2035-2045.
Lu XU, Yuanjin PAN, Yuchen WANG, Zhenyu CHEN, Wei JIANG, Jiangquan YU, Ruiqiang ZHENG. Xuebijing Injection ameliorates sepsis-associated acute respiratory distress syndrome in mice by upregulating phospholipid transfer protein to regulate macrophage JNK/c-JUN pathway[J]. Journal of Southern Medical University, 2026, 46(9): 2035-2045.
| siRNA | Sequence (5'-3') |
|---|---|
| siPLTP-sense | GGUUUCAAAUCUACUCAAATTT |
| siPLTP-antisense | UUUGAGUAGAUUUGAAACCTT |
| siNC-sense | UUCUCCGAACGUGUCACGUTT |
| siNC-antisense | ACGUGACACGUUCGGAGAATT |
表1 siRNA序列
Tab.1 siRNA sequences
| siRNA | Sequence (5'-3') |
|---|---|
| siPLTP-sense | GGUUUCAAAUCUACUCAAATTT |
| siPLTP-antisense | UUUGAGUAGAUUUGAAACCTT |
| siNC-sense | UUCUCCGAACGUGUCACGUTT |
| siNC-antisense | ACGUGACACGUUCGGAGAATT |
| Primer | Sequence (5'-3') |
|---|---|
| PLTP(mouse)-F | TTCCTCCTCAACCAGCAGATCT |
| PLTP(mouse)-R | CAGGAGGGAGTTGAGCAACAC |
| GAPDH(mouse)-F | TGACGTGCCGCCTGGAGAAA |
| GAPDH(mouse)-R | AGTGTAGCCCAAGATGCCCTTCAG |
表2 qPCR引物序列
Tab.2 qPCR primer sequences
| Primer | Sequence (5'-3') |
|---|---|
| PLTP(mouse)-F | TTCCTCCTCAACCAGCAGATCT |
| PLTP(mouse)-R | CAGGAGGGAGTTGAGCAACAC |
| GAPDH(mouse)-F | TGACGTGCCGCCTGGAGAAA |
| GAPDH(mouse)-R | AGTGTAGCCCAAGATGCCCTTCAG |
图1 血必净对脓毒症相关ARDS小鼠的保护作用
Fig.1 Protective effect of Xuebijing against sepsis-associated ARDS in mice. A: Schematic diagram of the experimental procedure. B: Four-day survival curves of the mice in each group (n=15). C: HE staining of mouse lung tissues. D: Lung injury scores (n=6). E: Lung wet/dry weight (W/D) ratio (n=10). ###P<0.001 vs Control group; ***P<0.001 vs LPS group.
图2 血必净改善脓毒症相关ARDS小鼠的炎症反应
Fig.2 Xuebijing ameliorates inflammatory response in mice with sepsis-associated ARDS. A: Expressions of TNF-α and IL-6 in CD31+ endothelial cells detected by immunofluorescence co-localization (Scale bar=50 μm). B: Expression of TNF‑α and IL-6 in F4/80+ macrophages detected by immunofluorescence co-localization (Scale bar=50 μm). C-H: Results of ELISA for measuring concentrations of TNF-α, IL-6, and IL-1β in serum (C-E) and bronchoalveolar lavage fluid (BALF) (F-H) of the mice (n=6). ###P<0.001 vs Control group; *P<0.05, **P<0.01, ***P<0.001 vs LPS group.
图3 血必净上调脓毒症相关ARDS小鼠的PLTP表达
Fig.3 Xuebijing upregulates PLTP expression in sepsis-associated ARDS mice. A: Transcriptomic sequencing analysis showing downregulation of PLTP by LPS and upregulation by Xuebijing. B: Heatmap of PLTP expression in different groups. C: Venn diagram of differentially expressed genes between LPS and XBJ groups (n=3). D: qRT-PCR analysis of PLTP mRNA levels in mouse lung tissue. E, F: Western blotting for detecting PLTP protein expression in mouse lung tissue. G: ELISA measurement of serum PLTP levels. H: Immunofluorescence co-localization analysis of PLTP (green) with CD31+ endothelial cells (orange) in mouse lung tissue (Scale bar=50 μm). I: Immunofluorescence co-localization analysis of PLTP (red) with F4/80+ macrophages (green) in mouse lung tissue (Scale bar=50 μm). #P<0.05, ##P<0.01, ###P<0.001 vs Control group; *P<0.05, ***P<0.001 vs LPS group.
图4 血必净增强RAW264.7细胞活力并抑制LPS诱导的炎症因子释放
Fig.4 Xuebijing enhances viability and suppresses LPS-induced inflammatory cytokine release in RAW264.7 cells. A: Effect of Xuebijing at different dilutions on RAW264.7 cell viability measured by CCK-8 assay (n=3). B: Morphological changes of RAW264.7 cells induced by LPS and pretreatment with different concentrations of Xuebijing observed under optical microscope (Scale bar=100 μm). C-E: ELISA measurement of TNF-α (C), IL-6 (D), and IL-1β (E) levels in the supernatant of RAW264.7 cells pretreated with different concentrations of Xuebijing and stimulated with LPS (n=3). ###P<0.001 vs Control group; **P<0.01,***P<0.001 vs LPS group.
图5 血必净通过调节PLTP介导的JNK/c-JUN和NF-κB通路激活发挥作用
Fig.5 Xuebijing regulates PLTP-mediated activation of JNK/c-JUN and NF-κB pathways. A: qPCR analysis of the effects of LPS and Xuebijing on PLTP mRNA expression in RAW264.7 cells (n=3). B, C: Western blotting for detecting PLTP protein expression in response to LPS stimulation (n=3). D, E: Western blotting for detecting PLTP protein expression following treatment with different concentrations of Xuebijing (n=4). F: KEGG enrichment analysis of differentially expressed genes between PLTP knockdown (siPLTP)+LPS group and control (siNC)+LPS group. G-K: Western blotting for analyzing the effects of LPS and Xuebijing treatment on the expressions of p-JNK, JNK, p-c-JUN, c-JUN, p-NF-κB p65, and NF-κB p65 proteins in RAW264.7 cells (n=3). #P<0.05, ##P<0.01 vs Control group; *P<0.05, **P<0. 01 vs LPS group.
图6 PLTP过表达增强血必净对炎症通路及因子产生的抑制作用
Fig.6 PLTP overexpression enhances the inhibitory effect of Xuebijing on inflammatory pathways and cytokine production. A-C: Validation of PLTP overexpression efficiency in RAW264.7 cells by qPCR (A) and Western blotting (B, C) (n=3; ##P<0.01,###P<0.001 vs Flag-Vector group). D-H: Western blotting for analyzing the effects of PLTP overexpression and Xuebijing treatment on LPS-induced expressions of p-JNK, JNK, p-c-JUN, c-JUN, p-NF-κB p65, and NF-κB p65 proteins (n=3). I-K: Results of ELISA for measurement of the levels of IL-6 (I), IL-1β (J), and TNF‑α (K) in the supernatant of LPS-stimulated RAW264.7 cells with PLTP overexpression and XBJ treatment (n=3). #P<0.05, ##P<0.01, ###P<0.001 vs Flag-Vector+LPS group; *P<0.05, **P<0.01, ***P<0.001 vs Flag-PLTP+LPS group.
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