凉血解毒化瘀方通过抑制cGAS-STING通路治疗小鼠慢加急性肝衰竭

doi:10.12122/j.issn.1673-4254.2024.12.04

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (12): 2291-2299.doi: 10.12122/j.issn.1673-4254.2024.12.04

• • 上一篇

凉血解毒化瘀方通过抑制cGAS-STING通路治疗小鼠慢加急性肝衰竭

唐巧¹^,²(), 周超¹(), 柏兆方¹, 姚清¹^,², 陈思敏¹, 温心茹¹, 何召云¹, 张瑾¹, 李瑞生¹, 宫嫚¹^,²()

^1.解放军总医院第五医学中心，北京 100039
^2.南方医科大学中医药学院，广东广州 510515

收稿日期:2024-06-24 出版日期:2024-12-20 发布日期:2024-12-26
通讯作者: 宫嫚 E-mail:tangqiao9347@163. com;379317021@qq.com;gongman302@ 163.com
作者简介:唐巧，在读硕士研究生，医师，E-mail: tangqiao9347@163. com；
周超，博士，主治医师，E-mail: 379317021@qq.com。
第一联系人：唐巧、周超共同为第一作者
基金资助:
十三五国家科技重大专项课题(2018ZX10725-506-002);国家自然科学基金青年项目(82305067)

Liangxue Jiedu Huayu Formula improves liver function of mice with acute-on-chronic liver failure by inhibiting excessive activation of the cGAS-STING signaling pathway

Qiao TANG¹^,²(), Chao ZHOU¹(), Zhaofang BAI¹, Qing YAO¹^,², Simin CHEN¹, Xinru WEN¹, Zhaoyun HE¹, Jin ZHANG¹, Ruisheng LI¹, Man GONG¹^,²()

^1.Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
^2.School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

Received:2024-06-24 Online:2024-12-20 Published:2024-12-26
Contact: Man GONG E-mail:tangqiao9347@163. com;379317021@qq.com;gongman302@ 163.com
Supported by:
Natural Science Foundation for the Youth (NSFY) of China(82305067)

摘要/Abstract

摘要：

目的基于cGAS-STING 信号通路，研究中药凉血解毒化瘀方（LXJDHYF）治疗小鼠慢加急性肝衰竭（ACLF）的可能作用机制。方法 30只C57BL/6小鼠随机分为空白组、模型组、凉血解毒化瘀方高剂量组、凉血解毒化瘀方低剂量组及cGAS-STING 信号通路特异性抑制剂H151组，6只/组。除空白组外，其余各组采用CCl₄诱导形成肝硬化，后予以脂多糖联合D-氨基半乳糖腹腔注射急性攻击形成ACLF小鼠模型。取小鼠肝组织进行HE以及TUNEL染色，生化法测定血清ALT、AST及TBil水平以检测凉血解毒化瘀方对小鼠肝功能的影响。同时采用RT-qPCR测定凉血解毒化瘀方对小鼠骨髓巨噬细胞（BMDMs）β干扰素（IFN-β）、干扰素刺激基因15（ISG15）、白细胞介素6（IL-6）及肿瘤坏死因子-α（TNF-α）mRNA表达水平的影响；Western blotting分析凉血解毒化瘀方对BMDMs干扰素调节因子3（IRF3）及干扰素基因刺激因子（STING）蛋白磷酸化水平的影响；结合RT-qPCR检测ACLF小鼠肝组织中上述mRNA表达水平，ELISA法检测ACLF小鼠血清 IL-6及TNF-α水平以观察凉血解毒化瘀方对cGAS-STING 信号通路的作用。结果 HE染色显示，凉血解毒化瘀方组肝细胞坏死及炎症浸润程度与模型组相比较轻，TUNEL染色提示凉血解毒化瘀方组凋亡阳性细胞比例低于模型组（P<0.001）。凉血解毒化瘀方组血清ALT、AST及TBil水平均低于模型组（P<0.001）。RT-qPCR结果表明，凉血解毒化瘀方能抑制BMDMs及小鼠肝组织IFN-β、ISG15、IL-6及TNF-α的mRNA表达（P<0.05）。Western blotting结果显示，随着凉血解毒化瘀方给药剂量增加，BMDMs细胞中IRF3及STING蛋白磷酸化水平降低（P<0.05），凉血解毒化瘀方组血清IL-6及TNF-α水平均低于模型组（P<0.05）。结论凉血解毒化瘀方能够改善ACLF小鼠肝功能，降低小鼠血清促炎细胞因子水平，其机制可能与抑制cGAS-STING 通路过度激活有关。

关键词: 慢加急性肝衰竭, cGAS-STING 信号通路, β干扰素, 干扰素刺激基因15, 凉血解毒化瘀方

Abstract:

Objective To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula (LXJDHYF) for acute-on-chronic liver failure (ACLF) in mice. Methods Thirty C57BL/6 mice were randomly divided into blank control group, model group, low- and high-dose LXJDHYF groups, and H151 (a specific cGAS-STING pathway inhibitor) group (n=6). In all but the control group, the mice were treated with CCl₄ to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF. After the treatments, the mouse livers were collected for HE and TUNEL staining, and serum levels of ALT, AST and TBil were determined. In bone marrow-derived macrophages (BMDMs) and liver tissues of ACLF mice, the expressions of cGAS-STING signaling pathway-related mRNAs including IFN‑β, ISG15, IL-6 and TNF-α were determined with RT-qPCR, and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting. Results Compared with the mice in the model group, the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis. LXJDHYF treatment also significantly lowered serum levels of ALT, AST, TBil, IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs. Conclusion LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.

Key words: acute-on-chronic liver failure, cGAS-STING signaling pathway, interferon?β, interferon stimulating gene 15, Liangxue Jiedu Huayu Formula

唐巧, 周超, 柏兆方, 姚清, 陈思敏, 温心茹, 何召云, 张瑾, 李瑞生, 宫嫚. 凉血解毒化瘀方通过抑制cGAS-STING通路治疗小鼠慢加急性肝衰竭[J]. 南方医科大学学报, 2024, 44(12): 2291-2299.

Qiao TANG, Chao ZHOU, Zhaofang BAI, Qing YAO, Simin CHEN, Xinru WEN, Zhaoyun HE, Jin ZHANG, Ruisheng LI, Man GONG. Liangxue Jiedu Huayu Formula improves liver function of mice with acute-on-chronic liver failure by inhibiting excessive activation of the cGAS-STING signaling pathway[J]. Journal of Southern Medical University, 2024, 44(12): 2291-2299.

图/表 8

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Sequence
β-ACTIN	P1	GGCTGTATTCCCCTCCATCG
β-ACTIN	P2	CCAGTTGGTAACAATGCCATGT
IFN-β	P1	TCCGAGCAGA GATCTTCAGGAA
IFN-β	P2	TGCAACCACCACTCATTCTGAG
TNF-α	P1	GGGCAGTTAGGCATGGGAT
TNF-α	P2	TGAGCCTTT TAGGCTTCCCAG
IL-6	P1	CACTTCA CAAGTCGGAGGCT
IL-6	P2	CTGCAAGTGCATCATCGTTGT
ISG15	P1	GGTGTCCGTGACTAACTCCAT
ISG15	P2	CTGTACCACTAG CATCACTGTG

图1 各组肝脏病理图像和肝组织 HE染色、TUNEL染色的代表图和TUNEL凋亡阳性细胞数比例

Fig.1 HE staining (Original magnification: ×200) and TUNEL staining (×20) for examining pathologies and hepatocyte apoptosis in mouse livers (A) and comparison of TUNEL-positive cell percentages among the groups (B). ***P<0.001 vs model group.

图2 各组小鼠肝功能指标

Fig.2 Serum levels of ALT (A), AST (B) and TBil (C) of the mice in each group. ***P<0.001 vs model group.

图3 ATP法测定不同浓度凉血解毒化瘀方作用于BMDMs细胞中的毒性

Fig.3 ATP assay for evaluating cytotoxicity of different concentrations of LXJDHYF in BMDMs. ***P<0.001 vs 0 mg/mL.

图4 不同浓度凉血解毒化瘀方在BMDMs中对ISD激活的 cGAS-STING 信号通路相关mRNA的表达(A-D)及其对不同激活剂激活STING通路对相关mRNA表达水平的影响(E-H)

Fig.4 Effects of different concentrations of LXJDHYF on expressions of IFN-β (A), ISG 15 (B), TNF-α (C) and IL-6 mRNAs (D) in BMDMs with ISD-induced cGAS-STING signaling pathway activation. E-H: Expressions of IFN-β (E), ISG 15 (F), TNF-α (G) and IL-6 mRNAs (H) in BMDMs pre-treated with LXJDHYF (1 mg/mL) prior to stimulation with different STING agonists. *P<0.05, **P<0.01, ***P<0.001 vs 0 mg/mL.

图5 不同浓度凉血解毒化瘀方在BMDMs中对ISD激活的 cGAS-STING 信号通路相关蛋白磷酸化水平的影响(A-C); 不同种类STING激活剂激活STING通路时凉血解毒化瘀方对相关蛋白磷酸化水平的影响(D-F)

Fig.5 Western blotting for detecting phosphorylation levels of STING and IRF3 proteins in BMDMs with ISD-induced cGAS-STING signaling pathway activation and treated with different concentrations of LXJDHYF (A-C). D-F: Phosphorylation levels of STING and IRF3 proteins in BMDMs pre-treated with LXJDHYF (1 mg/mL) and then stimulated with different STING agonists. *P<0.05, **P<0.01, ***P<0.001 vs 0 mg/mL.

图6 各组小鼠cGAS-STING信号通路相关mRNA相对表达水平

Fig.6 Expressions of IFN-β (A), ISG15 (B), TNF-α (C) and IL-6 mRNAs (D) in the liver tissues of the mice in each group. ***P <0.001 vs model group.

图7 各组小鼠血清炎症细胞因子水平

Fig.7 Serum levels of IL-6 (A) and TNF-α (B) of the mice in each group. *P<0.05, **P<0.01, ***P<0.001 vs model group.

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凉血解毒化瘀方通过抑制cGAS-STING通路治疗小鼠慢加急性肝衰竭

Liangxue Jiedu Huayu Formula improves liver function of mice with acute-on-chronic liver failure by inhibiting excessive activation of the cGAS-STING signaling pathway

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