Shengjiang San alleviated sepsis-induced lung injury through its bidirectional regulatory effect

Abstract

Background: Sepsis is a life-threatening organ dysfunction caused by dysregulated host responses to infection, for which effective therapeutic strategies are still absent. Shengjiang San (SJS), a well-known Traditional Chinese Medicine formula, has been widely used clinically. However, its role in sepsis-induced lung injury remains unclear.

Methods: To explore its specific mechanism, we firstly established a sepsis animal model using cecal ligation and puncture (CLP) and treated MH-S cells with LPS plus ATP. Then, UPLC/Q-TOF-MS/MS was utilized to identify its active ingredients. Network pharmacology analysis was performed to uncover the potential mechanism. HE staining and biochemical analysis were conducted to validate its therapeutic effect. ELISA was applied to detect the release of pro-inflammatory and anti-inflammatory cytokines. Western blot was utilized to detect the protein levels of GSDMD, NLRP3, P65, ASC and caspase-1.

Results: SJS could dramatically increase the survival rate of sepsis. In addition, it is able to inhibit the pro-inflammatory cytokines release at day 1 post CLP while promote their production at day 7, indicating SJS could attenuate uncontrolled inflammatory response in the early stage and improve immunosuppression in the late phase. Network pharmacology analysis showed that pyroptosis is the crucial action SJS exerted in the protection of sepsis-induced lung injury. Western blot data implicated SJS could attenuate pyroptosis in early sepsis while enhance in the late phase.

Conclusions: SJS acted to alleviate sepsis-induced lung injury through its bidirectional regulatory effect.

Keywords: Lung injury; Network pharmacology; Pyroptosis; Sepsis; Shengjiang San.

Fig. 1

SJS treatment protects mice against CLP-induced sepsis and modulates immune homeostasis. A: SJS treatment improved the survival rate of sepsis mice (n = 20); B: SJS alleviated the multiple organ injury induced by sepsis using HE staining (n = 4); C-F: ALT, AST, Scr and BUN contents in SJS-treated mice using biochemical analysis (n = 4). G-I: Pro-inflammaory cytokines contents in SJS-treated mice using biochemical analysis (n = 4); J: Anti-inflammaory cytokines contents in SJS-treated mice using biochemical analysis (n = 4), *P < 0.05, **P < 0.01, ^#P < 0.05, ^##P < 0.01, ^P < 0.05,^^ P < 0.01

Fig. 2

Chemical components identification using UPLC/Q-TOF–MS/MS. A–B: The total ion chromatograms (TICs) of SJS in a positive or negative ion mode; C:Chemical structures and secondary mass spectra of Gallic acid, Rheic acid, Emodin, Purpurin and Emodin-8-O-β-d-Gluc opyranoside

Fig. 3

The potential molecular mechanism of SJS to treat sepsis based on network pharmacology analysis. A: Veen diagram between the targets of SJS and disease. B: Ingredients—genes—diseases network diagram; C: PPI network diagram; D: KEGG pathway enrichment; E: Visual analysis of NF-kappa B signaling pathway; F: GO enrichment analysis; G: Tissue expression analysis of intersection genes

Fig. 4

Molecular docking between the active ingredients and pyroptosis-associated proteins. A: Binding affinity between the major active ingredients and pyroptosis-assocaited proteins; B–E: 3D docking diagram of Emodin with NLRP3 (B), Hydroxygenkwanin with caspase-1 (C), gallic acid with GSDMD (D) and NF-kB with Wedelolactone (D), the light dashed lines represent hydrogen bonds, the dark dashed lines demarcate π–π interactions

Fig. 5

SJS bidirectional modulated pyroptosis through the regulation of NF-kB/NLRP3 axis. A: The protein levels of P-P65, NLRP3, GSDMD, caspase-1 and ASC using western blot (n = 4). B–C: SJS modulated the translocation of p65 to the nucleus using immunofluorescence staining (n = 3) *P < 0.05, **P < 0.01, ^#P < 0.05, ^##P < 0.01

Fig. 6

SJS alleviated LPS-induced inflammation in alveolar macrophages by modulating pyroptosis through NF-kB/NLRP3 axis. A: IL-6, IL-1β and TNF-α contents in culture medium using ELISA assay (n = 4); B: P65 level (n = 4); C–D: P-P65, NLRP3, GSDMD, caspase-1 and ASC protein level (n = 4). *P < 0.05, **P < 0.01, ^#P < 0.05, ^##P < 0.01

Fig. 7

Shengjiang San alleviated sepsis-induced lung injury through its bidirectional regulatory effect. In the continuous inflammatory phase, SJS could inhibit the NF-kB/NLRP3 axis and suppress the pyroptosis, thus alleviating inflammatory response. As the disease progresses, immunosuppression was seen and SJS could enhance the NF-kB/NLRP3 axis and promote the pyroptosis, thus boosting immune response