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

SJS treatment protects mice against CLP-induced sepsis and modulates immune homeostasis

To explore the therapeutic effect of SJS exerted on sepsis, we subjected SJS- or NS-treated C57BL/6 mice to CLP and sham surgery and investigated its protective effects through survival rate, biochemical analysis and HE staining. Compared with the control group, significantly more C57BL/6 mice died after CLP surgery after 7 days, whereas the SJS-treated mice showed a strong decrease. Additionally, the moderate-dose group showed a better protective role than the low- or high- dose group during the whole sepsis progression (Fig. 1A). Therefore, 6.75 g/kg SJS was used in our further study.

Fig. 1figure 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

Next, we investigated the protective role moderate-dose of SJS exerted on sepsis-induced multiple organ injury. Our biochemical analysis showed that the contents of AST and ALT, rather than SCr and BUN, were significantly elevated at day 1 post CLP, compared to those of the sham group. Besides, all of these biomarkers were elevated significantly at day 7, which can be decreased by SJS treatment (Fig. 1B–E). HE staining results were highly similar to those obtained by biochemical analysis (Fig. 1F). Additionally, ELISA data from Fig. 2G–J showed that SJS could decrease the release of pro-inflammatory cytokines while increase the production of anti-inflammatory cytokines at day 1. Interestingly, when developed into the immunosuppression phase, SJS acted to modulate the cytokines release and boost immune response (Fig. 1G–J). Accumulately, our data demonstrated that SJS treatment protects mice against CLP-induced sepsis and modulates immune homeostasis.

Fig. 2figure 2

Chemical components identification using UPLC/Q-TOF–MS/MS. AB: 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

Chemical components identification

To make clear the potential mechanism, we firstly identified the chemical components of SJS using UPLC/Q-TOF–MS/MS analysis both in a positive and negative mode (Fig. 2A–B). A total of 96 compounds were observed, including 23 acids, 16 flavonoids, 10 anthraquinones, 11 esters, 6 sugars, 6 phenols, 5 aldehydes, 5 alcohols, 3 amino cids and 11 others (Additional file 6: Table S3). Previous studies showed demonstrated that Gallic acid, Rheic acid, Emodin, Purpurin and Emodin-8-O-β-D-glucopyranoside acted to inhibit inflammatory response and adjust immunity [18,19,20,21,22,23], which were the top 20 compounds in SJS.

Network pharmacology analysis

Through the TCMSP databases, a total of 23 active components were collected (Additional file 7: Table S4). Subsequently, disease-associated targets were obtained from the disease-associated database and the targets of the active ingredients were screened out via the databases of TCMSP and Swiss Target Prediction. After matching the 164 SJS-associated proteins with 910 sepsis-related targets, 59 shared targets were identified as potential targets for SJS to treat sepsis (Fig. 3A).

Fig. 3figure 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

To screen out the hub genes and crucial compounds of SJS against sepsis, we performed ingredients-genes-disease and PPI networks through Cytoscape 3.8. Figure 3B revealed the major active components of SJS,such as kaempferol, emodin, glycitein, and morin etc., suggesting that they were the crucial compounds of SJS to treat sepsis. Figure 3C uncovered the hub genes based on the degree value, including IL-1β (degree = 47), RELA (degree = 32) and so on, indicating these genes were the core targets.

To decipher the molecular mechanism of the identified potential targets, 59 overlapping targets were submitted to DAVID database for GO enrichment and KEGG pathway analysis. We totally obtained 146 pathways by KEGG enrichment analysis, and we further visualized the top 20 pathways (Fig. 3D), such as NF-kappa B signaling pathway (Fig. 3E). In addition, a total of 1158 terms in GO function enrichment analysis were acquired, including 51 terms in Molecular Function (MF), 18 in Cellular Component (CC) and 1089 in Biological Process (BP) Visual analysis revealed that MF mainly were involved in transcription factor binding, cytokines receptor binding, heme binding; CC were involved in cytoplasmic vesicle lumen, vesicle lumen, membrane raft; and BC were involved in molecule of bacterial origin, reactive oxygen species metabolic process, lipopolysaccharide (Fig. 3F) Multiple organ injury is leading cause of death induced by sepsis. Next, we imported the 59 overlapped genes into the Bio GPS database for tissue expression analysis. Our data demonstrated that the overlapped targets were mainly involved in immune organs and lung, suggesting that SJS may exert a protective role in sepsis-induced lung injury (Fig. 3G).

Molecular docking

Our bioinformatics analysis has validated that NF-kB and and IL-1β were the core genes, which also were the key components of pyroptosis. Therefore, we next wanted to explore the binding affinity between the major active ingredients and pyroptosis-assocaited proteins. The absolute binding energy value > 4.25 kcal/mol was considered a good binding activity. The heat map results of our molecular docking study implicated that the most ingredients have a strong affinity with the pyroptosis-associated proteins, such as NLRP3, Caspase-1, GSDMD and NF-kB (Fig. 4A–E). These data implicated that SJS perhaps exerted its therapeutical effect through the regulation of pyroptosis.

Fig. 4figure 4

Molecular docking between the active ingredients and pyroptosis-associated proteins. A: Binding affinity between the major active ingredients and pyroptosis-assocaited proteins; BE: 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

SJS bidirectional modulated pyroptosis through the regulation of NF-kB/NLRP3 axis

Our previous study revealed that SJS functioned to maintain immune homeostasis, but its potential molecular mechanism is still largely unknown. As our molecular docking results revealed SJS presented with a good affinity for the pyroptosis-associated proteins, next, we wanted to explore whether SJS could regulate pyroptosis homeostasis. We conducted western blot, immunofluorescence staining and qRT-PCR of the sorted murine lung at different time points (0, 1 and 7d) after sepsis. These three time points represented the major stages in the dynamic alternation of sepsis progression, corresponding to a continuous inflammatory stimuli and immunosuppression. As shown in Fig. 5A, SJS was able to decrease the levels of GSDMD, caspase-1, ASC in the continuous inflammatory phase. Further detection found SJS intervention up-regulated their expression in the immunosuppression stage. Our qRT-PCR results is highly similar with the data of western blot assay (Additional file 1: Fig. S1). Together, these results indicated SJS exerted a bidirectional regulatory effects in pyroptosis.

Fig. 5figure 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). BC: 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

Several studies reported that NF-kB/NLRP3 axis is an effective and functionally relevant regulator of pyroptosis. Therefore, we detected the protein levels of NLRP3 and NF-kB. Figure 5A, B demonstrated that NLRP3、P-P65 and P65 levels was increased in the early stage while decreased in the immunosuppression phase, all of which partially reversed by SJS treatment. Together, these data showed the ability of SJS to tune immune homeostasis back toward the center between the extremes of the excessive inflammatory response or immunosuppression stage through NF-kB/NLRP3/pyroptosis axis.

SJS alleviated LPS-induced inflammation in alveolar macrophages by modulating pyroptosis through NF-kB/NLRP3 axis

To validate the effects SJS exerted on pytoptosis, MH-S cells were used to evaluate its regulatory role. Firstly, we detected the cell viability of MH-S cells treated with different concentration of SJS (0, 500, 1000, 2000, 4000 ng/mL) under normal conditions using CCK8 assays. Our results revealed that 4000 ng/mL SJS could significantly decrease the cells viability (Additional file 2: Fig. S2). Therefore, 0, 500, 1000, and 2000 ng/mL of SJS were utilized in our follow-up study. Then, MH-S cells were treated with 5 µg/mL LPS together with 50 µmoL ATP followed by different concentrations of SJS. Our ELISA results uncovered the release of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6 were elevated significantly by LPS and ATP stimuli, which could be effectively reduced after the addition of SJS (Fig. 6A).

Fig. 6figure 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); CD: P-P65, NLRP3, GSDMD, caspase-1 and ASC protein level (n = 4). *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01

As we have verified that SJS is able to modulate pyroptosis in sepsis animal model, next, we detected the protein level of GSDMD, and caspase-1 of MH-S cells treated with LPS and ATP along with different concentrations of SJS. Our in vitro results demonstrated the expression levels of these proteins were dramatically increased under LPS and ATP stimuli, which can be reversed by different concentrations of SJS (Fig. 6D). The results of qRT-PCR are shown in Additional file 3: Fig. S3. Together, these findings suggested that SJS could modulate pyroptosis of alveolar macrophages at cellular level.

To elucidate the mechanism involved in lung injury in vitro, we cultured MH-S cells and detected the expression level of NF-kB/NLRP3 axis, a crucial regulator of pyroptosis. As shown in Fig. 6B–D, P-P65、P65 and NLRP3 expression levels were significantly increased under LPS and ATP stimuli, which was attenuated in SJS-treated MH-S cells. These data suggested SJS inhibited pyroptosis of MH-S cells through NF-kB/NLRP3 axis.

Fig. 7figure 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

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