Abstract

   Sepsis-induced acute lung injury (SALI) poses a significant threat with
   high incidence and mortality rates. Ginsenoside Rg1 (GRg1), derived
   from Ginseng in traditional Chinese medicine, has been found to reduce
   inflammation and protect lung epithelial cells against tissue damage.
   However, the specific roles and mechanisms by which GRg1 mitigates SALI
   have yet to be fully elucidated. In this context, we employed a
   relevant SALI mouse model, alongside network pharmacology, molecular
   docking, and molecular dynamics simulation to pinpoint GRg1's action
   targets, complemented by in vitro assays to explore the underlying
   mechanisms. Our research shows that GRg1 alleviates CLP-induced SALI,
   decreasing lung tissue damage and levels of serum proinflammatory
   factor IL-6, TNF-α, and IL-1β, also enhancing the survival rate of CLP
   mice. A total of 116 common targets between GRg1 and ALI, with specific
   core targets including AKT1, VEGFA, SRC, IGF1, ESR1, STAT3, and ALB.
   Further in vitro experiments assessed GRg1's intervention effects on
   MLE-12 cells exposed to LPS, with qRT-PCR analysis and molecular
   dynamics simulations confirming AKT1 as the key target with the
   favorable binding activity for GRg1. Western blot results indicated
   that GRg1 increased the Bcl-2/Bax protein expression ratio to reduce
   apoptosis and decreased the high expression of cleaved caspase-3 in
   LPS-induced MLE-12 cells. More results showed significant increases in
   the phosphorylation of PI3K and AKT1. Flow cytometric analysis using PI
   and Annexin-V assays further verified that GRg1 decreased the apoptosis
   rate in LPS-stimulated MLE-12 cells (from 14.85 to 6.54%, p < 0.05).
   The employment of the AKT1 inhibitor LY294002 confirmed these trends,
   indicating that AKT1’s inhibition negates GRg1’s protective effects on
   LPS-stimulated MLE-12 cells. In conclusion, our research highlights
   GRg1's potential as an effective adjunct therapy for SALI, primarily by
   inhibiting apoptosis in alveolar epithelial cells and reducing
   pro-inflammatory cytokine secretion, thus significantly enhancing the
   survival rates of CLP mice. These beneficial effects are mediated
   through targeting AKT1 and activating the PI3K-AKT pathway.

   Keywords: Ginsenoside Rg1, Sepsis, ALI, Apoptosis, Molecular dynamics
   simulation

   Subject terms: Drug discovery, Pharmacology

Introduction

   Sepsis is a life-threatening condition caused by an uncontrolled and
   dysregulated systemic immune response to microbial infections that
   poses a significant global health burden. The mortality rate for sepsis
   among hospitalized patients is alarmingly high, ranging from 30 to
   45%^[35]1. Acute lung injury (ALI), characterized by impaired gas
   exchange due to pneumonia and tissue damage, is one of the most common
   and severe manifestations that contributes to the increased sepsis
   morbidity and mortality rate^[36]2. The development of sepsis-induced
   ALI (SALI) often involves the apoptosis of alveolar epithelial cells
   (AECs) and the disruption of the alveolar-capillary barrier^[37]3. In
   cases of viral sepsis, especially those caused by COVID-19, the virus
   primarily targets the alveolar epithelial cells. The consequent cell
   apoptosis contributes to an uncontrolled inflammatory response, leading
   to the build-up of protein-laden edematous fluid within the lungs. Such
   accumulation aggravates pulmonary edema and can escalate into acute
   respiratory distress syndrome (ARDS)^[38]4. The current therapeutic
   approaches for SALI are primarily restricted to anti-infection measures
   and respiratory support^[39]5. Targeting the apoptosis of alveolar
   epithelial cells may emerge as a promising method to alleviate the
   severity of SALI^[40]6.

   Ginseng (Panax ginseng C. A. Mey.), a cornerstone in traditional
   Chinese medicine (TCM), is leveraged extensively for the management of
   sepsis. Formulations like the Shen-Fu injection, which incorporate
   ginseng as a key constituent, are officially recognized by the National
   Administration of TCM and are routinely employed in the clinical
   setting for the treatment of septic shock^[41]7. Ginsenoside Rg1
   (GRg1), a principal bioactive compound found in ginseng, is noted for
   its capacity to modulate a variety of biological targets while
   conferring minimal adverse effects^[42]8. The extant literature
   corroborates the anti-inflammatory, anti-apoptotic, and
   immunomodulatory properties of GRg1^[43]9. Earlier studies have
   reported that GRg1 is documented as significantly reducing the levels
   of pro-inflammatory cytokines, including TNF-α, IL-6, iNOS, and IL-1β,
   while simultaneously enhancing the synthesis of the anti-inflammatory
   cytokine IL-10^[44]10. In mouse models, the administration of GRg1 has
   been observed to suppress the release of pro-inflammatory cytokines and
   mitigate lung tissue damage^[45]11,[46]12. Although several studies
   have elucidated the protective role of GRg1 in SALI, the detailed
   molecular underpinnings of its action continue to be poorly understood.

   Therefore, this study aims to elucidate the anti-inflammatory effects
   of GRg1 and its mechanisms in preventing lung injury in SALI. Using
   animal models and MLE-12 cells to assess the regulatory role of GRg1 on
   SALI. Additionally, network pharmacology and molecular docking are
   employed to identify potential drug action targets, thereby clarifying
   the underlying mechanisms of GRg1's effects (Fig. [47]1).

Figure 1.

   [48]Figure 1
   [49]Open in a new tab

   Flow chart showing the experimental design.

Results

GRg1 attenuates acute lung injury resulting from cecal ligation and puncture
(CLP) in vivo

GRg1 improve survival rate in CLP-induced ALI mice model

   We implemented the cecal ligation and puncture (CLP) procedure to
   induce polymicrobial sepsis in a murine model. Subsequently, the mice
   were stratified into four distinct cohorts: the sham controls; the CLP
   group; the CLP plus GRg1 treatment at a dose of 30 mg/Kg (Rg1 30 mg/Kg
   group); and the CLP plus GRg1 treatment at a higher dose of 60 mg/Kg
   (Rg1 60 mg/Kg group). Mice that received GRg1 underwent bi-daily
   intraperitoneal administration of their respective doses for a total of
   seven days. The variance in survival rates between the groups receiving
   lower and higher doses of GRg1 did not reach statistical significance
   (p > 0.05). But, mice that received the higher dose of GRg1 exhibited a
   substantial decrease in seven-day mortality rates (p < 0.05), along
   with amelioration of weight loss within the initial 72 h following the
   CLP procedure (p < 0.05), as illustrated in Fig. [50]2A,B.

Figure 2.

   [51]Figure 2
   [52]Open in a new tab

   GRg1 could attenuate the lung injury induced by CLP. (A, B) The 7-day
   survival and 3-day weight changes of each group of mice were monitored.
   ***p  < 0.001 vs. sham, ****p  < 0.0001 vs. sham, ##p < 0.01 vs. CLP,
   ###p  < 0.001 vs. CLP and ####p  0.0001 vs. CLP.

GRg1 relieves pulmonary pathological damage and reduces apoptosis in
CLP-induced injury

   To further investigate the therapeutic effects of GRg1, mice with ALI
   were treated with GRg1, and lung tissue was collected 48 h post-surgery
   and stained with H&E. As expected, compared to the sham-operated group,
   LPS stimulation induced significant histological changes, including
   interstitial edema, thickening of the alveolar walls, and extensive
   infiltration of inflammatory cells in the alveolar spaces. However,
   GRg1 treatment significantly alleviated these CLP-induced pathological
   changes (p < 0.05) (Fig. [53]3A–D).

Figure 3.

   [54]Figure 3
   [55]Open in a new tab

   GRg1 could attenuate the lung injury induced by CLP. (A) H&E staining
   and TUNEL staining were used to assess GRg1's effect on lung histology.
   Scale magnification × 400 (n = 6); (B) Lung injury scores were measured
   in the lungs; (C) Lung wet/dry (W/D) ratio. Dissected lung tissues were
   weighed and oven dried at 80 ℃ for 48 h for calculation of W/D ratio.
   (D) The total protein concentration in supernatant was measured by the
   BCA method. (E–F) Apoptosis in CLP mice effectively alleviated with the
   treatment of GRg1. ***p  < 0.001 vs. sham, ****p  < 0.0001 vs. sham,
   ## p  0.01 vs. CLP, ###p  < 0.001 vs. CLP and #### p  0.0001 vs. CLP.

   To further evaluate the protective effect of GRg1 in experimental ALI,
   we quantitatively analyzed several parameters related to CLP-induced
   pulmonary edema and microvascular permeability. These parameters
   include the lung W/D weight ratio and protein concentration in BALF.
   GRg1 treatment essentially eliminated CLP-induced pulmonary edema and
   microvascular permeability, as evidenced by the significant reduction
   in the lung W/D weight ratio (p < 0.05) (Fig. [56]3C) and protein
   content in BALF (p < 0.05) (Fig. [57]3D). These results indicate that
   GRg1 strongly protects alveolar-vascular barrier integrity, reduces
   pulmonary edema, and mitigates LPS-induced cellular damage.
   Furthermore, TUNEL staining revealed a significant number of apoptotic
   cells in the lung tissue of CLP-induced ALI, whereas GRg1 effectively
   reduced the number of apoptotic cells (p < 0.05) (Fig. [58]3E,F).

GRg1 attenuates pro-inflammatory factors in vivo

   The levels of pro-inflammatory cytokines, including IL-6, TNF-α, and
   IL-1β, in the serum were measured in mice 48 h post-CLP procedure
   (Fig. [59]4A–C). The results highlighted a significant surge in the
   production of the aforementioned inflammatory cytokines in the CLP
   group compared to the sham group. Notably, the GRg1 treatment, at both
   dosage levels, was correlated with a graded decrease in the serum
   concentrations of these pro-inflammatory mediators when contrasted with
   the model group (p < 0.05).

Figure 4.

   [60]Figure 4
   [61]Open in a new tab

   GRg1 could attenuate the lung injury induced by CLP. (A–C) Influence of
   GRg1 on the concentrations of IL-6, TNF-α, and IL-1β in serum of mice
   with CLP-induced acute lung injury. ***p < 0.001 vs. sham,
   ****p < 0.0001 vs. sham, ##p < 0.01 vs. CLP, ###p  < 0.001 vs. CLP and
   ####p  < 0.0001 vs. CLP.

AKT1 as a key target for GRG1 to ameliorate SALI damage by Network Analysis

PPI network analysis and central target screening

   The two-dimensional structures of GRg1 are illustrated in Fig. [62]5A,
   and their pharmacological and molecular properties were obtained from
   the TCMSP database (Supplementary Materials Table [63]S1). GRg1 has a
   molecular weight of 801.14 and a drug-likeness (DL) score of 0.28. A
   drug-likeness weight of ≥ − 0.18 generally indicates favorable
   pharmacokinetic properties^[64]13. Our compilation of 155 targets
   associated with GRg1 was obtained through SwissTargetPrediction (with a
   probability threshold of > 0) and PharmMapper (with a Norm Fit
   of > 0.5). In the exploration of targets linked to ALI, 3702 targets
   were identified using the Genecards, OMIM, and TDD databases.
   Furthermore, we found 116 common targets (Fig. [65]5B and Supplementary
   Table [66]S2) between GRg1 and ALI.

Figure 5.

   [67]Figure 5
   [68]Open in a new tab

   The structure of GRg1 and identification of shared targets of GRg1 in
   combating ALI. (A) Two-dimensional 2D structure of GRg1; (B) Potential
   targets of GRg1 against ALI; (C) The PPI network is visualized by
   Cytoscape.

   Using the STRING tool, a comprehensive analysis was performed. The
   constructed network comprised 113 nodes and 910 edges, exhibiting an
   average node degree of 16.1, and was depicted as a protein–protein
   interaction (PPI) network through the use of Cytoscape software
   (Fig. [69]5C). To further refine the results, the core targets were
   identified using the CytoHubba extension (Supplementary Materials
   Figure [70]S1). By overlapping the targets identified in the four
   screening results, several central targets were detected, including
   AKT1, EGFR, VEGFA, STAT3, IGF1, ESR1, ALB, SRC, and HSP90AA1, among
   others. The prominent target genes identified in this research have
   crucial roles in the development and potential management of ALI,
   highlighting their significance in mediating the therapeutic effects of
   GRg1.

GO and KEGG enrichment analysis

   A total of 116 target genes were analyzed using Metascape, leading to
   the identification of the top 20 enriched terms through Gene Ontology
   (GO) analysis (Fig. [71]6A). Simultaneously, we conducted a KEGG
   pathway enrichment analysis, unveiling the top 20 potential signaling
   cascades highlighted in Fig. [72]6B. Foremost among the up-regulated
   biological processes (BPs) identified were those pertinent to the
   negative regulation of the immune system process, and the attenuated
   regulation of the apoptotic signaling pathway, among others. Regarding
   cellular components (CCs), the notable up-regulation involved entities
   such as vesicle lumen, extracellular matrix, and cytoplasmic vesicle
   lumen, as well as various membrane segments. On the molecular function
   (MF) front, an upsurge in activities was noted in protein kinase
   binding, kinase activity specific to proteins, and protein
   serine/threonine kinase activity. Within the realm of the KEGG
   pathways, significant enrichment was observed in pathways such as
   PI3K-Akt, MAPK, and Ras, which represent potential key pathways for
   treating SALI using GRg1.

Figure 6.

   Figure 6
   [73]Open in a new tab

   Enrichment analysis of GRg1 against ALI using GO and KEGG. (A)The top
   10 terms for biological processes (BP), cellular components (CC), and
   molecular functions (MF) in the GO enrichment analysis are represented
   by green, orange, and purple bars, respectively; (B) The bubble chart
   displays the top 20 pathways identified through KEGG enrichment
   analysis; (C) Component-Disease-Target-Pathway Interaction Network. Red
   represents GRg1, green represents ALI, orange represents the core
   targets, and blue represents the pathways.

   Finally, we constructed a component-Disease-Target-Pathway Interaction
   Network to visualize that GRg1 potentially influences ALI by modulating
   multiple targets and signaling pathways (Fig. [74]6C), including AKT1,
   EGFR, VEGFA, STAT3, IGF1, ESR1, ALB, SRC, the PI3K-Akt signaling
   pathway, the MAPK signaling pathway, and the Ras signaling pathway.

Molecular docking analysis between GRg1 and the key shared targets

   Molecular docking was performed to verify the binding activity of GRg1
   with core targets, including AKT1, EGFR, VEGFA, SRC, IGF1, ESR1, STAT3,
   and ALB, using AutoDockTools 1.5.6 (Fig. [75]7C; Table [76]S3). To
   delve into the complex binding interactions, visualizations were
   created using both PyMOL and Discovery Studio 2019, as depicted in
   Fig. [77]7A,[78]B. Our analysis indicated that the preponderance of the
   targeted proteins, encompassing AKT1, VEGFA, SRC, IGF1, ESR1, STAT3,
   and ALB, exhibited binding energies surpassing the benchmark of
   stability set at − 1.2 kcal/mol, except for EGFR. The outcome showed a
   stable conformational structure and strong binding affinity between
   GRg1 and these proteins. These findings further supported the potential
   of GRg1 as an active constituent capable of effectively interacting
   with the identified key targets.

Figure 7.

   [79]Figure 7

   [80]Figure 7

   Figure 7
   [81]Open in a new tab

   The complex conformations of GRg1 with eight proteins: (A) ALB, AKT1,
   EGFR, and VEGFA. (B) SRC, ESR1, IGF1, and STAT3; (C) Molecular docking
   binding energies. Each protein complex display consists of three parts:
   1. The three-dimensional conformation of GRg1 with each protein
   complex, with GRg1 depicted in green and the protein in blue; 2.
   Residues around GR1 involved in conventional hydrogen bonds are shown.
   Different atoms are labeled with distinct colors. The carbon (C) of
   GRg1 and the protein are colored purple and pink, respectively.
   Hydrogen (H), sulfur (S), oxygen (O), and nitrogen (N) atoms are
   represented in white, yellow, red, and blue, respectively.

Screening of potential targets for Grg1 effect on MLE-12 cells

GRg1-related targets in MLE-12 cells after LPS stimulation identified qRT-PCR

   To evaluate the therapeutic efficacy of GRg1 on the aforementioned
   targets in SALI, we established a cell-based assay using the murine
   lung epithelial cell line, MLE-12, which was stimulated with
   lipopolysaccharides (LPS). After LPS induction, the cells were treated
   with GRg1. We assessed the mRNA expression levels of AKT1, VEGFA, SRC,
   IGF1, ESR1, STAT3, and ALB through quantitative real-time PCR (qRT-PCR)
   analysis.

   Our study revealed that LPS stimulation resulted in an up-regulation of
   STAT3 expression (as depicted in Fig. [82]8E), alongside a
   down-regulation of VEGFA (Fig. [83]8A) and AKT1 (Fig. [84]8C)
   expression in MLE-12 cells, with these changes reaching statistical
   significance (p < 0.05). In contrast, the GRg1 treatment mitigated the
   effects induced by LPS, as it significantly decreased STAT3 levels
   while enhancing the expression of AKT1 and VEGFA (p < 0.05). There were
   no notable differences in the expression levels of ALB, IGF1, SRC, and
   ESR1 among the four study groups (Fig. [85]8G–J). These findings
   underscore the potential of STAT3, AKT1, and VEGFA as prospective
   therapeutic targets for GRg1 in treating SALI.

Figure 8.

   [86]Figure 8
   [87]Open in a new tab

   Analysis of GRg1 Key Target Expression in MLE-12 Cells via RT-qPCR and
   RMSD correlation with Critical Targets. (A) AKT1; (B) (6CCY)-GRg1; (C)
   VEGFA; (D) VEGFA (4KZN)-GRg1; (E) STAT3. (F) STAT3 (6NJS)-GRg1; (G)
   ALB; (H) IGF1; (I) SRC; (J) ESR1. ***p < 0.001 vs. Control,
   ****p < 0.0001 vs. Control, #p < 0.05 vs. Model, ##p < 0.01 vs. Model,
   ###p < 0.001 vs. Model and ####p < 0.0001 vs. Model. Control
   (untreated, n = 3); Model (LPS-treated, n = 3); GRg1L (GRg1L treated
   cell at 2.5 ug/ml, n = 3); GRg1L (GRg1L treated cell at 5 ug/ml,
   n = 3).

A potential interaction between GRg1 and AKT1 by Molecular dynamics
simulation

   The stability of the protein–ligand complexes was investigated using
   molecular dynamics simulations. The study involved 100 ns molecular
   dynamics simulations for the AKT1 (6CCY)-Rg1, STAT3 (6NJS)-Rg1, and
   VEGFA (4KZN)-Rg1 complexes, aiming to assess the dynamics,
   trajectories, structural characteristics, binding affinities, and
   conformational changes in the molecules based on the docking results
   and their validation. The root-mean-square deviation (RMSD) serves as a
   dependable measure for evaluating the stability of protein–ligand
   complexes, quantifying the extent to which atomic positions vary from
   their original configurations. A lower RMSD value indicates enhanced
   conformational stability^[88]14. The RMSD alterations in the complexes
   were analyzed. The RMSD of the AKT1 protein remained consistently
   within the range of 1.8–2.4 Å (Fig. [89]8B), and the RMSD of the STAT3
   protein remained relatively stable at approximately 28 Å (Fig. [90]8F);
   meanwhile, the RMSD fluctuation range of the VEGFA protein was between
   7.5 and 10.5 Å (Fig. [91]8D). AKT1 and GRg1 achieved a more stable
   binding conformation through molecular dynamics simulation, leading to
   increased affinity and stability in this specific conformation. Thus,
   AKT1 is a fundamental element of the PI3K-Akt signaling pathway, which
   is thought to be intrinsically linked to the apoptosis of alveolar
   epithelial cells.

GRg1 reduces alveolar epithelial cell apoptosis by engaging the PI3K-Akt
signaling pathway in vitro

GRg1 mitigate apoptosis in MLE-12 cells prompted by LPS exposure

   Cell apoptosis is implicated in the pathogenesis of SALI^[92]15. Within
   this framework, results in vivo suggested that GRg1 could mitigate
   pulmonary pathology and apoptosis in models of CLP-induced injury.
   Therefore, we investigated the anti-apoptotic effects of GRg1 on MLE-12
   cells. Initially, a cytotoxicity assessment was performed using
   different GRg1 concentrations (0, 0.1, 0.25, 0.5, 1, 2.5, and 5 µg/ml)
   (Fig. [93]9A). The findings indicated negligible cytotoxicity, with
   concentrations of 2.5 and 5 µg/ml GRg1 showing a notable promotion of
   cell proliferation (p < 0.05). Following this, an LPS-induced model of
   cellular stress was generated utilizing a concentration of 25 µg/ml of
   LPS. Treatment with GRg1, at both 5 µg/ml and 2.5 µg/ml, resulted in a
   significant reduction in LPS-mediated cellular activity inhibition
   (p < 0.05, Fig. [94]9B). Consequently, the investigation proceeded with
   2.5 µg/ml of GRg1 for the low-dose group and 5 µg/ml of GRg1 for the
   high-dose group.

Figure 9.

   [95]Figure 9
   [96]Open in a new tab

   GRg1 Attenuation of LPS-Induced Apoptosis in MLE-12 Cells. (A) CCK-8
   analysis was used to detect the viability of MLE-12 cells exposed to
   different concentrations of GRg1 for 24 h. ##p < 0.01, ###p < 0.001 vs
   0 mg/ml (n = 6); (B) The viability of MLE-12 cells stimulated with GRg1
   and LPS for 24 h was determined via CCK-8 analysis. ***p < 0.001 vs
   control, ##p < 0.01 vs LPS group (n = 6); (C) Bcl-xl, caspase-3,
   cleaved-caspase-3, and Bax expression in MLE-12 cell groups analyzed by
   Western blot analysis (n = 3), the samples derive from the same
   experiment and that blots were processed in parallel; (D) The
   expression of Bcl-xl; (E) The expression of Bax; (F) Bcl-xl/Bax ratio;
   (G) cleaved-caspase-3/ caspase-3 ratio; (H, I) Flow Cytometric
   Assessment of Early and Late Apoptosis in LPS-Treated MLE-12 Cells.
   *p < 0.05 vs. Control, ***p < 0.001 vs. Control, ****p < 0.0001 vs.
   Control, #p < 0.05 vs. Model, ##p < 0.01 vs. Model and ###p < 0.001 vs.
   Model.

   Further experiments involved subjecting MLE-12 cells to 25 µg/ml LPS
   for 24 h. Subsequent analysis centered on the ratio between the
   anti-apoptotic protein, Bcl-xl, and the pro-apoptotic protein, Bax,
   recognized as an apoptosis marker^[97]16. This was evaluated via
   Western blotting (WB). In parallel, caspase-3 activation, an apoptotic
   hallmark, was appraised. GRg1 treatment was found to significantly
   reverse both the LPS-induced decline in the Bcl-xl/Bax ratio and the
   elevation in cleaved caspase-3 levels (p < 0.05, Fig. [98]9C–G).

   In the final phase, apoptosis was quantified through flow cytometry by
   analyzing apoptosis rates in the Q2 regions via the PI and Annexin-V
   assay (F[99]ig. [100]9H–I). The data reflected a substantive increase
   in apoptosis levels due to LPS (p < 0.05), which GRg1 effectively
   ameliorated (p < 0.05). Apoptotic cell proportions were 4.22%, 14.85%,
   6.54%, and 5.82% for the control, model, LPS + Rg1 low-dose
   (2.5 µg/mL), and LPS + Rg1 high-dose (5 µg/mL) groups, respectively.

GRg1 effects in LPS-stimulated MLE-12 cells through the PI3K-Akt signaling
pathway

   The KEGG enrichment analysis results indicated that the PI3K-Akt
   signaling pathway was the most significant, apart from the
   cancer-related pathways. Molecular docking and dynamic simulations
   further corroborated a robust affinity and stable interaction between
   GRg1 and AKT1. Consequently, our investigation honed in on the Akt
   signaling cascade to discern the anti-apoptotic influence of GRg1 in
   the context of LPS-challenged MLE-12 cells.

   Commencing the analysis, we quantified the protein levels of
   phosphorylated PI3K (p-PI3K) and PI3K, and phosphorylated AKT1 (p-AKT1)
   and AKT1 post-treatment with either LPS or LPS alongside GRg1. Western
   blot analysis demonstrated that LPS exposure notably diminished the
   phosphorylation of PI3K and AKT1 compared to the control group, which
   was significantly restored by the addition of GRg1 (p < 0.05,
   Fig. [101]10A–C). These results endorse the notion that GRg1 may
   activate the PI3K-Akt signaling pathway within MLE-12 cells.

Figure 10.

   [102]Figure 10
   [103]Open in a new tab

   GRg1 exerts an anti-apoptotic function in MLE-12 cells via the Akt
   signaling pathway. (A–C) The level of phosphorylated Akt and
   phosphorylated PI3K proteins were assessed through immunoblotting and
   quantitatively analyzed in MLE-12 cells treated with LPS (25ug/ml) and
   GRg1 (2.5ug/ml and 5ug/ml); (D, E) Western blotting was employed to
   assess the expression of p-Akt in MLE-12 cells upon stimulation with
   LY294002 (20uM). &&p < 0.01 vs. control; (F–J) Western blotting was
   utilized to detect the protein expression of LPS (25ug/ml), GRg1 H
   (5ug/ml), and LY294002 (20uM) in stimulated MLE-12 cells, followed by
   quantitative analysis; (K, L) The impact of LPS (25ug/ml), GRg1 H
   (5ug/ml), and LY294002 (20uM) on the apoptosis rate of MLE-12 cells was
   evaluated using flow cytometry. Statistical analysis was performed, and
   the following significance levels were observed. *p < 0.05 vs. Control,
   ***p < 0.001 vs. Control, #p < 0.05 vs. Model, ##p < 0.01 vs. Model,
   ###p < 0.001 vs. Model and ####p < 0.0001 vs. Model; & p< 0.05 vs. GRg1
   H; &&&p < 0.001 vs. GRg1 H. The samples were derived from the same
   experiment and the blots were processed in parallel.

   Subsequent explorations focused on whether the anti-apoptotic effect of
   GRg1 on LPS-induced ALI was mediated via the PI3K-Akt pathway. To this
   end, MLE-12 cells underwent treatment with the specific Akt inhibitor
   LY294002 before Western blotting, which confirmed the successful
   suppression of AKT1 phosphorylation by LY294002 (p < 0.05,
   Fig. [104]10D–E). Thereafter, MLE-12 was subjected to treatment
   combinations of LPS, GRg1, and LY294002. Western blotting indicated
   that GRg1 countered the LPS-induced reduction in Bcl-xl levels and
   Bcl-xl/Bax ratio and the upregulation of Bax, which was then annulled
   by LY294002 concurrent with the inactivation of AKT1 (Fig. [105]10F–J).
   Consistent with these findings, flow cytometric analysis corroborated
   the exacerbation of apoptosis rates in MLE-12 cells stimulated with LPS
   and GRg1 when LY294002 was present (p < 0.05, Fig. [106]10K–L).

   Collectively, these observations illuminate the biological processes
   and molecular interactions potentially modulated by GRg1, offering
   substantive insights into its prospective therapeutic modality in the
   context of SALI management.

The role of GRg1 in CLP induced ALI through the PI3K/AKT signaling pathway

   To verify whether the protective effect of GRg1 on ALI is reversed when
   the PI3K/AKT signaling pathway is inhibited, we first examined the
   histopathological and immunohistochemical staining of the inhibitor
   LY294002. As expected, compared to the Con group, CLP significantly
   increased the number of inflammatory cells in the alveolar cavity,
   while GRg1 treatment significantly alleviated these CLP-induced
   pathological changes. However, after administering the inhibitor
   LY294002, there was a substantial infiltration of inflammatory cells in
   the lung tissue, with noticeable edema in the lung interstitium
   (Fig. [107]11A–D,[108]I). Immunohistochemical staining showed that GRg1
   promotes the expression of p-AKT, while LY294002 partially inhibits the
   expression of p-AKT (Fig. [109]11E–H,J). ELISA results for inflammatory
   factors IL-6, IL-1β, and TNF-α in serum indicated that LY294002 can
   reverse the inhibitory effect of GRg1 on these inflammatory
   factors(Fig. [110]11K–M). Further, WB analysis of lung tissue showed
   that, compared to the Con group, CLP significantly reduced the
   p-AKT/AKT ratio and the expression of the anti-apoptotic protein
   Bcl-xl, while increasing the ratio of apoptotic proteins Bax and
   CC3/C3. GRg1 treatment altered these results, but the combined use of
   the inhibitor LY294002 reversed the therapeutic effects of
   GRg1(Fig. [111]11N–R). These findings indicate that the LY294002
   inhibitor can interfere with the anti-ALI effect of GRg1 pretreatment,
   suggesting that this inhibitor may block the PI3K/ AKT signaling
   pathway, thus interfering with its protective effect on lung tissue.
   Therefore, PI3K/ AKT is likely the primary signaling pathway regulated
   by GRg1.

Figure 11.

   [112]Figure 11
   [113]Open in a new tab

   GRg1 attenuates CLP-induced ALI by PI3K/Akt pathway in vivo. (A–D) Lung
   tissue sections were stained with H&E for histopathologic analysis
   (magnification 400 × , n = 6); (E–H) Immunohistochemistry staining of
   p-Akt protein (magnification 400 × , n = 8). (I) lung injury score; (J)
   Representative images of p-Akt protein level in lung determined by
   immunohistochemistry; (K–M) GRg1 significantly reduces the production
   of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in serum through
   the PI3K/AKT pathway; (N) Western blotting was used to assess the
   levels of p-AKT,AKT,Bax,Bcl-xl,caspase-3 and cleaved caspase-3; (O–R)
   Densitometric quantification of the level of p-AKT/AKT,Bax,Bcl-xl and
   CC3/C3. Statistical analysis was performed, and the following
   significance levels were observed. *p < 0.05 vs. Control, ***p < 0.001
   vs. Control, #p < 0.05 vs. Model, ##p < 0.01 vs. Model and ###p < 0.001
   vs. Model; &p < 0.05 vs. GRg1; &&p < 0.01 vs. GRg1 and &&&p < 0.001 vs.
   GRg1. The samples were derived from the same experiment and the blots
   were processed in parallel.

Discussion

   The persistent inflammation characteristics of sepsis harbor the
   potential to escalate into acute lung injury (ALI), precipitating
   irreversible pulmonary damage. The association of sepsis-induced ALI
   with increased mortality rates in comparison to ALI from other
   etiologies is of particular note. ALI, and its more severe form, acute
   respiratory distress syndrome (ARDS), presents significant hindrances
   to survival, incited by various innate and extraneous factors. The
   current understanding is that the pathology encompasses widespread
   damage to the pulmonary interstitium and alveolar epithelial cells,
   with mortality reaching as high as 40% in affected individuals^[114]17.
   In this light, obstructing the progression of ALI is vital to
   ameliorating the severity of sepsis. Within the detrimental milieu of
   septic ALI, the process of apoptosis plays a noteworthy role^[115]18.
   Emerging studies have suggested that curbing apoptosis in pulmonary
   epithelial cells may offer a viable strategy to alleviate lung
   inflammation and injury^[116]19,[117]20. Ginsenoside Rg1 has surfaced
   in recent pharmacological discourse as a compound proficient in
   blunting inflammatory and apoptotic cascades^[118]12,[119]21,[120]22.
   However, its explicit function and the mechanisms by which it
   influences sepsis-associated ALI require further elucidation. Thus, our
   study sought to clarify the impacts and possible mechanisms of GRg1 on
   this condition.

   Our in vivo experiments, GRg1 was administered to septic mice,
   resulting in significantly reduced mortality and weight loss.
   Histopathological H&E staining, lung tissue W/D ratio, protein content
   in bronchoalveolar lavage fluid, and TUNEL staining provided evidence
   that GRg1 significantly reduced tissue damage and apoptosis. ELISA
   assays confirmed GRg1's ability to inhibit pro-inflammatory mediators,
   including IL-6, TNF-α, and IL-1β. These observations support the
   hypothesis that GRg1’s protective effect against SALI may operate
   through mechanisms that prevent apoptosis, thereby regulating the
   secretion of inflammatory factors.

   Our comprehensive approach, combining network pharmacology and
   molecular docking, delved into the complex mechanisms of GRg1 in ALI.
   Analysis of relevant databases revealed potential key targets and
   pathways associated with ALI, highlighting the regulation of immune
   responses, apoptosis, and inflammation. Proteins involved in these
   processes were further scrutinized, and PPI network analysis identified
   eight central targets. Subsequent molecular docking, in vitro
   validation, and dynamic simulation confirmed that AKT1 is the most
   stable binding target of GRg1. KEGG pathway enrichment emphasized the
   critical role of the PI3K-Akt pathway in cell survival.

   Our in vitro results demonstrated that GRg1 could reverse LPS-induced
   apoptosis in mouse lung epithelial cells (MLE-12), normalize the
   Bcl2/Bax ratio, and increase levels of cleaved caspase-3 post-LPS
   administration. Western blot analysis confirmed that GRg1 mitigates
   LPS-induced apoptosis in MLE-12 cells by modulating the PI3K-Akt
   pathway. To verify the crucial role of AKT1, we used the AKT-specific
   inhibitor LY294002, which blocked the AKT pathway and negated the
   anti-apoptotic effects of GRg1 on MLE-12 cells. This supports the
   notion that GRg1 alleviates lung injury via the PI3K-Akt pathway by
   inhibiting apoptosis.

   Further in vivo investigation into the effects of GRg1 on the PI3K/AKT
   pathway revealed that the anti-inflammatory and anti-apoptotic effects
   of GRg1 were reversed when the PI3K/AKT signaling pathway inhibitor
   LY294002 was administered. Therefore, these results demonstrate that
   GRg1 has a protective effect against sepsis-induced acute lung injury,
   mediated through the activation of the PI3K/AKT pathway.

Materials and methods

Animals

   Female Balb/c mice, aged 6 to 8 weeks and weighing between 20 to 25 g,
   were obtained from Vital River Laboratories, China. All experimental
   mice were housed at the Experimental Animal Center of Guangdong
   Provincial Hospital of Chinese Medicine (SYXK 2018–0094, Guangdong).
   The mice were kept in a controlled environment with a temperature
   maintained at 22 ± 1 °C, relative humidity at 50 ± 1%, and a 12-h
   light/12-h dark cycle. All animal handling procedures, including the
   euthanasia protocol, followed the standards and protocols of the
   Institutional Animal Care and Use Committee of Guangdong Provincial
   Hospital of Chinese Medicine and were in accordance with the principles
   set by AAALAC and IACUC. The animal research protocol was approved by
   the Animal Care and Use Committee of Guangdong Provincial Hospital of
   Chinese Medicine (Guangzhou, China) under permit number 2020071. Animal
   welfare and experimental protocols were conducted in accordance with
   the ARRIVE guidelines and strictly adhered to the Guide for the Care
   and Use of Laboratory Animals of the United States.

Establishment of the SALI mouse model

   The procedure of cecal ligation and puncture (CLP) was adopted as a
   model for simulating polymicrobial sepsis, with the methodology in this
   study aligned with previously described processes^[121]23. BALB/c mice
   were divided into four groups: Sham group, CLP group, and two CLP
   groups receiving either a high dose or low dose of GRg1 (n = 20/group).
   Before surgery, animals were fasted for at least 12 h and then
   anesthetized with inhaled isoflurane. In the SALI and GRg1 intervention
   groups, a midline laparotomy was performed to expose the cecum, which
   was then punctured twice with an 18-gauge needle and ligated below the
   ileocecal valve. The Sham group underwent the same surgical exposure
   without CLP. Postoperative subcutaneous saline infusion was provided to
   all cohorts. The designated treatment groups received daily
   intraperitoneal injections of GRg1 at 30 mg/kg for the low-dose group
   or 60 mg/kg for the high-dose group, starting 7 days before the CLP
   intervention^[122]21,[123]24,[124]25. The control group underwent CLP
   alone and received an equivalent volume of saline.

   Seven days post-CLP, survival rates and body weight changes were
   recorded. Additionally, lung tissue samples and serum were extracted
   for further analysis two hours after the second day's treatment. During
   the observation period following successful modeling, we assessed
   whether the mice exhibited intolerable pain or significant declines in
   mobility, which were defined as humane endpoints. Mice meeting these
   criteria were euthanized by inhalation of excess isoflurane, and the
   experiment was immediately terminated. At the end of the experiment,
   all mice were anesthetized with excess isoflurane and then euthanized.

   For signaling pathway elucidation assays, we used an inhibitor to block
   the PI3K/Akt signaling pathway. Male Balb/c mice were randomly divided
   into the following four groups (n = 8 per group): (1) Control group;
   (2) CLP group; (3) CLP + GRg1 (60 mg/kg/d; i.p.) group; (4)
   CLP + GRg1 + LY294002 group. Mice in the CLP + GRg1 group received
   intraperitoneal injections of GRg1 for 7 days prior to CLP modeling.
   The CLP + GRg1 + LY294002 group was pretreated with the PI3K/Akt
   pathway inhibitor LY294002 (1 mg/kg) one hour before CLP modeling. The
   inhibitor dose was predetermined in preliminary experiments and had no
   significant impact on lung function.

Histological studies

   The histopathological examination of lung tissues was conducted on the
   mice. The right upper lobe of the lung was collected and fixed in 4%
   paraformaldehyde for at least 24 h. Paraffin-embedded Sects. (4-5 μm)
   were prepared and stained with hematoxylin and eosin after
   deparaffinization and rehydration. The slides were blindly scored by
   experienced pathologists under an optical microscope using the
   following criteria: 0-normal tissue; 1-mild inflammation; 2-no
   significant damage to lung structure; 3-thickening of alveolar septa;
   4-formation of nodules or areas of distorted normal structure; and
   5-complete loss of the field of view^[125]26.

TUNEL staining

   The pulmonary tissues were preserved in 4% paraformaldehyde solution
   and then encased within paraffin blocks. TUNEL assay was performed
   using a one-step TUNEL apoptosis detection kit (Beyotime, Jiangsu,
   China) to detect apoptotic cells through TUNEL staining, and DAPI was
   used to stain the cell nucleus. Images were captured using a fully
   automated inverted fluorescence microscopy system (ECLIPSE Ti2-E;
   NIKON, Japan).

Immunohistochemical analysis

   The presence of phosphorylated protein kinase B (p-Akt) in
   paraffin-embedded sections was detected by immunohistochemical
   staining^[126]27. Hydrated paraffin sections were incubated in a
   blocking solution (10% normal rabbit serum + 5% skim milk + 3%
   BSA + 0.1% Triton X-100) for 10 min, then incubated with anti-p-AKT
   (#4060) overnight at 4 °C. After rinsing with PBS (pH 7.4), the
   sections were incubated with horseradish peroxidase (HRP)-conjugated
   goat anti-rabbit IgG antibody (1:200) at room temperature for 50 min.
   The sections were then incubated with diaminobenzidine on a chromogenic
   substrate, counterstained with hematoxylin, and observed using an
   Olympus IX73 microscope system (Tokyo, Japan). The extent of
   immunopositive staining was evaluated using ImageJ.

Serum levels of IL-6, TNF-α, and IL-1β were measured using enzyme-linked
immunosorbent assay (ELISA)

   Blood serum was extracted, and the quantities of inflammatory cytokines
   present in the serum of the mice were evaluated using an ELISA kit
   (CUSABIO, China).

Predicting potential gene targets of ginsenoside Rg1

   The chemical structure (canonical SMILES) and 2D structure of
   ginsenoside Rg1 were downloaded from PubChem
   ([127]https://pubchem.ncbi.nlm.nih.gov/). Subsequently, the chemical
   structure was imported into SwissTargetPrediction
   ([128]http://www.swisstargetprediction.ch/) and PharmMapper
   ([129]http://www.lilab-ecust.cn/pharmmapper/) to identify drug compound
   targets. To ensure accuracy, any duplicated targets were removed from
   the analysis.

ALI-associated target genes

   To screen disease gene targets associated with ALI, the keyword "acute
   lung injury" was used to search the Genecards database
   ([130]https://www.genecards.org/), the Online Mendelian Inheritance in
   Man (OMIM) database ([131]https://omim.org/) and the Therapeutic Target
   (TDD) Database ([132]http://db.idrblab.net/ttd/). The resulting gene
   targets associated with ALI were collected. Duplicate targets were
   carefully removed to ensure data accuracy and reliability.

Protein–protein interaction data

   To identify potential therapeutic targets for ALI associated with GRg1,
   we performed a gene target intersection analysis through Venny 2.1
   ([133]https://bioinfogp.cnb.csic.es/tools/venny/index.html)^[134]28.
   The common targets obtained from this analysis were further analyzed
   for their protein–protein interactions (PPI) using the String database
   ([135]https://cn.string-db.org/)^[136]29, with the species limited to
   "Homo sapiens". The PPI data were visualized using Cytoscape 3.9.1 to
   gain insights into the interactions between these targets.

Enrichment analysis and molecular docking

   To explore the signaling pathways involved with the application of GRg1
   in addressing ALI, we performed KEGG analysis and GO analysis by
   Metascape
   ([137]https://metascape.org/gp/index.html#/main/step1)^[138]30,[139]31.
   These analyses aimed to uncover the biological processes and pathways
   enriched with the common targets identified earlier. For molecular
   docking studies, AutoDockTools 1.5.6 and Discovery Studio 2019 were
   applied^[140]32,[141]33. The PDB files of the corresponding small
   molecular ligand substances can be obtained from the RCSB PDB PROTEIN
   DATA BANK ([142]https://www.rcsb.org/) for performing molecular docking
   experiments^[143]33. Additionally, the mol2 Format File for GRg1 was
   downloaded from the Traditional Chinese Medicine Systems Pharmacology
   Database and Analysis Platform (TCMSP,
   [144]https://old.tcmsp-e.com/tcmsp.php) to facilitate the molecular
   docking analysis^[145]34.

Component–disease-target–pathway interaction network construction

   To further clarify the potential relationship among components,
   disease, targets, and pathways, the selected core targets (AKT1, EGFR,
   VEGFA, SRC, IGF1, ESR1, ALB, and STAT3) and the top 20 enriched KEGG
   pathways were inputted into Cytoscape 3.9.1 software to construct a
   component—disease-target—pathway interaction network.

Molecular dynamics simulations

   The molecular dynamics simulations were executed utilizing the
   non-commercial edition of Desmond/Maestro software, version 2022.1, for
   molecular modeling and simulations^[146]35,[147]36. The systems were
   hydrated with the TIP3P water model and ionic balance was achieved with
   0.15 M NaCl. The systems underwent minimization and equilibration,
   followed by a 100 ns production run in the NPT ensemble, maintaining a
   temperature of 300 K and pressure at 1 bar. Snapshots of the trajectory
   were recorded at intervals of 100 ps. Analyses of the molecular
   dynamics were carried out using the Simulation Interaction Diagram
   feature available in Desmond.

Cell culture

   MLE-12 cell lines, procured from the American Type Culture Collection
   (ATCC), were maintained in Dulbecco’s Modified Eagle Medium (DMEM)
   enriched with 20% fetal bovine serum (FBS) sourced from Gibco (San
   Diego, California), and incubated at 37 °C within a moisture—saturated
   environment comprising 5% CO2 and 95% air. Cell subculturing was
   performed with 0.25% trypsin at a dilution ratio of 1:3. The MLE-12
   cells were categorized into four distinct groups for experimental
   purposes: one control group, two separate groups receiving differing
   doses of GRg1; and a group serving as the model.

Quantitative real-time polymerase chain reaction (qRT-PCR)

   Genomic RNA was extracted from MLE-12 cells using the SteadyPure
   Universal RNA Extraction Kit (AG21017; Accurate Biotechnology, Hunan,
   Co., Ltd.) following the manufacturer's protocol. Subsequently, the
   extracted RNA was reverse-transcribed into complementary DNA (cDNA)
   through the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2
   (AG11728, Accurate Biotechnology, Hunan, Co., Ltd.). We conducted
   qRT-PCR Utilizing the SYBR Green Premix Pro Taq HS qPCR Kit (ROX Plus)
   (AG11718, Accurate Biotechnology, Hunan, Co., Ltd.), and the 2-ΔΔCt
   method was employed for relative gene expression analysis. The primer
   sequences for qRT-PCR are provided in Table [148]S4.

Cell Viability analysis

   Cell viability was assessed using the CCK8 cell counting kit (New Cell
   & Molecular Biotech, China). MLE-12 cells were plated at a density of
   10,000 cells per well in a 96-well plate and allowed to incubate for
   24 h. The absorbance at 450 nm was measured using a microplate reader
   (Bio-TEK Instrument, USA), and cell viability was calculated using the
   following formula: cell viability = OD test/OD control, where OD refers
   to the optical density.

Western blot

   After cell collection, the protein concentrations were assessed
   utilizing the BCA Protein Assay Kit (Thermo Fisher, Rockford, IL).
   Individual protein samples were subsequently resolved through SDS‒PAGE
   electrophoresis and transferred onto PVDF membranes. These membranes
   were then immersed in TBST buffer containing 5% skim milk for one hour.
   Primary antibodies, including p-PI3K (#4228), PI3K (#4257), p-AKT1
   (#4060), AKT1 (#4691), Bcl-xl (#2764), Bax (#14,796), caspase-3
   (#9662), and cleaved caspase-3 (#9661), all diluted at 1:1000, and
   β-actin (#4970) diluted at 1:5000 and obtained from Cell Signaling
   Technology, were incubated with the membranes overnight at 4 °C. The
   membranes were then washed and incubated with the corresponding
   secondary antibodies for 1 h, and chemiluminescence detection was
   performed using a contact chemiluminescence instrument (e-BLOT,
   Shanghai, CN). Protein band quantification was carried out using ImageJ
   software, and statistical analysis was conducted based on at least
   three mean values obtained from three independent experiments.

Apoptosis analysis

   MLE-12 cells in the logarithmic growth stage were seeded at a density
   of 12 × 10^4 cells per well in 6-well plates. Following the incubation
   period, MLE-12 cells were collected, washed with PBS buffer, and
   digested with trypsin. The Annexin V-FITC/PI Apoptosis Detection Kit
   was used to treat the cells as per the protocol. Subsequently, a flow
   cytometry analyzer (ACEA, USA) was utilized to analyze apoptosis levels
   and collect corresponding data.

Statistical analysis

   The results are expressed as the mean ± SEM from a minimum of three
   independent experiments. Statistical analysis was conducted using
   one-way ANOVA followed by Bonferroni tests for multiple comparisons
   with GraphPad Prism software, version 8.0 for Windows (GraphPad
   Software Inc., USA). For data variables with normal distribution and
   independent samples, Student’s unpaired t-test was applied to compare
   the two groups. A significance threshold was set at p < 0.05.

Ethics approval

   The animal procedures were conducted following the approval of the
   Animal Ethics Committee of Guangdong Provincial Hospital of TCM, with
   ethics number 2020071.

Conclusions

   In our research, we employed both experimental models and network
   pharmacology approaches to forecast and substantiate the therapeutic
   efficacy of GRg1. The results indicated that GRg1 is an efficacious
   multi-target therapeutic agent for SALI. Our findings robustly
   corroborate the protective effect of GRg1 against SALI by reducing
   apoptosis of AECs via the PI3K-Akt signaling pathway (Fig. [149]12).

Figure 12.

   [150]Figure 12
   [151]Open in a new tab

   PI3K/Akt-pathway network of the predicted targets of GRg1 enriched in
   pathways correlated with SALI.

Supplementary Information

   [152]Supplementary Information 1.^ (517KB, pdf)
   [153]Supplementary Information 2.^ (404.7KB, pdf)
   [154]Supplementary Information 3.^ (337.7KB, pdf)
   [155]Supplementary Information 4.^ (788.6KB, pdf)

Acknowledgements