Application of aralia chinensis saponin A in preparation of medicine for treating septicopyemia lung injury

Abstract

The invention discloses application of aralia saponin A in preparation of a medicine for treating septicopyemia lung injury, and provides an experimental basis and a new strategy for clinically treating septicopyemia-induced acute lung injury and improving the life quality of a patient. According to the application, the effect of the aralia chinensis saponin A on treating septicopyemia lung injury is found for the first time, the molecular mechanism of the aralia chinensis saponin A on resisting inflammation is clarified through experiments, the experiments verify that the aralia chinensis saponin A effectively relieves inflammation, cell death and barrier loss of acute lung injury induced by septicopyemia, and finally the death rate is reduced. The research result also shows that the aralia elata saponin A recovers a PHD2 / HIF-1alpha signal axis in the proinflammatory activated macrophages, so that the protein level of the HIF-1alpha is reduced, the proinflammatory activation of the macrophages in the septicopyemia is inhibited, the inflammation development is resisted, and the lung injury of mice with the septicopyemia is relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of Aralia saponin A in preparing a medicine for treating lung injury caused by sepsis. Background Art

[0002] Acute lung injury is a common acute respiratory disease that can be triggered by factors such as sepsis, mechanical trauma, and inhaled foreign bodies, and manifests as lung inflammation, edema, and impaired respiratory function. It is worth noting that nearly half of sepsis patients experience lung injury, and the recovery of lung injury significantly affects their survival rate. The current clinical methods for treating sepsis-induced acute lung injury mainly include antibiotics, hormones, and nutritional support, and use mechanical assisted ventilation and, in severe cases, extracorporeal respiratory support. However, these interventions are not only expensive to treat, but also lead to the abuse of antibiotics and a large number of complications, such as respiratory trauma, postoperative bleeding, and liver and kidney dysfunction. Therefore, it is of great clinical significance to explore and establish innovative and effective methods for the treatment of sepsis-induced acute lung injury.

[0003] With the continuous advancement of modern medicine, a large number of new strategies for the treatment of septic lung injury have emerged, including therapies such as stem cell therapy and nanodrug delivery. Despite some progress, clinical interventions for septic lung injury are still limited and costly. In recent years, the application of plant extracts, herbs and their refined ingredients in the treatment of various clinical diseases (such as liver fibrosis, cardiovascular disease, etc.) has attracted increasing attention. Compared with traditional chemical synthetic drugs and biopharmaceuticals, these drugs not only ensure efficacy, but also have advantages such as easy availability, strong biocompatibility and low cost. Some Chinese herbal extracts have shown significant therapeutic effects in the field of sepsis research. For example, Fisetin has been shown to alleviate multi-organ dysfunction in sepsis by inhibiting p38 / MK2 signaling, while Cardamonin has been found to alleviate LPS-induced cardiac dysfunction through an Nrf2-regulated anti-oxidative stress mechanism.

[0004] Araliasaponin A (ARA) is extracted from Panax notoginseng, and there are few studies on its pharmacological effects. In previous studies, it has been reported to have antioxidant effects and improve gastrointestinal ulcers in rats, but it is still unknown whether it can treat septic lung injury. The present invention experimentally verifies the effect of Araliasaponin A in treating septic lung injury, and explores and establishes the molecular mechanism of its pharmacological effects, laying a foundation for its application in clinical treatment. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of Araliasaponin A, specifically the use of Araliasaponin A in preparing a medicine for treating septic lung injury.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides an application of aralia saponin A in preparing a medicine for treating sepsis-induced lung injury.

[0007] Furthermore, the drug for treating septic lung injury contains Aralia saponin A as the only active ingredient or one of the active ingredients.

[0008] Furthermore, the drug for treating septic lung injury improves septic lung injury by inhibiting the progression of inflammation.

[0009] Furthermore, the drug for treating septic lung injury inhibits pro-inflammatory activation of macrophages in sepsis.

[0010] Furthermore, the drug for treating septic lung injury reduces pro-inflammatory activation of macrophages by downregulating HIF-1α (hypoxia-inducible factor-1α).

[0011] Furthermore, the drug for treating septic lung injury restores the PHD2 / HIF-1α axis by enhancing egln1 gene expression, thereby reducing HIF-1α protein levels and inhibiting pro-inflammatory activation of macrophages.

[0012] Furthermore, the drug for treating septic lung injury also includes pharmaceutically acceptable excipients.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention first discovered the effect of Aralia saponin A in treating septic lung injury, and experimentally elucidated the molecular mechanism of Aralia saponin A in fighting inflammation. Experiments verified that Aralia saponin A effectively reduced inflammation, cell death and barrier loss in sepsis-induced acute lung injury, and ultimately reduced mortality. The results also showed that Aralia saponin A restored the PHD2 / HIF-1α signaling axis in pro-inflammatory activated macrophages, leading to downregulation of HIF-1α protein levels to inhibit pro-inflammatory activation of macrophages in sepsis, fight against the development of inflammation, and reduce lung injury in septic mice. This study established for the first time the anti-inflammatory mechanism of Aralia saponin A in sepsis-induced acute lung injury, positioning Aralia saponin A as a promising clinical treatment strategy for septic lung injury. Based on the efficacy of araliasaponin A in treating septic lung injury, the present invention provides an application of araliasaponin A in the preparation of a medicament for treating septic lung injury, and provides an experimental basis and a new strategy for clinical treatment of sepsis-induced acute lung injury and improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0015] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Figure 1 This is a diagram showing the results of the optimal dose screening experiment for Aralia saponin A to exert anti-inflammatory effects in cell experiments; Figure 2 This is a graph showing the experimental results of an in vivo experiment on mice, showing that Aralia saponin A reduced the mortality rate of septic mice; Figure 3 This is the experimental result that Aralia saponin A did not reduce the bacterial load in septic mice; Figure 4 This is the experimental result diagram showing that Aralia saponin A significantly improved septic lung injury in mice under the LPS model; Figure 5 This is the experimental result that Aralia saponin A significantly improved the inflammatory infiltration and endothelial barrier loss in the lungs of septic mice; Figure 6 This is the experimental result diagram showing that Aralia saponin A did not counteract the adhesion-promoting effect of endothelial cells and the loss of endothelial barrier under LPS stimulation; Figure 7 This is the experimental result that Aralia saponin A significantly reduced the levels of inflammatory factors in the lungs and peripheral blood of septic mice; Figure 8 This is a diagram showing the experimental results that Aralia saponin A did not improve liver dysfunction in septic mice; Fig. 9 This is a diagram showing the experimental results that Aralia saponin A did not improve the renal dysfunction of septic mice; Fig.10 This is the experimental result of Aralia saponin A inhibiting the pro-inflammatory activation of BMDM and RAW264.7 cells; Fig.11 This is the experimental result of Aralia saponin A reducing the number of pro-inflammatory activated macrophages in the lungs of septic mice; Fig.12 This is the experimental result diagram of the differential gene profile of BMDM under the action of Aralia saponin A; Fig.13 This is the experimental result diagram of the inflammatory factor genes and chemokine genes whose expression levels were downregulated under the action of Aralia saponin A; Fig.14 This is the experimental result that Aralia saponin A does not counteract the pro-inflammatory activation of macrophages through the NF-κB and MAPK signaling pathways; Fig.15 This is the experimental result diagram showing that the HIF-1α signaling pathway is inhibited under the action of Aralia saponin A; Fig.16This is the experimental result of Aralia saponin A down-regulating the protein level of HIF-1α in pro-inflammatory activated macrophages; Fig.17 This is the experimental result of Aralia saponin A against pro-inflammatory activation of macrophages by inhibiting the HIF-1α signaling pathway; Fig.18 The experimental results for constructing macrophage-specific HIF-1α overexpression mice; Fig.19 Araliasaponin A in LyzM-Cre DIO-hif1a Figure 1: Experimental results showing that mice are no longer anti-inflammatory; Fig. 20 Araliasaponin A in LyzM-Cre DIO-hif1a Figure 1: Experimental results showing that mice are no longer protected against septic lung injury. Fig.21 Araliasaponin A in LyzM-Cre DIO-hif1a Figure 2: Experimental results showing that cell death and endothelial barrier loss in the lungs were no longer improved in mice; Fig. 22 This is a diagram of the experimental results of the specific analysis of the effect of Aralia saponin A on the HIF-1 signaling pathway; Fig.23 This is the experimental result diagram showing that Aralia saponin A upregulates PHD2 and restores the PHD2 / HIF-1α signaling axis. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Materials and methods: Ethics Statement: In the present studies, the housing and handling of all experimental animals complied with the Guide for the Care and Use of Laboratory Animals. The Ethics Committee of Wenzhou Medical University also approved the protocols of the mouse sepsis model (LPS injection and CLP surgery) used in each experiment.

[0019] Mouse grouping and treatment: C57BL / 6 male mice (6-8 weeks old; weight 18-22 g) for the sepsis mouse model were purchased from the Model Animal Research Center of Nanjing University. After a week of rest, healthy mice were used for LPS-induced sepsis and divided into the following groups: (1) 0.9wt% saline group; (2) ARA (20 mg / kg) group; (3) LPS group; (4) LPS+ARA (10 mg / kg) group; (5) LPS+ARA (20 mg / kg) group; and (6) LPS+2ME2 (5 mg / kg) group. The LPS reagent used in this experiment is a lipopolysaccharide endotoxin purchased from Beijing Solarbio (L8880, Solarbio) at a dose of 10 mg / kg. For the survival test, the LPS dose was 20 mg / kg.

[0020] Mice used for CLP-induced sepsis protocol were divided into the following groups: (1) normal group; (2) ARA (20 mg / kg) group; (3) CLP group; (4) CLP+ARA (10 mg / kg) group; (5) CLP+ARA (20 mg / kg) group; and (6) CLP+2ME2 (5 mg / kg) group.

[0021] ARA (Aralia saponin A) was purchased from TargetMol (Cat. No. T4S0290) with a purity of 99.88%. 2ME2 (2-methoxyestradiol) was purchased from Selleck (Cat. No. S1233) with a purity of 99.95%. CLP surgery (cecal ligation and puncture surgery) was performed according to a previous protocol. Serum samples and lung samples for subsequent experiments were collected 12 h after LPS injection or 12 h after CLP. Lung samples were collected after serum samples were collected. The left ventricle of the mouse was perfused with 10 mL of 0.9 wt% saline for 2 min, and then the lung samples were carefully separated and rinsed twice with 0.9 wt% saline. The separated lung samples were placed in a culture dish (10 cm in diameter) and photographed. Then, the lung tissue was carefully removed from the culture dish and excess water on the surface was gently absorbed with absorbent paper. Subsequently, the lung tissue for Western Blotting and qPCR analysis was stored at -80 °C. At the same time, the lung tissue for immunofluorescence was immediately immersed in formaldehyde fixative.

[0022] Construction of macrophage-specific HIF-1α overexpression mice 1) Breeding LyzM-Cre mice Perform genetic identification and retain the offspring LyzM-Cre mice for subsequent breeding and genetic identification until the number of LyzM-Cre mice reaches the experimental requirements.

[0023] 2) AAV injection Twenty 3-4 week old LyzM-Cre mice were taken and divided equally into AAV-lung-vector injection group and AAV-lung-DIO-hif1a injection group. Subsequently, the two groups of mice were injected with the corresponding adeno-associated virus through the tail vein injection instrument. The injection titer of AAV is as follows: AAV-lung-vector was commissioned by Shanghai Heyuan Biotechnology Co., Ltd., injection titer: 5×10 12 vg / mL, AAV-lung-DIO-hif1a was commissioned to Shanghai Heyuan Biotechnology Co., Ltd., injection titer: 5×10 12 vg / mL.

[0024] 3) Stable overexpression After the injection, the two groups of mice were returned to the mouse cages for rest. Three weeks later, lung macrophages were extracted from four mice in each group, and the successful overexpression of HIF-1α was verified by Western Blotting.

[0025] Cell culture: BMDMs (mouse bone marrow-derived macrophages) and RAW264.7 cells (mouse mononuclear macrophage cell line) were used to verify macrophages. BMDMs were isolated from C57BL / 6 male mice in previous studies. RAW264.7 cells were purchased from ATCC (ATCC, catalog number TIB-71TM) and cultured in RPMI1640 basal medium (purchased from Gibco (C11875500BT)) containing 10wt% fetal bovine serum (Fetal Bovine Serum, Gibco, catalog number 10,437–028) and 1wt% penicillin-streptomycin solution (Procell, catalog number PB180120).

[0026] BMDMs and RAW264.7 cells were treated with LPS (1 μg / mL) for 12 h to induce pro-inflammatory activation. The ARA concentration used for the combination treatment experiment was 8 μM. Before LPS administration, BMDMs and RAW264.7 cells were pretreated with DMOG (dimethyloxalylaminoacetic acid, 100 μM, Selleck, Catalog No. S7483) for 12 h to verify the potential molecular mechanism.

[0027] ELISA to measure biochemical parameters: Before measuring biochemical parameters, peripheral blood was collected, allowed to settle overnight, centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was used to measure inflammatory cytokine levels. Mouse-IL-1β (mouse interleukin-1β, Proteintech, catalog number KE10003), Mouse-TNF-α Elisa (mouse tumor necrosis factor-α, Proteintech, catalog number KE10002), and Mouse-IL-6 (mouse interleukin-6, Proteintech, catalog number KE10007) were analyzed using a double antibody sandwich method according to the manufacturer's instructions.

[0028] Lung Histology Analysis: The lungs of each group of mice were sectioned into 5 μm sections and histologically analyzed using a hematoxylin-eosin staining kit (Solarbio, catalog number G1120). After blocking with neutral resin, images of the stained sections were taken under a microscope (Motic, PA53 BIO).

[0029] Immunoblot analysis: After measuring protein concentration using the BCA method (bicinchoninic acid method), 30 μg of protein from the samples were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) in Tris-Glycine Gel and transferred to PVDF membrane (polyvinylidene fluoride membrane), blocked with 5wt% skim milk (2h, room temperature), washed three times with 1× TBST (biochemical buffer, 5min each time), and then incubated with primary antibodies (4°C overnight or 37°C for 3h), followed by incubation with HRP goat anti-mouse (Abcam, catalog number ab205719) or HRP goat anti-rabbit (Abcam, catalog number ab205718) secondary antibodies (2h, room temperature). The membrane was washed three times with 1× TBST (5min each time) before visualization with chemiluminescent HRP substrate (Millipore, catalog number WBKLS0500). After visualization, proteins on the membrane were analyzed using ImageQuant800 (Amersham, Cytiva) and quantified using ImageQuant5.2 software (Molecular Dynamics, Sunnyvale, CA). Primary antibodies were raised against the following proteins: β-actin (β-actin, Cell Signaling Technology, catalog number 3700), iNOS (inducible nitric oxide synthase, Cell Signaling Technology, catalog number 13,120), COX2 (cyclooxygenase-2, HUABIO, catalog number RE6064), HIF-1α (hypoxia-inducible factor-1α, Affinity Biosciences, catalog number BF8002), and PHD2 (proline hydroxylase 2, HUABIO, catalog number R1510-40).

[0030] Immunofluorescence staining of lung sections: Lung sections (5 μm) were heated at 96°C for 5 min for antigen retrieval and then permeabilized with 0.5wt% Triton X-100 (Triton X-100, 30 min, room temperature). The sections were washed three times with 1×PBST (PBST is a widely used buffer in biological experiments, consisting of phosphate buffer PBS and non-ionic surfactant Tween-20, 5 min each time), blocked with 5wt%BSA (bovine serum albumin, 30 min, room temperature), and then incubated with primary antibodies at 4°C overnight. The next day, lung sections were washed three times with 1×PBST (5 min each time) and subsequently incubated with secondary antibodies (2 h, room temperature). Finally, the sections were washed three times with 1×PBST (5 min each time) and cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole, 30 min, room temperature). Primary antibodies included VE-cadherin (vascular endothelial cadherin, Abcam, catalog number ab33168), F4 / 80 (Cell Signaling Technology, catalog number 30,325), CD86 (Santa Cruz, catalog number sc-28,347) and HIF-1α (hypoxia-inducible factor-1α, Santa Cruz, catalog number sc-13,515). Secondary antibodies were goat anti-rabbit IgG H&L (Abcam, catalog number ab150077) and goat anti-mouse IgG H&L (Abcam, catalog number ab150115). Images of stained sections were taken under a ZEISS LSM 980 confocal microscope and analyzed by Airyscan2 (ZEISS, Germany).

[0031] Measurement of bacterial counts and cell numbers in BALF (bronchoalveolar lavage fluid) Blood samples were collected from each group of mice, and lungs were isolated. PLF (peritoneal lavage fluid) was collected by injecting 1 mL of PBS (phosphate buffered saline) into the peritoneal cavity under sterile conditions. The blood and PLF samples were then diluted (100 times) in sterile 0.9 wt% saline. Soybean casein agar plates (BD, catalog number BA-256, 665.02) were coated with diluted blood or PLF and then incubated at 37°C for 48 h. Colony counts were analyzed using the previous research method, and BALF was collected. BALF cells were collected by centrifugation at 2000g for 5 min at 37°C for further cell counting.

[0032] Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from BMDMs and RAW264.7 cells using TRI reagent (Sigma, catalog number T9424). After measuring the total RNA concentration of each sample, cDNA was synthesized from 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad, catalog number 1,708,890). Next, cDNA from BMDMs and RAW264.7 cells was amplified by qRT-PCR using primers.

[0033] Quantification of pulmonary hemorrhage Mouse lung tissues were rinsed twice with 0.9 wt% saline and then lysed in tissue lysis buffer for 2 h. Then, 200 μL of lysate was transferred to a 96-well plate at room temperature and the OD value was measured spectrophotometrically at 400 nm.

[0034] RNA interference in BMDMs (mouse bone marrow-derived macrophages) Si-Scramble (control siRNA, constructed by Limibio Biotechnology) or Si-egln1 (siRNA specifically targeting the egln1 gene, constructed by Limibio Biotechnology) was mixed with Lipofectamine 2000 (cationic liposome transfection reagent, Invitrogen, catalog number 11,668-019) in Opti-MEM (reduced serum medium, Gibco, catalog number 51,985,034) and incubated at 37°C for 20 min. Then, BMDMs were washed with PBS and incubated in the medium containing the siRNA mixture. After 6-8 h of culture, the Opti-MEM mixture was discarded and the cells were washed with PBS. Fresh medium was added and cultured for 48 h before use in subsequent experiments.

[0035] TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining of lung sections: Lung sections (5 μm) were subjected to antigen retrieval at 96°C for 5 min and then permeabilized with 0.5 wt% Triton X-100 (30 min, room temperature). They were blocked with 5 wt% BSA for 30 min, room temperature, and washed three times with 1× PBST (5 min each) before incubation with TUNEL reaction mixture (Promega, catalog number G3250) at 37°C for 1 h. Nuclei were then stained with DAPI (30 min, room temperature). Images of stained sections were taken on a ZEISS LSM 980 confocal microscope and analyzed by Airyscan2 (ZEISS, Germany).

[0036] Statistical analysis The long-rank test was used to evaluate the statistical differences in survival rate. The statistical significance of the differences between the two groups was analyzed by two-sided unpaired t-test. For experiments involving multiple groups, the statistical significance of any differences was evaluated by one-way analysis of variance. The results are expressed as mean ± standard error, and P < 0.05 was considered statistically significant. All statistical analyses were performed using Graph PadPrism 8 software.

[0037] In the in vivo experiment, we used intraperitoneal injection of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) to construct a mouse model of septic lung injury, and simultaneously gave mice arachidina saponin A by gavage. After 12 hours of modeling, lung tissues of mice in each group were collected for subsequent organ function evaluation experiments to verify the therapeutic effect of arachidina saponin A on septic lung injury. In the in vitro experiment, we exposed mouse bone marrow-derived macrophages (BMDM) to LPS to induce pro-inflammatory activation of macrophages in septic lung injury, and combined with arachidina saponin A treatment. After 12 hours of modeling, samples from each group were collected for RNA sequencing to clarify its detailed mechanism. The specific experimental results are as follows.

[0038] 1. Aralia saponin A has certain anti-inflammatory potential To test the anti-inflammatory potential of Araliasaponin A, we extracted BMDM from mice and established an in vitro macrophage pro-inflammatory activation model by applying LPS. By applying different concentrations of Araliasaponin A, it was found that the protein expression of iNOS (inducible nitric oxide synthase) in BMDM was significantly reduced with the increase of Araliasaponin A concentration, and it was most significant at 8μM and 16μM. Similarly, the concentration of NO (nitric oxide) in the BMDM cell culture medium of each group also gradually decreased significantly with the increase of Araliasaponin A concentration. Early research results on Araliasaponin A showed that high doses of Araliasaponin A often have a pro-apoptotic effect. Therefore, in order to avoid the influence of apoptosis on the experimental results, the cell activity of BMDM was detected using the CCK8 cell activity assay kit. The results showed that among the doses of Aralia saponin A that produced significant anti-inflammatory effects, only 8 μM did not significantly affect the cell activity of BMDM, while 16 μM, which had a stronger anti-inflammatory effect, significantly reduced the cell activity of BMDM ( Figure 1 , *P<0.05, **P<0.01, ***P<0.001, ns=no significant difference). Therefore, in order to prevent the impact on the subsequent experiments related to the mechanism exploration of Aralia saponin A, a dose that would not affect cell activity (8μM) was selected for subsequent cell experiments. The above data show that Aralia saponin A can inhibit the pro-inflammatory activation of macrophages and has certain anti-inflammatory potential. Based on this, we began to explore whether it has a therapeutic effect on sepsis.

[0039] 2. Aralia saponin A can improve the survival rate of septic mice After confirming that Aralia saponin A has certain anti-inflammatory potential, in order to explore the therapeutic potential of Aralia saponin A for sepsis, we applied two mouse sepsis models (LPS model and CLP model) and followed Figure 2 A was used for subsequent experiments. The results of the survival rate experiment of septic mice showed that high doses of Aralia saponin A (20 mg / kg) could significantly improve the survival rate of septic mice in both LPS and CLP models, while lower doses of Aralia saponin A (10 mg / kg) only showed the effect of improving the survival rate of septic mice in the CLP model ( Figure 2 At the same time, we found that the therapeutic effect of high-dose ARA was similar to that of the positive control drug 2ME2 (2-Methoxyestradiol, which has been proven to significantly improve mouse sepsis). Based on this, it was determined that ARA had a certain rescue effect on septic mice, and in subsequent experiments, the specific way and molecular mechanism of ARA's action were explored in depth.

[0040] 3. Aralia saponin A did not show significant antibacterial effects Since Aralia saponin A can significantly improve the survival rate of septic mice, we speculate that Aralia saponin A may play a therapeutic role by controlling the infection level in septic mice. However, after in vitro bacterial culture of peripheral blood, peritoneal lavage fluid, and tissue homogenate (lungs, liver, and kidneys) of each group of mice, we found that low-dose and high-dose Aralia saponin A and the positive control drug 2ME2 could not significantly reduce the total amount of bacteria in the peripheral blood, peritoneal lavage fluid, and tissue homogenate (lungs, liver, and kidneys) of CLP model mice ( Figure 3 ), that is, Araliasaponin A cannot control the infection level in septic mice. These results indicate that Araliasaponin A does not show obvious antibacterial effect in the CLP model, that is, Araliasaponin A may save the death of septic mice in other ways.

[0041] 4. Aralia saponin A can alleviate sepsis-induced lung injury Septic lung injury is the most common and most dangerous organ dysfunction in sepsis. Considering that Aralia saponin A has a certain therapeutic effect on sepsis, but has no obvious antibacterial effect, we speculate that Aralia saponin A may save septic mice by improving the lung function of septic mice. The results of pulmonary blood exudation detection and lung wet weight / dry weight experiment showed that, similar to the positive control 2ME2, Aralia saponin A can significantly reduce blood exudation in the lungs of septic mice and reduce the wet weight / dry weight ratio of lung tissue, and improve pulmonary edema under the LPS model. Similarly, in the CLP model, Aralia saponin A can also significantly reduce blood exudation in the lungs of septic mice and reduce the wet weight / dry weight ratio of lung tissue, and relieve pulmonary edema ( Figure 4 ). These experimental results indicate that Aralia saponin A can rescue septic mice by improving pulmonary edema.

[0042] 5. Aralia saponin A can improve inflammatory cell infiltration and endothelial barrier loss in the lungs of septic mice In the further pathological analysis of lung tissues of septic mice in each group, we evaluated the effect of Araliasaponin A by H&E staining, BALF cell counting and VE-cadherin immunofluorescence staining. The results of H&E staining showed that, similar to the positive control 2ME2, Araliasaponin A could significantly reduce the infiltration of inflammatory cells in the lungs of septic mice under the LPS model. The cell counting results of BALF also showed that both 2ME2 and high-dose Araliasaponin A could significantly reduce the number of cells in the BALF of septic mice. Similarly, under the CLP model, the results of H&E staining and the cell counting results of BALF also showed that Araliasaponin A could significantly inhibit the infiltration of inflammatory cells in the lungs of septic mice. Since Araliasaponin A could significantly improve pulmonary edema in septic mice, we speculated that Araliasaponin A may have a certain effect on maintaining the pulmonary barrier. At the same time, the results of VE-cadherin immunofluorescence staining showed that both the positive control 2ME2 and high-dose aralien saponin A could significantly rescue the loss of endothelial barrier protein VE-cadherin in the lungs of septic mice in both the LPS model and the CLP model ( Figure 5 These results suggest that the protective effect of Araliasaponin A may be related to endothelial cell-related pathological processes such as inflammatory cell infiltration and endothelial barrier.

[0043] 6. Aralia saponin A has no significant effect on inflammatory activation of endothelial cells To verify whether Araliasaponin A protects against septic lung injury by inhibiting the inflammatory activation of endothelial cells, we applied Araliasaponin A to EA.hy926 cells in vitro. It is well known that endothelial cells undergo inflammatory activation under inflammatory stimuli (such as LPS), that is, while recruiting immune cells through its adhesion-promoting effect, the endothelial barrier is lost (the amount of VE-cadherin protein is reduced). Therefore, we evaluated the effects of Araliasaponin A on these two phenotypes of inflammatory activation of endothelial cells. The monocyte adhesion experiment showed that EA.hy926 cells stimulated by LPS could promote the adhesion of a large number of THP-1 cells on their surface, but the application of Araliasaponin A did not significantly inhibit this phenomenon. Similarly, the results of VE-cadherin immunofluorescence staining experiments also showed that Araliasaponin A did not reverse the decrease in VE-cadherin protein in EA.hy926 cells stimulated by LPS ( Figure 6 ). These results indicate that Aralia saponin A has no significant improvement effect on the inflammatory activation of endothelial cells, that is, the effect of Aralia saponin A on alleviating inflammatory infiltration and endothelial barrier loss in the lungs of septic mice may not be achieved by directly acting on endothelial cells.

[0044] 7. Aralia saponin A can significantly inhibit the occurrence and development of lung inflammation in septic mice Since Aralia saponin A cannot protect septic lung injury by directly acting on endothelial cells, we speculated whether Aralia saponin A could reduce pulmonary endothelial injury by inhibiting the occurrence and development of pulmonary inflammation, and finally protect septic lung injury. By detecting the levels of inflammatory factors such as IL-1β (interleukin-1β), IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α) in the BALF of mice in each group, we found that, consistent with the positive control 2ME2, Aralia saponin A could significantly reduce the levels of inflammatory factors such as IL-1β, IL-6 and TNF-α in the BALF of septic mice, whether in the LPS model or the CLP model. At the same time, the results of TUNEL staining experiments also showed that as a subsequent effect of the occurrence and development of pulmonary inflammation, cell death in the lungs of septic mice was also significantly improved by the application of Aralia saponin A. Similarly, although the degree of downregulation was not as significant as in BALF, Aralia saponin A was still able to significantly reduce the levels of inflammatory factors such as IL-1β, IL-6 and TNF-α in the peripheral blood of septic mice in both the LPS model and the CLP model ( Figure 7 ). At the same time, the fold change of inflammatory factors in BALF down-regulated by Aralia saponin A was significantly higher than that in peripheral blood. Based on this, these results indicate that Aralia saponin A can inhibit the development of inflammation in the lungs of septic mice, thereby improving the damage in the lungs of septic mice (inflammatory infiltration, endothelial barrier loss, cell death, etc.), relieve pulmonary edema, and improve the survival rate of septic mice.

[0045] 8. Aralia saponin A has no significant protective effect on the liver and kidneys Since the development of sepsis is often accompanied by multiple organ dysfunction, we then explored whether Araliasaponin A has a certain protective effect on other organs in addition to protecting lung function. Considering the obvious therapeutic effect of high-dose Araliasaponin A in the above experimental results, we selected this dose in this part of the experiment to evaluate the protective effect of Araliasaponin A on other organs. By detecting the peripheral blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of each group of mice, we found that, whether in the LPS model or the CLP model, high-dose Araliasaponin A had little protective effect on the liver, only slightly down-regulated the ALT level in the peripheral blood, but had no significant effect on the AST level in the peripheral blood. Different from the results in BALF and peripheral blood, high doses of Aralia saponin A could no longer reduce the levels of inflammatory factors such as IL-1β, IL-6 and TNF-α in the liver of septic mice, whether in the LPS model or the CLP model ( Figure 8 ).

[0046] Subsequently, in the assessment of mouse renal function, we also found that, whether in the LPS model or the CLP model, high doses of Aralia saponin A had no significant effect on the significantly increased levels of blood urea nitrogen (BUN) and plasmacreatinine (serum creatinine) in the peripheral blood of septic mice, that is, it had no significant protective effect on the renal function of septic mice. Similarly, whether in the LPS model or the CLP model, high doses of Aralia saponin A could no longer reduce the levels of inflammatory factors such as IL-1β, IL-6 and TNF-α in the kidneys of septic mice ( Fig. 9 ). The above results show that the antagonistic effect of Aralia saponin A on the development of sepsis inflammation is only significant in the lungs, but ineffective in the liver and kidneys. At the same time, these results also explain the phenomenon that the fold change of inflammatory factors in BALF down-regulated by Aralia saponin A is significantly higher than that in peripheral blood.

[0047] 9. Aralia saponin A can inhibit LPS-induced pro-inflammatory activation of macrophages 8μM was selected as the action concentration of Araliasaponin A for subsequent cell experiments, and 20mg / kg was selected as the action concentration of Araliasaponin A for subsequent animal experiments. In the cell experiment, we found that in LPS-induced pro-inflammatory activated BMDM (bone marrow-derived macrophages), Araliasaponin A could significantly downregulate the expression of pro-inflammatory activated macrophage marker proteins such as iNOS and COX2, and could also inhibit the gene expression of inflammatory factors such as IL-1β, IL-6 and TNF-α. In addition, the detection results of NO, IL-6 and TNF-α in BMDM cell culture medium showed that Araliasaponin A significantly inhibited the secretion of NO, IL-6 and TNF-α by BMDM caused by LPS, and Araliasaponin A inhibited the pro-inflammatory activation of BMDM caused by LPS. Similarly, in the mouse macrophage cell line RAW264.7, we also found that Aralia saponin A can also significantly downregulate the expression of iNOS and COX2, and can inhibit the gene expression of inflammatory factors such as IL-1β, IL-6 and TNF-α in RAW264.7 cells. At the same time, the detection results of NO, IL-6 and TNF-α in the RAW264.7 cell culture medium also showed that Aralia saponin A can significantly inhibit the secretion of NO, IL-6 and TNF-α by RAW264.7 induced by LPS ( Fig.10 ). The above in vitro experimental results show that Aralia saponin A can significantly inhibit the pro-inflammatory activation of macrophages induced by LPS, and may fight against the development of pulmonary inflammation in septic mice in this way.

[0048] 10. Aralia saponin A can inhibit pro-inflammatory activation of macrophages in the lungs of septic mice To further prove our conclusions in in vivo experiments, we used CD86 (a marker of pro-inflammatory activated macrophages) and F4 / 80 (a marker of macrophages) immunofluorescence double staining to detect the effect of Aralia saponin A on pro-inflammatory activation of macrophages in the lungs (Note: CD86 and F4 / 80 immunofluorescence double-positive cells are pro-inflammatory activated macrophages). At the same time, combined with the above experimental results, we chose 20 mg / kg as the action concentration of Aralia saponin A for subsequent animal experiments. The results of CD86 and F4 / 80 immunofluorescence double staining showed that Aralia saponin A could significantly reduce the number of CD86 and F4 / 80 double-positive cells in the lungs, whether in the LPS-induced mouse septic lung injury model or the CLP-induced mouse lung injury model. Fig.11 ). In summary, these experimental results show that Aralia saponin A can inhibit the pro-inflammatory activation of macrophages, inhibit the occurrence and development of inflammation and ultimately improve septic lung injury.

[0049] The multi-organ dysfunction caused by systemic inflammatory response syndrome and a large number of infections in sepsis is the main reason for sepsis patients to enter the ICU to receive high-level critical care services, and is also the culprit for the high mortality and poor prognosis of sepsis patients. Therefore, clinical treatment for this acute event generally adopts antibacterial and anti-inflammatory treatments in parallel. In the above study, it was found that Aralia saponin A can improve septic lung injury in mice and significantly improve the survival rate of septic mice, and its therapeutic effect at high doses is basically the same as that of the positive drug 2ME2, which has been proven to be effective. At the same time, it was also found that Aralia saponin A did not affect the bacterial load in sepsis mice induced by the CLP model, that is, the therapeutic effect of Aralia saponin A on sepsis has no obvious correlation with the effect on the reproduction and expansion of pathogens in the organism. Combined with the subsequent experimental results, we believe that Aralia saponin A mainly exerts its counteracting effect on sepsis lung injury by inhibiting the pro-inflammatory activation of macrophages. However, the pro-inflammatory activation of macrophages is often closely related to their ability to phagocytose and kill pathogens. Therefore, whether long-term use of Araliasaponin A or the use of higher doses of Araliasaponin A will affect the ability of macrophages to phagocytose and kill pathogens while fighting inflammation, thereby causing immunosuppression in the later stages of sepsis, remains to be confirmed by further animal experiments. Improving this link in subsequent experiments can further optimize the application time and dosage of Araliasaponin A for the treatment of sepsis.

[0050] In the process of evaluating the level of multiple organ dysfunction in mice, by evaluating the functions of multiple organ systems (lungs, liver, kidneys, etc.) of mice, we found that the organ protective effect of high-dose araliasaponin A, which can improve the survival rate of septic mice, was only significant in the lungs. It had little effect in the liver, and even had no obvious effect in the kidneys. In the course of multiple organ dysfunction caused by sepsis, although the inflammatory response is the main factor affecting the function of most organs in sepsis, the structural and tissue differences of each organ also make the effects of systemic inflammatory response on them different. In addition, compared with the lungs, the main reason for the damage of the liver and kidneys in sepsis is the occurrence of tissue hypoperfusion rate, that is, hypoxic injury and nutrient deficiency injury. Therefore, fighting the inflammatory storm cannot directly and effectively rescue the abnormal liver and kidney function in sepsis. Studies have shown that the occurrence of tissue hypoperfusion rate is closely related to the pro-vasodilatory phenotype obtained by inflammatory activation of endothelial cells. However, in in vitro experiments, araliasaponin A did not inhibit the inflammatory activation of EA.hy926 cells. This may be the reason why Aralia saponin A has no obvious rescue effect on liver and kidney abnormalities in sepsis. In addition, in terms of inflammation, our results also showed that Aralia saponin A significantly reduced the level of inflammatory factors in the BALF of septic mice, but did not affect the levels of inflammatory factors in the liver and kidneys. This may be the main reason why Aralia saponin A cannot reduce the inflammatory factors in the peripheral blood of septic mice to levels close to those of normal mice. Due to the differences in tissue structure and cell composition, there are also certain differences in the levels of inflammation in different tissues and the main cells that cause inflammation. Compared with the lungs, which are damaged earlier, the occurrence and development of inflammation in the liver and kidneys are mainly related to endothelial cells and other parenchymal cells therein. This may also be the reason why Aralia saponin A only has a significant effect on the lungs, but has no obvious effect on the liver and kidneys.

[0051] In the course of septic lung injury, various types of cells in the lungs [pulmonary epithelial cells (type I alveolar epithelial cells, type II alveolar epithelial cells), endothelial cells, fibroblasts, immune cells (neutrophils, macrophages, lymphocytes, etc.) and smooth muscle cells] all play an indispensable and important role. Since we first observed that Aralia saponin A could significantly alleviate the infiltration of inflammatory cells and the loss of endothelial barrier in the lungs of septic mice, we also verified whether Aralia saponin A could affect the function of endothelial cells in an inflammatory environment. However, the results of in vitro experiments on endothelial cells showed that Aralia saponin A could not directly improve the recruitment of monocytes by EA.hy926 under LPS stimulation and the loss of its own endothelial barrier. Therefore, the effect of Aralia saponin A on alleviating the infiltration of inflammatory cells and the loss of endothelial barrier in the lungs of septic mice is not achieved directly by acting on endothelial cells. It is well known that fighting against the intense inflammatory response (inflammatory factor storm) in sepsis can also alleviate the inflammatory cell infiltration and endothelial barrier loss in the septic lung. Therefore, we shifted our research focus to the effect of Aralia saponin A on the inflammatory response in sepsis. The results showed that Aralia saponin A had a significant inhibitory effect on the inflammatory response in the septic lung. Macrophage-mediated inflammatory response is a hot topic in the field of septic lung injury research and is also the target of most natural compounds. Compared with epithelial cells, endothelial cells and neutrophils that mediate the initiation of inflammation in the early acute phase of septic lung injury, the inflammation caused by macrophages is later and more persistent and intense, which matches the time when most natural compounds take effect. In this study, we first explored the effect of Aralia saponin A on macrophage-mediated inflammatory response and successfully confirmed its inhibitory effect on pro-inflammatory activation of macrophages. However, it remains unclear whether Aralia saponin A can have positive / negative effects on other cells in the lung.

[0052] The experimental results of this stage showed that Aralia saponin A can inhibit the occurrence and development of inflammation in sepsis by inhibiting the pro-inflammatory activation of macrophages, thereby improving septic lung injury and increasing the survival rate of septic mice. In animal experiments, we gavaged mice with Aralia saponin A according to the commonly used doses in previous studies. However, unlike the phenomenon observed in cell experiments, the application of higher doses of Aralia saponin A to normal mice did not produce obvious negative effects. This may be because the physiological environment in mice is more complex than the single physiological environment in cell experiments. At the same time, the tolerance of the entire animal body to a single drug is obviously stronger than that of a single cell population.

[0053] In this study, we used 2ME2 as a positive control drug for the study of therapeutic drugs for septic lung injury. Although the application dose of araliasaponin A reached 20 mg / kg to produce similar therapeutic effects as 2ME2 at a dose of 5 mg / kg, araliasaponin A is more widely available than natural compounds and chemically synthesized drugs such as 2ME2. Although we have demonstrated that araliasaponin A can inhibit macrophage inflammatory activation and improve septic lung injury, the detailed anti-inflammatory mechanism of araliasaponin A has not yet been clarified. 2ME2, as an inhibitor of the HIF-1α signaling pathway, inhibits the nuclear accumulation of HIF-1α and the transcriptional activity of HIF-1α by depolymerizing microtubules. High doses of araliasaponin A showed similar therapeutic effects to 2ME2, suggesting that the mechanism of action of araliasaponin A may be related to the HIF-1α signaling pathway. In addition, whether Araliasaponin A can affect the classic inflammatory signaling pathway, cell pyroptosis, oxidative stress and other mechanisms still needs to be further explored in subsequent experiments. In the field of natural compound research, the mechanism of action of natural compounds is often explored through high-throughput analysis methods such as transcriptomics analysis, proteomics analysis and virtual binding site screening. The emergence and application of these technologies make the study of the mechanism of action of natural compounds more intuitive and clear. Therefore, we will try to use these technologies in subsequent experiments to explore the mechanism of action of Araliasaponin A against pro-inflammatory activation of macrophages, and provide a detailed theoretical basis for the clinical application of Araliasaponin A.

[0054] In the following experiments, transcriptomic analysis revealed that Aralia saponin A may inhibit pro-inflammatory activation of macrophages by regulating the HIF-1α signaling pathway. Dimethyloxalylglycine (DMOG) was used and macrophage-specific HIF-1α overexpression mice (LyzM-Cre DIO-hif1α ), we verified that Aralia saponin A inhibits macrophage pro-inflammatory activation by downregulating HIF-1α and counteracts septic lung injury. In subsequent further mechanism analysis, we found that Aralia saponin A restores the PHD2 / HIF-1α signaling axis in macrophages by upregulating the expression of the egln1 gene, thereby downregulating HIF-1α and counteracting inflammation.

[0055] 11. Transcriptomics analysis of the anti-inflammatory mechanism of Araliasaponin A In order to further explore the molecular mechanism of Aralia saponin A in inhibiting pro-inflammatory activation of macrophages, we conducted transcriptomic analysis on BMDM in the LPS-treated group and BMDM in the LPS+ARA-treated group. The results of transcriptomic analysis showed that compared with BMDM in the LPS-treated group, BMDM in the LPS+ARA-treated group had a total of 2877 differentially expressed genes (2373 genes were upregulated and 504 genes were downregulated). After enriching these differentially expressed genes for related diseases, we found that most of the genes affected by Aralia saponin A were related to infectious diseases (viral infection, bacterial infection, parasitic infection) and immune system diseases ( Fig.12 ).

[0056] In the differential gene heat map analysis, we found that, consistent with the conclusion of the previous study that Aralia saponin A inhibits pro-inflammatory activation of macrophages, the expression of inflammatory factor-related genes such as il-1b, il-1a, il-6, il-18, nos2, and chemokine-related genes such as ccl family and cxcl family in BMDM of the LPS+ARA treatment group was significantly reduced compared with BMDM of the LPS treatment group ( Fig.13 ). Therefore, the transcriptome analysis results also verified the conclusion that Aralia saponin A inhibits pro-inflammatory activation of macrophages. At the same time, under the premise that Aralia saponin A exhibits significant anti-inflammatory effects, it is reasonable to use the transcriptome analysis results to explore the molecular mechanism of action of Aralia saponin A.

[0057] In the exploration of the molecular mechanism of action of Aralia saponin A, we first focused our research on two classic inflammatory signaling pathways downstream of the LPS receptor TLR4, namely the NF-κB signaling pathway and the MAPK signaling pathway, to observe whether Aralia saponin A can affect the NF-κB signaling pathway and the MAPK signaling pathway. The results of transcriptomics analysis showed that Aralia saponin A could cause changes in 15 genes in the NF-κB signaling pathway, but the effect was not statistically significant (P=0.5859). Western Blotting and cell immunofluorescence results also showed that Aralia saponin A could not inhibit the nuclear entry effect of p65 in BMDM caused by LPS. Subsequently, similar to the results found in the NF-κB signaling pathway, the KEGG analysis results for the MAPK signaling pathway showed that Aralia saponin A could not inhibit the activation of the MAPK signaling pathway caused by LPS in BMDM. On the contrary, Aralia saponin A can activate the MAPK signaling pathway ( Fig.14 ). Based on this, we believe that Aralia saponin A cannot counteract the pro-inflammatory activation of macrophages by inhibiting the classical NF-κB signaling pathway and the MAPK signaling pathway.

[0058] Subsequently, in the signal pathway enrichment results of transcriptomics analysis, we found that aralia saponin A can significantly inhibit the activation of the HIF-1α signaling pathway in BMDM cells caused by LPS. According to previous studies, the protein level of HIF-1α is significantly upregulated in immune cells in an inflammatory state, and it is crucial for the inflammatory activation of immune cells such as neutrophils and macrophages. In addition to being able to directly control the expression of inflammatory factor-related genes such as il-1b and nos2 like the classic NF-κB signaling pathway and MAPK signaling pathway, HIF-1α can also promote metabolic reprogramming (oxidative phosphorylation to aerobic glycolysis) in the pro-inflammatory activation of neutrophils and macrophages by controlling the expression of glycolysis-related genes (such as pfkfb3, hk2, ldha, etc.) to meet the energy needs under inflammatory conditions, thereby promoting the occurrence and development of inflammation. Consistent with the pathway result analysis, in the gene heat map analysis of HIF-1α downstream target genes, we also found that aralia saponin A can significantly downregulate the expression levels of genes such as pfkfb3 and dll1 ( Fig.15 ). Based on this, we speculated that Aralia saponin A could inhibit the activation of the HIF-1α signaling pathway in BMDM after LPS treatment.

[0059] Subsequently, to verify this inference, we explored the effect of Aralia saponin A on HIF-1α protein levels in macrophages under inflammatory conditions in in vitro and in vivo experiments. First, consistent with the results of transcriptomics analysis, in BMDM and RAW264.7 cells, Aralia saponin A was able to significantly inhibit the increase in HIF-1α protein levels caused by LPS stimulation. Similarly, whether in LPS-induced septic lung injury or CLP-induced septic lung injury, we found that compared with septic mice in the modeling group, septic mice treated with Aralia saponin A had fewer HIF-1α-positive macrophages in their lungs ( Fig.16 ). The above results indicate that Aralia saponin A can downregulate the protein level of HIF-1α in pro-inflammatory activated macrophages, and may use this as a key mechanism for its anti-inflammatory effect against septic lung injury.

[0060] 12. Aralia saponin A counteracts pro-inflammatory activation of macrophages by inhibiting the HIF-1α signaling pathway In subsequent experiments, in order to verify that the effect of Aralia saponin A in inhibiting pro-inflammatory activation of macrophages originated from its inhibition of the HIF-1α signaling pathway, we pre-treated RAW264.7 cells and BMDM in vitro experiments with DMOG (an agonist of HIF-1α, which upregulates the amount of HIF-1α protein by inhibiting the activity of PHDs family proteins that regulate HIF-1α degradation). First, in LPS-treated BMDM, we found that the effects of Aralia saponin A in downregulating iNOS and COX2 protein levels and reducing NO, IL-6 and TNF-α levels in their culture medium were offset by DMOG. Similarly, in RAW264.7 cells, we also found that DMOG reversed the anti-inflammatory effect of Aralia saponin A, that is, the iNOS and COX2 protein levels in RAW264.7 cells and the NO, IL-6 and TNF-α levels in their culture medium significantly recovered ( Fig.17 These results indicate that the inhibitory effect of araliosaponin A on the pro-inflammatory polarization of macrophages was offset by DMOG, suggesting that the anti-inflammatory effect of araliosaponin A originates from the inhibition of the HIF-1α signaling pathway in pro-inflammatory activated macrophages.

[0061] 13. Construction of Macrophage-specific HIF-1α Overexpression Mice LyzM-Cre mice aged 3-4 weeks were selected through gene identification, and the successfully identified 3-4 week old LyzM-Cre mice were injected with AAV-lung-vector and AAV-lung-DIO-hif1a through the tail vein. After 3 weeks of stable overexpression, lung macrophages of the two groups of mice were extracted to detect the HIF-1α protein level. The results showed that the protein level of HIF-1α in lung macrophages of LyzM-Cre mice injected with AAV-lung-DIO-hif1a through the tail vein was significantly higher than that of LyzM-Cre mice injected with AAV-lung-vector through the tail vein, that is, macrophage-specific HIF-1α overexpression mice were successfully constructed ( Fig.18 ).

[0062] 14. Anti-inflammatory effect of Aralia saponin A in LyzM-Cre DIO-hif1a Failure in mice To verify in vivo that Aralia saponin A can counteract the inflammatory development of septic lung injury by downregulating HIF-1α in macrophages, we used LyzM-Cre DIO-hif1a Mice were induced with LPS and CLP respectively and treated with Aralia saponin A. DIO-vector Mice, namely LyzM-cre mice injected with control adeno-associated virus, were used as LyzM-Cre DIO-hif1aThe control mice were also subjected to the same modeling and combined drug treatment. First, the results of CD86-F4 / 80 immunofluorescence double staining showed that Aralia saponin A could significantly reduce LyzM-Cre in both the LPS model and the CLP model. DIO-vector The number of pro-inflammatory activated macrophages in the lungs of mice was significantly decreased, but this effect was not observed in LyzM-Cre DIO-hif1a Similarly, Aralia saponin A can significantly reduce the expression of LyzM-Cre after LPS modeling or CLP modeling. DIO-vector The levels of inflammatory factors such as IL-1β, IL-6 and TNF-α in the BALF of mice were significantly decreased, but in the LyzM-Cre DIO-hif1a In mice, Aralia saponin A was effective in downregulating inflammatory factors such as IL-1β, IL-6, and TNF-α. Due to the disappearance of the anti-inflammatory effect, Aralia saponin A was unable to increase LyzM-Cre expression in both the LPS model and the CLP model. DIO-hif1a The survival rate of mice ( Fig.19 These results suggest that the anti-inflammatory effect of Araliasaponin A is DIO-hif1a It was clearly ineffective in mice, indicating that Aralia saponin A counteracted the inflammatory development of septic lung injury by inhibiting HIF-1α in macrophages.

[0063] 15. Effect of Aralia saponin A on sepsis-induced lung injury in LyzM-Cre DIO-hif1a Failure in mice Due to the anti-inflammatory effect of Aralia saponin A in LyzM-Cre DIO-hif1a In subsequent experiments, we investigated the LyzM-Cre DIO-hif1a The septic lung injury of mice was detected. The results of the pulmonary blood exudation test and the lung wet weight / dry weight experiment showed that Aralia saponin A could not inhibit the septic lung injury in either the LPS model or the CLP model as in the LyzM-Cre model. DIO-vector In mice, LyzM-Cre DIO-hif1a In mice, aralienin A improved blood infiltration in the lungs of septic mice and reduced the wet weight / dry weight ratio of lung tissue. Similarly, the cell count results of BALF also showed that aralienin A could not reduce LyzM-Cre DIO-hif1a The number of cells in mouse BALF ( Fig. 20 ).

[0064] At the same time, the results of TUNEL staining experiments also showed that Aralia saponin A played an important role in the expression of LyzM-Cre in both LPS and CLP models. DIO-vector Effect of LyzM-Cre on reducing cell death in mouse lungs DIO-hif1aSimilarly, the results of VE-cadherin immunofluorescence staining showed that Aralia saponin A could not inhibit the expression of VE-cadherin in either the LPS model or the CLP model as in the LyzM-Cre model. DIO-vector In mice, LyzM-Cre DIO-hif1a Rescue loss of VE-cadherin, a barrier protein in the lungs, in mice Fig.21 These results suggest that the effect of Aralia saponin A against septic lung injury is important in LyzM-Cre DIO-hif1a The results showed that aralienin A improved septic lung injury by inhibiting HIF-1α in macrophages.

[0065] 16. KEGG analysis of the effect of Aralia saponin A on HIF-1α signaling pathway After confirming that Aralia saponin A improves septic lung injury by inhibiting HIF-1α in macrophages, we further explored the molecular mechanism of Aralia saponin A downregulating HIF-1α. In immune cells, as a key transcription factor that upregulates protein levels and promotes inflammatory activation, there are two main reasons for the increase in HIF-1α protein levels. First, activation of signaling pathways such as NF-κB and MAPK promotes the transcription of the hif1a gene. Second, the PHDs family proteins that control the degradation of the classical proteasome pathway of HIF-1α are inhibited. First, the gene heat map results of transcriptome analysis showed that Aralia saponin A did not affect the expression level of the hif1a gene in BMDM treated with LPS. At the same time, the results of qPCR experiments also verified that Aralia saponin A could not reduce the upregulation of the expression of the hif1a gene caused by LPS. KEGG analysis of the HIF-1α signaling pathway based on this transcriptomics analysis. The results showed that Aralia saponin A had a significant upregulation effect on PHDs family proteins that control HIF-1α degradation ( Fig. 22 ). Based on this, we speculated that the down-regulation of HIF-1α by Araliasaponin A originated from the up-regulation of PHDs family proteins. Therefore, in subsequent studies, we conducted research on the effects of Araliasaponin A on PHDs family proteins and verified the dependence of Araliasaponin A on PHDs family proteins in regulating HIF-1α.

[0066] The gene heat map results of transcriptome analysis showed that among the egln family (genes encoding PHDs family proteins), Araliasaponin A could significantly upregulate the expression of egln1 (encoding PHD2 protein) gene, without affecting egln2 (encoding PHD1 protein) and egln3 (encoding PHD3 protein). In the validation of egln family genes by qRT-PCR, we also found that LPS significantly inhibited the expression of egln1 and egln3 genes in BMDM, but had no significant effect on egln2. However, Araliasaponin A could only restore the gene expression of egln1 in BMDM of the LPS-treated group. Similarly, Araliasaponin A could also significantly upregulate the protein level of PHD2 in BMDM of the LPS-treated group. Subsequently, to prove that the effect of Araliasaponin A in downregulating HIF-1α to inhibit pro-inflammatory activation of macrophages depends on the upregulation of PHD2, we used siRNA (small interfering RNA) targeting egln1. First, we used three interference fragments of si-egln1 on BMDM and verified the effectiveness by Western Blotting experiments. The results showed that the three interference fragments of si-egln1 could significantly downregulate PHD2 in BMDM, so we selected the third interference fragment of si-egln1 for subsequent experiments. The results showed that in si-egln1-treated BMDM, the effect of aralia saponin A on downregulating HIF-1α was offset. Similarly, in si-egln1-treated BMDM, the effect of aralia saponin A on downregulating iNOS, COX2 protein levels and inflammatory factors such as NO, IL-6 and TNF-α in the cell culture medium disappeared ( Fig.23 ). In summary, these experimental results indicate that Aralia saponin A upregulates the expression of the egln1 gene, restores the PHD2 / HIF-1α axis that is blocked in pro-inflammatory activated macrophages, and counteracts the pro-inflammatory activation of macrophages.

[0067] In the course of septic lung injury, the inflammatory response mediated by pro-inflammatory activation of macrophages is the main cause of lung dysfunction (pulmonary edema, cell death in the lungs, and loss of the pulmonary barrier). In the pro-inflammatory activation of macrophages, a large number of signaling pathways and life activities play an important role (such as NF-κB signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway, oxidative stress, cell pyroptosis, metabolic transformation, and cell autophagy, etc.). In this paper, we used transcriptomics analysis to find that aralia saponin A can significantly inhibit the HIF-1α signaling pathway in pro-inflammatory activated macrophages. At the same time, we proved in vivo and in vitro experiments that aralia saponin A inhibits macrophage pro-inflammatory activation by downregulating HIF-1α to fight septic lung injury. In the subsequent in-depth exploration of the mechanism of action, we found that aralia saponin A can specifically upregulate the expression of the egln1 gene, thereby restoring the blocked PHD2 / HIF-1α axis in pro-inflammatory activated macrophages, thereby exerting an anti-inflammatory effect. This is the first time that the anti-inflammatory mechanism of Araliasaponin A has been improved, providing a detailed theoretical basis for its future clinical application.

[0068] In the exploration of the anti-inflammatory mechanism of A. araliasaponin A, we applied transcriptomic analysis to BMDM before and after A. Before identifying the HIF-1α signaling pathway as the anti-inflammatory mechanism of A. araliasaponin A, we first screened other classical inflammatory signaling pathways. A recent study previously showed that A. araliasaponin A could significantly inhibit the phosphorylation of p65 in the NF-κB signaling pathway in fibroblast-like synoviocytes. Therefore, we also speculated in the early stage of the experiment that A. araliasaponin A might also inhibit the activity of the NF-κB signaling pathway in macrophages to produce anti-inflammatory effects. However, our results showed that in BMDM, the P value of A. araliasaponin A for the classical pro-inflammatory pathway NF-κB signaling pathway was 0.5859, which means that there was no significant difference. After a detailed comparison of our experimental basal conditions with that study, we found the root of the problem. In the samples for transcriptomics detection, we used 8μM of Aralia saponin A (Aralia saponin A can show obvious anti-inflammatory effects at this dose but does not affect the cell activity of macrophages). For fibroblast-like synoviocytes, the dose combination used by the other party was a gradient dose of 4μM, 8μM, and 16μM. Although this dose of Aralia saponin A significantly inhibited the phosphorylation of p65 in the NF-κB signaling pathway, their CCK-8 experimental results also showed that Aralia saponin A at a dose gradient of 4μM, 8μM, and 16μM can significantly inhibit the cell activity of fibroblast-like synoviocytes. That is, the application dose of Aralia saponin A needs to reach the level that affects the cell activity of fibroblast-like synoviocytes in order to downregulate the phosphorylation of p65, but the drug dose that affects cell activity is bound to cause deviations in the analysis of changes in intracellular signaling pathways. At the same time, it is well known that IκBα is an inhibitory protein for the NF-κB signaling pathway, and its phosphorylation will lead to its own degradation, thereby releasing p65 to enter the nucleus and activate the NF-κB signaling pathway. What is confusing is that in the other party's experimental results, while Aralia saponin A down-regulated p65 phosphorylation in fibroblast-like synoviocytes, it also inhibited the phosphorylation of IκBα. This phenomenon is obviously contrary to the phenomenon that Aralia saponin A down-regulated the phosphorylation of p65. Therefore, the other party's conclusion that Aralia saponin A can significantly inhibit the NF-κB signaling pathway in fibroblast-like synoviocytes may be incomplete or even contradictory.

[0069] In addition, compared with the NF-κB signaling pathway, Aralia saponin A can show a significant effect on the MAPK signaling pathway, which is also a classic pro-inflammatory signaling pathway. A study on ARC (ARA homologue, antibody-coupled nuclide drug) protecting cardiomyocytes from hypoxia-reoxygenation injury also showed that ARC can rescue cardiomyocyte apoptosis by upregulating HSP90 and inhibiting ERK phosphorylation. However, in the KEGG analysis of the MAPK signaling pathway, we found that Aralia saponin A showed an upregulating effect on the ERK signaling pathway, JNK signaling pathway, and p38 signaling pathway that promote inflammation in the MAPK signaling pathway, which is contrary to its actual anti-inflammatory effect. Although the effect of Aralia saponin A on the MAPK signaling pathway in our study is opposite to that of ARC, it is similar to the conclusion of an earlier study evaluating the anti-cancer activity of olefin-type triterpenoid extracts of Panax stipuleanatus (Wild Notoginseng). In this study, compounds 1 and 2, which are similar in structure to compound 8 ARA methyl ester, showed a significant upregulation of ERK signaling. Based on this, we believe that the failure of Aralia saponin A to inhibit MAPK signaling as expected may be due to the difference in chemical structure between Aralia saponin A and ARC. In summary, we did not select the MAPK signaling pathway as our research object in the previous selection of possible downstream signaling pathways of Aralia saponin A.

[0070] After hitting a wall in exploring the classic pro-inflammatory signaling pathway, we turned to the HIF-1α signaling pathway, which also underwent significant changes. Unlike the classic inflammatory signaling pathway, the regulation of the HIF-1α signaling pathway on the pro-inflammatory activation of macrophages mainly depends on its direct transcription of inflammatory genes such as nos2 and il-1β, and by upregulating the expression of glycolysis-related genes such as pfkfb3, hk2, pkm2, and ldha to promote macrophages to complete metabolic transformation (oxidative phosphorylation to aerobic glycolysis). Under normal circumstances, HIF-1α is constantly modified by hydroxylation by PHD2 family proteins, causing it to bind to VHL, undergo ubiquitination, and be degraded through the proteasome pathway. In a hypoxic environment, this hydroxylation modification is inhibited, causing the HIF-1α signaling pathway to be activated. Under inflammatory conditions, in addition to tissue hypoxia caused by low perfusion rate in tissues, blocked cell autophagy, activated MAPK signaling pathway, activated NF-κB signaling pathway, accumulation of ROS and stimulation of succinate will lead to activation of HIF-1α signaling pathway in macrophages (HIF-1α protein accumulation caused by multiple pathways such as gene transcription, blocked proteasome degradation pathway, blocked autophagy lysosome degradation pathway, etc.), thereby promoting pro-inflammatory activation of macrophages. In this study, we found that Aralia saponin A down-regulated HIF-1α by restoring the PHD2 / HIF-1α axis, thereby counteracting the pro-inflammatory activation of macrophages, but Aralia saponin A may still achieve the down-regulation of HIF-1α by affecting the above-mentioned other signaling pathways or life activities. If we want to fully clarify the mechanism of action of Aralia saponin A in down-regulating HIF-1α in pro-inflammatory activated macrophages, we need to study these signaling pathways and life activities one by one in subsequent research and exploration.

[0071] In the exploration of detailed molecular mechanisms, we found that araliasaponin A restores the PHD2 / HIF-1α axis by directly promoting the expression of the egln1 gene. At the same time, araliasaponin A does not affect the expression of egln1 homologous genes egln2 and egln3. This shows that araliasaponin A is specific for the regulation of the egln1 gene. However, how araliasaponin A achieves specific regulation of the egln1 gene remains a mystery. To deconstruct and explore this mystery, we can start by looking for transcription factors and corresponding signal pathways that have specific differences between the egln1 gene and the egln2 and egln3 genes. This is of great significance for the future research based on araliasaponin A as the basis for drug structure research, designing more targeted egln1 gene transcription agonists to restore the PHD2 / HIF-1α axis, and for the treatment of sepsis or other HIF-1α signaling pathway-related diseases (angiogenesis in tumors, liver fibrosis, etc.).

[0072] Combined with the research content of the present invention, Aralia saponin A is established as a new natural compound drug for treating septic lung injury. At the same time, the detailed molecular mechanism of Aralia saponin A in inhibiting pro-inflammatory activation of macrophages by restoring the PHD2 / HIF-1α axis, counteracting the occurrence and development of inflammation and improving septic lung injury is elucidated, providing a theoretical basis and research foundation for the clinical research and future application of Aralia saponin A.

Claims

1. A use of Aralia saponin A in the preparation of a drug for treating septic lung injury, characterized in that: The drug for treating septic lung injury uses Aralia saponin A as the only active ingredient or one of the active ingredients.

2. The use according to claim 1, characterized in that: The drug for treating septic lung injury improves septic lung injury by inhibiting the progression of inflammation.

3. The application according to claim 2, characterized in that: The drug for treating septic lung injury inhibits the pro-inflammatory activation of macrophages in sepsis.

4. The use according to claim 3, characterized in that: The drug for treating septic lung injury reduces the pro-inflammatory activation of macrophages by downregulating HIF-1α.

5. The use according to claim 4, characterized in that: The drug for treating septic lung injury restores the PHD2 / HIF-1α axis by enhancing egln1 gene expression, thereby reducing HIF-1α protein levels and inhibiting pro-inflammatory activation of macrophages.

6. The use according to claim 1, characterized in that: The drug for treating septic lung injury also includes pharmaceutically acceptable excipients.

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