Songorine inhibits oxidative stress-related inflammation through PI3K/AKT/NRF2 signaling pathway to alleviate lipopolysaccharide-induced septic acute lung injury

Abstract Objective The present study aimed to investigate the protective action and mechanism of songorine on sepsis-induced acute lung injury (ALI). Methods The sepsis-induced ALI mouse and cell models were established by lipopolysaccharide (LPS) induction. Lung injury was assayed by hematoxylin and eosin staining, lung injury score, and lung wet-to-dry (W/D) weight ratio. Apoptosis in lung tissues was evaluated by TUNEL assay, and the expression of apoptosis-related markers (Bcl2, Bax, and caspase-3) was measured by western blotting. Levels of pro-inflammatory factors and oxidative stress markers in the bronchoalveolar lavage fluid (BALF) of mice were measured by ELISA and RT-qPCR. The expression of PI3K/AKT/NRF2 pathway-related proteins was analyzed by western blotting. Results Songorine treatment at 40 mg/kg mitigated sepsis-induced ALI, characterized by improved histopathology, lung injury score, and lung W/D weight ratio (p < 0.05). Moreover, songorine markedly attenuated sepsis-induced apoptosis in lung tissues; this was evidenced by an increase in Bcl2 levels and a decrease in Bax and caspase-3 levels (p < 0.01). Also, songorine reduced levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β and MPO) and oxidative stress regulators (SOD and GSH) in the BALF of LPS-induced sepsis mice and RAW264.7 cells (p < 0.05). In addition, songorine upregulated the PI3K/AKT/NRF2 pathway-related proteins in LPS-induced sepsis mice and RAW264.7 cells (p < 0.05). Furthermore, LY294002 (a PI3K inhibitor) treatment reversed the protective effect of songorine on sepsis-induced ALI. Conclusion Songorine inhibits oxidative stress-related inflammation in sepsis-induced ALI via the activation of the PI3K/AKT/NRF2 signaling pathway.


Introduction
Sepsis is joint systemic inflammation in response to severe infection and trauma, with high mortality and long-term morbidity [1,2].The lung is highly vulnerable during sepsis, owing to its inherent susceptibility to infection and inflammation; therefore, acute lung injury (ALI) is one of the most common and severe complications [3].ALI is initially characterized by pulmonary inflammation and microvascular permeability and eventually progresses to acute respiratory distress syndrome [4].To our knowledge, there is no effective drug and therapeutic approach for sepsis-induced ALI.
Oxidative stress and the resulting inflammation are closely implicated in the pathogenesis of ALI.The imbalance between the production of reactive oxygen species (ROS) and the antioxidants results in oxidative stress and further promotes inflammation [5].Songorine is a C 20 -diterpene alkaloid from the root of Aconitum carmichaelii, exhibiting anti-inflammatory effects and low toxicity [6,7].Songorine can inhibit the generation of inflammatory cytokines, including IL-6, IL-1β, and TNF-α [8].Unlike nonsteroidal anti-inflammatory drugs, songorine exerted antioxidative activity without ulcerogenic effect [6].A previous study has shown that songorine prevents lipopolysaccharide (LPS)-evoked mitochondrial ROS production in septic heart injury [9]-Songorine is a critical bioactive ingredient of Shenfu injection for the treatment of sepsis [10].These pieces of evidence suggest a potential therapeutic avenue of songorine in sepsis-induced ALI that warrants further exploration.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central controller of oxidative stress and inflammatory response [11].A previous study indicated that songorine fosters cardiac mitochondrial biogenesis through the induction of Nrf2 during sepsis [9].Moreover, Li et al. demonstrated that obacunone mitigates ferroptosis in the context of LPS-induced ALI through the enhancement of Nrf2-dependent antioxidant responses [12].PI3K/Akt signaling can activate Nrf2, and crosstalk between them protects cells against inflammatory and oxidative damage [13].A previous study has demonstrated that PI3K/Akt signaling via Nrf2 protects against inflammation in hyperoxia-induced ALI [14].Moreover, boosting PI3K/Akt/mTOR and Nrf2/HO-1 signaling alleviates oxidative stress to prevent cyclophosphamide-induced ALI [15].For these reasons, PI3K/ Akt/Nrf2 signaling pathway may be a potential therapeutic target for songorine against sepsis-induced ALI by suppressing oxidative stress and inflammation.
The current study was designed to explore the effect of songorine attenuating LPS-induced septic ALI.We aimed to confirm the therapeutic efficacy of songorine on LPS-induced septic ALI by inhibiting oxidative stress and inflammation.Additionally, we revealed the underlying mechanism through which songorine treats LPS-induced septic ALI, involving regulation of the PI3K/Akt/Nrf2 signaling pathway.This research elucidates the therapeutic potential of songorine in addressing sepsis-induced ALI through its modulation of the PI3K/ Akt/Nrf2 signaling pathway, highlighting its prospective application in lung injury therapy.

Animal model establishment and treatment
Animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of The First Affiliated Hospital of Ningbo University.Healthy male C57BL/6 mice (6-8 weeks old) were obtained from Hubei Province Experimental Animal Center (Wuhan, China).The choice of male C57BL/6 mice is informed by their well-documented immune responses, genetic consistency, and susceptibility to LPS-induced septic lung injury [16].Mice were housed under specific pathogen-free conditions.All mice were randomly divided into four groups (n = 6 for each group): sham, LPS, LPS + Son (20), and LPS + Son (40) groups.In the LPS group, mice were intraperitoneally instilled with 10 mg/kg LPS for establishing a septic ALI model.For the LPS + Son (20) and LPS + Son (40) groups, mice were pretreated with 20 mg/ kg and 40 mg/kg songorine by intraperitoneal injection before 2 h of stimulation with LPS.We referenced the study by Li et al. as a guideline for the dose selection of songorine, wherein similar doses of songorine were employed to achieve desired pharmacological outcomes [9].After 12 h, lung tissues were removed from mice for subsequent experiments.

Hematoxylin and eosin (HE) staining
HE staining for lung tissues of mice was performed as previously described [17].The fixed lung tissues with 10% neutral buffered formalin were embedded in paraffin and sectioned into 5-µm slices.The sections were stained with hematoxylin and eosin for observation under a microscope.

Lung injury score
The severity of lung injury was assessed by a semiquantitative scoring standard [18].Four indicators (alveolar septal thickening, inflammation, hemorrhage, and edema) reflecting the severity of lung injury were blindly scored.Alveolar septal thickening, hemorrhage, and edema were characterized as follows: absent (score = 0), mild (score = 1), moderate (score = 2), severe (score = 3), and extensive (score = 4).Inflammation was quantified based on the total number of inflammatory cells per ×100 field view.The overall pathological scores of lung injury were analyzed using the non-parametric Kruskal-Wallis test.

Lung wet-to-dry (W/D) weight ratio
The Lung W/D ratio was used to evaluate the severity of pulmonary edema.Lung tissues were weighed immediately after removal from mice (wet weight) and then placed into a 60 °C oven for drying.After 48 h, the dried lung was re-weighed as dry weight.The ratio of wet to dry weight was calculated.

TUNEL assay
The cell apoptosis in lung tissues was measured by TUNEL staining assay.Lung tissues were fixed in 10% phosphate-buffered formalin and embedded into paraffin for cutting into 5-μm sections.The sections were incubated with a cocktail containing terminal deoxynucleotidyl transferase enzyme, and DAPI was used for nuclear counterstaining.The fluorescence was detected using a Leica DMI-3000B phase-contrast fluorescence microscope.

Enzyme-linked immunosorbent assay (ELISA)
Bronchoalveolar lavage fluid (BALF) was collected from the right lung of mice by lavage with 500 µL of phosphate-buffered saline (PBS) immediately after the end of ventilation.Levels of inflammatory cytokines (TNF-α, IL-6, IL-1β, and MPO) in bronchoalveolar lavage fluid (BALF) were measured using commercial ELISA kits.Levels of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in lung tissues were also measured by corresponding commercial ELISA kits (Jiancheng Bioengineering Institute, China).

Immunohistochemistry (IHC)
After dewaxing and rehydration, lung tissues were blocked by 3% H 2 O 2 for 30 min and subjected to citric acid retrieval at 100 °C for 20 min.Then, the tissues were blocked by bovine serum albumin and incubated with the primary antibodies anti-CD68 (1:100; ZM-0060, China) overnight and further incubated with the secondary antibody for 1 h.Next, tissues were stained by 3,3′-Diaminobenzidine and hematoxylin, followed by observation under a microscope.medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin sulfate in a humidified atmosphere with 5% CO 2 .Cells were incubated with gradient concentrations of songorine (1-60 μM) for 4 h.Then, cells were treated with 1 μg/mL LPS for 24 h.Some cells were treated with songorine and/or LY294002 (a PI3K inhibitor), followed by 1 μg/mL LPS for 24 h.

Cell viability
RAW264.7 macrophages were seeded into a 96-well plate with 100 μL culture medium.Various concentrations of songorine (1-60 μM) were used to treat macrophages with 1 μg/mL LPS stimulation.Next, the CCK8 reagent (#CK04, Dojindo, Japan) was added into each well to incubate for 2 h.Cell viability was reflected by the optical density value using a microplate reader at the wavelength of 450 nm.The IC50 value for songorine treating RAW264.7 macrophages was calculated and used for subsequent cell experiments.

Western blotting
Total proteins were extracted from lung tissues and RAW264.7 macrophages using ice-cold RIPA buffer containing protease inhibitors.Protein concentration was measured by the bicinchoninic acid method, and 30 μg of protein was loaded into 10% SDS-PAGE gel for electrophoresis.Afterward, proteins were transferred to PVDF membranes and blocked with 5% nonfat milk for 1 h.Then, the PVDF membranes were incubated with primary antibodies (Table S1) overnight at 4 °C.After being washed with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 h.After being washed thrice with TBST, protein bands were visualized by FluorChem Q and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).β-actin was used as a reference protein to calculate the relative expression of target proteins.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNAs in BALF, lung tissues and RAW264.7 macrophages were extracted with the TRIeasyTM Total RNA Extraction Reagent (Yeasen, China).Then, extracted RNA was reverse transcribed into cDNA with the Hifair® III 1st Strand cDNA Synthesis.Next, RT-qPCR was performed with the SYBR Green Real-Time PCR Master Mix (Yeasen, China) and detected on an Applied Biosystems.The relative mRNA levels were calculated using the 2 −ΔΔCt formula [19].GAPDH was applied as the internal reference gene.Primer sequences are listed in Table S2.

Statistical analysis
All experiments were performed at least three times.Prior to conducting parametric statistical tests, the normality of data distribution was confirmed using the Kolmogorov-Smirnov test.The data were analyzed by GraphPad Prism (software version 7.0) and are expressed as mean ± standard deviation.For two-group comparisons, an unpaired Student's t-test was used.One-way ANOVA followed by the Tukey-Kramer post hoc test was employed for multiple group comparisons.p < 0.05 was significantly different.

Songorine alleviates sepsis-induced ALI
A mouse model of septic ALI was established through LPS induction and was treated with either 20 or 40 mg/kg of songorine.HE staining showed severe lung injury in the LPS group, whereas treatment with songorine alleviated the lung injury in model mice (Figure 1(A)).Lung injury scores presented the same results with HE staining (p < 0.05; Figure 1(B)).Meanwhile, LPS-induced model mice had a higher lung W/D ratio than sham mice (p < 0.01).Treatment with songorine significantly reduced the lung W/D ratio of model mice (p < 0.05; Figure 1(C)).Moreover, cell apoptosis was detected in the lung tissues of mice.
Observations revealed a pronounced increase in TUNEL-positive cells in LPS-treated mice, indicating heightened apoptosis (p < 0.01).However, upon treatment with songorine, a marked decline in TUNEL-positive cells was evident (p < 0.05; Figure 1(D)).Additionally, western blotting revealed that the expression of anti-apoptotic protein Bcl2 was downregulated, whereas pro-apoptotic proteins (Bax and caspase-3) were upregulated in LPS-induced model mice (p < 0.01).Treatment with songorine reduced apoptosis in the lung tissues of the model mice, as evidenced by increased Bcl2 expression and decreased levels of Bax and caspase-3 (p < 0.01; Figure 1(E)).Of note, 40 mg/ kg songorine treatment showed better mitigative efficacy on septic ALI than 20 mg/kg songorine (p < 0.05; Figure 1(A-E)).

Songorine treatment inhibits inflammation and oxidative stress in septic ALI mice
Further, the effects of songorine on inflammation and oxidative stress in septic ALI were investigated.ELISA and RT-qPCR analyses showed that the levels of inflammatory factors (TNF-α, IL-6, IL-1β, and MPO) were higher in the BALF of LPS-induced model mice compared to the sham mice (p < 0.01).Treatment with songorine significantly reduced levels of these inflammatory factors in LPS-induced model mice (p < 0.05; Figure 2(A,B)).Also, CD68 is a histochemical/cytochemical marker of inflammation [20], which showed higher expression in LPS-induced model mice than in sham mice.After treatment with songorine, the CD68 expression was reduced in the model mice (Figure 2(C)).Furthermore, oxidative stress in the lung tissues of mice was detected.ELISA showed that SOD and GSH level was decreased, and MDA (a lipid peroxidation biomarker) level was increased in LPS-induced model mice compared to sham mice (p < 0.01).Treatment with songorine increased the SOD and GSH levels but decreased the MDA level in model mice (p < 0.05; Figure 2(D)).Additionally, the expression of antioxidant reactive elements (SOD2 and CAT) was downregulated in LPS-induced model mice, which was reversed by the treatment with songorine (p < 0.05; Figure 2(E)).Also, 40 mg/ kg songorine treatment showed better mitigative efficacy on septic ALI-induced inflammation and oxidative stress than 20 mg/kg songorine (p < 0.05; Figure 2(A-E)).

Songorine suppresses inflammation and oxidative stress in LPS-induced RAW264.7 cells
The mitigative effects of songorine on inflammation and oxidative stress induced by septic ALI were validated in vitro.The mouse monocyte cell line RAW 264.7 was treated with 1-60 μM songorine and then stimulated by 1 μg/mL LPS.Using the CCK-8 assay, we determined that the IC50 value for songorine treatment in RAW 264.7 cells was 18.97 μM (Figure 4(A)).Subsequently, cells were treated with 15 μM songorine to detect inflammation and oxidative stress.As shown in Figure 4(B-D), levels of MDA, TNF-α, IL-1β, IL-6, and MPO have increased in LPS-induced RAW 264.7 cells, significantly decreased by treatment with songorine (p < 0.01; Figure 4(B,C)).For oxidative stress, we found that SOD2, GSH, and   CAT expression levels were reduced in LPS-induced RAW 264.7 cells, which was reversed by treatment with songorine (p < 0.01; Figure 4(B-D)).

Songorine represses inflammation and oxidative stress by activating the PI3K/AKT/NRF2 signaling pathway in LPS-induced RAW264.7 cells
Further, the mechanism of songorine protecting against septic ALI involved in the PI3K/AKT/NRF2 signaling pathway was verified in vitro.Consistent with the results in vivo, the expression of p-PI3K/PI3K, p-AKT/AKT, HO-1, NQO1, and NRF2 in the nucleus was downregulated in LPS-induced RAW 264.7 cells, while the expression of NRF2 was upregulated, which was reversed by treatment with songorine (p < 0.01; Figure 5(A)).Moreover, LY294002 (a PI3K inhibitor) addition offset the stimulatory effect of songorine on the PI3K/AKT/NRF2 signaling pathway in LPS-induced RAW264.7 cells (p < 0.01; Figure 5(B)).Functionally, the addition of LY294002 weakened the inhibitory effects on inflammation and oxidative stress in LPS-induced cells, as evidenced by increased levels of MDA, TNF-α, IL-1β, IL-6, and MPO, along with decreased levels of SOD2, GSH, and CAT (p < 0.01; Figure 5(C-E)).

Discussion
ALI is a critical indicator of mortality risk in patients with sepsis [21].Numerous studies have concentrated on ALI in sepsis.However, there remains no effective strategy for preventing or treating lung function impairment in septic conditions.This challenge primarily arises from the complex pathogenesis of sepsis [22].Therefore, the need for novel treatments for sepsis-induced ALI is pressing.Songorine has been shown to promote mitochondrial biogenesis and enhance antioxidant defense through NRF2 induction in sepsis [9].A previous study has demonstrated that inhibiting PI3K/AKT and CYP2E/NRF2/ROS signaling alleviates LPS-induced sepsis by suppressing oxidative stress-related inflammation [23].The current study found that songorine attenuated sepsis-induced ALI in mice via its anti-apoptotic, anti-inflammatory, and anti-oxidative effects through PI3K/AKT/ NRF2 signaling pathway.The key findings are as follows: first, songorine significantly alleviated lung injury in LPS-induced sepsis mice, as evidenced by the changes in histopathology, the decreased lung injury score, and W/D ratio; second, songorine reduced cell apoptosis in lung tissues of LPS-induced sepsis mice; third, the levels of proinflammatory cytokines in the BALF in LPS-induced sepsis mice and RAW264.7 cells were decreased by songorine treatment; fourth, songorine alleviated oxidative stress in LPS-induced sepsis mice and RAW264.7 cells; finally, songorine activated the PI3K/AKT/NRF2 signaling pathway in LPS-induced sepsis mice and RAW264.7 cells.
During the inflammatory response, the NRF2 pathway can be activated by multiple signal kinases.PI3K/AKT pathway is an essential upstream regulator of Nrf-2/HO-1 [11].The interaction of the PI3K/Akt and NRF2 signaling pathways can safeguard cells from inflammatory and oxidative harm.Also, the PI3K/AKT signaling pathway could mediate cell survival in many cell types, and many studies have shown that the PI3K/ AKT signaling pathway plays a vital role in treating ALI [24,25].These studies suggest that the PI3K/AKT/NRF2 signaling pathway is an essential regulator for ALI.
Studies have shown that songorine possesses antiinflammatory and antioxidant properties [7].Compared to other agents like buformin for treating sepsis-induced acute lung injury, the natural compound songorine offers higher safety, fewer side effects, and easier accessibility [9,26].Zyuz'kov et al. observed that songorine could stimulate the proliferation and differentiation of mesenchymal progenitor cells by activating PI3K signaling [27].However, the protective effect and mechanism involving PI3K signaling of songorine against ALI have yet to be elucidated.The current study used LPS to induce sepsis and ALI in mice.LPS-induced sepsis is a commonly used animal model for the pharmacological and pathological studies of sepsis treatment.LPS induction leads to an intense inflammatory response, culminating in the significant production and release of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β, which are directly implicated in the onset of septic ALI [28].Our study found that severe ALI was present after LPS induction, including pathological injury in lung tissue, increased lung W/D ratio, cell apoptosis, and inflammatory response.However, songorine administration markedly reduced LPS-induced lung injury, apoptosis, inflammation, and oxidative stress.The levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were decreased in LPS-induced sepsis mice treatment with songorine.Similar effects of songorine were also presented in LPS-induced sepsis mice and RAW264.7 cells.Meanwhile, we found that songorine upregulated the expression of PI3K/AKT/NRF2 signaling pathway-related proteins.Furthermore, LY294002 (a PI3K inhibitor) treatment reversed the protective effect of songorine on sepsis-induced ALI.These results indicated that songorine protects against sepsis-induced ALI via activating the PI3K/AKT/NRF2 signaling pathway.

Conclusion
Our study demonstrated that songorine significantly alleviated lung injury, inflammation, and oxidative stress in LPS-induced septic ALI.Moreover, our findings further delineate the activation of the PI3K/AKT/NRF2 signaling pathway by songorine, pointing toward its role in curtailing oxidative stress-induced inflammation.Despite these promising results, it is important to maintain a circumspective approach, considering that the research does not comprehensively address the possible adverse effects associated with songorine use.This aspect forms a crucial frontier for further research, seeking to ensure a balanced understanding of both its therapeutic efficacy and safety profile.Besides, we used only male mice to control for gender-related variations in the results.Future studies will consider including female mice to offer more comprehensive insights.This research serves as a foundational step in elucidating the protective roles orchestrated by songorine in the context of sepsis-induced ALI, highlighting a promising therapeutic avenue.In integrating these insights with existing literature, we pave the way for future investigations targeting the PI3K/AKT/NRF2 signaling axis in ALI.The upstream and downstream components of PI3K/AKT/ NRF2 pathway affected by songorine will be further explored.Consequently, songorine emerges as a promising therapeutic candidate for sepsis-induced ALI.

RAW264. 7
macrophages were obtained from Procell Life Science and Technology Co., Ltd.(China) with STR profile and cultured in Roswell Park Memorial Institute-(RPMI-) 1640

Figure 2 .
Figure 2. Songorine (Son) treatment inhibits inflammation and oxidative stress in septic acute lung injury mice.(a,B) Enzyme-linked immunosorbent assay (ElISa) and rt-qpcr for detecting the levels of inflammatory cytokines (tnF-α, Il-6, Il-1β, and mpo) in bronchoalveolar lavage fluid of mice.(c) Immunohistochemical staining revealing cd68 expression, a marker of inflammation, in lung tissues (scale bar = 200 μm).(d) ElISa for detecting oxidative stress-related biomarkers (Sod, gSh, and mda) in lung tissues of mice.(E) rt-qpcr analysis evaluated the relative mrna expression of key antioxidant genes, Sod2 and cat, in mice.the data are presented as means ± standard deviation (n = 6/group).two-group differences assessed by student's t-test; multiple group comparisons used one-way anoVa with tukey-Kramer post hoc.**p < 0.01 versus sham group; #p < 0.05 and ##p < 0.01 versus lpS group; ^p < 0.05 and ^^p < 0.01 versus lpS + Son (20) group.