Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2023 Mar 30;114:154753. doi: 10.1016/j.phymed.2023.154753

The effects and mechanisms of the anti-COVID-19 traditional Chinese medicine, Dehydroandrographolide from Andrographis paniculata (Burm.f.) Wall, on acute lung injury by the inhibition of NLRP3-mediated pyroptosis

Zhichen Pu a,b,c,1, Bangzhi Sui d,1, Xingwen Wang a,1, Wusuan Wang e, Lingling Li f,⁎, Haitang Xie a,⁎
PMCID: PMC10060206  PMID: 37084628

Abstract

Background

Dehydroandrographolide (Deh) from Andrographis paniculata (Burm.f.) Wall has strong anti-inflammatory and antioxidant activities.

Purpose

To explore the role of Deh in acute lung injury (ALI) of coronavirus disease 19 (COVID-19) and its inflammatory molecular mechanism.

Methods

Liposaccharide (LPS) was injected into a C57BL/6 mouse model of ALI, and LPS + adenosine triphosphate (ATP) was used to stimulate BMDMs in an in vitro model of ALI.

Results

In an in vivo and in vitro model of ALI, Deh considerably reduced inflammation and oxidative stress by inhibiting NLRP3-mediated pyroptosis and attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis through the suppression of ROS production by inhibiting the Akt/Nrf2 pathway. Deh inhibited the interaction between Akt at T308 and PDPK1 at S549 to promote Akt protein phosphorylation. Deh directly targeted PDPK1 protein and accelerated PDPK1 ubiquitination. 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP and 223-ASP may be the reason for the interaction between PDPK1 and Deh.

Conclusion

Deh from Andrographis paniculata (Burm.f.) Wall presented NLRP3-mediated pyroptosis in a model of ALI through ROS-induced mitochondrial damage through inhibition of the Akt/Nrf2 pathway by PDPK1 ubiquitination. Therefore, it can be concluded that Deh may be a potential therapeutic drug for the treatment of ALI in COVID-19 or other respiratory diseases.

Keywords: Dehydroandrographolide, COVID-19, PDPK1, Pyroptosis, Ubiquitination

Graphical abstract

Image, graphical abstract

Open in a new tab

Introduction

Over the past few decades, coronaviruses (CoVs) have posed a significant threat to global public health (Albert et al., 2021). Facing the rapid spread of novel coronavirus acute lung injury (ALI) (COVID-19) caused by the novel coronavirus SARS-CoV-2 since December 2019 has induced growing panic (Amit et al., 2021). Compared with previous outbreaks of SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), COVID-19 shows some uniqueness in addition to a similar genome, in vivo replication kinetics and biological properties. Pneumonia is a common respiratory disease, with the main manifestations of rapid onset, severe symptoms, and many complications (Fenollar et al., 2021; Bora et al., 2022). Moreover, pneumonia easily causes respiratory and circulatory system failure (Fenollar et al., 2021). Once pneumonia develops into severe pneumonia, it will seriously endanger the lives of children and even cause death (Fenollar et al., 2021; Li et al., 2022).

COVID-19 is a global pandemic disease that has affected hundreds of millions of people worldwide. Most patients with COVID-19 may have mild to moderate symptoms such as cough and shortness of breath in the first week, and some patients may show extensive pneumonia, ultimate organ failure and diffuse intravascular coagulation in approximately three weeks (Fenollar et al., 2021; Li et al., 2023). The main cause of death from COVID-19 is acute lung injury (ALI). Novel coronavirus (SARS-CoV-2) invades alveolar epithelial cells and is expressed in a large number of alveolar type II cells, leading to pathological changes in alveolar epithelial cells, immune overactivation leading to an inflammatory factor storm, and the occurrence of ALI.

ALI is a critical disease syndrome with a mortality of 40% ∼ 60%. The clinical features of ALI are refractory hypoxemia and progressive dyspnea, and its pathological features are severe inflammation, endothelial injury, pulmonary edema and extensive thrombosis of capillaries around the alveoli (Zhang et al., 2022a; Albert et al., 2021). ALI includes several pathological conditions, such as trauma, pneumonia and septic shock. Imaging data show that most patients have spotted and ground glass shadows in both lungs. IL-1β is considered a key factor in the pathogenesis of cytokine storms in lung injury in patients with COVID-19. Inflammation can also cause endothelial cell damage, leading to coagulation disorder, further enhancing the release of inflammatory factors, forming a positive feedback loop of inflammation coagulation, and further aggravating lung injury (Albert et al., 2021).

As one of the important diseases threatening human health, ALI refers to lung infections caused by pathogens such as bacteria and viruses (Bordas-Martinez et al., 2021). Its severity depends on the severity and spread of lung inflammation (Climans et al., 2022). Manifestations of severe ALI include severe hypoxemia, acute respiratory failure, hypotension, and shock (Gallo González et al., 2021). In severe ALI, inflammatory factors are produced in lung tissue, and the body's inflammatory response is overactivated, resulting in a systemic inflammatory response. Therefore, anti-inflammatory drugs have a positive effect on relieving severe ALI (Gallo González et al., 2021).

ALI has been shown to cause inflammation and excessive production of reactive oxygen species (ROS). (Bonaventura et al., 2022). Due to the activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, the release of intracellular inflammatory factors is promoted, which ultimately leads to pyroptosis, the programmed death of inflammatory cells discovered in recent years (Li et al., 2020a; Jung and Lee, 2022; Machado et al., 2022). Due to the activation of Caspase-1 or Caspase-4/5/11 and cleavage of GSDMD (Gasdermin D), cell membrane pores are formed, and the cell body gradually swells. Finally, the cell membrane ruptures, and the cell contents are released, causing cell death (Li et al., 2020a; Machado et al., 2022).

Protein 3-phosphoinositide-dependent protein kinase 1 (PDPK1) is a 67 kD silk/threonine protein kinase and an important member of the serine/threonine protein kinase family (Li et al., 2019). PDPK1 promotes Akt phosphorylation (p-Akt), activates or inhibits the expression of target genes in downstream pathways, and plays an important role in apoptosis (Jeong et al., 2019; Li et al., 2019). Inhibition of PDPK1 expression can regulate the PI3K/Akt signaling pathway, thereby regulating apoptosis (Li et al., 2019). PDPK1 contributes a pro-inflammatory response to primordial follicle activation (Xiao et al., 2017). Other studies have shown that the proliferation and remodeling of the pulmonary artery or ALI can be inhibited by downregulating the expression of PDPK1 (Xiao et al., 2017; Yang et al., 2021).

Andrographis paniculata (Burm.f.) Wall is a common variety of traditional Chinese medicine (TCM) (Che et al., 2019). It is mainly used for the treatment of upper respiratory tract infection, bronchial asthma, viral pneumonia and diarrhea (Che et al., 2022). Dehydroandrographolide (Deh, Andrographis paniculata (Burm.f.) Wall) is one of the main components of Andrographis paniculata (Burm.f.) Wall (Guo et al., 2020). Deh's pharmacological effect is considered stronger than that of Andrographis paniculata (Burm.f.) Wall in some aspects (Huo et al., 2021). Several experiments have confirmed that Deh has pharmacological effects, such as anti-inflammatory and antioxidant activities (Liu et al., 2019b; Guo et al., 2020; Huo et al., 2021). However, its pharmacological mechanism remains unclear. Here, we investigated the effects of Deh on ALI in COVID-19 and its molecular mechanisms of inflammation.

Material and methods

Animal experiment

C57BL/6 mice (male, 5−6 weeks, 18−20 g) were obtained from the Animal Testing Center of Qinglongshan (Nanjing, Suzhou, China) and approved by the Animal Care and Use Committee of Yijishan Hospital of Wannan Medical College (LLSC-2022-134). All mice were randomly assigned to experimental groups and then anesthetized using 50 mg/kg pentobarbital sodium. Mice in the sham group (n = 6) were injected with normal saline, and mice in the ALI model group (n = 10) were injected with LPS (2 mg/kg, Sigma‒Aldrich) for 24 h according to the literature (Pu et al., 2022). The lung injury score and W/D rate were measured according to the literature (Pu et al., 2022). Mice in the Deh group (n = 10) received 12.5, 25, or 50 mg/kg Deh (SMB00350, ≥98%, Sigma‒Aldrich) or 1 mg/kg dexamethasone (Dex, D1756, ≥98%, Sigma‒Aldrich) for 24 h and then induced into the ALI model and 12.5, 25, or 50 mg/kg Deh for 24 h.

Mice in the PI3K inhibitor group (n = 10), ROS agonist group (n = 10), mitochondrial damage agonist group, and NLRP3 agonist group (n = 10) were pretreated with Deh (25 mg/kg) for 24 h, and then the ALI model was induced by treatment with Deh (25 mg/kg), LY294002 (20 mg/kg), urolithin C (10 mg/kg), rotenone (1.5 mg/kg), and nigericin sodium salt (4 mg/kg) for 24 h.

In vitro model

Murine bone marrow-derived macrophage (BMDM) cells were extracted and induced into an in vitro model of ALI as described in the literature (Pu et al., 2022). BMDMs were isolated from C57BL/6 mice and induced with 30% L929 cell-conditioned medium and 20% FBS for 1 week. BMDMs were kept in RPMI 1640 containing 5% L929 cell-conditioned medium and 10% FBS for 16 h. In the Deh treatment group, BMDMs were induced with 10, 20 or 40 μM Deh for 2 h, LPS (500 ng/mL, Sigma) for 4 h, and then pulsed with ATP (1 mM, Sigma) for 30 min.

In the PI3K inhibitor group or PI3K agonist group, BMDMs were stimulated with 0.5 μM LY294002 +20 μM Deh for 2 h or 10 μM YS-49 monohydrate +20 μM Deh for 2 h and then induced into an in vitro model.

In the ROS agonist group or ROS inhibitor group, BMDMs were stimulated with 10 μM urolithin C and 20 μM Deh for 2 h or 1 µM fulvene-5 and 20 μM Deh for 2 h and then induced into an in vitro model.

In the mitochondrial damage agonist group or mitochondrial damage inhibitor group, BMDMs were stimulated with 2.5 μM rotenone and 20 μM Deh for 2 h or 10 μM BI-6C9 and 20 μM Deh for 2 h and then induced into an in vitro model.

In the NLRP3 agonist group or NLRP3 inhibitor group, BMDMs were stimulated with 10 nM nigericin and 20 μM Deh for 2 h or 5 μM INF39 and 20 μM Deh for 2 h and then induced into an in vitro model.

Histological examination and immunofluorescence, cell viability assay and LDH activity levels

After treatment, mice were anesthetized using 50 mg/kg pentobarbital sodium, peripheral blood was collected from the caudal vein, and then the mice were sacrificed using decapitate. Left lung tissue samples were fixed in 4% paraformaldehyde for 24 h. Histological examination and immunofluorescence were executed according to the literature (Pu et al., 2022). After treatment with CA at 0, 12, 24, 48 or 72 h, cell viability was determined by the MTT assay as described in the literature (Xiao et al., 2013). Absorbance was measured at 490 nm using a fluorescence reader (Synergy H1 Microplate Reader, Bio Tek, Winooski). LDH activity levels were determined by an LDH activity kit (C0016, Beyotime).

ELISA, ROS production, and JC-1 assay

Calcein-AM/CoCl2 (C2013FT, Beyotime), JC-1 Assay (C2003S, Beyotime), calcein/PI rate (C2015S, Beyotime) and ROS Production (S0033S, Beyotime) according to the literature (Pu et al., 2022). IL-1β (H002), IL-6 (H007-1-1), TNF-α (H052-1), MDA activity level (A003-1-2), SOD (A001-3-2), GSH (A001-3-2) and GSH-PX (A005-1-2) activity level kits were purchased from Nanjing Jiancheng Bioengineering Research Institute and executed according to the literature (Pu et al., 2022).

Flow cytometry and electron microscopy

Annexin V-FITC/PI kits (BB-4101) were purchased from Beibokit, and apoptotic cells were analyzed by a BD Accuri C6 plus flow cytometer (BD Biosciences, San Jose, USA). Flow cytometry and electron microscopy were executed according to the literature (Xu et al., 2021) using a Hitachi H7650 transmission electron microscope (Tokyo, Japan) at 80 kV.

Western blotting analysis and immunofluorescence

Western blotting analysis and immunofluorescence were executed as described in the literature (Pu et al., 2022). The membranes were p-Akt (Ser473, 4060, 1:1000, Cell Signaling Technology, Inc.), NLRP3 (sc-66846, 1:500, Santa Cruz), AKT (4691, Cell Signaling Technology, Inc.), caspase-1 (sc-1780, 1:500, Santa Cruz), Nrf2 (ab62352, 1:1000, Abcam), and β-actin (BS6007MH, 1:5000, Bioworld Technology, Inc.) at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5000, Santa Cruz) for 1 h at 37 °C. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using Image Lab 3.0 (Bio-Rad Laboratories, Inc.

Cells were incubated at 4 °C overnight after blocking with PBS supplemented with 5% BSA for 2 h at room temperature. The cells were incubated with Alexa Fluor® 488/555 secondary antibody for 2 h at room temperature and then stained with DAPI for 15 min in the dark.

Statistical analysis

P values lower than 0.05 were considered significant. Data were expressed as the mean ± standard error of the mean (SEM) using GraphPad Prism 8 and evaluated using Student's t test or one-way analysis of variance (ANOVA) followed by Tukey's posttest.

Results

Deh significantly reduced inflammation and oxidative stress to induce lung injury in vivo and in vitro

The effect of Deh on lung injury in a mouse model of ALI was first investigated. Exposure to Deh resulted in a dose-dependent decrease in the lung injury score and W/D rate in a mouse model of ALI (Fig. 1 A and B). Medium or high concentrations of Deh significantly decreased inflammatory factors (IL-1β, IL-6 and TNF-α), inhibited oxidative stress-related factors (MDA activity level), and increased antioxidative activities (SOD, GSH and GSH-PX activity levels) in a mouse model of ALI (Fig. 1C–I). According to the above results, Deh protected against lung injury in vivo and in vitro by inhibiting inflammation and oxidative stress.

Fig. 1.

Fig 1

Open in a new tab

Deh made considerabale reduction of inflammation and oxidative stress to present lung injury in vivo model of ALI. Lung injury and HE staining (A), W/D rate (B), IL-1β (C), IL-6 (D), TNF-α (E), MDA (F), SOD (G), GSH (H), GSH-PX (I) levels in the tissue. Sham, control mice group; Model, ALI mice group; Low/Med/High, ALI mice with 7.5, 15, 30 mg/kg of CA group; Dex, ALI mice with 2 mg/kg of Dexamethasone. **p < 0.01 compared with control mice group; ## p < 0.01 compared with ALI mice group. Number of experiments = 6.

Deh decreased pyroptosis in vivo and in vitro models of ALI by pyroptosis

In an in vivo or in vitro model of ALI, the function of Deh in programmed cell death was confirmed. The results showed that in cells treated with Deh, cell viability was increased, and the LDH activity level and apoptosis rate were reduced in a dose-dependent manner (Fig. 2 A–C). Meanwhile, treatment with increasing concentrations of Deh inhibited a dose-dependent increase in the IL-1α level and calcein/PI activation rate in an in vitro model of ALI (Fig. 2D and E). In addition, Deh suppressed the protein expression of GSDMD in both in vivo and in vitro models of ALI (Fig. 2F and G). These data suggested that Deh can inhibit programmed cell death in the ALI model, namely, pyroptosis. However, its mechanism is unclear.

Fig. 2.

Fig 2

Open in a new tab

Deh decreased programmed cell death in vivo or vitro model of ALI by pyroptosis. Cell viability (A), LDH activity (B), apoptosis rate (C), proportions of PI positive cells (D), IL-1α levels (E) in vitro model; GSDMD protein expression in mice model (F) and in vitro model (G). Sham, control mice group; Model, ALI mice group; Low/Med/High, ALI mice with 7.5, 15, 30 mg/kg of CA group. Control, control group; Control, control group; LPS+ATP, vitro model of ALI group; Low/Med/High, vitro model of ALI with 10, 20 or 40 μM of CA group. **p < 0.01 compared with control mice or control group; ## p < 0.01 compared with ALI mice or vitro model of ALI group. Number of experiments = 3.

Deh suppressed NLRP3-mediated pyroptosis in vivo and in vitro models of ALI

NLRP3 plays a role in the regulation of pyroptosis in a model of ALI (Chen et al., 2021; Sun et al., 2021; Jung and Lee, 2022). The experiment determined the role of NLRP3 in the Deh-induced reduction in pyroptosis in a model of ALI. According to Western blot and immunohistochemical analysis, Deh suppressed NLRP3 and Caspase-1 protein expression in the lung tissue of mice with ALI (Fig. 3 A and B). To confirm the involvement of NLRP3 in Deh-reduced pyroptosis, mice with ALI were treated with an NLRP3 agonist (4 mg/kg nigericin sodium salt) and Deh. The NLRP3 agonist induced the inhibition of NLRP3, Caspase-1 and GSDMD protein expression in the lung tissue of mice with ALI by Deh (Fig. 3C). Furthermore, the NLRP3 agonist Deh significantly increased the inhibitory effect on lung injury scores and the W/D ratio (Fig. 3D–F). Furthermore, the loss of inflammatory factors induced by Deh was significantly enhanced by the NLRP3 agonist (Fig. 3F–I).

Fig. 3.

Fig 3

Open in a new tab

Deh suppressed NLRP3-mediated pyroptosis in vivo or vitro model of ALI. NLRP3/Caspase-1 protein expression (A), NLRP3 expression (Immunohistochemistry, B) in ALI mice; NLRP3/Caspase-1/GSDMD protein expressions (C), HE staining (D), Lung injury (E), W/D rate (F), IL-1β (G), IL-6 (H), TNF-α (I) in ALI mice with CA+NLRP3 agonist; NLRP3/Caspase-1 protein expression (J) in vitro model; NLRP3/Caspase-1/GSDMD protein expressions (K), IL-1β (L), IL-6 (M), TNF-α (N) in vitro model by CA+ NLRP3 agonist/ NLRP3 inhibitor. Sham, control mice group; Model, ALI mice group; Low/Med/High, ALI mice with 7.5, 15, 30 mg/kg of CA group. Control, control group; Control, control group; LPS+ATP, vitro model of ALI group; Low/Med/High, vitro model of ALI with 10, 20 or 40 μM of CA group; Vector, control; NLRP3 a, NLRP3 agonist; Chicoric acid+Model, ALI mice with 15 mg/kg of CA; CA, 20 μM of CA group; NLRP3 a, NLRP3 agonist; NLRP3 i, NLRP3 inhibitor. **p < 0.01 compared with control mice or 15 mg/kg of CA group; ## p < 0.01 compared with ALI mice or 20 μM of CA+ vitro model group. Number of experiments = 3.

Moreover, Deh decreased the expression of NLRP3 and Caspase-1 proteins in an in vitro model of ALI (Fig. 3J). In the in vitro model by Deh, NLRP3 agonist (10 nM nigericin) or NLRP3 inhibitor (5 μM INF39) was added to identify the role of NLRP3-mediated pyroptosis in the effect of Deh on ALI. Conversely, treatment with the NLRP3 agonist resulted in significant decreases in the expression of Deh-suppressed NLRP3, Caspase-1 and GSDMD protein in an in vitro model (Fig. 3K). In the in vitro model, it was verified that the NLRP3 inhibitor increased the inhibitory effect of Deh on the protein expression of NLRP3, Caspase-1 and GSDMD (Fig. 3K), and the NLRP3 agonist also decreased the anti-inflammatory and antioxidative effects of Deh in the in vitro model (Fig. 3L–N). Nevertheless, the NLRP3 inhibitor enhanced the anti-inflammatory and antioxidative effects of Deh in an in vitro model (Fig. 3L–N). Deh inhibited NLRP3-mediated pyroptosis in a model of ALI.

Deh attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis

To further investigate the molecular mechanism of Deh in NLRP3-mediated pyroptosis in a model of ALI, the attenuation effect of Deh on mitochondrial damage in vivo or in vitro was studied. Deh significantly enhanced the level of JC-1 disaggregation and calcien-AM/CoCl2 and recovered the mitochondrial structure in an in vitro model of ALI (Fig. 4 A–C). As illustrated in Fig. 4D and E, Deh induced MFN2 protein expression and suppressed MCU protein expression in an in vitro model and mice with ALI.

Fig. 4.

Fig 4

Open in a new tab

Deh attenuated mitochondrial damage in vivo or vitro model of ALI. JC-1 disaggregation (A), calcein-AM/CoCl2 assay (B), mitochondrion (Electron microscope, C), MFN2 and MCU protein expression in vitro model (D); MFN2 and MCU protein expression in mice model (E). Sham, control mice group; Model, ALI mice group; Low/Med/High, ALI mice with 7.5, 15, 30 mg/kg of CA group. Control, control group; LPS+ATP, vitro model of ALI group; Low/Med/High, vitro model of ALI with 10, 20 or 40 μM of CA group. **p < 0.01 compared with control mice or control group; ## p < 0.01 compared with ALI mice or vitro model of ALI group. Number of experiments = 3.

The experiment elucidated the role of mitochondrial damage in the anti-inflammatory effects of Deh on ALI. A mitochondrial damage agonist (1.5 mg/kg rotenone) reduced the inhibitory effect of Deh on the protein expression of NLRP3, Caspase-1 and GSDMD in mice with ALI (Fig. 5 A). Additionally, rotenone markedly increased the lung injury score and W/D rate, increased inflammation factors and oxidative stress-related factors, and decreased antioxidative activities in mice with CA-induced ALI (Fig. 5B–G).

Fig. 5.

Fig 5

Open in a new tab

Deh attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis in vivo or vitro model of ALI. NLRP3/Caspase-1/GSDMD protein expressions (A), W/D rate (B), HE staining (C), Lung injury (D), IL-1β (E), IL-6 (F), MDA (G) in ALI mice with CA+ mitochondrial damage agonist; NLRP3/Caspase-1/GSDMD protein expressions (H), Cell viability (I), LDH activity (J), IL-1β (K), IL-6 (L), ROS production (M), apoptosis rate (N), calcein-AM/CoCl2 assay (O), proportions of PI positive cells (P), mitochondrion (Electron microscope, Q), JC-1 disaggregation (R). Vector, control; Agonist, mitochondrial damage agonist; Chicoric acid+Model, ALI mice with 15 mg/kg of CA; Control, control group; CA, 20 μM of CA group; Agonist, mitochondrial damage agonist; inhibitor, mitochondrial damage inhibitor; LPS+ATP, vitro model of ALI group; **p < 0.01 compared with ALI mice with 15 mg/kg of CA group or vitro model of ALI group; ## p < 0.01 compared with 20 μM of CA+ vitro model group. Number of experiments = 3.

After treatment with Deh, the addition of a mitochondrial damage inhibitor (10 μM BI-6C9) or mitochondrial damage agonist (2.5 μM rotenone) regulated mitochondrial damage in an in vitro model of ALI. Rotenone markedly reduced the anti-inflammatory effects of Deh on NLRP3/Caspase-1/GSDMD protein expression, inflammation and ROS-induced oxidative stress, mitochondrial damage and pyroptosis in an in vitro model (Fig. 5H–R). These results suggested that Deh prevented mitochondrial damage-dependent pyroptosis in an in vivo or in vitro model of ALI by suppressing the NLRP3 inflammasome.

Deh weakened ROS production in mitochondria to suppress NLRP3-mediated pyroptosis

ROS production in mitochondria plays a role in the regulation of pyroptosis (Wang et al., 2019; Wang et al., 2020; An et al., 2021), further elucidating the molecular mechanism of Deh-prevented pyroptosis. The ROS agonist (10 mg/kg of Urolithin C) suppressed NLRP3/Caspase-1/GSDMD protein expression, promoted the lung injury score and W/D rate, and increased inflammation in mice with ALI by Deh (Fig. 6 A and C–H).

Fig. 6.

Fig 6

Open in a new tab

Deh weakened ROS production in mitochondrion to suppress NLRP3-mediated pyroptosis. NLRP3/Caspase-1/GSDMD protein expression (A) in ALI mice with CA+ROS agonist; NLRP3/Caspase-1/GSDMD protein expression (B) in vitro model with CA+ROS agonist/CA+ROS inhibitor; Lung injury (C), HE staining (D), W/D rate (E), IL-1β (F), IL-6 (G), TNF-α (H) in ALI mice with CA+ROS agonist; cell viability (I), LDH activity (J), IL-1β (K), IL-6 (L), TNF-α (M) in vitro model with CA+ROS agonist/CA+ROS inhibitor. Vector, control; ROS a, ROS agonist; Chicoric acid+Model, ALI mice with 15 mg/kg of CA; Control, control group; CA, 20 μM of CA group; ROS a, ROS agonist; ROS i, ROS inhibitor; LPS+ATP, vitro model of ALI group; **p < 0.01 compared with ALI mice with 15 mg/kg of CA group or vitro model of ALI group; ## p < 0.01 compared with 20 μM of CA+ vitro model group. Number of experiments = 3.

Next, a ROS agonist (10 μM urolithin C) reduced the effect of Deh on NLRP3/Caspase-1/GSDMD protein expression, inflammation and ROS-induced oxidative stress, mitochondrial damage and pyroptosis in an in vitro model (Fig. 6B and I and M, and S3E–S3L). Furthermore, the ROS inhibitor (1 µM of Fulvene-5) promoted the effects of Deh on NLRP3/Caspase-1/GSDMD protein expression, inflammation, mitochondrial damage and pyroptosis in an in vitro model (Fig. 6B and J–M). According to these results, inhibition of ROS production suppressed mitochondrial damage induced by Deh, thereby restoring mitochondrial function in pyroptosis in ALI.

Deh inhibited the interaction between Akt and PDPK1 to promote phosphorylation of the Akt protein

The experiment investigated the anti-inflammatory mechanism by which Deh regulates the Akt/Nrf2 pathway. The protein level of PDPK1 was decreased, and p-Akt and Nrf2 protein expression was induced in a dose-dependent manner by Deh in the mouse model and in vitro model (Fig. 7 A and B). Immunohistochemistry revealed that Deh suppressed the expression of PDPK1 and induced the expression of PI3K in the lung tissue of ALI mice (Fig. 7C and D).

Fig. 7.

Fig 7

Open in a new tab

Deh inhibited the interaction between Akt and PDPK1 to promote phosphorylation of Akt protein. PDPK1/p-Akt/ Nrf2 protein expression (A) in mice model; PDPK1/p-Akt/ Nrf2 protein expression (B) in vitro model; PDPK1 expression in lung tissue of mice model (Immunohistochemistry, C); p-Akt expression in lung tissue of mice model (Immunohistochemistry, D); PDPK1 and p-Akt expression in vitro model (Immunofluorescence, E); the combination of PDPK1 and Akt in vitro (co-immunoprecipitation, F); the combination of Akt and PDPK1 in vitro (co-immunoprecipitation, G); CA inhibited the interaction between Akt and PDPK1 (H and I). Sham, control mice group; Model, ALI mice group; Low/Med/High, ALI mice with 7.5, 15, 30 mg/kg of CA group. Control, control group; Control, control group; LPS+ATP, vitro model of ALI group; Low/Med/High, vitro model of ALI with 10, 20 or 40 μM of CA group; **p < 0.01 compared with control mice or control group; ## p < 0.01 compared with ALI mice or vitro model of ALI group. Number of experiments = 3.

Therefore, the mechanism by which Deh regulates the Akt/Nrf2 pathway through PDPK1 was further explored. Additionally, immunofluorescence also revealed that Deh suppressed the expression of PDPK1 and induced the expression of PI3K in an in vitro model (Fig. 7E). A co-IP assay verified that the interaction of the PDPK1 protein and Akt protein was reduced by Deh in an in vitro model (Fig. 7F). Therefore, these results indicated that Deh suppressed the interaction of the PDPK1 protein and Akt protein.

In addition, when PDPK1 WT protein was combined with Akt protein, Deh induced p-Akt protein expression at the T308 site (Fig. 7H and I). When PDPK1 Mut protein (S549A or S549D) was combined with Akt protein, Deh did not affect p-Akt protein expression (Fig. 7H). Furthermore, when the PDPK1 protein was combined with the Akt Mut protein (T308S), Deh did not affect p-Akt protein expression (Fig. 7I). These findings indicated that Deh promoted the phosphorylation of AKT at T308 through the potent role of PDK1 at S549.

Deh promoted the Akt/Nrf2 signaling pathway to reduce ROS production in mitochondria

To gain additional insight into the requirement of Deh for ROS production in mitochondria, several studies have demonstrated that the PDPK1/Akt/Nrf2 pathway suppresses ROS production (Li et al., 2018; Shin et al., 2019; Zhuang et al., 2019; Wei et al., 2021).

Compared with mice with ALI treated with Deh, the PI3K/Akt/Nrf2 signaling pathway was inhibited and NLRP3/Caspase-1/GSDMD protein expression was suppressed in mice with ALI treated with Deh and a PI3K inhibitor (20 mg/kg LY294002) (Fig. 8 A). The PI3K inhibitor increased the lung injury score and W/D rate and promoted lung injury (HE staining) in mice with ALI treated with Deh (Fig. 8B–D). The levels of these inflammatory factors in the PI3K inhibitor combined with Deh group were lower than those in the Deh group (Fig. 8E and F).

Fig. 8.

Fig 8

Open in a new tab

Deh induced Akt/Nrf2 signaling pathway to reduce ROS production in mitochondrion. PI3K/Akt/Nrf2/NLRP3/Caspase-1/GSDMD protein expression (A), Lung injury (B), HE staining (C), W/D rate (D), IL-1β (E), IL-6 (F) in ALI mice with CA+PI3K inhibitor; PI3K/Akt/Nrf2/NLRP3/Caspase-1/GSDMD protein expression (G), apoptosis rate (H), IL-1β (I), IL-6 (J), ROS production (K), MDA (L), SOD (M), mitochondrion (Electron microscope, N). Vector, control; PI3K i, PI3K inhibitor; Chicoric acid+Model, ALI mice with 15 mg/kg of CA; Control, control group; CA, 20 μM of CA group; PI3K i, PI3K i; PI3K a, PI3K agonist; LPS+ATP, vitro model of ALI group; **p < 0.01 compared with ALI mice with 15 mg/kg of CA group or vitro model of ALI group; ## p < 0.01 compared with 20 μM of CA+ vitro model group. Number of experiments = 3.

In the in vitro model of ALI, Deh and a PI3K inhibitor (0.5 μM LY294002) suppressed the PI3K/Akt/Nrf2 signaling pathway and induced NLRP3/Caspase-1/GSDMD protein expression compared with the Deh group (Fig. 8G). Compared with the CA group, CA and a PI3K agonist (10 μM YS-49 monohydrate) induced the PI3K/Akt/Nrf2 signaling pathway and suppressed NLRP3/Caspase-1/GSDMD protein expression (Fig. 8G). Throughout the experiments, Deh and the PI3K inhibitor increased the levels of inflammation and ROS-induced oxidative stress and accelerated mitochondrial damage and pyroptosis in the in vitro model compared with the Deh group (Fig. 8H–N). Furthermore, Deh and PI3K agonists reduced the levels of inflammation and ROS-induced oxidative stress and inhibited mitochondrial damage and pyroptosis in an in vitro model (Fig. 8H–N). These results suggested that Deh alleviated mitochondrial damage in a model of ALI by inhibiting ROS production in mitochondria and inducing the PI3K/Akt/Nrf2 signaling pathway.

Deh directly targeted the PDPK1 protein and accelerated PDPK1 ubiquitination

To elucidate the mechanism by which Deh targets the PDPK1 protein, linkage analysis of the drug and protein demonstrated that Deh had a linkage effect with the PDPK1 protein (Fig. 9 A). Deh affected the thermophoretic motion of PDPK1. Upon binding to Deh, the melting temperature of PDPK1 increased from ∼55 °C to ∼60 °C (Fig. 9B and C). According to the CETSA results of HEK293T cells, Deh significantly improved the thermal stability of exogenous WT-PDPK1, while that of Mut-PDPK1 was not changed. Therefore, 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP and 223-ASP might be responsible for the interaction between PDPK1 and Deh (Fig. 9D–F). Deh accelerated PDPK1 ubiquitination in an in vitro model of ALI (Fig. 9G). PDPK1 directly targeted the PDPK1 protein and lessened PI3K ubiquitination, which may be a target of the effects of Deh in the ALI model.

Fig. 9.

Fig 9

Open in a new tab

Deh directly targeted PDPK1 protein and accelerated PDPK1 Ubiquitination. 3D image revealed that CA bond to the binding pocket and formed with PDPK1 (A); Microscale thermophoresis (MST) of CA, PDPK1 incubated with CA (B); TSA results in the presence or absence of CA (C); PDPK1 protein expression (D); the thermal stability of WT PDPK1 and Mut PDPK1 plasmid after treatment with CA using CETSA (E and F); PDPK1 Ubiquitination (G).

Discussion

People with COVID-19 usually have relatively mild symptoms, such as fever, dry cough and general malaise (Majumder and Minko, 2021). However, some patients’ conditions may quickly become severe, or they may initially appear to have severe ALI (Sidiq et al., 2020). Through the analysis of 4021 and 1099 cases of COVID-19 in China, it was found that the proportions of severe ALI were 25.5% and 15.7%, respectively (To et al., 2021). Moreover, severe ALI is more common in male patients aged 40–70 years (To et al., 2021). In the current study, we found that Deh presented lung injury in a mouse model of ALI. Gyebi et al. showed that six compounds (chicoric acid, luteolin and so on) exhibited the highest binding tendencies to the equilibrated conformers of COVID-19 (Gyebi et al., 2021). Together, these data suggest that Deh presented ALI, so much so that CA might be used to prevent and cure COVID-19 in further clinical treatment.

ALI is a lower respiratory tract disease caused primarily by pathogens that present clinically with fever, cough, expectoration, wheezing, and even death (Lv et al., 2022). COVID-19 infection can exhibit severe pulmonary edema, dyspnea, hypoxemia, and even acute respiratory distress syndrome. A study of standardized autopsy results and clinical data in the medical records of 13 patients who died of COVID-19 showed that secondary alveolar injury caused by focal capillary microthrombosis in the lung was the cause of death (Piñeiro Roncal et al., 2021). At present, the pathological mechanism of ALI induced by SARS CoV-2 is not completely clear. According to literature reports, the mechanism can be summarized as follows: First, IL-1 is considered to be the "culprit" of the lung injury cytokine storm and the key cause of cytokine release syndrome (Piñeiro Roncal et al., 2021; Visca et al., 2021).. Moreover, we found that Deh reduced inflammation and oxidative stress to present lung injury in vivo or in vitro. Li et al. indicated that Deh decreased inflammation and oxidative stress in lipopolysaccharide in a model of acute liver injury (Li et al., 2020b). Consistent with previous reports, Deh reduced inflammation and ROS-induced oxidative stress in the ALI model.

The canonical pyroptosis pathway plays a decisive role in inflammasome-activated Caspase-1 species (Liu et al., 2021; Zeng et al., 2021). As the pathogen invades the body, the inflammasome is activated to Caspase-1 after pro-Caspase-1 through special adaptor proteins. Caspase can cleave IL-1β, IL-18 and GSDMD, among which GSDMD is an important mediator of pyroptosis initiation (van Lieshout et al., 2018). The GSDMD-N-terminus can be inserted into the cell membrane to induce the formation of cell membrane pores (van Lieshout et al., 2018). As the cells gradually swell to rupture, a large amount of cellular contents is released, which causes an inflammatory response. Furthermore, Caspase cleaves the precursors of IL-1β and IL-18, activating them to recruit inflammatory cells and create an inflammatory response (Wu and Huang, 2017). Interestingly, we also observed that Deh decreased programmed cell death in an in vivo or in vitro model of ALI by inhibiting pyroptosis. Liu et al. showed that Deh improved nerve cell death against inflammation in SH-SY5Y cells by promoting mitochondrial function (Liu et al., 2019a). These findings support the conclusion that Deh reduced programmed cell death in lung cells in a model of ALI.

Based on related studies, the transcriptional activation of the NLRP3 inflammasome plays an important role in inflammation and apoptosis in acute lung injury (Lara et al., 2020). In addition, it is important in regulating the inflammatory response and the occurrence and development of acute infectious ALI (Liu et al., 2021). NLRP3 can induce lung injury through the activation of NF-κB-related pathways, which may become a new target for lung injury therapy (Sun et al., 2021). This study showed that Deh suppressed NLRP3-mediated pyroptosis in vivo and in vitro in a model of ALI. El-Twab et al. showed that Deh prevented kidney injury by suppressing NLRP3 inflammasome activation (Abd El-Twab et al., 2019). Therefore, it is speculated that NLRP3 mediates Deh-induced inflammation in ALI.

In addition to providing the energy required for vital cell activities, mitochondria are also involved in the regulation of cells, such as calcium ion concentration in the internal environment, cell signal transduction, and apoptosis (Paul et al., 2022). The involvement of mitochondria in the regulation of innate immunity has been a major discovery in recent years (Pu et al., 2022). Current studies suggest that cellular stress caused by external factors such as infection and stimulation can lead to mitochondrial dysfunction, which further regulates the activation of the NLRP3 inflammasome through different pathways (Zhang et al., 2021; Pu et al., 2022). Our results showed that Deh attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis in vivo and in vitro. Xiao et al. disclosed that Deh induced mitochondria-dependent apoptosis through ROS-mediated PI3K/Akt signaling pathways in 3T3-L1 preadipocytes (Xiao et al., 2013). Therefore, we hypothesized that Deh suppresses NLRP3-mediated pyroptosis in an in vivo or in vitro model of ALI through the prevention of mitochondrial damage, suggesting that NLRP3 is probably a specific pharmacological target for ALI in COVID-19 treated by Deh.

ROS are mainly derived from mitochondria (An et al., 2019). When the electron transport chain on the inner mitochondrial membrane is disrupted, ROS accumulate inside the cell and become toxic when reached at certain levels (Lin et al., 2019). Soluble uric acid and deoxycholic acid can induce and promote the production of ROS and further activate the NLRP3 inflammasome (Minutoli et al., 2016; Pu et al., 2020). Our experiment suggests that Deh weakened ROS production in mitochondria to suppress NLRP3-mediated pyroptosis. Lu et al. reported that Deh prevented vascular smooth muscle cell dedifferentiation by suppressing ROS signaling (Lu et al., 2018). Kim et al. suggested that Deh improved mitochondrial function to relieve impaired insulin sensitivity (Kim et al., 2018). These findings support that ROS might play a key role in regulating NLRP3-mediated pyroptosis in Deh-inhibited ALI.

Nrf2 is a key transcription factor that cells use to resist foreign bodies and oxidative damage (Li et al., 2018). Activated Nrf2 moves to the nucleus, induces a variety of genes to play an antioxidant role, and then regulates inflammatory cytokines, increases airway hyperresponsiveness, and further amplifies inflammation (Li et al., 2018). Many studies have confirmed that Nrf2 is one of the target proteins of Akt. The PI3K/Akt signaling pathway activates Nrf2 and inhibits ROS levels (Zhang et al., 2016; Liu et al., 2020). PDPK1 phosphorylates Akt Thr308, while Akt Ser473 is activated by integrin-linked proteins, thereby activating a variety of downstream target proteins (Mamidi et al., 2022; Zhang et al., 2022b). Our results showed that Deh suppressed PDPK1 protein expression and induced the Akt/Nrf2 signaling pathway to reduce ROS production in mitochondria. Deh inhibited the interaction between Akt Thr308 and PDPK1 Ser549 to promote phosphorylation of Akt protein. Deh directly targeted the PDPK1 protein and advanced PDPK1 ubiquitination. Xiao et al. showed that Deh induced mitochondria-dependent apoptosis through ROS-mediated PI3K/Akt signaling pathways in 3T3-L1 preadipocytes (Xiao et al., 2013). Therefore, we hypothesized that the PDPK1/Akt/Nrf2 signaling pathways play a key role in regulating mitochondrial damage to suppress NLRP3-mediated pyroptosis in vivo or in vitro in a model of ALI by Deh. Of note, in this study, an important finding is that Deh accelerates PDPK1 ubiquitination to promote the Akt/Nrf2 signaling pathway by inhibiting the interaction between Akt Thr308 and PDPK1 Ser549 in a model of ALI due to COVID-19. Finally, Deh might be self-cytotoxic, which is insufficiently explored in this study, and we will conduct further research regarding this issue in future experiments.

In conclusion, our study provided direct evidence that Deh from Andrographis paniculata (Burm.f.) Wall presented NLRP3-mediated pyroptosis in a model of ALI through mitochondrial damage by the inhibition of ROS production (Fig. 10 ). Deh accelerated PDPK1 ubiquitination to decrease ROS production in mitochondria through the induction of Akt/Nrf2 signaling pathways in a model of ALI (Fig. 10). Deh reduced the interaction between Akt Thr308 and PDPK1 Ser549 to promote the activity of Akt (Fig. 10). Therefore, structural optimization of Deh to increase its affinity for the PDPK1 protein will be carried out in further studies. The results collected from the present study suggest that Deh might be a potential therapeutic drug to treat or prevent ALI in COVID-19 or other respiratory diseases.

Fig. 10.

Fig 10

Open in a new tab

Deh form Andrographis paniculata (Burm.f.) Wall presented NLRP3-mediated pyroptosis in model of ALI of COVID-19 through ROS-induced mitochondrial damage by the inhibition of Akt/Nrf2 pathway by PDPK1 Ubiquitination.

Funding

This work was supported by National Natural Science Foundation of China (81173133); Nature Science Research Project of Anhui province (2108085QH3811); Key Natural Science Projects of the Department of Education of Anhui province (2022AH051239); Yijishan Hospital of Wannan Medical College (YR202005, YPF2019016) and Wannan Medical College (WK2021ZF11, WK2021ZF39).

Data availability of statement

The datasets used and/or analyzed of this study are from corresponding author upon reasonable request.

Authorship contribution statement

The effects and mechanisms of the anti-covid-19 traditional Chinese medicine, Dehydroandrographolide from Andrographis paniculata (Burm.f.) Wall presented acute lung injury by the inhibition of NLRP3-mediated pyroptosis.

Zhichen Pu and Haitang Xie conceived the study, designed the study and prepared the manuscript. Zhichen Pu, Bangzhi Sui, Xingwen Wang, Wusan Wang, Lingling Li and Haitang Xie conducted the experiments and data analysis, involved in preparation of the figures and manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Competing Interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

References

  1. Abd El-Twab S.M., Hussein O.E., Hozayen W.G., Bin-Jumah M., Mahmoud A.M. Chicoric acid prevents methotrexate-induced kidney injury by suppressing NF-κB/NLRP3 inflammasome activation and up-regulating Nrf2/ARE/HO-1 signaling. Inflamm. Res. 2019;68:511–523. doi: 10.1007/s00011-019-01241-z. [DOI] [PubMed] [Google Scholar]
  2. Albert E., Torres I., Bueno F., Huntley D., Molla E., Fernández-Fuentes M., Martínez M., Poujois S., Forqué L., Valdivia A., Solano de la Asunción C., Ferrer J., Colomina J., Navarro D. Field evaluation of a rapid antigen test (Panbio™ COVID-19 Ag Rapid Test Device) for COVID-19 diagnosis in primary healthcare centres. Clin. Microbiol. Infect. 2021;27:472. doi: 10.1016/j.cmi.2020.11.004. e7-e10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amit S., Beni S.A., Biber A., Grinberg A., Leshem E., Regev-Yochay G. Postvaccination COVID-19 among healthcare workers, Israel. Emerg. Infect. Dis. 2021;27:1220–1222. doi: 10.3201/eid2704.210016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. An H., Heo J.S., Kim P., Lian Z., Lee S., Park J., Hong E., Pang K., Park Y., Ooshima A., Lee J., Son M., Park H., Wu Z., Park K.S., Kim S.J., Bae I., Yang K.M. Tetraarsenic hexoxide enhances generation of mitochondrial ROS to promote pyroptosis by inducing the activation of caspase-3/GSDME in triple-negative breast cancer cells. Cell Death Dis. 2021;12:159. doi: 10.1038/s41419-021-03454-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. An Y., Zhang H., Wang C., Jiao F., Xu H., Wang X., Luan W., Ma F., Ni L., Tang X., Liu M., Guo W., Yu L. Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis. FASEB J. 2019;33:12515–12527. doi: 10.1096/fj.201802805RR. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonaventura A., Vecchié A., Dagna L., Tangianu F., Abbate A., Dentali F. Colchicine for COVID-19: targeting NLRP3 inflammasome to blunt hyperinflammation. Inflamm. Res. 2022;71:293–307. doi: 10.1007/s00011-022-01540-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bora H., Kamle M., Hassan H., Al-Emam A., Chopra S., Kirtipal N., Bharadwaj S., Kumar P. Exploration of potent antiviral phytomedicines from lauraceae family plants against SARS-CoV-2 main protease. Viruses. 2022;14:2783. doi: 10.3390/v14122783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bordas-Martinez J., Del Río B., Molina-Molina M. Multiple solid nodules at post-COVID-19 follow-up after mild pneumonia. Arch. Bronconeumol. 2021;16(21) doi: 10.1016/j.arbres.2021.06.017. S0300-2896(21)00204-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Che D., Hou Y., Zeng Y., Li C., Zhang Y., Wei D., Hu S., Liu R., An H., Wang Y., Zhang T. Dehydroandrographolide inhibits IgE-mediated anaphylactic reactions via calcium signaling pathway. Toxicol. Appl. Pharmacol. 2019;366:46–53. doi: 10.1016/j.taap.2019.01.019. [DOI] [PubMed] [Google Scholar]
  10. Che D., Zheng Y., Hou Y., Li T., Du X., Geng S. Dehydroandrographolide targets CD300f and negatively regulated MRGPRX2-induced pseudo-allergic reaction. Phytother. Res. 2022;36:2173–2185. doi: 10.1002/ptr.7445. [DOI] [PubMed] [Google Scholar]
  11. Chen X., Xiao Z., Jiang Z., Jiang Y., Li W., Wang M. Schisandrin B Attenuates airway inflammation and airway remodeling in asthma by inhibiting NLRP3 inflammasome activation and reducing pyroptosis. Inflammation. 2021;44:2217–2231. doi: 10.1007/s10753-021-01494-z. [DOI] [PubMed] [Google Scholar]
  12. Climans S.A., Grunfeld E., Mason W.P., Chan K.K.W. Effectiveness and safety of pneumocystis pneumonia prophylaxis for patients receiving temozolomide chemoradiotherapy. Neuro. Oncol. 2022;24:1738–1748. doi: 10.1093/neuonc/noac072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fenollar F., Bouam A., Ballouche M., Fuster L., Prudent E., Colson P., Tissot-Dupont H., Million M., Drancourt M., Raoult D., Fournier P.E. Evaluation of the panbio COVID-19 rapid antigen detection test device for the screening of patients with COVID-19. J. Clin. Microbiol. 2021:59. doi: 10.1128/JCM.02589-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gallo González V., López-Padilla D., Puente Maestú L. Pulmonary calcifications as COVID-19 pneumonia sequelae. Arch. 2021;58:257. doi: 10.1016/j.arbres.2021.04.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guo W., Liu J., Zhang Y., Ma H., Li Y., Gong Q., Cao Y., Hu G., Xie S., Fu S. Dehydroandrographolide inhibits mastitis by activating autophagy without affecting intestinal flora. Aging (Albany NY) 2020;12:14050–14065. doi: 10.18632/aging.103312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gyebi G.A., Elfiky A.A., Ogunyemi O.M., Ibrahim I.M., Adegunloye A.P., Adebayo J.O., Olaiya C.O., Ocheje J.O., Fabusiwa M.M. Structure-based virtual screening suggests inhibitors of 3-Chymotrypsin-Like Protease of SARS-CoV-2 from Vernonia amygdalina and Occinum gratissimum. Comput. Biol. Med. 2021;136 doi: 10.1016/j.compbiomed.2021.104671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huo Z., Wang Y., Feng X., He Y., Qiu F. Tracking the deprotonation site of dehydroandrographolide with electrospray ionization mass spectrometry by deuteration. Rapid Commun. Mass Spectrom. 2021;35:e9152. doi: 10.1002/rcm.9152. [DOI] [PubMed] [Google Scholar]
  18. Jeong S.G., Kim S., Kim H.G., Kim E., Jeong D., Kim J.H., Yang W.S., Oh J., Sung G.H., Hossain M.A., Lee J., Kim J.H., Cho J.Y. Mycetia cauliflora methanol extract exerts anti-inflammatory activity by directly targeting PDK1 in the NF-κB pathway. J. Ethnopharmacol. 2019;231:1–9. doi: 10.1016/j.jep.2018.11.013. [DOI] [PubMed] [Google Scholar]
  19. Jung E.M., Lee G.S. Korean Red Ginseng, a regulator of NLRP3 inflammasome, in the COVID-19 pandemic. J. Ginseng. 2022;46:331–336. doi: 10.1016/j.jgr.2022.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim J.S., Lee H., Jung C.H., Lee S.J., Ha T.Y., Ahn J. Chicoric acid mitigates impaired insulin sensitivity by improving mitochondrial function. Biosci. Biotechnol. Biochem. 2018;82:1197–1206. doi: 10.1080/09168451.2018.1451742. [DOI] [PubMed] [Google Scholar]
  21. Lara P.C., Macías-Verde D., Burgos-Burgos J. Age-induced NLRP3 inflammasome over-activation increases lethality of SARS-CoV-2 pneumonia in elderly patients. Aging Dis. 2020;11:756–762. doi: 10.14336/AD.2020.0601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li C., Li Y., Zhang H., Zhuo Y., Zhang L., Yang L., Gao Q., Tu Z., Shao R., Wang Y., Zhang J., Cui L., Zhang S. Xuanfei Baidu decoction suppresses complement overactivation and ameliorates IgG immune complex-induced acute lung injury by inhibiting JAK2/STAT3/SOCS3 and NF-κB signaling pathway. Phytomedicine. 2022;109 doi: 10.1016/j.phymed.2022.154551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li G., Zeng L., Cheng H., Han J., Zhang X., Xie H. Acupuncture administration improves cognitive functions and alleviates inflammation and nuclear damage by regulating phosphatidylinositol 3 kinase (PI3K)/Phosphoinositol-Dependent Kinase 1 (PDK1)/Novel Protein Kinase C (nPKC)/Rac 1 signaling pathway in senescence-accelerated prone 8 (SAM-P8) mice. Med. Sci. Monit. 2019;25:4082–4093. doi: 10.12659/MSM.913858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li L.L., Dai B., Sun Y.H., Zhang T.T. The activation of IL-17 signaling pathway promotes pyroptosis in pneumonia-induced sepsis. Ann. Transl. Med. 2020;8:674. doi: 10.21037/atm-19-1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li S.T., Dai Q., Zhang S.X., Liu Y.J., Yu Q.Q., Tan F., Lu S.H., Wang Q., Chen J.W., Huang H.Q., Liu P.Q., Li M. Ulinastatin attenuates LPS-induced inflammation in mouse macrophage RAW264.7 cells by inhibiting the JNK/NF-κB signaling pathway and activating the PI3K/Akt/Nrf2 pathway. Acta Pharmacol. Sin. 2018;39:1294–1304. doi: 10.1038/aps.2017.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li Z., Feng H., Han L., Ding L., Shen B., Tian Y., Zhao L., Jin M., Wang Q., Qin H., Cheng J., Liu G. Chicoric acid ameliorate inflammation and oxidative stress in Lipopolysaccharide and d-galactosamine induced acute liver injury. J. Cell Mol. Med. 2020;24:3022–3033. doi: 10.1111/jcmm.14935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li Z., Pan H., Yang J., Chen D., Wang Y., Zhang H., Cheng Y. Xuanfei Baidu formula alleviates impaired mitochondrial dynamics and activated NLRP3 inflammasome by repressing NF-κB and MAPK pathways in LPS-induced ALI and inflammation models. Phytomedicine. 2023;108 doi: 10.1016/j.phymed.2022.154545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lin Q., Li S., Jiang N., Shao X., Zhang M., Jin H., Zhang Z., Shen J., Zhou Y., Zhou W., Gu L., Lu R., Ni Z. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. Redox Biol. 2019;26 doi: 10.1016/j.redox.2019.101254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu B., Deng X., Jiang Q., Li G., Zhang J., Zhang N., Xin S., Xu K. Scoparone improves hepatic inflammation and autophagy in mice with nonalcoholic steatohepatitis by regulating the ROS/P38/Nrf2 axis and PI3K/AKT/mTOR pathway in macrophages. Biomed. Pharmacother. 2020;125 doi: 10.1016/j.biopha.2020.109895. [DOI] [PubMed] [Google Scholar]
  30. Liu F., Liu T., Sun M., Zhou J., Xue F., Chen S., Chen J., Zhang L. Maxing Shigan decoction mitigates mycoplasma pneumonia-induced pyroptosis in A549 cells via the NLRP3 Inflammasome. Infect. Drug Resist. 2021;14:859–867. doi: 10.2147/IDR.S292413. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  31. Liu Q., Fang J., Chen P., Die Y., Wang J., Liu Z., Liu X. Chicoric acid improves neuron survival against inflammation by promoting mitochondrial function and energy metabolism. Food Funct. 2019;10:6157–6169. doi: 10.1039/c9fo01417a. [DOI] [PubMed] [Google Scholar]
  32. Liu X., Fan Y., Xie J., Zhang L., Li L., Wang Z. Dehydroandrographolide inhibits osteosarcoma cell growth and metastasis by targeting SATB2-mediated EMT. Anticancer Agents Med. Chem. 2019;19:1728–1736. doi: 10.2174/1871520619666190705121614. [DOI] [PubMed] [Google Scholar]
  33. Lu Q.B., Wan M.Y., Wang P.Y., Zhang C.X., Xu D.Y., Liao X., Sun H.J. Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade. Redox Biol. 2018;14:656–668. doi: 10.1016/j.redox.2017.11.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lv N., Zhao Y., Liu X., Ye L., Liang Z., Kang Y., Dong Y., Wang W., Kolliputi N., Shi L. Dysfunctional telomeres through mitostress-induced cGAS/STING activation to aggravate immune senescence and viral pneumonia. Aging Cell. 2022:e13594. doi: 10.1111/acel.13594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Machado M.G., Patente T.A., Rouillé Y., Heumel S., Melo E.M., Deruyter L., Pourcet B., Sencio V., Teixeira M.M., Trottein F. Acetate improves the killing of streptococcus pneumoniae by alveolar macrophages via NLRP3 inflammasome and glycolysis-HIF-1α axis. Front. Immunol. 2022;13 doi: 10.3389/fimmu.2022.773261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Majumder J., Minko T. Recent developments on therapeutic and diagnostic approaches for COVID-19. AAPS J. 2021;23:14. doi: 10.1208/s12248-020-00532-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mamidi M.K., Mahmud H., Maiti G.P., Mendez M.T., Fernandes S.M., Vesely S.K., Holter-Chakrabarty J., Brown J.R., Ghosh A.K. Idelalisib activates AKT via increased recruitment of PI3Kδ/PI3Kβ to BCR signalosome while reducing PDK1 in post-therapy CLL cells. Leukemia. 2022;36:1806–1817. doi: 10.1038/s41375-022-01595-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Minutoli L., Puzzolo D., Rinaldi M., Irrera N., Marini H., Arcoraci V., Bitto A., Crea G., Pisani A., Squadrito F., Trichilo V., Bruschetta D., Micali A., Altavilla D. ROS-mediated NLRP3 inflammasome activation in brain, heart, kidney, and testis ischemia/reperfusion injury. Oxid. Med. Cell. Longev. 2016;2016 doi: 10.1155/2016/2183026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Paul O., Tao J.Q., West E., Litzky L., Feldman M., Montone K., Rajapakse C., Bermudez C., Chatterjee S. Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome. Respir. Res. 2022;23:25. doi: 10.1186/s12931-022-01944-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Piñeiro Roncal, M., García Luján, R., Siesto López, G.M., Tejedor Ortiz, M.T., Miguel Poch, E., 2021. Use of an endobronchial valve for management of a persistent air-leak in a child with necrotising pneumonia. Arch. Bronconeumol. 21, S0300-2896(21)00172-1. [DOI] [PubMed]
  41. Pu Z., Liu Y., Li C., Xu M., Xie H., Zhao J. Using network pharmacology for systematic understanding of geniposide in ameliorating inflammatory responses in colitis through suppression of NLRP3 inflammasome in macrophage by AMPK/Sirt1 dependent signaling. Am. J. Chin. Med. 2020;48:1693–1713. doi: 10.1142/S0192415X20500846. [DOI] [PubMed] [Google Scholar]
  42. Pu Z., Shen C., Zhang W., Xie H., Wang W. Avenanthramide C from oats protects pyroptosis through dependent ROS-induced mitochondrial damage by PI3K ubiquitination and phosphorylation in pediatric pneumonia. J. Agric. Food Chem. 2022;70:2339–2353. doi: 10.1021/acs.jafc.1c06223. [DOI] [PubMed] [Google Scholar]
  43. Shin J.H., Kim K.M., Jeong J.U., Shin J.M., Kang J.H., Bang K., Kim J.H. Nrf2-Heme oxygenase-1 attenuates high-glucose-induced epithelial-to-mesenchymal transition of renal tubule cells by inhibiting ROS-mediated PI3K/Akt/GSK-3β signaling. J. Diabetes Res. 2019;2019 doi: 10.1155/2019/2510105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sidiq Z., Hanif M., Dwivedi K.K., Chopra K.K. Benefits and limitations of serological assays in COVID-19 infection. Indian J. Tuberc. 2020;67:S163–S1S6. doi: 10.1016/j.ijtb.2020.07.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sun J., Mao S., Ji W. LncRNA H19 activates cell pyroptosis via the miR-22-3p/NLRP3 axis in pneumonia. Am. J. Transl. Res. 2021;13:11384–11398. [PMC free article] [PubMed] [Google Scholar]
  46. To K.K., Sridhar S., Chiu K.H., Hung D.L., Li X., Hung I.F., Tam A.R., Chung T.W., Chan J.F., Zhang A.J., Cheng V.C., Yuen K.Y. Lessons learned 1 year after SARS-CoV-2 emergence leading to COVID-19 pandemic. Emerg. Microbes Infect. 2021;10:507–535. doi: 10.1080/22221751.2021.1898291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. van Lieshout M.H.P., de Vos A.F., Dessing M.C., de Porto A., de Boer O.J., de Beer R., Terpstra S., Florquin S., Van't Veer C., van der Poll T. ASC and NLRP3 impair host defense during lethal pneumonia caused by serotype 3 Streptococcus pneumoniae in mice. Eur. J. Immunol. 2018;48:66–79. doi: 10.1002/eji.201646554. [DOI] [PubMed] [Google Scholar]
  48. Visca D., Migliori G.B., Dinh-Xuan A.T., Centis R., Belli S., Vitacca M., Aliani M., Zampogna E., Feci D., Pignatti P., Zappa M., Saderi L., Sotgiu G., Spanevello A. The role of blood gas analysis in the post-acute phase of COVID-19 pneumonia. Arch. Bronconeumol. 2021;21(21) doi: 10.1016/j.arbres.2021.06.003. S0300-2896(21)00185-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang H., Liu C., Zhao Y., Gao G. Mitochondria regulation in ferroptosis. Eur. J. Cell Biol. 2020;99 doi: 10.1016/j.ejcb.2019.151058. [DOI] [PubMed] [Google Scholar]
  50. Wang Y., Shi P., Chen Q., Huang Z., Zou D., Zhang J., Gao X., Lin Z. Mitochondrial ROS promote macrophage pyroptosis by inducing GSDMD oxidation. J. Mol. Cell Biol. 2019;11:1069–1082. doi: 10.1093/jmcb/mjz020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wei Y., Chen J., Cai G.E., Lu W., Xu W., Wang R., Lin Y., Yang C. Rosmarinic acid regulates microglial M1/M2 polarization via the PDPK1/Akt/HIF pathway under conditions of neuroinflammation. Inflammation. 2021;44:129–147. doi: 10.1007/s10753-020-01314-w. [DOI] [PubMed] [Google Scholar]
  52. Wu S., Huang J. Resveratrol alleviates Staphylococcus aureus pneumonia by inhibition of the NLRP3 inflammasome. Exp. Ther. Med. 2017;14:6099–6104. doi: 10.3892/etm.2017.5337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Xiao H., Wang J., Yuan L., Xiao C., Wang Y., Liu X. Chicoric acid induces apoptosis in 3T3-L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways. J. Agric. Food Chem. 2013;61:1509–1520. doi: 10.1021/jf3050268. [DOI] [PubMed] [Google Scholar]
  54. Xiao Y., Peng H., Hong C., Chen Z., Deng X., Wang A., Yang F., Yang L., Chen C., Qin X. PDGF promotes the warburg effect in pulmonary arterial smooth muscle cells via activation of the PI3K/AKT/mTOR/HIF-1α signaling pathway. Cell Physiol. Biochem. 2017;42:1603–1613. doi: 10.1159/000479401. [DOI] [PubMed] [Google Scholar]
  55. Xu W., Che Y., Zhang Q., Huang H., Ding C., Wang Y., Wang G., Cao L., Hao H. Apaf-1 pyroptosome senses mitochondrial permeability transition. Cell Metab. 2021;33 doi: 10.1016/j.cmet.2020.11.018. 424-36 e10. [DOI] [PubMed] [Google Scholar]
  56. Yang K., Li B., Chen J. Knockdown of phosphoinositide-dependent kinase 1 (PDK1) inhibits fibrosis and inflammation in lipopolysaccharide-induced acute lung injury rat model by attenuating NF-κB/p65 pathway activation. Ann. Transl. Med. 2021;9:1671. doi: 10.21037/atm-21-5476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zeng J., Wan X., Liu T., Xiong Y., Xiang G., Peng Y., Zhu R., Zhou Y., Liu C. Chlorogenic acid ameliorates Klebsiella pneumoniae-induced pneumonia in immunosuppressed mice via inhibiting the activation of NLRP3 inflammasomes. Food Funct. 2021;12:9466–9475. doi: 10.1039/d0fo03185b. [DOI] [PubMed] [Google Scholar]
  58. Zhang F., Guo F., Zhang Y., Xu H., Liu Y., Lin L., Li H., Yang H., Huang L. Huashibaidu formula attenuates sepsis-induced acute lung injury via suppressing cytokine storm: implications for treatment of COVID-19. Phytomedicine. 2022;109 doi: 10.1016/j.phymed.2022.154549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang J., Wang X., Vikash V., Ye Q., Wu D., Liu Y., Dong W. ROS and ROS-mediated cellular signaling. Oxid. Med. Cell. Longev. 2016;2016 doi: 10.1155/2016/4350965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhang W., Li L., Guo E., Zhou H., Ming J., Sun L., Hu G., Zhang L. Inhibition of PDK1 enhances radiosensitivity and reverses epithelial-mesenchymal transition in nasopharyngeal carcinoma. Head Neck. 2022;44:1576–1587. doi: 10.1002/hed.27053. [DOI] [PubMed] [Google Scholar]
  61. Zhang W., Wang W., Shen C., Wang X., Pu Z., Yin Q. Network pharmacology for systematic understanding of Schisandrin B reduces the epithelial cells injury of colitis through regulating pyroptosis by AMPK/Nrf2/NLRP3 inflammasome. Aging (Albany NY) 2021;13:23193–23209. doi: 10.18632/aging.203611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhuang Y., Wu H., Wang X., He J., He S., Yin Y. Resveratrol attenuates oxidative stress-induced intestinal barrier injury through PI3K/Akt-mediated Nrf2 signaling pathway. Oxid. Med. Cell. Longev. 2019;2019 doi: 10.1155/2019/7591840. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analyzed of this study are from corresponding author upon reasonable request.

Articles from Phytomedicine are provided here courtesy of Elsevier

RESOURCES