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

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.


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)  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 . 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 . PDPK1 promotes Akt phosphorylation (p-Akt), activates or inhibits the expression of target genes in downstream pathways, and plays an important role in apoptosis . Inhibition of PDPK1 expression can regulate the PI3K/Akt signaling pathway, thereby regulating apoptosis . 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. 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.

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.

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).

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  using a Hitachi H7650 transmission electron microscope (Tokyo, Japan) at 80 kV.
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.

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. 1A and B). Medium or high concentrations of Deh significantly decreased inflammatory factors (IL-1β, IL-6 and TNF-α), inhibited oxidative stressrelated 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.

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. 2A-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.

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 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. 3A 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).
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 antiinflammatory and antioxidative effects of Deh in the in vitro model (Fig. 3L-N). Nevertheless, the NLRP3 inhibitor enhanced the antiinflammatory 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 NLRP3mediated 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. 4A-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.
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. 5A). Additionally, rotenone markedly increased the lung injury score and W/D rate, increased inflammation factors and oxidative stressrelated factors, and decreased antioxidative activities in mice with CAinduced ALI (Fig. 5B-G).
After treatment with Deh, the addition of a mitochondrial damage Rotenone markedly reduced the anti-inflammatory effects of Deh on NLRP3/Caspase-1/GSDMD protein expression, inflammation and ROSinduced 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 NLRP3mediated pyroptosis
ROS production in mitochondria plays a role in the regulation of pyroptosis Wang et al., 2020;, 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. 6A and C-H).
Next, a ROS agonist (10 μM urolithin C) reduced the effect of Deh on NLRP3/Caspase-1/GSDMD protein expression, inflammation and ROSinduced 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. 7A 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). 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. 8A). 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).
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 ROSinduced 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. 9A). 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.

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 . Moreover, severe ALI is more common in male patients aged 40-70 years . 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 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 . NLRP3 can induce lung injury through the activation of NF-κB-related pathways, which may become a new target for lung injury therapy . 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 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 . 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 . 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 . 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.

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 NLRP3mediated 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.