Astaxanthin Prevents Tuberculosis-Associated Inflammatory Injury by Inhibiting the Caspase 4/11-Gasdermin-Pyroptosis Pathway

Pyroptosis is a programmed cell death caused by inflammation. Multiple studies have suggested that Mycobacterium tuberculosis infection causes tissue pyroptosis. However, there are currently no protective drugs against the inflammatory damage caused by pyroptosis. In this study, anti-pyroptotic effects of the natural compound astaxanthin (ASTA) were explored in a simulated pulmonary tuberculosis-associated inflammatory environment. The results showed that ASTA maintained the stability of MLE-12 lung epithelial cell numbers in the inflammatory environment established by lipopolysaccharide. The reason is not to promote cell proliferation but to inhibit lipopolysaccharide-induced pyroptosis. The results showed that ASTA significantly inhibited the expression of key proteins in the caspase 4/11-gasdermin D pathway and the release of pyroptosis-related inflammatory mediators. Therefore, ASTA inhibits inflammation-induced pyroptosis by inhibiting the caspase 4/11-gasdermin D pathway and has the potential to protect lung tissue from tuberculosis-related inflammatory injury. ASTA, a functional food component, is a promising candidate for protection against tuberculosis-associated inflammatory lung injury.


Introduction
Tuberculosis (TB) remains a leading cause of infectious death worldwide [1]. Pulmonary tuberculosis has obvious symptoms such as cough, sputum, and hemoptysis [2]. Tis is closely related to the lung damage caused by TB. TB presents as "latent" TB and "active" TB disease, and the treatment for each is diferent [3]. Isoniazid is excellent for the former, and rifampin is excellent for the latter. Pyrazinamide and ethambutol complete the frst-line regimen for drug-susceptible TB, each playing a specifc role [4,5]. At present, the inhibitory therapy against Mycobacterium tuberculosis (MTB) has achieved good results, but infammatory lung injury caused by TB is a key factor, afecting the quality of life of patients [6]. Importantly, the factors causing lung impairment remain unclear. Host immune responses probably play a dominant role in lung damage, as excessive infammation and elevated expression of lung matrix-degrading proteases are common during TB [7]. According to reports, MTB can cause pyroptosis through multiple pathways leading to infammation or infammatory damage [8][9][10]. TB causes caspase-1/NLRP3/ gasdermin D-mediated pyroptosis of human monocytes and macrophages [8].
Pyroptosis, also known as infammatory necrosis, is a type of programmed cell death, which is characterized by the continuous expansion of cells until the cell membrane ruptures, resulting in the release of cell contents and the activation of a strong infammatory response [11]. Pyroptosis is an important innate immune response in the body and plays an important role in fghting infections [12]. Several studies have confrmed that pyroptosis is an important cause of lung injury in the process of pulmonary infammatory diseases [13,14]. Terefore, regulation of pyroptosis may be the strategy to protect against TB-related infammatory lung injury.
Natural compounds can be used as references for drug development [15][16][17]. Astaxanthin (ASTA) is a keto carotenoid whose chemical name is 3, 3′-dihydroxy-4, 4′diketo-β, β′-carotene. It is widely found in shrimps, crabs, fsh, birds, some algae, fungi, and other organisms [18]. Te antioxidant and immune-promoting efects of astaxanthin can be used to prevent oxidative tissue damage [19]. However, studies on its regulatory role in infammation are limited. In a previous study, we confrmed that astaxanthin can efectively reduce lipopolysaccharide (LPS)-induced apoptosis. Based on the previous studies, this study explores the efect of ASTA on lung injury caused by TB-related infammation based on the pyroptotic pathway.

Dynamic Cell Morphology
Monitoring. Real-time cell morphology was monitored using a high content analysis system (PerkinElmer, Operetta CLS). MLE-12 cells were seeded in a 96-well plate (1 × 10 3 cells/well) (Corning Incorporated, Corning, MA) and cultured overnight with diferent compounds. Te shape of cells was dynamically monitored by using the content analyzer for 24 h. All experiments were independently repeated three times.

Cell Viability Assay.
Te inhibitory efect of ASTA on the viability of the MLE-12 cells was measured with the CCK-8 assay. In brief, the cells were seeded in 96-well plates (Corning Incorporated, Corning, MA) at a density of 5 × 10 3 cells/well in 200 μL of the culture medium and grown for 4 h at 37°C under 5% CO 2 . Next, they were treated with diferent concentrations of ASTA for 24 h. Subsequently, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for 1 h at 37°C in a 5% CO 2 atmosphere. Te optical density of each well was determined at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). All experiments were independently repeated three times.
2.6. Cell Cycle Analysis. MLE-12 cells in the logarithmic growth phase were seeded in a 6-wellfat-bottom plate at the standard of 2 mL cell suspension at 24 h. Te cell suspensions were centrifuged at 181 × g for 5 min, and after that, the cell pellets were collected. After using chilled PBS to wash twice, 600 µL of DNA staining solution and 10 µL of permeabilization bufer (Multi Sciences, Hangzhou, China) were added. After incubation in the dark for 30 min at 4°C, fow cytometric analysis was performed. All experiments were independently repeated three times.

Western Blot
Analysis. MLE-12 cells were cultured overnight in six-well plates (1 × 10 5 cells/well) and treated with LPS and other compounds. Protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred into nitrocellulose membranes. Te membranes were blocked at room temperature for 2 h with 5% skim milk in tris-bufered saline/tween 20 and then incubated overnight at 4°C with primary antibody specifcs for CCNA2, CCNB1, CCNE1, caspase 4, caspase 11, and gasdermin D. Goat antirabbit IgG was used as a secondary antibody to incubate the membranes at room temperature for 2 h. Te bands formed were visualized using the enhanced chemiluminescence detection system (BioRad ChemiDocTM XRS+), and specifc signals were quantifed using densitometric analysis (Image J software). All experiments were independently repeated three times.

Enzyme-Linked Immunosorbent Assay.
Te cell supernatants were collected after centrifugation. Next, IL-1β, IL-18, and LDHA contents of the supernatants were assayed using the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Cayman Chemicals, Ann Arbor, MI, USA). Te optical density of each well was determined using a microplate reader (Bio-Rad, Hercules, CA). All experiments were independently repeated three times.
2.9. Statistical Analysis. Analyses were performed using SPSS version 24.0 (USA). Te results are presented as a mean ± standard deviation (SD) of at least three independent experiments. One-way analysis of variance (ANOVA) and Student's t-test were used to compare groups.

ASTA Maintains the Stable Number of MEL-12 Cells.
From previous experiments, we found that 15 μmol/L is the minimum concentration of ASTA to promote the proliferation of MEL-12 cells and that 8 μg/L is the minimum concentration to inhibit the proliferation of MEL-12 cells. Terefore, we used 8 μg/L LPS to establish a pulmonary tuberculosis-related alveolar epithelial cell infammatory injury model and then intervened MEL-12 cells with 15 μmol/L ASTA.
In this study, we frst examined the experimental concentration of TETD. As shown in Figure 1(a), when the TETD concentration was higher than 7.5 μmol/L and cells were incubated for 24 h, cell proliferation decreased. Terefore, we determined the experimental concentration of TETD to be 7.5 μmol/L. To evaluate the efects of ASTA on MLE-12 cells with LPS, a high content analysis system was used to monitor the cell numbers. As shown in Figure 1(b), LPS inhibits the number of MLE-12 cells, whereas both ASTA and TETD maintain the stable number of MEL-12 cells.

ASTA Does Not Afect Cell Proliferation Inhibition by LPS.
Te results show that ASTA prevented LPS-induced cell number reductions. We believe that this result may be due to two reasons: one is that ASTA promotes cell proliferation and the other is that ASTA prevents LPS-induced cell death. We frst observed the efect of ASTA on MEL-12 cell proliferation in an infammatory environment. We examined the efect of ASTA on the cell cycle using fow cytometry. As shown in Figure 2(a), LPS induced a signifcant decrease in both the S phase and G2 phase of the MLE-12 cell cycle, suggesting that cell proliferation was inhibited. However, ASTA did not improve the inhibition of cell proliferation caused by LPS. We further detected the expression of cyclins CCNA2, CCNB1, and CCNE1 using western blotting and found the same change in the trend in the cell cycle (Figure 2(b)).

ASTA Inhibits MLE-12 Cell Pyroptosis.
We found that ASTA did not change the LPS-induced inhibition of MLE-12 cell proliferation. Terefore, the reason why ASTA maintained the stability of the cell number is most likely that it inhibited LPS-induced cell death. We confrmed in another study that ASTA can inhibit LPS-induced apoptosis of MLE-12 cells. However, some reports suggest that associated pulmonary infammatory injury may also be closely related to the pyroptosis of alveolar epithelial cells [20,21]. In addition, we also observed in the above experiments that the pyroptosis inhibitor TETD maintained the stability of the number of MLE-12 cells in the infammatory environment. It is suggested that LPS caused the pyroptosis of MLE-12 cells, and the number of MLE-12 cells was maintained after the inhibition of pyroptosis. Based on the above analysis, we further observed whether ASTA plays any role in LPSinduced pyroptosis through dynamic observation.
Macrophages are the key infammatory cells in infammation-induced pyroptosis [22]. Many infammatory factors secreted by macrophages can cause tissue cell pyroptosis. To observe more directly whether LPS induces infammatory cell aggregation and induces pyroptosis of alveolar epithelial cells, we established a coculture system of alveolar epithelial cells and macrophages. As shown in Figure 3, through dynamic observation, we found that LPS induced obvious changes in cell morphology--cells increasingly swell, cell membranes rupture, and cells die. However, the cell morphology in the ASTA and TETD groups did not change signifcantly after 24 h. Trough this phenomenon, we preliminarily confrmed the speculation that ASTA can inhibit the pyroptosis of MLE-12 cells.

ASTA Inhibits the Caspase 4/11-Gasdermin D Pyroptosis
Pathway. LPS usually induces pyroptosis through the nonclassical pathways. LPS does not directly enter the cytoplasm through receptors. Other caspase family members such as caspase 4, 5, and 11 are activated [23,24]. Te activated caspases 4, 5, and 11 cleave to gasdermin D and induce pyroptosis [25]. Terefore, we detected the expression of key proteins caspase 4, caspase 11, and gasdermin D in the pyroptosis pathway using western blotting. LPS signifcantly induced increased expression of caspase 4, caspase 11, and gasdermin D. ASTA reversed the changes in the levels of these proteins. TETD also signifcantly reduced the expression of gasdermin D (Figures 4(a) and 4(b)). Te above experiments suggest that ASTA inhibits LPS-induced pyroptosis of MLE-12 cells by inhibiting the caspase 4/11gasdermin D pathway.

ASTA Reduces Pyroptosis-Related Infammatory Mediator
Release. In addition to the direct induction of pyroptosis by LPS, IL-1β, IL-18, and LDHA produced by pyroptosis will further expand the infammatory response and cause signifcant infammatory damage [26,27]. Using the ELISA, we

Discussion
In another study, we focused on the protective efects of ASTA on TB-related infammatory injury, which was mainly explained by the apoptotic pathway. In our further literature study, we found that pyroptosis is also an important mechanism that causes pulmonary infammatory damage. Terefore, based on the previous discovery that ASTA has an inhibitory efect on infammatory damage, we further explored whether it also acts through the pyroptosis pathway. Pyroptosis, also known as cell infammatory necrosis, is a type of programmed cell death. Pyroptosis is a new type of programmed cell death discovered and confrmed in recent years, which is characterized by its dependence on infammatory caspases (mainly caspase 1, 4, 5, and 11) and accompanied with the release of many proinfammatory factors [23][24][25]. Te morphological characteristics, occurrence, and regulatory mechanisms of pyroptosis are diferent from other cell death methods such as apoptosis and necrosis. Research shows that pyroptosis is widely involved in infectious diseases [28].
First, we found that both ASTA and the pyroptosis inhibitor TETD, which target gasdermin D, maintained stable MLE-12 cell numbers in an LPS-mimicking infammatory environment. It may be due to two reasons: one is that ASTA and TETD promote cell proliferation and the other is that ASTA prevents LPS-induced cell death. Terefore, we further analyzed the efect of ASTA and TETD on the proliferation of MLE-12 by detecting the cell cycle. Te results show that ASTA did not promote MEL-12 cell proliferation in an infammatory environment. Tis suggests that ASTA maintains stable MLE-12 cell numbers in an infammatory environment by reducing cell death.
We found that the pyroptosis inhibitor TETD could also maintain the stability of MLE-12 cell numbers in the LPSinduced infammatory microenvironment. Tis provided a basis for our further exploration of the aspect of scorching death. Pyroptosis manifests as cell swelling and cell membrane rupture. In the established coculture system of MEL-12 cells and RAW246.7 macrophages, we found that LPS could signifcantly cause cell swelling and cell membrane rupture. However, the morphology of MLE-12 cells  Evidence-Based Complementary and Alternative Medicine did not change much after ASTA and TETD intervention. Te above results suggest that LPS induced pyroptosis in MLE-12 cells. Cell numbers can be stabilized by preventing pyroptosis. ASTA also inhibits pyroptosis. In the noncanonical pathway, caspase 4/11 can be activated through direct contact with bacterial LPS and then cleaved to gasdermin D, triggering pyroptosis. Next, we further investigated the anti-pyroptotic mechanisms of ASTA. We found that ASTA can inhibit the activation of the caspase 4/ 11-gasdermin D pathway. In addition, ASTA also signifcantly inhibited the production of pyroptosis-related infammatory mediators.

Conclusion
Based on the previous confrmation that ASTA can reduce LPS-induced MLE-12 cell apoptosis, we also confrmed in this study that ASTA can also inhibit LPS-induced pyroptosis through the caspase 4/11-gasdermin D pathway ( Figure 6). Terefore, we are more convinced that ASTA is a natural compound worthy of attention and development in the treatment of pulmonary tuberculosis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Tis project was approved by the Ethics Committee of First People's Hospital of Kashi.

Disclosure
Li Li, Zulipikaer Abudureheman, and XueMei Zhong are the co-frst authors. Evidence-Based Complementary and Alternative Medicine 7