Baicalin Induces Gastric Cancer Cell Pyroptosis through the NF-κB-NLRP3 Signaling Axis

Reagents

Baicalein (Purity ≥ 98% - Purchased from CHENGDU MUST BIO-TECHNOLOGY Co., Ltd), DMSO, CCK8 (Vazyme, China), NLRP3, Caspase-1, GSDMD, β-Actin, NF-κB, IL-18, IL-1β (Purchased from Abmart Shanghai Co., Ltd.).

Cell and cultures

AGS (Human gastric gland carcinoma cell), nurtured in a conducive environment comprising 10% fetal bovine serum (ABW) supplemented with 1% streptomycin-penicillin 1640 medium (keyGEN BioTECH), finds solace within the confines of a temperature-controlled incubator set at a balmy 37 °C.

Cell viability assay

Evaluating the vitality of cells assumes paramount importance, thus necessitating the performance of CCK8 assays to gauge their sensitivity to the drug at hand. Inoculating cells in 96-well plates at a density of 5*10^4 cells per well, we embark upon their nurturing journey. Following an overnight incubation period, the cells are subjected to varying concentrations of BA (6.25 uM/ml, 12.5 uM/ml, 25 uM/ml, 50 uM/ml, 100 uM/ml) over a span of 24 hours. To assess the cellular response, 10 ul of cck8 is introduced to each well, fostering a delicate symbiosis that endures a two-hour incubation at 37 °C, diligently protected from the prying grasp of light. Subsequent measurement of absorbance (OD) at 490 nm utilizing a Microplate Reader, with data representative of no less than three independent experiments.

Molecular Docking

The crystal structures of proteins with high resolution and relatively intact structures were selected by the PDB database ¹⁷. Detailed data are as follows: Caspase-1 (ID: 6MFQ; 7AEH), Caspase-4 (ID: 5GPP; 5GPQ), Caspase-11 (ID: 3NIR; 3P4J), NLRP3 (ID: 3VWD), GSDMD (ID: 6BZ9), ASC (ID: 3VLN), IKB (ID: 4F0Z), IKKB (ID: 4KIK), NFκB (ID: 6P0Z). Prior to the docking extravaganza, a meticulous dance of pre-treatments involving dehydration and hydrogenation commences, orchestrated with the aid of Auto Dock Tools software. Engaging Autogrid in a simulated docking affair enables us to unearth binding energies that guide our discerning gaze towards molecules showcasing remarkable affinity. Molecules with strong binding energies were selected and visualized by using pymol software.

Western blot

Cells were stimulated with different concentrations of baicalin for 24 h. Cellular extracted proteins were collected with the measurement of total protein concentration. The proteins were separated by electrophoresis with 10%, 12%, and 15% SDS-PAGE gel and transferred to PVDF membranes. After sealing the membranes with the skim milk powder, the membranes were incubated with primary antibodies (β-Actin, NLRP3, Caspase-1, GSDMD, IL-18, IL-1β, and NF-κB) at 4 °C overnight and secondary antibodies at room temperature for 2 hours. Visualize the strips using a luminescence imaging system (Chemi Doc XRS+).

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from cells using HIScript II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme) and cDNA was synthesized using ChamQ Universal SYBR qPCR Master Mix kit (Vazyme). The detailed sequences of the primers used for RT-PCR are as follows:

Relative expression levels were analyzed by the 2-ΔΔCt method. PCR primers were synthesized by Shanghai Bioengineering Technology (Shanghai, China).

Lactate dehydrogenase (LDH)release assay

An intriguing biochemical phenomenon, the release of lactate dehydrogenase (LDH), serves as a remarkable indicator of cell membrane rupture. To measure cellular LDH release, an LDH assay kit from the esteemed Nanjing Jiancheng Bioengineering Institute in China was employed. Tumor cells underwent stimulation with varying concentrations of BA (4 μM, 7 μM, 11 μM) for a duration of 24 hours. The inhibitor group received prior treatment with 20 μM MCC950 Sodium (MS) for a period of 2 hours, followed by incubation with diverse concentrations of BA for another 24 hours. The absorbance at 450 nm was meticulously detected utilizing a Microplate Reader.

Network Pharmacology Analysis

Delving deeper into the intricate realm of pharmacology, therapeutic targets associated with baicalin were meticulously amassed through the remarkable Traditional Chinese Medicine Database and Analysis Platform (TCMSP) ¹⁸. In order to procure targets relevant to gastric cancer, the esteemed DrugBank database ¹⁹ and the DisGeNET v6.0 database ²⁰ were diligently consulted. VENNY 2.1.0 online interactive software ²¹ was used to merge and de-duplicate the data. Further analysis was conducted employing the Metaspace database ²², specifically exploring the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology analysis- cellular component (GO-CC), and the multifaceted Gene Ontology biological process (GO-BP) and molecular function (GO-MF). The results were visualized by Cytospace to obtain the "Baicalin-Therapeutic Target-Pathway of Action" diagram.

ROS detection

Cells were collected from each experimental group, and the positive control group was first stimulated with Rosup (100 μM) for 2 hours. Probes were loaded on cells using the Reative Oxygen Species Assay kit (Yeasen Biotechnolo (Shanghai) Co.,Ltd). Cell sorting by using flow cytometry.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8 software, and comparison between two datasets demanded the meticulous employment of an independent samples t-test, where statistical significance was denoted by P<0.05. Furthermore, comparisons among multiple groups were astutely analyzed via Tukey's test, capitalizing on the foundation of one-way ANOVA.

BA promotes gastric cancer cell pyroptosis

The investigation commenced by subjecting gastric cancer cells to a diverse range of BA (The structure of baicalein is shown in Figure 1a) concentrations (6.25uM, 12.5uM, 25uM, 50uM, 100uM) for a duration of 24 hours, as visually represented in Figure 1b. Notably, it revealing that the administration of BA at a concentration of 6.25uM already elicited a pronounced inhibition of cell growth within the gastric cancer cell population.To further explore the potency of baicalin's action on gastric cancer cells, the 24-hour IC50 value was determined to be 12.05uM. Based on this critical threshold, subsequent investigation focused on administering baicalin at the doses of 4uM, 7uM, and 11uM.

Microscopic examination provided further insights into the cellular dynamics following a 24-hour exposure to baicalin. Notably, the cell volume exhibited a significant reduction compared to the blank group, accompanied by a transformation in cell morphology from irregular to round-like, vacuoles emerged within the cellular milieu, and certain cell membranes underwent rupture, leading to the release of cellular contents (Figure 1c).

Intriguingly, the inclusion of 20 μM MS failed to impede BA's capacity to induce tumor cell death and induce alterations in cellular morphology (Figure 2). Furthermore, an in-depth analysis of LDH levels, considered a consequential product of pyroptosis, revealed that the inhibition of NLRP3 expression by MS profoundly dampened the release of LDH. Conversely, baicalin exhibited a dose-dependent promotion of LDH release, surpassing the inhibitory effect induced by MS and resulting in higher levels of LDH release compared to baicalin alone (Figure 5a). These observations provide compelling evidence showcasing BA's ability to fuel the scorching of gastric cancer cells. It demonstrates that BA could promote gastric cancer cell pyroptosis.

BA regulates NLRP3-induced pyroptosis in gastric cancer cells

It is widely acknowledged within scholarly circles that the modicum of docking energy exhibited by the protein-ligand complex and the therapeutic drug in question serves as a pivotal determinant of the binding conformation's stability. The underlying principle posits that a lower docking energy corresponds to heightened stability in the binding interactions. Specifically, docking energies below the threshold of -4.25 kJ are deemed indicative of binding activity between the protein-ligand complex and the drug. Energies falling below -7.0 kJ signify favorable docking outcomes, while those below -9.0 kJ herald robust and potent docking activities.

To gain insights into the mechanistic nuances underpinning Baicalin's actions, molecular docking simulations were scrupulously conducted. This astute undertaking aimed to pinpoint the precise target with which Baicalin intricately interacts. The ensuing results unveiled noteworthy associations, as NLRP3、Caspase-1、GSDMD, and ASC emerged as prominent entities that engaged in docking interactions with Baicalin. Notably, the binding affinity was most pronounced with NLRP3, boasting an exquisite binding energy of -9.2 kJ (Table 2).

The experimental analysis employing Western blotting (WB) and quantitative polymerase chain reaction (Q-PCR) methodologies has yielded substantive evidence demonstrating the capacity of Baicalin to modulate the expression levels of NLRP3, GSDMD-N, Caspase-1 P20, ASC protein, and mRNA. Notably, the findings reveal that the medium concentration of 7 μM Baicalin exerts a significantly more pronounced stimulatory effect on NLRP3 levels compared to the alternative concentration groups of 4 μM and 11 μM (Figure 4a,4b,5b). Conversely, the levels of Caspase-1-P20 (Figure 4a,4c,5f) and GSDMD-N (Figure 4a, 4d,5c) witness substantial increments upon Baicalin administration, albeit without displaying any overt dependence on dosage. Moreover, intriguing dose-dependent variations are evident in the ASC levels stimulated by Baicalin treatment. Specifically, lower and medium concentrations of Baicalin provoke significant elevations in ASC levels, while higher concentrations thereof elicit negligible effects on ASC expression (Figure 4g). In addition, the consequential evaluation of pyroptosis products, namely IL-18 and IL-1β, attests to the ability of Baicalin to augment their levels. The experimental outcomes reveal that Baicalin induces a robust increase in IL-18 (Figure 4a, 4e, 5d) and IL-1β (Figure 4a, 4f, 5e) levels, with the medium concentration group (7 μM) eliciting the most prominent effect.

Furthermore, the effects of Baicalin under the pretreatment of MS were investigated, yielding observations. Remarkably, Baicalin exhibits a dose-dependent reversal of the inhibitory influence exerted by MS on NLRP3 expression levels in gastric cancer cells. Baicalin demonstrates augmented NLRP3, Caspase-1 P20, and ASC expression levels when subjected to MS pretreatment. Upon careful consideration of concentration factors, it is evident that under MS pretreatment, baicalin exhibited a stronger stimulatory effect on the expression of NLRP3 in the high concentration group (11 μM) as compared to the low concentration group (4 μM) and medium concentration group (7 μM). Moreover, all concentrations of Baicalin effectively counteract the inhibitory impact of MS on ASC in gastric cancer cells. Subsequent to subjecting scorched products IL-18 and IL-1β to pre-treatment with MS, notable insights of discernible nature arise. Specifically, an examination of the levels of IL-18 and IL-1β within the low and medium concentration groups reveals a lack of significant distinctions. However, in marked contrast, the high concentration group exhibits a substantial augmentation in both IL-18 and IL-1β levels, surpassing those induced by equivalent doses of Baicalin administered independently. This striking divergence in the response underscores the intricate interplay between MS pretreatment and Baicalin administration, thereby accentuating potential regulatory mechanisms underlying the modulation of these pyroptosis mediators.

BA affects NLRP3- caspase-1 via NF-κB to promote gastric cancer cell Pyroptosis

Baicalein, a flavonoid compound, was subjected to comprehensive evaluation using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database ¹⁸, which provided insightful information regarding its pharmacological attributes. Considerable drug similarity (DL: 0.21) and favorable oral absorption (OB: 33.52%) were uncovered, suggesting its potential as a therapeutic agent. In light of this promising discovery, thorough investigations were conducted to elucidate the intricate molecular interactions underlying BA's effects.

To discern the specific targets pertinent to gastric cancer, an amalgamation of data from the DisGeNET and GeneCards databases was undertaken, employing rigorous de-duplication procedures. Resulted in the identification of 32 unique co-action targets shared by BA and gastric cancer compounds (Figure 6a). These co-action targets were then subjected to comprehensive functional enrichment analyses utilizing the Meatscape database ²².

The outcome of these analyses unveiled compelling insights into the regulatory processes modulated by BA. Notably, among the enriched results derived from KEGG (Table 3) and GO (Table 4) analyses, the TNF signaling pathway emerged as a prominent pathway under the influence of BA (Figure 6b). This finding underscores the regulatory potential of BA in orchestrating crucial cellular responses mediated by tumor necrosis factor (TNF). Further exploration into the biological processes impacted by BA illuminated several noteworthy facets. Response to oxidative stress, reactive oxygen species metabolic process, and regulation of cellular catabolic process were among the intricate molecular cascades influenced by BA (Figure 6c). These findings collectively shed light on the diverse regulatory mechanisms through which BA exerts its influence within the complex milieu of gastric cancer cells.

Of particular significance was the identification of RELA protein as a key player associated with BA's effects. RELA, a pivotal component of the nuclear factor kappa B (NF-κB) pathway, displayed a robust association with BA based on docking studies involving NF-κB, IKB, and IKKB (Figure 7a,7b,7c). Molecular docking simulations revealed the strong affinities between BA and NF-κB (Table 5). This interaction underscores the potential role of BA in modulating NLRP3-caspase-1 signaling, thereby facilitating the release of inflammatory factors during tumor cell pyroptosis via the NF-κB pathway. Moreover, investigations into the concentration-dependent effects of BA on NF-κB (p65) mRNA levels uncovered intriguing stage-specific discrepancies (Figure 7d). Notably, low (4 μM) and medium concentrations (7 μM) of BA elicited dose-dependent increases in NF-κB (RELA) and IKKB mRNA levels. In contrast, the excessively high concentration of BA (11 μM) manifested reduced expression levels compared to the low- and medium-dose groups (Figure 7e,7f).

BA promotes a dramatic increase in ROS levels in gastric cancer cells

The impact of different concentrations of BA (4 μM, 7 μM, and 11 μM) on reactive oxygen species (ROS) levels within AGS cells was investigated over a 24-hour incubation period. Flow cytometry analysis was employed to quantify the changes in ROS levels under each condition (Figure 8). Notably, BA treatment resulted in a significant elevation in ROS levels within gastric cancer cells when compared to the positive control group (Rosup). Furthermore, no noticeable differences in ROS levels were observed among the various BA dose groups.