Baicalin (572667), MDL12330A (M182), disuccinimidyl suberate (S1885), Hoechst 33342 (B2261), propidium iodide (PI) (P4170), ATP (A6419), lipopolysaccharide (LPS) (Escherichia coli O111:B4) (L4391), dimethyl sulfoxide (DMSO) (D8418), Tween-80 (P8074), and Tween-20 (P1379) were bought from Sigma-Aldrich (St. Louis, MO, USA). Baicalin was dissolved in DMSO at 100 mM and stored at −20°C. H89 (S1643), cell lysis buffer for Western and IP (P0013), and phenylmethanesulfonyl fluoride (PMSF) (ST505) were obtained from Beyotime (Haimen, China). Nigericin (#tlrl-nig) was purchased from InvivoGen (San Diego, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) medium with high glucose, fetal bovine serum (FBS), streptomycin, and penicillin, Opti-MEM were products of Thermo Fisher/Gibco (Carlsbad, CA, USA). The anti-NLRP3 antibody (AG-20B-0014) was purchased from Adipogen AG (Liestal, Switzerland). The antibody against caspase-1p10 (M-20) (sc-514) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against phospho-(Ser/Thr) PKA substrate (#9621), IL-1β (#12242), ASC (#67824), HMGB1 (#3935), β-tubulin (#2128), and horse-radish peroxidase (HRP)-linked horse anti-mouse IgG (#7076), HRP-linked goat anti-rabbit IgG (#7074), and protein G agarose beads (#37478) were purchased from Cell Signaling Technology (Danvers, MA, USA). CF568 goat-anti-rabbit IgG (H + L), highly cross-adsorbed (#20103) and CF488A-conjugated goat-anti-mouse IgG, and highly cross-adsorbed (#20018) were obtained from Biotium (Hayward, CA, USA).

Female C57BL/6 mice (6–8 weeks of age) were bought from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All animals were acclimatized for 1 week before experiments under 12 h dark/12 h light cycle condition. Animal experiments were performed according to the guidelines for the care and use of animals approved by the Committee on the Ethics of Animal Experiments of Jinan University.

Mouse BMDMs were differentiated as reported previously (21, 39). Briefly, mice were sacrificed and bone marrows were collected from the femurs. Bone marrow cells were re-suspended in BM-Mac medium (80% DMEM medium containing 10% FBS plus 20% M-CSF-conditioned medium from L929 cells). Subsequently cells were seeded in 10-cm Petri dish with 10 ml BM-Mac medium and cultured at 37°C in a humidified incubator of 5% CO₂. BMDMs were ready for experiments after 6 days.

Cell death was measured by PI incorporation as described previously (39, 40). Cells were cultured in 24-well plates and primed with 500 ng/ml LPS in Opti-MEM for 4 h. Subsequently, cells were treated with indicated concentrations of baicalin in Opti-MEM for 1 h followed by stimulation with ATP (3 mM) for 30 min or nigericin (10 µM) for 1 h. The cells were stained with PI solution (2 µg/ml PI plus 5 µg/ml Hoechst 33342) for 10 min at room temperature and observed immediately by live imaging using Zeiss Axio Observer D1 microscope equipped with a Zeiss LD Plan-Neofluar 20×/0.4 Korr M27 objective lens (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Fluorescence images were captured with a Zeiss AxioCam MR R3 cooled CCD camera controlled with ZEN software (Carl Zeiss).

Immunofluorescence analysis was performed as previously described (41, 42). In brief, BMDMs were seeded in glass-bottomed dishes (5 × 10⁵ cells/dish) and cultured at 37°C overnight. Cells were primed with 500 ng/ml LPS in Opti-MEM for 4 h. Then the cells were treated with baicalin for 1 h, followed by treatment with ATP (3 mM) for 30 min or nigericin (10 µM) for 1 h in Opti-MEM. After fixation, permeabilization and blocking, cells were incubated with rabbit anti-ASC antibody (1:300) and mouse anti-NLRP3 antibody (1:300), followed by staining with CF568-conjugated goat-anti-rabbit IgG and CF488A-conjugated goat-anti-mouse IgG. After staining with Hoechst 33342 solution (5 µg/ml in PBS) to reveal the nuclei, the cells were observed under a Zeiss Axio Observer D1 microscope with a Zeiss LD Plan-Neofluar 40×/0.6 Korr M27 objective (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Fluorescence images were captured by a Zeiss AxioCam MR R3 cooled CCD camera controlled with ZEN software (Carl Zeiss).

The cross-linking of ASC oligomers was performed as previously described (43, 44). In brief, cells were seeded in 6-well plates at 1.0 × 10⁶ cells/well. After indicated treatments, cells were lysed with cold PBS containing 0.5% Triton-X 100, and the cell lysates were centrifuged at 6,000 × g for 15 min at 4°C. The pellets were washed twice with PBS and then re-suspended in 200 µl PBS. 2 mM disuccinimidyl suberate was added to the resuspended pellets, and the suspension was incubated at room temperature for 30 min with rotation. The cross-linked pellets were spun down at 6,000 × g for 15 min at 4°C and redissolved in 20 µl of 1× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Samples were boiled for 5 min and analyzed by Western blotting.

Cells were rinsed once with ice-cold PBS and lyzed with 0.5 ml ice-cold cell lysis buffer for Western blot and IP (containing 1 mM PMSF). The cell lysates were centrifuged at 13,000 × g for 10 min at 4°C, precleared with a 10% volume of Protein G agarose beads for 30 min at 4°C with gentle agitation, and then incubated with anti-NLRP3 antibody (0.5 µg antibody for 100 µg cell lysate) overnight at 4°C with gentle shaking. The antibody-NLRP3 complexes were collected with a 10% volume of Protein G agarose beads for 2 h at 4°C with gentle shaking. The beads were washed five times with cell lysis buffer, boiled for 5 min in 3× SDS-PAGE sample loading buffer, and resolved by Western blot analysis.

Soluble IL-1β in culture supernatants and sera was determined by cytometric bead array (CBA) mouse IL-1β Flex Set (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Data were acquired on a flow cytometer (FACSCalibur; Becton Dickinson, Mountain View, CA, USA) equipped with CELLQuest software (Becton Dickinson).

Soluble protein in culture supernatants was precipitated as previously described (21, 39). The precipitated proteins were dissolved in equal volume of 1× SDS-PAGE sample loading buffer and subjected to Western blot analysis of secreted mature IL-1β, caspase-1p10, and HMGB1.

Western blotting was performed essentially as previously described (39). Briefly, total proteins were separated by SDS-PAGE and electro-transferred to PVDF membranes (#03010040001; Roche Diagnostics GmbH, Mannheim, Germany). The membranes were blocked by blocking buffer (PBS containing 3% FBS and 0.1% Tween-20) for 1 h and incubated with indicated primary antibody overnight at 4°C, followed by incubation with appropriate HRP-linked secondary antibody (horse anti-mouse or goat anti-rabbit IgG). Bands were revealed with an enhanced chemiluminescence kit (BeyoECL Plus; Beyotime, Haimen, China) and recorded by X-ray films (Carestream, Xiamen, China). The blot images were captured by FluorChem8000 imaging system (AlphaInnotech, San Leandro, CA, USA). The gray values were analyzed by AlphaEaseFC 4.0.

The mouse model of bacterial sepsis was established as previously described (5, 42). E. coli (DH5α strain) was grown in Luria Broth (LB) media at 37°C overnight, and then reinoculated into fresh LB media and grown for 4 h at 37°C. The viable bacteria were collected by centrifugation at 2,600 × g for 10 min, washed with PBS, and then resuspended in appropriate volume of PBS. Bacterial density was measured by using an ultraviolet-visible spectrophotometer (NanoDrop2000, Thermo Scientific), and the corresponding colony-forming units (CFUs) were determined on LB agar plates. All mice were acclimated for 1 week, randomly divided into three groups, and intragastrically administered once with baicalin solution (100 or 200 mg/kg body weight) or vehicle (2% Tween-80 in PBS). Three hours later, viable E. coli cells (2 × 10⁹ CFU/mouse) in 0.5 ml of PBS were injected into the peritoneal cavity of each mouse. Mice were intragastrically administered once again with baicalin solution or vehicle 1 h after bacterial infection. Mouse survival was monitored every 6 h for five consecutive days. In another experiment, mice were treated similarly and were sacrificed at 4 and 8 h post bacterial infection. Their sera were collected, and serum IL-1β levels were measured by CBA mouse IL-1β Flex Set.

All experiments were performed three times independently, with one representative experiment shown. The data were expressed as mean ± SD and analyzed for statistical significance using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance followed by Tukey post hoc test and unpaired Student’s t-test were used to analyze the statistical significance among multiple groups and between two groups, respectively. Kaplan–Meier survival curves were adopted for analysis of mouse survival, and the statistical difference between two groups was determined using the log-rank (Mantel–Cox) test. P-values < 0.05 were considered statistically significant.

NOD-like receptor (NLR) family, pyrin containing domain 3 inflammasome activation in macrophages needs two signals: PAMP (including LPS) priming (signal 1) and DAMP activation (signal 2) (3). We sought to explore whether baicalin could influence the activation of NLRP3 inflammasome in BMDMs. Cells were first primed with LPS (signal 1) and then pretreated with graded doses of baicalin before stimulation with NLRP3 activator ATP (signal 2). Western blotting was used to detect the two commonly used markers of inflammasome activation: the enzymatically active p10 subunit of caspase-1 (caspase-1p10) and mature IL-1β (17 kDa) in the culture supernatants. Consistent with previous reports (45), our data showed that both pro-IL-1β and NLRP3 proteins were highly induced by LPS, whereas pro-caspase-1 and ASC were constitutively expressed in macrophages irrespective of LPS priming (Figure 1A). Upon ATP treatment, both active caspase-1p10 (indicative of caspase-1 activation) and mature IL-1β were released into the culture supernatants. However, baicalin robustly suppressed the release of activated caspase-1p10 and mature IL-1β into the culture supernatant (Figures 1A–C). Notably, without ATP treatment, baicalin alone neither changed the constitutive expression nor the induced expression (by LPS) of the inflammasome related proteins (Figure 1A). Besides, bead-based immunoassay (CBA) was also used to detect mature IL-1β in culture supernatants. As expected, baicalin reduced ATP-induced secretion of mature IL-1β (Figure 1D), confirming the result of Western blot analysis of IL-1β (Figure 1A).

Further, we tested whether baicalin could affect NLRP3 inflammasome activation by another NLRP3 activator nigericin (a microbial toxin derived from Streptomyces hygroscopicus, acting as a potassium ionophore). In line with the results derived from ATP stimulation, baicalin also markedly reduced caspase-1 activation and mature IL-1β secretion in a dose-dependent manner upon nigericin stimulation (Figures 2A–D), indicating the suppression of NLRP3 activation. Taken together, these results indicated that baicalin inhibited NLRP3 inflammasome activation in macrophages upon ATP or nigericin stimulation.

NOD-like receptor (NLR) family, pyrin containing domain 3 inflammasome triggering leads to the activation of caspase-1 rapidly culminating in an inflammatory form of cell death—pyroptosis (7, 8), which can be detected either by PI staining (40) or by measuring released cellular components including HMGB1 in the culture supernatants (46, 47). We thus explored whether baicalin inhibited ATP- or nigericin-induced cell death in BMDMs. LPS-primed cells were treated with ATP, and cell death was monitored by using fluorescence microscopy. As shown in Figure 3A, PI staining showed that cell death was rapidly induced by ATP. The proportion of ATP-induced cell death was ~45% (Figure 3B). Consistent with its effects on suppressing NLRP3 activation, baicalin markedly decreased the ratios of ATP-induced cell death in a dose-dependent manner (Figure 3B). Further supporting this, baicalin dose-dependently inhibited the release of HMGB1 into the culture supernatants (Figures 3C,D). Without ATP triggering, however, baicalin alone induced neither cell death nor HMGB1 release. Similarly, baicalin remarkably attenuated nigericin-induced cell death (Figures 4A,B) and the release of HMGB1 into the culture supernatants but left more HMGB1 in the cell lysates (Figures 4C,D). Taken together, these results indicated that baicalin suppressed ATP-or nigericin-induced NLRP3 inflammasome activation and cell death in macrophages.

Upon activation, NLRP3 recruits its downstream adaptor ASC to form large multi-protein complexes, which can be revealed as specks by either immunofluorescence microscopy or be evidenced as ASC oligomers by Western blotting after chemical cross-linking. We firstly analyzed the formation of ASC specks by fluorescence microscopy. Consistent with the Western blotting results (Figures 1A and 2A), immunofluorescence analysis demonstrated that NLRP3 was weakly detected in unstimulated macrophages but was highly expressed in LPS-primed cells, whereas ASC expression was unaffected by LPS priming (Figure S1 in Supplementary Material). Without stimulation of NLRP3, ASC and NLRP3 were diffusely distributed in cells regardless of baicalin and/or LPS treatment, whereas ATP or nigericin treatment led to ASC speck formation in ~40 or ~30% of the cells, respectively (Figures 5A,B and 6A,B). Notably, ASC specks were highly co-localized with NLRP3 dots in the cytoplasm (Figures 5A and 6A), indicating the recruitment of ASC and formation of NLRP3 inflammasomes in these macrophages. Remarkably, baicalin treatment before ATP or nigericin stimulation drastically decreased the percentages of cells containing ASC specks to 3 or 1%, respectively (Figures 5A,B and 6A,B), suggesting that baicalin could suppress the activation of NLRP3 inflammasome by blocking ASC speck formation.

As ASC specks formed after NLRP3 activation are insoluble in PBS containing 0.5% Triton-X 100 thus being collected as pellets and the ASC proteins within the pellets can be cross-linked by disuccinimidyl suberate to form oligomers (43), we examined the effect of baicalin on such ASC oligomerization by Western blot analysis. In LPS-primed macrophages, different ASC complexes (dimers, trimers, and higher oligomers) were observed in the samples treated with ATP or nigericin, whereas no ASC was detectable in the cells treated with vehicle or baicalin alone indicating that ASC was soluble in these cell lysates. Similar to the ASC speck assay, ATP or nigericin-induced ASC oligomerization was almost completely blocked by baicalin (Figures 5C and 6C). Together, these data further confirmed that baicalin treatment inhibited NLRP3 inflammasome activation through blocking ASC recruitment into the NLRP3 inflammasome.

In light of previous findings that the PKA negatively regulates NLRP3 activation (37, 38) and that baicalin may increase the PKA activity (48), we next explored whether baicalin suppressed the NLRP3 inflammasome activation by upregulating PKA signaling in macrophages. First, the adenyl cyclase inhibitor MDL12330A was used to block cyclic AMP (cAMP) production thus attenuating the PKA activity. As shown in Figures 7A,B, the effect of baicalin on suppressing mature IL-1β (17 kDa) release upon ATP stimulation was partly antagonized by MDL12330A treatment. Furthermore, MDL12330A not only increased ATP-induced cell death but also counteracted the effect of baicalin on suppressing the cell death (Figures 7C,D). The PKA inhibitor H89 had similar effects as MDL12330A did, counteracting the inhibitory effect of baicalin on ATP-induced cell death (Figures 8A,B) and IL-1β release (Figure 8C). Together, these results suggested that baicalin suppressed NLRP3 inflammasome activation at least in part through the PKA signaling.

As baicalin could attenuate ASC speck formation upon NLRP3 activation, we next explore whether the suppression of ASC speck formation by baicalin could also be reversed by blocking the PKA activity. The PKA inhibitor H89 increased the ASC speck formation induced by ATP, and it could partly reversed the inhibitory effect of baicalin on ATP-induced ASC speck formation (Figures 9A,B), further confirming that PKA signaling had been involved in the action of baicalin on suppressing NLRP3 inflammasome activation.

Therefore, we next explored whether baicalin could enhance the Ser/Thr phosphorylation of NLRP3 on PKA-specific sites, which has been reported to prevent NLRP3 inflammasome activation in macrophages (37, 38). To this end, NLRP3 was immunoprecipitated, and its phosphorylation on Ser/Thr residues of PKA substrate motifs was evaluated by Western blotting. Indeed, the PKA-specific phosphorylation on Ser/Thr residues of NLRP3 was greatly increased by baicalin pretreatment. The PKA inhibitor H89 blocked such phosphorylation, further verifying that baicalin-enhanced NLRP3 phosphorylation was mediated by PKA signaling (Figures 9C,D). Altogether, these results suggested that baicalin inhibited NLRP3 inflammasome activation at least partly through augmenting PKA signaling.

As ATP-induced NLRP3 inflammasome activation has critical roles in bacterial sepsis (5), we explored whether baicalin could ameliorate sepsis in a mouse model of bacterial infection in the peritoneal cavity. Mice were administered once intragastrically with baicalin solution (100 or 200 mg/kg body weight) or vehicle 3 h before intraperitoneal injection of viable E. coli (2 × 10⁹ CFU/mouse). One hour after the injection of bacteria, the mice were administered once again with baicalin or vehicle. Only 10% of mice survived the period of observation (120 h) in the vehicle group, whereas baicalin administration significantly increased their survival rates when compared with the vehicle group (Figure 10A). Moreover, baicalin treatment significantly decreased IL-1β levels in the sera of bacterial infected mice as compared with vehicle (Figure 10B), indicating that baicalin suppressed systemic inflammation in the mice, which was consistent with the in vitro studies showing baicalin’s inhibitory effects on NLRP3 activation. Therefore, this result highlights the potential application of baicalin in the treatment of inflammatory diseases including bacterial sepsis.