Reagents: Dulbecco’s modified eagle medium (DMEM; C11965500BT), fetal bovine serum (FBS, cat no. 10099141C), and trypsin (25200056) were from GIBCO (NY, USA), MC (HY-N2332A), nelonicline (HY-16748) and ponalrestat (HY-106697) were purchased from MedChemExpress (NJ, USA). LDH (ab102526) was from Abcam (Cambridge, UK). Trizol reagent (15596-018) was purchased from Invitrogen (Carlsbad, CA, USA). mRNA was detected using the All-in-one^TM cDNA synthesis kit (AORT-0060) and All-in-one^TM quantitative polymerase chain reaction (qPCR) mix (QP002) (Genecopoeia^TM, Rockville, MD, USA), respectively. Human interleukin (IL)-1β enzyme-linked immunosorbent assay (ELISA) Kit (KE00021) and Human Total IL-18 ELISA Kit (KE00193) were purchased from Proteintech (Chicago, IL, USA). D-Sorbitol Assay (Colorimetric) (ab118968) was purchased from Abcam (Cambridge, UK).

Antibodies: NLRP3(68102-1-Ig, 1:1500), apoptosis-associated speck-like protein containing a CARD (ASC; 67494-1-Ig, 1:2000), caspase-1(22915-1-AP, 1:2000), gasdermin D (GSDMD; 66387-1-Ig, 1:2000), GSDME (67731-1-Ig, 1:2000), interleukin (IL)-1β (16806-1-AP, 1:2000), IL-18 (10663-1-AP, 1:2000), AKR1B1 (67498-1-Ig, 1:2000), NF-κB (66535-1-Ig, 1:2000) and phosphor-NF-κB (39675, 1:2000) were from Proteintech (Chicago, IL). HRP-conjugated goat anti-rabbit (111-035-003, 1:3000) and goat anti-mouse (115-035-003, 1:3000) were purchased from Jackson Immunoresearch and α-tubulin (AC007, 1:4000) were purchased from Ray (Beijing, China).

HeLa and SiHa cells of cervical cancer cells were purchased from the China Center for Type Culture Collection (Beijing, China) and cultured in DMEM supplemented with 10% FBS. The α7nAChR antagonist MC was used to treat cells at final concertation of 20 µM for 48 h, while NF-κB inhibitor BAY 11-7082 (HY-13453, MCE) and AR inhibitor ponalrestat were used at 10 and 5 µM, respectively, for the treatment.

After digestion, cells were collected to re-seeded into a 96-well plate overnight and treated further. The LDH level was tested after treatment with or without MC for 48 h.

The level of IL-1β, IL-18, and sorbitol in the medium content from HeLa and SiHa cells received with or without stimulation were quantified by corresponding commercial kit.

After treatment, the whole cell was lysed to isolate total RNA to conduct a qPCR assay to detect the expression of indicated genes. The primer sequences used in this study are listed in Suppl. Table S1.

The whole cell protein was extracted, and western blotting was performed. Briefly, the whole cell lysate was separated and transferred onto polyvinylidene fluoride membranes, and blocked with phosphate-buffered saline with Tween 20 (PBST) and 5% milk for 1 h at room temperature. Indicated antibodies were diluted and added to the membrane to incubate overnight at 4°C. Next, another 1 h incubation was required for horseradish peroxidase-conjugated secondary antibodies at room temperature. The bands were visualized using a chemiluminescent imaging system after washing with PBST three times.

SiRNA targeting NF-κB (si-NF-κB) or control siRNA (si-CTL), purchased from genepharm (Shanghai, China), combined with luciferase plasmid containing AKR1B1 promoter and Renilla plasmid were transfected into cells for 24 h, and stimulated with or without MC/nelonicline for further 24 h. Relative luciferase unit (RLU) were examined using Duo-Lite Luciferase Assay System (Vazyme, cat no. DD1205-01).

After serum-starvation for 18 h, cells were incubated with or without MC/nelonicline for 1 h. After isolation of chromatin, chromatin was immunoprecipitated with anti- NF-κB using SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads; CST, cat no. 9003) After purification of the chromatin DNA fragments, qPCR was used to analyze the effect of α7nAChR inhibition/activation on NF-κB binding to AKR1B1 promoter with primer synthesized from.

All statistical differences were examined using GraphPad Prism 9 software. A p-value < 0.05 was significant.

α7nAChR has been demonstrated to act as an important anti-inflammatory agent in various disease, such as preeclampsia [22] and cancer [23]. The Gene Expression Profiling Interactive Analysis (GEPIA) dataset showed that no significant difference was observed in α7nAChR expression in cervical squamous cell carcinoma and endocervical adenocarcinoma in comparison with control, and the Kaplan–Meier survival analysis indicated there is no difference in overall survival for patients with high α7nAChR expression in comparison with those with low α7nAChR expression (Figs. 1A and 1B). To further uncover the possible role of α7nAChR in cervical cancer cells, HeLa and SiHa cells were employed to determine the possible function of α7nAChR in cell pyroptosis. Morphologically, an increased number of dead cells were obtained in response to MC stimulation compared with the DMSO group, and inhibition of α7nAChR in HeLa and SiHa cells could significantly increase relative cell death as confirmed by LDH assay (Figs. 1C and 1D). Moreover, IL-1β/IL-18 were drastically enhanced at mRNA levels in response to α7nAChR inhibition (Fig. 1E). Moreover, the quantified band from WB and ELISA analysis also demonstrated that a drastically enhanced IL-1β/IL-18 levels were obtained in HeLa and SiHa cells in response to α7nAChR inhibition (Fig. 1F, Suppl. Fig. S1A). These results suggested α7nAChR inhibition could induce cell pyroptosis in cervical cancer cells to aggravate inflammation.

Next, we sought to discuss the possible change of NLRP3 inflammasome activation in response to α7nAChR inhibition in cervical cancer cells. Real-time PCR analysis suggested a significant enhancement in NLRP3, ASC, caspase1, and GSDMD mRNA levels in HeLa cells subjected to the inhibition of α7nAChR (Fig. 2A), while α7nAChR activation caused by nelonicline could decrease NLRP3, ASC, and caspase 1 mRNA levels (Fig. 2B). Consistently, the further work displayed that the level of NLRP3and GSDMD proteins, were markedly ablated in HeLa cells after nelonicline treatment, while inhibition of α7nAChR by MC triggered NLRP3 inflammasome activation (Fig. 2C, Suppl. Fig. S1B). Collectively, these results suggest that α7nAChR inhibition in cervical cancer cells led to cell pyroptosis through activation of NLRP3 inflammasome.

Metabolic reprogramming is a critical factor in tumor development. Accumulating evidences demonstrated the obvious role of AKR1B1, a limited enzyme of the polyol pathway that catalyzes glucose into sorbitol, in the promotion of inflammation [24,25]. Therefore, we focused our attention to exploring whether the polyol pathway is involved in α7nAChR modulating cell pyroptosis. As shown in Fig. 3A, α7nAChR inhibition in HeLa and SiHa cells led to an upregulation of sorbitol production, which was attributed to enhanced expression of AKR1B1 mRNA and protein caused by α7nAChR inhibition (Figs. 3B–3C, Suppl. Fig. S1C). Most importantly, the addition of AR inhibitor ponalrestat (ARI, 5 µM) could reverse the effect of α7nAChR inhibitor MC on cell pyroptosis, resulting in decreasing IL-1β and IL-18 levels (Figs. 3D–3E, Suppl. Fig. S1D). Taken together, these results suggest that α7nAChR suppressed NLRP3 inflammasome activation by reducing AR-mediated sorbitol production.

Next, to unravel the mechanism through which α7nAChR modulated AKR1B1 expression. AR was confirmed to be a direct target of NF-κB [26], which led us to analyze the influence of NF-κB in α7nAChR-mediated AR transactivity. Luciferase reporter assay showed that α7nAChR inhibition led to a significant increase, while α7nAChR activation drastically decreased AKR1B1 transactivity. Loss of NF-κB expression could reverse the enhanced AKR1B1 luminescence caused by α7nAChR inhibition (Fig. 4A). Further work showed that α7nAChR inhibition increased, while activation of α7nAChR largely blocked the binding of NF-κB to AKR1B1 promoter (Fig. 4B).

Phosphorylation of NF-κB is a critical event for NLRP3 inflammasome activation [14]. To understand the driving forces controlling α7nAChR-mediated AKR1B1 transcription better, we focused our attention on analyzing the influence of α7nAChR on the phosphorylation of NF-κB. The inhibition of α7nAChR could increase the phosphorylation of NF-κB, while activation of α7nAChR in HeLa cells drastically reduced the phosphorylation of NF-κB (Fig. 4C). Taken together, these findings suggested α7nAChR regulated AR expression through NF-κB.

To test the hypothesis that α7nAChR inhibition contributed to NF-κB-mediated AR-dependent cell pyroptosis, we treated HeLa cells with NF-κB inhibitor BAY 11-7082 (10 µM) and analyzed cellular pyroptosis and polyol pathway. As expected, inhibition of NF-κB alone significantly decreased baseline cell pyroptosis and AR expression, and this NF-κB inhibition-mediated effect could reverse the promotion of α7nAChR inhibition on cell pyroptosis, including IL-1β and IL-18 levels, leading to reduced relative cell death (Figs. 5A–5C, Suppl. Fig. S1E), in addition, specific depletion of NF-κB expression could overcome the effect of MC on IL-1β and IL-18 level (Fig. 5D).