Sulfated Pelvetia siliquosa Polysaccharides Attenuate Pyroptosis via NF-κB Pathway Inhibition Against Calcium Oxalate Stone Formation

Abstract

Objective: The formation of calcium oxalate (CaOx) kidney stones is accompanied by the pyroptosis of renal epithelial cells. The risk of kidney stone formation can possibly be reduced through pyroptosis inhibition. Methods: Pyroptosis of HK-2 cells induced by 3 µm CaOx monohydrate (COM-3 µm) was inhibited by Pelvetia siliquosa polysaccharides before and after sulfation (PSP0 and PSP3, with −OSO₃− contents of 1.04% and 36.12%, respectively). The inhibitory efficiency and mechanism of PSP0 and PSP3 were evaluated via caspase-1/PI double staining and Western blot detection of pathway proteins in pyroptosis cells. The potential anti-stone effect of polysaccharides was evaluated through measurement of the extent of crystal adhesion on the cell surface. Results: The proportion of pyroptosis cells induced by COM-3 µm reached 17.87%. After protection by PSP0 and PSP3, the percentage of pyroptosis cells was reduced to 12.7% and 6.35%. The levels of NLRP3, ASC, gasdermin D, IL-1β, and IL-18 related to pyroptosis were downregulated. In addition, the activation of the NF-κB pathway was considerably inhibited. During inhibition of pyroptosis, reactive oxygen species and lactate dehydrogenase levels were decreased, the integrity of zonula occludens-1 protein was restored, and the expressions of CaOx-specific adhesion proteins (ANXA3 and CD44) were substantially decreased. As a result, the adhesion of COM crystals on the cell surface was reduced. PSP3 exhibited a higher protection energy efficiency than PSP0. Conclusions: PSP0 and PSP3 inhibited the pyroptosis of HK-2 cells through the NLRP3/ASC/caspase-1/IL-1β pathway, which caused the inhibition of cell inflammation and injury, reduced the expressions of adhesion proteins, and reduced the risk of CaOx crystal adhesion and stone formation. The biological activity of PSP0 and PSP3 after sulfation modification increased.

1. Introduction

Traditional preventive strategies for calcium oxalate (CaOx) stones exhibit certain advantages yet also have inherent limitations [1]. Thus, novel strategies for the treatment and prophylaxis of such stones need to be explored. Among the pharmacotherapies recommended by clinical guidelines for preventing the recurrence of calcium kidney stones, potassium citrate has proven preventive benefits yet is limited [2]. Alkali therapy is associated with gastrointestinal adverse effects and a potential risk of inducing calcium phosphate stones, necessitating concurrent monitoring of urinary pH and citrate levels in clinical application [3]. As first-line agents, thiazide diuretics have seen the clinical value of hydrochlorothiazide questioned due to methodological flaws, poor efficacy, a high incidence of adverse events and the absence of bone-protective effects [4]. The thiazide-like diuretics indapamide and chlorthalidone may offer superior efficacy, yet direct comparative studies between different types of these drugs are lacking, and their clinical use currently requires individualized decision-making, with an ongoing clinical trial addressing this research gap [5]. Allopurinol demonstrates a definite effect in preventing stone recurrence in patients with hyperuricosuria, but its application value for calcium kidney stone patients without hyperuricosuria remains unclear [6].

With the in-depth investigation into the mechanisms of cell death, intervention strategies targeting pyroptosis have gradually emerged as a novel research avenue; these strategies exhibit striking differences in intervention logic, action targets and core mechanisms, thereby providing diversified approaches for urolithiasis prevention.

Pyroptosis-targeted drugs offer distinct advantages over conventional therapies for CaOx stone prevention. First, they enable precise intervention: unlike non-specific traditional drugs regulating urinary electrolytes/metabolic indicators, these act directly on key pyroptosis molecular nodes linked to stone formation, inhibiting crystal-induced inflammation and cell damage to target the pathological root. Second, they have broader applicability, overcoming traditional drugs’ subgroup limitation because the formation of stones is closely related to the occurrence of pyroptosis, promising efficacy for more patients. Third, they boost safety: compared with alkali therapy (secondary stone risk) and thiazides (high adverse events), targeting core inflammation minimizes off-target metabolic effects, improving safety.

Studies have gradually highlighted the roles of cell pyroptosis in various diseases [7,8,9,10]. Pyroptosis of vascular endothelial cells destroys the integrity of the vascular wall, which results in local lipid deposition and atherosclerosis formation and indicates the close relation between pyroptosis and atherosclerosis [8]. Pyroptosis is involved in early amyloid β (Aβ) deposition and neuronal death and promotes the occurrence and progression of Alzheimer’s disease [9]. In addition, pyroptosis can affect tumor development [10], including those of colorectal cancer, gastric cancer, lung cancer, cervical cancer, and leukemia [10].

Pyroptosis can also promote the formation of CaOx stones because oxidative stress damage occurs during this process [11,12,13,14]. Redox signals modulate inflammasome activation (a key upstream step in pyroptosis); mitochondrial ROS drives NLRP3 inflammasome assembly, linking oxidative stress to caspase-1 activation and pyroptosis [15,16,17]. ROS induces NLRP3 activation and pro-inflammatory factor release in inflammation [18], with elevated ROS triggering NLRP3-dependent pyroptosis and ROS inhibition abrogating it [19,20], confirming ROS regulates pyroptosis via NLRP3 [21].

Sun et al. [11] observed that after CaOx (2 mmol/L) was coincubated with HK-2 cells for 12 h, the levels of reactive oxygen species (ROS), malondialdehyde, lactate dehydrogenase (LDH), Toll-like receptor (TLR) 4, nuclear factor-κB (NF-κB), NLR family pyrin domain-containing 3 (NLRP3), cleaved caspase-1, interleukin (IL)-1β, IL-18, IL-6, and TNF-α were increased. The level of superoxide dismutase in cells decreased substantially, which indicates that CaOx crystals caused strong oxidative stress and cellular inflammation. In vivo experimental studies have shown that not only were the proteins gasdermin D (GSDMD), NLRP3, caspase-1, and IL-1β, which are related to cell pyroptosis, highly expressed in kidney stone mouse models, but also remarkably the levels of COM crystal-specific adhesion proteins osteopontin (OPN) and CD44, which promoted the deposition of COM crystals [12]. Joshi et al. [14] showed that compared with those in normal mice, the expression levels of OPN, hyaluronate (HA), and CD44 and the deposition rate of CaOx crystals were greatly reduced in NLRP3 gene-deficient mice that received a high-oxalate diet; this finding indicates that the inhibition of the activation of pyroptosis-related inflammasome can inhibit the formation of CaOx stones.

CaOx crystals induce pyroptosis through a classical pathway [22], which depends on the NLRP3/apoptosis-associated speck-like protein containing a CARD (ASC)/caspase-1/IL-1β axis and the release of inflammatory mediators. Therefore, intervention in a certain link in the classical pyroptosis pathway is an effective strategy to inhibit its process. The inhibition of pyroptosis [23,24,25] has attracted considerable attention by inhibiting the activities of NLRP3 inflammasome [23] and caspase-1 protein [24,25]. Knocking out ASC or treatment with a caspase-1 inhibitor (VX-765) prevents the caspase-1-mediated activation of IL-1β, IL-18, and GSDMD [24], which results in the inhibition of pyroptosis.

Some Chinese herbs and plant extracts have been used to inhibit pyroptosis [12,26,27]. Shenhuangdan (Panax ginseng C.A. Mey. (Renshen), Rheum palmatum L. (Dahuang), and Salvia miltiorhiza Bge.) can extensively inhibit the protein expression levels of NLRP3, GSDMD, cleaved N-terminal GSDMD (GSDMD-N), cleaved caspase-1, caspase-1, and ASC in the lung tissue of sepsis mice and improve the lung injury induced by cecal ligation puncture [26]. Vitexin can reduce the levels of pyroptosis-related proteins GSDMD, NLRP3, caspase-1, and IL-1β (which are elevated in kidney stones in mice) [12]. The classical Chinese medicine Taohong Siwu Decoction can alleviate acute myocardial ischemia–reperfusion injury by inhibiting cell pyroptosis [27].

Plant polysaccharides can also inhibit pyroptosis [28,29,30,31]. Lipopolysaccharide-binding protein can downregulate the expressions of NLRP3, caspase-1, and GSDMD-N; inhibit the pyroptosis process of arising retinal pigment epithelia-19 cells activated by Aβ1–40; and reduce the pyroptosis of cells exposed to Aβ1–40 oligomers [28]. Antrodia camphorata polysaccharide can improve the neurobehavioral, motor, and coordination abilities of mice afflicted with Parkinson’s disease; the effect of Antrodia camphorata polysaccharide is related to the reduced activation of NLRP3 inflammasome and expressions of IL-1β, caspase-1, and pro-caspase-1 [29]. Plantaginis Semen polysaccharides downregulate the expression levels of NLRP3, ASC, and caspase-1 proteins, inhibit the release of downstream inflammatory factors, and therefore improve kidney inflammation in rats suffering from gouty nephropathy to prevent kidney injury [30]. Bletilla striata polysaccharides can reduce the levels of NLRP3/caspase-1/GSDMD and high-mobility group box 1/TLR4 in lung tissue and MH-S cells to inhibit cell pyroptosis and thus improve lipopolysaccharide-induced acute respiratory distress syndrome [31]. In conclusion, polysaccharides exert a good effect on the inhibition of pyroptosis induced by the activation of the NLRP3/ASC/caspase-1/IL-1β pathway. However, no pyroptosis inhibitors have been approved for clinical treatment.

We previously observed [32] the good antioxidant and anti-inflammatory effects of Pelvetia siliquosa polysaccharides (PSPs), which can inhibit CaOx crystal deposition in mice; however, studies on the related mechanism remain limited. This study aimed to explore the inhibitory effect of PSPs on the CaOx monohydrate (COM)-induced pyroptosis of cells to shed new light on the formation mechanism of kidney stones and develop antilithiasis drugs.

2. Results

2.1. PSP Characterization and Polysaccharide Concentration Selection

Fourier transform infrared spectroscopy (FT-IR) spectral results (Figure 1A) showed very similar FT-IR spectra of natural Pelvetia siliquosa polysaccharide (PSP0) and Pelvetia siliquosa polysaccharides after sulfation (PSP3), whose −OSO₃− contents were 1.04% and 36.12%, respectively, before and after sulfation, which indicates that the overall structure of the polysaccharides remained intact during sulfation. Compared with that of PSP0, the absorption peaks of PSP3 near 1250 and 820 cm⁻¹ were considerably improved, with the two peaks being attributed to the asymmetric stretching vibration of sulfuric acid group O=S=O [9] and S=O vibration at the C6 position [33]. This finding indicates a substantial increase in the −OSO₃− content at the C6 position of PSP3 (red −OSO₃− base in Figure 1B).

COM-3 µm crystals were synthesized and characterized, with the SEM image shown in Figure 1C and the particle size analysis presented in Figure 1D. In this study, a cellular pyroptosis model was established using COM-3 µm crystals, as our research group has previously verified that this particle size of COM crystals can effectively induce pyroptosis in HK-2 cells [34,35].

Figure 1E shows the protective effect of various concentrations of PSP0/PSP3 on COM-3 µm crystal-induced HK-2 cells. When 300 µg/mL COM damaged cells for 48 h, the cell viability decreased to 69.25%. Different concentrations of PSP0/PSP3 (30–240 µg/mL) increased the cell viability, and the maximum value increased to 93.75%, which indicates that PSP0/PSP3 can protect cells from COM crystal damage.

However, the protective capability of polysaccharides on cells did not increase stepwise with their concentration, although a good concentration value was obtained (180 µg/mL).

Compared with PSP0, PSP3 showed a stronger capability to protect cells. At 180 µg/mL, the cell viability of the PSP0 and PSP3 groups increased to 81.16% and 93.75%, respectively. Therefore, PSP0 and PSP3 with a concentration of 180 µg/mL were selected for subsequent protection studies.

2.2. PSPs Inhibit the Activation of the NF-κB Pathway

The NF-κB signaling pathway is assumed to be the first step in inflammasome activation. NF-κB p65 is normally present in the cytoplasm, and upon receipt of a stimulus signal, a large amount of NF-κB p65 is translocated to the nucleus [36]. Thus, whether the NF-κB signaling pathway is activated can be determined by detecting the location of NF-κB p65 (i.e., colocalization with the nucleus).

Figure 2A shows the distribution of NF-κB p65 throughout the control cells. Analysis of the cross-sectional fluorescence intensity of the normal group of cells (orange arrow in Figure 2A) revealed that the green fluorescence intensity remained at approximately 50 (Figure 2B). This result indicates that NF-κB p65 was distributed in the cytoplasm and did not reach the nucleus. In the COM-damaged group (Figure 2A), NF-κB p65 (green fluorescence) and nuclear (blue fluorescence) colocalization were observed; that is, green fluorescence was improved during nuclear colocalization (blue fluorescence). Analysis of cross-sectional fluorescence intensity (Figure 2A) revealed that the green fluorescence intensity of the cytoplasm was still around 50, but that of the nuclear region increased substantially to 120 (Figure 2B). This result implies that NF-κB p65 was ectopic to the nucleus under stimulation of COM crystals. After PSP0/PSP3 protection, the number of NF-κB p65-colocalized cells decreased, which means that the polysaccharides inhibited the nuclear ectopia of NF-κB p65 (Figure 2B). In addition, compared with PSP0, PSP3 showed a stronger capability to inhibit NF-κB p65 nuclear ectopia (Figure 2B).

2.3. PSPs Inhibit COM Crystal-Induced Pyroptosis

Figure 2C shows the comparison of the inhibitory capabilities of PSP0/PSP3 in COM crystal-induced cellular pyroptosis, which was detected via the triple staining method using caspase-1/propidium iodide (PI)/Hoechst 33342. The control cells did not express caspase-1, Hoechst 33342-labeled nuclei were dark, and no red cells labeled by PI appeared, which indicates that the cells were normal (orange arrow).

The COM-injured group expressed green caspase-1, and the cells stained red by PI were late pyroptosis cells (yellow arrow). The injured group included cells with green and dark blue fluorescence, that is, early pyroptosis cells (purple arrow), which implies that COM induced pyroptosis.

After being protected by 180 µg/mL polysaccharides, the number of cells with high expression of caspase-1 decreased, which indicates the decreased number of pyroptosis cells, pyroptosis of cells by PSP0/PSP3, and better inhibitory effect of PSP3 than PSP0.

To accurately determine the degree of pyroptosis, we used caspase-1/PI double staining for flow quantitative detection. A total of 97.7% of the normal cells were concentrated in Q4 in the lower left corner in Figure 2D. In the COM injury group, Q1, Q2, and Q3 cells accounted for 6.38%, 12.4%, and 5.47%, respectively (Figure 2D), which implies that 6.38% of cells underwent apoptosis and necrosis and 17.87% experienced pyroptosis (Q2 + Q3), among which 12.4% and 5.47% of the cells were in late and early pyroptosis, respectively. However, the percentage of pyroptosis cells in the PSP protection group decreased (Figure 2D). This finding indicates that PSPs inhibited pyroptosis cells, and the proportion of pyroptosis cells decreased to 6.35% after PSP3 protection.

2.4. PSPs Decreased the Expressions of Pyroptosis-Related Proteins

NLRP3 inflammasome performs an extremely important function in the classical pyroptosis pathway, and its expression promotes cellular inflammation and pyroptosis [22]. During the activation of NLRP3 inflammasome, the ASC protein, NLRP3, and pro-caspase-1 oligomerize to form the active NLRP3 inflammasome complex [37,38], which further promotes the expression of caspase-1. This complex mediates the production of pro-inflammatory factors (IL-18 and IL-1β) and the cleavage of cell membrane by GSDMD, which results in cell membrane damage and the release of inflammatory factors [37]. Immunofluorescence and Western blot were used in the detection of proteins associated with the classical pyroptosis pathway of HK-2 cells (Figure 3).

(a) Inhibition of the activation of NLRP3 inflammasome

Figure 3A shows the findings of immunofluorescence detection involving NLRP3 inflammasome. Normal cells maintained a low level of NLRP3 expression; that is, a weak green fluorescence was observed. More cells in the COM-injured group showed a strong green fluorescence, which indicates their higher NLRP3 expression level. After the intervention with PSP0 or PSP3 in the injured group, NLRP3 exhibited a reduced expression (Figure 3B), and PSP3 showed a stronger capability to inhibit NLRP3 expression than PSP0 (Figure 3B).

(b) Inhibition of ASC protein expression

Figure 3C shows the expression level of intracellular ASC detected by immunofluorescence. The control group presented a lower level of ASC, whereas more cells in the COM-injured group showed a strong green fluorescence, which indicates their higher ASC expression. In addition, oligomerization of ASC was observed; that is, evident green, fluorescent spots were observed in the fluorescence diagram, which represent the oligomerization assembly of ASC protein, NLRP3, and pro-caspase-1; these findings indicate the activation of NLRP3 inflammasome [38]. After the injured-group cells were protected by PSP0/PSP3, the number of cells with a high ASC level was considerably reduced, and oligomerization of ASC was also reduced (Figure 3D). In addition, compared with PSP0, PSP3 exhibited a stronger capability to inhibit ASC expression and oligomerization.

(c) Inhibition of GSDMD protein expression

Figure 3E shows the GSDMD protein bands expressed in HK-2 cells measured by Western blotting. The control group showed a lower expression level of GSDMD, compared with the COM-injured group (Figure 3F), which indicates that COM crystals induced the pyroptosis of HK-2 cells. After PSP0/PSP3 intervention protection, the expression level of GSDMD decreased, and the capability of PSP3 to inhibit GSDMD expression became stronger than that of PSP0 (Figure 3F).

(d) Inhibition of the expression of pro-inflammatory cytokines IL-1β and IL-18

IL-1β and IL-18 proteins are pro-inflammatory cytokines. In pyroptosis, activated caspase-1 participates in the maturation of pro-IL-1β and pro-IL-18 and subsequently generates IL-1β and IL-18 [37]; it then triggers inflammation and the damage of surrounding cells, which lead to additional cell death [39]. According to its protein band in Figure 3E, the expression level of IL-1β in the COM-injured group was 2.64 times that in the normal group (Figure 3G), which implies that COM crystals induced cellular inflammation. After PSP0/PSP3 intervention protection, the expression level of IL-1β decreased (Figure 3G), which implies that the polysaccharides can inhibit the expression of IL-1β. In addition, PSP3 exhibited stronger anti-inflammatory activity than PSP0.

In the COM-injured group, the secretion of IL-18 was as high as 11.68 pg/mL, which was more than 8 times that of the normal group (1.41 pg/mL) (Figure 3H). This finding indicates that COM crystals induced the secretion of IL-18 and aggravated cellular inflammation. After the intervention by 180 µg/mL PSP0 or PSP3 protection, the secretion of IL-18 was substantially reduced (Figure 3H), and stronger anti-inflammatory activity was observed in the sulfation-modified PSP3 group (3.84 pg/mL).

2.5. PSPs Decreased ROS and LDH Levels and Increased the Expression of Cell Tight Junction Protein ZO-1

ROS, as the upstream signal of NLRP3 inflammasome activation, play an important role in NLRP3 inflammasome activation [40]. Zonula occludens-1(ZO-1) protein, as a cell-binding protein, can reflect the state of adherent cells and cell membrane integrity. When the cell membrane structure is destroyed, the LDH in the cytoplasm is released into the culture medium [38,41]. Therefore, the release of ROS, ZO-1 protein, and LDH can be detected to indirectly reveal pyroptosis.

The control cells presented low ROS levels and the lighter color of Hoechst 33342-labeled nuclei (Figure 4A,B). The cells in the COM-injured group showed a strong green fluorescence and more nuclei exhibited a bright blue fluorescence, which imply the release of more ROS and the presence of more dead cells. After PSP0/PSP3 protection, the ROS levels and number of dead cells decreased remarkably, and PSP3 exhibited a stronger capability to clear excess ROS than PSP0.

Figure 4C shows the expression of ZO-1 protein observed via laser confocal microscopy. The control group showed normal ZO-1 expression, tight adhesion between cells, and a clear cell outline. However, the expression of ZO-1 was decreased in the cells of the COM crystal group. In addition, seriously damaged adhesion between cells and a blurred cell outline were observed. After PSP0/PSP3 protection, adhesion between cells showed improvement, and the protective effect of PSP3 was better than that of PSP0.

Figure 4D shows the results on LDH release. The control group attained a considerably lower LDH release (2.67%) compared with the COM-injured group (26.38%). After PSP0 and PSP3 protection, the LDH release decreased from 26.38% to 19.51% and 9.46%, respectively.

2.6. PSPs Inhibited the Expressions of Crystal Adhesion Proteins ANXA3 and CD44

Cell injury can lead to the increased expressions of adhesion proteins/molecules, such as annexin A3 (ANXA3) and CD44, on the cell surface [12,14,42], which increased cell adhesion to the COM crystals. As shown in Figure 5A, more cells in the COM-injured group showed a strong red fluorescence compared with those in the control group, which indicates the sharp increase in ANXA3 expression. Protection by PSPs inhibited ANXA3 expression, with PSP3 showing stronger inhibition than PSP0 (Figure 5B).

Similarly, control cells showed a lower expression level of CD44; that is, their red fluorescence was weaker. The COM-injured group exhibited stronger CD44 expression (Figure 5C). After polysaccharide protection, the number of cells with high CD44 expression was substantially reduced (Figure 5D).

2.7. PSPs Reduced COM Crystal Adhesion After the Inhibition of Cell Pyroptosis

(a) Scanning electron microscopy (SEM) qualitative observation

First, qualitative observation of the adhesive crystals was achieved via SEM (Figure 6A). No COM crystal adhesion was detected in the control group and many COM crystals were attached to the COM-injured group, which indicate that cell injury exacerbated the adhesion of COM crystals. After the intervention using PSP0/PSP3, the amount of COM crystal adhesion was considerably reduced, and a better effect of PSP3 on the inhibition of COM adhesion was observed.

(b) Inductively coupled plasma (ICP) quantitative detection

Figure 6B displays the amount of crystal adhesion on the cell surface of each group quantitatively detected through ICP. The amount of COM crystal adhesion in the control cells was theoretically zero. However, the actual ICP test results (Figure 6B) revealed a Ca Ca²⁺ concentration of 1.10 ppm, which was caused by the presence of Ca²⁺ in the cells. The amount of COM crystal adhesion in the COM damage group was 199.39 µg. After the protection by PSP0 and PSP3, the amount of COM crystal adhesion was reduced to 135.37 and 71.01 µg, respectively, which indicates that the protection offered by polysaccharides, especially PSP3, can reduce the adhesion of COM crystals.

3. Discussion

3.1. NF-κB Pathway Pyroptosis Mediated by the NLRP3 Inflammasome

As a classic inflammation-regulating transcription factor, NF-κB serves as a core upstream regulatory node mediating the activation of the NLRP3/ASC/caspase-1/IL-1β pyroptosis pathway [43]. NF-κB signaling is a necessary prerequisite for proper NLRP3 activation [43], and the human NLRP3 gene harbors a putative NF-κB binding site upstream of its transcriptional start site [43]. When cells are stimulated by CaOx crystals, the intracellular ROS level elevates and the NF-κB pathway is activated, leading to the phosphorylation and nuclear translocation of the p65 subunit in the cytoplasm [44]; this in turn initiates the transcription and expression of downstream pyroptosis-related genes including NLRP3 and pro-IL-1β. Upon binding to the adaptor protein ASC, NLRP3 recruits and activates caspase-1. Activated caspase-1 not only cleaves the pro-IL-1β precursor to generate the mature pro-inflammatory factor IL-1β for extracellular release but also cleaves the pyroptosis executor protein GSDMD, thereby inducing the formation of cellular pores and the occurrence of pyroptosis [45]. Collectively, this forms the NF-κB/NLRP3/ASC/caspase-1/IL-1β pyroptosis regulatory axis, which acts as a crucial molecular pathway mediating crystal-induced pyroptosis of renal tubular epithelial cells and the formation of stones.

Owing to the low toxicity and multi-target anti-inflammatory regulatory properties, polysaccharides from Chinese herbal medicines have emerged as potential natural bioactive substances for intervening in the NF-κB-mediated pyroptosis pathway. A study by Gong et al. [46] demonstrated that L-Fucose, a sulfated polysaccharide with anti-inflammatory and antioxidant properties, mitigated cardiac inflammation, pyroptosis and mitochondrial injury by inhibiting the TLR4/MyD88/NF-κB pathway, which highlighted its potential as a therapeutic agent for obesity-associated cardiac injury. Zhan et al. [47] found that polysaccharides from garlic reduced the elevated levels of LPS, IL-1β, IL-18, NLRP3, GSDMD, caspase-1, ASC, TLR4, MyD88, NF-κB and phospho-NF-κB, while increasing IL-10 levels in the livers of mice, indicating that these polysaccharides alleviated secondary liver injury by suppressing inflammation and pyroptosis. Yuan et al. [48] revealed that the therapeutic effect of Polygonatum sibiricum polysaccharides significantly alleviated the symptoms of inflammatory bowel disease by inhibiting the Toll-like receptor 4/nuclear factor-κB signaling pathway and attenuating NLRP3/ASC/caspase-1/GSDMD-mediated cellular pyroptosis and mitochondrial damage in the colon.

3.2. PSPs Inhibit NLRP3/ASC/Caspase-1/IL-1β Axis-Mediated Pyroptosis by Inhibiting the NF-κB Pathway

The NF-κB signaling pathway is considered a key pathway in the regulation of inflammatory response [49]. The NF-κB pathway indirectly regulates the activation of caspase-1 and GSDMD and the release of pro-inflammatory factors [50]. Through inhibition of the activation of the NF-κB pathway, the expression level of NLRP3 inflammatory body and the release of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-18 can be downregulated, and the expression of pyroptosis-related proteins caspase-1 and GSDMD can be reduced [51].

Numerous studies conducted on the treatment of disease involved blocking the activation of the NF-κB pathway to inhibit pyroptosis [52,53,54,55]. Ge et al. [52] reported that punicalagin can inhibit pyroptosis by inhibiting the activation of the NF-κB signaling pathway and downregulating the expressions of NLRP3, caspase-1, IL-1β, and IL-18; this condition improved joint inflammation, cartilage injury, and systemic bone destruction in arthritic (collagen-induced arthritis) mice. Dai et al. [53] showed that treatment with BAY 11-7082 (NF-κB inhibitor) can inhibit cell pyroptosis and reduce neuronal damage, which improved neurocognitive impairment in rats. Angelica polysaccharides showed a protective effect on sepsis-induced acute lung injury by inhibiting the activation of the NF-κB pathway and NLRP3 inflammasome in mice [54]. Scutellaria baicalensis Georgi polysaccharides alleviated acute colitis in mice by inhibiting the activation of the NF-κB pathway and NLRP3 inflammasome [55].

In this study, PSPs inhibited the activation of the NF-κB pathway (Figure 2A), which resulted in the downregulated expressions of pyroptosis-related proteins NLRP3, ASC, GSDMD, IL-1β, and IL-18 (Figure 3) and decreased expressions of pyroptosis (Figure 2C–D) and adhesion proteins ANXA3 and CD44 (Figure 5A–D). Thus, the adhesion and deposition of CaOx crystals (Figure 6) and the risk of stone formation were reduced.

The inhibition of pyroptosis by PSPs can be attributed to the following reasons: (a) Good antioxidant activity. ROS are one of the upstream signals activated by NLRP3 inflammasome [40], and elevated intracellular ROS levels can promote cell pyroptosis [56]. However, PSPs can remove excess ROS to a certain extent [57,58]. (b) PSP3 contains abundant polyanion groups (−OSO₃−), which can bind to Ca²⁺ on the COM crystal plane [59,60] and prevent COM from contacting HK-2 cells, which results in the reduced pyroptosis induced by COM [61].

PSP-mediated downregulation of intracellular ROS levels is the core molecular basis for its inhibitory effect on NLRP3 inflammasome. As a critical upstream signal of NLRP3 inflammasome activation, excessive ROS accumulation can activate the NF-κB pathway to upregulate the transcriptional expression of NLRP3 and pro-IL-1β [15,16,17,18,19,20,21,62,63,64] and then induce pyroptosis. Our research indicates that PSP inhibits the aforementioned process induced by COM crystals.

3.3. PSPs Inhibited the Adhesion of CaOx Crystals After Pyroptosis

Adhesion of CaOx crystals to renal tubular epithelial cells is a key step in kidney stone formation [65]. Cell damage increases the expressions of crystal adhesion proteins (CD44, ANXA2, ANXA1, OPN, etc.) and adhesion molecules (PS and HA) [33,66], which improves cell adhesion to CaOx crystals and increases the risk of lithoblast.

In this study, after the pyroptosis induced by COM crystals, the expressions of adhesion proteins ANXA3 and CD44 on the surface of HK-2 cells were considerably higher than those of normal cells (Figure 5). However, the expressions of ANXA3 and CD44 can be substantially reduced after PSP3 protection. This condition reduced crystal adhesion (Figure 6) and the damage of COM crystals to HK-2 cells.

3.4. Reasons for Increased Activity of Polysaccharides Modified by Sulfation

Compared with PSP0, the sulfuric acid-modified polysaccharide PSP3 showed a better capability to inhibit pyroptosis of cells due the following reasons: First, PSP3, with an increased content of −OSO₃−, contained more negative charge and had a greater solubility, which enhanced its biological activity (such as antioxidant, antitumor, and immune activity) [67]. Second, the −OSO₃− group in PSP3 can be adsorbed onto the surface of COM crystals, which prevented COM from contacting HK-2 cells and thereby reduced COM-induced pyroptosis [61]. Figure 7 shows the mechanism by which PSP3 reduced the adhesion of CaOx crystals to HK-2 cells, that is, by inhibiting NF-κB signaling and reducing cell damage caused by pyroptosis and the overexpression of adhesion proteins.

3.5. Pyroptosis-Mediated Anti-Stone Mechanism of PSPs: Findings, Limitations and Perspectives

The structures of PSPs were characterized, including the determination of sulfate group content, monosaccharide composition analysis, 1D and 2D NMR analyses, FT-IR analysis, XPS analysis, and zeta potential measurement [32]. We also investigated their antioxidant activity and preliminarily verified their phenotypic protective effects against CaOx stone formation in mice, such as inhibiting CaOx crystallization and deposition, alleviating oxidative damage and non-specific inflammation, and suppressing apoptosis [32]. However, mechanistic research into PSPs’ anti-stone effects was limited to superficial phenotypic observations, with no investigation of pyroptosis-related molecular events. Here, we aimed to explore PSPs’ inhibitory effect on COM-induced cellular pyroptosis, elucidate its underlying molecular mechanism, and provide new insights for kidney stone research and antilithiasis drug development. For the first time, we demonstrate that PSPs attenuate CaOx stone formation by inhibiting ROS-mediated NLRP3/GSDMD-dependent pyroptosis via the NLRP3 inflammasome pathway, extending our prior phenotypic findings to mechanistic elaboration and uncovering a novel pyroptosis-targeted regulatory axis of PSPs in CaOx nephrolithiasis prevention.

This study encountered various shortcomings. First, a huge difference was observed between the external and internal environments. In vitro cell experiments are only a basic part of drug development. After a drug enters a complex biological environment in vivo, certain metabolic problems still need to be considered. Therefore, whether PSP3 can be used as a pyroptosis inhibitor or a drug to prevent and treat kidney stones must be verified in vivo. Second, the proteins that PSP3 binds to and the underlying mechanism must be determined. Third, the specific molecular binding sites and detailed action mechanisms underlying PSP-mediated regulation of pyroptosis have not been elucidated in this study, and this is proposed as a key direction for follow-up research to further investigate the precise molecular mechanism of PSPs in the specific regulation of cellular pyroptosis.

4. Materials and Methods

4.1. Reagents and Equipment

Materials: Human kidney proximal tubular epithelial cells (HK-2) were obtained from Shanghai Cell Bank, Chinese Academy of Sciences, China. DMEM/F-12 medium, trypsin, fetal bovine serum, and penicillin–streptomycin were purchased from Gibco Biochemical Products Co., Ltd. (New York, NY, USA) Phosphate buffer solution (PBS); 4% paraformaldehyde; 4,6-diamidino-2-phenylindole (DAPI) staining solution; NF-κB p65 antibody (rabbit polyclonal antibody) and 2′,7′ -dichlorofluorescein diacetate (DCFH-DA) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). The CCK-8 cell proliferation assay kit, human IL-18 enzyme-linked immunosorbent assay (ELISA) kit and LDH assay kit were purchased from Beijing solarbio Technology Co., Ltd. (Beijing, China). Gasdermin D (GSDMD) primary antibodies were purchased from Cell Signaling Technology. The Immunochemistry FAM-FLICA^® Caspase-1 (YVAD) Assay Kit was purchased from ImmunoChemistry Technologies (Davis, CA, USA). Primary antibodies for CD44, ANXA3, zonula occludens-1 (ZO-1), NLRP3, ASC, and IL-1β, and sheep anti-mouse and sheep anti-rabbit fluorescent secondary antibodies were purchased from Proteintech (Wuhan, China). CaCl₂, Na₂C₂O₄ and other chemical reagents were analytically pure and were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

PSP0 was purchased from Shaanxi Ciyuan Biotechnology Co., Ltd. (Baoji, China), and purified with reference to [32]. PSP0 (−OSO₃− content 1.04%) was sulfated by the sulfur trioxide–pyridine method to obtain PSP3 (36.12%).

Synthesis and characterization of calcium oxalate monohydrate (COM-3 μm) was carried out with crystals with sizes of 3 μm [68].

Instruments: Fourier-transform infrared spectrometer (FT-IR, Nicolet, MN, USA); field emission scanning electron microscope (ULTRA55, Zeiss, Oberkochen, Germany); microplate reader (Safire2, Tecan, Männedorf, Switzerland); laser confocal microscopy (LSM510 META DUO SCAN, ZEISS, Oberkochen, Germany); fluorescence inverted microscope (Leica DMRA2, Heerbrugg, Germany); flow cytometry (Beckman, Gallios, Brea, CA, USA); transmission electron microscopy (TEM, H-7650, Hitachi, Tokyo, Japan); electron-coupled plasma atomic emission spectrometer (OptimA-2000DV, Perkin Elmer, Waltham, Ma, USA); multifunctional chemiluminescence imager (Odyssey, Wakefield, MA, USA).

4.2. Experimental Methods

4.2.1. Cell Culture

HK-2 cells were cultured in DMEM-F12 medium containing 10% serum and 100 U/mL penicillin–streptomycin at 37 °C and 5% CO₂ at saturated humidity. The cells were passaged by trypsin digestion.

4.2.2. Determination of Cell Viability

The experimental models were divided into three groups:

(1) Normal control (NC) group: serum-free DMEM-F12 medium was added and incubated for 48 h.

(2) Damage control (DC) group: serum-free medium containing a final concentration of 300 μg/mL COM was added and incubated for 48 h.

(3) Polysaccharide protection group: serum-free medium containing a final concentration of 300 μg/mL COM and a final concentration of 30, 60, 90, 120, 150, 180, 210, and 240 μg/mL PSP0/PSP3 was added and incubated for 48 h.

After the predetermined time was reached, the Optical Density (OD) value was measured at 450 nm with a microplate reader according to the test method of the CCK-8 kit. Then cell viability (%) was calculated according to Formula (1):

$$\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)={\displaystyle \frac{\mathrm{O}\mathrm{D}\left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}\right)}{\mathrm{O}\mathrm{D}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}\right)}}\times 100\%$$

(1)

4.3. Effects of PSPs on NF-κB p65 Pathway Signaling and Pyroptosis of Cells

4.3.1. Caspase-1/PI Double Staining for Flow Quantitative Analysis

The cell seed plate and grouping were the same as Section 4.2.2, but only 180 μg/mL PSP0/PSP3 was retained in the polysaccharide protection group. After incubation time was reached, the cells were washed 3 times with PBS. The cells were collected and rehung in the solution of FLICA, incubated at 37 °C away from light for 1 h, cleaned 3 times with PBS, and further rehung in the dyeing solution containing PI. The percentage of pyroptosis was detected by flow cytometry [69].

4.3.2. Qualitative Observation of Caspase-1/PI/Hoechst 33342 Triple Staining

The cell seed plate and grouping were the same as Section 4.3.1. After the incubation time was reached, the cells were washed twice with PBS and stained with caspase-1 working solution for 1h. The staining solution was removed and the cells were washed with PBS 3 times. Then 300 μL Hoechst 33342 working solution (10 μg/mL) was added to stain for 10 min, the staining solution was removed, and the cells were washed with PBS 3 times. Then 300 μL PI working solution (10 μg/mL) was added to stain for 10 min, the staining solution was removed, and the cells were washed with PBS 3 times.

Then 300 μL PI working solution (10 μg/mL) was added to stain for 10 min, the staining solution was removed, and the cells were washed with PBS for 3 times. The cells can be observed by laser confocal microscopy.

4.3.3. Nuclear Localization of NF-κB p65 Was Observed by Immunofluorescence

The cell seed plate and grouping were the same as Section 4.3.1. After the cells were washed twice with PBS, they were fixed with paraformaldehyde for 15 min, then closed with sheep serum for 20 min, and then incubated at 4 °C overnight with NF-κB p65 primary antibody (1:100). After the incubation time was reached, the cells were cleaned with PBS 3 times, FITC secondary antibody was added, and they were incubated at 37 °C for 0.5 h without light. Finally, the nuclei were labeled with DAPI staining for 10 min. The expression of NF-κB p65 was qualitatively observed under laser confocal microscopy, and the fluorescence intensity was semi-quantitatively analyzed by ImageJ 1.54p software.

4.4. Detection and Analysis of the Signaling Pathway of Pyroptosis

4.4.1. NLRP3 Expression Was Determined by Immunofluorescence Method

The cell seed plate and grouping were the same as Section 4.3.1. Cells were fixed with paraformaldehyde for 15 min, then closed with sheep serum for 20 min, and then NLRP3 primary antibody (1:100) was added and they were incubated at 4 °C overnight. After the incubation time was reached, the cells were cleaned with PBS 3 times, FITC secondary antibody was added, and they were incubated at 37 °C for 0.5 h without light. Finally, the nuclei were labeled with DAPI staining for 10 min. The expression of NLRP3 was qualitatively observed under laser confocal microscopy, and the fluorescence intensity was semi-quantitatively analyzed by ImageJ software.

4.4.2. The Expression Levels of GSDMD and IL-1β Were Determined by Western Blotting

The experimental grouping was the same as Section 4.3.1. Add 100 µL of pre-cooled lysate (strong) to make full contact with the cells to ensure complete lysis. Scrape off all the cells with a cell scraper and collect them. Centrifuge at 4 °C (12,000 rpm) for 15 min, quantitatively absorb the supernatant and record the volume. Then determine the protein concentration and balance the protein concentration of the sample and denature the protein boiling at 100 °C for 10 min.

Regarding glue preparation, the electrophoresis tank was assembled (water was added and stood for 10 min without leakage) and the electrophoresis solution was added. Samples were added and SDS-PAGE electrophoresis was performed. The membrane was transferred immediately after electrophoresis and the membrane was enclosed for 30 min. After closure, the membrane was washed with tris-buffered saline with Tween 20 (TBST) 3 times (10 min/time), incubated with IL-1β and GSDMD primary antibody at 4 °C slowly overnight, then recovered from the primary antibody and washed with TBST 3 times (10 min/time). Finally, the second antibody was diluted 5000 times with TBST, incubated at room temperature for 2 h away from light, cleaned by TBST 4 times (10 min/time), and developed with a multifunctional chemiluminescence imager.

4.4.3. ELISA Was Used to Detect the Secretion of IL-18

The seed plate and group of cells were the same as Section 4.3.1. After the incubation time was reached, the secretion of IL-18 was measured by the ELISA kit.

The specific process is as follows:

(1) Collection of the samples to be tested: move the cell medium to a sterile centrifuge tube, centrifuge at 12,000 rpm for 10 min, and then divide the supernatant into a small EP tube in equal parts and store below −20 °C (the test can be stored at 2–8 °C within 24 h) to avoid repeated freeze–thaw.

(2) Preparation of standard products: prepare standard solutions with concentrations of 375, 187.5, 93.75, 46.87, 23.43, 11.71, 5.85 and 0 pg/mL with standard products with a concentration of 1500 pg/mL.

(3) IL-18 detection: take out the kit and restore to room temperature, add 300 μL washing solution to clean the enzyme label plate 3 times and pat dry. Add 100 μL standard substance/test sample into the reaction hole, seal the plate and incubate at 37 °C for 90 min. Wash the board 4 times and pat dry. Add 100 μL biotinylated antibody working solution to each well, seal the plate and incubate at 37 °C for 60 min. Wash the plate 4 times. Add 100 μL enzyme conjugate working liquid, seal the plate and incubate for 30 min. Wash the plate 5 times, add 100 μL color-developing substrate, and develop color at 37 °C for 15 min. Add 50 μL termination solution and measure the OD value at 450 nm immediately.

4.5. Effects of PSPs on Cell Damage

4.5.1. ROS Determination

The seed plate and grouping of cells were the same as Section 4.3.1. After the action time was reached, DCFH-DA was diluted with 500 µL serum-free culture solution at 1:1000 and incubated at 37 °C for 30 min. The nuclei were then labeled with DAPI staining for 10 min. The green fluorescence intensity of the cells was observed under laser confocal microscopy. The fluorescence was analyzed semi-quantitatively by ImageJ software.

4.5.2. Determination of LDH

The seed plate and grouping of cells were the same as Section 4.3.1. After the predetermined time was reached, LDH release was measured by the LDH kit.

4.5.3. Determination of ZO-1

The seed plate and grouping of cells were the same as Section 4.3.1. After the cells were washed twice with PBS, they were fixed with paraformaldehyde for 15 min, then permeated with permeant for 10 min, and closed with sheep serum for 30 min, and then ZO-1 primary antibody (1:100) was added and the cells were incubated at 4 °C overnight. After the incubation time was reached, the cells were cleaned with PBS 3 times and then the FITC secondary antibody was added away from light and incubated at 37 °C for 0.5 h. Finally, the nuclei were labeled with DAPI staining for 10 min. The expression of ZO-1 was qualitatively observed under laser confocal microscopy.

4.6. Effects of PSPs on COM-Induced Adhesion Proteins

4.6.1. Determination of ANXA3

The seed plate and grouping of cells were the same as Section 4.3.1. After the cells were washed twice with PBS, they were fixed with paraformaldehyde for 15 min, then permeated with immunostaining permeant (Triton X-100) for 10 min, closed with sheep serum for 30 min, and then incubated at 4 °C overnight with ANXA3 primary antibody (1:100). The cells were washed with PBS 3 times, then the cells were incubated with FITC secondary antibody at 37 °C for 0.5 h, and finally the nuclei were labeled with DAPI staining for 10 min. The expression of ANXA3 was qualitatively observed under laser confocal microscopy, and the fluorescence was semi-quantitatively analyzed by ImageJ software.

4.6.2. Determination of CD44

The seed plate and grouping of cells were the same as Section 4.3.1. After the cells were washed twice with PBS, they were fixed with paraformaldehyde for 15 min, then permeated with Triton X-100 for 10 min, closed with sheep serum for 30 min, and then incubated at 4 °C overnight with CD44 primary antibody (1:100). The cells were washed with PBS 3 times, then the cells were incubated with FITC secondary antibody at 37 °C for 0.5 h, and finally the nuclei were labeled with DAPI staining for 10 min. The expression of CD44 was qualitatively observed under laser confocal microscopy, and the fluorescence was semi-quantitatively analyzed by ImageJ software.

4.7. Effects of PSPs on COM Crystal Adhesion After Pyroptosis Inhibition

4.7.1. SEM Was Used to Observe the Adhesion Quantity Qualitatively

The seed plate and grouping of cells were the same as Section 4.3.1. After washing the cells with PBS 3 times, the cells were fixed at 4 °C with 2.5% glutaraldehyde for 24 h, washed with PBS, and dehydrated with gradient ethanol (30%, 50%, 70%, 90%, and 100%), and then gold was sprayed after CO₂ critical point drying and SEM observation.

4.7.2. ICP Quantitative Measurement of Crystal Adhesion

The seed plate and grouping of cells were the same as Section 4.3.1. The cells were washed 3 times with PBS to remove the unbound crystals. The sample was then transferred to a 25 mL beaker, digested with 4.0 mL concentrated HNO₃ and 1.0 mL HClO₄ solution, and heated until smoke appeared and stopped, and the remaining heat was used to dry the solution. After cooling, 6 mL 2% HNO₃ was added. The Ca²⁺ concentration was measured using an electron-coupled plasma atomic emission spectrometer, which was then converted to determine the amount of crystal adhesion. The control group was treated with the same method to determine the interference of intracellular Ca²⁺.

4.8. Statistical Analysis

The experimental results were statistically analyzed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA), and the Tukey test was used to analyze the differences between the means of each experimental group and the control group. * indicates p < 0.05, ** indicates p < 0.01, and p > 0.05 indicates no significant difference.

5. Conclusions

PSPs downregulate the expressions of NLRP3-, ASC-, GSDMD-, IL-1β-, and IL-18-related proteins through the ROS/NF-κB pathway and inhibit COM-3 µm-mediated pyroptosis through the NLRP3/ASC/caspase-1/IL-1β pathway. After the inhibition of pyroptosis, PSPs not only reduced the levels of ROS and LDH and maintained the integrity of ZO-1 protein but also substantially reduced the expression of CaOx-specific adhesion proteins (ANXA3 and CD44). As a result, the adhesion and deposition of COM crystals and the risk of CaOx stone formation were reduced. Compared with PSP0, PSP3 showed a better protection capability. Thus, PSP3 has the potential to be developed as a pyroptosis inhibitor and used in the inhibition of related diseases caused by pyroptosis.