NLRP3 inflammasome activation plays an important role in the development of pancreatic fibrosis. However, it is unclear whether the activation of the NLRP3 inflammasome is directly involved in the activation of Pancreatic stellate cells (PSCs). The aim of this study was to investigate the role and mechanism of the NLRP3 inflammasome in the activation of PSCs. In vivo, a rat model of chronic pancreatitis (CP) was induced by intravenous injection of dibutyltin dichloride (DBTC). In vitro, rat primary PSCs were isolated from pancreatic tissues and incubated with the NLRP3 inflammasome activator LPS, the NLRP3 inhibitor MCC950, or NLRP3 siRNA. The results showed that the expression of NLRP3, pro-Caspase-1, Caspase-1 and IL-18 was increased in the rat model of CP and during PSCs activation. LPS increased the protein levels of NLRP3, ASC, Caspase-1, IL-1β and IL-18 accompanied by the upregulation of α-SMA, Col I and FN expression. Moreover, MCC950 or NLPR3 siRNA decreased the expression of α-SMA, Col I, FN, TGF-β1 and p-Smad3. Furthermore, MCC950 reversed the LPS-induced upregulation of α-SMA, FN and Col Ⅰ expression in PSCs. This study revealed that the NLRP3 inflammasome is directly involved in the activation of PSCs in vivo and in vitro. Inhibiting NLRP3 suppresses the activation of PSCs through the TGF-β1/Smad3 pathway.

Atherosclerosis (AS) is an inflammatory disease caused by multiple factors. Multiple circRNAs are involved in the development of AS. The present study focusses on delineating the role of circ_0090231 in AS. Human aortic endothelial cells (HAECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct an in vitro AS model. Real-time quantitative polymerase-chain reaction (RT-qPCR) was used to detect the levels of circ_0090231, IL-1β, and IL-18 transcripts. CircRNA/target gene interactions were predicted using StarBase and TargetScan and confirmed using an RNA pull-down assay and dual-luciferase reporter assay. Further, 3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH) release assays were performed to evaluate cell viability and damage in the AS model, respectively. Cell pyroptosis and protein expression were determined using flow cytometry and western blotting respectively. The treatment of HAECs with ox-LDL not only led to significant increase in the levels of circ_0090231 but also resulted in improved cell viability as well as reduced cell injury and pyroptosis as compared to that in non-treated cells. The circ_0090231 was also identified to function as a sponge for miR-635, knockdown of which reverses the effects of circ_0090231 inhibition. Furthermore, our results revealed that levels of NLRP3, a miR-635 target, are not only augmented in the AS model but its overexpression also weakens the miR-635 regulatory effects in the AS development. Taken together, the circ_0090231/miR-635/NLRP3 axis affects the development of AS by regulating cell pyroptosis, thus providing new insights into the mechanism of AS development.

We aimed to clarify the therapeutic effects of imperatorin (IMP) on knee osteoarthritis (KOA). Thirty 3-month-old SD male rats were randomly divided into Normal, monosodium iodoacetate (MIA) and MIA+IMP groups. Their synovial tissues were subjected to histopathological analysis. Primary synovial fibroblasts obtained from additional normal rats were treated by lipopolysaccharide (LPS) and then IMP. The mRNA and protein expressions of factors related to synovitis and synovial fibrosis were detected by qRT-PCR and Western blotting, respectively. The concentrations of inflammatory factors IL-1β and IL-18 were measured by ELISA. IMP reduced HIF-1α, NLR family pyrin domain-containing 3 inflammasome expression and IL-1β, IL-18 production in synovial fibroblasts induced by LPS. IMP also downregulated synovial fibrosis markers. In vitro study revealed that MIA-induced synovitis and synovial fibrosis were relieved by IMP. IMP relieves the inflammation associated with synovitis and synovial fibrosis. It reduces the production of pro-inflammatory mediators and cytokines and inhibits TGF-β1, TIMP-1 and VEGF expressions that promote synovial fibrosis.

Intracerebral hemorrhage (ICH) is an incurable neurological disease. Microglia activation and its related inflammation contribute to ICH-associated brain damage. FERM domain containing kindlin 1 (FERMT1) is an integrin-binding protein that participates in microglia-associated inflammation, but its role in ICH is unclear. An ICH model was constructed by injecting 50 µl of autologous blood into the bregma of rats. FERMT1 siRNA was injected into the right ventricle of the rat for knockdown of FERMT1. A significant striatal hematoma was observed in ICH rats. FERMT1 knockdown reduced the water content of brain tissue, alleviated brain hematoma and improved behavioral function in ICH rats. FERMT1 knockdown reduced microglia activity, inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activity and decreased the expression of inflammatory factors including IL-1β and IL-18 in the peri-hematoma tissues. BV2 microglial cells were transfected with FERMT1 siRNA and incubated with 60 µM Hemin for 24 h. Activation of NLRP3 inflammasome induced by hemin were reduced in microglia when FERMT1 was knocked down, leading to decreased production of inflammatory factors IL-1β and IL-18. In addition, knockdown of FERMT1 prevented the activation of nuclear factor kappa B (NF-κB) signaling pathway in vivo and in vitro. Our findings suggested that down-regulation of FERMT1 attenuated microglial inflammation and brain damage induced by ICH via NLRP3/NF-κB pathway. FERMT1 is a key regulator of inflammatory damage in rats after ICH.

NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology. Long noncoding RNAs (lncRNAs) are active participators of diabetic nephropathy (DN). X inactive specific transcript (XIST) expression has been reported to be elevated in the serum of DN patients.

Pectic polysaccharides (PPs) could exert functions on ulcerative colitis (UC), which is classified as a nonspecific inflammatory disorder. This study investigated the molecular mechanism of PPs derived from Rauwolfia in UC. First, the dextran sodium sulfate (DSS)-induced mouse colitis models and lipopolysaccharide (LPS)-treated colonic epithelial cell (YAMC) models were established and treated with PP. Subsequently, the effects of PPs on mucosal damages in DSS mice were detected, and the levels of inflammatory cytokines, pyroptosis-related factors, oxidative stress-related markers, and the tight junction-related proteins in the tissues or cells were examined, and the results suggested that PPs ameliorated colonic mucosal damages and cell pyroptosis in DSS mice, and limited colonic epithelial cell pyroptosis in in vitro UC models. Subsequently, the binding relations of retinol-binding protein 4 (RBP4) to miR-124-3p and NLR pyrin domain-containing 3 (NLRP3) were analyzed. miR-124-3p targeted RBP4 and reduced the binding of RBP4 to NLRP3, thus inhibiting NLRP3-mediated pyroptosis. Finally, functional rescue experiments revealed that miR-124-3p suppression or RBP4 overexpression promoted colonic epithelial cell pyroptosis. Collectively, Rauwolfia-derived PPs limited miR-124-3p and targeted RBP4 and reduced the binding potency of RBP4 to NLRP3 to inhibit NLRP3-mediated pyroptosis, resulting in the alleviation of colonic epithelial cell pyroptosis and mucosal damages in UC.

Sepsis brain injury (SBI) is a major cause of death in critically ill patients. The present study aimed to investigate the role of emodin in SBI development. Human astrocyte 1321N1 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) to establish an SBI model in vitro. Flow cytometry was performed to measure the cell pyroptosis. The protein expression levels of syndecan-1 (SDC-1), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and the N-terminal fragment of gasdermin D (GSDMD-N) were measured using Western blotting. Interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α levels in cells were measured using enzyme-linked immunosorbent assay kits. The N6-methyladenosine (m6A) modification was analyzed using the methylated RNA immunoprecipitation assay. NLRP3 activator, nigericin, was used to overexpress NLRP3. LPS treatment significantly enhanced the pyroptosis in 1321N1 cells, increased the levels of TNF-α, IL-1β, and IL-6, and decreased the levels of IL-10. The protein expression levels of NLRP3, SDC-1, GSDMD-N, and Caspase-1 were also increased. Emodin treatment decreased the levels of TNF-α, IL-1β, IL-6, NLRP3, SDC-1, GSDMD-N, and Caspase-1, while increasing the levels of IL-10 in LPS-treated 1321N1 cells. Nigericin reversed the effects of emodin. Furthermore, emodin upregulated m6A levels in NLRP3 by increasing the expression of methyltransferase-like 3 (METTL3). Meanwhile, knockdown of METTL3 reversed the effects of emodin on the mRNA expression and stability of NLRP3. Therefore, emodin inhibits the inflammation and pyroptosis of LPS-treated 1321N1 cells by inactivating METTL3-mediated NLRP3 expression.

BACKGROUND Galangin is believed to exert antioxidant effects by inhibition of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which has been linked to chemotherapy sensitivity in cancers. In this study, we explored the synergistic effect of galangin in combination with the chemotherapy agent 5-fluorouracil (5-FU) in esophageal cancer cells and xenografts. MATERIAL AND METHODS The esophageal squamous epithelium cell line Het-1A and 2 human esophageal cancer cell lines (Eca109, OE19) were used to investigate the effect of galangin with or without 5-FU in vitro through proliferation and invasion analyses, while apoptosis was analyzed in cancer cells. Furthermore, a subcutaneous xenograft tumor model in mice was used to study cancer development in vivo. RESULTS Compared with 5-FU monotherapy, combined galangin and 5-FU treatment reduced human esophageal cancer cell growth activities and invasion abilities. The results suggested that galangin had a chemotherapy-sensitized synergistic antitumor effect induced by 5-FU. The susceptibility of cancer cells to apoptosis, which is linked with chemotherapy sensitivity, was induced by 5-FU and further enhanced by galangin. NLRP3 was identified as being significantly activated by 5-FU, but galangin treatment reversed the effect and inhibited NLRP3 expression, which was accompanied by downregulated interleukin-1b levels. Further investigation showed that the induced apoptotic cascade can be mostly reversed by incubation with an NLRP3 activator, irrespective of AKT signaling. Using xenograft mouse models, we found that galangin exposure further restrained cancer development after 5-FU treatment and increased sensitivity to chemotherapy by suppressing the NLRP3 inflammasome pathway. CONCLUSIONS Our results indicated that galangin played a synergistic anticancer role through NLRP3 inflammasome inhibition when paired with FU-5.

Myocardial fibrosis (MF) is the result of persistent and repeated aggravation of myocardial ischemia and hypoxia, leading to the gradual development of heart failure of chronic ischemic heart disease. Triptolide (TPL) is identified to be involved in the treatment for MF. This study aims to explore the mechanism of TPL in the treatment of MF. The MF rat model was established, subcutaneously injected with isoproterenol and treated by subcutaneous injection of TPL. The cardiac function of each group was evaluated, including LVEF, LVFS, LVES, and LVED. The expressions of ANP, BNP, inflammatory related factors (IL-1β, IL-18, TNF-α, MCP-1, VCAM-1), NLRP3 inflammasome factors (NLRP3, ASC) and fibrosis related factors (TGF-β1, COL1, and COL3) in rats were dete cted. H&E staining and Masson staining were used to observe myocardial cell inflammation and fibrosis of rats. Western blot was used to detect the p-P65 and t-P65 levels in nucleoprotein of rat myocardial tissues. LVED and LVES of MF group were significantly upregulated, LVEF and LVFS were significantly downregulated, while TPL treatment reversed these trends; TPL treatment downregulated the tissue injury and improved the pathological damage of MF rats. TPL treatment downregulated the levels of inflammatory factors and fibrosis factors, and inhibited the activation of NLRP3 inflammasome. Activation of NLRP3 inflammasome or NF-κB pathway reversed the effect of TPL on MF. Collectively, TPL inhibited the activation of NLRP3 inflammasome by inhibiting NF-κB pathway, and improved MF in MF rats.

Uncoupling protein 2 (UCP2) may be critical for intestinal barrier function which may play a key role in the development of sepsis, and insulin has been reported to have anti-inflammatory effects. Male Sprague-Dawley rats were randomly allocated into five groups: control group, cecal ligation and puncture (CLP) group, sham surgery group, CLP plus glucose-insulin-potassium (GIK) group, and CLP plus glucose and potassium (GK) group. Ileum tissues were collected at 24 h after surgery. Histological and cytokine analyses, intestinal permeability tests, and western blots of intestinal epithelial tight junction component proteins and UCP2 were performed. Compared with CLP group, the CLP + GIK group had milder histological damage, lower levels of cytokines in the serum and ileum tissue samples, and lower UCP2 expression, whereas the CLP + GK group had no such effects. Moreover, the CLP + GIK group exhibited decreased epithelial permeability of the ileum and increased expression of zonula occludens-1, occludin, and claudin-1 in the ileum. The findings demonstrated that the UCP2 and NLR family-pyrin domain-containing 3/caspase 1/interleukin 1β signaling pathway may be involved in intestinal barrier injury and that GIK treatment decreased intestinal barrier permeability. Thus, GIK may be a useful treatment for intestinal barrier injury during sepsis.

BACKGROUND Protein kinase R (PKR) is implicated in the inflammatory response to bacterial infection while the role of PKR in sepsis-induced acute kidney injury (AKI) is largely unknown. This study aimed to investigate the effects of the specific PKR inhibitor C16 (C13H8N4OS) on lipopolysaccharide (LPS)-induced AKI, and its mechanisms of action. MATERIAL AND METHODS C57BL/6J mice were injected intraperitoneally with C16 or vehicle 1 h before the LPS challenge and then injected intraperitoneally with LPS or 0.9% saline. After the LPS challenge, histopathological damage, renal function, and levels of proinflammatory cytokines were assessed. All the related signaling pathways were analyzed. RESULTS C16 effectively inhibited LPS-induced renal elevation of proinflammatory cytokines and chemokines. C16 prevented NF-kappaB activation and suppressed the PKR/eIF2alpha signaling pathway in AKI after the LPS challenge. Furthermore, C16 significantly inhibited pyroptosis during AKI, as evidenced by decreased renal levels of apoptosis-associated speck-like protein; NACHT, LRR, NLR Family Pyrin Domain-Containing 3; caspase-1; interleukin (IL)-1ß; and IL-18. CONCLUSIONS Our findings suggest that inhibition by C16 ameliorated LPS-induced renal inflammation and injury, at least partly through modulation of the pyroptosis signal pathway in the kidney.

Diabetic nephropathy (DN) is a serious complication of diabetes and can cause an increased mortality risk. It was previously reported that NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in the pathogenesis of diabetes. However, the underlying mechanism is not clearly understood. In the present study, the effects of spleen tyrosine kinase (Syk) and c‑Jun N‑terminal kinase (JNK) on the NLRP3 inflammasome were examined in vivo and in vitro. Sprague‑Dawley rats were injected intraperitoneally with streptozotocin (65 mg/kg) to induce diabetes. HK2 cells and rat glomerular mesangial cells (RGMCs) were examined to detect the expression of JNK and NLRP3 inflammasome‑associated proteins following treatment with a Syk inhibitor or Syk‑small interfering (si)RNA in a high glucose condition. In the present study, it was revealed that the protein and mRNA expression levels of NLRP3 inflammasome‑associated molecules and the downstream mature interleukin (IL)‑1β were upregulated in vivo and in vitro. The Syk inhibitor and Syk‑siRNA suppressed high glucose‑induced JNK activation, and subsequently downregulated the activation of the NLRP3 inflammasome and mature IL‑1β in HK2 cells and RGMCs. Furthermore, high glucose‑induced apoptosis of HK2 cells was reduced by the Syk inhibitor BAY61‑3606. Therefore, the present results determined that high glucose‑induced activation of the NLRP3 inflammasome is mediated by Syk/JNK activation, which subsequently increased the protein expression level of IL‑1β and mature IL‑1β. The present study identified that the Syk/JNK/NLRP3 signaling pathway may serve a vital role in the pathogenesis of DN.

Neuroinflammation plays a key role in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have shown that metformin exerts anti-inflammatory effects and promotes functional recovery in various central nervous system diseases. We designed this study to investigate the effects of metformin on EBI after SAH. Our results indicate that the use of metformin alleviates the brain edema, behavioral disorders, cell apoptosis, and neuronal injury caused by SAH. The SAH-induced NLRP3-associated inflammatory response and the activation of microglia are also suppressed by metformin. However, we found that the blockade of AMPK with compound C weakened the neuroprotective effects of metformin on EBI. Collectively, our findings indicate that metformin exerts its neuroprotective effects by inhibiting neuroinflammation in an AMPK-dependent manner, by modulating the production of NLRP3-associated proinflammatory factors and the activation of microglia.

Intracerebral hemorrhage (ICH) is a devastating disease without effective treatment. As a key component of the innate immune system, the NOD-like receptor (NLR) family, NLRP3 (pyrin domain-containing protein 3) inflammasome, when activated after ICH, promotes neuroinflammation and brain edema. MCC950 is a potent, selective, small-molecule NLRP3 inhibitor that blocks NLRP3 activation at nanomolar concentrations. Here, we examined the effect of MCC950 on brain injury and inflammation in 2 models of ICH in mice.

BACKGROUND Sepsis-induced acute kidney injury (AKI) is threatening the patients with sepsis, and nucleotide-binding oligomerization domain-like receptors (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is considered to play a critical role in this complication of sepsis and might be regulated by sirtuin1 (SIRT1). Thus, we explored the roles of NLRP3 and SIRT1 in the lipopolysaccharide (LPS)-induced AKI in the HK-2 cell line. MATERIAL AND METHODS Cell viability was assessed by Cell Counting Kit-8 (CCK-8). Apoptosis rate was measured by flow cytometry. Protein levels of interleukin (IL)-1ß and IL-18 were tested by enzyme-linked immunosorbent assay (ELISA) and NLRP3, cleaved caspase-1, caspase-1 were tested by western blot. The mRNA levels of IL-1ß, IL-18, and SIRT1 were quantified by qPCR. RESULTS LPS could decrease cell viability and the expression of SIRT1 and elevate the expressions of IL-1ß, IL-18, NLRP,3 and cleaved caspase-1. However, the overexpression of SIRT1 could upregulate cell viability and expression of caspase-1 and downregulate apoptosis rate, expressions of NLRP3, IL-1ß, IL-18, and cleaved caspase-1. CONCLUSIONS NLRP3 inflammasome could act as a critical regulator promoting the process of AKI induced by LPS, and the overexpression of SIRT1 might be able to suppress the activation of NLRP3 and therefore resist the kidney injury, showing promise to be used as a target in the treatment of sepsis-induced AKI.

A classical hallmark of Parkinson's disease (PD) pathogenesis is the accumulation of misfolded alpha-synuclein (αSyn) within Lewy bodies and Lewy neurites, although its role in microglial dysfunction and resultant dopaminergic (DAergic) neurotoxicity is still elusive. Previously, we identified that protein kinase C delta (PKCδ) is activated in post mortem PD brains and experimental Parkinsonism and that it participates in reactive microgliosis; however, the relationship between PKCδ activation, endoplasmic reticulum stress (ERS) and the reactive microglial activation state in the context of α-synucleinopathy is largely unknown. Herein, we show that oxidative stress, mitochondrial dysfunction, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, and PKCδ activation increased concomitantly with ERS markers, including the activating transcription factor 4 (ATF-4), serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1α (p-IRE1α), p-eukaryotic initiation factor 2 (eIF2α) as well as increased generation of neurotoxic cytokines, including IL-1β in aggregated αSynagg-stimulated primary microglia. Importantly, in mouse primary microglia-treated with αSynagg we observed increased expression of Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the thioredoxin (Trx) pathway, a major antioxidant protein system. Additionally, αSynagg promoted interaction between NLRP3 and TXNIP in these cells. In vitro knockdown of PKCδ using siRNA reduced ERS and led to reduced expression of TXNIP and the NLRP3 activation response in αSynagg-stimulated mouse microglial cells (MMCs). Additionally, attenuation of mitochondrial reactive oxygen species (mitoROS) via mito-apocynin and amelioration of ERS via the eIF2α inhibitor salubrinal (SAL) reduced the induction of the ERS/TXNIP/NLRP3 signaling axis, suggesting that mitochondrial dysfunction and ERS may act in concert to promote the αSynagg-induced microglial activation response. Likewise, knockdown of TXNIP by siRNA attenuated the αSynagg-induced NLRP3 inflammasome activation response. Finally, unilateral injection of αSyn preformed fibrils (αSynPFF) into the striatum of wild-type mice induced a significant increase in the expression of nigral p-PKCδ, ERS markers, and upregulation of the TXNIP/NLRP3 inflammasome signaling axis prior to delayed loss of TH+ neurons. Together, our results suggest that inhibition of ERS and its downstream signaling mediators TXNIP and NLRP3 might represent novel therapeutic avenues for ameliorating microglia-mediated neuroinflammation in PD and other synucleinopathies.

Sebocytes express Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which participate in the innate immune response of the skin. Although the roles of TLRs and NLR family pyrin domain-containing 3 (NLRP3) in inflammatory responses in sebocytes have been reported, the expression and functions of other NLR members, such as NOD protein-1 and -2 (NOD1 and NOD2, respectively), remain unclear. In this study, we showed that, in sebocytes, the expression of NOD1 is higher than that of NOD2, and that NOD1 is involved in inflammatory responses, such as the secretion of proinflammatory cytokines. A NOD1 agonist, L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid (Tri-DAP) induced the expression and secretion of interleukin-8 (IL-8) and activated the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways. On the other hand, a NOD2 agonist, muramyl dipeptide, did not. Either inhibition with a NOD1 inhibitor, ML130, or knockdown of NOD1 expression abolished Tri-DAP-induced inflammatory responses, suggesting that NOD1 is involved in the immunogenic signaling system of sebocytes. Furthermore, Tri-DAP and an agonist of TLR2 or TLR4 additively increased IL-8 expression compared with each agonist alone. Our results reveal the role of NOD1 in the inflammatory responses of sebocytes and may provide a novel therapeutic target for sebaceous gland inflammatory diseases, such as acne vulgaris.

Nerve damage often leads to nervous system dysfunction and neuropathic pain. The serine-threonine kinases receptor-interacting protein 1 (RIP1) and 3 (RIP3) are associated with inflammation and cell necrosis. This study aimed to explore the role of RIP1 and RIP3 in sciatic nerve chronic constriction injury (CCI) in mice. On a total of thirty mice, sciatic nerve CCI was performed. The paw withdrawal threshold was measured using Von Frey filaments. The mRNA expression and protein levels of inflammatory factors RIP1 and RIP3 in the dorsal root ganglion (DRG), spinal cord (SC) and hippocampus (HIP) were also determined. We found that paw withdrawal threshold was significantly reduced from the second day after the operation, and the levels of tumour necrosis factor-α and interferon-γ in DRG, SC and HIP were significantly increased on the eighth and 14th days in CCI mice. Furthermore, the downstream signalling molecules of RIP1 and RIP3, GTPase dynamin-related protein-1, NLR family pyrin domain containing-3 (NLRP3) and nuclear factor κB-p65 were upregulated. Increased protein levels of programmed cell death protein 1, which indicate cell death of peripheral and central nervous tissue, were induced by CCI of the sciatic nerve. Overall, this study showed that RIP1 and RIP3 were highly expressed in DRG, SC and HIP of the sciatic nerve in CCI mice and may be involved in chronic neuroinflammation and neuronecrosis.

Chronic cadmium (Cd) exposure contributes to the progression of cardiovascular disease (CVD), especially atherosclerosis (AS), but the underlying mechanism is unclear. Since mitochondrial homeostasis is emerging as a core player in the development of CVD, it might serve as a potential mechanism linking Cd exposure and AS. In this study, we aimed to investigate Cd-mediated AS through macrophage polarization and know the mechanisms of Cd-caused mitochondrial homeostasis imbalance. In vitro, flow cytometry shows that Cd exposure promotes M1-type polarization of macrophages, manifested as the increasing expressions of nuclear Factor kappa-light-chain-enhancer of activated B (NF-kB) and NLR family pyrin domain containing 3 (NLRP3). Mitochondrial homeostasis tests revealed that decreasing mitochondrial membrane potential and mitophage, increasing the mitochondrial superoxide (mROS), and mitochondrial fission are involved in the Cd-induced macrophage polarization. The upregulated expressions of receptor-interacting protein kinase 3 (RIPK3) and pseudokinase-mixed lineage kinase domain-like protein (p-MLKL) were observed. Knocking out RIPK3, followed by decreasing the expression of p-MLKL, improves the mitochondrial homeostasis imbalance which effectively reverses macrophage polarization. In vivo, the oil red O staining showed that Cd with higher blood significantly aggravates AS. Besides, M1-type polarization of macrophages and mitochondrial homeostasis imbalance were observed in the aortic roots of the mice through immunofluorescence and western blot. Knocking out RIPK3 restored the changes above. Finally, the administered N-acetyl cysteine (NAC) or mitochondrial division inhibitor-1 (Mdivi-1), which decreased the mROS or mitochondrial fission, inhibited the expressions of RIPK3 and p-MLKL, attenuating AS and macrophage M1-type polarization in the Cd-treated group. Consequently, the Cd exposure activated the RIPK3 pathway and impaired the mitochondrial homeostasis, resulting in pro-inflammatory macrophage polarization and subsequent AS. Knocking out RIPK3 provided a potential therapeutic target for Cd-caused macrophage polarization and subsequent AS.

Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficient ob/ob (+/? and -/-) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1β were upregulated in 16 L and downregulated in ob/ob. Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation in ob/ob-/-. We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.