Glycated serum protein is independently associated with progressive infarction in patients with acute ischemic stroke.

OBJECTIVE: This study investigated the relationship between glycated serum protein (GSP) and progressive infarction (PI). METHODS: From April 2017 to December 2020, we recruited 477 patients within 48 hours after the onset of acute ischemic stroke into this case-control study. Demographic characteristics, clinical information, and laboratory and neuroimaging data were recorded after admission. RESULTS: PI occurred in 144 (30.8%) patients. Patients with PI had higher initial National Institute of Health Stroke Scale (NIHSS) scores, higher discharge NIHSS scores, higher modified Rankin scale scores at 3 months after onset, higher GSP levels, lower prothrombin times, and lower creatinine levels than patients without PI. The likelihood of PI increased with increases in the GSP quartile. Multiple regression analysis revealed that high GSP levels (>2.14 mmol/L) were independently associated with PI. Subgroup analyses identified high GSP levels as an independent predictor of PI in patients with large artery atherosclerosis (third quartile: odds ratio [OR] = 3.793; 95% confidence interval [CI] = 1.555-9.250; fourth quartile: OR = 2.675; 95% CI = 1.056-6.776) and anterior circulation small vessel occlusion (fourth quartile: OR = 13.859; 95% CI = 2.024-94.885). CONCLUSIONS: GSP might be an independent predictor for PI in certain patients with acute ischemic stroke.

Enhancer-mediated FOXO3 expression promotes MSC adipogenic differentiation by activating autophagy.

BACKGROUND: Mesenchymal stem cells (MSCs) are pluripotent stem cells capable of differentiating into osteocytes, adipocytes and chondrocytes. However, in osteoporosis, the balance of differentiation is tipped toward adipogenesis and the key mechanism is controversial. Researches have shown that, as upstream regulatory elements of gene expression, enhancers ar involved in the expression of identity genes. In this study, we identified enhancers-mediated gene FOXO3 promoting MSC adipogenic differentiation by activating autophagy. METHODS: We integrated data of RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC-sequencing (ATAC-seq) to find the identity gene FOXO3. The expression of FOXO3 protein, adipogenic transcription factors and the substrate of autophagy were measured by western blotting. The Oil Red O (ORO) staining was used to visualize the adipogenesis of MSCs. Immunohistochemistry was used to visualize the FOXO3 expression in adipocytes in bone marrow. Immunofluorescence was used to detect the expression of PPARγ and LC3B. RESULTS: During adipogenesis, enhancers redistribute to genes associated with adipogenic differentiation, among which we identified the pivotal identity gene FOXO3. FOXO3 could promote the expression of the adipogenic transcription factors PPARγ, CEBPα, and CEBPß during adipogenic differentiation, while PPARγ, CEBPα, and CEBPß could in turn bind to FOXO3 and continue to promote FOXO3 expression to form a positive feedback loop. Consistently elevated FOXO3 expression promotes autophagy by activating the PI3K-AKT pathway which mediates adipogenic differentiation. CONCLUSIONS: Pivotal identity gene FOXO3 promotes autophagy by activating PI3K-AKT pathway, which provokes adipogenic differentiation of MSCs. Enhancer-regulated adipogenic identity gene FOXO3 could be an attractive treatment for osteoporosis.

Ischemic patterns and their angiographic risk factors in adult patients with moyamoya disease.

OBJECTIVE: The present study aims to determine whether angiographic differences increase the risk of ischemic pattern among adult patients with moyamoya disease (MMD). METHODS: From January 2020 to December 2021, we retrospectively enrolled 123 ischemic or asymptomatic adult patients diagnosed as MMD. Angiographic changes including Suzuki stage, moyamoya vessels, anterior choroidal artery (AChoA) dilatation, lenticulostriate artery (LSA) dilatation, posterior communicating artery (PcomA) dilatation, and posterior cerebral artery (PCA) involvement were evaluated for all patients. RESULTS: Among the 123 participants, 35 ischemic patients and 88 asymptomatic patients were analyzed. There was no significant difference of Suzuki stage, AChoA dilatation, LSA dilatation, and PcomA dilatation between ischemic group and asymptomatic group. The grading of moyamoya vessels differed significantly but was not a factor associated with ischemic pattern after adjusting multiple related confounders. However, the frequency of PCA steno-occlusive changes in ischemic patients was statistically higher than that in asymptomatic patients (54.3% vs 34.1%, p = 0.039). Furthermore, PCA involvement was a risk factor associated with ischemic form and remained statistically significant after the multivariate adjustment (p = 0.033, 95% CI 1.092-8.310). INTERPRETATION: PCA involvement is closely related to the presentation of ischemic stroke but other angiographic features had no association with ischemic pattern in adult MMD.

Silencing circSERPINE2 restrains mesenchymal stem cell senescence via the YBX3/PCNA/p21 axis.

Increasing evidence indicates that circular RNAs (circRNAs) accumulate in aging tissues and nonproliferating cells due to their high stability. However, whether upregulation of circRNA expression mediates stem cell senescence and whether circRNAs can be targeted to alleviate aging-related disorders remain unclear. Here, RNA sequencing analysis of differentially expressed circRNAs in long-term-cultured mesenchymal stem cells (MSCs) revealed that circSERPINE2 expression was significantly increased in late passages. CircSERPINE2 small interfering RNA delayed MSC senescence and rejuvenated MSCs, while circSERPINE2 overexpression had the opposite effect. RNA pulldown followed by mass spectrometry revealed an interaction between circSERPINE2 and YBX3. CircSERPINE2 increased the affinity of YBX3 for ZO-1 through the CCAUC motif, resulting in the sequestration of YBX3 in the cytoplasm, inhibiting the association of YBX3 with the PCNA promoter and eventually affecting p21 ubiquitin-mediated degradation. In addition, our results demonstrated that senescence-related downregulation of EIF4A3 gave rise to circSERPINE2. In vivo, intra-articular injection of si-circSerpine2 restrained native joint-resident MSC senescence and cartilage degeneration in mice with aging-related osteoarthritis. Taken together, our findings provide strong evidence for a regulatory role for the circSERPINE2/YBX3/PCNA/p21 axis in MSC senescence and the therapeutic potential of si-circSERPINE2 in alleviating aging-associated syndromes, such as osteoarthritis.

Galangin mitigates glucocorticoid-induced osteoporosis by activating autophagy of BMSCs via triggering the PKA/CREB signaling pathway.

Glucocorticoid-induced osteoporosis (GIOP), one of the most common and serious adverse effects associated with glucocorticoid administration, manifests as decreased bone formation and increased bone resorption, eventually culminating in bone loss. Galangin (GAL) is a flavonoid extracted from the medicinal herbal galangal that possesses a variety of pharmacological activities and can inhibit osteoclastogenesis. However, the effects of GAL on GIOP remain unclear. Our study aims to explore the effects of GAL on GIOP in mice and the underlying mechanism. Our results show that GAL markedly mitigates the severity of dexamethasone (Dex)-induced osteoporosis in mice and potentiates osteogenic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). Furthermore, GAL also significantly counteracts Dex-mediated suppression of osteogenic differentiation and autophagy in human BMSCs. GAL augments PKA/CREB-mediated autophagic flux in BMSCs and the bones of osteoporotic mice. GAL-mediated osteogenic differentiation in Dex-treated BMSCs is significantly decreased by the PKA inhibitor H89 and autophagy inhibitor 3-methyladenine. Collectively, our data indicate that GAL can ameliorate GIOP, partly by augmenting the mineralization of BMSCs by potentiating PKA/CREB-mediated autophagic flux, highlighting its potential therapeutic use in treating glucocorticoid-related osteoporosis.

A novel Anti-ROS osteoblast-specific delivery system for ankylosing spondylitis treatment via suppression of both inflammation and pathological new bone formation.

Ankylosing spondylitis (AS) is a common rheumatic disorder distinguished by chronic inflammation and heterotopic ossification at local entheses sites. Currently available medications, including nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs) and TNF inhibitors, are limited by side effects, high costs and unclear inhibitory effects on heterotopic ossification. Herein, we developed manganese ferrite nanoparticles modified by the aptamer CH6 (CH6-MF NPs) that can efficiently scavenge ROS and actively deliver siRNA into hMSCs and osteoblasts in vivo for effective AS treatment. CH6-MF NPs loaded with BMP2 siRNA (CH6-MF-Si NPs) effectively suppressed abnormal osteogenic differentiation under inflammatory conditions in vitro. During their circulation and passive accumulation in inflamed joints in the Zap70mut mouse model, CH6-MF-Si NPs attenuated local inflammation and rescued heterotopic ossification in the entheses. Thus, CH6-MF NPs may be an effective inflammation reliever and osteoblast-specific delivery system, and CH6-MF-Si NPs have potential for the dual treatment of chronic inflammation and heterotopic ossification in AS.

The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting monocyte chemoattractant protein 1 (MCP1). However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, the N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through the m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed greater ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

Alpinetin alleviates osteoporosis by promoting osteogenic differentiation in BMSCs by triggering autophagy via PKA/mTOR/ULK1 signaling.

Osteoporosis, a systemic bone disease that is characterized by a reduction in bone mass and destruction of bone microstructure, is becoming a serious problem worldwide. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into bone-forming osteoblasts, and play an important role in maintaining homeostasis of bone metabolism, thus being a potential therapeutic target for osteoporosis. Although the phytochemical alpinetin (APT) has been reported to possess a variety of pharmacological activities, it is still unclear whether APT can influence the osteogenic differentiation of on BMSCs and if it can improve osteoporosis. In this study, we found that APT treatment was able to enhance osteogenic differentiation levels of human BMSCs in vitro and mouse ones in vivo as revealed by multiple osteogenic markers including increased alkaline phosphatase activity and osteocalcin expression. Mechanistically, the protein kinase A (PKA)/mTOR/ULK1 signaling was involved in the action of APT to enhance the osteogenic differentiation of BMSCs. In addition, oral administration of APT significantly mitigated the bone loss in a dexamethasone-induced mouse model of osteoporosis through strengthening PKA signaling and autophagy. Altogether, these data demonstrate that APT promotes osteogenic differentiation in BMSCs by augmenting the PKA/mTOR/ULK1 autophagy signaling, highlighting its potential therapeutic application for treating osteoporotic diseases.

LncRNA MRF drives the regulatory function on monocyte recruitment and polarization through HNRNPD-MCP1 axis in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

Impairment of APPL1/Myoferlin facilitates adipogenic differentiation of mesenchymal stem cells by blocking autophagy flux in osteoporosis.

An imbalance of human mesenchymal stem cells (hMSCs) adipogenic and osteogenic differentiation is crucial in the pathogenesis of osteoporosis, and elucidation of the underlying mechanism is urgently needed. APPL1, an adaptor protein of the adiponectin receptor, was recently shown to be closely related to bone mass. However, the role of APPL1 in the imbalance of hMSC differentiation in osteoporosis is unclear. Therefore, we aimed to explore the mechanisms by which APPL1 alters hMSCs adipogenic differentiation in osteoporosis. Here, we found that APPL1 expression was downregulated in elderly patients with osteoporosis and in mouse osteoporosis model. APPL1 negatively regulated hMSC adipogenic differentiation in vivo and in vitro. Mechanistically, by enhancing ubiquitination-mediated Myoferlin degradation, downregulated APPL1 expression increased the risk of lysosome dysfunction during hMSCs adipogenic differentiation. Lysosomal dysfunction inhibited autophagy flux by suppressing autophagosome degradation and promoted hMSC differentiation towards the adipocyte lineage. Our findings suggest that APPL1/Myoferlin downregulation promoted hMSCs adipogenic differentiation by inhibiting autophagy flux, further impairing the balance of hMSCs adipogenic and osteogenic differentiation in osteoporosis; the APPL1/ Myoferlin axis may be a promising diagnostic and therapeutic target for osteoporosis.

RNA Sequencing Reveals the Expression Profiles of circRNAs and Indicates Hsa_circ_0070562 as a Pro-osteogenic Factor in Bone Marrow-Derived Mesenchymal Stem Cells of Patients With Ankylosing Spondylitis.

Recent studies have reported that circular RNAs (circRNAs) play a crucial regulatory role in a variety of human diseases. However, the roles of circRNAs in pathological osteogenesis in ankylosing spondylitis (AS) remain unclear. We conducted circRNA and miRNA expression profiling of osteogenically differentiated bone marrow-derived mesenchymal stem cells (BMSCs) of patients with AS compared with those of healthy donors (HDs) by RNA sequencing (RNA-seq). Results showed that a total of 31806 circRNAs were detected in the BMSC samples, of which 418 circRNAs were significantly differentially expressed (DE) with a fold change ≥2 and p value <0.05. Among these, 204 circRNAs were upregulated, and 214 were downregulated. GO and KEGG analyses demonstrated that the DE circRNAs were mainly involved in the regulation of biological processes of the cell matrix adhesion and the TGF-beta signaling pathway, which are closely related to AS. circRNA-miRNA interaction networks related to the TGF-beta signaling pathway were established. The results of qRT-PCR showed that has_circ_0070562 was significantly up-regulated in AS-MSCs. In vitro experiments showed that silencing of has_circ_0070562 weakened osteogenesis of AS-BMSCs. In conclusion, we identified numerous circRNAs that were dysregulated in AS-BMSCs compared with HD-BMSCs. Bioinformatic analyses suggested that these dysregulated circRNAs might play important functional roles in AS-BMSCs osteogenesis. Circ_0070562 functioned as a pro-ostegenic factor and might serve as a potential biomarker and a therapeutic target for AS.

DAPK1 Interacts with the p38 Isoform MAPK14, Preventing Its Nuclear Translocation and Stimulation of Bone Marrow Adipogenesis.

Bone marrow (BM) adipose tissue (BMAT), a unique adipose depot, plays an important role in diseases such as osteoporosis and bone metastasis. Precise control of mesenchymal stem cell (MSC) differentiation is critical for BMAT formation and regeneration. Here, we show that death associated protein kinase 1 (DAPK1) negatively regulates BM adipogenesis in vitro and in vivo. Prx1creDapk1loxp/loxp mice showed more adipocytes in the femur than Dapk1loxp/loxp mice. Further mechanistic analyses revealed that DAPK1 inhibits p38 mitogen-activated protein kinase (MAPK) signaling in the nucleus by binding the p38 isoform MAPK14, decreasing p38 nuclear activity, which subsequently inhibits BM adipogenesis. The inhibitory effect of DAPK1 against MAPK14 was independent of its kinase activity. In addition, the decreased DAPK1 was observed in the BM-MSCs of ageing mice. Our results reveal a previously undescribed function for DAPK1 in the regulation of adipogenesis and may also reveal the underlying mechanism of BMAT formation in ageing.

Effects of long-term culture on the biological characteristics and RNA profiles of human bone-marrow-derived mesenchymal stem cells.

Expansion in vitro prior to mesenchymal stem cells (MSCs) application is a necessary process. Functional and genomic stability has a crucial role in stem-cell-based therapies. However, the exact expression and co-expressed profiles of coding and non-coding RNAs in human bone marrow (BM)-MSCs in vitro aging are still lacking. In the present studies, the change of morphology, immunophenotype, and capacity of proliferation, differentiation, and immunoregulation of MSCs at passage (P) 4, P6, P8, P10, and P12 were investigated. RNA sequencing identified that 439 mRNAs, 65 long noncoding RNAs (lncRNAs), 59 microRNAs (miRNAs), and 229 circular RNAs (circRNAs) were differentially expressed (DE) in P12 compared with P4, with a similar trend in P6. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) identified several significant biological processes and pathways, including binding, ossification, and Wnt and PPAR signaling pathways. Interaction and co-expression/localization analyses were performed for DE mRNAs and lncRNAs, and several key lncRNAs, circRNAs, and important pathways like autophagy and mitophagy were identified in the competing endogenous RNA (ceRNA) network. Some key RNAs found in the bioinformatics analysis were validated. Our studies indicate that replicative senescence of MSCs is a continuous process, including widespread alterations in biological characteristics and global gene expression patterns that need to be considered before therapeutic applications of MSCs.

Caspase-3-mediated GSDME activation contributes to cisplatin- and doxorubicin-induced secondary necrosis in mouse macrophages.

OBJECTIVE: Induction of secondary necrosis/pyroptosis contributes to the toxicity of chemotherapeutic drugs, in which gasdermin E (GSDME) plays critical roles. This study aimed to explore whether GSDME is involved in mediating the cytotoxic effects of cisplatin and doxorubicin on mouse macrophages. METHODS: RAW 264.7 cells and bone marrow-derived macrophages (BMDMs) were treated with cisplatin or doxorubicin. Propidium iodide staining was used to assay necrosis, and immunoblotting was performed to detect protein expression. GSDME was knocked down by using small interfering RNA. Mice were injected intraperitoneally to evaluate toxicity to macrophages in vivo. Flow cytometry and immunofluorescence microscopy were adopted to analyse phenotypes of peritoneal cells. Cytokine levels were assayed by cytometric bead array. RESULTS: Both cisplatin and doxorubicin dose-dependently induced necrosis in mouse RAW 264.7 macrophages and BMDMs. Accompanying this, multiple caspases were activated, concomitant with the cleavage of poly (ADP-ribose) polymerase. Consistent with caspase-3 activation, GSDME was cleaved to generate its N-terminal fragment (GSDME-NT), thus leading to secondary necrosis/pyroptosis. Inhibition of caspase-3 significantly attenuated the generation of GSDME-NT concurrently with decreased necrosis in macrophages. GSDME knockdown also evidently decreased the necrosis in RAW 264.7 and BMDMs. Besides, cisplatin administration depleted peritoneal macrophages in mice, which was associated with caspase-3 activation and GSDME-NT generation. Consistent with the macrophage depletion, cisplatin administration significantly decreased survival of mice with bacterial infection. CONCLUSION: Chemotherapeutic cisplatin and doxorubicin exerted their cytotoxicity on macrophages partly by inducing caspase-3/GSDME-mediated secondary necrosis.

ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3 pathway-blocked murine macrophages.

ATP acts as a canonical activator to induce NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation in macrophages, leading to caspase-1/gasdermin D (GSDMD)-mediated pyroptosis. It remains unclear whether ATP can induce pyroptosis in macrophages when the NLRP3 pathway is blocked by pathogenic infection. In this study, we used cellular models to mimic such blockade of NLRP3 activation: bone marrow-derived macrophages (BMDMs) treated with NLRP3-specific inhibitor MCC950 and RAW264.7 cells deficient in ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression. The results showed that ATP treatment induced lytic cell death morphologically resembling canonical pyroptosis in both MCC950-treated BMDMs and RAW264.7 cells, but did not cause the activation of caspase-1 (by detecting caspase-1p10 and mature interleukin-1ß) and cleavage of GSDMD. Instead, both apoptotic initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspases were evidently activated and gasdermin E (GSDME) was cleaved to generate its N-terminal fragment (GSDME-NT) which executes pyroptosis. The GSDME-NT production and lytic cell death induced by ATP were diminished by caspase-3 inhibitor. In BMDMs without MCC950 treatment, ATP induced the formation of ASC specks which were co-localized with caspase-8; with MCC950 treatment, however, ATP did not induced the formation of ASC specks. In RAW264.7 cells, knockdown of GSDME by small interfering RNA attenuated ATP-induced lytic cell death and HMGB1 release into culture supernatants. Collectively, our results indicate that ATP induces pyroptosis in macrophages through the caspase-3/GSDME axis when the canonical NLRP3 pathway is blocked, suggestive of an alternative mechanism for combating against pathogen evasion.

Evodiamine Augments NLRP3 Inflammasome Activation and Anti-bacterial Responses Through Inducing α-Tubulin Acetylation.

Evodiamine is a major ingredient of the plant Evodia rutaecarpa, which has long been used for treating infection-related diseases including diarrhea, beriberi and oral ulcer, but the underlying mechanism is unclear. Here we aimed to explore whether evodiamine influenced NLRP3 (NLR family, pyrin containing domain 3) inflammasome activation in macrophages, which is a critical mechanism for defending the host against pathogenic infections. We uncovered that evodiamine dose-dependently enhanced NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, as indicated by increased interleukin (IL)-1ß production and caspase-1 cleavage, accompanied by increased ASC speck formation and pyroptosis. Mechanistically, evodiamine induced acetylation of α-tubulin around the microtubule organization center (indicated by γ-tubulin) in lipopolysaccharide-primed macrophages. Such evodiamine-mediated increases in NLRP3 activation and pyroptosis were attenuated by activators of α-tubulin deacetylase, resveratrol and NAD+, or dynein-specific inhibitor ciliobrevin A. Small interfering RNA knockdown of αTAT1 (the gene encoding α-tubulin N-acetyltransferase) expression, which reduced α-tubulin acetylation, also diminished evodiamine-mediated augmentation of NLRP3 activation and pyroptosis. Evodiamine also enhanced NLRP3-mediated production of IL-1ß and neutrophil recruitment in vivo. Moreover, evodiamine administration evidently improved survival of mice with lethal bacterial infection, accompanied by increased production of IL-1ß and interferon-γ, decreased bacterial load, and dampened liver inflammation. Resveratrol treatment reversed evodiamine-induced increases of IL-1ß and interferon-γ, and decreased bacterial clearance in mice. Collectively, our results indicated that evodiamine augmented the NLRP3 inflammasome activation through inducing α-tubulin acetylation, thereby conferring intensified innate immunity against bacterial infection.

Chemotherapeutic paclitaxel and cisplatin differentially induce pyroptosis in A549 lung cancer cells via caspase-3/GSDME activation.

Gasdermin E (GSDME) has an important role in inducing secondary necrosis/pyroptosis. Upon apoptotic stimulation, it can be cleaved by activated caspase-3 to generate its N-terminal fragment (GSDME-NT), which executes pyroptosis by perforating the plasma membrane. GSDME is expressed in many human lung cancers including A549 cells. Paclitaxel and cisplatin are two representative chemotherapeutic agents for lung cancers, which induce apoptosis via different action mechanisms. However, it remains unclear whether they can induce GSDME-mediated secondary necrosis/pyroptosis in lung A549 cancer cells. Here we showed that both paclitaxel and cisplatin evidently induced apoptosis in A549 cells as revealed by the activation of multiple apoptotic markers. Notably, some of the dying cells displayed characteristic morphology of secondary necrosis/pyroptosis, by blowing large bubbles from the cellular membrane accompanied by caspase-3 activation and GSDME-NT generation. But the ability of cisplatin to induce this phenomenon was much stronger than that of paclitaxel. Consistent with this, cisplatin triggered much higher activation of caspase-3 and generation of GSDME-NT than paclitaxel, suggesting that the levels of secondary necrosis/pyroptosis correlated with the levels of active caspase-3 and GSDME-NT. Supporting this, caspase-3 specific inhibitor (Ac-DEVD-CHO) suppressed cisplatin-induced GSDME-NT generation and concurrently reduced the secondary necrosis/pyroptosis. Besides, GSDME knockdown significantly inhibited cisplatin- but not paclitaxel-induced secondary necrosis/pyroptosis. These results indicated that cisplatin induced higher levels of secondary necrosis/pyroptosis in A549 cells than paclitaxel, suggesting that cisplatin may provide additional advantages in the treatment of lung cancers with high levels of GSDME expression.

Paclitaxel Enhances the Innate Immunity by Promoting NLRP3 Inflammasome Activation in Macrophages.

Microtubules play critical roles in regulating the activation of NLRP3 inflammasome and microtubule-destabilizing agents such as colchicine have been shown to suppress the activation of this inflammasome. However, it remains largely unknown whether paclitaxel, a microtubule-stabilizing agent being used in cancer therapy, has any influences on NLRP3 inflammasome activation. Here we showed that paclitaxel pre-treatment greatly enhanced ATP- or nigericin-induced NLRP3 inflammasome activation as indicated by increased release of cleaved caspase-1 and mature IL-1ß, enhanced formation of ASC speck, and increased gasdermin D cleavage and pyroptosis. Paclitaxel time- and dose-dependently induced α-tubulin acetylation in LPS-primed murine and human macrophages and further increased ATP- or nigericin-induced α-tubulin acetylation. Such increased α-tubulin acetylation was significantly suppressed either by resveratrol or NAD+ (coenzyme required for deacetylase activity of SIRT2), or by genetic knockdown of MEC-17 (gene encoding α-tubulin acetyltransferase 1). Concurrently, the paclitaxel-mediated enhancement of NLRP3 inflammasome activation was significantly suppressed by resveratrol, NAD+, or MEC-17 knockdown, indicating the involvement of paclitaxel-induced α-tubulin acetylation in the augmentation of NLRP3 inflammasome activation. Similar to paclitaxel, epothilone B that is another microtubule-stabilizing agent also induced α-tubulin acetylation and increased NLRP3 inflammasome activation in macrophages in response to ATP treatment. Consistent with the in vitro results, intraperitoneal administration of paclitaxel significantly increased serum IL-1ß levels, reduced bacterial burden, dampened infiltration of inflammatory cells in the liver, and improved animal survival in a mouse model of bacterial infection. Collectively, our data indicate that paclitaxel potentiated NLRP3 inflammasome activation by inducing α-tubulin acetylation and thereby conferred enhanced antibacterial innate responses, suggesting its potential application against pathogenic infections beyond its use as a chemotherapeutic agent.

Scutellarin Suppresses NLRP3 Inflammasome Activation in Macrophages and Protects Mice against Bacterial Sepsis.

The NLRP3 inflammasome plays a critical role in mediating the innate immune defense against pathogenic infections, but aberrant activation of NLRP3 inflammasome has been linked to a variety of inflammatory diseases. Thus targeting the NLRP3 inflammasome represents a promising therapeutic for the treatment of such diseases. Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant.) Hand.-Mazz. and has been reported to exhibit potent anti-inflammatory activities, but the underlying mechanism is only partly understood. In this study, we aimed to investigate whether scutellarin could affect the activation of NLRP3 inflammasome in macrophages. The results showed that scutellarin dose-dependently reduced caspase-1 activation and decreased mature interleukin-1ß (IL-1ß) release in lipopolysaccharide (LPS)-primed macrophages upon ATP or nigericin stimulation, indicating that scutellarin inhibited NLRP3 inflammasome activation in macrophages. Consistent with this, scutellarin also suppressed pyroptotic cell death in LPS-primed macrophages treated with ATP or nigericin. ATP or nigericin-induced ASC speck formation and its oligomerization were blocked by scutellarin pre-treatment. Intriguingly, scutellarin augmented PKA-specific phosphorylation of NLRP3 in LPS-primed macrophages, which was completely blocked by selective PKA inhibitor H89, suggesting that PKA signaling had been involved in the action of scutellarin to suppress NLRP3 inflammasome activation. Supporting this, the inhibitory effect of scutellarin on NLRP3 inflammasome activation was completely counteracted by H89 or adenyl cyclase inhibitor MDL12330A. As NLRP3-dependent release of IL-1ß has a critical role in sepsis, the in vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis, which was established by intraperitoneally injection of a lethal dose of viable Escherichia coli. Oral administration of scutellarin significantly improved the survival of mice with bacterial sepsis. In line with this, scutellarin treatment significantly reduced serum IL-1ß levels and attenuated the infiltration of inflammatory cells in the liver of E. coli-infected mice. These data indicated that scutellarin suppressed NLRP3 inflammasome activation in macrophages by augmenting PKA signaling, highlighting its potential therapeutic application for treating NLRP3-related inflammatory diseases.