Mutability landscape guided engineering of a promiscuous microbial glycosyltransferase for regioselective synthesis of salidroside and icariside D2.

Microbial glycosyltransferases efficiently synthesize glucosides and have garnered increasing interest. However, limited regioselectivity has impeded their broad application, particularly in the pharmaceutical industry. In this study, the UDP-glycosyltransferase YjiC from Bacillus licheniformis (BlYjiC) was engineered to achieve the bidirectional regioselective glycosylation of tyrosol and its derivatives. Initially, site-directed saturation mutagenesis was performed on two newly identified substrate-binding cavities in the acceptor pocket of BlYjiC to provide a comprehensive blueprint of the interplay between mutations and function (mutability landscape). Iterative saturation mutagenesis was performed, guided by the mutability landscape. Two highly regioselective mutants M6 (M112L/I325Y/L70R/Q136E/I67E/M77R) and M2' (M112D/I62L) were generated, exhibiting >99 % regioselectivity toward the alcoholic and phenolic hydroxyl of tyrosol, respectively, compared with the wild-type (product mixture: 51:49 %). Both mutants exhibited excellent regioselectivity toward several dihydroxy phenolic substrates, offering valuable biocatalysts for the regioselective synthesis of glucosides. Their application was confirmed in a short synthesis of salidroside (3.6 g/L) and icariside D2 (2.4 g/L), which exhibited near-perfect regioselectivity. This study provides valuable insights into future protein engineering of similar enzymes and opens new avenues for their practical applications.

TiCl4-mediated deoxygenative reduction of aromatic ketones to alkylarenes with ammonia borane.

A rapid and mild protocol for the exhaustive deoxygenation of various aromatic ketones to corresponding alkanes was described, which was mediated by TiCl4 and used ammonia borane (AB) as the reductant. This reduction protocol applies to a wide range of substrates in moderate to excellent yields at room temperature. The gram-scale reaction and syntheses of some key building blocks for SGLT2 inhibitors demonstrated the practicability of this methodology. Preliminary mechanistic studies revealed that the ketone is first converted into an alcohol, which then undergoes a carbocation to give the alkane via hydrogenolysis.

Highly efficient biosynthesis of salidroside by a UDP-glucosyltransferase-catalyzed cascade reaction.

OBJECTIVE: Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). RESULTS: The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana, which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. CONCLUSION: This is the highest salidroside titer ever reported so far using biocatalytic approach.

Improvement in organic solvent resistance of keratinase BLk by directed evolution.

Keratinase, a vital enzyme in hair degradation, requires enhanced stability for industrial applications in the harsh reaction environment used for keratin hydrolysis. Previous studies have focused on improving keratinase thermostability. In this study, directed evolution was applied to enhance the organic solvent stability of the keratinase BLk from Bacillus licheniformis. Three mutants were identified, exhibiting significant enhanced stability in various solvents, although no similar improvements were observed in terms of thermostability. The identified mutations were located on the enzyme surface. The half-lives of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to that of the wild type (WT). Similarly, in the presence of 50% (v/v) acetone, the half-lives of these mutants increased by 22-, 27-, and 27-fold compared to that of the WT enzyme. Notably, the proteolytic activity of all the selected mutants was similar to that of the WT enzyme. Furthermore, molecular dynamics simulation was used to assess the possible reasons for enhanced solvent stability. These results suggest that heightened intramolecular interactions, such as hydrogen bonding and hydrophobic interactions, contribute to improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.

Separation of Flavonoids and Purification of Chlorogenic Acid from Bamboo Leaves Extraction Residues by Combination of Macroporous Resin and High-Speed Counter-Current Chromatography.

Flavonoids are major active small-molecule compounds in bamboo leaves, which can be easily obtained from the bamboo leaves extraction residues (BLER) after the polysaccharides extraction. Six macroporous resins with different properties were screened to prepare and enrich isoorientin (IOR), orientin (OR), vitexin (VI), and isovitexin (IVI) from BLER, and the XAD-7HP resin with the best adsorption and desorption performance was selected for further evaluation. Based on the static adsorption experiments, the experimental results showed that the adsorption isotherm fitted well with the Langmuir isotherm model, and the adsorption process was better explained by the pseudo-second-order kinetic model. After the dynamic trial of resin column chromatography, 20 bed volume (BV) of upload sample and 60% ethanol as eluting solvent was used in a lab scale-up separation, and the results demonstrated that the content of four flavonoids could be increased by 4.5-fold, with recoveries between 72.86 and 88.21%. In addition, chlorogenic acid (CA) with purity of 95.1% was obtained in water-eluted parts during dynamic resin separation and further purified by high-speed countercurrent chromatography (HSCCC). In conclusion, this rapid and efficient method can provide a reference to utilize BLER to produce high-value-added food and pharmaceutical products.

T helper 17 (Th17) cell responses to the gut microbiota in human diseases.

The gut microbiota colonizing the gastrointestinal tract, is an indispensable "invisible organ" that affects multiple aspects of human health. The gut microbial community has been assumed to be an important stimulus to the immune homeostasis and development, and increasing data support the role of the gut microbiota-immunity axis in autoimmune diseases. Host's immune system requires recognition tools to communicate with the gut microbial evolutionary partners. Among these microbial perceptions, T cells enable the widest spectrum of gut microbial recognition resolution. Specific gut microbiota direct the induction and differentiation of Th17 cells in intestine. However, the detailed links between the gut microbiota and Th17 cells have not been well established. In this review, we describe the generation and characterization of Th17 cells. Notably, we discuss the induction and differentiation of Th17 cells by the gut microbiota and their metabolites, as well as recent advances in our understanding of interactions between Th17 cells and the gut microbiota in human diseases. In addition, we provide the emerging evidences in support of interventions targeting the gut microbes/Th17 cells in human diseases.

Rational engineering of a metalloprotease to enhance thermostability and activity.

The rational design of enzymes with enhanced thermostability is efficient. Solvent-tolerant metalloprotease from Pseudomonas aeruginosa PT121 presents high Z-aspartame (Z-APM) synthesis activity, but insufficient thermostability. In this study, we enhanced enzyme thermostability using a rational strategy. Molecular dynamics (MD) simulation was applied to rapidly identify that the D28 and D116 mutations are likely to exhibit increased thermostability, and experimentation verified that the D28N and D116N mutants were more stable than the wild-type (WT) enzyme. In particular, the Tm of the D28N and D116N mutants increased by 6.1 °C and 9.2 °C, respectively, compared with that of the WT enzyme. The half-lives of D28N and D116N at 60 °C were 1.07- and 1.8-fold higher than that of the WT, respectively. Z-APM synthetic activities of the mutants were also improved. The potential mechanism of thermostability enhancement rationalized using MD simulation indicated that increased hydrogen bond interactions and a regional hydration shell were mostly responsible for the thermostability enhancement. Our strategy could be a reference for enzyme engineering, and our mutants offer considerable value in industrial applications.

Bioartificial livers: a review of their design and manufacture.

Acute liver failure (ALF) is a rapidly progressive disease with high morbidity and mortality rates. Liver transplantation and artificial liver (AL) support systems, such as ALs and bioartificial livers (BALs), are the two major therapies for ALF. Compared to ALs, BALs are composed of functional hepatocytes that provide essential liver functions, including detoxification, metabolite synthesis, and biotransformation. Furthermore, BALs can potentially provide effective support as a form of bridging therapy to liver transplantation or spontaneous recovery for patients with ALF. In this review, we systematically discussed the currently available state-of-the-art designs and manufacturing processes for BAL support systems. Specifically, we classified the cell sources and bioreactors that are applied in BALs, highlighted the advanced technologies of hepatocyte culturing and bioreactor fabrication, and discussed the current challenges and future trends in developing next-generation BALs for large-scale clinical applications.

A novel prognostic nomogram for hepatocellular carcinoma after thermal ablation.

It remains impossible to accurately assess the prognosis after thermal ablation in patients with hepatocellular carcinoma (HCC). Our aim was to build a nomogram to predict the survival rate of HCC patients after thermal ablation. We developed and validated a nomogram using data of 959 HCC patients after thermal ablation from two centers. Harrell's concordance index (C-index), calibration plot and Decision curve analysis (DCA) were used to measure the performance of the nomogram, and we compared it with the Barcelona Clinic Liver Cancer (BCLC) staging system and a previous nomogram. Six variables including age, serum albumin, operation method, risk area, tumor number and early recurrence were selected to construct the nomogram. In the training cohort, internal validation cohort, and external validation cohort, the nomogram all had a higher C-index to predict survival rate than both the BCLC staging system and the previous nomogram (0.736, 0.558 and 0.698, respectively; 0.763, 0.621 and 0.740, respectively; and 0.825, 0.551 and 0.737, respectively). Calibration plots showed a high degree of consistency between prediction and actual observation. Decision curve analysis (DCA) presented that compared with BCLC system and the previous nomogram, our nomogram had the highest net benefit. In all three cohorts, the nomogram could accurately divide patients into three subgroups according to predicted survival risk. A nomogram was developed and validated to predict survival of HCC patients who underwent thermal ablation, which is helpful for prognostic prediction and individual surveillance in clinical practice.

Effectiveness of a Clinical Pathway for Hepatic Cystic Echinococcosis Surgery in Kashi Prefecture, Northwestern China: A Propensity Score Matching Analysis.

INTRODUCTION: Surgical treatment for hepatic cystic ehinococcosis (CE) is not standardized in Kashi Prefecture. Previous evidence identified effectiveness of a clinical pathway in the field of liver surgery. However, proof of a clinical pathway program, especially for CE patients, is lacking. This study aimed to assess the validity of a clinical pathway for hepatic CE surgery performed on patients from Kashi Prefecture. METHODS: A clinical pathway was developed and implemented by a multidisciplinary team for patients undergoing hepatic CE surgery. Two groups were formed from patients undergoing hepatic CE surgery during a defined period before and after implementing a clinical pathway. Additionally, a propensity score matching analysis was performed. RESULTS: In the overall analysis (n = 258) as well as the matched analysis (n = 166), after implementing the clinical pathway, hospital stay was significantly reduced from 13 to 10 days and from 14 to 10 days, respectively (P < 0.05). Postoperative morbidity did not increase. Cost analysis showed a significant decrease in median costs of medication and nursing in favor of the clinical pathway (medication: 5400 CNY vs. 6400 CNY, P = 0.038; nursing: 3200 CNY vs. 4100 CNY, P = 0.02). CONCLUSION: Implementing the clinical pathway for hepatic CE surgery is feasible and safe. The clinical pathway achieved significant reduction of hospital stay without compromising postoperative morbidity. Costs of medication and nursing are significantly reduced. The clinical pathway program is valid and propagable to a certain extent, especially in remote, poor-resourced medical centers in endemic areas.

Gingiva-Derived Mesenchymal Stem Cells: Potential Application in Tissue Engineering and Regenerative Medicine - A Comprehensive Review.

A unique subpopulation of mesenchymal stem cells (MSCs) has been isolated and characterized from human gingival tissues (GMSCs). Similar to MSCs derived from other sources of tissues, e.g. bone marrow, adipose or umbilical cord, GMSCs also possess multipotent differentiation capacities and potent immunomodulatory effects on both innate and adaptive immune cells through the secretion of various types of bioactive factors with immunosuppressive and anti-inflammatory functions. Uniquely, GMSCs are highly proliferative and have the propensity to differentiate into neural cell lineages due to the neural crest-origin. These properties have endowed GMSCs with potent regenerative and therapeutic potentials in various preclinical models of human disorders, particularly, some inflammatory and autoimmune diseases, skin diseases, oral and maxillofacial disorders, and peripheral nerve injuries. All types of cells release extracellular vesicles (EVs), including exosomes, that play critical roles in cell-cell communication through their cargos containing a variety of bioactive molecules, such as proteins, nucleic acids, and lipids. Like EVs released by other sources of MSCs, GMSC-derived EVs have been shown to possess similar biological functions and therapeutic effects on several preclinical diseases models as GMSCs, thus representing a promising cell-free platform for regenerative therapy. Taken together, due to the easily accessibility and less morbidity of harvesting gingival tissues as well as the potent immunomodulatory and anti-inflammatory functions, GMSCs represent a unique source of MSCs of a neural crest-origin for potential application in tissue engineering and regenerative therapy.

An evaluation of the cut-off value of methamphetamine in hair samples via HPLC-MS/MS.

Methamphetamine (MAMP) is one of the most commonly abused illicit drugs in Asian countries, which belongs to the amphetamine-type stimulant class of substances. To detect the chronic drug misuse, human hairs have often been used as analytical specimens due to their long detection windows and easy accessibility. However, there is no investigation regarding the cut-off value of MAMP detection used in black-hair Chinese populations. Based on the analytical data obtained from 563 MAMP users, the cut-off value was found to be 0.97 ng/mg for the simultaneous detection of MAMP and amphetamine (AMP) ≥0.004 ng/mg (LOD). Through the established HPLC-MS/MS analytical method, the limits of detection and quantification of MAMP were 0.004 and 0.01 ng/mg, respectively. The cut-off value was optimized by AMP detection rate and receiver operating characteristic analysis, and the results were consistent with the previously reported MAMP/AMP ratio.

Effect of 3-nitropropionic acid inducing oxidative stress and apoptosis of granulosa cells in geese.

The mechanism of action by which oxidative stress induces granulosa cell apoptosis, which plays a vital role in initiating follicular atresia, is not well understood. In the present study, the effect of 3-nitropropionic acid (3-NPA) on oxidative stress and apoptosis in granulosa cells in geese was investigated. Our results showed that treatment with 3-NPA at 5.0 mmol/l for 24 h increased intracellular reactive oxygen species (ROS) production by 25.4% and decreased granulosa cell viability by 45.5% (P<0.05). Catalase and glutathione peroxidase gene expression levels in granulosa cells treated with 3-NPA were 1.32- and 0.49-fold compared with those of the control cells, respectively (P <0.05). A significant decrease in the expression level of B-cell lymphoma 2 (Bcl-2) protein and remarkable increases in the levels of Bax, p53 and cleaved-Caspase 3 proteins and the ratio of Bax/Bcl-2 expression in granulosa cells treated with 3-NPA were observed (P<0.05). Furthermore, a 38.43% increase in the percentage of early apoptotic cells was also observed in granulosa cells treated with 3-NPA (P<0.05). Moreover, the expression levels of NF-κB, Nrf2, Fhc, Hspa2 and Ho-1 in granulosa cells treated with 3-NPA were elevated 4.36-, 1.63-, 3.62-, 27.54- and 10.48-fold compared with those of the control cells (P<0.05), respectively. In conclusion, the present study demonstrates that treatment with 3-NPA induces ROS production and apoptosis and inhibits the viability of granulosa cells in geese. Furthermore, 3-NPA triggers increases in the expression of cleaved-Caspase 3 protein and the ratio of Bax/Bcl-2 expression, and induces the early apoptosis of granulosa cells.

Neural Crest Stem-Like Cells Non-genetically Induced from Human Gingiva-Derived Mesenchymal Stem Cells Promote Facial Nerve Regeneration in Rats.

Non-genetic induction of somatic cells into neural crest stem-like cells (NCSCs) is promising for potential cell-based therapies for post-traumatic peripheral nerve regeneration. Here, we report that human gingiva-derived mesenchymal stem cells (GMSCs) could be reproducibly and readily induced into NCSCs via non-genetic approaches. Compared to parental GMSCs, induced NCSC population had increased expression in NCSC-related genes and displayed robust differentiation into neuronal and Schwann-like cells. Knockdown of the expression of Yes-associated protein 1 (YAP1), a critical mechanosensor and mechanotransducer, attenuated the expression of NCSC-related genes; specific blocking of RhoA/ROCK activity and non-muscle myosin II (NM II)-dependent contraction suppressed YAP1 and NCSC-related genes and concurrently abolished neural spheroid formation in NCSCs. Using a rat model of facial nerve defect, implantation of NCSC-laden nerve conduits promoted functional regeneration of the injured nerve. These promising findings demonstrate that induced NCSCs derived from GMSCs represent an easily accessible and promising source of neural stem-like cells for peripheral nerve regeneration.

Characterization of goose SPMS: Molecular characterization and expression profiling of SPMS in the goose ovary.

Spermine synthase (SPMS), which converts spermidine into spermine, is essential for normal cell growth and development processes in humans and other mammals, but the molecular characterization and expression profiling of the SPMS gene remain undetermined in goose tissues and ovarian follicles. In this study, the SPMS cDNA sequence of the Sichuan white goose was cloned and analysed, and SPMS mRNA expression was profiled in various tissues and ovarian follicles. The results showed that the open reading frame of the SPMS cDNA sequence was 1092 bp in length, encoding 363 amino acids with a molecular weight of 41 kDa. Among all the examined tissues, SPMS expression was highest in the spleen and cerebrum and lowest in the breast and thigh muscles. SPMS expression in the F1 follicle was significantly higher than that in the POF (except for POF2) (P < 0.05). Our results indicate that SPMS might play an important role in follicular development and ovulation.

Neural Progenitor-Like Cells Induced from Human Gingiva-Derived Mesenchymal Stem Cells Regulate Myelination of Schwann Cells in Rat Sciatic Nerve Regeneration.

Regeneration of peripheral nerve injury remains a major clinical challenge. Recently, mesenchymal stem cells (MSCs) have been considered as potential candidates for peripheral nerve regeneration; however, the underlying mechanisms remain elusive. Here, we show that human gingiva-derived MSCs (GMSCs) could be directly induced into multipotent NPCs (iNPCs) under minimally manipulated conditions without the introduction of exogenous genes. Using a crush-injury model of rat sciatic nerve, we demonstrate that GMSCs transplanted to the injury site could differentiate into neuronal cells, whereas iNPCs could differentiate into both neuronal and Schwann cells. After crush injury, iNPCs, compared with GMSCs, displayed superior therapeutic effects on axonal regeneration at both the injury site and the distal segment of the injured sciatic nerve. Mechanistically, transplantation of GMSCs, especially iNPCs, significantly attenuated injury-triggered increase in the expression of c-Jun, a transcription factor that functions as a major negative regulator of myelination and plays a central role in dedifferentiation/reprogramming of Schwann cells into a progenitor-like state. Meanwhile, our results also demonstrate that transplantation of GMSCs and iNPCs consistently increased the expression of Krox-20/EGR2, a transcription factor that governs the expression of myelin proteins and facilitates myelination. Altogether, our findings suggest that transplantation of GMSCs and iNPCs promotes peripheral nerve repair/regeneration, possibly by promoting remyelination of Schwann cells mediated via the regulation of the antagonistic myelination regulators, c-Jun and Krox-20/EGR2. Stem Cells Translational Medicine 2017;6:458-470.

A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue.

In the oral cavity, the tongue is the anatomic subsite most commonly involved by invasive squamous cell carcinoma. Current treatment protocols often require significant tissue resection to achieve adequate negative margins and optimal local tumor control. Reconstruction of the tongue while preserving and/or restoring its critical vocal, chewing, and swallowing functions remains one of the major challenges in head and neck oncologic surgery. We investigated the in vitro feasibility of fabricating a novel combinatorial construct using porcine small intestinal submucosa extracellular matrix (SIS-ECM) and human gingiva-derived mesenchymal stem cells (GMSCs) as a GMSC/SIS-ECM tissue graft for the tongue reconstruction. We developed a rat model of critical-sized myomucosal defect of the tongue that allowed the testing of therapeutic effects of an acellular SIS-ECM construct versus a GMSC/SIS-ECM construct on repair and regeneration of the tongue defect. We showed that the GMSC/SIS-ECM construct engrafted at the host recipient site, promoted soft tissue healing, and regenerated the muscular layer, compared to the SIS-ECM alone or nontreated defect controls. Furthermore, our results revealed that transplantation of the GMSC/SIS-ECM construct significantly increased the expression of several myogenic transcriptional factors and simultaneously suppressed the expression of type I collagen at the wounded area of the tongue. These compelling findings suggest that, unlike the tongue contracture and fibrosis of the nontreated defect group, transplantation of the combinatorial GMSC/SIS-ECM constructs accelerates wound healing and muscle regeneration and maintains the overall tongue shape, possibly by both enhancing the function of endogenous skeletal progenitor cells and suppressing fibrosis. Together, our findings indicate that GMSC/SIS-ECM potentially served as a myomucosal graft for tongue reconstruction postsurgery of head and neck cancer.

EGF induces epithelial-mesenchymal transition and cancer stem-like cell properties in human oral cancer cells via promoting Warburg effect.

"Warburg effect", the enhanced glycolysis or aerobic glycolysis, confers cancer cells the ability to survive and proliferate even under stressed conditions. In this study, we explored the role of epidermal growth factor (EGF) in orchestrating Warburg effect, the epithelial-mesenchymal transition (EMT) process, and the acquisition of cancer stem-like cell properties in human oral squamous cell carcinoma (OSCC) cells. Our results showed that EGF induces EMT process in OSCC cells, which correlates with the acquisition of cancer stem-like properties, including the enrichment of CD44+/CD24- population of cancer cells and an increased expression of CSC-related genes, aldehyde dehydrogenase-1 (ALDH1) and Bmi-1. We also showed that EGF concomitantly enhanced L-lactate production, while blocking glycolysis by 2-deoxy-D-glucose (2-DG) robustly reversed EGF-induced EMT process and CSC-like properties in OSCC cells. Mechanistically, we demonstrated that EGF promoted EMT process and CSC generation through EGFR/PI3K/HIF-1α axis-orchestrated glycolysis. Using an orthotopic tumor model of human OSCC (UM-SCC1) injected in the tongue of BALB/c nude mice, we showed that treatment with 2-DG in vivo significantly inhibited the metastasis of tumor cells to the regional cervical lymph nodes and reduced the expression of ALDH1 and vimentin in both in situ tumors and tumor cell-invaded regional lymph nodes. Taken together, these findings have unveiled a new mechanism that EGF drives OSCC metastasis through induction of EMT process and CSC generation, which is driven by an enhanced glycolytic metabolic program in OSCC cells.

Targeting Cancer Stem Cells in Oral Cancer.

The concept of tumor development driven by a unique subpopulation of cancer stem cells (CSCs), or the CSCs hypothesis, may help to explain the high mortality, low response to treatment and tendency of developing multiple tumors in oral cancer. We will review current knowledge of the CSCs hypothesis in oral cancer and the traits displayed by CSCs, focusing on the resistance to therapy and attempts being made to treat oral cancer by specifically targeting CSCs.

Bisphosphonate Induces Osteonecrosis of the Jaw in Diabetic Mice via NLRP3/Caspase-1-Dependent IL-1ß Mechanism.

Diabetes mellitus is an established risk factor associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase-1, and IL-1ß in macrophages residing at local wounds. Constitutively, bone marrow-derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL-1ß than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL-1ß by db/db BMDMs was 1.77-fold higher than that by db/+ BMDMs (p < 0.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen-containing bisphosphonate, resulted in a 1.86- and 1.63-fold increase in NLRP3/caspase-1-dependent IL-1ß secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (p < 0.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase-1 and NLRP3 activation, an antagonist for P2X7 R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol-enhanced IL-1ß release from macrophages in response to NLRP3 activation (p < 0.001). Our findings suggest that diabetes-associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages.