Construction of nanoparticles from blueberry anthocyanins-lecithin/gum Arabic improves lipid droplet accumulation and gut microbiota disturbance in HFD-induced obese mice.

The digestive instability of anthocyanins (ACNs) limits their application in food nutrition, especially precision nutrition. Blueberry ACNs-loaded nanoparticles (Lipo/GA-ACNs NPs) were prepared using gum arabic (GA) as the delivery carrier and liposomal vesicles (Lipo) prepared from soy lecithin as the targeting scaffold. The average particle size of the NPs was 99.4 nm, and the polydispersion index (PDI) was 0.46. The results showed that the presence of the Lipo-GA matrix enhanced the NPs' in vitro stability and antioxidant activity. In addition, the in vitro biocompatibility, uptake ability, lipid-lowering activity, and free-radical scavenging ability were improved to a certain extent. In a high-fat diet (HFD)-induced obese mouse model, oral administration of ACNs-LNP (LNP, liver-targeted nanoparticle) showed better effects on body weight, liver injury, and lipid droplet accumulation in the liver than ACNs. In addition, ACNs-LNP also played a role in regulating HFD-induced gut microbiota imbalance. These results provide a promising ACNs delivery strategy with the potential to be developed into a functional food that targets the liver to prevent fatty liver.

FHL2 regulates microglia M1/M2 polarization after spinal cord injury via PARP14-depended STAT1/6 pathway.

Neuronal apoptosis and inflammation exacerbate the secondary injury after spinal cord injury (SCI). Four and a half domains 2 (FHL2) is a multifunctional scaffold protein with tissue- and cell-type specific effects on the regulation of inflammation, but its role in SCI remains unclear. The T10 mouse spinal cord contusion model was established, and the mice were immediately injected with lentiviruses carrying FHL2 shRNA after SCI. The results showed that FHL2 expression was increased following SCI, and then gradually decreased. Moreover, FHL2 depletion aggravated functional impairment, neuronal necrosis, and enlarged lesion cavity areas in the injured spinal cord. FHL2 deficiency facilitated neuronal apoptosis by elevating cleaved caspase 3/9 expression, neuroinflammation by regulating microglia polarization, and bone loss. Indeed, FHL2 deficiency increased the secretion of TNF-α and IL-6, M1 microglia polarization, and the activation of STAT1 pathway but decreased the secretion of IL-10 and IL-4, M2 microglia polarization, and the activation of the STAT6 pathway in the spinal cord. In vitro, FHL2 silencing promoted LPS + IFN-γ-induced microglia M1 polarization through activating the STAT1 pathway and alleviated IL-4-induced microglia M2 polarization via inhibiting the STAT6 pathway. FHL2 positively regulated the expression of poly (ADP-ribose) polymerase family member 14 (PARP14) by promoting its transcription. PARP14 overexpression inhibited FHL2 silencing-induced microglia M1 polarization and relieved the inhibitory effect of FHL2 silencing on microglia M2 polarization. Collectively, the study suggests that FHL2 reduces the microglia M1/M2 polarization-mediated inflammation via PARP14-dependent STAT1/6 pathway and thereby improves functional recovery after SCI.

Acid sphingomyelinase mediates ferroptosis induced by high glucose via autophagic degradation of GPX4 in type 2 diabetic osteoporosis.

BACKGROUND: Ferroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), although the specific underlying mechanisms remain largely unknown. This study aimed to clarify the role and possible mechanism of acid sphingomyelinase (ASM)-mediated osteoblast ferroptosis in T2DOP. METHODS: We treated hFob1.19 cells with normal glucose (NG) and different concentrations of high glucose (HG, 26.25 mM, 35 mM, or 43.75 mM) for 48 h. We then measured cell viability and osteogenic function, quantified ferroptosis and autophagy levels, and measured the levels of ASM and ceramide in the cells. To further investigate the specific mechanism, we examined these indicators by knocking down ASM expression, hydroxychloroquine (HCQ) treatment, or N-acetylcysteine (NAC) treatment. Moreover, a T2DOP rat model was induced and microcomputed tomography was used to observe the bone microstructure. We also evaluated the serum levels of iron metabolism-associated factors, ceramide and lipid peroxidation (LPO) and measured the expression of ASM, LC3 and GPX4 in bone tissues. RESULTS: HG inhibited the viability and osteogenic function of osteoblasts by inducing ferroptosis in a concentration-dependent manner. Furthermore, the expression of ASM and ceramide and autophagy levels were increased by HG treatment, and these factors were required for the HG-induced reactive oxygen species (ROS) generation and LPO. Similarly, inhibiting intracellular ROS also reduced HG-induced ASM activation and autophagy. ASM-mediated activation of autophagy was crucial for HG-induced degradation of GPX4, and inhibiting ASM improved osteogenic function by decreasing HG-induced autophagy, GPX4 degradation, LPO and subsequent ferroptosis. We also found that inhibiting ASM could alleviated ferroptosis and autophagy and improved osteogenic function in a T2DOP rat model. CONCLUSION: ASM-mediated autophagy activation induces osteoblast ferroptosis under HG conditions through the degradation of GPX4, providing a novel mechanistic insight into the treatment and prevention of T2DOP.

Permanganate activation with Mn oxides at different oxidation states: Insight into the surface-promoted electron transfer mechanism.

The development of new strategies to improve the removal of organic pollutants with permanganate (KMnO4) is a hot topic in water treatment. While Mn oxides have been extensively used in Advanced Oxidation Processes through an electron transfer mechanism, the field of KMnO4 activation remains relatively unexplored. Interestingly, this study has discovered that Mn oxides with high oxidation states including γ-MnOOH, α-Mn2O3 and α-MnO2, exhibited excellent performance to degrade phenols and antibiotics in the presence of KMnO4. The MnO4- species initially formed stable complexes with the surface Mn(III/IV) species and showed an increased oxidation potential and electron transfer reactivity, caused by the electron-withdrawing capacity of the Mn species acting as Lewis acids. Conversely, for MnO and γ-Mn3O4 with Mn(II) species, they reacted with KMnO4 to produce cMnO2 with very low activity for phenol degradation. The direct electron transfer mechanism in α-MnO2/KMnO4 system was further confirmed through the inhibiting effect of acetonitrile and the galvanic oxidation process. Moreover, the adaptability and reusability of α-MnO2 in complicated waters indicated its potential for application in water treatment. Overall, the findings shed light on the development of Mn-based catalysts for organic pollutants degradation via KMnO4 activation and understanding of the surface-promoted mechanism.

Hypoglycemic Coma Caused by Methimazole-Induced Insulin Autoimmune Syndrome, a Case Study.

Islet autoimmune syndrome (IAS) is an autoimmune disease caused by high concentrations of insulin autoantibodies (IAA) in the blood. It is characterized by hyperinsulinemia and spontaneous hypoglycemia. The incidence of IAS is low, and the hypoglycemia symptom is usually mild. Hence, the severe manifestations (up to seizures and coma) are rarely reported. Here, we reported two cases of Graves' disease who developed insulin autoimmune syndrome after methimazole treatment. The patients exhibited sudden hypoglycemic coma after receiving methimazole treatment for approximately 2 or 6 months. The patients' serum glucose levels were below 2.8 mmol/L, and laboratory tests showed high levels of serum insulin and high titers of insulin autoantibodies. Patient 1 discontinued methimazole treatment and the hypoglycemic symptoms disappeared after 7 days. However, patient 2 experienced severe hypoglycemia after discontinuation of methimazole, and the patient condition improved after glucocorticoid therapy. He developed thyroid storm during the treatment, and his condition improved after receiving standard treatment procedures for thyroid storm. To the best of our knowledge, this is the first case report of IAS in Graves' disease with thyroid storm.

Poly(ADP-ribose) polymerase family member 14 promotes functional recovery after spinal cord injury through regulating microglia M1/M2 polarization via STAT1/6 pathway.

Poly(ADP-ribose)polymerase family member 14 (PARP14), which is an intracellular mono(ADP-ribosyl) transferase, has been reported to promote post-stroke functional recovery, but its role in spinal cord injury (SCI) remains unclear. To investigate this, a T10 spinal cord contusion model was established in C57BL/6 mice, and immediately after the injury PARP14 shRNA-carrying lentivirus was injected 1 mm from the injury site to silence PARP14 expression. We found that PARP14 was up-regulated in the injured spinal cord and that lentivirus-mediated downregulation of PARP14 aggravated functional impairment after injury, accompanied by obvious neuronal apoptosis, severe neuroinflammation, and slight bone loss. Furthermore, PARP14 levels were elevated in microglia after SCI, PARP14 knockdown activated microglia in the spinal cord and promoted a shift from M2-polarized microglia (anti-inflammatory phenotype) to M1-polarized microglia (pro-inflammatory phenotype) that may have been mediated by the signal transducers and activators of transcription (STAT) 1/6 pathway. Next, microglia M1 and M2 polarization were induced in vitro using lipopolysaccharide/interferon-γ and interleukin-4, respectively. The results showed that PARP14 knockdown promoted microglia M1 polarization, accompanied by activation of the STAT1 pathway. In addition, PARP14 overexpression made microglia more prone to M2 polarization and further activated the STAT6 pathway. In conclusion, these findings suggest that PARP14 may improve functional recovery after SCI by regulating the phenotypic transformation of microglia via the STAT1/6 pathway.

Association of maternal pre-pregnancy dietary intake with adverse maternal and neonatal outcomes: A systematic review and meta-analysis of prospective studies.

This study aimed to summarize the evidence regarding the effects of dietary intake before conception on pregnancy outcomes by performing a systematic review and meta-analysis of prospective studies. Electronic databases were searched from inception up to August 2021. Overall, 65 studies involving 831 798 participants were included and 38 studies were quantitatively pooled. With regard to maternal outcomes, pre-pregnancy intake of fried food, fast food, red and processed meat, heme iron and a low-carbohydrate dietary pattern was positively associated with the risk of gestational diabetes mellitus (GDM) (all P < 0.05). However, a high dietary fiber intake and folic acid supplementation were negatively associated with GDM risk (both P < 0.05). With regard to neonatal outcomes, maternal caffeine intake before pregnancy significantly increased the risk of spontaneous abortion, while folic acid supplementation had protective effects on total adverse neonatal outcomes, preterm birth, and small-for-gestational age (SGA, all P < 0.05). However, no significant associations were found between adverse pregnancy outcomes (i.e., GDM and SGA) and the pre-pregnancy dietary intake of sugar-sweetened beverages, potato, fish, and carbohydrates and the Healthy Eating Index. Our study suggests that maintaining a healthy diet before conception has significant beneficial effects on pregnancy outcomes.Supplemental data for this article is available online at https://doi.org/10.1080/10408398.2021.1989658.

Modified birnessite MnO2 as efficient Fenton-like catalysts through electron transfer process between the simultaneously surface-activated peroxymonosulfate and pollutants.

The development of efficient and eco-friendly Mn-based hybrids for the degradation of biorefractory organic pollutants via peroxymonosulfate (PMS) activation is highly desired. In this study, a novel graphite nanosheet (GNs)-based Fe-Mn bimetallic oxide (Fe doped birnessite MnO2, FeMn/GNs) was synthesized under mild conditions. Compared with monometallic Fe or Mn oxide on GNs, FeMn/GNs exhibited a higher surface area, decreased Mn oxidation states, stronger interaction with GNs, and more active sites for PMS adsorption. Among different Fe/Mn ratios, Fe2Mn1/GNs showed the optimum performance for bisphenol A (BPA) degradation with the first-order rate constant of 0.22 min-1, which was about 8.5 and 12.9 times higher than that of Mn/GNs and Fe/GNs, respectively. Different from the pollutant-catalyst-PMS electron transfer mechanism for Mn/GNs, the direct two-electron transfer in FeMn/GNs+PMS system, was mainly processed between the simultaneously activated BPA and PMS. This was probably based on the double adsorption sites of Fe and Mn species on the same catalyst: PMS was adsorbed by Fe species through hydroxyl groups, while BPA was mainly coordinated with Mn species due to the layered structure and hydrophobicity of the Mn oxide. This study is expected to provide the rational design of efficient Mn-based hybrids for PMS activation.

Activation of sulfite by metal-organic framework-derived cobalt nanoparticles for organic pollutants removal.

Sulfite (SO32-) activation is one of the most potential sulfate-radical-based advanced oxidation processes, and the catalysts with high efficiency and low-cost are greatly desired. In this study, the cobalt nanoparticles embedded in nitrogen-doped graphite layers (Co@NC), were used to activate SO32- for removal of Methyl Orange in aqueous solution. The Co@NC catalysts were synthesized via pyrolysis of Co2+-based metal-organic framework (Co-MOF), where CoO was firstly formed at 400℃ and then partially reduced to Co nanoparticles embedded in carbon layers at 800℃. The Co@NC catalysts were more active than other cobalt-based catalysts such as Co2+, Co3O4 and CoFe2O4, due to the synergistic effect of metallic Co and CoxOy. A series of chain reaction between Co species and dissolved oxygen was established, with the production and transformation of SO3•-, SO52-, and subsequent active radicals SO4•- and HO•. In addition, HCO3- was found to play a key role in the reaction by complexing with Co species on the surface of the catalysts. The results provide a new promising strategy by using the Co@NC catalyst for SO32- oxidation to promote organic pollutants degradation.

Effect of Proprioception and Balance Training Combined with Continuous Nursing on BBS Score and HSS Score of Patients Undergoing Total Knee Arthroplasty.

Background: Total knee arthroplasty is one of the most effective methods for the treatment of end-stage knee osteoarthritis, but 10% of patients still show insufficient function, strength, and mobility. Continuous nursing service plays an important role in the rehabilitation of patients undergoing total knee arthroplasty. For discharged and convalescent patients, the traditional follow-up model cannot solve the nursing problems of discharged patients. How to meet the health needs of discharged patients under the limited nursing resources has become an existing problem. Objective: To explore the effect of proprioception and balance training combined with continuous nursing on Berg balance scale (BBS) score and Hospital for Special Surgery (HSS) score of patients undergoing total knee arthroplasty (TKA) is the objective of this study. Methods: Sixty patients undergoing TKA in our hospital from December 2019 to April 2021 were enrolled. The patients were randomly assigned into the control group and the study group. The control group received continuous nursing, and the study group received proprioception and balance training combined with continuous nursing. Results: The nursing satisfaction of the study group was higher than that of the control group (P < 0.05). The HSS scores at discharge, 1 month, 3 months, and 6 months after discharge in the study group were higher than those in the control group (P < 0.05). It was higher in the study group than in the control group at 1 month, 3 months, and 6 months after discharge (P < 0.05). The pain catastrophizing score of the study group at discharge was lower than that of the control group at 1 month, 3 months, and 6 months after discharge (P < 0.05). The BBS scores at discharge, 1 month, 3 months, and 6 months after discharge in the study group were higher than those in the control group (P < 0.05). The Lindmark balance scores at discharge, 1 month, 3 months, and 6 months after discharge in the study group were lower than those in the control group (P < 0.05). Conclusion: Proprioception and balance training combined with continuous nursing can effectively promote the recovery of knee joint function of patients after TKA, enhance patients' pain catastrophizing grade, enhance patients' quality of life, effectively promote patients' knee joint function and BBS score, and promote the improvement of disease.

Different frequencies of repetitive transcranial magnetic stimulation combined with local injection of botulinum toxin type A for post-stroke lower limb spasticity: study protocol for a prospective, single-center, non-randomized, controlled clinical trial.

No definite consensus has currently been reached regarding the safety and efficacy of low- or high-frequency repetitive transcranial magnetic stimulation in the treatment of post-stroke muscle spasticity. The latest research indicates that when combined with local injections of botulinum toxin type A, it is more effective on post-stroke muscle spasticity than local injections of botulinum toxin type A alone. We designed a prospective, single-center, non-randomized, controlled clinical trial to investigate the safety and efficacy of different frequencies of repetitive transcranial magnetic stimulation combined with local injections of botulinum toxin type A in treating post-stroke lower limb muscle spasticity to determine an optimal therapeutic regimen. This trial will enroll 150 patients with post-stroke muscle spasticity admitted to the Department of Rehabilitation Medicine at the First Affiliated Hospital of China Medical University. All enrolled patients will undergo routine rehabilitation training and will be divided into five groups (n = 30 per group) according to the particular area of cerebral infarction and treatment methods. Group A: Patients with massive cerebral infarction will be given local injections of botulinum toxin type A and low-frequency (1 Hz) repetitive transcranial magnetic stimulation on the contralateral side; Group B: Patients with non-massive cerebral infarction will be given local injections of botulinum toxin type A and high-frequency (10-20 Hz) repetitive transcranial magnetic stimulation on the affected side; Group C: Patients with massive/non-massive cerebral infarction will be given local injections of botulinum toxin type A; Group D: Patients with massive cerebral infarction will be given low-frequency (1 Hz) repetitive transcranial magnetic stimulation on the contralateral side; and Group E: Patients with non-massive cerebral infarction will be given high-frequency (10-20 Hz) repetitive transcranial magnetic stimulation on the affected side. The primary outcome measure of this trial is a modified Ashworth scale score from 1 day before treatment to 12 months after treatment. Secondary outcome measures include Fugl-Meyer Assessment of Lower Extremity, Visual Analogue Scale, modified Barthel index, and Berg Balance Scale scores for the same time as specified for primary outcome measures. The safety indicator is the incidence of adverse events at 3-12 months after treatment. We hope to draw a definite conclusion on whether there are differences in the safety and efficacy of low- or high-frequency repetitive transcranial magnetic stimulation combined with botulinum toxin type A injections in the treatment of patients with post-stroke lower limb spasticity under strict grouping and standardized operation, thereby screening out the optimal therapeutic regimen. The study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of China Medical University (approval No. [2021] 2021-333-3) on August 19, 2021. The trial was registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR2100052180) on October 21, 2021. The protocol version is 1.1.

Geriatric nutritional risk index as a prognostic factor in patients with hepatocellular carcinoma following transarterial chemoembolization: A retrospective study.

The geriatric nutritional risk index (GNRI) has been shown to be associated with the prognosis of cancer patients except for hepatocellular carcinoma (HCC) patients after transarterial chemoembolization (TACE). Our aim is to examine the association between the GNRI and long-term prognosis in patients with HCC who underwent TACE. Patients with HCC who underwent TACE were enrolled. The relationship between the patient characteristics and GNRI were compared, and the independent prognostic factors were investigated. Nomogram performance was assessed via the concordance index (C-index) and calibration plots. Decision curve analysis (DCA) was performed to evaluate the net benefit of the nomogram. A total of 235 patients met the inclusion criteria. Compared with the parameters of the high GNRI group, low GNRI was significantly associated with hypertension, ascites, body mass index, tumor size, anemia, Child-Turcotte-Pugh class. The univariate analysis demonstrated that overall survival (OS) was inferior when GNRI < 98, tumor size ≥ 5cm, vascular invasion, alpha-fetoprotein level ≥ 400, Barcelona clinical liver cancer stage B to C and TACE times < 3. The multivariate analysis revealed that GNRI < 98, tumor size ≥ 5cm, tumor number ≥ 2, alpha-fetoprotein level ≥ 400 and TACE times < 3 were independent predictors of a poor OS. In the validation step, OS was shown to be well calibrated (C-index = 0.724), and a satisfactory clinical utility was proven by DCA. Low GNRI score was associated with a shorter OS in patients undergoing TACE.

Thiosulfate enhanced degradation of organic pollutants in aqueous solution with g-C3N4 under visible light irradiation.

Developing new strategies to design more practicable and efficient g-C3N4 based photocatalysts is important to solve the environmental issues. Thiosulfate (STS) is a common residual product found in wastewater and removal of STS remains a matter of great environmental concern. In this work, however, STS is activated by g-C3N4 under visible light irradiation, resulting in a fast degradation of Rhodamine B (RhB) and other pollutants. The performance of g-C3N4 prepared from urea was much higher than that from melamine, due to the higher surface area and more negative conduction band potential of the former catalyst. In addition, comparison with other oxidants and reductants such as peroxymonosulfate, peroxydisulfate, hydrogen peroxide and sulfite, the use of STS in g-C3N4/Vis system showed the highest efficiency for RhB degradation. During ten successive cycles, the excellent reusability of the catalyst was also obtained. The effect of different concentrations of STS and g-C3N4, and initial solution pH on the performance of the system were also studied. The mechanism study suggests that STS is first oxidized to S2O3- radicals by photohole, which will be transformed to other oxysulfur radicals such as SO3- and finally to SO42- ions. At the same time, the rate of O2 reduction by photoelectrons to O2- radicals as well as RhB degradation increases. The finding of this study provides a promising advanced oxidation process for organic pollutants degradation via STS activation.

An improved passive shimming approach to design correction iron pieces for high field MRI.

This paper proposes a new passive shimming method to design correction iron pieces for compensating field impurities generated by the main magnets of high field magnetic resonance imaging (MRI) for human body imaging. First, the relationship formula of a magnetic dipole to any field point in space is established. Then, a sensitivity matrix in the form of spherical harmonic expansions can be set up based on the contribution of each shim piece to each field point. Next, an optimization procedure of linear programming is applied to determine the location and thickness of the ferromagnetic shim pieces. This is different from the previous methods that consider that all magnetic moments for each magnetized iron piece are located only at its central position, which may cause some intrinsic errors. This method takes the shim piece's volume into account and gives more accurate results in a sensitivity matrix by means of integration in the azimuthal and axial directions while the radial (thickness) direction remains constant. Finally, a case study combining the analytical method and a 3D finite element analysis simulation demonstrates that the new approach provides better results in terms of homogeneity. The method presented can also be used to design passive shims for various practical MRI applications.

Research hotspots and effectiveness of repetitive transcranial magnetic stimulation in stroke rehabilitation.

Repetitive transcranial magnetic stimulation, as a relatively new type of rehabilitation treatment, is a painless and non-invasive method for altering brain excitability. Repetitive transcranial magnetic stimulation has been widely used in the neurorehabilitation of stroke patients. Here, we used CiteSpace software to visually analyze 315 studies concerning repetitive transcranial magnetic stimulation for stroke rehabilitation from 1999 to 2019, indexed by Web of Science, to clarify the research hotspots in different periods and characterize the gradual process of discovery in this field. We found that four main points were generally accepted: (1) repetitive transcranial magnetic stimulation has a positive effect on motor function recovery in patients with subcortical stroke; (2) it may be more advantageous for stroke patients to receive low-frequency repetitive transcranial magnetic stimulation in the unaffected hemispheres than to receive high-frequency repetitive transcranial magnetic stimulation in affected hemisphere; (3) low-frequency repetitive transcranial magnetic stimulation has become a potential therapeutic tool for patients with non-fluent aphasia after chronic stroke for neurological rehabilitation and language recovery; and (4) there are some limitations to these classic clinical studies, such as small sample size and low test efficiency. Our assessment indicates that prospective, multi-center, large-sample, randomized controlled clinical trials are still needed to further verify the effectiveness of various repetitive transcranial magnetic stimulation programs for the rehabilitation of stroke patients.

Ipr1 Regulation by Cyclic GMP-AMP Synthase/Interferon Regulatory Factor 3 and Modulation of Irgm1 Expression via p53.

Intracellular pathogen resistance 1 (Ipr1) has been found to be a mediator to integrate cyclic GMP-AMP synthase (cGAS)-interferon regulatory factor 3 (IRF3), activated by intracellular pathogens, with the p53 pathway. Previous studies have shown the process of Ipr1 induction by various immune reactions, including intracellular bacterial and viral infections. The present study demonstrated that Ipr1 is regulated by the cGAS-IRF3 pathway during pathogenic infection. IRF3 was found to regulate Ipr1 expression by directly binding the interferon-stimulated response element motif of the Ipr1 promoter. Knockdown of Ipr1 decreased the expression of immunity-related GTPase family M member 1 (Irgm1), which plays critical roles in autophagy initiation. Irgm1 promoter characterization revealed a p53 motif in front of the transcription start site. P53 was found to participate in regulation of Irgm1 expression and IPR1-related effects on P53 stability by affecting interactions between ribosomal protein L11 (RPL11) and transformed mouse 3T3 cell double minute 2 (MDM2). Our results indicate that Ipr1 integrates cGAS-IRF3 with p53-modulated Irgm1 expression.

Activation of cannabinoid receptor type 2-induced osteogenic differentiation involves autophagy induction and p62-mediated Nrf2 deactivation.

BACKGROUND: Dysfunction in survival and differentiation of osteoblasts commonly occurs in patients with osteoporosis. Cannabinoid receptor type 2 (CNR2) is a major receptor of endocannabinoid system that is crucial for bone mass homeostasis. Our group prior demonstrated that activation of CNR2 signaling promoted osteogenic differentiation of bone marrow derived mesenchymal stem cells in vitro. Autophagy is reported to participate in osteoblastic differentiation. Whether autophagy is regulated by CNR2-mediated cannabinoid signaling is unknown, and how the autophagy-CNR2 interaction affects osteoblastic differentiation requires further elucidation. METHODS: hFOB 1.19 osteoblasts were treated with CNR2 agonists HU308 (5, 10, 25, 50 or 100 nM) and JWH133 (1, 2, 5, 10 or 20 µM) in presence or absence of autophagy inhibitor 3-Methyladenine (3-MA). The differentiation of hFOB 1.19 cells was determined via evaluating their alkaline phosphatase (ALP) activity and mineralization ability (Alizarin red staining). Alterations in autophagy-related molecules and osteogenic markers were analyzed via real-time PCR and/or immunoblotting assays. RESULTS: hFOB 1.19 cells spontaneously differentiated towards mature osteoblasts under 39 °C, during which CNR2 expression increased, and autophagy was activated. The strongest autophagy flux was observed at 192 h post differentiation─LC3I to LC3II conversion was enhanced and Beclin 1 expression was upregulated considerably, while p62 expression was downregulated. Treatment of HU308 and JWH133 promoted autophagy in a dose-dependent manner, and suppressed mTOR signaling pathway in hFOB 1.19 cells. In CNR2-silenced cells, HU308's and JWH133's effects on autophagy were weakened. HU308 and JWH133 enhanced the ALP activity and mineralization, and upregulated the expression of osteogenic markers, osteopontin and osteocalcin, in hFOB 1.19 cells. Intriguingly, such pro-osteogenic effects induced by CNR2 activation were markedly mitigated by 3-MA. In addition to provoking autophagy, CNR2 agonists also reduced nuclear Nrf2 accumulation and increased Keap1 expression. Further, re-expression of p62 inhibited CNR2 agonists-induced Nrf2 degradation. CONCLUSIONS: Osteogenic differentiation induced by CNR2 signaling activation involves autophagy induction and p62-mediated Nrf2 deactivation.