Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis.

Diabetes mellitus (DM) affects bone metabolism and leads to osteoporosis; however, its pathogenetic mechanisms remain unknown. We found that high glucose (HG) conditions induced the production of reactive oxygen species (ROS) and the expression of proteins related to MAPKs [phosphorylated (p)-ERK, p-JNK, and p-p38], NF-κB (NF-κB, p-IκB, and IKK), and NACHT-LRR-PYD domains-containing protein 3 (NALP3) (NLRP3) [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), caspase-1, IL-18, IL-1ß, and NLRP3] in osteoclasts (OCs) in vitro. Further analysis showed that in HG-induced OCs, ROS is an upstream signal for MAPKs, NF-κB, and the NLRP3 inflammasome. Moreover, MAPKs mediated the activation of NF-κB and NLRP3, whereas NF-κB up-regulated the NLRP3 inflammasome response. Interestingly, HG inducement enhanced the bone resorption of OCs but inhibited their efferocytosis, whereas insulin and lipoxin A4 (4) treatment reversed this phenomenon. In streptozotocin-induced diabetic rats in vivo, the numbers and the bone-resorption capacity of OCs as well as the serum levels of TRACP-5b were significantly increased, and the expression of MAPK-, NF-κB-, and NLRP3 inflammasome-related proteins in the proximal tibia were also significantly elevated; however, treatment with insulin and LXA4 reversed this elevation. Together, these results demonstrated that the activation of ROS/MAPKs/NF-κB/NLRP3 and the inhibition of efferocytosis in OCs are the main causes of osteoporosis in DM.-An, Y., Zhang, H., Wang, C., Jiao, F., Xu, H., Wang, X., Luan, W., Ma, F., Ni, L., Tang, X., Liu, M., Guo, W., Yu, L. Activation of ROS/MAPKs/NF-κB/NLRP3 and inhibition of efferocytosis in osteoclast-mediated diabetic osteoporosis.

Effects of CwlM on autolysis and biofilm formation in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Tuberculosis is a highly infectious disease and of high incidence in low-income countries that is caused by Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis can form biofilms in vitro and in vivo, and the cells in the biofilm can survive at high concentrations of antibiotics. CwlM is a peptidoglycan hydrolase (amidase) and can hydrolyze bacterial cell walls, and the effects of CwlM on autolysis and biofilms is worthy of in-depth study. In this study, we successfully constructed an in vitro biofilm model of M. tuberculosis and Mycobacterium smegmatis (M. smegmatis). Reverse transcription followed by real-time quantitative PCR (qPCR) revealed that the expression of cwlM in M. tuberculosis and M. smegmatis was significantly up-regulated during the middle stage of biofilm formation. Treatment with recombinant CwlM enhanced the autolytic ability of M. tuberculosis and M. smegmatis and reduced the formation of their biofilms. As M. smegmatis is a model bacterium of M. tuberculosis, we built the M. smegmatis cwlM-deletion strain MSΔ6935, whose autolytic ability, biofilm production, and eDNA and eRNA content were determined to be lower than those of its parental strain. In conclusion, the cwlM gene plays a key regulatory role in biofilm formation in M. tuberculosis and M. smegmatis. This study provided a theoretical basis for using peptidoglycan hydrolase as a target for the inhibition of biofilms.

Comparative proteomics analysis between biofilm and planktonic cells of Mycobacterium tuberculosis.

Tuberculosis is highly persistent and displays phenotypic resistance to high concentrations of antimicrobials. Recent reports exhibited that Mycobacterium tuberculosis biofilm was implicated to its pathogenicity and drug resistance. In this study, there were 47 kinds of differential proteins in the biofilm of M. tuberculosis H37Rv cells compared with the planktonic bacteria, and 37 proteins were nonredundant and identified by proteomics approach, such as 2DE and LC-MS/MS. Moreover, six kinds of proteins were identified as HspX, which were conservative and highly expressed in biofilm. Note that 47 differential proteins were divided into seven categories, such as cell wall and cell processes, conserved hypotheticals, intermediary metabolism and respiration, and so on by TUBERCULIST. The Gene Ontology classification results showed that the largest protein group involved in metabolism, binding proteins, and catalytic function accounts for 30% and 57% of all identified proteins, respectively. Moreover, the protein interaction network analyzed by STRING showed that the minority proteins such as RpoA, SucC, Cbs, Tuf, DnaK, and GroeL in the interaction network have high network connectivity. These results implied that the proteins involved in metabolic process and catalytic function and the minority proteins mentioned above may play an important role in M. tuberculosis biofilm formation. To our knowledge, this is the first report about differential proteins between biofilm and planktonic M. tuberculosis, which provided the potential antigens for vaccines and target proteins for anti-mycobacterial drugs.

NAFLD Induction Delays Postoperative Liver Regeneration of ALPPS in Rats.

BACKGROUND: Associating liver partition and portal vein ligation (ALPPS) is a promising two-step hepatectomy that is beneficial for accumulative regeneration of the future liver remnant (FLR) and avoids postoperative liver failure. AIMS: Our study aimed to evaluate whether nonalcoholic fatty liver disease affected the liver regeneration induced by ALPPS. METHODS: Sprague-Dawley rats fed a high-fat diet were used to construct the NAFLD model. ALPPS were performed, and blood and future liver remnant samples were collected at postoperative days 1 (POD1), POD3, and POD7. RESULTS: The hepatic regeneration rate (HRR) of ALPPS was higher than that of portal vein ligation (PVL) at POD3 and POD7 (p < 0.05), and the number of Ki-67-positive hepatocytes (POD3) and CD68-positive Kupffer cells (POD7) per visual field was higher in the ALPPS group than in the PVL group (p < 0.05). The serum TNF-α, hepatocyte growth factor protein, and the serum IL-6 level were higher in the ALPPS group than in the PVL group at POD3 and POD7. Compared with those of the standard laboratory diet (SLD)-fed rats, the rats with NAFLD exhibited a decrease in the HRR, Ki-67-positive hepatocytes, and CD68-positive Kupffer cells in the FLR. The number of CD68-positive Kupffer cells was lower in rats with NAFLD than that in SLD-fed rats; noteworthily, the serum level of IL-6 and TNF-α changed dramatically after surgeries. CONCLUSIONS: NAFLD induction delayed liver regeneration induced by the ALPPS procedure, which might be associated with hepatocyte proliferation and the number of Kupffer cells.

Cordycepin reduces weight through regulating gut microbiota in high-fat diet-induced obese rats.

BACKGROUND: An increasing number of studies have shown that obesity is the key etiological agent of cardiovascular diseases, nonalcoholic fatty liver disease, type 2 diabetes and several kinds of cancer and that gut microbiota change was one of the reasons suffering from obesity. At present, the gut microbiota has gained increased attention as a potential energy metabolism organ. Our recent study reported that cordycepin, a major bioactive component separated from Cordyceps militaris, prevented body weight gain in mice fed a high-fat diet directly acting to adipocytes, however, the effect of cordycepin regulating gut microbiota keeps unknown. METHODS: In this research, we synthesized cordycepin (3-deoxyadenosine) by chemical methods and verified that cordycepin reduces body weight gain and fat accumulation around the epididymis and the kidneys of rats fed a high-fat diet. Furthermore, we used high-throughput sequencing on a MiSeq Illumina platform to test the species of intestinal bacteria in high-fat-diet-induced obese rats. RESULTS: We found that cordycepin modifies the relative abundance of intestinal bacteria in high-fat-diet-induced obese rats. However, cordycepin did not alter the variety of bacteria in the intestine. Cordycepin treatment dramatically reversed the relative abundance of two dominant bacterial phyla (Bacteroidetes and Firmicutes) in the high-fat-diet-induced obese rats, resulting in abundance similar to that of the chow diet group. CONCLUSION: Our study suggests that cordycepin can reduce body weight and microbiome done by cordycepin seems be a result among its mechanisms of obesity reduction.

Study on the Mechanism of the Combination of Methotrexate and Leflunomide in the Treatment of Rheumatoid Arthritis Based on Network Pharmacology, Molecular Docking, and in vitro Experimental Verification.

BACKGROUND: To date, disease-modifying antirheumatic drugs (DMARDs) are widely used as the primary first-line treatment option for patients with rheumatoid arthritis (RA), and the curative effect of methotrexate (MTX) and leflunomide (LEF; MTX + LEF) is greater than that of single-agent MTX therapy, but the synergistic mechanism of MTX + LEF is unclear. METHODS: First, we explored the mechanism of action of MTX + LEF in RA through network pharmacology and molecular docking. Venn diagram analysis revealed 97 overlapping gene targets of MTX + LEF-RA and STRING, along with Cytoscape plug-in MOCDE and cytoHubba; and GO enrichment analysis revealed that the functions of 97 synergistic targets were related to 123 molecular functions (MF), 63 cell components (CC), and 1,068 biological processes (BP). The Cytoscape plug-in ClueGO demonstrated that these targets were enriched in KEGG pathways of 52 terms, whereas 9 pivotal genes were mainly involved in the signaling pathways of estrogen, Ras, Rap1, PI3K-Akt, relaxin, TNF, AMPK, FoxO, prolactin, IL-17, and adherens junction. Finally, CETSA and DARTS validated the direct binding of MTX or LEF to the selected target proteins EGFR, PPARG, MMP9, and SRC in RAW264.7 cells. RESULTS: We identified 292 MTX targets and 247 LEF targets from 7 databases. Furthermore, 2,814 potential targets of RA were identified by merging 1,925 targets from 7 databases and 999 differentially expressed genes (DEGs) between normal controls and patients with RA extracted from 5 GEO databases. Nine pivotal genes, ESR1, ALB, CASP3, EGFR, HSP90AA1, SRC, MMP9, PPARG, and IGF1, were identified. Molecular docking verified that both MTX and LEF strongly bind to most of the 9 pivotal proteins except ESR1 and IGF1. CONCLUSION: These results contribute to our understanding of the enhancement mechanism of MTX combined with LEF and provide a targeted basis for the clinical treatment of RA.

[Prognosis analysis of multi-indicator combined with sequential organ failure assessment in patients with sepsis].

OBJECTIVE: To explore the prognostic value of early multiple detection indicators in combination with sequential organ failure assessment (SOFA) in sepsis patients. METHODS: A retrospective analysis was conducted. Patients with sepsis admitted to the department of critical care medicine of Huanggang Central Hospital of Yangtze University from May 2020 to May 2022 were selected as the research subjects. Coagulation indicators, inflammatory factors, blood routine, liver and kidney function, and blood gas analysis were collected at admission. Organ dysfunction was assessed based on the SOFA score within 24 hours after admission. Patients were divided into a survival group and a death group according to the outcome of 28 days in ICU. Differences in the above indicators between the two groups were compared. Multifactorial Logistic regression analysis was used to analyze prognostic factors of 28-day mortality in sepsis patients. Receiver operator characteristic curve (ROC curve) was drawn to analyze the predictive performance of various indicators, the SOFA score, and the combine model for the 28-day outcome in patients with sepsis. RESULTS: A total of 101 patients with sepsis were enrolled, 56 patients survived and 45 patients died. Compared to the survival group, patients in the death group were older, the proportion of patients with septic shock was larger, the SOFA score, and the proportion of pulmonary infection were higher, the prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly prolonged, the prothrombin activity (PTA) was significantly shortened, and antithrombin (AT) was significantly decreased, the levels of hypersensitivity C-reactive protein (hs-CRP), blood urea nitrogen (BUN), total bilirubin (TBil), and lactic acid (Lac) were significantly increased, while the platelet count (PLT) was significantly decreased. Multifactorial Logistic regression analysis showed that pulmonary infection [odds ratio (OR) = 0.010, 95% confidence interval (95%CI) was 0.001-0.164, P = 0.001], AT (OR = 0.944, 95%CI was 0.910-0.978, P = 0.002), hs-CRP (OR = 1.008, 95%CI was 1.001-1.015, P = 0.017), Lac (OR = 1.619, 95%CI was 1.195-2.193, P = 0.002), and SOFA score (OR = 1.363, 95%CI was 1.076-1.727, P = 0.010) were independent prognostic factors for 28-day mortality in patients. A combined model was constructed using pulmonary infection, AT, hs-CRP, Lac, and SOFA score. ROC curve analysis showed that the area under the ROC curve (AUC) for the combine model in predicting sepsis prognosis was 0.936 (95%CI was 0.869-0.975, P < 0.001), which was higher in value compared to single indicators (AUC of AT, hs-CRP, Lac, and SOFA score were 0.775, 0.666, 0.802, 0.796, respectively, all P < 0.05). CONCLUSIONS: The predictive ability of the SOFA score for sepsis patient outcomes is limited. The combine model combining infection site, AT, hs-CRP, and Lac shows better predictive ability.

The combination of insulin and linezolid ameliorates Staphylococcus aureus pneumonia in individuals with diabetes via the TLR2/MAPKs/NLRP3 pathway.

Diabetes mellitus (DM) complicated with Staphylococcus aureus (S. aureus) infection lacks effective treatment strategies. In this study, we found that insulin combined with linezolid has potential to deal with the thorny problem. In vitro, our drug sensitivity assay, bacterial growth curve and hemolytic tests showed that a combination of insulin and linezolid exerted good antibacterial and anti-α-hemolysin activity, CCK8 experiment, glucose content and glycogen content determination showed that the combination of insulin and linezolid increased murine macrophage survival rate and reduced the extracellular glucose level of high glucose-treated MH-S cells and intracellular glycogen level, and Western blot showed that the combination inhibited TLR2/MAPKs/NLRP3-related inflammatory pathways in MH-S cells. The results of in vivo experiments showed that the combination therapy stabilized glucose level, remained body weight, ameliorated lung injury including improving pulmonary edema and decreasing lung wet/dry weight ratio, reduced the CFUs and inflammation in the lung tissue in a mouse model of diabetes with S. aureus pneumonia, and inhibited the expression of TLR2, MAPKs and NLRP3 inflammatory pathway. Overall, the combination of insulin and linezolid as autolytic inhibitor exhibited the effects of significant antibacterial and improving glucose level in vitro and in vivo, and also has an anti-inflammation activity via the TLR2/MAPKs/NLRP3 pathway, this paves the way for new treatments for diabetes mellitus complicated with S. aureus infection.

Corosolic acid inhibits tumour growth without compromising associating liver partition and portal vein ligation-induced liver regeneration in rats.

BACKGROUND: Associating liver partition and portal vein ligation (ALPPS) technique is a promising strategy for unresectable primary liver tumours without sufficient future liver remnants (FLRs). OBJECTIVE: Our study explored the effect of corosolic acid (CA) on inhibiting tumour growth without compromising ALPPS-induced liver regeneration. METHODS: The ALPPS procedure was performed in Sprague-Dawley rats with orthotopic liver cancer. Blood, tumour, and FLR samples were collected, and the effect of CA on the inhibition of tumour progression and ALPPS-induced liver regeneration, and its possible mechanism, were investigated. RESULTS: The tumour weight in the implantation/ALPPS group was higher than in the implantation without ALPPS group (p < .05), and the tumour weight in the implantation/ALPPS/CA group was lower than in the implantation/ALPPS group (p < .05). On postoperative day 15, the hepatic regeneration rate, and the expression of Ki67+ hepatocytes in the FLRs had increased significantly in the group that underwent ALPPS. The number of cluster of differentiation (CD) 86+ macrophages markedly increased in the FLRs and in the tumours of groups that underwent the ALPPS procedure. Additionally, the number of CD206+ macrophages was higher than the number of CD86+ macrophages in the tumours of the implantation and the implantation/ALPPS groups (p < .01, respectively); however, the opposite results were observed in the CA groups. The administration of CA downregulated the expression of transforming growth factor-beta (TGF-ß), CD31, and programmed cell death protein 1 (PD-1) but increased the number of CD8+ lymphocytes in tumours. CONCLUSION: Corosolic acid inhibits tumour growth without compromising ALPPS-induced liver regeneration. This result may be attributed to the CA-induced downregulation of PD-1 and TGF-ß expression and the increased CD8+ lymphocyte infiltration in tumour tissue associated with the suppression of M2 macrophage polarisation. Key MessagesThis study aimed to investigate the effect of CA on ALPPS-induced liver regeneration and hepatic tumour progression after ALPPS-induced liver regeneration.Corosolic acid inhibits tumour growth without compromising ALPPS-induced liver regeneration. This result may be attributed to the CA-induced downregulation of PD-1 and TGF-ß expression and the increased CD8+ lymphocyte infiltration in tumour tissue associated with the suppression of M2 macrophage polarisation.

The combination of cloxacillin, thioridazine and tetracycline protects mice against Staphylococcus aureus peritonitis by inhibiting α-Hemolysin-induced MAPK/NF-κB/NLRP3 activation.

Staphylococcus aureus (S. aureus) infection is difficult to fight, previous experimental reports have demonstrated thioridazine (TZ) and tetracycline (TC) is an inhibitor of S. aureus efflux pump NorA and autolysin Atl, respectively, here, by means of molecular docking and molecular dynamics simulation, we observed that thioridazine (TZ) and tetracycline (TC) blocked the binding of substrates to NorA and Atl, respectively, and reduced their activities, and our antibacterial susceptibility test and three-dimensional checkerboard method showed that the three-drug combination of antibiotic cloxacillin (CXN), TZ and TC had a synergistic anti-Staphylococcal activity in vitro, and α-Hemolysin tests and scanning electron microscopy showed that the three-drug combination and the subinhibitory concentration of the combination significantly inhibited the secretion of α-hemolysin relative to the number of membrane-derived vesicles produced by S. aureus. Whereas Western blot and pharmacological inhibition assays showed that the three-drug combination significantly inhibited the expression of MAPK/NF-κB/NLRP3 proteins in macrophages induced with S. aureus α-hemolysin. In vivo, the drug combination significantly reduced bacterial colony-forming unit counts in the viscera of a mouse peritonitis model of S. aureus infection, therapy reduced the primary inflammatory pathology and the bacteria-stimulated release of cytokines such as IL-1ß and TNF-α, and inhibited the expression of MAPK/NF-κB/NLRP3 proteins in peritoneal macrophages. Thus, the combination of efflux pump inhibitor, autolysis inhibitor and antibiotic, is a novel anti-Staphylococcal and anti-inflammatory strategy who owning good antibacterial activity and significant inhibiting staphylococcal α-hemolysin and inflammation.

Akt2 mediates glucocorticoid resistance in lymphoid malignancies through FoxO3a/Bim axis and serves as a direct target for resistance reversal.

Glucocorticoids (GCs) are widely used drugs in the treatment of lymphoid malignancies; resistance of GCs in lymphocytes confers poor prognosis and the mechanisms are poorly understood. Here, we found T-acute lymphoblastic leukemia (T-ALL) cells acquire resistance to dexamethasone (DEX)-mediated killing through abnormal activation of Akt, resulting in inhibition of the FoxO3a/Bim pathway. The resistant state was reported to be associated with increased glycolysis, NOTCH1 activating mutations and activated PI3K/ serum GS regulated kinases (SGK) pathway. Use of aforementioned pathway inhibitors blocked FoxO3a-phosphorylation and partially improved DEX-mediated killing of GC-resistant T-ALL cells, further revealing the essential role of the FoxO3a/Bim pathway in the development of GC resistance. Inhibition of Akt is most effective at restoring sensitivity to DEX of GC-resistant lymphocytes in vitro and in vivo, but shows significant hepatotoxicity in vivo. A significantly elevated expression of Akt2 not Akt1 in intrinsically, secondarily GC-resistant lymphocytes and relapsed/refractory ALL patients implicates a more specific target for GC resistance. Mechanistically, Akt2 has a stronger binding capacity with FoxO3a compared to Akt1, and acts as a direct and major negative regulator of FoxO3a activity driving GC resistance. Pharmacologic inhibition of Akt2 more effectively restores sensitivity to GCs than inhibition of Akt1 in vitro, shows higher synergistic effect acting with DEX, and reverses GC resistance in GC-resistant T- or B- lymphoid tumors in vivo with reduced liver toxicity. In summary, these results suggest that Akt2 might serve as a more direct and specific kinase mediating GC resistance through FoxO3a/Bim signaling pathway, and Akt2 inhibition may be explored as a promising target for treating GC-resistant hematopoietic malignancies.

Inhibition of Drug Resistance of Staphylococcus aureus by Efflux Pump Inhibitor and Autolysis Inducer to Strengthen the Antibacterial Activity of ß-lactam Drugs.

This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 - 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 ­ 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.

The TRAPs From Microglial Vesicles Protect Against Listeria Infection in the CNS.

Previous studies have demonstrated that T cells and microglia could fight against cerebral Listeria monocytogenes (Listeria); however, their synergistic anti-Listeria mechanisms remain unknown. Following Listeria infection in a culture system, we found that microglia, but not nerve cells, could release extracellular traps (ETs) which originated from microglial vesicles. Specific inhibitor analysis showed that extracellular DNA (eDNA), matrix metallopeptidases (MMP9 and MMP12), citrullinated histone H3, and peptidyl arginine deiminase 2 were the major components of microglial ETs (MiETs) and were also the components of vesicles. Systematic analysis indicated that Listeria-induced MiETs were cytosolic reactive oxygen species (ROS)- and NADPH oxidase (NOX)-dependent and involved ERK. MiETs were exhibited in Listeria-infected mouse brain and might protected against Listeria infection via bacterial killing in a mouse meningitis model, and MiETs existed in cerebrospinal fluid (CSF) from Listeria meningitis patients in vivo and in vitro. Additionally, interferon-γ could induce MiET formation in Listeria-infected microglia in vitro that was mediated by NOX, and there was a positive relationship between the elevated level of IFN-γ and eDNA and nucleosomes in the brain homogenates and CSF of Listeria meningitis model mice and in the CSF before treatment in clinical Listeria meningitis patients. Together, this is the first report of MiET formation, these findings pave the way for deeper exploration of the innate immune response to pathogens in CNS.

Cordycepin regulates body weight by inhibiting lipid droplet formation, promoting lipolysis and recruiting beige adipocytes.

OBJECTIVE: To explore the effect of cordycepin on reducing lipid droplets in adipocytes. METHODS: Rats were fed a 60% high-fat diet to construct a hyperlipidaemia animal model and then treated with cordycepin at different concentrations for 8 weeks. Adipocytes were extracted, and BODIPY staining was used to detect the size of the lipid droplets. The adipocyte membrane proteins ASC-1, PAT2 and P2RX5 were assessed to determine the transformation of white adipocytes to beige and brown adipocytes. In an in vitro study, 3T3-L1 cells were cultured, and Western blotting was used to determine the expression of the lipid droplet-related genes Fsp27, perilipin 3, perilipin 2, PPAR-γ, Rab5, Rab7, Rab11, perilipin 1, ATGL and CGI-58. RESULTS: We found that cordycepin could promote the transformation of white adipocytes into beige and brown adipocytes. Cordycepin also downregulated the lipid droplet-associated genes Fsp27, perilipin 3, perilipin 2, Rab5, Rab11 and perilipin 1. Moreover, cordycepin reduced the expression of protein CGI-58, which inhibits lipid droplet degradation. In addition, cordycepin significantly increased the expression of ATGL, suggesting that cordycepin might stimulate lipolysis by upregulating the expression of ATGL instead of CGI-58 and by downregulating the expression of perilipin 1. CONCLUSIONS: Cordycepin could blockade lipid droplet formation and promote lipid droplet degradation.

Cordycepin Modulates Body Weight by Reducing Prolactin Via an Adenosine A1 Receptor.

BACKGROUND: Cordycepin is an extract from the insect fungus Cordyceps. militaris with various biological function. In previous studies, cordycepin has demonstrated an excellent anti-obesity effect, but the mechanism is unclear. It was also demonstrated that prolactin played an important role in body weight regulation and hyperprolactinemia can promote appetite and accelerate fat deposition. In this study, we explored the molecular mechanism of the anti-obesity effect of cordycepin. METHODS: In Vivo, the obese rat model was induced by high fat diet for five weeks, and the serum and liver lipid levels coupled with the serum prolactin levels were reduced following cordycepin treatment (P<0.01). RESULTS: The results suggested that cordycepin is a potential drug that lowers blood and liver lipid levels and reduces body weight related to prolactin. Cordycepin also protects adipocytes from enlargement and hepatocytes from lipotoxicity-induced inflammation. In vitro, cordycepin inhibited prolactin secretion in GH3 cells via upregulating the expression of adenosine A1 receptor, and the inhibition effect was blocked by an antagonist of adenosine receptor A1 DPDPX, demonstrating that cordycepin may work as an adenosine agonist. Additionally, cordycepin inhibited the ERK/AKT/PI3K pathway in GH3 cells. At the same time, cordycepin blocked prolactininduced upregulation of lipogenesis genes PRLR, and phosphorylation of JAK2 in 3T3-L1 cells. In an in vivo study, cordycepin downregulated the expression of prolactin receptor (PRLR) but not the phosphorylation of JAK2. CONCLUSION: Thus, it was proved that cordycepin modulates body weight by reducing prolactin release via an adenosine A1 receptor.

Biochanin a Enhances the Defense Against Salmonella enterica Infection Through AMPK/ULK1/mTOR-Mediated Autophagy and Extracellular Traps and Reversing SPI-1-Dependent Macrophage (MΦ) M2 Polarization.

A novel treatment regimen for bacterial infections is the pharmacological enhancement of the host's immune defenses. We demonstrated that biochanin A (BCA), an isoflavone constituent in some plants, could enhance both intra- and extracellular bactericidal activity of host cells. First, BCA could induce a complete autophagic response in nonphagocytic cells (HeLa) or macrophages (MΦ) via the AMPK/ULK1/mTOR pathway and Beclin-1-dependent manner, and BCA enhanced the killing of invading Salmonella by autophagy through reinforcing ubiquitinated adapter protein (LRSAM1, NDP52 and p62)-mediated recognition of intracellular bacteria and through the formation of autophagolysosomes. Second, we demonstrated that BCA could enhance the release of MΦ extracellular traps (METs) to remove extracellular Salmonella also via the AMPK/ULK1/mTOR pathway, not through reactive oxygen species (ROS) pathway. Furtherly, in a Salmonella-infected mouse model, BCA treatment increased intra- and extracellular bactericidal activity through the strengthening autophagy and MET production, respectively, in peritoneal MΦ, liver and spleen tissue. Additionally, our findings showed that BCA downregulated SPI-1 (Salmonella pathogenicity island 1) expression during Salmonella infection in vitro and in vivo to reverse the MΦ M2 polarization, which was different from the MΦ M1 phenotype caused by most of bacteria infection. Together, these findings suggest that BCA has an immunomodulatory effect on Salmonella-infected host cells and enhances their bactericidal activity in vitro and in vivo through autophagy, extracellular traps and regulation of MΦ polarization.