Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Biomechanical evaluation of modified and traditional cortical bone trajectory technique on adjacent segment degeneration in transforaminal lumbar interbody fusion-finite element analysis.

OBJECTIVES: Modified cortical bone trajectory (MCBT) technique was proposed by our team in previous studies, but its biomechanical properties at adjacent segments have not been discussed yet. Therefore, the purpose of this study is to investigate the biomechanical properties of modified cortical bone trajectory (MCBT) technique on adjacent segment degeneration (ASD) in transforaminal intradiscal lumbar disc fusion (TLIF) compare to traditional bone trajectory (TT) technique and cortical bone trajectory (CBT) technique. METHODS: The four human cadaveric lumbar specimens were provided by the anatomy teaching and research department of Xinjiang Medical University and four intact finite element models of the L1-S1 segment were generated. For each of these, three transforaminal lumbar interbody fusion procedures with three different fixation techniques were reconstructed at the L4-L5 segment, as follows: TT-TT (TT at both L4 and L5 segments), CBT-CBT (CBT at both L4 and L5 segments), MCBT-MCBT (MCBT at both L4 and L5 segments). The range of motion and von Mises stress of the intervertebral disc of the L3-L4 and L5-S1 segments were recorded with a 400N compressive load and 7.5 Nm moments in flexion, extension, left-right bending, and left-right rotation. RESULTS: The peak ROM of the L3-L4 segment in the MCBT-MCBT group was reduced by 10.5%, 6.1%, 12.2%, 4.1%, and 1.5% in flexion, extension, left-right bending, and left rotation compared to the TT-TT group and reduced by 1.8%, 5.5%, 10.0%, 12.8%, and 8.8% in flexion, left-right bending, and left-right rotation compared to the CBT-CBT group, respectively. The MCBT-MCBT group has the lowest peak ROM of the L3-L4 segment in flexion, left bending, and right rotation, the lowest peak ROM of the L5-S1 segment in extension and right rotation, and the lowest peak von Mises stress of the intervertebral disc at the L5-S1 segment in right rotation compared to the TT-TT and CBT-CBT group. In addition, the peak von Mises stress at the L3-L4 segment was lowest and more dispersed in all motions, the MCBT-MCBT group exhibited lower peak ROM of the L5-S1 segment in flexion, extension, and right rotation, and showed lower peak von Mises stress of the disc at the L5-S1 segment in flexion, extension, and right rotation compared with the TT-TT group. CONCLUSION: The modified cortical bone trajectory technique may have a beneficial effect on reducing the incidence of ASD in the L4-L5 TLIF model compared to the traditional bone trajectory technique and cortical bone trajectory technique.

Inhibition of Smad3 in macrophages promotes Aß efflux from the brain and thereby ameliorates Alzheimer's pathology.

Impaired amyloid-ß (Aß) clearance is believed to be a primary cause of Alzheimer's disease (AD), and peripheral abnormalities in Aß clearance have recently been linked to AD pathogenesis and progression. Data from recent genome-wide association studies have linked genetic risk factors associated with altered functions of more immune cells to AD pathology. Here, we first identified correlations of Smad3 signaling activation in peripheral macrophages with AD progression and phagocytosis of Aß. Then, manipulating the Smad3 signaling regulated macrophage phagocytosis of Aß and induced switch of macrophage inflammatory phenotypes in our cell cultures. In our mouse models, flag-tagged or fluorescent-dye conjugated Aß was injected into the lateral ventricles or tail veins, and traced. Interestingly, blocking Smad3 signaling efficiently increased Aß clearance by macrophages, reduced Aß in the periphery and thereby enhanced Aß efflux from the brain. Moreover, in our APP/PS1 transgenic AD model mice, Smad3 inhibition significantly attenuated Aß deposition and neuroinflammation, and ameliorated cognitive deficits, probably by enhancing the peripheral clearance of Aß. In conclusion, enhancing Aß clearance by peripheral macrophages through Smad3 inhibition attenuated AD-related pathology and cognitive deficits, which may provide a new perspective for understanding AD and finding novel therapeutic approaches.

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling.

BACKGROUND: Alzheimer's disease (AD) is a major clinical problem, but there is a distinct lack of effective therapeutic drugs for this disease. We investigated the potential therapeutic effects of zerumbone, a subtropical ginger sesquiterpene, in transgenic APP/PS1 mice, rodent models of AD which exhibit cerebral amyloidosis and neuroinflammation. METHODS: The N9 microglial cell line and primary microglial cells were cultured to investigate the effects of zerumbone on microglia. APP/PS1 mice were treated with zerumbone, and non-cognitive and cognitive behavioral impairments were assessed and compared between the treatment and control groups. The animals were then sacrificed, and tissues were collected for further analysis. The potential therapeutic mechanism of zerumbone and the signaling pathways involved were also investigated by RT-PCR, western blot, nitric oxide detection, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and flow cytometry analysis. RESULTS: Zerumbone suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype from the classic inflammatory phenotype to the alternative anti-inflammatory phenotype by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B signaling pathway in vitro. After a treatment period of 20 days, zerumbone significantly ameliorated deficits in both non-cognitive and cognitive behaviors in transgenic APP/PS1 mice. Zerumbone significantly reduced ß-amyloid deposition and attenuated pro-inflammatory microglial activation in the cortex and hippocampus. Interestingly, zerumbone significantly increased the proportion of anti-inflammatory microglia among all activated microglia, potentially contributing to reduced ß-amyloid deposition by enhancing phagocytosis. Meanwhile, zerumbone also reduced the expression of key molecules of the MAPK pathway, such as p38 and extracellular signal-regulated kinase. CONCLUSIONS: Overall, zerumbone effectively ameliorated behavioral impairments, attenuated neuroinflammation, and reduced ß-amyloid deposition in transgenic APP/PS1 mice. Zerumbone exhibited substantial anti-inflammatory activity in microglial cells and induced a phenotypic switch in microglia from the pro-inflammatory phenotype to the anti-inflammatory phenotype by inhibiting the MAPK signaling pathway, which may play an important role in its neuroprotective effects. Our results suggest that zerumbone is a potential therapeutic agent for human neuroinflammatory and neurodegenerative diseases, in particular AD.

Zaocys type II collagen regulates the balance of Treg/Th17 cells in mice with collagen-induced arthritis.

OBJECTIVES: Zaocys type II collagen is an active collagen extracted from Zaocys that has been used to treat rheumatoid arthritis in China for over 1000 years. However, the mechanism still remains unknown. Therefore, we set out to investigate the inhibitory effect and possible mechanism of action of zaocys type II collagen on collagen-induced arthritis. METHODS: Collagen-induced arthritis was induced in C57BL/6 mice by immunisation with type II collagen. After immunisation, the mice were treated with Zaocys type II collagen. Clinical and histological scores were assessed and the cytokine levels in the serum and lymphocytes supernatant from the spleen and mesenteric lymph node were determined by enzyme-linked immune sorbent assay. The T-helper 17 cell and regulatory-T cell frequencies were analysed by flow cytometry and the expression of interest markers was examined by direct immuno-fluorescence. RESULTS: The arthritis score and severity of histological inflammation and cartilage destruction were dose-dependently reduced after treatment. The analysis results indicated that Zaocys type II collagen significantly increased the proportion of regulatory-T cells and lowered the T-helper 17 cells, it also increased the number of regulatory-T cells and conversely decreased the T-helper 17 cells in synovial tissue compared with the model group. Treatment also caused a higher level of transforming growth factor-ß and a decreased production of interleukin -17A. CONCLUSIONS: The oral administration of Zaocys type II collagen potently suppressed the severity of collagen-induced arthritis by repairing the imbalance between regulatory-T cells and T-helper 17 cells, suggesting that it might be a promising candidate for the treatment of rheumatoid arthritis.

[Identification of Zaocys type II collagen and its effect on arthritis in mice with collagen-induced arthritis].

OBJECTIVE: To analyze the homology of Zaocys type 1I collagen ( ZC II ) with the C II collagen from other species, and to investigate the effect of ZC II on arthritis in mice with collagen-induced arthritis (CIA). METHODS: ZC II was purified with restriction pepsin digestion. Then SDS-PAGE gel electrophoresis and UV spectrophotometry were used to identify the protein,the homology of the ZC II peptide was analyzed with Mass Spectrometry. The model of CIA mice were induced by subcutaneous injection of Chicken C II into male C57BL/6 mice from the base of the tails. After immunization,ZC II [H,M,L:40,20 and 10 µg/(kgd) ]was administered orally to mice from day 21 to 28 accordingly. The severity of the arthritis in each limb was evaluated using a macroscopic scoring system, and his- topathological change of joint was observed by light microscope with HE staining. RESULTS: The molecular weight of ZC II protein deter- mined by SDS-PAGE gel electrophoresis was between 110 kD and 140 kD, and UV absorption peak appeared at around 230 nm in wave- length. The peptide mass fingerprinting(PMF) of the purified protein by Mass Spectrometry analysis showed that it had at least 4 peptides matched with other species,and the protein score was greater than 95%. Compared with normal group,the CIA model group had significantly higher scores for arthritis and histopathological changes (P <0. 05). Meanwhile,the same scores for the ZC II peptide-treated mice with CIA were significantly lower than the mice from CIA model group(P <0. 05). CONCLUSION: Results of Mass Spectrometry analysis demonstrate that ZC II has high homology with the C II from other species. Oral administration of ZC II can suppress arthritis in mice with CIA and ameliorate the histopathological changes of the joint.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin ß1/FAK signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PURPOSE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin ß1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin ß1, FAK, ß-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin ß1, ß-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin ß1/FAK pathway. CONCLUSION: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin ß1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

DKK-1 and Its Influences on Bone Destruction: A Comparative Study in Collagen-Induced Arthritis Mice and Rheumatoid Arthritis Patients.

Dickkopf-1 (DKK-1) has been considered a master regulator of bone remodeling. As precursors of osteoclasts (OCs), myeloid-derived suppressor cells (MDSCs) were previously shown to participate in the process of bone destruction in rheumatoid arthritis (RA). However, the role of DKK-1 and MDSCs in RA is not yet fully understood. We investigated the relevance between the level of DKK-1 and the expression of MDSCs in different tissues and joint destruction in RA patients and collagen-induced arthritis (CIA) mouse models. Furthermore, the CIA mice were administered recombinant DKK-1 protein. The arthritis scores, bone destruction, and the percentage of MDSCs in the peripheral blood and spleen were monitored. In vitro, the differentiation of MDSCs into OCs was intervened with recombinant protein and inhibitor of DKK-1. The number of OCs differentiated and the protein expression of the Wnt/ß-catenin signaling pathway were explored. The level of DKK-1 positively correlates with the frequency of MDSCs and bone erosion in RA patients and CIA mice. Strikingly, recombinant DKK-1 intervention significantly exacerbated arthritis scores and bone destruction, increasing the percentage of MDSCs in the peripheral blood and spleen in CIA mice. In vitro experiments showed that recombinant DKK-1 promoted the differentiation of MDSCs into OCs, reducing the expression of ß-catenin and TCF4 and increasing the expression of CyclinD1. In contrast, the DKK-1 inhibitor had the opposite effect. Our findings highlight that DKK-1 promoted MDSCs expansion in RA and enhanced the differentiation of MDSCs into OCs via targeting the Wnt/ß-catenin pathway, aggravating the bone destruction in RA.

Internalisation of neutrophils extracellular traps by macrophages aggravate rheumatoid arthritis via Rab5a.

OBJECTIVES: Although elevated levels of neutrophil extracellular traps (NETs) have been reported in patients with rheumatoid arthritis (RA), the role of NETs in RA and the relationship between NETs and macrophages in the pathogenesis of RA requires further research. Here, we sought to determine the role of NETs in RA pathogenesis and reveal the potential mechanism. METHODS: Neutrophil elastase (NE) and myeloperoxidase (MPO)-DNA were measured in human serum and synovium. NETs inhibitor GSK484 was used to examine whether NETs involved with RA progression. We stimulated macrophages with NETs and detected internalisation-related proteins to investigate whether NETs entry into macrophages and induced inflammatory cytokines secretion through internalisation. To reveal mechanisms mediating NETs-induced inflammation aggravation, we silenced GTPases involved in internalisation and inflammatory pathways in vivo and in vitro and detected downstream inflammatory pathways. RESULTS: Serum and synovium from patients with RA showed a significant increase in NE and MPO, which positively correlated to disease activity. Inhibiting NETs formation alleviated the collagen-induced arthritis severity. In vitro, NETs are internalised by macrophages and located in early endosomes. Rab 5a was identified as the key mediator of the NETs internalisation and inflammatory cytokines secretion. Rab 5a knockout mice exhibited arthritis alleviation. Moreover, we found that NE contained in NETs activated the Rab5a-nuclear factor kappa B (NF-κB) signal pathway and promoted the inflammatory cytokines secretion in macrophages. CONCLUSIONS: This study demonstrated that NETs-induced macrophages inflammation to aggravate RA in Rab 5a dependent manner. Mechanically, Rab5a mediated internalisation of NETs by macrophages and NE contained in NETs promoted macrophages inflammatory cytokines secretion through NF-κB-light-chain-enhancer of activated B cells signal pathway. Therapeutic targeting Rab 5a or NE might extend novel strategies to minimise inflammation in RA.

Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/ß-Catenin and Akt/NF-κB signaling pathways.

BACKGROUND: Surgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PURPOSE: This study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. METHODS: The potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. RESULTS: Network pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/ß-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. CONCLUSION: Our study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/ß-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Sn-doped BiOI assisted the excellent photocatalytic performance of multi-shelled ZnO microspheres under simulated sunlight.

To deal with the environmental pollution and energy crises, it is indispensable to find green and efficient means to overcome these challenges. Herein, the Sn-doped BiOI modified multi-shelled ZnO heterojunction composite with a high performance are designed and prepared. The results prove the formation of heterojunction structure in the composites and the morphology is shown as the multi-shelled microsphere. The performances of the composites are evaluated by different kinds of antibiotic degradation and H2-evolution under simulation sunlight irradiation. The measurements present that the Sn-BiOI/ZnO (SBZs) could completely remove ciprofloxacin (CIP) within 100 min, which is 4.18 times that of ZnO in kinetics. Typically, the degradation rate of CIP for SBZ6 is over 99.9%, which is more than 25% higher than that of pure ZnO microspheres. In addition, the rate of H2 production could reach 3.08 mmol g-1∙ h-1, which is 1.79 times of the pure ZnO microspheres. The boosted performance of the composites may originate from the enhanced electronic transmission efficiency and improved separation and recombination efficiency of electrons/holes. The charge transfer mode in the SBZs heterojunction composites is proposed and verified as the Z-scheme by the active species experimental and the possible electron transfer path analysis. Therefore, Sn-doped BiOI is introduced into multi-shelled ZnO microsphere to form contact heterojunction interfacial, which greatly improves the photocatalytic performances of the SBZs. Furthermore, this work supplies a strategy for designing and preparing highly active ZnO-based heterojunction composites, which could effectively address the challenges of environmental remediation and clean energy production.

Natural Killer Cells Infiltration in the Joints Exacerbates Collagen-Induced Arthritis.

Introduction: The role of natural killer (NK) cells in rheumatoid arthritis remains controversial. We aimed to assess the role of NK cells in the pathogenesis of rheumatoid arthritis. Materials and Methods: The percentage of NK cells in the peripheral blood, spleen, lymph nodes and inflamed paws from collagen-induced arthritis mice were examined through the disease progression. Correlation between the proportion of NK cells and subsets with arthritis score, histopathological changes, and bone destruction were evaluated. Adoptive cell transfer was performed to determine the effect of NKp46+NK cells on arthritis development, and the role of receptor NKp46 was explored with NKp46 knockout mice. Results: The percentage of NK cells in peripheral blood decreased at the late stage of the disease and negatively correlated with arthritis score. NK cells increased in the inflamed paws during arthritis development and were positively associated with arthritis score, histopathological change, and bone destruction. Adoptive transfer of NKp46+NK cells before disease onset resulted in increased NK cells infiltration in the joints, higher incidence of arthritis, more severe clinical symptoms, and more pronounced joint inflammation and bone damage. NKp46 deficiency had no significant influence on the incidence and severity of arthritis in collagen-induced arthritis mice. Conclusions: NK cell infiltration in the joints positively correlates with arthritis progression, inflammation, and bone destruction. The pathogenic role of NK cells in rheumatoid arthritis may be independent of the receptor NKp46.

Resveratrol Relieves Gouty Arthritis by Promoting Mitophagy to Inhibit Activation of NLRP3 Inflammasomes.

BACKGROUND: Gouty arthritis (GA) is a common inflammatory disease with pain caused by the deposition of monosodium urate (MSU) crystals into joints and surrounding tissues. Resveratrol (Res), derived from grapes and peanuts and the traditional Chinese medicine (TCM) Reynoutria japonica for GA, acts against oxidation and inflammation. The present study aimed to investigate the therapeutic effect and mechanism of Res on GA. METHODS: Arthritis rat models, MSU-induced peritonitis mouse models, and inflammatory models of mouse bone marrow-derived macrophage (BMDM) were used in this study. Enzyme-linked immunosorbent assay (ELISA), JC-1, histopathological, immunofluorescence, flow cytometry, Western blot methods were applied to observe the effects of resveratrol on NLRP3 inflammasomes and mitophagy. RESULTS: Res significantly improves the gait score and synovitis of rats with GA and inhibits the peritoneal inflammation induced by MSU. Res inhibits the MSU-induced activation of NLRP3 inflammasomes by reducing the levels of IL-1ß, IL-18, and Caspase-1 and the pyroptosis of macrophages. In addition, Res raises the level of mitochondrial membrane potential, inhibits the expression of P62 and Pink1, enhances the expressions of LC3B-II, Parkin, and TOMM20, and promotes mitophagy, while mitophagy inhibitors reverse the inhibitory effect of Res on the activation of NLRP3 inflammasomes. CONCLUSION: Res significantly improves GA, and the underlying mechanism might be inhibiting the activation of NLRP3 inflammasomes by triggering the Pink1/Parkin pathway to promote mitophagy.

Prevalence of basal core promoter and precore mutations in Chinese chronic hepatitis B patients and correlation with serum HBeAG titers.

The A1762T and G1764A mutations in the basal core promoter (BCP) region and the G1896A mutation in the precore (PC) region of hepatitis B virus (HBV) genome are found commonly in HBeAg-negative patients. Experiments in vitro suggest that BCP and PC mutation reduce and abolish HBeAg expression, respectively. In the present study, the prevalence of the BCP and PC mutations were determined in 207 patients with HBeAg positive chronic hepatitis B from China and correlated with the titers of serum HBeAg. None of the patients received antiviral therapy. The HBV genotype was determined by direct sequencing of the HBsAg gene. The BCP and PC mutations were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by DNA sequencing. The HBeAg titer was measured by the microparticle enzyme immunoassay. Fifty-one of the 207 patients (24.6%) were infected with genotype B and the remainder with genotype C. The BCP mutations were detected in 103 patients (50%) while the PC mutation was present in 43 (20.8%). Thirteen patients (6.3%) harbored both BCP and PC mutations. No significant difference in the titers of HBeAg was found between patients infected with the two HBV genotypes, but the presence of either the BCP or PC mutation was associated with reduced HBeAg titer (P < 0.05). The presence of both the BCP and PC mutations was accompanied by even lower HBeAg titer (P < 0.05). These findings confirm that in patients with HBeAg, the BCP and PC mutations reduced the expression of HBeAg.

Spontaneous HBeAg seroconversion and loss of hepatitis B virus DNA after acute flare due to development of drug resistant mutants during entecavir monotherapy.

AIMS: Patients with chronic hepatitis B virus (HBV) infection under entecavir (ETV) treatment develop resistant mutants with viral rebound. Here, we report an interesting case of spontaneous loss of HBV-DNA and seroconversion following an acute flare after the development of ETV-resistant mutants. This patient received ETV after lamivudine breakthrough. METHODS: Cloning and sequence analysis of the HBV reverse transcriptase (RT) region were performed with seven samples during ETV therapy. In addition, two full-length HBV genomes derived from samples before and after the emergence of ETV resistance were sequenced. RESULTS: ETV resistant mutants appeared at week 228, with virological and biochemical rebound at the same time. Unexpectedly, HBeAg seroconversion occurred 8 weeks later. The viral load decreased and became undetectable from week 252. Analysis of HBV isolates in the patient at week 124 revealed that wild-type HBV was predominant at that time and ETV resistant mutants were not found among 20 clones. Interestingly, a new mutant type with rtL180M+rtT184L was found alongside rtL180M+rtT184L+rtM204V/I at week 228 and appeared to develop independently, according to the sequence analysis. In contrast to the previously identified ETV resistant mutants, it did not carry the rtM204V/I mutations. CONCLUSION: The data presented here indicates that the flare following the emergence of ETV resistant mutants may reflect immune-mediated control of HBV infection, leading to a spontaneous loss of HBV-DNA and seroconversion.

[The effect of the type II collagen protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis].

OBJECTIVE: To investigate the effect of the type II collagen (C II) protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis (AA). METHODS: Type II collagen protein was abstracted and purificated from Zaocys. Adjuvant arthritis (AA) was induced by a single intradermal injection of 0.1 mL of complete Freund's adjuvant into the left hind paw. Synoviocytes' supernatants were harvested and synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Tumor necrosis factor-alpha (TNF-alpha) activity was measured by L929 cytotoxicity bioassay and Interleukin (IL)-1beta activity was measured by MTT dye reduction. The synoviocytes' supernatants cytokines' levels were detected by ELISA. RESULTS: Each concentration of C II from Zaocys had no effect on IL-1beta and TNF-alpha production by synoviocytes in vitro. Middle concentration of C II suppressed the activity of IL-1beta and TNF-alpha production by synoviocytes-PP cells coculture system (P < 0.05). Treating with low and high dose of C II suppressed the activity of TNF-alpha and IL-1beta producing by synoviocyte (P < 0.05), significantly suppressed in the group of AA rats treated with middle dose of C II (P < 0.01). Treating with middle and high dose of C II decreased the level of synoviocytes' supernatants TNF-alpha (P < 0.05), the level of synoviocytes' supernatants IL-1beta decreased in all treating groups (P < 0.05). Treating with middle dose of C II increased the level of serum TGF-beta (P < 0.05). Middle concentration of C II suppressed the activity of IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.05). CONCLUSIONS: C II from Zaocys has no direct effect on the activity of IL-1beta and TNF production by synoviocytes in vitro. Oral administration of type II collagen protein from Zaocys can effectively suppressed the activity and level of the cytokines production by synoviocytes in rats with adjuvant arthritis (AA).

Taxol alleviates collagen-induced arthritis in mice by inhibiting the formation of microvessels.

The objective of the present study is to evaluate the inhibitory effects of taxol (PTX) on angiogenesis in a collagen-induced arthritis (CIA) mouse model. Collagen II (C II) and complete Freund's adjuvant (CFA) were used in C57BL/6 (H-2b) mice to generate the CIA model. Random grouping was performed in the normal control group, CIA model group, PTX 1.5 mg/kg group, PTX 1.0 mg/kg group, and PTX 0.5 mg/kg group. Arthritis index scores, tissue pathology scores, and synovium microvessel density (MVD) analysis were performed. Immunohistochemistry and enzyme-linked immunosorbent assay were used to detect the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-α (HIF-1α). The correlation between MVD and pathological scores and between MVD and the expression of VEGF as well as HIF-1α in the synovium were also evaluated. After PTX treatment, the three intervention group arthritis index scores were reduced when compared with the CIA group. The total histological scores in the three PTX treatment groups were lower than those in the CIA group. Similarly, PTX significantly alleviated the scores for synovitis, pannus formation, and bone destruction. Compared with the CIA group, the MVD of the three intervention groups decreased in a dose-dependent manner. The expression of VEGF and HIF-1α in synovial tissues and serum also significantly decreased after PTX treatment. Further analysis showed that MVD and pathological scores and MVD and expression of VEGF as well as HIF-1α in the synovium were positively correlated. PTX may alleviate CIA by suppressing angiogenesis, providing new insights into the treatment of rheumatoid arthritis (RA). VEGF and HIF-1α may be targets for PTX suppression of microvessel formation.

Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.

BACKGROUND: The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in patients with chronic hepatitis B virus (HBV) infection has been explained by the presence of viral escape mutants. Yet, no systematic analysis of such patients has been performed. We analyzed both the HBV strains and the nature of anti-HBs in such patients. METHODS: Four hundred eleven patients with chronic HBV infection were tested for the presence of anti-HBs. The sequences of the HBsAg coding region were analyzed. Anti-HBs were purified and examined in commercial assays alone and with 3 different HBsAg subtypes. RESULTS: Twenty patients had positive results for anti-HBs. This serological status remained stable for 12 months (as tested thus far). Amino acid substitutions and/or variations on HBsAg were found in 13 patients, and the HBV isolates from 4 others were wild types. Importantly, no significant difference in the occurrence of amino acid substitutions within the HBsAg was found in HBV isolates from patients with and without anti-HBs. Purified immunoglobulin fractions from serum samples from patients were reactive to HBsAg but had a lower specific activity, compared with those taken from immunized persons. Anti-HBs in patients were directed to the HBsAg subtypes other than the coexisting one. No circulating immune complex could be detected in these patients. CONCLUSION: HBsAg and anti-HBs with an unmatched specificity coexisted in 4.9% of patients. The presence of anti-HBs was not associated with the appearance of specific HBV mutants in patients with chronic infection. Apparently, the presence of anti-HBs in patients with chronic HBV infection did not lead to a selection of HBV escape mutants.

[The rate of hepatitis B virus resistance to adefovir dipivoxil (ADV) and the evolution of hepatitis B virus in lamivudine-resistant chronic hepatitis B patients with ADV monotherapy].

OBJECTIVE: To study the resistant rate of hepatitis B virus (HBV) to ADV and the dynamic evolution of HBV in lamivudine (Lam)-resistant chronic hepatitis B (CHB) patients. METHODS: Twenty-three Lam-resistant CHB patients were assigned to a 10mg/d ADV monotherapy for 68-116 weeks. The baseline and different time point blood samples after ADV monotherapy were analyzed for ADV-resistant mutations using direct sequencing of PCR products; the evolution of HBV mutations was examined by clonal analysis of serial samples from one patient infected with ADV-associated resistant HBV strains. RESULTS: The cumulative incidence of genotypic ADV resistance at weeks 48 and 96 was 4.3% and 10.5% respectively respectively. The evolution analysis of HBV mutant strains in an ADV-resistant CHB patient showed that the proportion of YMDD mutants gradually decreased with rtA181S mutants increasing over time after ADV monotherapy, and that rtA181S+N236T mutants became the predominant strains during prolonged ADV monotherapy. The addition of Lam to the ongoing ADV treatment had poorer antiviral response in the patient with rtA181S or rtA181S+N236T mutant infection; one clone with multi-drug resistant mutations was selected during Lam and ADV combination therapy. CONCLUSION: Increased risk of adefovir resistance and selection of multi-drug resistant mutations are associated with long-term ADV monotherapy in patients with Lam-resistant chronic hepatitis B.