Deficiency of leucine-rich repeat kinase 2 aggravates thioacetamide-induced acute liver failure and hepatic encephalopathy in mice.

BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.

Occurrence and Exposure Assessment of Lipophilic Shellfish Toxins in the Zhejiang Province, China.

Although lipophilic shellfish toxins (LSTs) pose a significant threat to the health of seafood consumers, their systematic investigation and risk assessment remain scarce. The goals of this study were as follows: (1) analyze LST levels in commercially available shellfish in Zhejiang province, China, and determine factors influencing LST distribution; (2) assess the acute dietary risk of exposure to LSTs for local consumers during the red tide period; (3) explore potential health risks of LSTs in humans; and (4) study the acute risks of simultaneous dietary exposure to LSTs and paralytic shellfish toxins (PSTs). A total of 546 shellfish samples were collected. LSTs were detected in 89 samples (16.3%) at concentrations below the regulatory limits. Mussels were the main shellfish species contaminated with LSTs. Spatial variations were observed in the yessotoxin group. Acute exposure to LSTs based on multiple scenarios was low. The minimum tolerable exposure durations for LSTs calculated using the mean and the 95th percentile of consumption data were 19.7 and 4.9 years, respectively. Our findings showed that Zhejiang province residents are at a low risk of combined exposure to LSTs and PSTs; however, the risk may be higher for children under 6 years of age in the extreme scenario.

Microglial LRRK2-mediated NFATc1 attenuates α-synuclein immunotoxicity in association with CX3CR1-induced migration and the lysosome-initiated degradation.

Synucleinopathies refer to a range of neurodegenerative diseases caused by abnormal α-synuclein (α-Syn) deposition, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Their pathogenesis is strongly linked to microglial dysfunction and neuroinflammation, which involves the leucine-rich-repeat kinase 2 (LRRK2)-regulated nuclear factor of activated T-cells (NFAT). Of the NFAT family, NFATc1 has been found to be increasingly translocated into the nucleus in α-syn stimulation. However, the specific role of NFATc1-mediated intracellular signaling in PD remains elusive in regulating microglial functions. In the current study, we crossbred LRRK2 or NFATc1 conditional knockout mice with Lyz2Cre mice to generate mice with microglia-specific deletion of LRRK2 or NFATc1, and by stereotactic injection of fibrillary α-Syn, we generated PD models in these mice. We found that LRRK2 deficiency enhanced microglial phagocytosis in the mice after α-Syn exposure and that genetic inhibition of NFATc1 markedly diminished phagocytosis and α-Syn elimination. We further demonstrated that LRRK2 negatively regulated NFATc1 in α-Syn-treated microglia, in which microglial LRRK2-deficiency facilitated NFATc1 nuclear translocation, CX3CR1 upregulation, and microglia migration. Additionally, NFATc1 translocation upregulated the expression of Rab7 and promoted the formation of late lysosomes, resulting in α-Syn degradation. In contrast, the microglial NFATc1 deficiency impaired CX3CR1 upregulation and the formation of Rab7-mediated late lysosomes. These findings highlight the critical role of NFATc1 in modulating microglial migration and phagocytosis, in which the LRRK2-NFATc1 signaling pathway regulates the expression of microglial CX3CR1 and endocytic degradative Rab7 to attenuate α-synuclein immunotoxicity.

Periodontitis-induced neuroinflammation impacts dendritic spine immaturity and cognitive impairment.

OBJECTIVE: This study investigated the spinal changes in ligature-induced periodontitis and the role of periodontitis in cognitive impairment. METHODS: Twenty mice were randomized into the control and chronic periodontitis (CP) groups, with the latter receiving ligature-induced periodontitis. Cognitive performance was assessed by fear conditioning test. Periodontal inflammation and alveolar bone resorption were evaluated by micro-computed tomography and histopathology. The hippocampal microglial activation was evaluated by immunohistochemistry (IHC). The expressions of hippocampal cytokines (TNF-α, iNOS, IL-1ß, IL-4, IL-10, and TREM2) were measured by reverse transcription-polymerase chain reaction. The morphology and density of the dendritic spines were determined by Golgi-Cox staining. RESULTS: The CP mice reported significant inflammatory cell infiltration and alveolar bone resorption, with marked increases in cytokine levels (TNF-α, iNOS, IL-1ß, and TREM2) in the brain. Moreover, the CP mice showed significantly reduced freezing to the conditioned stimulus in the cued and contextual tests, indicating impaired memory. Further analyses revealed, in the hippocampus of the CP mice, enhanced microglial activation, decreased dendritic spine density, and increased proportion of thin dendritic spines. CONCLUSIONS: Periodontitis-induced neuroinflammation may impair the cognitive function by activating hippocampal microglia and inducing dendritic spine immaturity.

Tracking contaminants in groundwater flowing across a river bottom within a complex karst system: Clues from hydrochemistry, stable isotopes, and tracer tests.

Tracking contaminants in karst aquifers is challenging because of the high heterogeneity encountered in carbonate rocks. Multi-tracer tests, combined with chemical and isotopic analyses, were conducted to solve a groundwater contamination incident within a complex karst aquifer in Southwest China. Results showed that: (1) the wastewater from a paper mill, public sewers, and septic tanks were the three main potential contaminant sources identified by chemical and isotopic methods; (2) a direct effect of the paper mill wastewater with high Na+ (up to 2230.5 mg/L) and chemical oxygen demand (COD) concentrations on spring water quality was confirmed by multi-tracer tests, which changed the water type from Ca-HCO3 in the 1970s to Ca-Na-HCO3 in the present study and resulted in a depleted carbon isotope value (-16.5‰); and (3) the studied aquifer is a highly complex karst system, due to two conduits crossed each other without mixing, contaminants traveled a long distance (up to 14 km) within the lower conduit, paper mill-contaminated groundwater flowed across a river bottom and discharged to the opposite bank, and an active subsurface divide occurred. After several months of operation, the groundwater restoration measure based on karst hydrogeologic conditions proved that cutting off contaminant sources for karst aquifer self-restore was effective in practice, which contributed to the decline in NH4+ (from 7.81 mg/L to 0.04 mg/L), Na+ (from 50.12 mg/L to 4.78 mg/L), and COD (from 16.42 mg/L to 0.9 mg/L) concentrations coupled with an increase in δ13C-DIC value (from -16.5‰ to -8.4‰) in the earlier contaminated karst spring. This study's integrated method is expected to screen and confirm contaminant sources within complex karst systems rapidly and effectively, thereby contributing to karst groundwater environmental management.

Eco-friendly strategy for preparation of high-purity silica from high-silica IOTs using S-HGMS coupling with ultrasound-assisted fluorine-free acid leaching technology.

Iron ore tailings (IOTs), a typical hazardous solid waste, seriously threaten human health and the ecological environment. However, the abundance of quartz, particularly in high-silica IOTs, renders them useful. Yet, state-of-the-art technologies have rarely reported the preparation of high-purity silica from high-silicon IOTs. Thus, this study proposed an eco-friendly technology for producing high-purity silica from high-silica IOTs through the coupling of superconducting high gradient magnetic separation (S-HGMS) preconcentration with leaching followed by the use of ultrasound-assisted fluorine-free acid solution. Following an analysis of the separation index and chemical composition, the optimum conditions for the quartz preconcentration were determined as a magnetic flow ratio of 0.068 T s/m, a slurry flow velocity of 500 mL/min, and a pulp concentration of 40 g/L. Consequently, the SiO2 grade increased from 69.32% in the raw sample to 93.12% in quartz concentrate following the application of S-HGMS, with the recovery reaching 45.24%. X-ray diffraction, vibrating sample magnetometer, and scanning electron microscope analyses indicated that quartz was effectively preconcentrated from the tailings by S-HGMS. Subsequently, employing the "ultrasound-assisted fluorine-free acid leaching process," impurity elements were removed and high-purity silica was produced. Under optimal leaching conditions, the SiO2 purity of silica sand increased to 97.42%. Following a three-stage acid leaching process with 4 mol/LHCl +2 mol/LH2C2O4, the removal efficiency of Al, Ca, Fe, and Mg exceeded 97% for all cases, and the SiO2 purity in high-purity silica reached 99.93%. Thus, this study proposes a new strategy for the preparation of high-purity quartz from IOTs, which facilitated the effective realization of the high-value utility of the tailings. Furthermore, it provides a theoretical basis for the industrial application of IOTs, which is of great scientific significance and practical application value.

[Occurrence and exposure assessment of fumonisins from grains and grain products in Zhejiang Province in 2018-2019].

OBJECTIVE: To monitor fumonisins(FBs) in grains and grain products in Zhejiang and assess the exposure risks of FBs to local residents. METHODS: Liquid chromatography coupled with tandem mass spectrometry method was used to determine the occurrence of FBs in rice, millet, dried noodles, instant noodles, and maize grains, and food frequency questionnaires were used to collect the food consumption data of Zhejiang population. Then, the simple probability distribution model was used to assess the exposure risk. RESULTS: The levels of FBs in rice, millet, dried noodles and instant noodles were relatively low. The occurrence of FB_1, FB_2 and FB_3 in these foods was 0-23.7%, 0-16.7% and 0-5.4%, respectively, and the mean levels were not detected(ND)-22.36, ND-20.63 and ND-7.19 µg/kg correspondingly. However, the levels of FBs in maize grains were relatively high. The occurrence of FB_1, FB_2, and FB_3 in maize grains was 100%, 93.6% and 90.3%, respectively, and the mean levels were 638.99, 103.54 and 59.69 µg/kg correspondingly. In 12.9% of the maize grain samples, the levels of FBs were higher than the standard reference. The residents were at low exposure risk overall. The mean estimated daily intake(EDI) of FBs was far lower than the provisional maximum tolerable daily intake of 2 µg/(kg·BW·d). However, 0.30% of the residents were at high risk. Among people of different ages, the mean EDI of children, adults, and elderly were 0.43, 0.28 and 0.29 µg/(kg·BW·d) respectively, and children were in the highest exposure levels of FBs. Among the tested five foodstuffs, rice and maize grains were the main sources of FBs exposure. CONCLUSION: Except for maize grains, the levels of FBs in grains and grain products were relatively low, and Zhejiang residents were at low FBs exposure risk generally.

Lipoxin A4 regulates M1/M2 macrophage polarization via FPR2-IRF pathway.

Lipoxin A4 (LXA4) has been shown to have anti-inflammatory activity, but its underlying molecular mechanisms are not clear. Herein, we investigated the potential role of LXA4 in macrophage polarization and elucidated its possible molecular mechanism. The RAW264.7 macrophage cell line was pretreated with LXA4 with or without lipopolysaccharides (LPSs) and interleukin-4 (IL-4). In cultured macrophages, LXA4 inhibited LPS-induced inflammatory polarization, thereby decreasing the release of proinflammatory cell factors (IL-1ß, IL-6, TNF-α) and increasing the release of anti-inflammatory cytokines (IL-4 and IL-10). Notably, the inhibitory effect of LXA4 on inflammatory macrophage polarization was related to the downregulation of p-NF-κB p65 and IRF5 activity, which reduced the LPS-induced phenotypic and functional polarization of M1 macrophages via the FPR2/IRF5 signaling pathway. Moreover, LXA4 also induced the IL-4-induced polarization of M2 macrophages by promoting the FPR2/IRF4 signaling pathway. Therefore, LXA4 regulates M1/M2 polarization of macrophages via the FPR2-IRF pathway.

MiR-15a Functions as a Diagnostic Biomarker for Coronary Artery Disease.

BACKGROUND: The present study aimed to explore the diagnostic value of miR-15a in coronary artery disease (CAD). METHODS: After recruiting all the participants, peripheral blood samples were obtained according to the instructions. miR-15a expression was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and IL-6, TNF-α, and hs-CRP expressions were detected using ELISA kits. RESULTS: The results elucidated a significantly decreased expression of miR-15a in peripheral blood samples from CAD patients compared to non-CAD controls (p < 0.01). Meanwhile, miR-15a expression was negatively correlated with LDL-C and Gensini score (p = 0.0059, 0.0243, respectively). Moreover, miR-15a expression was negatively correlated with inflammatory cytokines IL-6, TNF-α, and hs-CRP (p = 0.0009, 0.0178, 0.0005, respectively). The receiver operating curve (ROC) curve analysis demonstrated that miR-15a was a promising biomarker for early diagnosis of CAD with an area under the curve (AUC) of 0.9368. CONCLUSIONS: The present study elucidated a decreased miR-15a expression in CAD patients and showed negative correlations with LDL-C, Gensini score, and inflammatory cytokines. miR-15a may serve as a promising biomarker for early diagnosis of CAD.

miR-142 is a Sensitive Biomarker for the Diagnosis and Prognosis of Acute Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary arteries. AMI is one of the most common diseases in European countries and over 1.5 million AMI patients die of it in the United States annually. A collection of studies proposed that certain micro-RNAs play crucial roles in the onset and development of AMI. METHODS: Ninety-four AMI patients and 83 non-AMI healthy controls were recruited from Zhongda Hospital, Southeast University between July 2015 and September 2017. Serum samples were collected at admission and the expression of miR-142 was detected using real-time quantitative polymerase chain reaction (RT-qPCR) assays. RESULTS: miR-142 expression was markedly elevated in serum samples of AMI patients compared with the 83 non-AMI healthy controls. miR-142 expression was positively correlated with creatine kinase-KB (CK-MB; r = 0.6731, p = 0.0021) and troponin (r = 0.7138, p = 0.0013). The area under the curve (AUC) of miR-142, CK-MB, and troponin for the diagnosis of AMI were 0.9185, 0.8172, and 0.8717, respectively. Overall survival analysis implied that high miR-142 expression may predict poor survival (log-rank test, p = 0.0146). CONCLUSIONS: miR-142 may be a diagnostic and prognostic indicator for AMI, and therefore, it may contribute to AMI clinicopathologic prediction.

[Lead pollution status and exposure level in cereals and related products in Zhejiang Province from 2014 to 2019].

OBJECTIVE: To investigate the level of lead in cereals and related products from Zhejiang market, and to provide the preliminary assessment. METHODS: Lead was detected in 4855 samples of cereals and related products during 2014-2019, food consumption data was taken from Zhejiang residents' consumption survey in 2015-2016. RESULTS: The total detection rate of lead in cereals and related products was 59. 03%, and the acceptance rate was 99. 3% when compared to the national food safety standards(GB 2762-2017). The average lead level in cereals and related products was 0. 031 mg/kg, high mean level was found in millet with 0. 049 mg/kg, low mean level was in grain cans with 0. 020 mg/kg. The average dietary exposure to lead via cereals and related products was 0. 16 µg/(kg·d), and 0. 45 µg/(kg·d) for high consumer(P97. 5). CONCLUSION: Lead in cereals and related products are at very low contamination levels, residents' average daily intake of lead from grains and products was low in Zhejiang Province, but measures are still needed to reduce lead in cereal products considering the adverse health effects of lead.

[Analysis of multi-minerals in seafoods in Zhejiang Province].

OBJECTIVE: To analyze the contents of Ca, K, Na, Mg, Fe, Zn, Cu and Mn in 33 kinds of marine products, such as fish, shrimp, crab, shellfish and mollusk. METHODS: The national standard GB 5009. 268-2016 Determination of multi elements in foods was used for determination. The correlation between multi elements was statistically analyzed. RESULTS: The highest level of Ca was found in clam(510. 2 mg/100 g edible), K in mackerel(444. 4 mg/100 g edible), Na in clam(487. 6 mg/100 g edible), Mg in conch(132. 7 mg/100 g edible). The highest level of Fe and Mn was in conch(37. 35 and 2. 6 mg/100 g edible). The highest level of Zn and Cu was in oyster(15. 92 and 8. 58 mg/100 g edible). The correlation analysis showed that Mn-Ca was highly correlated in fish and shrimp(r=0. 9438 and 0. 8585, P < 0. 05), while Cu-Mg, Cu-Zn, Zn-Na were highly correlated in shellfish(r=-0. 9102, 0. 8501 and 0. 8428, P < 0. 05). CONCLUSION: There are rich mineral elements in different seafood, which can provide reference for the reasonable diet of residents.

DNA methylation regulates α-smooth muscle actin expression during cardiac fibroblast differentiation.

Cardiac fibroblast (CF) differentiation to myofibroblasts expressing α-smooth muscle actin (α-SMA) plays a key role in cardiac fibrosis. Therefore, a study of the mechanism regulating α-SMA expression is a means to understanding the mechanism of fibroblast differentiation and cardiac fibrosis. Previous studies have shown that DNA methylation is associated with gene expression and is related to the development of tissue fibrosis. However, the mechanisms by which CF differentiation is regulated by DNA methylation remain unclear. Here, we explored the epigenetic regulation of α-SMA expression and its relevance in CF differentiation. In this study, we demonstrated that α-SMA was overexpressed and DNMT1 expression was downregulated in the infarct area after myocardial infarction. Treatment of CFs with transforming growth factor-ß1 (TGF-ß1 ) in vitro upregulated α-SMA expression via epigenetic modifications. TGF-ß1 also inhibited DNMT1 expression and activity during CF differentiation. In addition, α-SMA expression was regulated by DNMT1. Conversely, increasing DNMT1 expression levels rescued the TGF-ß1 -induced upregulation of α-SMA expression. Finally, TGF-ß1 regulated α-SMA expression by inhibiting the DNMT1-mediated DNA methylation of the α-SMA promoter. Taken together, our research showed that inhibition of the DNMT1-mediated DNA methylation of the α-SMA promoter plays an essential role in CF differentiation. In addition, DNMT1 may be a new target for the prevention and treatment of myocardial fibrosis.

Baicalin Suppresses the Proliferation and Migration of Ox-LDL-VSMCs in Atherosclerosis through Upregulating miR-126-5p.

Atherosclerosis (AS) is a chronic inflammatory disease threatening human health, and vascular smooth muscle cells (VSMCs) are involved in AS processes. Baicalin is a flavonoid compound, which has anti-atherosclerotic effect. The aim of our study was to explore the molecular mechanism of baicalin on AS. The expression of miR-126-5p was measured in peripheral blood of AS patients and healthy control. We found miR-126-5p expression was decreased in AS. Then, high-mobility group box 1 (HMGB1) was verified as a target of miR-126-5p and its expression was increased in AS. Similarly, miR-126-5p and HMGB1 expression was downregulated and upregulated in oxidized low-density lipoprotein treated VSMCs (ox-LDL-VSMCs), respectively. Furthermore, baicalin upregulated miR-126-5p and downregulated HMGB1 expression. Functionally, baicalin significantly inhibited ox-LDL-VSMCs proliferation and migration, and miR-126-5p targets HMGB1 to enhance the inhibition induced by baicalin. Taken together, baicalin is able to prevent AS, which suppressed the proliferation and migration of ox-LDL-VSMCs through upregulating miR-126-5p by targeting HMGB1. These findings suggested that baicalin is an effective drug to alleviate AS, and miR-126-5p is a novel therapeutic target for AS.

LRRK2 modulates microglial activity through regulation of chemokine (C-X3-C) receptor 1 -mediated signalling pathways.

Multiple missense mutations in Leucine-rich repeat kinase 2 (LRRK2) have been linked to Parkinson's disease (PD), the most common degenerative movement disorder. LRRK2 is expressed by both neurons and microglia, the residential immune cells in the brain. Increasing evidence supports a role of LRRK2 in modulating microglial activity, of which Lrrk2-null rodent microglia display less inflammatory response to endotoxin lipopolysaccharide (LPS). The underlying molecular mechanism, however, remains elusive. Chemokine (C-X3-C) receptor 1 (CX3CR1), predominantly expressed by microglia, suppresses microglial inflammation while promotes migration. Using whole-genome microarray screening, we found that Cx3cr1 mRNA levels were substantially higher in microglia derived from Lrrk2 knockout (Lrrk2-/-) mice. The total and cell surface levels of CX3CR1 proteins were also remarkably increased. In correlation with the enhanced CX3CR1 expression, Lrrk2-null microglia migrated faster and travelled longer distance toward the source of fractalkine (CX3CL1), an endogenous ligand of CX3CR1. To investigate the impact of CX3CR1 elevation in vivo, we compared LPS-induced inflammation in the striatum of Lrrk2-/- knockout mice with Cx3cr1 heterozygous and homozygous knockout background. We found that a complete loss of Cx3cr1 restored the responsiveness of Lrrk2-/- microglia to LPS stimulation. In conclusion, our findings reveal a previously unknown regulatory role for LRRK2 in CX3CR1 signalling and suggest that an increase of CX3CR1 activity contributes to the attenuated inflammatory responses in Lrrk2-null microglia.

The Epigenetically-Regulated microRNA-378a Targets TGF-ß2 in TGF-ß1-Treated Hepatic Stellate Cells.

BACKGROUND/AIMS: In liver fibrosis, the activation of hepatic stellate cells (HSCs) is considered as a pivotal event. It is well known that transforming growth factor-ß1 (TGF-ß1) is the main stimuli factor responsible for HSC activation. microRNAs (miRNAs), regulating various biological processes, have recently been shown to be involved in HSC activation. A recent study reported that deficiency of miR-378a contributes to cardiac fibrosis via TGF-ß1-dependent paracrine mechanism. However, the involvement of miR-378a and its roles in TGF-ß1-induced HSC activation remains largely unknown. METHODS: miR-378a expression was detected in TGF-ß1-treated cells and patients with cirrhosis. Then, effects of miR-378a overexpression on cell proliferation and HSC activation were analyzed. We also analyzed the binding of miR-378a to the 3'-untranslated region of TGF-ß2. RESULTS: In response to TGF-ß1, miR-378a expression was down-regulated in a dose-dependent manner. miR-378a overexpression suppressed both cell proliferation and cell cycle in TGF-ß1-treated LX-2 cells. Moreover, miR-378a overexpression inhibited TGF-ß1-induced HSC activation including the reduction of α-smooth muscle actin (α-SMA) and type I collagen. Similarly, miR-378a resulted in a reduction in cell proliferation, and the expressions of α-SMA and Col1A1 in TGF-ß1-treated primary HSCs. Notably, TGF-ß2 was confirmed as a target of miR-378a by luciferase reporter assays. Interestingly, miR-378a promoter methylation may be responsible for miR-378a down-regulation in TGF-ß1-treated LX-2 cells and TGF-ß1-treated primary HSCs. Further studies confirmed that reduced miR-378a was associated with promoter methylation in patients with cirrhosis compared with healthy controls. CONCLUSION: Our results demonstrate that miR-378a expression is associated with its methylation status in TGF-ß1-treated cells, and epigenetically-regulated miR-378a inhibits TGF-ß1-induced HSC activation, at least in part, via TGF-ß2.

Association of apolipoprotein E (APOE) polymorphisms with warfarin maintenance dose in a northern Han Chinese population.

BACKGROUND: Apolipoprotein E (apoE) induces the uptake of vitamin K-rich lipoproteins by the liver, which likely affects inter-individual variation of warfarin dosing requirements. Associations between APOE polymorphisms and warfarin dosing were previously reported inconsistently among different ethnic groups, so the present study investigated this association in northern Han Chinese patients with mechanical heart valve prosthesis. METHODS: A total of 186 patients who underwent mechanical heart valve replacement and attained a stable warfarin dose were included. APOE single nucleotide polymorphisms (SNPs) rs7412 and rs429358 were genotyped using Illumina SNP GoldenGate Assay. Genotyping results were confirmed by direct sequencing. PHASE v2.1 software was used to construct rs7412 and rs429358 haplotypes. The effects of different APOE genotypes on warfarin dose were analyzed statistically. RESULTS: The mean warfarin maintenance dose was 3.10 ± 0.96 mg/day, and the mean international normalized ratio (INR) was 2.09 ± 0.24. APOE E2, E3, and E4 allele frequencies were 11.6 %, 82.5 %, and 5.9 %, respectively. No E2/E2 or E4/E4 genotypes were detected in this population. E2/E3, E3/E3, E2/E4, and E3/E4 genotype frequencies were 21.0 %, 67.2 %, 2.2 %, and 9.7 %, respectively. Significant differences in warfarin dose requirements were observed among patients with E2/E3, E3/E3, and E3/E4 genotypes (p < 0.05). In post hoc comparison, daily warfarin maintenance doses were significantly higher in E2/E3 heterozygotes compared with E3/E3 homozygotes (p < 0.05), but no differences in dose requirements were found between E3/E4 and E3/E3, or E2/E3 and E3/E4 (p > 0.05). Patients were divided into low-intensity anticoagulant treatment group (1.6 ≤ INR <2.0) and relatively high-intensity anticoagulant treatment group (2.0 ≤ INR ≤ 2.5), and significantly higher warfarin dose requirements were observed in E2/E3 heterozygotes compared with E3/E3 homozygotes in both subgroups (p < 0.05). Multivariable analysis adjusting for other confounders showed that E2/E3 genotype was associated with a significantly higher warfarin dose compared with E3/E3 genotype (p < 0.05). CONCLUSIONS: APOE allele and genotype frequencies in the northern Han Chinese population appear to differ from other racial groups or populations living in other regions of China. The APOE E2 variant was associated with a significantly higher warfarin maintenance dose. Thus, APOE polymorphisms could be one of the predictors influencing warfarin doses in this population.

Tripchlorolide improves cognitive deficits by reducing amyloid ß and upregulating synapse-related proteins in a transgenic model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by early impairments in memory and progressive neurodegeneration. Disruption of synaptic plasticity processes that underlie learning and memory contribute partly to this pathophysiology. Tripchlorolide (T4 ), an extract from a traditional Chinese herbal Tripterygium wilfordii Hook F, has been shown to be neuroprotective in animal models of Parkinson's disease and to improve cognitive deficits in senescence-accelerated mouse P8. In this study, we investigated the effect of T4 on cognitive decline and synaptic plasticity in five times familial AD (5XFAD) mice co-expressing mutated amyloid precursor protein and presenilin-1. Five-month-old 5XFAD mice and wild type littermates were intraperitoneally injected with T4 , 5 µg/kg or 25 µg/kg, every other day for 60 days. T4 treatment significantly improved spatial learning and memory, alleviated synaptic ultrastructure degradation, up-regulated expression of synapse-related proteins, including synaptophysin, post-synaptic density-95, N-methyl-D-aspartate receptor subunit 1, phosphorylation of calcium/calmodulin dependent protein kinase II α, and phosphorylation of cyclic AMP-response element binding protein, and promoted activation of the phophoinositide-3-kinase-Akt-mammalian target of rapamycin signaling pathway in 5XFAD mice. Accumulation of amyloid ß (Aß) may contribute to synapse dysfunction and memory impairment in AD. We found that T4 treatment significantly reduced cerebral Aß deposits and lowered Aß levels in brain homogenates. These effects coincided with a reduction in cleavage of ß-carboxyl-terminal amyloid precursor protein (APP) fragment, levels of soluble APPß, and protein expression of ß-site APP cleaving enzyme 1. Taken together, our findings identify T4 as a potent negative regulator of brain Aß levels and show that it significantly ameliorates synaptic degeneration and cognitive deficits in a mouse model of AD.

Tripchlorolide ameliorates experimental autoimmune encephalomyelitis by down-regulating ERK1/2-NF-κB and JAK/STAT signaling pathways.

Tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, has been found to possess anti-inflammatory and immunosuppressive actions. In the current study, these actions were evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis by scoring the clinical signs, observing the infiltration of inflammatory cells and myelin sheath in the lumbar spinal cord of EAE mice. The results demonstrated that T4 (at a dose of 40 µg/kg) significantly reduced the severity of EAE and slowed down the ongoing EAE. Further analysis showed that T4 suppressed the mRNA and protein levels of the transcription factors T-bet and RoRrt and mRNA levels of IFN-γ and IL-17 in the spinal cords. Furthermore, T4 down-regulated the ERK1/2-NF-κB and JAK/STAT signaling pathways. At 40 µg/kg, T4 did not induce side effects on hematological parameters. These findings suggest that T4 ameliorates EAE by immunosuppression, providing a new insight into T4 application in multiple sclerosis treatment.

MiRNA-30a-mediated autophagy inhibition sensitizes renal cell carcinoma cells to sorafenib.

Chemotherapy-induced autophagy activation often contributes to cancer resistance. MiRNA-30a (miR-30a) is a potent inhibitor of autophagy by downregulating Beclin-1. In this study, we characterized the role of miR-30a in sorafenib-induced activity in renal cell carcinoma (RCC) cells. We found that expression of miR-30a was significantly downregulated in several human RCC tissues and in RCC cell lines. Accordingly, its targeted gene Beclin-1 was upregulated. Sorafenib activated autophagy in RCC cells (786-0 and A489 lines), evidenced by p62 degradation, Beclin-1/autophagy protein 5 (ATG-5) upregulation and light chain (LC)3B-I/-II conversion. Exogenously expressing miR-30a in 786-0 or A489 cells inhibited Beclin-1 expression and enhanced sorafenib-induced cytotoxicity. In contrast, knockdown of miR-30a by introducing antagomiR-30a increased Beclin-1 expression, and inhibited sorafenib-induced cytotoxicity against RCC cells. Autophagy inhibitors, including chloroquine, 3-methyaldenine or Bafliomycin A1, enhanced sorafenib activity, causing substantial cell apoptosis. Meanwhile, knockdown of Beclin-1 or ATG-5 by targeted siRNAs also increased sorafenib-induced cytotoxicity in above RCC cells. These findings indicate that dysregulation of miR-30a in RCC may interfere with the effectiveness of sorafenib-mediated apoptosis by an autophagy-dependent pathway, thus representing a novel potential therapeutic target for RCC.