A novel heterozygous deletion in ABCB4 gene in a Chinese family with intrahepatic cholestasis of pregnancy, neonatal hyperbilirubinemia, and cholelithiasis: Case reports and literature review.

BACKGROUND: ABCB4 gene (OMIM *171060) variant is associated with a wide clinical spectrum of hepatobiliary diseases, including familial intrahepatic cholestasis of pregnancy (ICP), progressive familial intrahepatic cholestasis type 3 (PFIC3), and neonatal hyperbilirubinemia due to impaired protection of the bile duct. The majority of reported cases, however, were missense or nonsense variants, with few deletion variant findings in the Chinese population. METHOD: We performed whole genome sequencing and confirmed it with Sanger sequencing of the proband infant and his families. Clinical courses and laboratory results were documented and collected from the proband infant and his mother. We also reviewed other published cases related to genetic variants in ABCB4 in the Chinese population. RESULTS: A 26-year-old Chinese female (II.2) who had recurrent intrahepatic cholestasis of pregnancy and her 49-day-old son (III.4) who had hyperbilirubinemia, both presented with extremely elevated total bile acid, cholestatic dominant pattern liver function abnormalities. They were able to stay relatively stable with mild pruritus on ursodeoxycholic acid treatment. After ruling out other possibilities, genetic sequencing revealed a diagnosis of heterozygous deletion variant NM_018849.3:c.1452_1454del (NP_061337.1:p.Thr485del) in ABCB4, which was not reported before, in the symptomatic mother (II.2), index patient (III.4), and the symptomatic grandmother (I.2). This variant resulted in clinical spectrums of ICP, neonatal hyperbilirubinemia, and cholelithiasis in our pedigree. CONCLUSION: We reported a novel heterozygous deletion variant of the ABCB4 gene in a Chinese family, as well as a literature review of ABCB4-related disorders. We aim to facilitate healthcare professionals to better understand genetic factors as an uncommon cause of hepatobiliary diseases, as well as improve therapeutic strategies in challenging clinical situations such as pregnancy and neonatal care.

Dihydromyricetin alleviates hippocampal ferroptosis in type 2 diabetic cognitive impairment rats via inhibiting the JNK-inflammatory factor pathway.

Type 2 diabetes mellitus (T2DM) is frequently associated with diabetic cognitive impairment (DCI), and recent studies have shown a strong association between DCI and hippocampal ferroptosis. In this study, we administered dihydromyricetin (DHM) or JNK inhibitor SP600125, to T2DM rats and monitored changes in blood glucose levels, conducted behavioral tests, and detected changes in JNK, inflammatory factors and ferroptosis-related indicators. Our findings demonstrated that T2DM rats displayed signs of cognitive impairment (CI), with ferrozine assays indicating elevated iron content in the hippocampus. Concurrently, there was an increase in p-JNK activity and inflammatory factors IL-6 and TNF-α in the hippocampal region of these rats. Furthermore, we observed elevated levels of Fe2+, MDA, ROS, LPO, and ACSL4, along with a decrease in GPX4 and GSH, suggesting the occurrence of hippocampal ferroptosis. SP600125 application reversed these changes in the T2DM rats, although it exhibited no significant effects in the control group. Treatment with high and low doses of DHM led to a reduction in p-JNK expression, inflammatory factor-related proteins, and iron accumulation in the hippocampal region, effectively alleviating hippocampal ferroptosis in T2DM rats. No notable effects of DHM were observed in the control group. To conclude, our study suggests that DHM can potentially alleviate hippocampal ferroptosis of T2DM cognitive impairment rats, primarily by suppressing the JNK-inflammatory factor pathway in the hippocampus.

Encephalomyelitis Caused by Balamuthia mandrillaris in a Woman With Breast Cancer: A Case Report and Review of the Literature.

Balamuthia mandrillaris is one cause of a rare and severe brain infection called granulomatous amoebic encephalitis (GAE), which has a mortality rate of >90%. Diagnosis of Balamuthia GAE is difficult because symptoms are non-specific. Here, we report a case of Balamuthia amoebic encephalomyelitis (encephalitis and myelitis) in a woman with breast cancer. She sustained trauma near a garbage dump 2 years ago and subsequently developed a skin lesion with a Mycobacterium abscessus infection. She experienced dizziness, lethargy, nausea and vomiting, inability to walk, and deterioration of consciousness. Next-generation sequencing of cerebrospinal fluid (CSF) samples revealed B. mandrillaris, and MRI of both brain and spinal cord showed abnormal signals. T-cell receptor (TCR) sequencing of the CSF identified the Top1 TCR. A combination of amphotericin B, flucytosine, fluconazole, sulfamethoxazole, trimethoprim, clarithromycin, pentamidine, and miltefosine was administrated, but she deteriorated gradually and died on day 27 post-admission.

Immune dysfunction following COVID-19, especially in severe patients.

The coronavirus disease 2019 (COVID-19) has been spreading worldwide. Severe cases quickly progressed with unfavorable outcomes. We aim to investigate the clinical features of COVID-19 and identify the risk factors associated with its progression. Data of confirmed SARS-CoV-2-infected patients and healthy participants were collected. Thirty-seven healthy people and 79 confirmed patients, which include 48 severe patients and 31 mild patients, were recruited. COVID-19 patients presented with dysregulated immune response (decreased T, B, and NK cells and increased inflammatory cytokines). Also, they were found to have increased levels of white blood cell, neutrophil count, and D-dimer in severe cases. Moreover, lymphocyte, CD4+ T cell, CD8+ T cell, NK cell, and B cell counts were lower in the severe group. Multivariate logistic regression analysis showed that CD4+ cell count, neutrophil-to-lymphocyte ratio (NLR) and D-dimer were risk factors for severe cases. Both CT score and clinical pulmonary infection score (CPIS) were associated with disease severity. The receiver operating characteristic (ROC) curve analysis has shown that all these parameters and scores had quite a high predictive value. Immune dysfunction plays critical roles in disease progression. Early and constant surveillance of complete blood cell count, T lymphocyte subsets, coagulation function, CT scan and CPIS was recommended for early screening of severe cases.

Characterization of cytokine profile to distinguish latent tuberculosis from active tuberculosis and healthy controls.

BACKGROUND: Tuberculosis (TB) is an infectious disease and its mortality rate ranks first. Latent tuberculosis infection (LTBI) means that a patient is infected with Mycobacterium tuberculosis, but has no relative clinical symptoms. It has been estimated that approximately 10% of patients with LTBI would develop into active tuberculosis. Therefore, it was urgent to search for more efficient biomarkers to discriminate LTBI from healthy population. METHODS: The Luminex assay was employed to detect the quantity of cytokines secreted by mononuclear cells from peripheral blood stimulated with the ESAT6 protein among TB, LTBI and healthy controls. The cytokine profile was analyzed by principal components analysis and the receiver operating characteristic curve analysis. RESULTS: The principal components analysis indicated that LTBI and TB were clearly separated from healthy controls, and that LTBI was also successfully differentiated from healthy controls. The cytokine profiling method to distinguish LTBI from healthy controls has a sensitivity and specificity of 100%. Nine potential biomarkers, including IL-23, IL-21, HGF, Bngf, IL-27, IL-31, IL-1ß, IL-22 and IL-18, were identified, and these cytokines were considered as a potential cytokine complex for more effectively discriminating LTBI from healthy controls. CONCLUSION: IL-23, IL-21, HGF, Bngf, IL-27, IL-31, IL-1ß, IL-22 and IL-18 were demonstrated to be the potential cytokine complex for the assessment between LTBI and healthy controls.

[Effects of dihydromyricetin on high fat diet induced obesity in mice and its mechanism].

OBJECTIVE: To observe the effects of dihydromyricetin (DHM) on obesity induced by high-fat diet in mice, and to explore whether its mechanism of action is related to the promotion of WAT browning. METHODS: Sixty c57bl/6j mice were randomly divided into 6 groups (n=10): ①normal control group (ND group): normal feed feeding; ②Normal control + low dose DHM group (ND+L-DHM group): normal feed feeding was treated with low dose DHM (125 mg/(kg·d)); ③Normal control + high dose DHM group (ND+H-DHM group): normal feed feeding was treated with high dose DHM (250 mg/(kg·d)); ④High-fat diet group (HFD): high-fat diet; ⑤high-fat diet + low-dose DHM group (HFD+L-DHM group): high-fat diet feeding with low-dose DHM; ⑥High-fat diet + high-dose DHM group (HFD+H-DHM group): High-fat diet was treated with high-dose DHM. After 16 weeks, the mice were fasted overnight, blood samples were collected for fasting blood glucose and blood lipids, then the animals were sacrificed, body length was measured, and Lee's index was calculated. After weighing the adipose tissue in the scapula, groin and epididymis, formaldehyde fixation and HE staining were used to observe the fat cells size, immunohistochemistry was used to detect the expression of uncoupling protein 1 (UCP1). The body weight was measured every 4 weeks during the experiment. RESULTS: Compared with the ND group, the body weight of the mice in the HFD group was increased significantly, suggesting that the obese mouse model replicated successfully. In addition, the body fat weight, fat cell diameter, Lee's index and blood glucose of the HFD group were increased significantly, and the expression of UCP1 in the adipocytes was increased. Body weight, fat cell diameter, Lee's index and blood glucose of HFD mice treated with L-DHM and H-DHM were reversed significantly, while the expression of UCP1 in adipocytes was more significantly increased; however, L-DHM and H-DHM had no significant effects on the above indicators in normal mice. CONCLUSION: Dihydromyricetin inhibited high fat diet induced mouse obesity; the mechanism might be associated with promoting WAT browning.

Characteristics and outcomes of acute-on-chronic liver failure patients with or without cirrhosis using two criteria.

The aim of the study was to identify the characteristics and outcomes in acute-on-chronic liver failure (ACLF) patients with or without cirrhosis using two criteria. Patients with acute deterioration of chronic hepatic disease or acute decompensation of cirrhosis were included retrospectively from April 10, 2016 to April 10, 2019. European Association for the Study of the Liver-chronic liver failure (EASL-CLIF) criterion except for consideration of cirrhosis and Chinese Group on the Study of Severe Hepatitis B (COSSH) criterion were used. Clinical features, laboratory data and survival curves were compared between the ACLF patients with and without cirrhosis. A total of 799 patients were included. Among them, 328 had COSSH and EASL ACLF, 197 had COSSH alone, and 104 had EASL alone. There were 11.6% more ACLF with COSSH criterion. Furthermore, EASL ACLF patients with non-cirrhosis vs. cirrhosis had different laboratory characteristics: ALT (423 vs. 154, p < 0.001), AST (303 vs. 157, p < 0.001), γ-GT (86 vs. 75, p < 0.01), and INR (2.7 vs. 2.6, p < 0.001) were significantly higher but creatinine (71 vs. 77, p < 0.01) were significantly lower; but importantly there was no statistical changes between non-cirrhosis and cirrhosis in EASL ACLF patients on 28-day (p = 0.398) and 90-day (p = 0.376) survival curves. However, 90-day (p = 0.030) survival curve was different between non-cirrhosis and cirrhosis in COSSH ACLF patients. COSSH ACLF score (auROC = 0.778 or 0.792, 95%CI 0.706-0.839 or 0.721-0.851) displayed the better prognostic ability for EASL ACLF patients with non-cirrhosis, but CLIF-C ACLF score (auROC = 0.757 or 0.796, 95%CI 0.701-0.807 or 0.743-0.843) still was the best prognostic scoring system in EASL ACLF patients with cirrhosis. In conclusions, EASL definition exhibited better performance on homogeneous identification of ACLF regardless of cirrhosis or non-cirrhosis. And COSSH ACLF score displayed the better prognostic ability for EASL ACLF patients without cirrhosis.

Cellular context- and protein level-dependent interaction of pluripotency factor OCT4A with multiple octamer motifs of the same target gene.

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.

Short-Course Rather Than Low-Dose Amphotericin B May Exert Potential Influence on Mortality in Cryptococcal Meningitis Patients Treated With Amphotericin B Plus Flucytosine Alone or in Combination With Fluconazole.

BACKGROUND: The influence of Amphotericin B (AmB) dose and the addition of fluconazole (Flu) on the AmB + 5-flucytosine (5FC) regimen for cryptococcal meningitis (CM) treatment remain debatable. METHODS: A retrospective study was conducted to compare 44 CM patients treated with AmB + 5FC and 78 CM patients treated with AmB + 5FC + Flu using the propensity score matching method. The effects of AmB dosage, AmB course and Flu addition on the cerebrospinal fluid (CSF) chemical profile recovery, adverse effects, and 90-day mortality were compared between the groups. RESULTS: No differences in adverse effects, the rate of the 14-day CSF chemical profile recovery and 90-day cumulative survival rate (91.2% vs. 87.5%, P = 0.637) were observed between the AmB + 5FC group and the AmB + 5FC + Flu group. However, the incidence rates of hypokalemia (33.9%) and creatinine elevation (7.1%) in patients treated with an AmB dosage of 0.4-0.5 mg/kg/d were less than those (53.0 and 22.7%, respectively) treated with an AmB dosage of 0.6-0.7 mg/kg/d (P = 0.034 and P = 0.018, respectively). The 90-day cumulative survival rate was 70.1% for patients treated with AmB for <14 days and 96.4% for patients treated with AmB for ≥14 days (log-rank P < 0.001). Multivariate Cox proportional hazards models suggested the hazard ratio was 26.8 (95% CI: 3.9-183.2) for patients treated with AmB < 14 days than those treated with AmB ≥ 14 days (P = 0.001). CONCLUSION: Treatment with AmB less than 14 days was associated with a higher 90-day mortality in CM patients. A relative lower dosage but prolonged course of AmB in the +5FC ± Flu regimen led to favorable trends of fewer adverse effects and comparable clinical efficacy.

Multi-perspective predictive modeling for acute kidney injury in general hospital populations using electronic medical records.

OBJECTIVES: Acute kidney injury (AKI) in hospitalized patients puts them at much higher risk for developing future health problems such as chronic kidney disease, stroke, and heart disease. Accurate AKI prediction would allow timely prevention and intervention. However, current AKI prediction researches pay less attention to model building strategies that meet complex clinical application scenario. This study aims to build and evaluate AKI prediction models from multiple perspectives that reflect different clinical applications. MATERIALS AND METHODS: A retrospective cohort of 76 957 encounters and relevant clinical variables were extracted from a tertiary care, academic hospital electronic medical record (EMR) system between November 2007 and December 2016. Five machine learning methods were used to build prediction models. Prediction tasks from 4 clinical perspectives with different modeling and evaluation strategies were designed to build and evaluate the models. RESULTS: Experimental analysis of the AKI prediction models built from 4 different clinical perspectives suggest a realistic prediction performance in cross-validated area under the curve ranging from 0.720 to 0.764. DISCUSSION: Results show that models built at admission is effective for predicting AKI events in the next day; models built using data with a fixed lead time to AKI onset is still effective in the dynamic clinical application scenario in which each patient's lead time to AKI onset is different. CONCLUSION: To our best knowledge, this is the first systematic study to explore multiple clinical perspectives in building predictive models for AKI in the general inpatient population to reflect real performance in clinical application.

Dynamic changes of key metabolites during liver fibrosis in rats.

BACKGROUND: Fibrosis is the single most important predictor of significant morbidity and mortality in patients with chronic liver disease. Established non-invasive tests for monitoring fibrosis are lacking, and new biomarkers of liver fibrosis and function are needed. AIM: To depict the process of liver fibrosis and look for novel biomarkers for diagnosis and monitoring fibrosis progression. METHODS: CCl4 was used to establish the rat liver fibrosis model. Liver fibrosis process was measured by liver chemical tests, liver histopathology, and Masson's trichrome staining. The expression levels of two fibrotic markers including α-smooth muscle actin and transforming growth factor ß1 were assessed using immunohistochemistry and real-time polymerase chain reaction. Dynamic changes in metabolic profiles and biomarker concentrations in rat serum during liver fibrosis progression were investigated using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The discriminatory capability of potential biomarkers was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: To investigate the dynamic changes of metabolites during the process of liver fibrosis, sera from control and fibrosis model rats based on pathological results were analyzed at five different time points. We investigated the association of liver fibrosis with 21 metabolites including hydroxyethyl glycine, L-threonine, indoleacrylic acid, ß-muricholic acid (ß-MCA), cervonoyl ethanolamide (CEA), phosphatidylcholines, and lysophosphatidylcholines. Two metabolites, CEA and ß-MCA, differed significantly in the fibrosis model rats compared to controls (P < 0.05) and showed prognostic value for fibrosis. ROC curve analyses performed to calculate the area under the curve (AUC) revealed that CEA and ß-MCA differed significantly in the fibrosis group compared to controls with AUC values exceeding 0.8, and can clearly differentiate early stage from late stage fibrosis or cirrhosis. CONCLUSION: This study identified two novel biomarkers of fibrosis, CEA and ß-MCA, which were effective for diagnosing fibrosis in an animal model.

Comprehensive Analysis of Lysine Acetylome Reveals a Site-Specific Pattern in Rapamycin-Induced Autophagy.

Protein acetylation reportedly acts as a key regulator of autophagy. However, up to now, the relationship between acetylome and autophagy has remained unclear. Here stable isotope labeling of amino acids in cell culture and high-throughput quantitative mass spectrometry were used to perform an acetylome analysis of rapamycin-induced autophagy in vitro. Our data revealed that 2135 sites were quantified on 1081 proteins. During autophagy, 421 sites were significantly regulated on 296 proteins, with 80.8% of sites downregulated and 19.2% upregulated. Motif enrichment analysis revealed five main motifs. Most of the downregulated sites conformed to the classical functional motif of p300/CBP [G-AcK]. Furthermore, acetylation targeted proteins involved mainly in ribosomes, spliceosomes, and AcCoA-related metabolic process. In-depth analysis indicated that most of the acetylation sites were in the critical domain, were functional sites, or could change their enzymatic activity by acetylation, highlighting the importance of site-specific acetylation patterns. Subsequently, we demonstrated that K1549 of p300 was also a functional site that could regulate the autophagic process in vitro. In conclusion, our data reveal a deacetylation-preponderant profile with autophagy. The specificity of the related motifs and the identification of site-specific acetylation patterns will assist searches for potential targets or subsequent mechanism-focused studies to elucidate site-specific protein networks in autophagy.

Endogenous authentic OCT4A proteins directly regulate FOS/AP-1 transcription in somatic cancer cells.

OCT4A is well established as a master transcription factor for pluripotent stem cell (PSC) self-renewal and a pioneer factor for initiating somatic cell reprogramming, yet its presence and functionality in somatic cancer cells remain controversial and obscure. By combining the CRISPR-Cas9-based gene editing with highly specific PCR assays, highly sensitive immunoassays, and mass spectrometry, we provide unequivocal evidence here that full-length authentic OCT4A transcripts and proteins were both present in somatic cancer cells, and OCT4A proteins were heterogeneously expressed in the whole cell population and when expressed, they are predominantly localized in cell nucleus. Despite their extremely low abundance (approximately three orders of magnitude lower than in PSCs), OCT4A proteins bound to the promoter/enhancer regions of the AP-1 transcription factor subunit c-FOS gene and critically regulated its transcription. Knocking out OCT4A in somatic cancer cells led to dramatic reduction of the c-FOS protein level, aberrant AP-1 signaling, dampened self-renewal capacity, deficient cell migration that were associated with cell growth retardation in vitro and in vivo, and their enhanced sensitivity to anticancer drugs. Taken together, we resolve the long-standing controversy and uncertainty in the field, and reveal a fundamental role of OCT4A protein in regulating FOS/AP-1 signaling-centered genes that mediate the adhesion, migration, and propagation of somatic cancer cells.

Causal risk factor discovery for severe acute kidney injury using electronic health records.

BACKGROUND: Acute kidney injury (AKI), characterized by abrupt deterioration of renal function, is a common clinical event among hospitalized patients and it is associated with high morbidity and mortality. AKI is defined in three stages with stage-3 being the most severe phase which is irreversible. It is important to effectively discover the true risk factors in order to identify high-risk AKI patients and allow better targeting of tailored interventions. However, Stage-3 AKI patients are very rare (only 0.2% of AKI patients) with a large scale of features available in EHR (1917 potential risk features), yielding a scenario unfeasible for any correlation-based feature selection or modeling method. This study aims to discover the key factors and improve the detection of Stage-3 AKI. METHODS: A causal discovery method (McDSL) is adopted for causal discovery to infer true causal relationship between information buried in EHR (such as medication, diagnosis, laboratory tests, comorbidities and etc.) and Stage-3 AKI risk. The research approach comprised two major phases: data collection, and causal discovery. The first phase is propose to collect the data from HER (includes 358 encounters and 891 risk factors). Finally, McDSL is employed to discover the causal risk factors of Stage-3 AKI, and five well-known machine learning models are built for predicting Stage-3 AKI with 10-fold cross-validation (predictive accuracy were measured by AUC, precision, recall and F-score). RESULTS: McDSL is useful for further research of EHR. It is able to discover four causal features, all selected features are medications that are modifiable. The latest research of machine learning is employed to compare the performance of prediction, and the experimental result has verified the selected features are pivotal. CONCLUSIONS: The features selected by McDSL, which enable us to achieve significant dimension reduction without sacrificing prediction accuracy, suggesting potential clinical use such as helping physicians develop better prevention and treatment strategies.

Dihydromyricetin improves type 2 diabetes-induced cognitive impairment via suppressing oxidative stress and enhancing brain-derived neurotrophic factor-mediated neuroprotection in mice.

Type 2 diabetes mellitus (T2DM) leads to cognitive impairment (CI), but there have been no effective pharmacotherapies or drugs for cognitive dysfunction in T2DM. Dihydromyricetin (DHM) is a natural flavonoid compound extracted from the leaves of Ampelopsis grossedentata and has various pharmacological effects including anti-oxidant and anti-diabetes. Thus, we investigated the effects of DHM on CI in T2DM mouse model and its possible mechanism. To induce T2DM, mice were fed with high-sugar and high-fat diet for 8 weeks, followed by a low dose streptozotocin (STZ) administration. After the successful induction of T2DM mouse model, mice were treated respectively with equal volume of saline (T2DM group), 125 mg/kg/d DHM (L-DHM group), or 250 mg/kg/d DHM (H-DHM group). After 16 weeks of DHM administration, the body weight (BW), fasting blood glucose, blood lipids, intraperitoneal glucose tolerance (IPGT), and cognitive function were determined. Then, alterations in the expressions of oxidative stress markers and brain-derived neurotrophic factor (BDNF) in the hippocampus were investigated. Our findings demonstrated that DHM could significantly ameliorate CI and reverse aberrant glucose and lipid metabolism in T2DM mice, likely through the suppression of oxidative stress and enhancement of BDNF-mediated neuroprotection. In conclusion, our results suggest that DHM is a promising candidate for the treatment of T2DM-induced cognitive dysfunction.

[The effects of arecoline on the lipid metabolism of 3T3-L1 adipocytes].

OBJECTIVE: To observe the effects of arecoline on lipid metabolism in 3T3-L1 adipocytes and explore its possible mechanisms. METHODS: 3T3-L1 pre-adipocytes were induced into adipocytes with the classic "cocktail" method, subsequently, adipocytes were treated with arecoline at the concentrations of 0, 25, 50 and 100 µmol/L for 72 hours. After 72 hours, cell vability was measured with MTT method, lipid droplet accumulation in the cytoplasm was observed with oil red O staining, the protein expression of fatty acid synthase (FAS), adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were detected by Western blot assay. RESULTS: There were a large number of lipid droplets in the cytoplasm in the differentiated 3T3-L1 adipocytes. MTT results showed that 0~100 µmol/L arecoline had no significant effect on cell vability; oil red O staining found arecoline reduced lipid amount in 3T3-L1 adipocytes; Western blot results showed that compared with 0 µmol/L arecoline group (the control group), arecoline significantly reduced the protein level of FAS and increased the protein levels of ATGL and HSL, and 50 µmol/L arecoline group was the most significant. CONCLUSIONS: Arecoline significantly increased lipolysis of 3T3-L1 adipocyte, which might be associated with decreased the FAS expression of key enzyme of lipid synthesis and increased the ATGL and HSL expression of key enzyme of adipolysis.

[The effects of dihydromyricetin on cognitive dysfunction in type 2 diabetes mice].

OBJECTIVE: To observe the effects of dihydromyricetin(DHM) on cognitive dysfunction and expression of brain derived neurotrophic factor(BDNF) protein in hippocampus of type 2 diabetic mice(T2DM). METHODS: Forty C57BL/6J mice were randomly divided into two groups, normal control group (n=8):normal diet feeding; T2DM model group (n=32):high-glucose and high-fat combined with 100 mg/kg streptozocin(STZ) treatment (five mice died during modeling and three failed). Twenty-four diabetic mice were modeled successfully and divided into three groups (T2DM group, T2DM+L-DHM group and T2DM+H-DHM group). Three groups mice were fed with high-glucose and high-fat diet, and treated with equal volume of normal saline, 125 mg/(kg·d) DHM or 250 mg/(kg·d) DHM for 16 weeks respectively. The control mice were fed with normal diet and treated with equal volume of saline (once a day, gavage) for 16 weeks. After 16 weeks, the body weight and fasting blood glucose were measured, intraperitoneal glucose tolerance test and related behavioral experiment were performed. Finally, the expression of BDNF protein in hippocampus of mice was detected by Western blot. RESULTS: The model of type 2 diabetes mellitus was established successfully with high-glucose and high-fat combined with 100 mg/kg STZ. After 16 weeks, the body weight of T2DM group was significantly decreased, the fasting blood glucose was significantly increased and the glucose tolerance was significantly abnormal compared with the normal control group. Compared with T2DM group, the body weight of T2DM+DHM groups mice was increased, while the levels of fasting blood glucose were decreased. And H-DHM could significantly improve the abnormal glucose tolerance of T2DM mice. Behavior test results showed that the ability of learning and memory of T2DM mice was significant decreased compared with control group, but these phenomena were improved in T2DM+DHM groups mice, and T2DM+H-DHM group was more obvious. Western blot analysis showed that the expression of BDNF protein in hippocampus of T2DM group was significantly lower than that of control group, while T2DM+DHM group was significant increased compared with T2DM mice. CONCLUSIONS: Dihydromyricetin can improve the cognitive dysfunction in type 2 diabetic mice. The mechanism may be through hypoglycemic effect and activation of BDNF protein expression in hippocampus.

[Ameliorative effects and the mechanism of lyceum barbarum polysaccharide on insulin resistance of HepG2 cell].

OBJECTIVE: To observe the effects of lyceum barbarum polysaccharide (LBP) on insulin resistance of HepG2 cells and investigate its possible mechanism. METHODS: IR-HepG2 cell model was induced with high glucose and high insulin in combination for 24 hours,with 104/vaccination in the 96-well plates, hole density after adherent cells (30 µg/ml、100 µg/ml、300 µg/ml) LBP cultivate 48 h, 200 µl/hole, each all had four holes. The effects of LBP at different concentrations on HepG2 cell activity and insulin resistance were tested. Intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected. The expressions of related proteins in insulin signal transduction pathways such as insulin receptor substrate-2(IRS-2), phosphatidylinositol-3-kinase(PI3-K), protein kinase B(Akt) and glucose transport-2(GLUT2) were determined. RESULTS: Compared with normal control group, the content of MDA was increased significantly and the activity of SOD and the expression levels of IRS-2,PI-3K,Akt and GLUT2 were decreased significantly in the IR model group. Compared with IR model group, medium and high concentrations of LBP decreased the content of MDA and increased the activity of SOD and the expression levels of IRS-2, PI-3K, Akt and GLUT2 in insulin-resistant HepG2 cells. MTT showed that at the same time, the OD value gradually decreased with the increase of LBP's concentration; under the same concentration of LBP, the OD value also gradually decreased with the extension of time, which indicated that LBP inhibited the proliferation of HepG2 cells with time and concentration-dependent manner. Glucose consumption experiment indicated that medium and high concentration of LBP could increase the glucose consumption of insulin-resistant HepG2 cells significantly, but low concentration of LBP had no significant impacted on glucose consumption of insulin-resistant HepG2 cells. CONCLUSIONS: Medium and high concentration of LBP can improve insulin resistance of HepG2 cell, its mechanisns may be associated with decreasing the level of oxidative stress and increasing the protein expressions of insulin signaling pathway.

Slow reduction of IP-10 Levels predicts HBeAg seroconversion in chronic hepatitis B patients with 5 years of entecavir treatment.

The aim of this study was to determine the correlation between dynamic changes in serum cytokine/chemokine expression levels in response to entecavir (ETV) treatment and HBV e antigen (HBeAg) seroconversion in patients with chronic hepatitis B (CHB). Four cytokines (interleukin [IL]-4, IL-6, IL-8, and interferon-γ) and five chemokines (macro-phage inflammatory protein [MIP]-1α, MIP-1ß, platelet derived growth factor-BB, and interferon-inducible protein 10 [IP-10]) before ETV therapy and at 3, 6, 12, 24, 36 and 60 months during therapy in 105 CHB patients were analyzed. The results showed that the low decrease rate of IP-10 levels after 1 year of ETV treatment was an independent predictor of HBeAg seroconversion at year 5 (Hazard ratio = 0.972). The area under the receiver operating characteristic curves for the decrease rate of IP-10 levels after 1 year of treatment to discriminate a year-5 HBeAg seroconversion was 0.752 (p = 0.005). The results indicate that higher IP-10 level at year one of ETV treatment is associated with an increased probability of HBeAg seroconversion. Quantification of IP-10 during ETV treatment may help to predict long-term HBeAg seroconversion in patients with CHB.

[Effect of arecoline on proliferation and apoptosis of MCF-7 human breast cancer cells].

OBJECTIVE: To observe the effects of arecoline on proliferation and apoptosis of MCF-7 human breast cancer cells and to ex-plore its possible mechanism. METHODS: Human breast cancer MCF-7 cells were treated with arecoline at the concentrations of 0,10,30,50, 100,300,500µmol/L, the cell proliferation were detected by MTT assay, cell apoptosis were analyzed by Hoechst 33342 staining and flow cy-tometry, the protein expression of Bax,Bcl-2 and P53 were detected by Western blot. RESULTS: Low concentration(0,10,30, 50 µmol/L) arecoline had no effect on the proliferation and apoptosis of MCF-7. However, high concentration(100,300,500µmol/L) arecoline inhibited proliferation and induced apoptosis of MCF-7 cells in a concentration-dependent manner, arecoline also significantly increased P53 and Bax protein expression and decreased Bcl-2 protein expression. CONCLUSIONS: High concentration arecoline inhibited the proliferation and induced the apoptosis of MCF-7 cells, the mechanism was probably corrected with increasing P53 and Bax protein expression and decreasing Bcl-2 pro-tein expression.