Differences in MicroRNA Expression in Chronic Hepatitis B Patients with Early Liver Fibrosis Based on Traditional Chinese Medicine Syndromes.

The aim of this study was to determine if microRNA (miRNA) expression is different among chronic hepatitis B (CHB) patients with early liver fibrosis classified according to traditional Chinese medicine (TCM) syndromes. Eighteen CHB-fibrosis patients and 12 CHB patients without fibrosis were enrolled. The CHB-fibrosis group included 9 patients with the TCM syndrome of Ganyu Pixu Xueyu (GYPXXY), characterized by liver stagnation, spleen deficiency, and blood stasis, and 9 patients with the TCM syndrome of Qixu Xueyu (QXXY), characterized by deficiency of qi, blood, and blood stasis. Agilent miRNA microarray was performed first in liver specimens to determine whether miRNA expression is different in patients with these two TCM syndromes of CHB-fibrosis. Gene Ontology (GO) analysis and KEGG analysis were applied to determine the roles of the differentially expressed miRNAs. QRT-PCR was performed to validate the Agilent miRNA microarray results. Compared with GYPXXY patients, 6 differentially expressed miRNAs were upregulated (miR-144-5p, miR-18a-5p, miR-148b-3p, miR-654-3p, miR-139-3p, and miR-24-1-5p) and 1 was downregulated (miR-6834-3p) in QXXY patients. According to qRT-PCR data, miR-144-5p and miR-654-3p were confirmed as upregulated in CHB-liver fibrosis patients compared to CHB patients without fibrosis, whereas the other 4 miRNAs were not significantly different. More importantly, miR-654-3p was confirmed to be significantly upregulated in QXXY patients compared with values in GYPXXY patients, whereas no significant difference was found in miR-144-5p. Moreover, the pathways of central carbon metabolism in cancer and cell cycle related to miR-654-3p and the target genes of PTEN and ATM were found to be different between QXXY patients and GYPXXY patients. These results indicate that there are different miRNAs, pathways, and target genes between QXXY patients and GYPXXY patients. However, due to the limited sample, whether miR-654-3p and the target genes PTEN and ATM could be molecular markers to differentiate TCM syndromes could not be established.

Metformin Ameliorates Aß Pathology by Insulin-Degrading Enzyme in a Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease. The accumulation of amyloid beta (Aß) is the main pathology of AD. Metformin, a well-known antidiabetic drug, has been reported to have AD-protective effect. However, the mechanism is still unclear. In this study, we tried to figure out whether metformin could activate insulin-degrading enzyme (IDE) to ameliorate Aß-induced pathology. Morris water maze and Y-maze results indicated that metformin could improve the learning and memory ability in APPswe/PS1dE9 (APP/PS1) transgenic mice. 18F-FDG PET-CT result showed that metformin could ameliorate the neural dysfunction in APP/PS1 transgenic mice. PCR analysis showed that metformin could effectively improve the mRNA expression level of nerve and synapse-related genes (Syp, Ngf, and Bdnf) in the brain. Metformin decreased oxidative stress (malondialdehyde and superoxide dismutase) and neuroinflammation (IL-1ß and IL-6) in APP/PS1 mice. In addition, metformin obviously reduced the Aß level in the brain of APP/PS1 mice. Metformin did not affect the enzyme activities and mRNA expression levels of Aß-related secretases (ADAM10, BACE1, and PS1). Meanwhile, metformin also did not affect the mRNA expression levels of Aß-related transporters (LRP1 and RAGE). Metformin increased the protein levels of p-AMPK and IDE in the brain of APP/PS1 mice, which might be the key mechanism of metformin on AD. In conclusion, the well-known antidiabetic drug, metformin, could be a promising drug for AD treatment.

Sodium tanshinone IIA sulfonate protects against Aß-induced cell toxicity through regulating Aß process.

Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aß-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aß (10 µmol/L)-induced cell toxicity in a dose-dependent manner in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL-1ß, IL-6 and TNF-α) in the SH-SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin-degrading enzyme were up-regulated in the SH-SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aß. ELISA and qPCR results indicated that STS could increase α-secretase (ADAM10) activity and decrease ß-secretase (BACE1) activity. In conclusion, STS could protect against Aß-induced cell damage by modulating Aß degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.

Quercetin protects against diabetic encephalopathy via SIRT1/NLRP3 pathway in db/db mice.

Epidemiological studies have found that diabetes and cognitive dysfunction are closely related. Quercetin has been certified with the effect on improving diabetes mellitus (DM) and cognitive impairment. However, the effect and related mechanism of quercetin on diabetic encephalopathy (DE) are still ambiguous. In this study, we used the db/db mice (diabetic model) to discover whether quercetin could improve DE through the Sirtuin1/NLRP3 (NOD-, LRR- and pyrin domain-containing 3) pathway. Behavioural results (Morris water maze and new object recognition tests) showed that quercetin (70 mg/kg) improved the learning and memory. Furthermore, quercetin alleviated insulin resistance and the level of fasting blood glucose. Besides, Western blot analysis also showed that quercetin increased the protein expressions of nerve- and synapse-related protein, including postsynapticdensity 93 (PSD93), postsynapticdensity 95 (PSD95), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of db/db mice. Quercetin also increased the protein expression of SIRT1 and decreased the expression of NLRP3 inflammation-related proteins, including NLRP3, the adaptor protein ASC and cleaved Caspase-1, the pro-inflammatory cytokines IL-1ß and IL-18. In conclusion, the present results indicate that the SIRT1/NLRP3 pathway may be a crucial mechanism for the neuroprotective effect of quercetin against DE.

Autoantibodies in patients with interleukin 12 receptor beta 1 deficiency.

OBJECTIVE: Interleukin 12 receptor beta 1 (IL-12Rß1) deficiency is a primary immunodeficiency that exposes affected individuals to an augmented risk of intracellular pathogen-mediated infections. The paradoxical presence of autoimmune manifestations in immune-deficient patients has been recognized, but the basis of this phenomenon is unclear, with the role of frequent infections being a possible trigger to break tolerance. Our study aimed to analyze extensively a profile of autoantibodies in a clinically well-defined case series of patients with IL-12Rß1 deficiency. METHODS: Eight patients with IL-12Rß1 deficiency referred to Children's Medical Center in Tunis, Tunisia, during 1995-2012 were enrolled in the study. Sixteen age- and gender-matched blood donors served as controls. Serum, liver-related autoantibodies immunoglobulin (Ig)G, IgM, IgA were tested by ELISA and by standard indirect immunofluorescence on Hep-2 cells. RESULTS: We found a significant prevalence of liver autoantibodies in the study group. Regarding primary biliary cholangitis (PBC), two of eight patients were positive for MIT3 autoantibodies, both confirmed by immunofluorescence, and one patient was positive for PBC-specific antinuclear antibodies, sp100. Moreover, two patients had significantly increased gamma-glutamyltransferase levels and one had IgM levels twice the upper limit of normal. Intriguingly two patients were positive for anti-actin antibodies; a typical feature of autoimmune hepatitis type 1, along with a significant increase in IgG levels. CONCLUSIONS: This is the first report of a serological analysis in patients with an IL-12Rß1 deficiency. Despite the difficulty in interpreting the role of the IL-12, the evidence of liver-specific autoantibodies confirms the importance its signal in liver autoimmunity.

CGY-1, a biflavonoid isolated from cardiocrinum giganteum seeds, improves memory deficits by modulating the cholinergic system in scopolamine-treated mice.

Certain biflavonoids have been proven to protect against cognitive dysfunction. A new biflavonoid, CGY-1, isolated from Cardiocrinum giganteum seeds, has not yet been reported to have any neuroprotective effect. In this study, a scopolamine-induced memory deficit model was used to explore the neuroprotective effect of CGY-1. Behavioral experiments, such as tests using the Morris water maze, the Y-maze and the fear conditioning test, were conducted. The results revealed that oral administration of CGY-1 (20 and 40 mg/kg) and donepezil shortened the escape latency, improved the percentage of spontaneous alternation, and increased the freezing times, respectively. CGY-1 decreased the levels of reactive oxygen species and malondialdehyde and increased the activities of superoxide dismutase and glutathione peroxidase in the hippocampus. In addition, CGY-1 decreased the activity of acetylcholinesterase and increased the activities of choline acetyltransferase and acetylcholine in the hippocampus. Furthermore, qPCR and western blot results revealed that the expressions of neurotrophic factors, brain-derived neurotrophic factor and nerve growth factor were upregulated in the hippocampus after CGY-1 treatment. In conclusion, CGY-1 could be a promising candidate for the treatment of cognitive dysfunction.

Sodium tanshinone IIA sulfonate ameliorates hepatic steatosis by inhibiting lipogenesis and inflammation.

Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic disease in adults and children worldwide. Importantly, there are currently no approved treatments available for NAFLD. This study aims to investigate the potential applications of sodium tanshinone IIA sulfonate (STS) on improving the NAFLD condition using both in vitro and in vivo approaches. The results showed that STS markedly inhibited lipid accumulation in oleic acid (OA) and palmitic acid (PA) treated HepG2 and primary immortalized human hepatic (PIH) cells. STS suppressed lipogenesis by inhibiting expression of sterol regulatory element binding transcription factor 1 (SREBF1), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD). In addition, STS reduced inflammation in cells treated with OA-PA, shown by decreased transcriptional levels of tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1) and interleukin 1 beta (IL1B). Consistently, protective effects on hepatic steatosis in db/db mice were observed after STS administration, demonstrated by decreased lipid accumulation in mouse hepatocytes. This protective effect might be associated with STS induced activation of sirtuin 1 (SIRT1)/protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1) pathways. Our findings suggest a potential therapeutic role for STS in the treatment of NAFLD.

Serological thymidine kinase 1 is a biomarker for early detection of tumours--a health screening study on 35,365 people, using a sensitive chemiluminescent dot blot assay.

Serological thymidine kinase 1 (STK1) is a reliable proliferation marker for prognosis, monitoring tumour therapy, and relapse. Here we investigated the use of STK1 in health screening for early detection of pre-malignant and malignant diseases. The investigation was based on 35,365 participants in four independent health screening studies in China between 2005-2011. All participants were clinically examined. The concentration of STK1 was determined by a sensitive chemiluminescent dot blot ECL assay. The ROCvalue of the STK1 assay was 0.96. At a cut-off STK1 value of 2.0 pM, the likelihood (+) value was 236.5, and the sensitivity and the specificity were 0.78 and 0.99, respectively. The relative number of city-dwelling people with elevated STK1 values (≥2.0 pM) was 0.8% (198/26,484), while the corresponding value for the group of oil-field workers was 5.8% (514/8,355). The latter group expressed significantly higher frequency of refractory anaemia, fatty liver, and obesity, compared to the city dwellers, but no cases of breast hyperplasia or prostate hyperplasia. Furthermore, people working in oil drilling/oil transportation showed higher STK1 values and higher frequency of pre-malignancies and benign diseases than people working in the oil-field administration. In the STK1 elevated group of the city-dwelling people, a statistically significantly higher number of people were found to have malignancies, pre-malignancies of all types, moderate/severe type of hyperplasia of breast or prostate, or refractory anaemia, or to be at high risk for hepatitis B, compared to people with normal STK1 values (<2.0 pM). No malignancies were found in the normal STK1 group. In the elevated STK1 group 85.4% showed diseases linked to a higher risk for pre-/early cancerous progression, compared to 52.4% of those with normal STK1 values. Among participants with elevated STK1 values, 8.8% developed new malignancies or progress in their pre-malignancies within 5 to 72 months, compared to 0.2% among people with normal STK1 values. People who showed elevated STK1 values were at about three to five times higher risk to develop malignancies compared to a calculated risk based on a cancer incidence rate of 0.2-0.3%. We conclude that serological TK1 protein concentration is a reliable marker for risk assessment of pre/early cancerous progression.

[Investigation of the effect of 2-methoxyestradiol and arsenic trioxide on the apoptosis-associated gene expression profile of myeloma cells].

OBJECTIVE: To explore the effect of 2-methoxyestradiol (2ME2) and arsenic trioxide (As2O3) on the apoptosis related gene expression in multiple myeloma cell line CZ-1. METHODS: Total RNA was isolated from CZ-1 cells which had been treated with 2ME2 and As2O3 at an apoptosis-inducing concentration and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP, and then hybridized it with a microarray containing up to 96 key genes involved in apoptosis. The gene expression profile of the 2ME2 and As2O3 treated CZ-1 cells were analyzed with GEArray Analyzer software. The microarray results were confirmed by RT-PCR. RESULTS: As2O3 treatment caused the alteration in the expression of 52 genes (54.2% of total genes on microarray). Among them, 42 (80.8%) were upregulated and 10 (19.2%) were downregulated. The upregulated genes were mainly involved in caspases family, P53 and ATM pathway, death effector domain family, TNF receptor family and CIDE family. 2ME2 treatment resulted in the alteration of 42 genes (43.8% of the total genes on microarray). Of them, 32 (76.2%) were downregulated and 10 (23.8%) were upregulated. The downregulated genes mainly belonged to bcl-2 family, inhibitor of apoptosis protein family (IAP), TRAF family, TNF ligand family, and CARD family. CONCLUSION: 2ME2 and As2O3 induce the CZ-1 cells apoptosis by different pathways. As2O3 mainly induces upregulation of proapoptotic genes, and 2ME2 downregulation of anti-apoptotic genes expression.

A clinical study on the treatment of chronic pelvic inflammation of Qi-stagnation with blood stasis syndrome by Penyanqing capsule.

OBJECTIVE: To observe the clinical efficacy of Penyanqing Capsule (PYQC) in treating pelvic inflammation of Qi-stagnation with blood stasis syndrome. METHODS: The randomized, single blinded, parallel positive drug controlled method was adopted, with 82 patients assigned into two groups by envelop method. The 42 patients in the treated group received PYQC 3 times a day, 4 capsules each time taken orally; the 40 patients in the control group were given orally Fuyankang tablets (FYKT) 3 times a day, 6 tablets each time. The therapeutic course for both groups was 2 months, and 2 courses of treatment were given successively to observe the comprehensive effect, changes of symptoms and signs before and after treatment. The effects of PYQC on hemorrheological character in part of the patients and on the pathogenetic chlamydia and mycoplasma were also observed. RESULTS: The total effective rate in the treated group was 83.3%, which was insignificantly different from that in the control group (77.5%, P > 0.05). However, PYQC could significantly lower the hemorrheologic indexes in patients and showed definite influence on the pathogenetic chlamydia and mycoplasma. CONCLUSION: PYQC has good therapeutic effect in treating chronic pelvic inflammation of Qi-stagnation with blood stasis syndrome, and showed definite effect on chlamydia and mycoplasma.

[Genetic association between interleukins gene polymorphisms with primary biliary cirrhosis in Chinese population].

OBJECTIVE: To determine the relationship between polymorphisms in the genes encoding IL-1, IL-6, and IL-10 with primary biliary cirrhosis (PBC) in Chinese population. METHODS: Whole-blood samples were taken from 77 patients with PBC and 160 healthy controls. DNA was extracted and the polymorphisms at positions IL-1 +3953, IL-1RN intron 2, IL-6 -174, and IL-10 -1082, -819, and -592 were determined by using sequence-specific polymerase chain reaction (SSP) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequency of IL-1RN1,1 allele in PBC group was significantly higher than in control group (90.9% vs 79.4%, P=0.026), and the frequency of IL-1RN1,2 in PBC group was significantly lower than in control group (6.5% vs 18.8%, P=0.013). There was no significant difference in the frequence of IL-1RN*2 allele between PBC group and control group (P=0.06). Of the 77 patients with PBC, 4 patients were IL-6 -174GC, 73 were IL-6 174GG. All the 160 health controls are IL-6 -174GG (P=0.0036). The frequence of IL-6 -174C allele in PBC group was significantly higher than that in control group (P=0.0038). No significant differences of polymorphisms for IL-1 +3953 and IL-10 (-1082, -819 and -592) were found between PBC group and control group. CONCLUSION: The polymorphisms of IL-1RN and IL-6 -174G/C appear to be associated with PBC, and the polymorphisms of IL-1 +3953 and IL-10 promoter gene are not associated with PBC in a Chinese population.

[Study on the relationship of CTLA-4 -318, +49 polymorphisms with autoimmune hepatitis and primary biliary cirrhosis in a Chinese population].

OBJECTIVE: To investigate the association between Chinese patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and the polymorphisms of cytotoxic T lymphocyte -associated antigen-4 (CTLA-4) gene promoter (-318) and exon 1 (+49). METHODS: The CTLA-4 promoter (-318 T/C) and exon 1 (+49A/G) polymorphisms were genotyped via restriction fragment length polymorphism methods in 62 Chinese AIH patients, 77 Chinese PBC patients and 160 healthy controls. RESULTS: There was no difference in the distribution of CTLA-4 promoter -318 T/C polymorphisms between AIH patients and controls, but the C allele frequency was significantly increased in patients with AIH, compared to controls (P=0.02, OR=2.43). The distribution of CTLA-4 gene exon 1 49 A/G genotypes exhibited significant difference between PBC patients and controls (P=0.006), and the frequency of G allele showed a significant increase in PBC group as compared with controls (P=0.0046, OR=1.8). Although the genotype distribution of the CTLA-4 exon 1-promoter gene displayed no significant difference between AIH and PBC patients and controls, the occurrence of GG-CC was increased in the patients of the two groups (AIH: 32.3%, PBC: 37.7%; control: 22.5%). CONCLUSION: The above findings suggest that the polymorphisms of CTLA-4 gene probably confer susceptibility to AIH and PBC in the Chinese population.

[A study on the relationship between interleukin-10 promoter polymorphism and autoimmune liver disease].

OBJECTIVE: To investigate whether three biallelic polymorphisms at the position -592, -819 and -1082 in the promoter region of the IL-10 gene were associated with the incidence of autoimmune liver disease. METHODS: The IL-10 -592 and IL-10-1082 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphisms analysis (PCR-RFLP), while polymerase chain reaction- sequence specific primer (PCR-SSP) assay was used to detect IL-10 -819 polymorphisms. RESULTS: Among 54 Chinese patients with AIH or 77 Chinese patients with PBC versus healthy controls, the frequency of AA, GA genotypes at IL-10 gene promoter -1082 position was 87.0% or 83.1% versus 90.0%, 13.0% or 16.9% versus 10.0%, respectively (P > 0.05), the GG genotype in Chinese populations is absent; the frequency of CC, CT, TT genotypes at IL-10 gene promoter -819 position was 11.11% or 9.1% versus 8.1%, 44.4% or 53.3% versus 45.0%, 44.4% or 37.7% versus 46.9%, respectively (P > 0.05); the frequency of CC, CA, AA genotypes at IL-10 gene promoter -592 position was 4.9% or 14.3% versus 10.0%, 51.2% or 53.3% versus 51.9%, 43.9% or 32.5% versus 38.1%, respectively (P > 0.05). No alleles differed significantly in each groups. CONCLUSION: There were no association between IL-10 gene polymorphisms and autoimmune liver disease