GW4064, a farnesoid X receptor agonist, upregulates adipokine expression in preadipocytes and HepG2 cells.

AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells. METHODS: The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064. RESULTS: The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064. CONCLUSION: The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.

miR-96 and miR-330 overexpressed and targeted AQP5 in lipopolysaccharide-induced rat lung damage of disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a severe clinical condition that can lead to or aggravate the development of multiple organ dysfunction syndrome. Of all types of organ damage, lung damage is the most frequent and most severe. In DIC patients, lung damage is primarily characterized by pulmonary edema. Aquaporin (AQP) 5 is the chief AQP in the lungs and it plays a key role in many processes, including water transport in normal and abnormal lungs. Here we demonstrate that expression of AQP5 and two microRNAs, miR-96 and miR-330, in rat lung of lipopolysaccharide (LPS)-induced DIC. We also show that both miR-96 and miR-330 can regulate the expression of AQP5 by binding with its 3'-untranslated region (UTR) by luciferase activity assay. These results suggest that microRNAs are involved in lung damage in LPS-induced rat DIC and can be a potential target for molecular therapy.

[Effects of farnesoid X receptor agonist on adiponectin and its receptors].

OBJECTIVE: To investigate the effects of GW4064, a farnesoid X receptor (FXR) agonist, on adiponectin and its receptors during the differentiation of 3T3-L1 preadipocytes and on adiponectin receptors in HepG2 cells. METHODS: The mRNA expressions of FXR, PPARγ2, adiponectin, AdipoR1, and AdipoR2 and the protein levels of adiponectin on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes treated with GW4064 were detected by fluorescent real-time PCR and ELISA, respectively. The mRNA expressions of AdipoR1 and AdipoR2 in HepG2 cells were also examined at 0, 12, 24, and 48 h after GW4064 treatment. RESULTS: The mRNA expressions of FXR, PPARγ2, adiponectin, and AdipoR2 in 3T3-L1 preadipocytes and AdipoR2 in HepG2 cells treated with GW4064 was significantly increased compared with the control group (all P<0.05). The protein level of adiponectin was also significantly increased after GW4064 treatment. The expression of AdipoR1 in either 3T3-L1 preadipocytes or HepG2 cells showed no significant changes after GW4064 treatment. CONCLUSION: GW4064 can up-regulate the expressions of FXR, PPARγ2, adiponectin, AdipoR2 in 3T3-L1 preadipocytes and AdipoR2 in HepG2 cells. As adiponectin and its receptors are two important factors in the treatment of non-alcoholic fatty liver disease, FXR agonist may potentially produce therapeutic effect on non-alcoholic fatty liver disease and can regulate adipocytes via up-regulating PPARγ during adipocyte differentiation.

[Application of genome-wide genechip for screening and identifying genes related to CD133(+)CD200(+) colorectal cancer stem cells].

OBJECTIVE: To screen and identity genes related to CD133(+)CD200(+) colorectal cancer stem cells. METHODS: The two subpopulations of colorectal cancer cells, namely CD133(+)CD200(+) and CD133(-)CD200(-) cells, were sorted and verified by flow cytometry. The gene expression profiles of CD133(+)CD200(+)and CD133(-)CD200(-) colorectal cancer cells were examined using Affymetrix Human U133 Plus2.0 genome-wide genechip. The differentially expressed genes between the two cell subpopulations were analyzed to identify the genes responsible for the main effect in association with colorectal cancer stem cells. Real-time quantitative PCR was performed to confirm some of the differentially expressed genes identified by genechip. RESULTS: The genechip result showed that 655 genes were differentially expressed in CD133(+)CD200(+) colorectal cancer stem cells by at least 3 folds, including 290 up-regulated and 365 down-regulated ones. Bioinformatics analysis and gene co-expression network building identified 3 genes (MDM2, PRKACG, and CACNA1G) with specific expression in CD133(+)CD200(+) colorectal cancer stem cells, and this result was confirmed by real-time quantitative PCR analysis. CONCLUSION: A specific gene expression profile of colorectal cancer stem cells has been established through screening and identifying genes related to CD133(+)CD200(+)colorectal cancer stem cells by gene genechip technique, which provides a basis for further study of gene targeting therapy of colorectal cancer.

Down-regulated expression of AQP5 on lung in rat DIC model induced by LPS and its effect on the development of pulmonary edema.

Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by the widespread activation of coagulation, which leads to failure of multiple organs in the body. DIC of rat with lipopolysaccharide (LPS) is associated with subsequent pulmonary edema. Lung tissue is highly water permeable and expresses several aquaporins (AQPs). We therefore explored whether AQP5 involved in the pathogenesis of LPS-induced lung edema. The rats were intravenously infused with LPS (30 mg/kg) for 4 h, 6 h, 8 h, 10 h, and 12 h to induce DIC. Platelets count (PLT), D-Dimer (DD), fibrinogen (FIB), prothrombin time (PT), and activated partial thromboplastin time (APTT) were determined. Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of AQP5. Lung samples were stained with hematoxylin-eosin and lung wet/dry weight (W/D) ratios were measured. Here, we demonstrated that PLT and FIB values were significant decreased, the values for DD, PT, and APTT were marked increased, microthrombus was observed in lung specimens, and simultaneously with the AQP5 showed down-regulated expression following LPS infused from 4 h to 12 h. However, histopathological changes such as pulmonary edema and the increased lung W/D weight ratio were observed after LPS infused from 6 h to 12 h. These results indicated that the decreased expression of AQP5 maybe induce liquid transport obstacles between alveolar and capillary, and provides the report of AQP5 gene regulation, revealing the pathogenesis of pulmonary edema in DIC model of rat.

Position 22 of the V3 loop is associated with co-receptor usage and disease progression in HIV-1 subtype B isolates.

Our goal in this study was to analyze position 22 of the V3 loop associated with co-receptor usage and disease progression in human immunodeficiency virus type 1 (HIV-1) subtype B infection. Bioinformatics approaches were used to compare the amino acid sequence and secondary structure of the V3 loop of the CCR5-tropic virus and CXCR4-tropic virus in HIV-1 subtype B. HIV-1 subtype B V3 amino acid sequence files in the FASTA format were collected from the HIV Sequence Database. The amino acid sequences of different tropism were multiple-aligned with CLUSTAL W program, and the frequencies of the amino acids at each position of the V3 loop sequences of two groups were calculated and sorted in descending order. The secondary structure of the consensus V3 amino acid sequences from CCR5-tropic and CXCR4-tropic viruses were predicted with the APSSP2 method. The amino acids at positions 11, 22, and 25 of V3 were different between the CCR5-tropic virus and CXCR4-tropic virus. The consensus amino acid frequencies were found to be 71.9% S, 66.7% A, and 56.0% D for the CCR5-tropic virus and 50.0% R, 57.1% T, and 26.2% Q for the CXCR4- tropic virus at positions 11, 22, and 25, respectively. There was a strong association between the identity of the residues at position 11, 22, and 25 of the V3 loop amino acid sequence and CD4+ T cell counts of different patients. The change of the residue at position 22 in the R5-tropic or X4-tropic viruses is expected to likely change the secondary structure to be similar to the X4-tropic or R5-tropic viruses. Our study indicates that position 22 of the V3 loop amino acid sequence is significantly associated with viral tropism and disease progression in HIV-1 subtype B.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells.

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR). RESULTS: siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dose-dependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantly, combination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially on cccDNA amplification.

Influenza virus infection and risk of acute myocardial infarction.

Increasing evidences have shown that pathogens might promote atherosclerosis and trigger acute myocardial infarction (AMI). But the conclusions from various studies on the correlation between previous influenza virus (IV) infection and AMI were inconsistent. We conducted a case-control study to assess the association of previous IV infection and AMI. Questionnaire survey was conducted to collect information about demographic characteristics and heart disease risk factors. Fasting blood sample was obtained to measure IgG antibodies to influenza virus A(IV-A), influenza virus B(IV-B), cytomegalovirus (CMV), herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2), adenovirus (ADV), rubella virus (RV) and Chlamydia pneumoniae (CP) and measure the level of some biochemistry markers. Compared to controls, cases were more likely to have positive IgG antibodies to IV-A and IV-B (IV-A: OR, 3.3; 95%CI, 1.5 to 7.4; IV-B: OR, 17.2; 95%CI, 7.7 to 38.0). After adjustment for potential confounding variables, the risk of AMI was still associated with the presence of IgG antibodies to IV-A (adjusted OR, 7.5; 95%CI, 1.3 to 43.0) and IV-B (adjusted OR, 27.3; 95%CI, 6.6 to 113.8). The study supported the hypothesis that previous IV infection took part in the development of atherosclerosis and trigger the occurrence of AMI.

Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells. METHODOLOGY: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR. RESULTS: Our data demonstrated that three used siRNAs showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification. CONCLUSION: Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.

Effects of biological variations on platelet count in healthy subjects in China.

The effects of biological variations on platelet counts were investigated in 694 healthy subjects aged 18 to 60 years living in three cities including Chengdu (Sichuan Province), Suzhou (Jiangsu Province) and Harbin (Heilongjang Province) in China. Platelet counts in healthy subjects were significantly lower in Chengdu (52-202 x 10(9)/L) and Suzhou (60-259 x 10(9)/L) than in Harbin (154-348 x 10(9)/L) (p <0.0001), but the mean platelet volume (MPV) determined concurrently was negatively correlated with platelet count, the MPV values were significantly higher in Chengdu (11.8-15.6 fl) and Suzhou (10.9-15.8 fl) than in Harbin (9.5 approximately 12.9 fl) (p < 0.0001). Platelet counts were significantly higher in summer (73-289 x 10(9)/L) than in winter (52-202 x 10(9)/L) (p <0.0001), but the MPV values were lower in summer (11.2-14.7 fl) than in winter (11.8-15.6 fl) (p <0.05) in Chengdu. Platelet associated immunoglobulin (PA-IgG) in Chengdu was revealed to be significantly higher in the low platelet count group (<150 x 10(9)/L, 13.5 +/- 7.1 ng/10(7) PLT) than in the normal platelet count group (> or =150 x 10(9)/L, 8.3 +/- 2.7 ng/10(7) PLT) (p <0.0001). Similar results were observed in Suzhou for the reticulated platelet ratio, which was significantly higher in the low platelet count group (19.5 +/- 7.1%) than in the normal platelet count group (11.6 +/- 2.7%) (p <0.01). The bleeding time in Chengdu showed a significantly longer time in the low platelet count group (8.6 +/- 2.3 min) than in the normal platelet count group (6.0 +/- 1.2 min)(p <0.01). With regard to the effects of lipids on platelet counts, the HDL values were significantly higher in the normal platelet count group (1.60 +/- 0.76 mmol/L) than the low platelet count group (1.23 +/- 0.31 mmol/L) (p <0.01); but no significant differences in cholesterol and triglycerides values between the normal and low platelet count groups (p >0.05) were recorded. These findings suggest that the platelet counts could be greatly influenced in healthy subjects by biological variations such as geographical, seasonal, and lipid variations.

Studies on reference intervals for platelet counts in three cities in China and one in Japan.

Hematological parameters including platelet counts, etc. were determined in 1,140 healthy subjects living in four cities: Suzhou (Jiangsu Province), Chengdu (Sichuan Province) and Harbin (Heilongjang Province) in China, and Kobe in Japan. Then, the reference intervals for platelet counts were calculated and compared. The reference interval for platelet count of subjects aged between 18 and 60 years was 60-259 x 10(9)/L in Suzhou and 52-202 x 10(9)L in Chengdu, and subjects with platelet counts of 100 x 10(9)/L or less accounted for about 30% of the subjects examined in these cities. The reference intervals in Harbin and Kobe were within the range of 150-350 x 10(9)/L, and no subject having a platelets count of 100 x 10(9)/L or less was detected. Mean platelet volume (MPV) determined concurrently was negatively correlated with platelet count, and the reference intervals for MPV in Chengdu and Suzhou were higher than those in Harbin and Kobe.

[Effect of local mild hypothermia on treatment of acute intracerebral hemorrhage, a clinical study].

OBJECTIVE: To observe the effect of local mild hypothermia on treatment of acute intracerebral hemorrhage. METHODS: Forty patients with acute intracerebral hemorrhage were matched according to age and state of illness at a ratio of one to one and then randomly divided into two groups of 20 patients: routine group (dehydrant, antihypertensive agent, etc were given) and hypothermia group (in addition to the general treatment, controllable semiconductor brain-protecting freezer was used to produce local hyperthermia at temperature of 6 degrees C for 48 hours). Immediately after admission and one week and 2 weeks after treatment cranium CT was conducted and the volume of cerebral edema was calculated by Tada's formula. RESULTS: There was no significant difference in the volume of edema and scores according to European Stroke Scale between the two groups at admission (P > 0.05). The volumes of edema one week and 2 weeks after therapy were 17.4 +/- 6.2 and 13.1 +/- 5.8 milliliter respectively in hypothermia group, both significantly lower than those in the routine group (33.8 +/- 16.0 and 22.4 +/- 12.2 milliliter respectively, P < 0.05). The scores according to European Stroke Scale one week and 2 weeks after therapy were 62.1 +/- 10.8 and 70.3 +/- 10.7 in hypothermia group, both significantly higher than those in the routine group (52.8 +/- 10.9 and 60.5 +/- 10.9 respectively, P < 0.05). No side effect caused by local hyperthermia was observed during the therapy. CONCLUSION: A safe and reliable treatment, local mild hypothermia combined with routine therapy is effective in protecting the brain of patients with intracerebral hemorrhage.