Untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease induced by high-temperature-processed feed in Sprague-Dawley rats.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases in the world. In our early clinical data and questionnaire analysis of NAFLD, it was found that the body mass index of some patients did not meet the diagnostic criteria for overweight or obesity. The consumption of high-temperature-processed foods such as fried food, hot pot and barbecue is closely related to the occurrence of nonobese NAFLD. Reducing the intake of this kind of food can reduce disease severity and improve prognosis. AIM: To explore the untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease in Sprague-Dawley rats induced by high-temperature-processed feed. METHODS: Fifty-four male Sprague-Dawley rats were divided into three groups: The control group received a standard diet; the nonfried soybeans (NDFS) group received 60% NDFS and 40% basic feed and the dry-fried soybeans (DFS) group received 60% DFS and 40% basic feed. Six rats were sacrificed at week 4, 8, and 12 in each group. The food intake, body weight, Lee's index, liver index, serological index and hepatic histopathology were assessed. Untargeted metabolomics characteristics were used to analyze the changes in liver metabolites of rats at week 12. Correlations between metabolites and pathology scores between the DFS and control groups and between the DFS and NDFS groups were analyzed. We selected some of the metabolites, both within the pathway and outside of the pathway, to explain preliminarily the difference in liver pathology in the three groups of rats. RESULTS: There were no statistically significant differences in the food intake, body weight, Lee's index or serological index between the DFS group and the control group (P > 0.05). At week 8 and week 12, the steatosis scores in the DFS group were significantly higher than those in the other two groups (P < 0.05). At week 12, the liver index of the DFS group was the lowest (NDFS group vs DFS group, P < 0.05). The fibrosis score in the DFS group was significantly higher than those in the other two groups (P < 0.05). The correlation analysis of the liver pathology score and differential metabolites in the DFS and NDFS groups showed that there were 10 strongly correlated substances: Five positively correlated substances and five negatively correlated substances. The positively correlated substances included taurochenodeoxycholate-3-sulfate, acetylcarnitine, 20a,22b-dihydroxycholesterol, 13E-tetranor-16-carboxy-LTE4 and taurocholic acid. The negatively correlated substances included choline, cholesterane-3,7,12,25-tetrol-3-glucuronide, nicotinamide adenine dinucleotide phosphate, lysoPC [16:1 (9Z)] and glycerol 3-phosphate. The correlation analysis of the liver pathology score and differential metabolites in the DFS and control groups showed that there were 13 strongly correlated substances: Four positively correlated substances and 9 negatively correlated substances. The positively correlated substances included 4-hydroxy-6-eicosanone, 3-phosphoglyceric acid, 13-hydroxy-9-methoxy-10-oxo-11-octadecenoic acid and taurochenodeoxycholate-3-sulfate. The negatively correlated substances included lysoPC [16:1(9Z)], S-(9-hydroxy-PGA1)-glutathione, lysoPC [20:5 (5Z, 8Z, 11Z, 14Z, 17Z)], SM (d18:1/14:0), nicotinamide adenine dinucleotide phosphate, 5,10-methylene-THF, folinic acid, N-lactoyl-glycine and 6-hydroxy-5-methoxyindole glucuronide. CONCLUSION: We successfully induced liver damage in rats by using a specially prepared high-temperature-processed feed and explored the untargeted metabolomics characteristics.

[Effect of xiaotan huayu liqiao traditional Chinese medicine compound on myocardial fibrosis in rats with chronic intermittent hypoxia and its mechanism].

Objective: To explore the role of transforming growth factor-ß (TGF-ß) signaling pathway in xiaotan huayu liqiao traditional Chinese medicine compound (XC)'s anti-myocardial fibrosis in chronic intermittent hypoxia (CIH) rats. Methods: Forty SD rats were randomly divided into normoxia group, oxygen + traditional Chinese medicine compound group ( TCMC), Chronic intermittent hypoxia model group (CIH), TCMC + CIH, 10 in each group. CIH cabin was built by filling with nitrogen and oxygen. Firstly, the volume fraction of oxygen in the cabin reduced from 21% to 9% in 90 s by filling the cabin with nitrogen. And then it gradually rose to 21% by reoxygenating in 90s, as a cycle. CIH and TCMC+CIH group rats were placed in the CIH device, while normoxia and TCMC group rats were placed in the normal oxygen chamber. In addition, rats in TCMC +CIH group and TCMC group were treated with XC crude drug (24 g/kg) daily by gavage, while rats in CIH group and normoxia group were given equal volume normal saline. Using sirius red staining, the collagen in myocardial interstitium was visualized. The protein expressions of collagen I, collagen III and fibronectin were detected by Western blot, p-Smad3, p-Smad2 and TGF-ß protein in the TGF-ß/Smads signaling pathway were also analyzed by Western blot. The mRNA expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase -2(TIMP-2) were measured by real-time quantitative polymerase chain reaction (PCR). Results: Compared with the rats exposed to normoxia, the CIH rats showed obvious collagen deposition, protein expressions of collagen I, collagen III and fibronectin were significantly increased in the myocardial tissue (P＜0.01). The protein expression levels of TGF-ß, p-smad2 and p-smad3 in the myocardial tissue of the CIH rats were also significantly increased (P＜0.01). The up-regulation of TIMP-2 mRNA in the myocardial tissues resulted in the decrease of MMP-2 mRNA(P＜0.01). XC reduced myocardial fibrosis of CIH rats and inhibited the expressions of collagen I and collagen III and fibronectin protein (P＜0.05,P＜0.01,P＜0.05, respectively). The further mechanism study showed that XC inhibited the expression of TGF-ß (P＜0.01), which down-regulated the expressions of p-smad2, p-smad3 and TIMP-2 (P＜0.05). Conclusion: XC could reduce the expression of TGF-ß and smad2/3 phosphorylation, down-regulate the expression of TIMP-2, which would inhibit the formation of myocardial fibrosis in CIH rats, and improve the myocardial function of CIH rats.

[Early cardiac injury in patients with obstructive sleep apnea].

OBJECTIVE: To evaluate the early cardiac injury caused by obstructive sleep apnea (OSA) before the development of cardiovascular symptoms of OSA. METHODS: Ninety-two patients without any known cardiovascular disorders who underwent polysomnography (PSG) were enrolled in the study. Subjects were divided into mild, moderate, and severe OSA groups by their apnea hypopnea index (AHI), and 25 healthy individuals were identified as controls. After PSG examination, fasting blood samples for the evaluation of N-terminal pro-brain natriuretic peptide (NT-proBNP) and heart-type fatty acid binding protein (h-FABP) were collected in the morning, and left ventricular(LV) functions were assessed by using echocardiographic methods. Thirty moderate and severe OSA patients were treated with continuous positive airway pressure respectively (CPAP). RESULTS: The levels of h-FABP and NT-proBNP were obviously higher in all OSA groups than those in the control group (P<0.01), and were positively correlated with AHI (P<0.01). The Em/Am values of all OSA groups and E/A values of the moderate and severe OSA groups were significantly reduced (P<0.01). The difference in Em/Am values among the groups was statistically significant (P<0.01). Compared with those before treatment, h-FABP and NT-BNP levels in serum of OSA patients after CPAP treatment were significantly reduced (P<0.01), and Em/Am and E/A values were significantly increased (P<0.01). CONCLUSIONS: Left ventricular diastolic dysfunction and early myocardial microtrauma are major manifestations of early heart damage in patients with OSA. CPAP therapy could significantly improve early cardiac damage in OSA patients.

Association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma in the Chinese population.

OBJECTIVE: The objective of this study was to evaluate the association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma (HCC). METHODS: A total of 689 HCC patients and 680 cancer-free subjects were enrolled. Human MDR1 gene polymorphisms were investigated by created restriction site- polymerase chain reaction (CRS-PCR) and DNA sequencing methods. Multiple logistic regression models were applied to estimate the association between MDR1 gene polymorphisms and susceptibility to HCC. RESULTS: We detected a novel c.4125A>C polymorphism and our findings suggested that this variant was significantly associated with susceptibility to HCC. A significantly increased susceptibility to HCC was noted in the homozygote comparison (CC versus AA: OR=1.621, 95% CI 1.143-2.300, χ2=7.4095, P=0.0065), recessive model (CC versus AC+AA: OR=1.625, 95% CI 1.167-2.264, χ2=8.3544, P=0.0039) and allele contrast (C versus A: OR=1.185, 95% CI 1.011-1.389, χ2=4.4046, P=0.0358). However, no significant increase was observed in the heterozygote comparison (AC versus AA: OR=0.995, 95% CI 0.794-1.248, χ2=0.0017, P=0.9672) and dominant model (CC+AC versus AA: OR=1.106, 95% CI 0.894-1.369, χ2=0.8560, P=0.3549). CONCLUSIONS: These findings suggest that the c.4125A>C polymorphism of the MDR1 gene might contribute to susceptibility to HCC in the Chinese population. Further work will be necessary to clarify the relationship between the c.4125A>C polymorphism and susceptibility to HCC on larger populations of diverse ethnicity.

Physical and mental health status of soldiers responding to the 2008 Wenchuan earthquake.

OBJECTIVE: While internationally major disasters occur frequently, for any one country they are unusual events. In this project we aimed to identify public health issues arising from the physical and mental health symptoms suffered by the soldier volunteers deployed in an emergency relief task during the Wenchuan earthquake. METHODS: Health problems identified in other emergency volunteer populations guided the development of a questionnaire. A cohort of 1,187 soldier volunteers completed the questionnaire, which measured physical and mental health symptoms during their rescue mission. The results were compared with a population norm of soldiers, although baseline data of the respondents were unavailable. RESULTS: Half the respondents reported suffering from skin and mucous membrane problems, followed by respiratory symptoms (38%), digestive (29%) and nervous (22%) symptoms. Despite a low response rate (53%) to the mental health component, nearly half (49%) of those who did respond reported mental health problems. The incidence of the above symptoms were significantly higher than the general soldier population. CONCLUSIONS: Health complaints were common in the soldiers, who had not received any formal training in rescue operations. IMPLICATIONS: Non-professional rescue workers who are not appropriately prepared for the role may suffer more than their professional counterparts. Attention needs to be paid to the health and safety of non-professional rescue workers, which has been ignored in most disaster management plans. These findings can be used to enhance the understanding of emergency response programs within and outside China, where this particular disaster occurred.

[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

[An epidemiological study on physical and mental health of inhabitants who experienced a catastrophic event of "5.12 serious earthquake in Wenchuan county"].

OBJECTIVE: To investigate the influence of a catastrophic event to the physical and mental health of inhabitants in "5.12 serious earthquake in Wenchuan county". METHODS: The analysis of descriptive epidemiology on the spot was made in 3006 servicemen who were living in the disaster area of Wenchuan county and Dujiangyan city. RESULTS: The diseases were mainly found in both psychological symptoms and respiratory system, and the incidences were 62.16% and 51.78%, respectively. It was obviously showed that these children and juveniles less than 14 years had the most apparent psychological symptoms, and the incidence was 85%. On the contrary, the diseases were testified to be low in the incidence of digestive system, and the incidences were only 31.21%. CONCLUSIONS: The physical and mental health of inhabitants who experienced a catastrophic earthquake disaster was harmed in various degree, and the corresponding measures should be made in the medication or mental intervention.

[Preparation of antibody against Memo protein and analysis of expression profile of Memo in mouse tissues].

AIM: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice. METHODS: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot. RESULTS: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot. CONCLUSION: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein].

BACKGROUND: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. METHODS: Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. CONCLUSION: Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

[Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors].

OBJECTIVE: To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. METHODS: Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. CONCLUSION: After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

[Replication and encapsidation of HBV mutants with the truncated C gene].

OBJECTIVE: To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. METHODS: The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. RESULTS: The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. CONCLUSION: The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

[Approach to transforming hepatitis B virus as a gene therapeutic vector].

OBJECTIVE: To evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy. METHODS: A fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot. RESULTS: The HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon. CONCLUSION: It is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.