Effects of bone marrow-derived mesenchymal stem cells on the autophagic activity of alveolar macrophages in a rat model of silicosis.

The aim of the present study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of the autophagy-associated proteins, microtubule-associated protein light chain 3 (LC-3) and autophagy-related gene Beclin-1 (Beclin-1), in alveolar macrophages (AMs) in a rat model of silicosis. Furthermore, the study investigated the molecular mechanisms underlying the effects of BMSC treatment. A population of 60 adult female Sprague-Dawley (SD) rats were allocated at random into three groups, namely the control, model and BMSC treatment groups (n=20 per group). BMSCs were isolated from five male SD rats (age, 6-8 weeks) and cultured in vitro. The silicosis model was established using a single 1.0-ml infusion of silicon dioxide suspension administered via non-exposed tracheal intubation. Rats in the BMSC treatment group received a 1.0-ml transplantation of BMSCs (1×106/ml). The rats were sacrificed on days 1, 7, 14 and 28 after modeling, and AMs were extracted from the rats using bronchoalveolar lavage. Third-generation BMSCs were identified using flow cytometry with fluorescein isothiocyanate staining, and the morphological characteristics of the AMs were observed using hematoxylin and eosin staining. The expression levels of LC-3 and Beclin-1 were determined using immunocytochemistry sand western blot analysis. The expression levels of LC-3 and Beclin-1 were found to be increased at all the time points in the model group. LC-3 and Beclin-1 levels began to increase at day 1, peaked at day 14 and decreased after day 28; however, the levels remained elevated compared with the basal expression levels. The AMs of the BMSC treatment group exhibited significantly alleviated pathological symptoms compared with the model group AMs, as indicated by significantly decreased expression levels of LC-3 and Beclin-1 at each time point. Therefore, the results indicated that autophagy was promoted in the AMs of the silicosis model rats. Furthermore, treatment with BMSCs was demonstrated to reduce the expression levels of LC-3 and Beclin-1, subsequently inhibiting autophagic activity and mitigating the damage associated with silicosis.

High Physical Activity is Associated with an Improved Lipid Profile and Resting Heart Rate among Healthy Middle-aged Chinese People.

OBJECTIVE: To investigate the effects of physical activity (PA) on dyslipidemia and elevated resting heart rate (RHR) in a large-scale cross-sectional study in China. METHODS: We recruited community-based individuals who were 40-60 years old using a cluster sampling method. The PA levels of the participants were classified as low, moderate, or high, using the International Physical Activity Questionnaire. Dyslipidemia was defined as the detection of abnormalities in lipid indicators, and 4 lipid parameters were evaluated using fasting blood samples. Multivariate logistic regression analyses were used to evaluate the associations of PA with dyslipidemia and RHR. RESULTS: A total of 10,321 participants (38.88% men) were included in this study. The percentages of individuals with high, moderate, and low PA levels were 46.5%, 43.9%, and 9.6%, respectively. In both men and women, high PA provided odds ratios of 0.88 [95% confidence interval (CI): 0.83, 0.94] for dyslipidemia and 0.82 (95% CI: 0.73, 0.92) for elevated RHR, compared to participants with low PA. CONCLUSION: Our data suggested that substantial health benefits (related to dyslipidemia and elevated RHR) occurred at higher intensity PA, with greater energy consumption, in middle-aged Chinese people, and particularly in men.

N-acetylcysteine alleviated silica-induced lung fibrosis in rats by down-regulation of ROS and mitochondrial apoptosis signaling.

Reactive oxygen species (ROS) is a normal metabolic product of cellular respiration, but too much ROS can induce cell apoptosis. Here, we used N-acetylcysteine (NAC) to inhibit ROS activity to explore the effects of NAC on silica-induced pulmonary fibrosis in rats and provide evidence for study on the mechanism of silicosis. 24 adult male Sprague-Dawley rats weighing 180-220 g were randomly divided into three groups with eight rats in each group. Silicosis model group and NAC group were adopted non-tracheal exposure method of disposable intrapulmonary injection of 50 g/L, silica suspension 1 mL to establish animal silicosis model, NAC group treated with 600 mg/kg NAC by gavage from the right day of modeling, all animals were sacrificed after 28 days. The level of ROS contents and mitochondrial transmembrane potential changes of AM, the mRNA expression level of type I and type III procollagen, cytochrome C, cysteinyl aspartate specific protease-9 and caspase-3 were detected. The severity of pathological changes and pulmonary fibrosis were observed by pathologic specimens. It was showed that ROS contents and MTP changes were lower in the NAC group compared with the silicosis model group, other indexes were lower in the NAC group than the model group, but higher than those of the control group, the degree of lung fibrotic lesions observed from the pathological slices showed the same trend. These data indicated that NAC can reduce ROS content of AM in silica exposure rats, the mitochondrial apoptosis pathway can also be inhibited, the severity of pulmonary fibrosis alleviated as a result.

[Autophagy in lung tissue of rats exposed to silica dust].

OBJECTIVE: To investigate the autophagy of effector cells in lung tissue at different time points when rats were exposed to free SiO2 dust. METHODS: Sixty Wistar rats (220∼230 g) were selected and allocated to experimental group (n = 30) and control group (n = 30). In the experimental group, a rat silicosis model was established by infusing SiO2 suspension into the trachea of rats. Six rats in each group were sacrificed on days 1, 7, 14, 21, or 28 of dust exposure. Lung tissue samples were collected to prepare lung tissue sections. The pulmonary inflammation and fibrosis were observed by HE staining. The proautophagosome, autophagosome, and autophagolysosome in lung tissue sections were observed under a transmission electron microscope. RESULTS: On day 1 of dust exposure, many proautophagosomes and autophagosomes were seen in both experimental group and control group. On day 7 of dust exposure, the experimental group had more autophagosomes in lung tissue than the control group. On day 14 of dust exposure, the experimental group had fewer autophagosomes than the control group. On days 21 and 28, autophagolysosomes were seen in macrophage plasma in both experimental group and control group; the autophagolysosomes in experimental group showed cloudy swelling and expansion, and some were vacuolated, and these changes were more significant on day 28. CONCLUSION: Free SiO2 dust can induce autophagy in the lung tissue of rats, with varying degrees at different time points of dust exposure.

[Effect of CYP11B2 gene -344T/C polymorphism on renin-angiotensin-aldosterone system activity and blood pressure response to hydrochlorothiazide].

OBJECTIVE: To evaluate the effect of CYP11B2 gene -344T/C polymorphism on renin-angiotensin-aldosterone system (RAAS) activity and blood pressure in response to hydrochlorothiazide (HCTZ) treatment in Han Chinese patients with essential hypertension. METHODS: Eight hundred and twenty-nine patients with mild/moderate essential hypertensive were enrolled. All subjects had their antihypertensive medications withdrawn. After two weeks of wash-out period with placebo, each patient was given 12.5 mg of HCTZ per day for the next six weeks. Physical, biochemical measurements, and the activity of RAAS were taken at the end of the wash-out period (baseline) and 6-week diuretic therapy period. Changes in systolic and diastolic blood pressure were analyzed for association with interaction between genotypes at CYP11B2 -344T/C polymorphism and gender. RESULTS: A total of 776 patients completed the study. 17.5% of subjects have achieved blood pressure normalization after six weeks treatment. For male patients, the aldosterone level with CC genotype was significantly higher than that of those with TT or TC genotype. Following the HCTZ treatment, the blood pressure response in patients with CC genotype was less obvious than that in others, whilst the increase of aldosterone level was greater. For female patients, no association was found between CYP11B2 -344T/C polymorphism and aldosterone level. Following the HCTZ treatment, the blood pressure response in patients with CC genotype was greater than others, whilst the increase of aldosterone activity was less apparent. CONCLUSION: In males, the -344T/C polymorphism of CYP11B2 gene is associated with aldosterone level, and the change of aldosterone level was greater, the blood pressure response was weaker after HCTZ treatment. In females, there was no association between this polymorphism and aldosterone level. The change of aldosterone level and blood pressure response to HCTZ were different from that in males.

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis.

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.

Smoking and COX-2 functional polymorphisms interact to increase the risk of gastric cardia adenocarcinoma in Chinese population.

BACKGROUND: Over-expression and increased activity of cyclooxygenase (COX)-2 induced by smoking has been implicated in the development of cancer. This study aimed to explore the interaction between smoking and functional polymorphisms of COX-2 in modulation of gastric cardia adenocarcinoma (GCA) risk. METHODS AND FINDINGS: Three COX-2 polymorphisms, including -1195G>A (rs689466), -765G>C (rs20417), and 587Gly>Arg (rs3218625), were genotyped in 357 GCA patients and 985 controls. In the multivariate logistic regression analysis, we found that the -1195AA, -765GC, and 587Arg/Arg genotypes were associated with increased risk of GCA (OR = 1.50, 95% CI = 1.05-2.13; OR = 2.06, 95% CI = 1.29-3.29 and OR = 1.67, 95% CI = 1.04-2.66, respectively). Haplotype association analysis showed that compared with G(-1195)-G(-765)- G(Gly587Arg), the A(-1195)-C(-765)-A(Gly587Arg) conferred an increased risk of GCA (OR = 2.49, 95% CI = 1.54-4.01). Moreover, significant multiplicative interactions were observed between smoking and these three polymorphisms of -1195G>A, -765G>C, and 587Gly>Arg, even after correction by false discovery rate (FDR) method for multiple comparisons (FDR-P(interaction) = 0.006, 5.239×10(-4) and 0.017, respectively). Similarly, haplotypes incorporating these three polymorphisms also showed significant interaction with smoking in the development of GCA (P for multiplicative interaction = 2.65×10(-6)). CONCLUSION: These findings indicated that the functional polymorphisms of COX-2, in interaction with smoking, may play a substantial role in the development of GCA.

[Macrophage apoptosis and the levels of interleukin-1 and interleukin-8 in the rats exposed to silica].

OBJECTIVE: To study the roles of macrophage apoptosis, IL-1, and IL-8 in the pathogenesis of rat pulmonary fibrosis induced by silica. METHODS: Forty eight male Wistar rats were divided into the 4 control groups (24 rats) and 4 experimental groups (24 rats). Rats in the control groups were treated with 1 ml normal saline by trachea instillation, whereas the rats in experimental groups were exposed 1 ml silica suspension (100 mg/ml) by trachea instillation for 1, 7, 14 and 28 days, respectively. Six rats of each group were sacrificed, then the bronchoalveolar lavage fluid and lung tissues were collected, respectively. Pulmonary inflammation, fibrosis and other pathological changes were detected with H.E. staining. Morphological changes of the early stage apoptosis in macrophages were detected with transmission electron microscope (TEM). The early apoptosis rates of macrophages in BALF were also assessed using Annexin V-FITC/PI kit. The IL-1 and IL-8 levels of serum were measured with the ELISA. RESULTS: The apoptotic rates (11.48% +/- 0.24%, 16.03% +/- 0.68%, 15.53% +/- 1.07%, 18.92% +/- 2.70%, respectively) of macrophage in the experimental groups increased obviously with time, as compared to the controls (5.47% +/- 2.06%, 6.39% +/- 0.215, 9.07% +/- 0.61% and 8.54% +/- 0.16%, Respectively) (P < 0.05). The IL-1 levels of serum in the experimental groups were 23.64 +/- 0.84, 23.38 +/- 1.10, 22.21 +/- 0.86 and 24.29 +/- 1.31 pg/ml, respectively, which were significantly higher than those (18.52 +/- 1.23, 18.40 +/- 1.6, 17.92 +/- 2.21 and 18.53 +/- 2.64 pg/ml, respectively) in the control groups (P < 0.05) without time-effect relationship. The serum IL-8 levels on the 1st, 7th and 14th days in the experimental groups were 21.32 +/- 1.44, 21.90 +/- 2.08 and 22.00 +/- 2.80 pg/ml, respectively, which were significantly higher than those (17.69 +/- 1.09, 16.98 +/- 2.09 and 17.54 +/- 1.62 pg/ml, respectively) in the control groups (P < 0.05). CONCLUSION: The early macrophage apoptosis and changes of IL-1 and IL-8 may in lungs may play an important role in the development of pulmonary fibrosis induced by silica.

Association of TNF-alpha-238G/A and 308 G/A gene polymorphisms with pulmonary tuberculosis among patients with coal worker's pneumoconiosis.

OBJECTIVES: Tumor necrosis factor-alpha (TNF-alpha) may play an important role in host's immune response to mycobacterium tuberculosis (M. tuberculosis) infection. This study was to investigate the association of TNF-alpha gene polymorphism with pulmonary tuberculosis (TB) among patients with coal worker's pneumoconiosis (CWP). METHODS: A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-alpha gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism (PCR-RFLP). Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher's exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplicative model with combined OR. All data were analyzed using SAS version 8.2 software. RESULTS: No significant difference in frequency of the TNF-alpha-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls (chi2 = 5.44, P = 0.07). But difference in frequency of the TNF-alpha-308 A allele was identified between them (chi2 = 5.14, P = 0.02). No significant difference in frequencies of the TNF-alpha-238 genotype and allele (P = 0.23 and P = 0.09, respectively) was found between cases and controls either, with combined (GG and AA) OR of 3.96 (95% confidence interval of 1.30-12.09) at the -308 locus of the TNF-alpha gene, as compared to combination of the TNF-alpha-238 GG and TNF-alpha-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-alpha-238 GG and TNF-alpha-308 GA genotypes was 1.98 (95% CI of 1.06-3.71) for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-alpha-308 GG genotype and body mass index (OR = 4.92), as well as an interaction between the TNF-alpha-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-alpha-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. CONCLUSIONS: TNF-alpha-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-alpha-238 A allele was otherwise.

[Construction and effect identification of MiR RNAi eukaryotic expression vectors of prohibitin].

AIM: To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection. METHODS: The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting. RESULTS: The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1. CONCLUSION: A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.

[Study on the association of INT4 and 3'UTR polymorphism of natural-resistance-associated macrophage protein 1 gene with susceptibility to pulmonary tuberculosis].

OBJECTIVE: To investigate the association between natural-resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms and susceptibility to pulmonary tuberculosis (TB) in Han nationality from Northern part of China. METHODS: A 1:1 matched case-control study was adopted. Polymerase chain reaction and restriction fragment length polymorphism technique were used to type the two NRAMP1 polymorphisms: INT4 and 3'UTR. Information on environmental-related risk factors and pathological changes of tuberculosis was collected using a pre-tested standard questionnaire. Univariate and multivariate conditional logistic analyses were conducted using SPSS for window software. RESULTS: A sample consisting 124 pairs of cases and controls was studied. Univariate analysis demonstrated that the 3'UTR TGTG+/del genotype occurred more frequently in the cases than in the controls, with crude OR (95% CI) being 2.923 (1.557 - 5.487). No significant association was observed between TB and INT4 polymorphism. In multivariate analysis, associations of TB and 3'UTR TGTG+/del genotype remained, after adjusting for scar of bacillus Calmette-Guérin vaccine, marriage status, body mass index and exposure history. Adjusted OR (95% CI) was 2.955 (1.369 - 6.381). Again, no significant association between INT4 polymorphism and TB was found. Among different INT4 genotypes, the pathological characters of pulmonary tuberculosis were also found different (chi(2) = 9.634, P < 0.05). CONCLUSION: Polymorphism of 3'UTR locus in NRAMP1 gene might affect their susceptibility to TB in Han nationality living in the northern part of China, and polymorphism of INT4 might affect the pathological characters of tuberculosis.

[A study of chronic hepatitis B infection superinfected with hepatitis E infection].

OBJECTIVES: To compare the influence of hepatitis E virus (HEV) infection on the replication of hepatitis B virus (HBV) by observing clinical features, the relationship between the number of HBV DNA copies in serum, the degree of hepatic function impairments and prognosis of chronic hepatitis B patients with hepatitis E superinfection. METHODS: One hundred and fifteen chronic hepatitis B (CHB) patients and 115 CHB patients with hepatitis E (HE) superinfection were studied and compared. 74 liver tissue biopsy samples of the CHB and 51 of the CHB-HE sufferers were obtained. HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, anti-HBc-IgG, anti-HBc-IgM, HBV DNA and anti-HEVIgM were detected respectively by enzyme linked immunosorbent assay and polymerase chain reaction. RESULTS: The number of HBV DNA copies in sera in the CHB-HE group and the severity of the clinical features were significantly higher than those in the CHB group. The former group had a poorer prognosis with a 49.6% occurrence rate of severe hepatitis and a 25.2% death rate while in the latter group the corresponding rates were just 4.4% and 1.7%. CONCLUSION: Re-infection with HEV, in those CHB patients, severely damaged their hepatic functions and increased their mortality; the older the patient and the higher the level of HBV DNA, the poorer the prognosis.

[Study of mechanism on immune response in goats by different immunization with Helicobacter pylori and analyse of its prospects for using].

OBJECTIVES: To observe the effect of three different routes on the induction of immunogenicity to whole bacterial antigen of Vac A+ Helicobacter pylori strain (NCTC11637) and explore its mechanism. METHODS: Two milk goats and one pregnant goat were immunized by different routes respectively with whole bacterial of H. pylori (6 x 10(9) cfu/ml) cultivated by solid culture medium. At the 1st, 14th, 21st, 28th day, two goats were immunized four times by intranasal or subcutaneous injection. The other pregnant goat was immunized four times at a interval of two weeks before and after one month of lamb birth by muscular injection. Serum and milk samples were collected and assayed by indirect enayme-linked immunosorbent assay (ELISA). The levels of anti-Hp of IgG and IgA in serum and milk were determined by reading the optical density (A). RESULTS: Three immune routes all induced systemic immune response. The optical density (A) of ELISA proved that the specific IgG in serum increased while IgA didn't increase significantly, and that the specific IgA and IgG in milk all increased to a greater or lesser extent compared with control group. CONCLUSION: Three immune routes all induce systemic immune response, resulting in increases of anti-Hp of IgG/IgA in milk and IgG in serum. Among them, intranasal inoculation can induce systemic immune response and local immune response in different mucosal sites, which may be a safe and effective immunization route.

[Study on the determinants of Helicobacter pylori infection among coal miners].

OBJECTIVE: To study the prevalence and determinants of Helicobacter pylori (H. pylori) infection among coal miners and to seek for competent preventive measures. METHODS: 425 coal miners from three coal mines, Tangshan, Daxing, and baodian were chosen under stratified random cluster sampling. Face to face interview was conducted to fill the unified questionnaires by trained interviewers. 306 subjects underwent gastroenduoscopy to detect the situation of the gastroenduodenal diseases, according to the Sydney System of diagnosis. Mucosa biopsies were also undertaken according to the regulated location for culture of H. pylori and for pathological examination. Blood samples were obtained to detect the anti-HpU-IgG by enzyme-linked immunosorbent assay (ELISA). H. pylori infection was determined through culture and ELISA but confirmed under the standards set at the National Congress on Gastroduodenal Diseases in 1999. RESULTS: Among 425 eligible coal miners being tested, 297 (69.9%) were H. pylori positive and the rate for those working underground (74.0%) was higher than that of those working on ground (P=0.004). No difference was found among coal miners between the three mines (P >0.05). Age, living conditions in childhood, number of current family members, the amount of alcohol intake and ways of eating at home were strongly associated with the status of H. pylori infection. CONCLUSIONS: Difference of H. pylori infection prevalences between the underground and the aboveground coal miners was noticed. Determinants that influencing the H. pylori infection would include socioeconomic factors, individual habits and ways of eating at home.