Effects of 4% paraformaldehyde and modified Davidson's fluid on the morphology and immunohistochemistry of Xiang pig testes.

Modified Davidson's fluid (mDF) is a good fixative for morphological and antigen preservation. However, recent studies have shown that 4% paraformaldehyde (PFA) can better preserve the actin structure in rodent testes. It remains controversial which of these fixatives is best for testicular tissue. This study investigated the effects of both mDF and 4% PFA on the morphology and antigen preservation of Xiang pig testes using hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). The stronger testis penetration of mDF compared with that of 4% PFA was primarily manifested as testicular color change and decrease in tissue weight loss. Testes fixed with 4% PFA displayed a severe shrinkage of both the tubular and interstitial compartments and the seminiferous tubule area decreased by 12.02% compared with that in mDF-fixed tissues. In contrast, IHC results showed that 4% PFA fixation achieved better IHC-positive performance than mDF fixation for antigens specifically expressed in germ cells, Leydig cells and Sertoli cells. Due to this improved antigen preservation by 4% PFA fixation, the relative immunoreactions intensity significantly increased by 39.8%, 27.8%, and 76.4%, respectively, compared with that in mDF fixation. In summary, fixation of Xiang pig testes with mDF was suitable for HE staining, while fixation with 4% PFA was more suitable for IHC.

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease.

BACKGROUND: Stem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats. METHODS: The MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson's trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay. RESULTS: rAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions. CONCLUSIONS: Vacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.

Establishment of surfactant-associated protein A suicide gene system and analysis of its activity.

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

[Effect of Shugan Jianpi Recipe on LXRα/FAS signaling pathway mediated hepatocyte fatty deposits in NAFLD rats].

OBJECTIVE: To explore the effect of Shugan Jianpi Recipe (SJR) on LXRα/FAS signaling pathway mediated hepatocyte fatty deposits in nonalcoholic fatty liver disease (NAFLD) rats. METHODS: Totally 75 SPF grade male SD rats were randomly divided into 5 groups, i.e., the normal control group, the model group, the Shugan Recipe (SR) treatment groups, the Jianpi Recipe (JR) treatment group, and the SJR group. Except rats in the normal control group, the NAFLD rat model was duplicated using high fat diet (HFD). SR (Chaihu Shugan Powder) was administered to rats in the SR group. JR (Shenlin Baizhu Powder) was administered to rats in the JR group. SJR (Chaihu Shugan Powder plus Shenlin Baizhu Powder) was administered to rats in the SJR group. Changes of liver fat were analyzed using automatic biochemical analyzer. Liver cells were separated by low-speed centrifugation. Their activities and purities were identify using Typan blue and flow cytometry (FCM). Expression levels of LXRα and FAS mRNA in hepatocytes detected by Real-time quantitative PCR. Expression levels of LXRα and FAS protein were detected by Western blot. RESULTS: (1) Pathological results showed in the model group, hepatocytes were swollen with nucleus locating at the cell edge after oil red O staining; unequal sized small vacuoles could be seen inside cytoplasm. Some small vacuoles merged big vacuoles. All these indi- cated a NAFLD rat model was successfully established by high fat diet. Pathological structural changes could be impaired to some degree in all medicated groups, especially in the SR group. (2) Compared with the normal control group, expression levels of LXRα and FAS genes and proteins obviously increased in the model group (P < 0.01). Compared with the model group, their expression levels were obviously down-regulated in the JR group and the SR group (P < 0.01, P < 0.05). CONCLUSIONS: LXRα/FAS signaling pathway was an important signaling pathway for mediating lipid metabolism disorders of NAFLD rats. SJR could make hepatocyte fatty deposits tend to repair by adjusting the LXRα/FAS signaling pathway in NAFLD rats, which might be one of important mechanisms for SJR to prevent and cure NAFLD.

[Effects of soothing liver and invigorating spleen recipes on SREBP-1c, SCD-1 mRNA and proteins expression in hepatocytes of NAFLD rats].

OBJECTIVE: To observe the effects of soothing liver and invigorating spleen recipes on the expression levels of SREBP-1c, SCD-1 mRNA and proteins in hepatocytes of NAFLD rats,and to explore its possible mechanisms of prevention and treatment of NAFLD. METHODS: 75 SD male rats were randomly divided into 5 groups: normal group, model group, oothing liver group (administrated with 9.6 g/kg), invigorating spleen group (administrated with 30 g/kg)and integrated group (administrated with 39.6 g/kg). The rats of NASH model were induced by feeding a high-fat diet. After treatment for 8 weeks,9 rats were randomly taken to detect liver function, TC, TG and pathological changes in liver tissue. The other 6 rats of each group were taken respectively and collagenase (Type IV) was perfused to digest liver tissue with the circulation in vitro to separate hepatocytes. Real-time Q-PCR and Western Blot were used to detect the expression levels of SREBP-1c, SCD-1 mRNA and proteins. RESULTS: Compared with the model group,the different decrease levels of SREBP-1c, SCD-1 genes and proteins were found in all drug therapy groups (P < 0.05 or P < 0.01), as well different degrees that liver lipid and pathological changes became better, especially that of in soothing liver group. Comparison between the all drug therapy groups,the hepatocytes expression levels of SREBP-1c and SCD-1 mRNA in soothing liver group were lower than that of in invigorating spleen group (P < 0.05), but expression levels of the proteins had no statistical significances. CONCLUSION: Soothing liver and invigorating spleen recipes prevent and treat NAFLD,its mechanism may be related to inhibiting the activation of SREBP-1c/SCD-1 signal pathway in hepatocytes to down-regulate TC and TG synthesis and reduce hepatic lipid deposition. SREBP-1c, SCD-1 mRNA and proteins may be the effective targets.

Change of extracellular signal-regulated kinase expression in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease.

BACKGROUND: Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease (COPD) by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell (PASMC) proliferation. However, the molecular mechanism underlying this process remains poorly understood. OBJECTIVES: The purpose of this study was to investigate the role of extracellular signal-regulated kinase (ERK) in pulmonary arteries from smokers with normal lung function and smokers with mild to moderate COPD. METHODS: The peripheral lung tissues were obtained from 14 nonsmokers with normal lung function, 18 smokers with normal lung function, and 16 smokers with mild to moderate COPD. The morphological changes of pulmonary arteries were observed by hematoxylin-eosin (HE) staining. Primary cultured human pulmonary artery smooth muscle cells (HPASMCs) were exposed to cigarette smoke extract (CSE). Cell proliferation was determined by cell counting and Methyl thiazolyl tetrazolium assay. Protein expression was analyzed by western blotting. RESULTS: Morphometrical analysis showed that the pulmonary vessel wall thickness in smoker group and COPD group was significantly greater than that in nonsmoker group (P < .01). The protein level of ERK was significantly increased in smoker group and COPD group as compared with nonsmoker group (P < .01). The expression of ERK was significantly increased in HPASMCs at protein levels when HPASMCs were treated with 5% CSE (P < .01), which significantly promoted the proliferation of HPASMCs (P < .01). CONCLUSIONS: Increased expression of ERK might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with and without COPD.

Relationship between the microsatellite D2S388-5 and D2S2232 polymorphisms and chronic obstructive pulmonary disease in the Chinese Kazakh population.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is influenced by multiple genetic and environmental factors. The role of genetic susceptibility in the pathogenesis of COPD has recently gained more attention. The surface lung surfactant protein B plays an important role in COPD pathogenesis. Microsatellite DNA has been characterized in the surfactant protein B alleles D2S388-5 and D2S2232. The aim of this research was to investigate the distribution of the D2S388-5 and D2S2232 microsatellite polymorphisms in smokers of the Kazakh ethnic group in Xinjiang, China, with and without COPD to assess whether such polymorphisms are associated with COPD susceptibility. METHODS: DNA was extracted from the blood of 197 smokers with COPD and 236 control smokers of Kazakh ethnicity. The smokers diagnosed with COPD were registered at the Department of Respiratory Medicine from four different hospitals. The control group was recruited at the medical examination centre from the same area. The polymorphisms of the D2S388-5 and D2S2232 microsatellite loci were measured by multiple short tandem repeat amplification using fluorescence-labelled polymerase chain reaction and capillary electrophoresis. RESULTS: Nine alleles and 32 genotypes were identified in D2S388-5, while 9 alleles and 31 genotypes were identified in D2S2232. Both genotype distributions in control smokers were in accordance with Hardy-Weinberg equilibrium. The frequency of the 254 bp allele from the D2S388-5 locus was significantly higher in the COPD group versus the control (P < 0.001, odds ratio = 5.942). CONCLUSIONS: D2S388-5 microsatellite polymorphism may be associated with susceptibility to COPD in Xinjiang Kazakhs.

Mannose-binding lectin two gene polymorphisms and tuberculosis susceptibility in Chinese population: a meta-analysis.

Numerous studies have been done to explore the association between mannose-binding lectin two (MBL2) gene polymorphisms and the risk of tuberculosis (TB). However, the results are inconsistent. We performed a meta-analysis to investigate whether polymorphisms in the MBL2 gene were associated with TB risk. Databases including PubMed, Medline, Chinese Biomedicine Database, China National Knowledge Infrastructure, Wanfang Database, and Weipu Database were searched to find relevant articles published up to 2 October, 2012. Odds ratio (OR) with 95% confidence interval (CI) was used to evaluate the strength of association. All statistical tests were performed by using Revman 5.1 software and STATA 11.0 software. Six case-control studies including 1106 cases and 1190 controls were accepted in the meta-analysis. The results indicated that individuals carrying the MBL2 codon 54 B allele may have an increased risk of TB as compared with AA homozygotes (BB+AB vs. AA: OR=1.52, 95% CI: 1.22-1.88), whereas MBL2 +4 P/Q was possibly not associated with TB susceptibility in Chinese population.

ERK1/2 promotes cigarette smoke-induced rat pulmonary artery smooth muscle cells proliferation and pulmonary vascular remodeling via up-regulating cycline1 expression.

This study investigated the potential role of ERK1/2-cyclinE1 signaling pathway in rat pulmonary artery smooth muscle cells (rPASMCs) proliferation and pulmonary vascular remodeling induced by cigarette smoke exposure. A total of 24 male Wistar rats were randomly divided into 4 groups: control group (C group), S-1M, S-3M and S-6M groups (animals in the groups were exposed to smoke for 1, 3, and 6 months, respectively). HE staining and anti-α-smooth muscle actin antibody staining were performed to observe the degree of pulmonary vascular remodeling. Immunohistochemistry and Western blotting were performed to evaluate ERK1/2 and cyclinE1 expression in pulmonary vessels. Primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) were exposed to cigarette smoke extract (CSE). ERK inhibitor (PD98059) and cyclinE1 siRNA were used to verify the role of ERK1/2 and cyclinE1 in CSE-induced rPASMCs proliferation. Cell proliferation was assessed by cell counting and 5-bromo-2-deoxyuridine (BrdU) incorporation. Our results showed that abnormal pulmonary vascular remodeling was found in cigarette smoked rats. Compared to C group, activated ERK1/2 and cyclinE1 expression was significantly increased in smoke-exposure groups. This up-regulated expression was positively correlated with the severity of pulmonary vascular remodeling, and there was positive correlation between the expression of ERK1/2 and cyclinE1. PD98059 and cyclinE1 siRNA inhibited the proliferation of rPASMCs. The expression of cyclinE1 could be down-regulated by PD98059. Our data demonstrated that increased expression of ERK1/2 and cyclinE1 might be involved in the pathogenesis of abnormal rPASMCs proliferation and rat pulmonary vascular remodelling induced by cigarette smoke exposure.

Effect of dexamethasone on expression of AGR2 protein in asthmatic mice.

This study examined the expression of the anterior gradient-2 (AGR2) protein and Muc5ac protein in the lung tissues of asthmatic mice and the effect of dexamethasone, with an attempt to explore the role of AGR2 in the over-secretion of mucus in the airway. Eighteen BALB/c mice were divided into asthma group, control group and dexamethasone group. In dexamethasone group, dexamethasone was intraperitoneally administered. Expression of AGR2 protein and Muc5ac protein in the murine lung tissues was immunohistochemically detected. IL-13 level was determined in the bronchoalveolar lavage fluid (BALF) by ELISA. The results exhibited that the expression of AGR2 protein in asthma group (0.522±0.041) was significantly higher than that in normal controls (0.361±0.047) (P<0.01) and bore a positive linear relationship to the expression of Muc5ac protein (r=0.873, P<0.05) and IL-13 level (r=0.828, P<0.05). Expression of AGR2 protein in the dexamethasone group (0.456±0.049) was significantly lower than that in the asthma group. It was concluded that: (1) the expression of AGR2 protein was significantly higher in asthmatic mice as compared with their normal counterparts; (2) the expression was obviously related to the expression of Muc5ac protein and IL-13; (3) dexamethasone could down-regulate the expression of AGR2 protein. Our findings suggested that AGR2 might be involved in the over-secretion of mucus in the airway in asthma.

Effect of NADPH oxidase inhibitor apocynin on the expression of hypoxia-induced factor-1α and endothelin-1 in rat carotid body exposed to chronic intermittent hypoxia.

The effects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin on the enhanced hypoxia induced factor-1α (HIF-1α) and endothelin-1 (ET-1) expression, elevated systolic blood pressure under chronic intermittent hypoxia (CIH) condition and its action mechanism were investigated. Thirty healthy 8-week old Sprague-Dawley (SD) male rats were randomly divided into three groups (n=10 each): sham group, CIH group, and apocynin-treated CIH group. Tail artery systolic blood pressure was measured by tail-cuff method. Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of HIF-1α and ET-1 in the carotid body, and the HIF-1α protein expression was examined by using Western blotting. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by using colorimetric method. In addition, the plasma ET-1 and HIF-1α levels were measured by using enzyme-linked immunosorbent assay. It was found that CIH exposure was associated with increased MDA levels, and apocynin-treated CIH animals showed reduction in MDA levels. Apocynin treatment prevented CIH-induced hypertension as well as CIH-induced decrease in SOD. The increases of HIF-1α and ET-1 mRNA along with HIF-1α protein expression in the carotid body, and elevated circulating HIF-1α and ET-1 levels were observed in CIH-exposed animals. Treatment with apocynin significantly decreased the ET-1 mRNA, HIF-1α protein expression and circulating HIF-1α level in CIH-exposed animals, and there was no statistically significant difference in the HIF-1α mRNA expression between CIH group and apocynin-treated group. These results indicated that apocynin alleviated CIH-induced hypertension by inhibiting NADPH oxidase, further leading to the reduced vasoconstrictor ET-1 level and oxidative stress. HIF-1α/ET-1 system signal pathway may interact with CIH-induced NADPH oxidase-dependent oxidative stress. Inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for prevention and treatment of obstructive sleep apnea hypopnea syndrome-induced hypertension.

Inhibitory effect of dexamethasone on expression of cysteine-rich 61 protein in airway epithelial cells of allergic mouse models.

In order to study whether cysteine-rich 61 protein (cyr61) is involved in the pathogenesis of asthma and its relation to airway inflammation, the effect of dexamethasone (Dxm) on the expression of cyr61 in the lung tissues of asthmatic mice was investigated. Forty BALB/c mice were divided into asthma group (n=15), control group (n=10) and Dxm group (n=15). The asthma group was sensitized and challenged by ovalbumin (OVA). The mice in Dxm group were intraperitoneally administered with Dxm after OVA challenge. The expression of cyr61 in the lung tissues was detected by using immunohistochemistry, and that of eotaxin protein in the bronchoalveolar lavage fluid (BALF) by using enzyme-linked immunosorbent assay (ELISA). The number of inflammatory cells in BALF was also analyzed. The results showed that the cyr61 expression was highest in asthma group (P<0.05), followed by Dxm group (P<0.05) and control group. The cyr61 had a positive correlation with the total nucleated cells (r=0.867, P<0.05), especially eosinophils (r=0.856, P<0.05), and eotaxin level (r=0.983, P<0.05) in the BALF. Our findings suggested that cyr61 is expressed in airway epithelial cells and has a positive correlation with eotaxin and number of airway infiltrating eosinophils.

Genotoxicity and reduced heat shock protein 70 in human airway smooth muscle cells exposed to cigarette smoke extract.

Cigarette smoke is associated with the development of several diseases, such as chronic obstructive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (Hsp70) in human airway smooth muscle cells (HASMCs) exposed to cigarette smoke extract (CSE). HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined by using HPLC-ECD, the DNA damage was analyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The production of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In particular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.

[Correlation between the serum level of advanced oxidation protein products and the cognitive function in patients with obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: To observe the change of the cognitive function and the serum level of advanced oxidation protein products (AOPP) in patients with obstructive sleep apnea hypopnea syndrome (OSAHS), and then to investigate the correlation between them. METHODS: Sixty seven patients with OSAHS, 20 healthy controls with matched age, BMI, and education, 15 patients with OSAHS after effective treatment of continuous positive airway pressure (CPAP) with matched age, BMI, and education were enrolled. Polysomnography (PSG), Epworth sleepiness scale (ESS), mini-mental state examination (MMSE), and clock drawing test (CDT) were performed in these groups. The serum level of AOPP, superoxide dismutase (SOD), and malondialdehyde (MDA) were measured. RESULTS: The MMSE and CDT scores of patients with OSAHS were decreased compared to those in healthy controls [(4.73 ± 0.81) vs (2.69 ± 1.38), (2.85 ± 0.61) vs (1.92 ± 0.62)], but the scores improved after effective CPAP treatment. The serum levels of AOPP [(78 ± 20) vs (117 ± 20) µmol/L] and MDA [(2.9 ± 1.0) vs (6.1 ± 3.0) µmol/L] in patients with OSAHS were increased compared to those in healthy controls, but the levels decreased after effective CPAP treatment. The serum SOD level in patients with OSAHS was decreased compared to that in healthy controls [(89 ± 8) vs (57 ± 9) U/ml], but it was increased after effective CPAP treatment. The MMSE and CDT scores of all the subjects including the 2 groups (the OSAHS group and the effective CPAP-treatment group) were correlated with the results of PSG (baseline SaO2, lowest SaO2, AHI, LA/HT, SLT90%). The serum levels of AOPP, MDA and SOD of all the subjects were also correlated with the results of PSG (lowest SaO2, AHI, LA/HT, SLT90%). The serum levels of AOPP, MDA and SOD of all the subjects were correlated with the MMSE and CDT scores. The serum level of AOPP of all the subjects was also correlated with the serum levels of MDA and SOD. CONCLUSIONS: Cognitive impairment in patients with OSAHS is correlated with the severity of the disease. AOPP is a useful marker for oxidative stress and protein injury, and closely correlated with the cognitive impairment in patients with OSAHS.

[Effects of soothing liver and invigorating spleen recipes on NF-kappaB signal pathway related genes and proteins in primary hepatocytes of rats with NASH].

OBJECTIVE: To observe the effects of soothing liver and invigorating spleen recipes on NF-kappaB signal pathway related genes and proteins in primary hepatocytes of rats with NASH. METHODS: SD male rats were randomly divided into 8 groups: normal, model, high/low-dose soothing liver group, high/low-dose invigorating spleen group, high/low-dose integrated group. 15 rats in each group. The NASH model rats were induced by feeding high-fat diet (HFD). The treatment lasted for 16 weeks. Then TC, TG in the liver tissue and serum were determined with automatic biochemical analyzer. HE staining and oil red O staining were operated to observe the pathological changes. Another 6 rats of each group were taken respectively and collagenase (Type IV) was perfused to digest liver tissue with the circulation in vitro to separate hepatocytes. The expression levels of IKK(beta), NF-kappaB mRNA, proteins and phosphorylated IKK(beta) protein in hepatocytes of rats from each group were detected by Real-time Q-PCR and Western Blotting, respectively. RESULTS: Compared with normal group, liver histopathology was changed and levels of TC and TG were elevated in model group indicating hepatocytes had lipid accumulation and lipid metabolic disturbance obviously; The levels of serum TC, and hepatic homogenate TC, TG as well as the expression of IKK(beta) NF-kappa-B mRNA, proteins and phosphorylated IKK(beta) protein in hepatocytes were dramatically increased in model group (P < 0.01). Compared with the model group, the levels of IKK(beta), NF-kappaB mRNA expression were decreased most significanly in the invigorating spleen (with high dose) group and the integrated group (with high dose) (P < 0.01 or P < 0.05). The expression levels of the IKK(beta), NF-kappaB proteins and the phosphorylated IKK(beta) protein in hepatocytes were decreased significaniy in the treatment groups (P < 0.01 or P < 0.05), especially for the invigorating spleen (with high dose) group and the integrated (with high dose). CONCLUSION: Soothing liver and invigorating spleen recipes have effect on NASH rats induced by HFD and its mechanism may be related to the suppression of IKK(beta)/NF-kappaB signal pathway related genes and proteins. And the effect probably has a dose response relationship.

CCN2 promotes cigarette smoke-induced proliferation of rat pulmonary artery smooth muscle cells through upregulating cyclin D1 expression.

Cigarette smoke has been demonstrated to induce pulmonary vascular remodeling, which is characterized by medial thickening of the pulmonary arteries mainly resulting from the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the molecular mechanism underlying this process is still unclear. In the present study, we investigated whether CCN2 regulated rat PASMCs (rPASMCs) proliferation induced by cigarette smoke extract (CSE) and nicotine by upregulating cyclin D1 in vitro. CCN2 siRNA or cyclin D1 siRNA were transfected to rPASMCs which were then exposed to CSE and nicotine. Both mRNA and protein expressions of CCN2 were significantly increased in rPASMCs treated with 2% CSE or 1 µM nicotine, which markedly promoted the proliferation of rPASMCs. CCN2 siRNA inhibited the proliferation of rPASMCs induced by CSE or nicotine. Furthermore, CCN2 siRNA markedly suppressed the mRNA and protein expressions of cyclin D1 in rPASMCs and led to cell cycle arrest in G0/G1 phase resulting in reduced rPASMCs proliferation. These findings suggest that CCN2 contributes to the CSE and nicotine-induced proliferation of rPASMCs at least in part by upregulating cyclin D1 expression.

[The expression of macrophage migration inhibition factor in pulmonary tissues of smokers with or without chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the expression of macrophage migration inhibition factor (MIF) in pulmonary tissues of the smokers with and without chronic obstructive pulmonary disease (COPD). METHODS: The subjects were assigned into three groups: non-smokers without COPD (control group, n = 12), smokers without COPD (smoker group, n = 13) and smokers with COPD (COPD group, n = 16). The specimens were obtained from lung tissues as far away from cancer focus as possible (> 5 cm). Real-time quantitative PCR and immunohistochemistry were used to investigate the expression and distribution of MIF in pulmonary tissues. The relationship between the severity of airflow obstruction and the differential expressions of MIF in lung tissues of the smokers with or without COPD was analyzed. RESULTS: (1) MIF mRNA expression in COPD group (4.87 ± 1.79) was higher than that in the smoker group (2.16 ± 0.72; P < 0.01), which was higher than that in the control group (1.09 ± 0.48; P < 0.01). (2) Immunohistochemistry analysis showed that MIF protein expression in lung tissues of the COPD group (0.277 ± 0.025) was higher than that in the smokers group (0.199 ± 0.034; P < 0.01), which was significantly higher than that in control group (0.130 ± 0.021; P < 0.01). (3) Correlation analysis of MIF mRNA expression in the lung tissues and pulmonary function parameters of forced expired volume in one second (FEV(1)) percentage of predicted (FEV(1) pred)and ratio of FEV(1) to forced vital capacity (FEV(1)/FVC) suggested that MIF mRNA expression in the lung tissues was negatively related with FEV(1) pred (r = -0.578, P < 0.01) and FEV(1)/FVC (r = -0.607, P < 0.01). CONCLUSIONS: MIF expression significantly increases in the smokers with COPD, and MIF level in the lung is positively correlated with airflow limitation. The results suggest that MIF may play an important role in the pathogenesis of smoking-induced COPD.

[Expression of ß-catenin in human pulmonary tissues of smokers with and without chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the expression of ß-catenin in pulmonary tissues of smokers with and without chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary tissues were obtained from patients who had underwent pneumonectomy in Tongji Hospital. The subjects were assigned into non-smokers without COPD (control group), smokers without COPD (smoker group) and smokers with COPD (smoker + COPD group) based on their pulmonary functions and smoking history, with 12 subjects each group. The specimens were obtained as far from the tumor focus (> 5 cm) as possible. Immunofluorescence staining, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were used to investigate the expression and localization of ß-catenin in pulmonary tissues. Numerical data were expressed as the mean ± standard deviation, and were assessed for significance by one-way analysis of variance followed by a Student-Newman-Keuls test for multiple comparisons. The difference of enumeration data was detected by Chi-Square test. Relationship was estimated by Pearson correlation. RESULTS: Immunofluorescence analysis revealed that ß-catenin mainly expressed in the cell membrane of epithelial cells. There was also a positive expression in the cytoplasm and the nuclei of the epithelial cells. The number of alveolar epithelial cells with ß-catenin expressed in the cytoplasm and(or) nucleus was (1.2 ± 0.6)/HP in smokers + COPD group. And the protein and mRNA expression of ß-catenin in pulmonary tissues in smokers + COPD group were 0.26 ± 0.11 and 0.351 ± 0.129, respectively, which were significantly less than those of the smoker group and the control group [(5.0 ± 2.5)/HP and (8.4 ± 3.5)/HP, 0.62 ± 0.23 and 1.00 ± 0.50, 0.60 ± 0.14 and 1.03 ± 0.27]. The differences among the 3 groups were significant (F = 12.809 - 38.776, P < 0.05). Correlation analysis between ß-catenin expression and pulmonary function suggested that the protein and mRNA expression of ß-catenin positively related with FEV(1)%pred (P < 0.05) and FEV(1)/FVC (r = 0.402 - 0.558, P < 0.05). CONCLUSION: ß-catenin expression significantly was decreased in smokers with COPD, and ß-catenin level in the lungs was positively correlated with pulmonary function.

[Airway remodeling assessed by high-resolution computed tomography in patients with asthma: relationship to biological markers in induced sputum].

OBJECTIVE: To explore the significance of assessing asthma control by high-resolution computed tomography (HRCT) and biological markers in induced sputum. METHODS: Forty-eight patients with asthma (asthma group) and 10 healthy subjects (control group) were retrospectively analyzed. The asthma patients were divided into 4 groups based on severity: 6 with near-fatal attacks, 12 with severe, 14 with moderate and 16 with mild asthma. These patients received step therapy for 6 months based on the guidelines for the prevention and treatment of asthma. After achieving asthma control or partial control, HRCT, lung function and cytokine levels in induced sputum were measured. The ratio of wall area to total airway area (WA%), the ratio of 2 airway wall thickness to outer diameter (2T/D) and lung densities in both the inspiratory and expiratory phases were measured. Matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transformation growth factor-ß(1) (TGF-ß(1)) levels in the sputum were assessed by enzyme-linked immunosorbent assay. RESULTS: There were significant differences in forced vital capacity and forced expiratory volume in 1 second as the percentage of predicted value (FVC% and FEV(1)%, respectively), the ratio of FEV(1)/FVC, and diffusing capacity of the lung for carbon monoxide (D(LCO)) among groups (F = 5.526, 15.064, 16.326, 2.945, respectively, P < 0.05). Sputum levels of MMP-9, TIMP-1 and TGF-ß(1) were significantly increased in the near-fatal asthma, severe asthma, moderate asthma and mild asthma groups [MMP-9: (80 ± 16), (70 ± 9), (59 ± 6), and (52 ± 7) µg/L, respectively; TIMP-1: (212 ± 95), (258 ± 167), (28 ± 51), and 98 ± 60 µg/L, respectively; TGF-ß(1): (586 ± 81), (513 ± 54), (401 ± 45) and (351 ± 57) µg/L, respectively]compared with the control group [MMP9: (46 ± 5) µg/L; TIMP: (19 ± 13) µg/L; and TGF-ß(1): (258 ± 29) µg/L]. These parameters were progressively increased in the asthma groups with the severity of disease (F = 11.179, 49.914, 9.286, respectively, P < 0.05). The ratio of MMP-9/TIMP-1 in sputum was decreased in the near-fatal attack, severe, moderate and mild asthma groups (0.50 ± 0.28, 0.34 ± 0.13, 0.53 ± 0.22, and 0.87 ± 0.75, respectively) compared with the control group (2.93 ± 1.13). The MMP-9/TIMP-1 ratio in the severe asthma group was lowest among the asthma groups (F = 43.335, P < 0.05). 2T/D and WA% were higher in both the near-fatal asthma group (0.51 ± 0.01 and 0.75 ± 0.01, respectively) and the severe asthma group (0.53 ± 0.03 and 0.77 ± 0.03, respectively) as compared to the moderate asthma group (0.43 ± 0.04 and 0.67 ± 0.04, respectively) or the mild group (0.42 ± 0.04 and 0.66 ± 0.04, respectively). 2T/D and WA% were higher in the asthma groups than in the control group (0.35 ± 0.03 and 0.57 ± 0.04, respectively), (F = 40.224, 41.294, respectively, P < 0.05). Lung densities in both the inspiratory and expiratory phases were lower in the near-fatal attack group as compared to those in the other asthma groups or the control group; and the lung density differences between the two phases in the near-fatal attack group were smaller than those in the other asthma groups or the control group (F = 5.048, 13.247, 11.541, respectively, P < 0.05). 2T/D and WA% were correlated positively with MMP-9, TIMP-1 and TGF-ß(1) levels, but negatively with the MMP-9/TIMP-1 ratio, respectively. CONCLUSIONS: HRCT and biological markers in induced sputum could be used to accurately evaluate asthma control. These findings suggest that the severity of asthma, especially, near-fatal attack of asthma, is correlated not only with the degree of airway remodeling, but also with the degree of air trapping.

Cigarette smoke extract induced rat pulmonary artery smooth muscle cells proliferation via PKCα-mediated cyclin D1 expression.

Cigarette smoke could induce pulmonary smooth muscle cells (PASMCs) proliferation. Although our previous study had implied the involvement of protein kinase Cα (PKCα), the molecular mechanism underlying PKCα pathway in this process is still unknown. In this study, rat PASMCs were stimulated by cigarette smoke extract (CSE) or PMA (a special activator to PKCα). Two percent CSE and PMA significantly enhanced cyclin D1 expression and cells proliferation. But cyclin D1-specific siRNA successfully inhibited DNA synthesis in CSE-treated or PMA-treated cells. On the other hand, PKCα-specific siRNA significantly suppressed cyclin D1 expression in CSE-treated cells. Moreover, PKCα-specific siRNA resulted in a cell-cycle arrest in G0/G1 and decreased cells number significantly. We conclude that CSE induced rat PASMCs proliferation at least partly via PKCα-mediated cyclin D1 expression.