Expression and regulation of HIF-1a in hypoxic pulmonary hypertension: Focus on pathological mechanism and Pharmacological Treatment.

Hypoxia inducible factor-1(HIF-1), a heterodimeric transcription factor, is composed of two subunits (HIF-1α and HIF-1ß). It is considered as an important transcription factor for regulating oxygen changes in hypoxic environment, which can regulate the expression of various hypoxia-related target genes and play a role in acute and chronic hypoxia pulmonary vascular reactions. In this paper, the function and mechanism of HIF-1a expression and regulation in hypoxic pulmonary hypertension (HPH) were reviewed, and current candidate schemes for treating pulmonary hypertension by using HIF-1a as the target were introduced, so as to provide reference for studying the pathogenesis of HPH and screening effective treatment methods.

A Lipid Perspective on Regulated Pyroptosis.

Pyroptosis is a novel pro-inflammatory cell programmed death dependent on Gasdermin (GSMD) family-mediated membrane pore formation and subsequent cell lysis, accompanied by the release of inflammatory factors and expanding inflammation in multiple tissues. All of these processes have impacts on a variety of metabolic disorders. Dysregulation of lipid metabolism is one of the most prominent metabolic alterations in many diseases, including the liver, cardiovascular system, and autoimmune diseases. Lipid metabolism produces many bioactive lipid molecules, which are important triggers and endogenous regulators of pyroptosis. Bioactive lipid molecules promote pyroptosis through intrinsic pathways involving reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, lysosomal disruption, and the expression of related molecules. Pyroptosis can also be regulated during the processes of lipid metabolism, including lipid uptake and transport, de novo synthesis, lipid storage, and lipid peroxidation. Taken together, understanding the correlation between lipid molecules such as cholesterol and fatty acids and pyroptosis during metabolic processes can help to gain insight into the pathogenesis of many diseases and develop effective strategies from the perspective of pyroptosis.

Celastrol: The new dawn in the treatment of vascular remodeling diseases.

Evidence is mounting that abnormal vascular remodeling leads to many cardiovascular diseases (CVDs). This suggests that vascular remodeling can be a crucial target for the prevention and treatment of CVDs. Recently, celastrol, an active ingredient of the broadly used Chinese herb Tripterygium wilfordii Hook F, has attracted extensive interest for its proven potential to improve vascular remodeling. Substantial evidence has shown that celastrol improves vascular remodeling by ameliorating inflammation, hyperproliferation, and migration of vascular smooth muscle cells, vascular calcification, endothelial dysfunction, extracellular matrix remodeling, and angiogenesis. Moreover, numerous reports have proven the positive effects of celastrol and its therapeutic promise in treating vascular remodeling diseases such as hypertension, atherosclerosis, and pulmonary artery hypertension. The present review summarizes and discusses the molecular mechanism of celastrol regulating vascular remodeling and provides preclinical proof for future clinical applications of celastrol.

CHOP overexpression sensitizes human non-small cell lung cancer cells to cisplatin treatment by Bcl-2/JNK pathway.

C/EBP homologous protein (CHOP), a 29 kDa cellular protein, plays a role in regulating tumor proliferation, differentiation, metabolism, cell death, and in tumor resistance to chemotherapy. Non-small cell lung cancer (NSCLC) is a tumor of the respiratory system and drug resistance is prevalent among NSCLC clinical cell cultures. Herein, our study elucidated the effect of CHOP on NSCLC cells with cisplatin resistance and its mechanism. In a NSCLC cell line with cisplatin-resistance, CHOP expression was decreased, compared with A549 cells. Overexpression of CHOP decreased the cell viability and enhanced cell apoptosis in the cells treated with cisplatin. Expression of CHOP also inhibited the cell proliferation and metastasis. CHOP increased the therapeutic effect of cisplatin on NSCLC cells through the Bcl-2/JNK pathway. In summary, CHOP regulated cisplatin resistance in cells of NSCLC by promoting the expression of apoptotic proteins and inhibiting the Bcl-2/JNK signaling pathway, indicating the antitumor effects of CHOP.

α1-Antitrypsin alleviates inflammation and oxidative stress by suppressing autophagy in asthma.

BACKGROUND: Asthma is considered an incurable disease, although many advances have been made in asthma treatments in recent years. Therefore, elucidating the pathological mechanisms and seeking novel and effective therapeutic strategies for asthma are urgently needed. METHODS: Airway resistance was measured by whole-body plethysmography. H&E staining was used to observe the morphological changes in the lung. Oxidative stress was assessed by measuring the levels of MDA, CAT and SOD. Gene expression was analysed by western blotting and RT-qPCR. ELISA was used to analyse the concentrations of IL-4, IL-5 and IFN-γ. RESULTS: In the present study, we successfully established in vivo and in vitro asthma models. OVA administration led to elevated lung resistance, cell counts in BALF, and cytokine secretion, impaired airway structure and enhanced oxidative stress and autophagy in a mouse model of asthma, while IL-13 induced inflammation, oxidative stress and autophagy in BEAS-2B cells. A1AT reduced lung resistance and cell counts in BALF and suppressed inflammation, oxidative stress and autophagy in a mouse model of asthma and IL-13-induced BEAS-2B cells. Mechanistic investigations revealed that autophagy activation compromised the protective effect of A1AT on IL-13-induced BEAS-2B cells. Further mechanistic studies revealed that A1AT alleviated inflammation and oxidative stress by inhibiting autophagy in the context of asthma. CONCLUSION: We demonstrated that A1AT could alleviate inflammation and oxidative stress by suppressing autophagy in the context of asthma and thus ameliorate asthma. Our study revealed novel pathological mechanisms and provided novel potential therapeutic targets for asthma treatment.

PVAT targets VSMCs to regulate vascular remodelling: angel or demon.

Vascular remodelling refers to abnormal changes in the structure and function of blood vessel walls caused by injury, and is the main pathological basis of cardiovascular diseases such as atherosclerosis, hypertension, and pulmonary hypertension. Among them, the neointimal hyperplasia caused by abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of vascular remodelling. Perivascular adipose tissue (PVAT) can release vasoactive substances to target VSMCs and regulate the pathological process of vascular remodelling. Specifically, PVAT can promote the conversion of VSMCs phenotype from contraction to synthesis by secreting visfatin, leptin, and resistin, and participate in the development of vascular remodelling-related diseases. Conversely, it can also inhibit the growth of VSMCs by secreting adiponectin and omentin to prevent neointimal hyperplasia and alleviate vascular remodelling. Therefore, exploring and developing new drugs or other treatments that facilitate the beneficial effects of PVAT on VSMCs is a potential strategy for prevention or treatment of vascular remodelling-related cardiovascular diseases.

KLF2 regulates neutrophil migration by modulating CXCR1 and CXCR2 in asthma.

Neutrophils are key inflammatory cells in the immunopathogenesis of asthma. Neutrophil migration can be initiated through activation of the CXCR1 and CXCR2 receptors by CXC chemokines, such as IL-8. Although transcription factor KLF2 has been found to maintain T cell migration patterns through repression of several chemokine receptors, whether KLF2 can regulate neutrophil migration via modulation of CXCR1 and CXCR2 is unknown. Here, we aimed to explore the functions of KLF2, CXCR1 and CXCR2 in neutrophil migration in asthma and to establish a regulatory role of KLF2 for CXCR1/2. We demonstrate that with asthma aggravation, the percentages and migration rates of peripheral blood neutrophils gradually increased in asthmatic patients and the guinea pig asthma model. Correspondingly, both the KLF2 mRNA and protein levels in neutrophils were gradually reduced. While CXCR1 and CXCR2 expression was negatively correlated with KLF2. In vitro knockdown of KLF2 dramatically increased the migration of HL-60-drived neutrophil-like cells, which was accompanied by an increase in the CXCR1 and CXCR2 mRNA and protein expression levels. Taken together, our results indicate that decreased KLF2 aggravates asthma progression by promoting neutrophil migration, which is associated with the transcriptional upregulation of CXCR1 and CXCR2. The KLF2 and/or CXCR1/2 expression levels may represent an indicator of asthma severity.

Endoplasmic Reticulum Stress Induces HRD1 to Protect Alveolar Type II Epithelial Cells from Apoptosis Induced by Cigarette Smoke Extract.

BACKGROUND/AIMS: Cigarette smoking is a major risk factor of chronic obstructive pulmonary disease. This study aimed to examine the effects of cigarette smoke extract (CSE) on alveolar type II epithelial cells (AECII) and investigate the underlying mechanism. METHODS: Primary AECII were isolated from rat lung tissues and exposed to CSE. Apoptosis was detected by flow cytometry. Protein expression was detected by Western blot analysis. RESULTS: Primary rat AECII maintained morphological and physiological characteristic after 3 passages. CSE increased the expression of ER specific pro-apoptosis factors CHOP and caspase 12, and the phosphorylation of JNK in AECII. CSE activated ER stress signaling and increased the phosphorylation of PERK, eIF2α and IRE1. Furthermore, CSE induced the expression of Hrd1, a key factor of ER-associated degradation, in AECII. Knockdown of Hrd1 led to more than 2 fold increase of apoptosis, while overexpression of Hrd1 attenuated CSE induced apoptosis of AECII. CONCLUSIONS: Our results suggest that ER stress induces HRD1 to protect alveolar type II epithelial cells from apoptosis induced by CSE.

[Allergens-induced sensitization alters airway epithelial adhesion molecules expression in mice].

To explore the relationship between the epithelial adhesion molecules and immune responses of airway epithelium, we observed the expression of integrin ß4 and intercellular adhesion molecule-1 (ICAM-1) in the mice airway epithelium after sensitization with allergens. BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) or house dust mite (HDM) and then developed airway hyper-responsiveness as determined by barometric whole-body plethysmography. Both OVA and HDM sensitization led to increases of the number of peripheral leukocytes as well as inflammatory cells infiltration in lungs. OVA sensitized mice showed more severe inflammatory cells infiltration than HDM sensitized mice. Immunohistochemistry analysis of mice lung tissues revealed that sensitization with both allergens also led to a decrease of integrin ß4 expression and an increase of ICAM-1 expression in airway epithelia. OVA sensitized mice showed a more significant increase of ICAM-1 expression compared with HDM sensitized mice. siRNA mediated silencing of integrin ß4 gene in 16HBE cells resulted in an up-regulation of ICAM-1 expression. Our results indicate a possible role of airway epithelial adhesion molecules in allergen-induced airway immune responses.

Bevacizumab induces A549 cell apoptosis through the mechanism of endoplasmic reticulum stress in vitro.

AIMS: To observe the effect of bevacizumab on human A549 cells and explore its mechanism. METHODS: After different concentrations (0 µM, 1 µM, 5 µM, 25 µM) of bevacizumab treating in A549 cells, CCK8 assay detect the impact of bevacizumab on A549 cell proliferation and flow cytometry determine the effect of bevacizumab on human A549 cells apoptosis. Real-time PCR and Western blotting detect the changing expression of the target gene (CHOP, caspase-4, IRE1, XBP-1) on mRNA and Protein level. RESULTS: Treatment with bevacizumab for 24-hr have induced cell death in a does-dependent manner dramatically (P<0.05). In terms of the mRNA level, expression of XBP-1 has increased obviously in each group (1 µM, 5 µM, 25 µM) (P<0.01); the expression of CHOP (25 µM) and caspase-4 (5 µM) have increased slightly (P<0.05). In terms of the protein level, the expression of CHOP has increased obviously in each group (1 µM, 5 µM, 25 µM) when compared with the control group (0 µM) (P<0.05). As for caspase-4 (5 µM, 25 µM), the expression have increased slightly when compared with the control group (0 µM) (P<0.05). CONCLUSION: Bevacizumab can induce A549 cell apoptosis through the mechanism of endoplasmic reticulum stress.

[Sublingual gland amyloidosis causing obstructive sleep apnea hypopnea syndrome: a case report and review of the literatures].

OBJECTIVE: To improve the understanding of sublingual gland amyloidosis causing obstructive sleep apnea hypopnea syndrome(OSAHS). METHODS: A case of sublingual gland amyloidosis causing OSAHS diagnosed in april 2012 was reported and the related literatures were reviewed. The literature review was carried out respectively with "amyloidosis, sublingual gland, obstructive sleep apnea hypopnea syndrome", as the search terms in Wanfang Data and PubMed by November 2012. RESULTS: A case of 74 year-old male patient was admitted to the hospital because of snoring for 5 years, sleep apnea for 1 year and arousal for 1 month. After admission, polysomnography showed severe OSAHS, physical examination showed redundant the sublingual gland. Enhanced CT scanning showed soft tissue masses at the sublingual gland. Abdominal B ultrasonic and CT also showed a spaces-occupying lesion in the left retroperitoneal. B-guided core needle biopsy was performed in the left retroperitoneal. Pathology report showed amyloidosis. Subsequently, sublingual gland mass resection was performed. Pathology report after operation showed amyloid deposits staining with Congo red, which gives it a characteristic green birefringence in polarised light. Accordingly, it was diagnosed as sublingual gland amyloidosis. The symptoms of snore and sleep apnea were disappeared after operation.So far, there was no local recurrence with 10 months follow-up. A total of 3 literatures were received in Wanfang Data, including 2 of macroglossia amyloidosis causeing OSAHS case report and one of retrospective study. There were no reports about sublingual gland amyloidosis in Wanfang Data. A total of 5 literatures were received in Pubmed, including 2 of sublingual gland amyloidosis case report, 2 of macroglossia amyloidosis causeing OSAHS case report and one of retrospective study. However, there were no reports about sublingual gland amyloidosis causing OSAHS. CONCLUSIONS: Amyloidosis rarely occurred in the sublingual gland and is easy to be misdiagnosed and missed diagnosed, which can causing severe OSAHS. To make a definite diagnosis, histopathology and staining with Congo red are needed and a characteristic green birefringence in polarised light is a reliable marker for diagnosis. After sublingual gland mass resection, the patient had good prognosis.

[The expression of hypoxia-inducible factor-1alpha and its hydroxylases in pulmonary arteries of patient with chronic obstructive pulmonary disease].

OBJECTIVE: To observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD). METHODS: Pulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively. RESULTS: WA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein. CONCLUSION: PHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression

[The expression and possible role of SENP1 in the pulmonary vascular wall of rat during the development of hypoxic pulmonary hypertension].

OBJECTIVE: To investigate the dynamic expression and role of SENP1 (SUMO-specific proteases-1) in the pulmonary vascular wall of rat during the development of hypoxic pulmonary hypertension (HPH). METHODS: Forty adult male Wistar rats were randomly divided into 5 groups (n = 8), and exposed to normoxia (Control group) or exposed to hypoxia for 3, 7, 14 or 21 d, respectively. The HPH models were established by normobaric intermittent hypoxia. Mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), and vessel morphometry were measured. Reverse transcriptase-polymerase chain reaction(RT-PCR) and in situ hybridization were used to determine the mRNA expression of SENP1. Immunohistochemistry and Western blot were used to determine the protein expression of SENP1. RESULTS: The hypoxic rats developed pulmonary vascular remodeling in pulmonary arterioles after 7 d of hypoxia exposure. Pulmonary vascular remodeling in pulmonary arterioles significantly increased after 14 d of hypoxia. The level of mPAP in hypoxic rats increased significantly after 7 d of hypoxia, reached its peak after 14 d of hypoxic exposure. RVHI was markedly increased after 14 d of hypoxia. In situ hybridization and immunohistochemical analysis showed that SENP1 mRNA and protein were positively stained in control. SENP1 mRNA expression had little changes after exposure to hypoxia compared with the control, however, SENP1 protein expression was declined gradually after 7 d of hypoxia. The results of RT-PCR and Western blot showed that the same dynamic expression of SENP1 mRNA and protein in lung tissues of rats. Linear correlation analysis showed that SENP1 protein were negatively correlated with mPAP, pulmonary vascular remodeling index and RVHI. CONCLUSION: Under chronic hypoxia, SENP1 protein can be degradated. The dynamic expression of SENP1 protein may play a role in implicating in the development of HPH.

[The effect of transcription factor KLF2 in expression of gamma-GCS depend on regulation by Nrf2 in lung of rats with chronic obstructive pulmonary disease].

OBJECTIVE: To study the expression of lung Krüppel-like transcription factor (KLF2/LKLF) in lung tissues of rats with chronic obstructive pulmonary disease (COPD) and the relationship between KLF2 and NF-E2-related factor 2 (Nrf2), and make further explore the effects of KLF2 on the expression of gamma-glutamylcysteine synthetase (gamma-GCS). METHODS: Twenty-two male SD rats were randomly divided into a COPD group (n = 10) and a normal control group (n = 11). The rat model of COPD established by cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS), and lung tissues were obtained. The expressions of KLF2, Nrf2, gamma-GCS mRNA and protein in lung tissues were measured by immunohistochemistry (IHC), Western blot, in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR). To explore the relationship between KLF2 and Nrf2 protein,we utilize the method of co-immunoprecipitation (CO-IP). RESULTS: IHC and Western blot showed that protein expressions of KLF2, Nrf2, gamma-GCS were higher in the lung tissues from rats with COPD than those in the control groups (all P < 0.05). The levels of KLF2, gamma-GCS mRNA were markedly increased in the COPD group (all P < 0.01) while Nrf2 mRNA expression in COPD group had no significant difference with that in control group ( P > 0.05). CO-IP result showed that KLF2 were obviously present in immunoprecipitates of Nrf2 (P < 0.01) . Linear correlation analysis showed that the level of KLF2 protein was positively correlated with the level of Nrf2 protein (P < 0.05), and KLF2, Nrf2 proteins were positively correlated with gamma-GCS mRNA and protein (all P < 0.05). CONCLUSION: The expression of KLF2 is significantly up-regulated in COPD, which maybe up-regulate gamma-GCS mRNA expression by increasing Nrf2 expression and nuclear translocation against oxidative stress.

[Effects of feixin decoction on the contents of hypoxia-inducible factor-1alpha and vascular endothelial growth factor in the rat model of hypoxic pulmonary hypertension].

OBJECTIVE: To explore the effects of Feixin Decoction (FXD) on the hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in the rat model of hypoxic pulmonary hypertension (HPH), and to study its mechanisms for treating HPH. METHODS: Forty healthy male SD rats were randomly divided into four groups, i. e., the normal control group, the HPH model group, the FXD group, and the Nifedipine group, 10 rats in each group. The HPH rat model was prepared using normal pressure intermittent hypoxia method. Except the normal control group, rats in the rest groups were fed in a self-made hypoxic plexiglass cabin, with the poor oxygen condition for 8 h daily for 14 successive days. Then the distilled water (at 30 mL/kg) was given by gastrogavage to rats in the normal control group and the HPH model group. FXD (at 28 g/kg) and Nifedipine (at 20 mg/kg) were given by gastrogavage to rats in the FXD group and the Nifedipine group respectively, once daily, for 14 successive days. Besides, hypoxia was continued for 14 days while medicating. The mean pulmonary artery pressure (mPAP) was detected on the second day after the last medication. The morphology of the pulmonary arteriole was detected. The ratio of pulmonary artery wall area and tube area (WA%) was determined. The protein and mRNA expressions of HIF-1alpha and VEGF were detected using immunohistochemistry and in situ hybridization technique. RESULTS: Compared with the normal control group, mPAP, WA%, and the protein and mRNA expressions of HIF-1alpha and VEGF significantly increased in the model group (P < 0.01, P < 0.05). Compared with the HPH model group, mPAP, WA%, and the protein and mRNA expressions of HIF-1alpha and VEGF significantly decreased in the FXD group (P < 0.01, P < 0.05). CONCLUSIONS: FXD down-regulated the expression of VEGF through decreasing the expression of HIF-1alpha. One of its mechanisms for treating HPH might be partially due to reversing the remodeling of pulmonary vascular smooth muscle.

[The roles of PPAR-gamma/PGC-1alpha to Nrf2/gamma-GCS-h in lung of guinea pigs with bronchial asthma].

OBJECTIVE: To investigate the expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h in lung of guinea pigs with bronchial asthma, and to explore the roles of them. METHODS: Forty adult male guinea pigs were randomly divided into 4 groups: the control group (group A), asthmatic group ( group B), dexamethasone group (group C) and rogridone group (group D), 10 guinea pigs in each group. The asthmatic model was established by the ovalbumin challenge method. Expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h mRNA in lung tissue were assayed by in situ hybridization. Expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h protein were detected by immunohischemistry and by Western blot. RESULTS: In situ hybridization showed that the expressions of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h mRNA in lung tissue were the lowest in group B and the comparison among groups showed statistical significant (all P < 0.01). Immunohistochemistry and Western blot indicated that the value of PPAR-gamma/PGC-1alpha and Nrf2/gamma-GCS-h protein in lung tissue were the lowest in group B, and expressed primarily in nucleus, the differences being statistically significant (all P < 0.01). There was positive correlation between PPAR-gamma and PGC-1. gamma-GCS-h mRNA also positively correlated between PPAR-gamma/PGC-1alpha and Nrf2 in nucleus, and the expression of Nrf2 was also positively correlated with PPAR-gamma/ PGC-1alpha. CONCLUSION: In acute asthmatic models induced by ovalbumin, the expressions of PPAR-alpha/PGC-1alpha and Nrf2/gamma-GCS-h were decreased, and PPARgamma/PGC-1alpha could up-regulate the expressions of Nrf2/gamma-GCS-h to increase the antioxidant defense of tissues, thus being implicated that PPARgamma/PGC-1alpha might play important roles in the pathogenesis and prevention of asthma.

[The pathway of PI3k/Akt-aPKCiota/zeta-Nrf2 regulating the expression of gamma-glutamylcysteine synthetase in the bronchial epithelial cells of rats].

OBJECTIVE: To observe the effect of the signal pathway of phosphoinositol-3-kinase (PI3K)/Akt-antypical protein kinase C(iotazeta) (aPKC(iotazeta))-Nuclear factor-E2 related factor (Nrf2) on gamma-glutamylcysteine synthetase (gamma-GCS) of the bronchial epithelial cells of rats after exposure to cigarette smoke extracts (CSE). METHODS: gamma-GCS, Nrf2, p-Akt and p-aPKC(iotazeta) proteins were semi-quantified by Western blot. gamma-GCS protein expression was assessed by immunocytochemistry. gamma-GCS mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Nrf2 protein was observed by immunofluorescence. The rate of the cells expressed p-Akt were analyzed by flow cytometry. GSH content and gamma-GCS activity were measured. RESULTS: GSH content, Nrf2 protein of nucleus, p-aPKC(iotazeta) protein, p-Akt protein and positive cells, gamma-GCS protein and mRNA and activity were significantly increased after exposure to CSE for 3 hours. aPK(iotazeta) inhibitor RO813220 significantly reduced the expression of p-aPKC(iotazeta) protein, gamma-GCS protein and mRNA and activity, but enhanced Nrf2 protein of cytoplasm expression, had no effect on p-Akt. p-Akt inhibitor LY294002 and RO813220 + LY294002 decreased p-aPKC(iotazeta) protein, p-Akt protein and positive cells, gamma-GCS protein and mRNA and activity expression, increased Nrf2 protein of cytoplasm expression. The correlation analysises demonstrated that there were a positive correlation between Nrf2 and gamma-GCS, p-Akt, p-aPKC(iotazeta), between p-Akt and Nrf2, p-aPKC(iotazeta), gamma-GCS, between p-aPKC(iotazeta) and Nrf2, p-Akt, gamma-GCS. CONCLUSION: CSE might upregulate gamma-GCS expression through PI3K/Akt-aPKC(iotazeta)-Nrf2 signaling pathway in the bronchial epithelial cells of rats.

[The cooperation of OS-9 and PHDs in hypoxia-induced pulmonary hypertension of rats].

AIM: To investigate the dynamic expression of hypoxia-inducible factor 1alpha, PHDs and OS-9 in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. METHODS: SD rats were randomly divided into 5 groups (n = 8) and exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. RT-PCR and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of protein. RESULTS: HIF-1alpha protein was poorly positive in control, markedly up-regulated after 3 d and 7 d of hypoxia (P < 0.05, vs group C), and then declined slightly after 14 d and 21 d of hypoxia. HIF-1alpha mRNA increased dramatically after 14 d of hypoxia (P < 0.05, vs group C). PHD1, PHD2 mRNA and protein was positive in group C. PHD2 mRNA and protein were up-regulated after 3 d of hypoxia (P < 0.05, vs group C), reaching its peak after 14 d of hypoxia while PHD1 protein declined after 14 d of hypoxia (P < 0.05, vs group C) without statistic mRNA changing. PHD3 mRNA and protein were detected at low level in control, markedly up-regulated after 3 d of hypoxia (P < 0.05, vs group C), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14 d of hypoxia (P < 0.05, vs 7 d). OS-9 mRNA was positively in control, markedly decreased after 3 d of hypoxia (P < 0.05, vs group C), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that OS-9 protein was positively correlated with OS-9 mRNA (r = 0.82, P < 0.01) and HIF-1alpha protein (r = 0.57, P < 0.01). CONCLUSION: HIF-1alpha, PHDs and OS-9 are all involved in the pathogenesis of hypoxic pulmonary hypertension in rats. OS-9 may interact with both HIF-1alpha and PHDs to promote PHD-mediated hydroxylation of HIF-1alpha.