Differences in pulmonary nodular consolidation and pulmonary cavity among drug-sensitive, rifampicin-resistant and multi-drug resistant tuberculosis patients: the Guangzhou computerized tomography study.

Background: Pulmonary nodular consolidation (PN) and pulmonary cavity (PC) may represent the two most promising imaging signs in differentiating multidrug-resistant (MDR)-pulmonary tuberculosis (PTB) from drug-sensitive (DS)-PTB. However, there have been concerns that literature described radiological feature differences between DS-PTB and MDR-PTB were confounded by that MDR-PTB cases tend to have a longer history. This study seeks to further clarify this point. Methods: All cases were from the Guangzhou Chest Hospital, Guangzhou, China. We retrieved data of consecutive new MDR cases [n=46, inclusive of rifampicin-resistant (RR) cases] treated during the period of July 2020 and December 2021, and according to the electronic case archiving system records, the main PTB-related symptoms/signs history was ≤3 months till the first computed tomography (CT) scan in Guangzhou Chest Hospital was taken. To pair the MDR-PTB cases with assumed equal disease history length, we additionally retrieved data of 46 cases of DS-PTB patients. Twenty-two of the DS patients and 30 of the MDR patients were from rural communities. The first CT in Guangzhou Chest Hospital was analysed in this study. When the CT was taken, most cases had anti-TB drug treatment for less than 2 weeks, and none had been treated for more than 3 weeks. Results: Apparent CT signs associated with chronicity were noted in 10 cases in the DS group (10/46) and 9 cases in the MDR group (10/46). Thus, the overall disease history would have been longer than the assumed <3 months. Still, the history length difference between DS patients and MDR patients in the current study might not be substantial. The lung volume involvement was 11.3%±8.3% for DS cases and 8.4%±6.6% for MDR cases (P=0.022). There was no statistical difference between DS cases and MDR cases both in PN prevalence and in PC prevalence. For positive cases, MDR cases had more PN number (mean of positive cases: 2.63 vs. 2.28, P=0.38) and PC number (mean of positive cases: 2.14 vs. 1.38, P=0.001) than DS cases. Receiver operating characteristic curve analysis shows, PN ≥4 and PC ≥3 had a specificity of 86% (sensitivity 25%) and 93% (sensitivity 36%), respectively, in suggesting the patient being a MDR cases. Conclusions: A combination of PN and PC features allows statistical separation of DS and MDR cases.

Triptolide inhibits PDGF-induced proliferation of ASMCs through G0/G1 cell cycle arrest and suppression of the AKT/NF-κB/cyclinD1 signaling pathway.

Abnormal proliferation of airway smooth muscle cells (ASMCs) is a hallmark of airway remodeling. Platelet-derived growth factor (PDGF) is known to be a major stimulus inducing the proliferation of ASMCs. It has been reported that triptolide demonstrates protective effects against airway remodeling. In this study, we investigated the antiproliferative effects of triptolide on PDGF-induced ASMCs and its underlying mechanisms. Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Quantitative real-time PCR and Western blot analysis were employed to detect the expression of proliferating cell nuclear antigen (PCNA), cyclinD1 and cyclin dependent kinase 4 (CDK4). Proteins involved in the protein kinase B (AKT) and nuclear factor kappa B (NF-κB) signaling pathways were evaluated using Western blot analysis. Triptolide could significantly inhibit cell proliferation, induce cell cycle arrest in the G0/G1 phase, and reduce the expression of PCNA, cyclinD1, and CDK4 in PDGF-treated ASMCs. Levels of phosphorylated AKT, p65 and NF-κB inhibitor α (IκBα) stimulated by the presence of PDGF were markedly suppressed after triptolide treatment. Moreover, triptolide cotreatment with the phosphatidylinositol 3 kinase (PI3k) inhibitor, 2-(4-morpholinyl)-8-phenylchromone (LY294002), could further suppress the proliferation, NF-κB activation and cyclinD1 expression. Similar results were observed after triptolide cotreatment with the NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC). Our results suggest that triptolide could inhibit the PDGF-induced proliferation of ASMCs through G0/G1 cell cycle arrest and suppression of the AKT/NF-κB/cyclinD1 signaling pathway.

MiR-23b controls TGF-ß1 induced airway smooth muscle cell proliferation via direct targeting of Smad3.

BACKGROUND: MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. Here we report that miR-23b inhibited airway smooth muscle cells (ASMCs) proliferation through directly targeting of Smad3. METHODS: We obtained ASMCs by laser capture microdissection of normal and asthmatic mice lung tissues. Mice ASMCs were cultured and induced by TGF-ß1. The implication between TGF-ß1 and miR-23b in ASMCs were detected by RT-PCR. The effects of miR-23b on ASMCs proliferation and apoptosis were assessed by transient transfection of miR-23b mimics and inhibitor. The expression of Smad3 in ASMCs were detected by RT-PCR and Western blotting analysis. Dual-Luciferase Reporter Assay System will be applied to identify whether Smad3 is a target gene of miR-23b. RESULTS: TGF-ß1 and miR-23b mRNA expression of in-situ bronchial ASMCs collected by laser capture microdissection were increased in asthmatic mice compared to non-asthma controls. This is accompanied by an increase in miR-23b mRNA expression in TGF-ß1 induced ASMCs. miR-23b up-regulation significantly inhibited TGF-ß1-induced ASMCs proliferation and promoted apoptosis. MiR-23b negatively regulates the expression of Smad3 in ASMCs. Dual-Luciferase Reporter Assay System demonstrated that Smad3 was a direct target of miR-23b. CONCLUSIONS: MiR-23b may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via direct targeting of Smad3.

Airway smooth muscle cells from ovalbumin-sensitized mice show increased proliferative response to TGFß1 due to upregulation of Smad3 and TGFßRII.

OBJECTIVE: This study aimed to elucidate the role of Transforming growth factor (TGF)-ß1 signaling in the proliferation of airway smooth muscle cells (ASMCs). BACKGROUND: TGF-ß1 is an important cytokine in airway remodeling in asthma. However, results of studies focusing on the effect of TGFß1 on proliferation of ASMCs are controversial. METHODS: An allergic model that mimics airway remodeling in chronic asthma was established and primary ASMCs were cultured. Cell proliferation was detected by viable cell counting and Cell Counting Kit (CCK)-8 analysis. Expression and phosphorylation of Smad3, type 1 TGFß receptor (TGFßRI), type 2 TGFß receptor (TGFßRII), extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), C-Jun N-terminal kinase (JNK) and AKT were detected by western blot. siRNAs were used to knock down Smad3 and TGFßRII. RESULTS: Smad3 and TGFßRII were up-regulated in primary ASMCs isolated from ovalbumin (OVA)-sensitized mice as compared with ASMCs isolated from unsensitized control mice, which persisted for at least four passages. TGFß1 stimulated proliferation of ASMCs isolated from OVA-sensitized mice, which was inhibited by specific siRNA targeting Smad3 or TGFßRII. However ASMCs from control mice showed no proliferative response to TGFß1. TGFß1-induced proliferation of ASMCs from OVA-sensitized mice was markedly attenuated by PD-98059, a specific ERK1/2 inhibitor. TGFß1 induced ERK1/2 phosphorylation within 15 minute, which was partially blocked by specific inhibitor of Smad3 (SIS3). CONCLUSIONS: ASMCs isolated from OVA-sensitized mice showed hyper-proliferation upon TGFß1 stimulation. This might have been associated with up-regulated Smad3 and TGFßRII and mediated by ERK1/2 downstream to Smad3.

Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke.

Proliferation and synthetic function (i.e. the capacity to release numerous chemokines and cytokines) of airway smooth muscle cells (ASMCs) are important in airway remodeling induced by cigarette smoke exposure. However, the molecular mechanism has not been clarified. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is expressed ubiquitously and is crucial for the cellular physiological function of many cell types. The present study aimed to detect the expression of TRPM7 in ASMCs from smoke­exposed rats and determine the importance of TRPM7 in proliferation and interleukin­8 (IL­8) release. ASMCs were isolated and cultured from smoke­exposed rats. Expression levels of TRPM7 were determined by reverse transcription­polymerase chain reaction, western blot analysis and immunofluorescence. TRPM7 was silenced with TRPM7­short hairpin RNA lentivirus vector. DNA synthesis, cell number and IL­8 release of ASMCs induced by cigarette smoke extract (CSE) and tumor necrosis factor­α (TNF­α) were assessed using [3H]-thymidine incorporation assay, hemocytometer and enzyme­linked immunosorbent assay, respectively. It was determined that mRNA and protein expression levels of TRPM7 were increased in ASMCs from smoke­exposed rats. Stimulation with CSE or TNF­α elevated DNA synthesis, cell number and IL­8 release were more marked in ASMCs from smoke­exposed rats. Silencing of TRPM7 reduced DNA synthesis, cell number and IL­8 release induced by CSE or TNF­α in ASMCs from smoke-exposed rats. In conclusion, expression of TRPM7 increased significantly in ASMCs from smoke­exposed rats and the upregulation of TRPM7 led to augmented cell proliferation and IL-8 release in ASMCs from rats exposed to cigarette smoke.

MiR-23b controls TGF-ß1 induced airway smooth muscle cell proliferation via TGFßR2/p-Smad3 signals.

BACKGROUND: Abnormal proliferation of ASM (airway smooth muscle) directly contributes to the airway remodeling during development of lung diseases such as asthma. Here we report that a specific microRNA (miR-23b) controls ASMCs proliferation through directly inhibiting TGFßR2/p-Smad3 pathway. METHODS: The expression of miR-23b in ASMCs was detected by quantitative real-time polymerase chain reaction (RT-PCR). The effects of miR-23b on cell proliferation and apoptosis of ASMCs were assessed by transient transfection of miR-23b mimics and inhibitor. The target gene of miR-23b and the downstream pathway were further investigated. RESULTS: Overexpression of miR-23b significantly inhibited TGF-ß1-induced ASMCs proliferation and promoted apoptosis. RT-PCR and Western blotting analysis showed miR-23b negatively regulates the expression of TGFßR2 and p-Smad3 in ASMCs. Subsequent analyses demonstrated that TGFßR2 was a direct and functional target of miR-23b, which was validated by the dual luciferase reporter assay. CONCLUSIONS: MiR-23b may function as an inhibitor of airway smooth muscle cells proliferation through inactivation of TGFßR2/p-Smad3 pathway.

Triptolide inhibits TGF-ß1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling.

BACKGROUND: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-ß1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. METHODS: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-ß1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. RESULTS: Triptolide significantly inhibited TGF-ß1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-ß1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. CONCLUSIONS: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway.

Triptolide suppresses airway goblet cell hyperplasia and Muc5ac expression via NF-κB in a murine model of asthma.

BACKGROUND: We have reported that triptolide inhibited pulmonary inflammation in patients with steroid-resistant asthma. In the present study, we investigated whether suppresses airway remodeling and goblet cell hyperplasia, studied the mechanism of triptolide on mucin5ac (Muc5ac) expression in a murine model of asthma. METHODS: BALB/c mice were sensitized to intraperitoneal ovalbumin (OVA) followed by repetitive ovalbumin challenge for 6 weeks. Treatments included triptolide (40 µg/kg) and dexamethasone (2mg/kg). The area of bronchial airway (WAt/Pbm), smooth muscle (WAm/Pbm) and mucus index were assessed 24h after the final OVA challenge. Levels of Muc5ac were assessed by ELISA, immunohistology and real-time PCR. Western blot was performed to analyze the phosphorylation of NF-κB p65. RESULTS: Triptolide and dexamethasone significantly reduced allergen-induced increases in the thickness of bronchial airway, smooth muscle and goblet cell hyperplasia. Levels of lung Muc5ac and Muc5ac mRNA were significantly reduced in mice treated with triptolide and dexamethasone. Phosphorylation of NF-κB p65 was significantly reduced in mice treated with triptolide and dexamethasone. CONCLUSION: Triptolide may inhibit airway goblet cell hyperplasia and Muc5ac expression in asthmatic mice via NF-κB. It may be a potential drug for the treatment of patients with severe asthma.

Triptolide inhibits TGF-ß1 induced proliferation and migration of rat airway smooth muscle cells by suppressing NF-κB but not ERK1/2.

BACKGROUND: Airway remodeling contributes to increased mortality in asthma. We have reported that triptolide can inhibit airway remodeling in a mouse asthma model. In this study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation, migration and the possible mechanism. METHODS: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentrations of triptolide before stimulated by TGF-ß1. Cell proliferation was evaluated by cell counting and MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle. Migration was measured by Transwell analysis. Signal proteins (NF-κB p65 and ERK1/2) were detected by western blotting analysis. LDH releasing test and flow cytometry analysis of apoptosis were also performed to explore the potential cytotoxic or pro-apoptotic effects of triptolide. RESULTS: Triptolide significantly inhibited TGF-ß1 induced ASMC proliferation and migration (p<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. Western blotting analysis showed TGF-ß1 induced NF-κB p65 phosphorylation was inhibited by triptolide pretreatment, but ERK1/2 was not affected. No cytotoxic or pro-apoptotic effects were detected under the concentration of triptolide we used. CONCLUSIONS: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation and migration through inactivation of NF-κB pathway. This article is protected by copyright. All rights reserved.

Magnesium affects the cytokine secretion of CD4⁺ T lymphocytes in acute asthma.

INTRODUCTION: Magnesium (Mg) administration has been shown to promote bronchodilation and to improve lung function in asthma. It also plays an additional role in modulating the immune responses. This study was initiated to explore if Mg supplementation could affect the secretion of cytokines in acute asthmatic CD4⁺ T cells. METHODS: Total serum Mg concentrations of the acute asthmatic patients and healthy controls were determined. CD4⁺ T cells were isolated from the blood of the acute asthmatic patients. They were cultured in various concentrations of Mg-supplemented (0.8, 5, 10, 15, and 20 mmol/l) medium. Cytokine (IL-5, IL-13, and IFN-γ) levels were determined by Enzyme-Linked Immunosorbnent Assay (ELISA). RESULTS: Serum Mg concentration was lower in the acute asthmatic patients than that in the healthy controls (p < .05). The secretion of IL-5 and IL-13 was decreased, while the acute asthmatic CD4⁺ T cells were cultured in 10 and 15 mmol/l Mg-supplemented medium, respectively, as compared to the 0.8 mmol/l Mg group (p < .05). The secretion of IFN-γ increased in the 10 mmol/l Mg group (p < .05). CONCLUSION: Mg supplementation was able to modulate the immune responses of acute asthmatic CD4⁺ T cells and decrease the secretion of type 2 CD4⁺ T lymphocytes cytokines.

Effects of antireflux treatment on bronchial hyper-responsiveness and lung function in asthmatic patients with gastroesophageal reflux disease.

AIM: To investigate the effects of antireflux treatment on bronchial hyper-responsiveness and lung function in asthmatic patients with gastroesophageal reflux disease (GERD). METHODS: Thirty asthmatic patients with GERD were randomly divided into two groups (group A and group B). Patients in group A (n=15) only received asthma medication including inhaled salbutamol 200 microg four times a day and budesonide 400 microg twice a day for 6 weeks. Patients in Group B (n=15) received the same medication as group A, and also antireflux therapy including oral omeprazole 20 mg once a day and domperidone 10 mg three times a day for 6 weeks. Pulmonary function tests and histamine bronchoprovocation test were performed before and after the study. RESULTS: There was no significant difference in the baseline values of pulmonary function and histamine PC(20-FEV1) between the two groups. At the end of the study, the mean values for VC, VC%, FVC, FVC%, FEV(1), FEV(1)%, PEF, PEF%, PC(20-FEV1) were all significantly improved in group B, compared with group A. CONCLUSION: Antireflux therapy may improve pulmonary function and inhibit bronchial hyper-responsiveness in asthmatic patients with GERD.

[Expression of cyclin D1 and vascular endothelial growth factor(VEGF) in non-small cell lung carcinoma and their association with the prognosis].

BACKGROUND & OBJECTIVE: The occurrence and development of tumors are controled by oncogene, antioncogene and tumor metastasis-related gene. Cyclin D1 is the expressive product of CCND1(a kind of oncogen). Vascular endothelial growth factor (VEGF) regulates the growth of neoplastic angiogenesis, which plays a role in the invasion and metastasis of tumor. The objective of this study was to detect the expression of Cyclin D1 and VEGF in non-small cell lung cancer (NSCLC), and to explore their association with the prognosis of NSCLC. METHODS: Immunohistochemistry was used to detect the expression of Cyclin D1 and VEGF in pathological tissue sections of 55 cases of NSCLC and 10 cases of non-malignant tissue from lung lesions. The relationship between the expression of Cyclin D1 and VEGF in 55 NSCLC sections and the age, sex, smoke, size of tumor, histopathological type, differentiation, stage, lymph node metastasis and survival time were analyzed statistically. RESULTS: The expression rates of Cyclin D1 and VEGF in the 55 NSCLC tissue sections were 61.82% and 74.55%, respectively. In 10 cases of non-malignant tissue sections, cyclin D1 expression was not detected and VEGF expression (+/-) was only in 2 cases. The expression of Cyclin D1 and VEGF showed: (1) there was no significant difference among age, sex, histopathological type, stage, differentiation, tumor size and smoking level; (2) there were significant differences between the patients with and without lymph node metastasis; (3) there were significant differences of survival time between positive and negative expression groups. It meant Cyclin D1 and VEGF would be the poor prognostic factors. CONCLUSION: The expression of Cyclin D1 and VEGF occurred in more than 60% cases of NSCLC, which may play a role in the biologic behavior and prognosis of NSCLC.