Pulmonary fibrosis model of mice induced by different administration methods of bleomycin.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of the lung. How to build a typical human mimicking animal model has been a challenge. Thus, to reveal the mechanism and to make it useful for IPF clinical treatment, a different type of mice model and inspection methods are used to evaluate which one is applicable for the study of IPF. METHOD: 69 Twelve-weeks-old C57BL/6 mice were divided into 3 type groups (n = 7 for each control group, n = 8 for each BLM-induced pulmonary fibrosis groups), as intraperitoneal injection, intratracheal administration, and intravenous administration of bleomycin (BLM) to initiate lung fibrosis. Changes of the lung function measured through mice Pulmonary function test (PFT). Morphological changes in mice were observed by PET/CT, Masson and Picro-Sirius staining, Transmission electron microscopy (TEM). Biochemical changes were tested by Enzyme-linked immunosorbent assay (Elisa). RESULTS: PET/CT of BLM-receiving mice showed an increase in fibrotic consolidations and an increase in non-aerated lung area in BLM-treated mice compared with that in controls. TGF-b1, TNF-a, IL-6, GM-CSF in BALF and serum. PAI-1, HYP in the lung tissue of mice were significantly different in each BLM groups than those in the controls. The results of Masson staining in mice indicate that the lung tissues of all BLM received groups, the intratracheal groups, the intravenous groups, and the intraperitoneal groups have a higher degree of pulmonary septal thickening and collagen fiber consolidation compare to saline control. Picro-Sirius staining results are consistent with the results of Masson staining. Compared with the saline control group, the ratio of Col 1/Col 3 was significantly increased in each BLM group. TEM results found that in BLM group, type I alveolar epithelial cells were degenerated. Exfoliated endothelial cells were swelling, and type II alveolar epithelial cells were proliferated, the shape of the nucleus was irregular, and some tooth-like protrusions were seen. CONCLUSIONS: With three different methods of animal model construction, high dose of each show more compliable, and BLM can successfully induce animal models of pulmonary fibrosis, however, certain differences in the fibrosis formation sites of them three, and tail vein injection of BLM induced PF model is closer to the idiopathic pulmonary interstitial fibrosis.

Baicalin ameliorates cigarette smoke-induced airway inflammation in rats by modulating HDAC2/NF-κB/PAI-1 signalling.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease distinguished by airway remodelling and progressive inflammation. PAI-1 is an important regulator of fibrosis. Recent studies have shown that PAI-1 seems to be involved in COPD progression. Elevated levels of PAI-1 have been found in the lungs of patients with acute inflammation. PAI-1 has been shown to regulate the levels of proinflammatory cytokines in the lungs, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, indicating that PAI-1 may play a fundamental role during inflammation. In the present study, we investigated the anti-inflammatory role of baicalin, the main active component of Scutellaria baicalensis, against cigarette smoke (extract) (CS/CSE)-induced airway inflammation in vivo and in vitro. For the in vivo study, SD rats were exposed to CS for 1 h/day, 6 days/week, for 24 weeks and treated with baicalin (40, 80 and 160 mg/kg) or budesonide (0.2 mg/kg). For this study, HBE cells were pretreated with baicalin (10, 20, 40 µM) or dexamethasone (10-7 M) and then exposed to CSE. We found that baicalin treatment could ameliorate CS-induced airway inflammatory infiltration in rats and decrease PAI-1 expression. The ELISA results showed that baicalin significantly inhibited the levels of TNF-α and IL-1ß in CS/CSE-exposed rats and cells. Mechanistic studies showed that baicalin enhanced histone deacetylase 2 (HDAC2) protein expression and inhibited the expression of NF-κB and its downstream target PAI-1, and these effects were reversed by the HDAC2 inhibitor CAY-10683. In conclusion, baicalin ameliorated CS-induced airway inflammation in rats, and these effects were partially attributed to the modulation of HDAC2/NF-κB/PAI-1 signalling.

Loki zupa alleviates inflammatory and fibrotic responses in cigarette smoke induced rat model of chronic obstructive pulmonary disease.

BACKGROUND: Loki zupa formula is kind of a traditional medicines which used to treat airway diseases, especially those caused by abnormal phlegm, such as cough, asthma and chronic bronchitis. The study aim was to explore the anti-inflammatory and anti-remodeling effects of Loki zupa by using a cigarette-smoke induced rat model of chronic obstructive pulmonary disease. METHODS: The rats were divided into five groups: the normal group, the model group, the LZ 4 g/kg and LZ8g/kg group, and the positive control group. Rats were exposed to cigarette smoke for 24 weeks to induce a COPD rat model. Lung function was assessed. Histopathological changes were recorded using Haematoxylin-eosin and Masson's trichrome staining. Mucus hypersecretion was evaluated by PAS staining. Inflammatory factors were measured in blood serum and bronchial alveolar lavage fluid using an enzyme-linked immunosorbent assay. Malondialdehyde and superoxide dismutase and glutathione S-transferase levels were tested by biochemical methods. Gene expression patterns were evaluated using GN-GeneChip Clariom S Array for rat from Affymetrix. And top upregulated and downregulated genes validated by qPCR. And these genes was also compared with gene transcriptomic data from smoker patients with emphysema and non-smokers in GEO dataset. IL-6/PLAGA2A signalling protein expression was assessed by western blot and immunohistochemistry. TGF-ß1and smad2/3 signalling expressions were analysed by western Blot. RESULTS: Loki zupa improved COPD rats lung function as compared to the model group and pathological changes including inflammatory cell infiltration and goblet cell metaplasia was alleviated in rats treated with Loki zupa Inflammatory factors IL-6, TNF-α, IL-1ß and TGF-ß1 decreased while significant increase was observed in blood serum IL-10 content in rats treated with Loki zupa. And IL-6 and TNF-α level in bronchial alveolar lavage fluid showed same expression trend in blood serum, while there was no change in MMP-9 content. It also increased antioxidant enzyme SOD and GPX activity while reducing the lipid peroxidation. Gene microarray analysis showed that there were 355 differentially expressed gene in LZ treated COPD rat lung as compared to model group. Both microarray and qPCR results showed that top differentially expressed genes nxt1 (up regulated) and pla2g2a (down regulated) expression were also reversed by LZ treatment. And protein expression level of IL-6 and pla2g2a was also elevated in CS exposed rats while significant reduction was observed in LZ treated rats. Accordingly, Loki zupa inhibited Collagen-1 upstream protein expression of TGF-ß/smad2/3 signalling pathway. CONCLUSION: These results demonstrated that Loki zupa showed protective effects in the lung of the COPD rat model. This mainly because of Loki zupa exerts anti-inflammatory effects by blocking IL-6/pla2g2a signalling and inhibiting inflammatory gene expression and attenuates fibrotic responses by inhibiting TGF-ß/smad2/3 signalling pathway.

Osthole attenuates ovalbumin­induced lung inflammation via the inhibition of IL­33/ST2 signaling in asthmatic mice.

Asthma is a common chronic inflammatory airway disease. Recent studies have reported that interleukin (IL)­33 is a potential link between the airway epithelium and Th2­type inflammatory responses, which are closely related to the progression of asthma. The IL­33 receptor, ST2, is highly expressed in group 2 innate lymphoid cells (ILC2s), Th2 cells, mast cells, eosinophils and natural killer (NK) cells. Cnidii Fructus is a Chinese herb with a long history of use in the treatment of asthma in China. Osthole is one of the major components of Cnidii Fructus. The present study examined the anti­asthmatic effects of osthole in mice and aimed to elucidate the underlying mechanisms involving the IL­33/ST2 pathway. BALB/c mice were sensitized and challenged with ovalbumin and then treated with an intraperitoneal injection of osthole (25 and 50 mg/kg). Subsequently, the airway hyper­responsiveness (AHR) and inflammation of the lungs were evaluated. The amounts of IL­4, IL­5, IL­13, interferon (IFN)­Î³ and IL­33 in the bronchoalveolar lavage fluid (BALF) were measured by Luminex assay and their mRNA levels in the lungs were measured by reverse transcription­quantitative PCR. The histopathology of the lungs was performed with H&E, PAS and Masson's staining. The expression of ST2 in the lungs was evaluated by immunohistochemistry. The data demonstrated that osthole markedly reduced AHR and decreased the number of eosinophils and lymphocytes in BALF. It was also observed that osthole significantly inhibited the release of Th2­type cytokines (IL­4, IL­5 and IL­13) and upregulated the IFN­Î³ level in BALF. Moreover, osthole significantly attenuated the IL­33 and ST2 expression in the lungs of asthmatic mice. On the whole, osthole attenuated ovalbumin­induced lung inflammation through the inhibition of IL­33/ST2 signaling in an asthmatic mouse model. These results suggest that osthole is a promising target for the development of an asthma medication.

Bu-Shen-Fang-Chuan formula attenuates cigarette smoke-induced inflammation by modulating the PI3K/Akt-Nrf2 and NF-κB signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory disease. Unlike asthma, COPD is insensitive to glucocorticoid treatment; thus, it is of great importance to find alternative medications, including Chinese medicine, to suppress inflammation. Bu-Shen-Fang-Chuan formula (BSFCF) is commonly used for the treatment of COPD in China. However, the mechanisms of BSFCF in COPD treatment are still unclear. AIM OF THE STUDY: To verify the anti-inflammatory efficacy of BSFCF in COPD and to explore the possible mechanisms underlying its anti-inflammatory efficacy based on the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)-Nuclear factor erythroid 2-related factor 2 (Nrf2) and Nuclear factor (NF)-κB signalling pathways. MATERIALS AND METHODS: A rat model of COPD was established by chronic exposure to cigarette smoke (CS) for 6 months. Bronchoalveolar lavage fluid (BALF) and blood were obtained to detect inflammatory cytokines. Lung samples were harvested, and part of each sample was fixed for subsequent H&E staining and immunohistochemical (IHC) analysis. The remaining lung tissues were used for RNA sequencing analysis and western blotting. RESULTS: BSFCF significantly reduced inflammatory infiltration in the lungs of CS-exposed rats and decreased the concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both the BALF and serum. Additionally, BSFCF evidently attenuated NF-κB activation and downregulation of glucocorticoid receptor (GR) caused by CS. Furthermore, BSFCF increased the activation of PI3K/Akt-Nrf2 signalling in response to CS. CONCLUSIONS: BSFCF attenuated CS-induced inflammation in COPD, which was partially achieved through the PI3K/Akt-Nrf2 and NF-κB signalling pathways.

Bu-Shen-Fang-Chuan formula attenuates T-lymphocytes recruitment in the lung of rats with COPD through suppressing CXCL9/CXCL10/CXCL11-CXCR3 axis.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by irreversible airflow limitation. The current medications show limited effects on the decline of pulmonary function in COPD. Our multicenter clinical trial found that Bu-Shen-Fang-Chuan fomula (BSFCF), a Chinese herbal formula, markedly reduced the frequencies of acute exacerbation of COPD and delayed lung function decline. However, the underlying mechanisms are still unclear. In this study, we established a COPD rat model through a 6-month exposure to cigarette smoke (CS) and found that BSFCF (7.2 g/kg) effectively improved CS-induced reduction in pulmonary function and remarkably decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid (BALF). Importantly, BSFCF treatment notably prevented the accumulation of T-lymphocytes (especially CD8+ T-cells) in the lung of COPD rats. RNA sequencing analysis of lung tissue demonstrated that CXCL9/CXCL10/CXCL11-CXCR3 chemokine axis in the lung of CS-exposed rats was significantly suppressed by BSFCF. Moreover, our Real-time PCR data verified that BSFCF evidently inhibited the mRNA expressions of CXCL9, CXCL10, CXCL11 and CXCR3. Conclusively, BSFCF markedly improved pulmonary function and attenuated CD8+ T-cells recruitment in the lung of CS-exposed rats, which were partially through inhibition of CXCL9/CXCL10/CXCL11-CXCR3 axis.

Curcumin inhibits cigarette smoke-induced inflammation via modulating the PPARγ-NF-κB signaling pathway.

Peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated nuclear hormone receptor, is considered to be a potential target for the anti-inflammatory treatment of COPD, and its anti-inflammatory efficacy is likely related to the inhibition of transcriptional regulation of nuclear factor (NF)-κB. Curcumin, a dietary polyphenol isolated from the rhizome of turmeric, has been found to have therapeutic benefits in chronic obstructive pulmonary disease (COPD) via inhibiting NF-κB. However, whether the anti-inflammatory efficacy of curcumin in COPD is associated with PPARγ has scarcely been investigated before. The purpose of this study was to validate the relationship between PPARγ and NF-κB in cigarette smoke (CS)-induced COPD models, and then to investigate whether the therapeutic effect of curcumin on COPD is achieved through modulating the PPARγ-NF-κB signaling pathway. Our experiments in vitro illustrated that PPARγ might be upstream of NF-κB in cigarette smoke extract (CSE)-treated Beas-2B cells, and that curcumin could significantly ameliorate CSE-induced cell viability reduction and inflammation though up-regulating PPARγ and inhibiting NF-κB activation. Importantly, we found that the inhibitory effect on NF-κB by curcumin was dependent on PPARγ in T0070907-treated or PPARγ shRNA-transfected Beas-2B cells, indicating that curcumin inhibited CSE-induced inflammation partially through modulating the PPARγ-NF-κB pathway. Furthermore, we also observed that the effect of curcumin on PPARγ protein expression and NF-κB activation in CS-exposed rats was consistent with the results from experiments in vitro, and curcumin effectively attenuated pulmonary function decline and inflammatory responses in CS-exposed rats. In conclusion, all the results revealed that curcumin attenuated CS-induced inflammation both in vivo and in vitro, presumably by modulating the PPARγ-NF-κB pathway.

Scutellaria Flavonoids Effectively Inhibit the Malignant Phenotypes of Non-small Cell Lung Cancer in an Id1-dependent Manner.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the world. Inhibitor of differentiation 1 (Id1) is overexpressed in NSCLC and involved in promoting its progression and metastasis. Identifying natural compounds targeting Id1 may have utility in NSCLC treatment. Here, we sought to determine whether the anti-tumor activities of Scutellaria flavonoids (SFs) were related to Id1. We reported that three SFs (baicalin, baicalein and wogonin) exhibited strong antitumor activity in NSCLC cells in vitro and in vivo. Id1 played a pivotal role on blockage of migration and invasion by SFs. Abrogation of invasion and migration mediated by baicalin, baicalein and wogonin were totally abolished by ectopic overexpression of Id1. Mechanistically, baicalin, baicalein and wogonin activated Rap1-GTP binding and dephosphorylated Akt and Src by suppressing a7nAChR, consequently triggering inhibition of Id1. Then attenuation of its downstream mediators, VEGF-A, N-cadherin, vimentin, combined with augment of E-cadherin led to the blockage of proliferation, EMT and angiogenesis of NSCLC. Overall, our data shed light on heretofore-undescribed role of SFs as modulators of Id1, which may be a useful strategy in the treatment of NSCLC.

Baicalein Inhibits Orthotopic Human Non-Small Cell Lung Cancer Xenografts via Src/Id1 Pathway.

Non-small cell lung cancer (NSCLC) is one of the most lethal cancers worldwide. Inhibitor of differentiation 1 (Id1) is the member mostly linked to tumorigenesis in Id family and a potential molecular target in cancer therapy. In the current study, we established an orthotopic lung cancer model by injecting athymic nude mice with A549 cells and evaluated the antitumor effect of baicalein and expression of Id1-related proteins in vivo and in vitro. Micro-CT images showed that tumor volume in baicalein group was significantly reduced. Western blot analysis revealed that baicalein suppressed the expression of Id1 protein, epithelial-to-mesenchymal transition (EMT) related molecules (N-Cadherin, vimentin), and angiogenesis related protein (VEGF-A), accompanied by upregulation of epithelial markers (such as E-cadherin). In addition, phosphorylation of upstream molecular Src was significantly restrained after baicalein treatment. This study firstly demonstrates that baicalein inhibits tumor growth in orthotopic human NSCLC xenografts via targeting Src/Id1 pathway.

Dual role of autophagy/mitophagy in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common lung disease characterised by progressive, irreversible airflow limitation. Multiple regulatory pathways are involved in COPD pathogenesis. Emerging evidence from clinical and basic medical research has suggested that autophagy-a highly conserved catabolic process mediated under various cellular stress conditions-plays a role in the development and prognosis of COPD. Nevertheless, precise function of autophagy remains debatable owing to its beneficial as well as detrimental consequences. In this review, we summarised the 'double-edged sword' functions of autophagy in COPD and aimed to distinguish and classify these functions on the basis of various factors, such as different airway cell types and autophagy stimulators and modulators. Moreover, we determined the biological-functional consequences of autophagy. In particular, we discussed mitophagy-also termed mitochondrial autophagy-which is a critical process in cellular energy homeostasis. We hope that our findings will shed new light on future therapeutic strategies for COPD.

Kidney-Qi deficiency diagnosed by Deng's diagnosis standard was not correlated with aging based on clinical observation of 90 participants.

OBJECTIVE: To verify the Traditional Chinese Medicine (TCM) theory that kidney-Qi deficiency (KQD) is considered to be the main cause of aging using cross-sectional study. METHODS: Demographic and lifestyle characteristics of 90 healthy participants were collected with a self-administered questionnaire. KQD syndrome was diagnosed according to Deng's diagnosis standard. Creatinine-adjusted urinary 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and 8-isomeric-prostaglandin2α (8-iso-PGF2α), salivary advanced oxidation protein products (AOPPs), malondialdehyde (MDA) and dehydroepiandrosterone-sulfate (DHEA-S) were selected as aging markers and measured using enzyme-linked immunosorbent assay. RESULTS: No significant differences were observed in participant characteristics between the KQD group and non-KQD (NKQD) group (P > 0.05). Levels of 8-OH-dG, 8-iso-PGF2α, AOPPs, and MDA increased with age, except for a slight decrease in 8-OH-dG in the older group. The increase in 8-iso-PGF2α was significant (P < 0.05). DHEA-S significantly decreased with increasing age (P < 0.01). 8-OH-dG levels were higher in the KQD group compared with the NKQD group. Levels of urinary 8-iso-PGF2α, salivary AOPPs, and MDA in the KQD group were lower than in the NKQD group. Salivary DHEA-S was higher in the KQD group compared with the NKQD group. However, differences between KQD group and NKQD group were not significant. CONCLUSION: The current results suggested that KQD syndrome, as diagnosed by Deng's standard, does not underlie the aging phenotype.

Baicalin Exerts Anti-Airway Inflammation and Anti-Remodelling Effects in Severe Stage Rat Model of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a worldwide epidemic. Current approaches are disappointing due to limited improvement of the disease development. The present study established 36-week side stream cigarette smoke induced rat model of COPD with advanced stage feature and evaluted the effects of baicalin on the model. Fifty-four Sprague-Dawley rats were randomly divided into six groups including room air control, cigarette smoke exposure, baicalin (40 mg/kg, 80 mg/kg, and 160 mg/kg), and budesonide used as a positive control. Rats were exposed to cigarette smoke from 3R4F research cigarettes. Pulmonary function was evaluated and pathological changes were also observed. Cytokine level related to airway inflammation and remodelling in blood serum, bronchoalveolar lavage fluid, and lung tissue was determined. Blood gases and HPA axis function were also examined, and antioxidant levels were quantified. Results showed that, after treatment with baicalin, lung function was improved and histopathological changes were ameliorated. Baicalin also regulated proinflammatory and anti-inflammatory balance and also airway remodelling and anti-airway remodelling factors in blood serum, bronchoalveolar lavage fluid, and lung tissue. Antioxidant capacity was also increased after treatment with baicalin in COPD rat model. HPA axis function was improved in baicalin treated groups as compared to model group. Therefore, baicalin exerts lung function protection, proinflammatory and anti-inflammatory cytokine regulation, anti-airway remodelling, and antioxidant role in long term CS induced COPD model.

Establishment and Evaluation of a Rat Model of Sidestream Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a common cause of mortality worldwide. The current lack of an animal model that can be established within a certain time frame and imitate the unique features of the disease is a major limiting factor in its study. The present study established and evaluated an animal model of COPD that represents the early and advanced stage features using short-, middle-, and long-term sidestream cigarette smoke (CS) exposure. One hundred and nine Sprague-Dawley rats were randomly divided into 10 groups for different periods of sidestream CS exposure or no exposure (i.e., normal groups). The rats were exposed to CS from 3R4F cigarettes in an exposure chamber. Histological analysis was performed to determine pathological changes. We also conducted open-field tests, lung function evaluations, and cytokine analysis of the blood serum, bronchoalveolar lavage fluid, and lung tissue. The lung tissue protein levels, blood gases, and were also analyzed. As the CS exposure time increased, the indicators associated with oxidative stress, inflammatory responses, and airway remodeling were greater in the CS exposure groups than in the normal group. At 24 and 36 weeks, the COPD model rats displayed the middle- and advanced-stage features of COPD, respectively. In the 8-week CS exposure group, after the CS exposure was stopped for 4 weeks, inflammatory responses and oxidative responses were ameliorated and lung function exacerbation was reduced compared with the 12-week CS exposure group. Therefore, we established a more adequate rat model of sidestream CS induced COPD, which will have great significance for a better understanding of the pathogenesis of COPD and drug effectiveness evaluation.

The Analysis of Constitutions of Traditional Chinese Medicine in Relation to Cerebral Infarction in a Chinese Sample.

OBJECTIVE: To investigate the relationships between the constitutions of Traditional Chinese Medicine (TCM) and patients with cerebral infarction (CI) in a Chinese sample. METHODS: A total of 3748 participants with complete data were available for data analysis. All study subjects underwent complete clinical baseline characteristics' evaluation, including a physical examination and response to a structured, nurse-assisted, self-administrated questionnaire. A population of 2010 neutral participants were used as the control group. Multiple variable regression (MLR) were employed to estimate the relationship between constitutions of TCM and the outcome. DESIGN: A cross-sectional study was conducted to evaluate the association of body constitution of TCM and CI. SETTINGS/LOCATION: Communications and healthcare centers in Shanghai. SUBJECTS: A total of 3748 participants with complete data were available for data analysis. OUTCOME MEASURES: All study subjects underwent complete clinical baseline characteristics' evaluation, including a physical examination and response to a structured, nurse-assisted, self-administrated questionnaire. A population of 2010 neutral participants were used as the control group. MLR were employed to estimate the relationship between constitutions of TCM and the outcome. RESULT: The prevalence of CI was 2.84% and 4.66% in neutral participants and yang-deficient participants (p = 0.012), respectively. Univariate analysis demonstrated a positive correlation between yang deficiency and CI. After adjustment for relevant potential confounding factors, the MLR detected significant associations between yang deficiency and CI (odds ratio = 1.44, p = 0.093). CONCLUSION: A yang-deficient constitution was significantly and independently associated with CI. A higher prevalence of CI was found in yang-deficient participants as compared with neutral participants.