Deficiency of CARMA3 attenuates the development of bleomycin induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) has attracted more and more attention due to its irreversibility and high mortality rate. Currently, there is no effective treatment option is available to reverse the disease. Caspase recruitment domain-containing membrane-associated guanylate kinase protein (CARMA3) has been recognized as a proinflammatory molecule involved in many lung diseases, such as Allergic airway inflammation and lung cancer. Bleomycin (Bleo), as an alkaline sugar peptide antibiotics, is often used as a first-line anti-tumor agent. Its toxic effect is to induce pulmonary fibrosis (PF) and its clinical symptoms, so it has been widely used in the construction of pulmonary fibrosis model. METHODS: Wild type mice (WT, n = 20) and CARMA3 knockout mice (CARMA3-KO, n = 20) were generated and injected with bleomycin or saline via trachea. The severity of fibrosis was evaluated by fibrosis markers and lung histological morphology. Furthermore, the amount of alveolar epithelial cells and inflammation in lung tissue were examined. Finally, epithelial-mesenchymal transition was further investigated. RESULTS: We found CARMA3 expression in the mice alveolar epithelial cells. And compared with WT mice, CARMA3-KO mice showed reduced deposition of collagen fibers, inflammation and destruction of alveolar epithelial cells in lung tissue. In addition, after bleomycin induction, the expressions of proinflammatory factors and collagen-related factors in CARMA3-KO mice were much lower than those in WT mice. The epithelial-mesenchymal transformation phenotype was also improved in CARMA3-KO mice compared to WT mice. CONCLUSION: Our Results shows that CARMA3 plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. CARMA3 could alleviate the fibrosis by improving inflammation, deposition of collagen and damage of alveolar epithelial cells, which revealed that CARMA3 may be a potential target for pulmonary fibrosis.

Vildagliptin, a CD26/DPP4 inhibitor, ameliorates bleomycin-induced pulmonary fibrosis via regulating the extracellular matrix.

BACKGROUND: Idiopathic pulmonary fibrosis is a debilitating lung disease. CD26/DPP4 plays promotive roles in pulmonary damage and fibrosis. This study aimed to explore the roles of vildagliptin in bleomycin-induced pulmonary fibrosis, and to address its ameliorative effect on the extracellular matrix (ECM). METHODS: Idiopathic pulmonary fibrosis mice models were induced by intratracheal injection of bleomycin. DPP4 activity was evaluated, and the fibrosis was investigated by Hematoxylin-eosin, Masson's trichrome staining and hydroxyproline assay. Expression of extracellular matrix proteins including α-SMA, collagen IV, collagen I, FN and TGF-ß were analyzed by immunochemistry and western blot. Percentages of the numbers of monocytes, leukocytes, basophils and lymphocytes were classified, and inflammatory factors in plasma as well as lung tissues were examined by enzyme-linked immunosorbent assay and western blot. The influences of vildagliptin on TGF-ß1-induced cell proliferation, differentiation and inflammatory factors in MRC-5 cells were detected. RESULTS: Vildagliptin effectively attenuated inflammation and fibrosis in bleomycin-induced pulmonary tissue via inhibiting the activity of CD26/DPP4. extracellular matrix proteins were suppressed by vildagliptin. Thus, lung tissue fibrosis was efficiently alleviated by vildagliptin. CONCLUSION: As an inhibitor of CD26/DPP4, Vildagliptin could be a promising therapeutic candidate for idiopathic pulmonary fibrosis.

IKK Epsilon Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice by Inhibiting Inflammation, Oxidative Stress, and Apoptosis.

Abdominal aortic aneurysm (AAA) is a vascular disorder that is considered a chronic inflammatory disease. However, the precise molecular mechanisms involved in AAA have not been fully elucidated. Recently, significant progress has been made in understanding the function and mechanism of action of inhibitor of kappa B kinase epsilon (IKKε) in inflammatory and metabolic diseases. The angiotensin II- (Ang II-) induced or pharmacological inhibitors were established to test the effects of IKKε on AAA in vivo. After mice were continuously stimulated with Ang II for 28 days, morphologically, we found that knockout of IKKε reduced AAA formation and drastically reduced maximal diameter and severity. We also observed a decrease in elastin degradation and medial destruction, which were independent of systolic blood pressure or plasma cholesterol concentrations. Western blot analyses and immunohistochemical staining were carried out to measure IKKε expression in AAA tissues and cell lines. AAA phenotype of mice was measured by ultrasound and biochemical indexes. In zymography, immunohistology staining, immunofluorescence staining, and reactive oxygen species (ROS) analysis, TUNEL assay was used to examine the effects of IKKε on AAA progression in AAA mice. IKKε deficiency significantly inhibited inflammatory macrophage infiltration, matrix metalloproteinase (MMP) activity, ROS production, and vascular smooth muscle cell (VSMC) apoptosis. We used primary mouse aortic VSMC isolated from apolipoprotein E (Apoe) -/- and Apoe-/-IKKε -/- mice. Mechanistically, IKKε deficiency blunted the activation of the ERK1/2 pathway. The IKKε inhibitor, amlexanox, has the same impact in AAA. Our results demonstrate a critical role of IKKε in AAA formation induced by Ang II in Apoe-/- mice. Targeting IKKε may constitute a novel therapeutic strategy to prevent AAA progression.

Effect of Dandelion Extracts on the Proliferation of Ovarian Granulosa Cells and Expression of Hormone Receptors.

BACKGROUND: In the current society, infertility related to age has become a social problem. The in vitro fertilization (IVF) success rate in women with poor ovarian response (POR) is very low. Dandelion extract T-1 (DE-T1) is an effective component of the extract from the leaves and stems of Taraxacum officinale, which is one of the medicines used in some patients with POR, but its molecular mechanism remains unclear. METHODS: Following IVF, ovarian granulosa cells (GCs) of sixty patients were extracted and divided into normal ovarian response (NOR) and POR groups. GCs were cultured in a dose-dependent and time-dependent manner with DE-T1, proliferation of GCs was determined by Cell Counting Kit-8 assay, and mRNA levels of insulin-like growth factor 1 receptor (IGF-1R), luteotropic hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), LHR, and CYP19A1 (aromatase) were determined by quantitative polymerase chain reaction. Progesterone and estradiol (E2) concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: The cell viability gradually increased with the progressive increase in the DE-T1 concentration. Compared with the control group (without DE-T1), the mRNA expressions of FSHR, LHR, IGF-1R, and CYP19A1 were upregulated after the addition of DE-T1, especially in the 2.5% DE-T1 group (P < 0.01). The expression of IGF-1R was upregulated approximately 25 times (24.97 ± 4.02 times) in the POR group with 2.5% DE-T1. E2 and progesterone levels increased with the increasing DE-T1 concentration. There were highly significant differences in the E2 and progesterone secretion between the NOR and POR groups (P < 0.01). CONCLUSION: DE-T1 may promote steroid hormone synthesis by promoting GC proliferation and upregulating GC receptor expression, thereby improving ovarian endocrine function.

Deficiency of IKKε inhibits inflammation and induces cardiac protection in high-fat diet-induced obesity in mice.

Immune response and metabolic regulation have been recognized as a central homeostatic mechanism, the dysfunction of which can trigger a cluster of chronic metabolic disorders, particularly obesity, type Ⅱ diabetes and cardio-vascular disease. Serine/threonine kinase IκB kinase (IKK) ε is a multifunctional regulator that participates in immune regulation, cell proliferation and transformation, and oncogenesis. In the present study, we investigated the role of IKKε in cardiovascular disorders using murine models of apolipo-protein E­deficient [ApoE(-/-)] mice and ApoE/IKKε double­knockout [ApoE(-/-)/IKKε(-/-)]mice, which were fed a normal diet (ND) and high-fat diet (HFD) for 12 weeks, respectively. Results of this study showed that mouse obesity correlated in vivo with an increased expression of IKKε. Additionally, chronic low­grade inflammation in cardiac tissue was evident in ApoE(-/-) mice, but was markedly reduced in ApoE(-/-)/IKKε(-/-) mice. However, serum lipid levels in the ApoE(-/-) mice group were not significantly higher than those of the ApoE(-/-)/IKKε(-/-) group. Furthermore, immunofluorescence and western blot analysis demonstrated evident increases in the expression of nuclear factor-κB (NF-κB) pathway components and downstream factors in the ApoE(-/-) mice group, while these increases were blocked in the ApoE(-/-)/IKKε(-/-) group. Taken together, these data indicate that deficiency of IKKε prevented obesity and inflammatory response in the murine hearts in ApoE(-/-) and ApoE(-/-)/IKKε(-/-) mice fed an ND and HFD, respectively, suggesting that IKKε may play a role in HFD-induced inflammation in hearts of obese mice and may serve as a novel target for the treatment of a variety of metabolism-associated cardiovascular diseases.

[Expression and localization of vascular endothelial growth factor in the radial artery of the coronary artery bypass grafting patients with diabetes].

OBJECTIVE: To evaluate the quality of the radial artery for coronary artery bypass grafting (CABG) from patients with diabetes by observing the morphology of the radial artery and detecting the expression of vascular endothelial growth factor (VEGF) which may attribute to the long-term patency rate of the coronary artery bypass grafting. METHODS: Samples from 20 cases of diabetic and non-diabetic patients were prospective collected from June 2009 to December 2010. HE staining technique was used to test the morphology of radial artery through the observation of 20 cases of diabetic and 20 cases of non-diabetic patients who undergone CABG. The intimal thicken of the radial artery in the two groups of patients was compared. Western blot and immunofluorescence were then used to test the expression and location of VEGF in the two groups of patients. RESULTS: The radial artery endothelial thickening index and intima/media ratio were significantly higher in the diabetic patients when compared with non-diabetic patients (0.90 ± 0.28 vs. 0.29 ± 0.25, t = 7.27, P < 0.01; 0.90 ± 0.21 vs. 0.37 ± 0.18, t = 8.57, P < 0.01). The expression of VEGF in diabetic patients was significantly higher than non-diabetic patients as revealed by Western blot (1.20 ± 0.21 vs. 0.67 ± 0.15, t = 6.49, P < 0.01). Immunofluorescence showed that VEGF distributed in the cytoplasm of the endothelial cells of diabetic patients radial artery. CONCLUSIONS: Diabetic patient's radial artery intimal thickness is significantly higher than non-diabetic patient's. VEGF may be an important inflammatory cytokine which is leading the radial artery intima thickening in the diabetic patients. The choice of the radial artery grafts in diabetic patients for CABG should be careful.

Effects of IKKɛ on oxidised low-density lipoprotein-induced injury in vascular endothelial cells.

BACKGROUND: Oxidised low-density lipoprotein (ox-LDL) led to endothelial dysfunction, which was an initial step in the formation of atheroma. The role of IKKɛ in Ox-LDL-induced endothelial cell injury was investigated in this study. METHODS: Primary cultures of endothelial cell were identified by the immunofluorescent detection of von Willebrand's factor (vWF). Wild-type and IKKɛ knockout endothelial cells were treated with ox-LDL and effects on ultrastructural morphology were evaluated by transmission electron microscopy. Western blotting analysis was conducted to evaluate effects on IL-18, VEGF, IKKα, IKKß, IKKɛ, p65 and p50 expression. RESULTS: From the ultrastructural morphology of endothelial cells and the expression of inflammatory factor such as IL-18 and VEGF, it was suggested that IKKɛ knockout endothelial cells showed less severe ox-LDL-induced injury compared to wild-type endothelial cells. Ox-LDL increased IKKɛ and phosphorylated IKKɛ expression in wild-type endothelial cells, while no significant change in IKKα and IKKß levels was observed. Ox-LDL significantly increased the expression of p65(or p50) and phosphorylated p65(or p50) in wild-type endothelial cells, while this effect was completely blocked in IKKɛ knockout endothelial cells. CONCLUSIONS: IKKɛ might play an important role in ox-LDL-induced injury of endothelial cells.