MicroRNA-31-5p Exacerbates Lipopolysaccharide-Induced Acute Lung Injury via Inactivating Cab39/AMPKα Pathway.

Acute lung injury (ALI) and the subsequent acute respiratory distress syndrome remain devastating diseases with high mortality rates and poor prognoses among patients in intensive care units. The present study is aimed at investigating the role and underlying mechanisms of microRNA-31-5p (miR-31-5p) on lipopolysaccharide- (LPS-) induced ALI. Mice were pretreated with miR-31-5p agomir, antagomir, and their negative controls at indicated doses for 3 consecutive days, and then they received a single intratracheal injection of LPS (5 mg/kg) for 12 h to induce ALI. MH-S murine alveolar macrophage cell lines were cultured to further verify the role of miR-31-5p in vitro. For AMP-activated protein kinase α (AMPKα) and calcium-binding protein 39 (Cab39) inhibition, compound C or lentiviral vectors were used in vivo and in vitro. We observed an upregulation of miR-31-5p in lung tissue upon LPS injection. miR-31-5p antagomir alleviated, while miR-31-5p agomir exacerbated LPS-induced inflammation, oxidative damage, and pulmonary dysfunction in vivo and in vitro. Mechanistically, miR-31-5p antagomir activated AMPKα to exert the protective effects that were abrogated by AMPKα inhibition. Further studies revealed that Cab39 was required for AMPKα activation and pulmonary protection by miR-31-5p antagomir. We provide the evidence that endogenous miR-31-5p is a key pathogenic factor for inflammation and oxidative damage during LPS-induced ALI, which is related to Cab39-dependent inhibition of AMPKα.

Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice: Involvement of TGF-ß1/Smads Signaling Pathway.

Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease (COPD), its underlying mechanisms were not clear until now. We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract (CSE). Twenty SPF C57BL/6 mice were randomly divided into PBS control group, COPD model group, low-dose berberine group and high-dose berberine group, 5 mice in each group. The neutrophils and macrophages were examined by Wright's staining. The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The expression levels of TGF-ß1, Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting. It was found that CSE increased the number of inflammation cells in BALF, elevated lung inflammation scores, and enhanced the TGF-ß1/Smads signaling activity in mice. High-dose berberine restrained the alterations in the COPD mice induced by CSE. It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice. TGF-ß1/Smads signaling pathway might be involved in the mechanism. These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.

Potential role of store-operated Ca2+ entry in Th2 response induced by histamine in human monocyte-derived dendritic cells.

Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway of dendritic cells (DCs). DCs primed with histamine facilitate Th2 immune response via different types of histamine receptors. Histamine induces DCs to release Ca(2+) from internal store. Therefore, we wonder that whether histamine could activate SOCE in DCs through its receptors, and what's the functional relevance of the Ca(2+) influx through SOCE induced by histamine in Th(2) response. We certificate that histamine induced a transient Ca(2+) release followed by pronounced Ca(2+) influx after re-addition of external Ca(2+) which could be inhibited by SOCE blockers SKF-96365 and BTP-2. Moreover, the percentages of DCs that showed an obvious Ca(2+) release response to histamine were decreased in the presence of histamine 1 (H1) receptor antagonist pyridylethylamine (Pyr) or histamine 4 (H4) receptor antagonist JNJ7777120 (JNJ). Histamine up-regulated the mRNA expression of STIM1 in DCs, one of the two major proteins of SOCE channel. SOCE blocker BTP-2 and histamine receptor antagonists JNJ and Pyr inhibited the increase of CD86 induced by histamine on DCs. Histamine increased the level of IL-10 and decreased the level of IL-12p70 secreted by DCs. SOC blockers SKF and BTP-2 inhibited the level of both IL-10 and IL-12p70 secreted by DCs. Pretreatment of SOC blockers and H1, H4 receptor antagonists with DCs inhibited the Th2 polarization of T helper cells induced by histamine in mixed lymphocyte responses (MLR). We demonstrated that SOCE was involved in histamine-induced maturation and Th(2) response of DCs which was through histamine 1 and 4 receptor.

Role of STIM1/Orai1-mediated store-operated Ca²âº entry in airway smooth muscle cell proliferation.

Hyperplasia of airway smooth muscle cells (ASMCs) is a characteristic change of chronic asthma patients. However, the underlying mechanisms that trigger this process are not yet completely understood. Store-operated Ca(2+) (SOC) entry (SOCE) occurs in response to the intracellular sarcoplasma reticulum (SR)/endoplasmic reticulum (ER) Ca(2+) store depletion. SOCE plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs. Stromal interaction molecule (STIM)1 has been proposed as an ER/SR Ca(2+) sensor and translocates to the ER underneath the plasma membrane upon depletion of the ER Ca(2+) store, where it interacts with Orai1, the molecular component of SOC channels, and brings about SOCE. STIM1 and Orai1 have been proved to mediate SOCE of ASMCs. In this study, we investigated whether STIM1/Orai1-mediated SOCE is involved in rat ASMC proliferation. We found that SOCE was upregulated during ASMC proliferation accompanied by a mild increase of STIM1 and a significant increase of Orai1 mRNA expression, whereas the proliferation of ASMCs was partially inhibited by the SOC channel blockers SKF-96365, NiCl(2), and BTP-2. Suppressing the mRNA expression of STIM1 or Orai1 with specific short hairpin RNA resulted in the attenuation of SOCE and ASMC proliferation. Moreover, after knockdown of STIM1 or Orai1, the SOC channel blocker SKF-96365 had no inhibitory effect on the proliferation of ASMCs anymore. These results suggested that STIM1/Orai1-mediated SOCE is involved in ASMC proliferation.

Promoting effects of IL-13 on Ca2+ release and store-operated Ca2+ entry in airway smooth muscle cells.

Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca(2+) signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca(2+) entry (SOCE) plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca(2+) fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca(2+) ([Ca(2+)](i)) level, Ca(2+) release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting beta2 adrenergic agonist (beta2 agonist) salbutamol suppressed IL-13-augumented basal [Ca(2+)](i), Ca(2+) release and SOCE, whereas the long-acting beta2 agonist salmeterol had no effect on altered Ca(2+) signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca(2+) release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca(2+) channel blockers SKF-96365 and NiCl(2), by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca(2+) release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and beta2 agonists in inhibiting Ca(2+) signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.