Effect of prolonged mechanical ventilation on cough function and TRPV1 expression.

Cough is a pivotal airway protective reflex, yet the effects of prolonged mechanical ventilation (PMV) on cough function are unknown. This study compared the cough function in subjects with PMV (≥ 21 days, n = 29) and those with short-term mechanical ventilation (SMV, ≤ 7 days, n = 27). Cough reflex sensitivity was measured by capsaicin provocation concentrations after extubation. The cough strength of respiratory muscles was assessed by involuntary cough peak expiratory flow (iCPEF). The mRNA expression of transient receptor potential vanilloid 1 (TRPV1), a cough sensor activated by capsaicin, in tracheal tissues was determined. We found that cough reflex sensitivity and iCPEF were significantly lower in the PMV group than in the SMV group. The tracheal expression of TRPV1 was similar in both groups, suggesting that changes in TRPV1 expression may not be a contributing factor. Our finding regarding the cough dysfunction after PMV highlights the need to implement effective airway clearance management and rehabilitation in this population.

Airway hyperresponsiveness induced by intermittent hypoxia in rats.

This study investigated whether intermittent hypoxia (IH) induces airway hyperresponsiveness (AHR) and associated with lung inflammation. Male Brown Norway rats were exposed to 14-day IH or room air (RA) for 6 h/day. One day after the last exposure, total lung resistance to various doses of methacholine was measured as an index of bronchoconstrictive responses. Compared with RA controls, methacholine significantly induced an augmented bronchoconstriction in IH-exposed rats. Moreover, IH exposure evoked lung inflammation which was reflected by increased inflammatory cell infiltration, concentrations of interleukin-6 and prostaglandin E2 in bronchoalveolar lavage fluid, and lung lipid peroxidation. IH-induced AHR and lung inflammation were completely abolished by daily intraperitoneal injection of N-acetylcysteine (an antioxidant) or ibuprofen (a cyclooxygenase inhibitor), but not by apocynin (an inhibitor of NADPH oxidase) or vehicle. In conclusion, AHR and lung inflammation occur after 14-day IH exposure, with endogenous reactive oxygen species and cyclooxygenase metabolites being responsible for these responses.

Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging.

Studying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, the current 2PM typically relies on a slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating a gradient-index (GRIN) lens and a tunable acoustic GRIN (TAG) lens into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an ∼ 1-cm long GRIN lens allows imaging relay from any target region of a mouse brain, while a TAG lens provides a sub-second volume rate via a 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia.

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,ß-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17ß-estradiol (E2) at a low concentration (30 µg/ml) but not at high concentrations (50 and 150 µg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells.

BACKGROUND: Tanshinone IIA (TIIA), a diterpene quinone from the medicinal plant Salvia miltiorrhiza Bunge (Lamiaceae) was shown to possess apoptotic and TRAIL-sensitizing effects. Still, the molecular mechanisms whereby TIIA induces apoptosis remain largely unknown. PURPOSE: The role of survivin, an inhibitor of apoptosis protein, in TIIA-induced apoptosis has never been addressed before and hence was the primary goal of this study. METHODS: In this study, we explored the anticancer effect of TIIA in TOV-21G, SKOV3, and OVCAR3 ovarian carcinoma cells. Cytotoxicity was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of related signaling proteins. RESULTS: Our results illustrated that TIIA's cytotoxic effect was caused by apoptosis with the involvement of caspases activity. Moreover, TIIA downregulated survivin in a concentration-dependent manner without affecting the expression of Bcl-2, Bcl-xL, and Bax. TIIA-induced survivin downregulation is regulated by both transcriptional processes and proteasomal degradation. Using TOV-21G cells as our cellular model, we demonstrated that TIIA-induced survivin downregulation requires p38 MAPK activation. Importantly, genetic overexpression of survivin rendered cells more resistant to TIIA, indicating an essential role of survivin downregulation in TIIA-induced apoptosis. This TRAIL sensitization effect of TIIA is ascribed to survivin downregulation because the effect was abrogated in cells that overexpressed survivin. CONCLUSION: Our findings provide new insights into the action modes of TIIA-mediated anticancer effects and further implicate a rational design for cancer therapeutic regimens by combining TIIA-sensitized TRAIL via downregulating survivin to elicit ovarian cancer cell death.

Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines.

Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DR5 in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by a dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, clonogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DR5 protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DR5 plays an essential role in TIIA-induced TRAIL sensitization and that induction of DR5 by TIIA is mediated through the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DR5 up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TIIA promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5. In addition, the quenching of ROS using NAC prevented the induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DR5 receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.

Proapoptotic and TRAIL-sensitizing constituents isolated from Salvia militiorrhiza (Danshen).

Natural compounds isolated from medicinal plants are invaluable resources for drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent unique by its cancer cell-specific proapoptotic action, but its potential is heavily curbed by acquired resistance. We herein reported for the first time the identification of cytotoxic and TRAIL-sensitizing components of Salvia miltiorrhiza (Danshen), a traditional medicinal plant effective for treating cardiovascular disorders. Specifically, we found that the ethanol extract and its group 5 fraction of S. miltiorrhiza showed evident cytotoxicity against the human lung adenocarcinoma cell line A549 and ovarian adenocarcinoma cell line TOV-21G in a concentration-dependent manner. Likewise, a dose-dependent cytotoxicity was exerted by the standard solutions of cryptotanshinone, tanshinone I and tanshinone IIA, the major components of the group 5 fraction, where tanshinone IIA were most potent and displayed an IC50 of 2.00 ± 0.36 µM and 2.75 ± 0.23 µM for A549 and TOV-21G, respectively. Induction of apoptosis represents an essential mechanism underlying tanshinone IIA-mediated cytotoxic action, as evidenced by the proteolytic processing of PARP upon tanshinone IIA stimulation and, importantly, a marked rescue of the viability of tanshinone IIA-treated cells when co-treatment with the pan-caspase inhibitor z-VAD-fmk. Noteworthy, stimulation with cryptotanshinone, tanshinone I or tanshinone IIA all effectively potentiated TRAIL to reduce viability and inhibit the colony formation capacity of TRAIL-resistant TOV-21G and SKOV3. Collectively, we revealed the proapoptotic and TRAIL-sensitizing components of S. miltiorrhiza and further implicated the potential of developing these active compounds as monotherapeutic agent or TRAIL-based therapy for cancer chemoprevention or chemotherapy.