PDK1 upregulates PINK1-mediated pulmonary endothelial cell mitophagy during hypoxia-induced pulmonary vascular remodeling.

BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a complication of lung diseases with pulmonary vascular remodeling, although the underlying molecular mechanisms have not been fully elucidated. This study investigated the underlying molecular events by using a rat HPH model and primary pulmonary microvascular endothelial cells (PMVECs). METHODS AND RESULTS: This study first established a rat HPH model and cultured PMVECs for transmission electron microscopic analysis and manipulation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or phosphatase and tensin homolog-induced kinase 1 (PINK1) expression in vitro. After that, the cell viability was assessed and the expression of different proteins was assayed using cell viability and western blot assays, respectively. Reactive oxygen species production, apoptosis, NLR family pyrin domain containing 3 (NLRP3) expression, and the levels of interleukin (IL)-1ß, IL-6, and IL-8 were also assessed, while the interaction of PDK1 and PINK1 was determined using co-immunoprecipitation/western blot assays. Hypoxia induced mitophagy in the PMVECs and upregulated PINK1/Parkin expression, whereas knockdown of PINK1 expression under hypoxic conditions inhibited cell proliferation but induced endothelial cell apoptosis in vitro, decreased reactive oxygen species production and NLRP3 expression, and reduced the levels of inflammatory factors in PMVECs. However, hypoxia induced PDK1 expression, whereas knockdown of PDK1 downregulated PINK1 expression. Furthermore, treatment of the model rats with the PDK1 inhibitor dichloroacetate (DCA) was able to decrease PINK1 expression. In addition, the PDK1 and PINK1 proteins could interact with each other in the mitochondria of PMVECs to regulate the cell viability. CONCLUSIONS: This study revealed that PDK1 induced PMVEC proliferation but inhibited their apoptosis to participate in pulmonary vascular remodeling, ultimately leading to HPH through regulation of PINK1-mediated mitophagy signaling. Therefore, PINK1 is a novel therapeutic target for the control of HPH.

Clinical Features of COVID-19 Patients in Xiaogan City.

On February 6, 2020, Xiaogan City became the second most seriously affected city with coronavirus disease 2019 (COVID-19), outside Wuhan district, Hubei Province, China. The objectives are to study the clinical features of COVID-19 patients and assess the relationship between the severity of COVID-19, age, and C-reactive protein (CRP) levels. The retrospective data of 134 COVID-19 patients hospitalized in 3 hospitals of Xiaogan City, between February 1 and March 1, 2020, was collected. This study documented COVID-19 patients. Clinical data in terms of body temperature, history of travel, and direct contact with COVID-19 patients, and incubation period was collected. Out of the 134 patients, only 5 required intensive care. Moreover, 2 patients succumbed during this period. The median age of patients was 45 (33-56) years. The most common symptoms at the onset of disease were fever (66.4%), cough (33, 6%), and sore throat (14.7%). Amongst the medicines used, antiviral agents (92.3%) followed by the traditional Chinese medicine (89.5%) were most commonly used. In both the crude and adjusted (I to III) models, odds ratio and its 95% confidence interval for both age and CRP levels were > 1. Moreover, the smooth curve fitting graph reflected that the severity of COVID-19 was positively correlated with both age and CRP levels (all P value < 0.05). The signs and symptoms of COVID-19 patients were fairly moderate. The health care professionals treating the COVID-19 patients should be aware of the increased likelihood of progression to severe COVID-19 in elderly patients and those with high CRP levels.

Reversal of Hypoxic Pulmonary Hypertension by Hypoxia-Inducible Overexpression of Angiotensin-(1-7) in Pulmonary Endothelial Cells.

Hypoxia-induced pulmonary vascular constriction and structure remodeling are the main causes of hypoxic pulmonary hypertension. In the present study, an adeno-associated virus vector, containing Tie2 promoter and hypoxia response elements, was designed and named HTSFcAng(1-7). Its targeting, hypoxic inducibility, and vascular relaxation were examined in vitro, and its therapeutic effects on hypobaric hypoxia-induced pulmonary hypertension were examined in rats. Transfection of HTSFcAng(1-7) specifically increased the expression of angiotensin-(1-7) in endothelial cells in normoxia. Hypoxia increased the expression of angiotensin-(1-7) in HTSFcAng(1-7)-transfected endothelial cells. The condition medium from HTSFcAng(1-7)-transfected endothelial cells inhibited the hypoxia-induced proliferation of pulmonary artery smooth muscle cells, relaxed the pulmonary artery rings, totally inhibited hypoxia-induced early contraction, enhanced maximum relaxation, and reversed phase II constriction to sustained relaxation. In hypoxic pulmonary hypertension rats, treatment with HTSFcAng(1-7) by nasal drip adeno-associated virus significantly reversed hypoxia-induced hemodynamic changes and pulmonary artery-wall remodeling, accompanied by the concomitant overexpression of angiotensin-(1-7), mainly in the endothelial cells in the lung. Therefore, hypoxia-inducible overexpression of angiotensin-(1-7) in pulmonary endothelial cells may be a potential strategy for the gene therapy of hypoxic pulmonary hypertension.

Bioinformatics analysis reveals novel core genes associated with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent liver disease and associated with a wide spectrum of hepatic disorders ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). NASH is projected to become the most common indication for liver transplantation, and the annual incidence rate of NASH-related HCC is 5.29 cases per 1000 person-years. Owing to the epidemics of NAFLD and the unclear mechanism of NAFLD progression, it is important to elucidate the underlying NAFLD mechanisms in detail. NASH is mainly caused by the development of NAFL Therefore, it is also of great significance to understand the mechanism of progression from NAFL to NASH. Gene expression chip data for NAFLD and NASH were downloaded from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between NAFLD and normal controls (called DEGs for NAFLD), as well as between NASH and normal tissue (called DEGs for NASH-Normal), and between NASH and NAFL tissue (called DEGs for NASH-NAFL). For DEGs for the NAFLD group, key genes were identified by studying the form of intersection. Potential functions of DEGs for NASH were then analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein-protein interaction network (PPI) was constructed using the STRING database. A total of 249 DEGs and one key gene for NAFLD were identified. For NASH-Normal, 514 DEGs and 11 hub genes were identified, three of which were closely related to the survival analysis of HCC, and potentially closely related to progression from NASH to HCC. One key gene for NASH-NAFL (AKR1B10) was identified. These genes appear to mediate the molecular mechanism underlying NAFLD and may be promising biomarkers for the presence of NASH.

A simpler diagnostic formula for screening nonalcoholic fatty liver disease.

OBJECTIVE: To increase the accuracy of non-invasive diagnosis of nonalcoholic fatty liver disease (NAFLD), clinical and laboratory NAFLD indicators were integrated into a diagnostic formula. METHODS: A total of 141 patients with clinically diagnosed NAFLD and 30 healthy controls were enrolled. We collected case history, body weight, height and mass index (BMI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase, blood urea nitrogen and blood uric acid (UA), serum creatinine, plasma total cholesterol, triglyceride, low density lipoprotein, glycosylated hemoglobin, fasting plasma glucose, fasting insulin, ultrasonic tests, Fibroscans, and other data. Linear correlation, multiple linear regressions, and receiver operating characteristic (ROC) curve methods were used to process and analyze the collected data. The performance of Fibroscan and our diagnostic formula was compared in reference to the findings of liver biopsy. RESULTS: The identified NAFLD diagnostic indices consisted of BMI, ALT, AST and UA. A regression formula was proposed as: CAP = 113.163 + 0.252 * ALT + 6.316 * BMI. Diagnosis of the area under the ROC curve was 0.927, the sensitivity was 87.68%, and specificity was 90%. The cutoff was 277.67 (p < 0.01). The accuracy of the NAFLD diagnosis with the proposed formula was significantly higher than FibroScan (82.6% vs 69.6%; p = 0.005). CONCLUSIONS: NAFLD diagnosis with the proposed formula demonstrated both high sensitivity and specificity, and its accuracy was significantly higher than FibroScan. This formula only utilized non-invasive clinical and laboratory findings and the calculation was simple. It can be conveniently used for clinical diagnosis of NAFLD.

Hypoxia induces endothelial­mesenchymal transition in pulmonary vascular remodeling.

It is well established that hypoxia induces epithelial­mesenchymal transition in vitro and in vivo. However, the role of hypoxia in endothelial­mesenchymal transition (EndMT), an important process in the pathogenesis of pulmonary hypertension, is not well­characterized. The present study demonstrated a significant downregulation of the endothelial marker CD31 and its co­localization with a mesenchymal marker, α­smooth muscle actin (α­SMA), in the intimal layer of small pulmonary arteries of rats exposed to chronic hypoxia. These results suggest a possible role of hypoxia in inducing EndMT in vivo. Consistent with these observations, pulmonary microvascular endothelial cells (PMVECs) cultured under hypoxic conditions exhibited a significant decrease in CD31 expression, alongside a marked increase in the expression of α­SMA and two other mesenchymal markers, collagen (Col) 1A1 and Col3A1. In addition, hypoxia promoted the proliferation and migration of α­SMA­expressing mesenchymal­like cells, but not of PMVECs. Of note, knockdown of hypoxia­inducible factor 1α (HIF­1α) effectively inhibited hypoxic induction of α­SMA, Col1A1 and the transcription factor Twist1, while rescuing hypoxic suppression of CD31; these results suggest that HIF­1α is essential for hypoxia­induced EndMT and that it serves as an upstream regulator of Twist1. Mechanistically, HIF­1α was identified to bind to the promoter of the Twist1 gene, thus activating Twist1 transcription and regulating EndMT. Collectively, the present results indicate that the HIF­1α/Twist1 pathway has a critical role in mediating the effect of hypoxia­induced EndMT in pulmonary arterial remodeling.

Automatic regulation of NF-κB by pHSP70/IκBαm to prevent acute lung injury in mice.

Controlling target gene expression is a vital step in the procedure of gene therapy upon acute lung injury (ALI). Excessive activation of nuclear factor-kappa B (NF-κB) has been the key point of the inflammation overwhelming process in onset of ALI. We designed and tested a variety of plasmid named pHSP70/IκBαm which conditionally carries a mutant inhibitor of kappa B (IκB) transgene to regulate the activity of NF-κB signaling pathway in its response to an inflammatory stimulus that causes acute lung injury. Results recorded along our experiments showed that pHSP70/IκBαm was able to control mutant IκB expression in RAW264.7 cells with reference to the level of inflammatory response induced by LPS, thereby inhibiting NF-κB activation and downstream inflammatory cytokine expression. Vivo experiments revealed that construction naming pHSP70/IκBαm reduced LPS-induced lung injury and the secretion of inflammatory factors from lungs, hearts, and livers of sample mice in a LPS dose-dependent manner. In conclusion, the promoter heat shocking protein 70(HSP70) regulatory sequence of the construction was shown to drive mutant IκB expression so that its levels were positively associated with the dose of LPS used to induce acute lung injury. NF-κB activation and the downstream expression of inflammatory factors were therefore down-regulated in along an efficient path and ameliorating the damage as a consequence of LPS-induced acute lung injury.

Leptin attenuates lipopolysaccharide or oleic acid-induced acute lung injury in mice.

Leptin is reported to be involved in acute lung injury (ALI). However, the role and underlying mechanisms of leptin in ALI remain unclear. The aim of this study was to determine whether leptin deficiency promoted the development of ALI. LPS or oleic acid (OA) were administered to wild-type and leptin deficient (ob/ob) mice to induce ALI. Leptin level, survival rate, and lung injury were examined. Results showed that leptin levels were predominantly increased in the lung, but also in the heart, liver, kidney, and adipose tissue after LPS adminiatration. Compared with wild-type mice, LPS- or OA-induced lung injury was worse and the survival rate was lower in ob/ob mice. Moreover, leptin deficiency promoted the release of proinflammatory cytokines. Exogenous administration of leptin reduced lethality in ob/ob mice and ameliorated lung injury partly through inhibiting the activation of NF-κB, p38, and ERK pathways. These results indicated that leptin deficiency contributed to the development of lung injury by enhancing inflammatory response, and a high level of leptin improved survival and protected against ALI.

Osthole improves acute lung injury in mice by up-regulating Nrf-2/thioredoxin 1.

Inhibiting reactive oxygen species (ROS) has been viewed as a therapeutic target for the treatment of acute lung injury (ALI). Osthole, an active component in Chinese herbal medicine, has drawn increasing attention because of its various pharmacological functions, including anti-inflammatory and anti-oxidative activities. The aim of the present study was to examine the effects of osthole on ALI induced by lipopolysaccharide (LPS) through intratracheal instillation. The mRNA and protein expression levels of thioredoxin 1 (Trx1) and the nuclear factor erythroid-2 related factor 2 (Nrf2) were detected by real-time PCR, reverse transcription PCR (RT-PCR) and Western blot, respectively. ROS production was measured by flow cytometry. Our results showed that osthole treatment improved the mice survival rates in the middle and high dosage groups, compared with the untreated LPS group. Moreover, osthole treatment significantly improved LPS-induced lung pathological damage, and it decreased the lung injury scores, lung wet/dry ratios and the total protein level in Bronchoalveolar lavage fluid (BALF). Osthole treatment dramatically reduced the H2O2, MDA and OH levels in the lung homogenates. LDH and ROS were markedly reduced in the osthole+LPS group in vitro. Furthermore, osthole increased Nrf2 and Trx1 expression in terms of mRNA and protein in vivo and in vitro. Nrf2 siRNA (siNrf2) could suppress the beneficial effects of osthole on ALI. In conclusion, the current study demonstrates that osthole exerted protective effects on LPS-induced ALI by up-regulating the Nrf-2/Trx-1 pathway.

Insulin reduces LPS-induced lethality and lung injury in rats.

Insulin is a main glucose homeostatic hormone in the body. Previous reports showed that insulin also exerted anti-inflammatory actions and attenuated systemic inflammatory response. Here, we observed the effects and the underlying mechanisms of insulin on lipopolysaccharide (LPS)-induced acute lung injury (ALI). As revealed by survival study, insulin reduced mortality of rats and prolonged their survival time. Meanwhile, insulin significantly reduced the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and high mobility group box 1 (HMGB1) in bronchoalveolar lavage fluid (BALF). Besides, insulin markedly inhibited the expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB). Taken together, these data provided information that insulin attenuated LPS-induced ALI may attribute partly to the inhibition of the production of cytokines, and the expression of TLR2, TLR4 and NF-κB.

Role of macrophage migration inhibitory factor in the proliferation of smooth muscle cell in pulmonary hypertension.

Pulmonary hypertension (PH) contributes to the mortality of patients with lung and heart diseases. However, the underlying mechanism has not been completely elucidated. Accumulating evidence suggests that inflammatory response may be involved in the pathogenesis of PH. Macrophage migration inhibitory factor (MIF) is a critical upstream inflammatory mediator which promotes a broad range of pathophysiological processes. The aim of the study was to investigate the role of MIF in the pulmonary vascular remodeling of hypoxia-induced PH. We found that MIF mRNA and protein expression was increased in the lung tissues from hypoxic pulmonary hypertensive rats. Intensive immunoreactivity for MIF was observed in smooth muscle cells of large pulmonary arteries (PAs), endothelial cells of small PAs, and inflammatory cells of hypoxic lungs. MIF participated in the hypoxia-induced PASMCs proliferation, and it could directly stimulate proliferation of these cells. MIF-induced enhanced growth of PASMCs was attenuated by MEK and JNK inhibitor. Besides, MIF antagonist ISO-1 suppressed the ERK1/2 and JNK phosphorylation induced by MIF. In conclusion, the current finding suggested that MIF may act on the proliferation of PASMCs through the activation of the ERK1/2 and JNK pathways, which contributes to hypoxic pulmonary hypertension.

Macrophage migration inhibitory factor contributes to hypoxic pulmonary vasoconstriction in rats.

BACKGROUND: Hypoxic pulmonary vasoconstriction may lead to pulmonary hypertension, but the underlying mechanisms of persistent vasoconstriction are still unclear. There is evidence that pulmonary inflammation contributes to the abnormalities of function in the pulmonary artery (PA) following chronic hypoxia exposure. Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory cytokine, and we found that expression of MIF was increased in the smooth muscle of PA from hypoxic pulmonary hypertensive rats. Therefore, the aim of the study was to investigate the role of MIF in modulating vasoreactivity of isolated PA rings. METHODS: Sprague-Dawley rats were challenged by intermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models. Subsequently, immunohistochemistry and western blot assay were used to examine the MIF expression in pulmonary artery. Moreover, isometric force displacement was measured in isolated intrapulmonary artery. RESULTS: In the isolated PA, our results showed that MIF mediated the enhanced pulmonary arterial vasoconstriction in response to chronic hypoxia, and the delayed hypoxic constriction in a biphasic pattern of constriction occurs in response to acute hypoxia. We also present the finding that MIF had no effect on force on its own, but concentration-dependently potentiated constrictions pre-evoked by phenylephrine under normoxic condition. The potentiation was independent of the endothelium. MIF-induced potentiation of phenylephrine-evoked constriction was partially inhibited by PKC inhibitor chelerythrine, p38 inhibitor SB 203580, ERK1/2 inhibitor U0126, respectively. CONCLUSIONS: Our results suggested that MIF enhanced vasoconstriction of pulmonary artery elicited by agonist through PKC, p38 and ERK1/2 signal pathways, which may contributes to hypoxic pulmonary vasoconstriction.

Antiinflammatory effects of matrine in LPS-induced acute lung injury in mice.

Matrine is one of the main active components of Chinese herb Sophora flavescens Ait (Kushen), which has been demonstrated to be effective in suppressing inflammation. The aim of the present study is to investigate the effect of matrine on LPS-induced lung injury. Lung injury was assessed by histological study and wet to dry weight ratios, as well as cell count and protein content in bronchoalveolar lavage fluid. We also detected MPO activity reflecting neutrophil infiltration and MDA activity examining oxidative stress in lung tissues. Cytokines and ROS production in cells were monitored by ELISA and flow cytometry, respectively. The results showed that high dose of matrine significantly reduced the mortality rate of mice with LPS administration. Treatment with matrine improved LPS-induced lung histopathologic changes, alleviated pulmonary edema and lung vascular leak, inhibited MPO and MDA activity,and reduced the production of inflammatory mediators including TNF-α, IL-6 and HMGB1. In vitro, matrine administration reduced the production of ROS and inflammatory factors, which was possibly associated with inhibition of NF-κB. In conclusion, the current study demonstrated that matrine exhibited a protective effect on LPS-induced acute lung injury by inhibiting of the inflammatory response, which may involve the suppression of ROS and tissue oxidative stress.

Beta-estradiol attenuates hypoxic pulmonary hypertension by stabilizing the expression of p27kip1 in rats.

BACKGROUND: Pulmonary vascular structure remodeling (PVSR) is a hallmark of pulmonary hypertension. P27(kip1), one of critical cyclin-dependent kinase inhibitors, has been shown to mediate anti-proliferation effects on various vascular cells. Beta-estradiol (ß-E2) has numerous biological protective effects including attenuation of hypoxic pulmonary hypertension (HPH). In the present study, we employed ß-E2 to investigate the roles of p27(kip1) and its closely-related kinase (Skp-2) in the progression of PVSR and HPH. METHODS: Sprague-Dawley rats treated with or without ß-E2 were challenged by intermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models, which resemble moderate severity of hypoxia-induced PH in humans. Subsequently, hemodynamic and pulmonary pathomorphology data were gathered. Additionally, pulmonary artery smooth muscle cells (PASMCs) were cultured to determine the anti-proliferation effect of ß-E2 under hypoxia exposure. Western blotting or reverse transcriptional polymerase chain reaction (RT-PCR) were adopted to test p27(kip1), Skp-2 and Akt-P changes in rat lung tissue and cultured PASMCs. RESULTS: Chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of right ventricle/left ventricle plus septum (RV/LV+S) ratio, medial width of pulmonary arterioles, accompanied with decreased expression of p27(kip1) in rats. Whereas, ß-E2 treatment repressed the elevation of RVSP, RV/LV+S, attenuated the PVSR of pulmonary arterioles induced by chronic hypoxia, and stabilized the expression of p27(kip1). Study also showed that ß-E2 application suppressed the proliferation of PASMCs and elevated the expression of p27(kip1) under hypoxia exposure. In addition, experiments both in vivo and in vitro consistently indicated an escalation of Skp-2 and phosphorylated Akt under hypoxia condition. Besides, all these changes were alleviated in the presence of ß-E2. CONCLUSIONS: Our results suggest that ß-E2 can effectively attenuate PVSR and HPH. The underlying mechanism may partially be through the increased p27(kip1) by inhibiting Skp-2 through Akt signal pathway. Therefore, targeting up-regulation of p27(kip1) or down-regulation of Skp-2 might provide new strategies for treatment of HPH.

Tanshinone IIA modulates pulmonary vascular response to agonist and hypoxia primarily via inhibiting Ca2+ influx and release in normal and hypoxic pulmonary hypertension rats.

The present study was designed to investigate the vascular effects and underlying mechanisms of tanshinone IIA on isolated rat pulmonary artery. Isometric tension was recorded in the arteries from normal and hypoxic pulmonary hypertension rats under normoxia or hypoxia condition. The results showed that tanshinone IIA exerted a biphasic effect on rat pulmonary artery. The constriction was attenuated by endothelium-denudation but was enhanced by inhibition of nitric oxide synthase. Pretreatment with tetraethylammonium (Ca2+-activated K+ channel inhibitor) upward shifted the concentration-response curve without affecting the maximum dilatation. Pretreatment with zinc protoporphyrin IX (heme oxygenase-1 inhibitor), 4-aminopyridine (KV channel inhibitor), glibenclamide (KATP channel inhibitor) or BaCl2 (inwardly rectifying K+ channel inhibitor) did not affect the vasoreactivity. Meanwhile, tanshinone IIA almost abolished vasoconstriction induced by extracellular Ca2+. Under hypoxia condition, tanshinone IIA eliminated acute hypoxia-induced initial contraction, potentiated following vasorelaxation, attenuated and reversed sustained contraction to relaxation in pulmonary artery from normal rats, and reversed phenylephrine-induced sustained constriction to sustained relaxation in remodeled pulmonary artery from hypoxic pulmonary hypertension rats. We concluded that the mild constrictive effect induced by tanshinone IIA was affected by integrity of endothelium and production of nitric oxide, while the potent dilative effect was endothelium-independent and produced primarily by inhibiting extracellular Ca2+ influx and partially by inhibiting intracellular Ca2+ release, as well as activating Ca2+-activated K+ channels. The modulation of tanshinone IIA on pulmonary vasoreactivity under both acute and chronic hypoxia condition may provide a new insight for curing hypoxic pulmonary hypertension.

Effects of sodium tanshinone II A sulphonate on hypoxic pulmonary hypertension in rats in vivo and on Kv2.1 expression in pulmonary artery smooth muscle cells in vitro.

AIM OF THE STUDY: To investigate the effect of sodium tanshinone IIA sulphonate (STS), a water-soluble derivative of tanshinone II A, on hypoxic pulmonary hypertension (HPH) in rats and its underlying mechanisms. MATERIALS AND METHODS: Rats were exposed to hypoxia for two or three weeks, pretreated with or without STS. We detected mean pulmonary arterial pressure (mPAP), the ratio of right ventricle weight to left ventricle with septum weight [RV/(LV+S)], wall thickness and voltage-activated potassium channel (Kv) 2.1 mRNA level of pulmonary arteries (PAs), respectively, and the in vitro effects of STS on proliferation and Kv2.1 expression of cultured pulmonary smooth muscle cells (PASMCs) from normal rats. Cell proliferation was determined by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazoliumbromiede (MTT) assay and direct cell counting. Kv2.1 mRNA and protein level were evaluated by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS: Chronic hypoxia increased values of mPAP and RV/(LV+S) and inhibited Kv2.1 mRNA level in PAs. Three weeks' daily STS pretreatment inhibited the hypoxia-induced increased mPAP and RV/(LV+S), pulmonary arterial thickening and up-regulated Kv2.1 mRNA level in PAs. Further study in vitro showed that STS suppressed significantly hypoxia-induced PASMCs proliferation and inhibition of Kv2.1 expression in PASMCs. CONCLUSIONS: STS might play protective effects on HPH through decreasing mPAP, V/(LV+S) and inhibiting structural remodeling in distal PAs. The mechanism of these effects may be attributed to inhibiting PASMCs proliferation and stimulating Kv2.1 expression.

Tanshinone IIA reduces lethality and acute lung injury in LPS-treated mice by inhibition of PLA2 activity.

Tanshinone IIA (TIIA) is one of the main active components from Chinese herb danshen. Previous reports showed that TIIA reduced the production of pro-inflammatory mediators stimulated with lipopolysaccharide (LPS). However, the effects of TIIA on LPS-induced acute lung injury are not fully understood. Here, we observed the effects of TIIA on mortality and lung injury in LPS-treated mice and on LPS-induced pulmonary epithelial cell injury, and further studied the underlying mechanism. As revealed by survival study, pretreatment with TIIA reduced mortality of mice and prolonged their survival time. Meanwhile, TIIA pretreatment significantly improved LPS-induced lung histopathologic changes, decreased lung wet-to-dry and lung-to-body weight ratios, inhibited lung myeloperoxidase activity and reduced protein leakage. TIIA also alleviated LPS-induced pulmonary epithelial cell injury, as proved by methyl thiazolyl tetrazolium (MTT) and lactic dehydrogenase assay. Furthermore, TIIA suppressed LPS-induced phospholipase A2 (PLA2) activity in both lung homogenate and bronchoalveolar lavage fluid. TIIA also inhibited the metabolites of PLA2, which was confirmed by results of thromboxane B2, prostaglandin E2 and leukotriene B4 detection. Besides, TIIA in vitro inhibited LPS-induced PLA2 activity in a dose-dependent manner. Western blotting showed that TIIA markedly inhibited the activation of nuclear factor kappa B (NF-kappaB) in LPS-treated mice. Taken together, these data firstly provided the novel information that the protective role of TIIA against LPS-induced lung injury may attribute partly to the inhibition of PLA2 activity and NF-kappaB activation.