OBJECTIVETo explore the effect of hypoxia and hypoxia-inducible factor-1alpha (HIF-1alpha) on the expression of multidrug resistance gene-1 (mdr-1) and its coded p-glyeoprotein (P-gp) as well as the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism.

METHODSThe mRNA and protein levels of HIF-1alpha, mdr-1 and p-gp were studied by immunocytochemistry, semiquantitative reverse transcription-ploymerase chain reaction (RT-PCR) and Western blot analysis in human ovarian cancer cells (A2780) in 5% CO2 + 1% O2 hypoxic culture and 21% O2 normoxic culture, respectively. Methyl thiazolyl tetrazolium (MIT) was used to evaluate the chemotherapeutic sensitivity of A2780 cells to paclitaxel by inhibition rate. RNA interference technique was used and small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed as pSiHIF-1alpha, and transfected into A2780 cells. RT-PCR and Western blot were used to detect gene silencing effect on HIF-1alpha, the expressions of mdr-1 and p-gp. The inhibition rate was observed after HIF-1alpha gene silence.

RESULTSHIF-1alpha at both mRNA and protein levels was induced significantly under hypoxia. The HIF-1alpha expression at mRNA level was oxygen gradient-independent, while HIF-1alpha expression at protein level was oxygen gradient-dependent. The inhibition rate of paclitaxel to hypoxic A2780 cells in 5% CO2 + 1% O2 was significantly lower than that in normoxic A2780 cells (P <0.05). The shRNAs plasmid targeting HIF-1alpha was constructed successfully and HIF-1alpha gene was silenced in A2780 cells efficiently followed by mdr-1 and p-gp down-regulation. The inhibition rate was greatly increased in hypoxic A2780/siHIF-1alpha cells.

CONCLUSIONHypoxia can decrease the chemotherapeutic sensitivity of human ovarian cancer A2780 cells to paclitaxel through HIF-1alpha regulating the expression of mdr-1 and p-gp.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Antineoplastic Agents, Phytogenic ; pharmacology ; Blotting, Western ; Cell Hypoxia ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Paclitaxel ; pharmacology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Accumulating evidence demonstrates that the nucleus tractus solitarii (NTS) neurons serve as central respiratory chemoreceptors, but the underlying molecular mechanisms remain undefined. The present study investigated the expression of acid-sensitive ether-à-go-go-gene-like (Elk, Kv12) channels in the NTS of mice. Immunofluorescence staining was used to observe the distribution and cellular localization of the Kv12 channels in NTS neurons. Western blot and quantitative real-time PCR (qPCR) were used to evaluate protein and mRNA expression levels of Kv12 channels. The results showed that all of the three members (Kv12.1, Kv12.2, Kv12.3) of the Kv12 channel family were expressed in NTS neurons, and their expressions were co-localized with paired-like homeobox 2b gene (Phox2b) expression. The expression of Kv12.1 mRNA was the largest, whereas the expression of Kv12.3 was the least in the NTS. The results suggest Kv12 channels are expressed in Phox2b-expressing neurons in the NTS of mice, which provides molecular evidence for pH sensitivity in Phox2b-expressing NTS neurons.
Animals ; Mice ; Neurons ; Potassium Channels, Voltage-Gated ; Solitary Nucleus ; Transcription Factors/genetics*

OBJECTIVETo investigate the renal protective effect of sodium ferulate magnetic nanoparticle targeting therapy in septic rats receives norepinephrine treatment.

METHODSFirst we constructed sodium ferulate magnetic nanoparticle, Wistar male rats were randomly divided into 4 groups (n = 6): control group, septic group, norepinephrine treatment group (NE treatment group) and NE plus sodium ferulate magnetic nanoparticle treatment group (NE + SF group), septic rat model was reproduced by intravenous injection of lipolysaccharide (IPS) in rats in NE treatment group norepinephrine were used to elevate the blood pressure of septic rats, in NE + SF group sodium ferulate magnetic nanoparticle was injected via tail vein, magnetic field was placed near renal region. Urinary concentration of N-acetyl-beta-D-glucosaminidase (NAG), kidney injury molecule (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), renal tissue concentration of malonaldehyde (MDA), superoxide dismutase (SOD), and serum concentration of blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were measured 3 hours after treatment.

RESULTSAfter injected LPS via tail vein, systolic blood pressure, pH value, PaO2 and PaCO2 of arterial blood of septic rats decreased significantly. Systolic blood pressure, pH value, PaO2 and PaCO2 of arterial blood returned to baseline approximately after norepinephrine treatment, sodium ferulate magnetic nanoparticle targeting therapy did not change hemodynamic effects of norepinephrine. Compared with control group, urine NAG, KIM-1 and NGAL of sepsis group were increased significantly (P < 0.01), after treatment with norepinephrine, urine NAG, KIM-1 and NGAL of NE treatment group were elevated rapidly compared with those of sepsis group (P < 0.01), combined with sodium ferulate magnetic nanoparticles targeting treatment, urine NAG, KIM-1 and NGAL of NE + SF group were decreased significantly compared with those of sepsis group and NE treatment group (P < 0.01). Compared with control group, renal tissue MDA levels of septic rats increased significantly (P < 0.01), NE treatment could notably raise MDA levels compared with those of sepsis group (P < 0.01), renal tissue MDA levels of NE + SF group were decreased significantly compared with those of sepsis group and NE treatment group (P < 0.01). Compared with control group, renal tissue activities of SOD of sepsis group and NE treatment group were decreased significantly (P < 0.01), after targeted treatment with sodium ferulate magnetic nanoparticle, renal tissue SOD activities of NE + SF group increased significantly compared with those of sepsis group and NE treatment group (P < 0.01 vs sepsis group and NE treatment group). Serum BUN, Cr, ALT, AST levels did not significantly change in each groups.

CONCLUSIONSodium ferulate magnetic nanoparticle targeting therapy can effectively decrease urine NAG, KIM-1, NGAL and renal tissue MDA level, increase tissue SOD activity of sepsis group and NE treatment group rats, thus protect renal function of septic rats.

Animals ; Coumaric Acids ; administration & dosage ; pharmacology ; Kidney ; drug effects ; pathology ; Magnetics ; Male ; Nanoparticles ; Rats ; Rats, Sprague-Dawley ; Sepsis ; pathology ; urine

ABSTRACT OBJECTIVE：To evaluate the in vitro and in vivo genotoxicity of emodin-8-O-β-D-glucoside（EG），and to compare the difference of in vitro cell test and in vivo test of rats. METHODS：2D and 3D hepatocyte models were established by in vitro two-dimensional（2D）and three-dimensional（3D）cell culture. After modeling，2D and 3D hepatocyte were divided into blank control group（0.5％ DMSO），mitomycin C group（positive control，0.1 μg/mL），EG low-dose，medium-dose and high-dose groups（10，50，200 μg/mL），respectively. The micronucleus ratio and tail DNA％ of HepaRG cells were detected. SD rats were divided into blank control group（0.5％ sodium carboxymethyl cellulose），ethyl methanesulfonate group（positive control，200 mg/kg），EG low-dose，medium-dose and high-dose groups（100，300，1 000 mg/kg），with 6 rats in each group. They were given medicine intragastrically for consecutive 15 d，once a day. 15 days later，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were measured. RESULTS：In the in vitro 2D HepaRG hepatocyte model，compared with blank control group，the micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01）. There was no statistical significance in micronucleus formation rate and tail DNA％ of HepaRG cell among EG groups（P＞0.05）. In 3D HepaRG cell model， compared with blank control group， micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01 or P＜0.001）， while tail DNA％ of HepaRG cell wasincreased significantly in EG high-dose group（P＜0.01）. In the in vivo test，compared with blank control group，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were all increased significantly in ethyl methanesulfonate group（P＜0.01）. Tail DNA％ of peripheral blood lymphocytes was increased significantly in EG high-dose group （P＜0.01）. There was no statistical significance in the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of hepatocytes among EG groups（P＞0.05）；with the increase of dose，there was an increasing trend. CONCLUSIONS：The results of this study suggest that in 2D cell model，EG not lead to chromosome breakage and DNA damage，but the long-term administration and repeated administration in vivo of 3D cell model show that EG has a certain risk of DNA damage，so the evaluation results of 3D HepaRG cell model are more similar to those of rats in vivo. KEYWORDS Emodin-8-O-β-D-glucoside；Genotoxicity；Two-dimensional culture；Three-dimensional culture；Rat；Micronucleus test

italic>Lamiophlomis rotata is an important medicinal plant species endemic to the Tibetan Plateau, which is prone to strong climate change impacts on its habitable range due to the high sensitivity of the Tibetan Plateau to climate change. Accurate quantification of species vulnerability to climate change is essential for assessing species extinction risk and developing effective conservation strategies. Therefore, we carried out the α-shape analysis to determine the habitat of L. rotata. We then carried out the climate-niche factor analysis (CNFA) to assess the vulnerability of L. rotata to climate change based on five climate variables (i.e., mean diurnal range, temperature seasonality, mean temperature of warmest quarter, precipitation of driest month and precipitation of warmest quarter) in the context of two shared socioeconomic pathways (i.e., SSP126 and SSP585) and three global climate models (CMCC-ESM2: Centro Euro-Mediterraneo sui Cambiamenti Climatici-Earth System Model version 2; HadGEM3-GC31-LL: Hadley Global Environment Model version 3-Global Coupled configuration 3.1; IPSL-CM6A-LR: Institut Pierre Simon Laplace-Climate Model version 6) during two different periods (2041-2060 and 2081-2100). The vulnerability of L. rotata to climate change was calculated by integrating the sensitivity and exposure indices of L. rotata to five climate variables. The results showed that L. rotata had the highest vulnerability to the precipitation of warmest quarter. Its vulnerability within its habitat range generally showed a spatial pattern of high value in the southern region and low in the northern region, high in the western region and low in the eastern region. In general, the vulnerability of L. rotata under the SSP585 scenario was higher than that under the SSP126 scenario. The climate data of different global climate models have some influence on the results, while the resulted uncertainty can be reduced by data integration methods. As a result of climate change, the pressure on the survival of L. rotata in the future will be intensified in the low-altitude areas such as the Yarlung Zangbo River, Yigongzangbu River, Zayu River, and Jiaomuzu River, etc., while the highly weathered scree flats or stony alpine meadows in the high-altitude zones, such as the eastern Tanggula Mountain Range, the northern part of Hengduan Mountain Range, and the western part of the Qinling Mountains, may become its refuge. It is necessary to focus on and strengthen the protection and management of L. rotata resources in these vulnerble and critical areas.