Discovery of Natural Products Alleviating Renal Fibrosis with a Viscosity-Responsive Molecular Probe.

Renal fibrosis poses a significant threat to individuals suffering from chronic progressive kidney disease. Given the absence of effective medications for treating renal fibrosis, it becomes crucial to assess the extent of fibrosis in real time and explore the development of novel drugs with substantial therapeutic benefits. Due to the accumulation of renal tissue damage and the uncontrolled deposition of fibrotic matrix during the course of the disease, there is an increase in viscosity both intracellularly and extracellularly. Therefore, a viscosity-sensitive near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe, BDP-KY, was developed to detect aberrant changes in viscosity during fibrosis. Furthermore, BDP-KY has been applied to screen the effective components of herbal medicine, rhubarb, resulting in the identification of potential antirenal fibrotic compounds such as emodin-8-glucoside and chrysophanol 8-O-glucoside. Ultrasound, PA, and NIRF imaging of a unilateral uretera obstruction mice model show that different concentrations of emodin-8-glucoside and chrysophanol 8-O-glucoside effectively reduce viscosity levels during the renal fibrosis process. The histological results showed a significant decrease in fibrosis factors α-smooth muscle actin and collagen deposition. Combining these findings with their pharmacokinetic characteristics, these compounds have the potential to fill the current market gap for effective antirenal fibrosis drugs. This study demonstrates the potential of BDP-KY in the evaluation of renal fibrosis, and the two identified active components from rhubarb hold great promise for the treatment of renal fibrosis.

A novel method for evaluating pseudoallergy based on ß-hexosaminidase and its application for traditional Chinese medicine injections.

Pseudoallergy is a typical and common adverse drug reaction to injections, especially in traditional Chinese medicine injections (TCMIs). At present, the evaluation methods for pseudoallergy include cell methods in vitro and animal methods in vivo. The mast cell evaluation method based on the ß-hexosaminidase (ß-Hex)-catalyzed substrate, 4-nitrophenyl-ß-N-acetyl-D-glucosaminide (4-NPG), is an important method for the evaluation of drug-induced pseudoallergy, but it is prone to false positive results and has insufficient sensitivity. In this study, a novel ß-Hex evaluation system with rat basophilic leukemia-2H3 cells based on high-performance liquid chromatography-fluorescence detection (HPLC-FLD) was established, which effectively increased the sensitivity and avoided false positive results. Cell viabilities were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. In addition, a method for the determination of histamine, which is another indicator in the development of pseudoallergy, was established to validate the above method. The results of this novel method indicated that two TCMIs (Shuxuening injection and Shenqi Fuzheng injection), which were considered to be pseudoallergenic using 4-NPG, were not pseudoallergenic. Overall, the novel ß-Hex/HPLC-FLD evaluation system using Rat basophilic leukemia-2H3 cells established was effective and precise. It could be used for the evaluation of pseudoallergic reactions caused by TCMIs and other injections.

A ternary correlation multi-symptom network strategy based on in vivo chemical profile identification and metabolomics to explore the molecular basis of Ephedra herb against viral pneumonia.

Ephedra herb (EH), an important medicine prescribed in herbal formulas by Traditional Chinese Medicine practitioners, has been widely used in the treatment of viral pneumonia in China. However, the molecular basis of EH in viral pneumonia remains unclear. In this study, a ternary correlation multi-symptom network strategy was established based on in vivo chemical profile identification and metabolomics to explore the molecular basis of EH against viral pneumonia. Results showed that 143 compounds of EH and 70 prototype components were identified in vivo. EH could reduce alveolar-capillary barrier disruption in rats with viral pneumonia and significantly downregulate the expression of inflammatory factors and bronchoalveolar lavage fluid. Plasma metabolomics revealed that EH may be involved in the regulation of arachidonic acid, tryptophan, tyrosine, nicotinate, and nicotinamide metabolism. The multi-symptom network showed that 12 compounds have an integral function in the treatment of viral pneumonia by intervening in many pathways related to viruses, immunity and inflammation, and lung injury. Further verification demonstrated that sinapic acid and frambinone can regulate the expression of related genes. It has been shown to be a promising representative of the pharmacological constituents of ephedra.

Target Cell Extraction and Spectrum-Effect Relationship Coupled with BP Neural Network Classification for Screening Potential Bioactive Components in Ginseng Extract with a Protective Effect against Myocardial Damage.

Cardiovascular disease has become a common ailment that endangers human health, having garnered widespread attention due to its high prevalence, recurrence rate, and sudden death risk. Ginseng possesses functions such as invigorating vital energy, enhancing vein recovery, promoting body fluid and blood nourishment, calming the nerves, and improving cognitive function. It is widely utilized in the treatment of various heart conditions, including palpitations, chest pain, heart failure, and other ailments. Although numerous research reports have investigated the cardiovascular activity of single ginsenoside, there remains a lack of systematic research on the specific components group that predominantly contribute to cardiovascular efficacy in ginseng medicinal materials. In this research, the spectrum-effect relationship, target cell extraction, and BP neural network classification were used to establish a rapid screening system for potential active substances. The results show that red ginseng extract (RGE) can improve the decrease in cell viability and ATP content and inhibit the increase in ROS production and LDH release in OGD-induced H9c2 cells. A total of 70 ginsenosides were identified in RGE using HPLC-Q-TOF-MS/MS analysis. Chromatographic fingerprints were established for 12 batches of RGE by high-performance liquid chromatography (HPLC). A total of 36 common ingredients were found in 12 batches of RGE. The cell viability, ATP, ROS, and LDH of 12 batches RGE were tested to establish gray relationship analysis (GRA) and partial least squares discrimination analysis (PLS-DA). BP neural network classification and target cell extraction were used to narrow down the scope of Spectral efficiency analysis and screen the potential active components. According to the cell experiments, RGE can improve the cell viability and ATP content and reduce the oxidative damage. Then, seven active ingredients, namely, Ginsenoside Rg1, Rg2, Rg3, Rb1, Rd, Re, and Ro, were screened out, and their cardiovascular activity was confirmed in the OGD model. The seven ginsenosides were the main active substances of red ginseng in treating myocardial injury. This study offers a reference for quality control in red ginseng and preparations containing red ginseng for the management of cardiovascular diseases. It also provides ideas for screening active ingredients of the same type of multi-pharmacologically active traditional Chinese medicines.

Preparation of Ultra-High Temperature Resistant Cyclodextrin-Based Filtration Loss Reducer for Water-Based Drilling Fluids.

In the development of ultra-deep wells, extremely high temperatures can lead to inefficiency of additives in drilling fluids. Hence, there is a need to prepare additives with a simple preparation process and good effects at ultra-high temperatures to ensure stable drilling fluid performance. In this study, a high temperature resistant filtration loss polymer (LY-2) was prepared using γ-methacryloyloxypropyltrimethoxysilane (KH570), N,N-dimethylallyl ammonium chloride (DMDAAC), sodium p-styrenesulfonate (SSS), and ß-cyclodextrin (ß-CD). The impact of the different monomer ratios on particle size, rheology, and filtration performance was systematically investigated. Infrared spectroscopy afforded the structural features. Thermogravimetric Analysis detected the temperature stability, and scanning electron microscopy characterized the polymer micromorphology. LY-2 was completely decomposed at a temperature above 600 °C. Experiments showed FLAPI of the drilling fluid containing 3% LY-2 aged at 260 °C/16 h was only 5.1 mL, which is 85.4% lower compared to the base fluid. This is attributed to the synergistic effect of the polymer adsorption through chemical action at high temperatures and the blocking effect of carbon nanoparticles on the filter cake released by cyclodextrin carbonization at high temperatures. Comparing LY-2 with commercial filter loss reducers shows that LY-2 has excellent temperature resistance, which exhibited five times higher filtration performance and relatively low cost, making it possible to be applied to ultra-high temperature drilling operations in an industrial scale-up.

[Value of SOX1 and PAX1 Gene Methylation Detection in Secondary Triage of High-Grade Cervical Lesions].

Objective To evaluate the value of SOX1 and PAX1 gene methylation detection in the secondary triage of high-grade cervical lesions.Methods Exfoliated cervical cells were collected from 122 patients tested positive for human papilloma virus (HPV) and subjected to thin-prep cytologic test (TCT) and SOX1/PAX1 gene methylation tests.Results The HPV test combined with TCT showed the sensitivity of 95.24% and the specificity of 23.75% for detecting cervical intraepithelial neoplasia (CIN) grade 2 and above (CIN2+).After the addition of the SOX1/PAX1 gene methylation detection in secondary triage,the sensitivity for detecting CIN2+ was 83.33%,which had no statistically significant difference from the sensitivity of TCT combined with HPV test (P=0.078).However,the specificity reached 77.50%,which was significantly higher than that of HPV test combined with TCT (P<0.001).The SOX1/PAX1 gene methylation level in the CIN2+ group was higher than those in the normal cervical tissue and the CIN1 group(P<0.001).The cut-off values of SOX1 and PAX1 gene methylation for CIN2+ detection were -11.81 and -11.98,respectively.Conclusion Adding the detection of SOX1/PAX1 gene methylation in secondary triage significantly improves the efficiency and accuracy of CIN2+ detection.

Maintaining Antibacterial Activity against Biofouling Using a Quaternary Ammonium Membrane Coupling with Electrorepulsion.

Antibacterial modification is a chemical-free method to mitigate biofouling, but surface accumulation of bacteria shields antibacterial groups and presents a significant challenge in persistently preventing membrane biofouling. Herein, a great synergistic effect of electrorepulsion and quaternary ammonium (QA) inactivation on maintaining antibacterial activity against biofouling has been investigated using an electrically conductive QA membrane (eQAM), which was fabricated by polymerization of pyrrole with QA compounds. The electrokinetic force between negatively charged Escherichia coli and cathodic eQAM prevented E. coli cells from reaching the membrane surface. More importantly, cathodic eQAM accelerated the detachment of cells from the eQAM surface, particularly for dead cells whose adhesion capacity was impaired by inactivation. The number of dead cells on the eQAM surface was declined by 81.2% while the number of live cells only decreased by 49.9%. Characterization of bacteria accumulation onto the membrane surface using an electrochemical quartz crystal microbalance revealed that the electrorepulsion accounted for the cell detachment rather than inactivation. In addition, QA inactivation mainly contributed to minimizing the cell adhesion capacity. Consequently, the membrane fouling was significantly declined, and the final normalized water flux was promoted higher than 20% with the synergistic effect of electrorepulsion and QA inactivation. This work provides a unique long-lasting strategy to mitigate membrane biofouling.

Inhibition of TRPC6 suppressed TGFß-induced fibroblast-myofibroblast transdifferentiation in renal interstitial NRK-49F cells.

Renal fibrosis is a global health concern with limited curative treatment. Canonical transient receptor potential channel 6 (TRPC6), a nonselective cation channel, has been shown to regulate the renal fibrosis in murine models. However, the molecular mechanism is unclear. Fibroblast-myofibroblast transdifferentiation is one of the critical steps in the progression of renal fibrosis. In the present study, we demonstrate that transforming growth factor (TGF)-ß1 exposure significantly increases the TRPC6 expression in renal interstitial fibroblast NRK-49F cells. Pharmacological inhibition of TRPC6 and knockdown of Trpc6 by siRNA alleviate TGF-ß1-increased expression levels of α-smooth muscle actin (α-SMA) and collagen I, two key markers of myofibroblasts. Although direct activation of TRPC6 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) does not affect the expression of α-SMA and collagen I, OAG potentiates TGF-ß1-induced fibroblast-myofibroblast transdifferentiation. Further study demonstrates that TGF-ß1 exposure increases the phosphorylation level of p38 and Yes-associated protein (YAP) translocation into the nuclei. Inhibition of p38 and YAP decreases TGF-ß1-enhanced TRPC6 and α-SMA expression. In conclusion, we demonstrate that TRPC6 is a key regulator of TGF-ß1-induced fibroblast-myofibroblast transdifferentiation and provides the mechanism of how TGF-ß1 exposure regulates TRPC6 expression in NRK-49F fibroblasts.

Ultrasound-guided microwave ablation for tertiary hyperparathyroidism in patients with renal transplantation.

OBJECTIVE: To explore the safety and efficacy of ultrasound-guided microwave ablation (MWA) for tertiary hyperparathyroidism (THPT) in patients with renal transplantation (RT). METHODS: In total, fifteen patients with THPT after renal transplantation who underwent MWA were enrolled in the study. The pre- and post-MWA intact parathyroid hormone (iPTH), serum calcium, phosphorus, creatinine, urea nitrogen and estimated glomerular filtration rate (eGFR) values were compared. RESULTS: A total of 38 parathyroid hyperplastic nodules in 15 RT patients were treated with ultrasound-guided MWA. The mean (median, range) size of the hyperplastic parathyroid nodules was 11.5 mm (11 mm, 5-25 mm), and the average (median, range) ablation time was 163.5s (121 s, 44-406 s). The average levels of serum iPTH and calcium at 1 d, 7 d, 1 month, 3 months, 6 months, 1 year post-MWA and at the end of follow-up were significantly lower than those pre-MWA (all p < 0.05). Compared with the pre-MWA value (0.76 mmol/L), the serum phosphorus levels at 1 d post-MWA (0.63 mmol/L) were significantly decreased, and those at 7 d, 1 month, 3 months, 6 months, 1 year post-MWA and at the end of follow-up were significantly increased, but all were within the normal range. There was no significant difference in serum creatinine and eGFR pre-MWA and post-MWA. No major MWA-related complications occurred. CONCLUSION: Ultrasound-guided MWA shows potential as a viable treatment for THPT in RT patients. However, further studies are required to confirm its safety and effectiveness in larger cohorts of longer duration.

Decomposition and comparative analysis of health inequities between the male and female older adults in China: a national cross-sectional study.

BACKGROUND: This study aimed to examine the factors influencing self-rated health (SRH) among Chinese older adults by gender differences and provide suggestions and theoretical references to help make policies for older adults' health concerns by government agencies. METHODS: Chinese Longitudinal Health Longevity Survey (CLHLS) in 2018 was adopted, the chi-squared test and the logistic regression analysis were performed to analyse self-rated health reported by Chinese female and male older adults and its influencing factors. In addition, Fairlie decomposition analysis was performed to quantify the contribution level of different influencing factors. RESULTS: Among older adults, males (48.0%) reported a significantly higher level of good self-rated health than females (42.3%). Residence, body mass index (BMI), self-reported income, smoking, drinking, exercise, and social activity were the factors that influenced SRH reported by male and female respondents, with age, marital status and education reaching the significance level only in women. The Fairlie decomposition model can explain the underlying reasons for 86.7% of the gender differences in SRH, with self-reported income (15.3%), smoking (32.7%), drinking (42.5%), exercise (17.4%), social activity (15.1%) and education (-14.6%) being the major factors affecting gender differences in SRH. CONCLUSIONS: The study results can help promote the implementation of the Healthy China Initiative, inform intervention measures, and offer new proposals on creating policies for older adults' health issues by the Chinese government to improve health equity.

Small-Molecule Photoacoustic Imaging Probe with Aggregation-Enhanced Amplitude for Real-Time Visualization of Acute Kidney Injury.

Acute kidney injury (AKI) has become a growing issue for patients with the extensive use of all kinds of drugs in clinic. Photoacoustic (PA) imaging provides a noninvasive and real-time imaging method for studying kidney injury, but it has inherent shortages in terms of high background signal and low detection sensitivity for exogenous imaging agents. Intriguingly, J-aggregation offers to tune the optical properties of the dyes, thus providing a platform for developing new PA probes with desired performance. In this study, a small-molecule PA probe (BDP-3) was designed and synthesized. We serendipitously discovered that BDP-3 can transform into renal clearable nanoaggregates under physiological conditions. The hydrodynamic diameter of the BDP-3 increased from 0.64 ± 0.11 to 3.74 ± 0.39 nm when the content of H2O increased from 40 to 90%. In addition, it was surprising that such a transforming process can significantly enhance its PA amplitude (2.06-fold). On this basis, PA imaging with BDP-3 was applied as a new method for the noninvasive detection of AKI induced by anticancer drugs, traditional Chinese medicine, and clinical contrast agents in animal models and exhibited higher sensitivity than the conventional serum index test, demonstrating great potential for further clinical diagnostic applications.

Omentin1 ameliorates myocardial ischemia-induced heart failure via SIRT3/FOXO3a-dependent mitochondrial dynamical homeostasis and mitophagy.

BACKGROUND: Adipose tissue-derived adipokines are involved in various crosstalk between adipose tissue and other organs. Omentin1, a novel adipokine, exerts vital roles in the maintenance of body metabolism, insulin resistance and the like. However, the protective effect of omentin1 in myocardial ischemia (MI)-induced heart failure (HF) and its specific mechanism remains unclear and to be elucidated. METHODS: The model of MI-induced HF mice and oxygen glucose deprivation (OGD)-injured cardiomyocytes were performed. Mice with overexpression of omentin1 were constructed by a fat-specific adeno-associated virus (AAV) vector system. RESULTS: We demonstrated that circulating omentin1 level diminished in HF patients compared with healthy subjects. Furthermore, the fat-specific overexpression of omentin1 ameliorated cardiac function, cardiac hypertrophy, infarct size and cardiac pathological features, and also enhanced SIRT3/FOXO3a signaling in HF mice. Additionally, administration with AAV-omentin1 increased mitochondrial fusion and decreased mitochondrial fission in HF mice, as evidenced by up-regulated expression of Mfn2 and OPA1, and downregulation of p-Drp1(Ser616). Then, it also promoted PINK1/Parkin-dependent mitophagy. Simultaneously, treatment with recombinant omentin1 strengthened OGD-injured cardiomyocyte viability, restrained LDH release, and enhanced the mitochondrial accumulation of SIRT3 and nucleus transduction of FOXO3a. Besides, omentin1 also ameliorated unbalanced mitochondrial fusion-fission dynamics and activated mitophagy, thereby, improving the damaged mitochondria morphology and controlling mitochondrial quality in OGD-injured cardiomyocytes. Interestingly, SIRT3 played an important role in the improvement effects of omentin1 on mitochondrial function, unbalanced mitochondrial fusion-fission dynamics and mitophagy. CONCLUSION: Omentin1 improves MI-induced HF and myocardial injury by maintaining mitochondrial dynamical homeostasis and activating mitophagy via upregulation of SIRT3/FOXO3a signaling. This study provides evidence for further application of omentin1 in cardiovascular diseases from the perspective of crosstalk between heart and adipose tissue.

The myosin II inhibitor, blebbistatin, ameliorates pulmonary endothelial barrier dysfunction in acute lung injury induced by LPS via NMMHC IIA/Wnt5a/ß-catenin pathway.

Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS), is a severe inflammatory pulmonary process triggered by varieties of pathophysiological factors, among which endothelial barrier disruption plays a critical role in the progression of ALI/ARDS. As an inhibitor of myosin II, blebbistatin inhibits endothelial barrier damage. This study aimed to investigate the effect of blebbistatin on lung endothelial barrier dysfunction in LPS induced acute lung injury and its potential mechanism. Mice were challenged with LPS (5 mg/kg) by intratracheal instillation for 6 h to disrupt the pulmonary endothelial barrier in the model group. Blebbistatin (5 mg/kg, ip) was administrated 1 h before LPS challenge. The results showed that blebbistatin could significantly attenuate LPS-induced lung injury and pulmonary endothelial barrier dysfunction. And we observed that blebbistatin inhibited the activation of NMMHC IIA/Wnt5a/ß-catenin pathway in pulmonary endothelium after LPS treatment. In murine lung vascular endothelial cells (MLECs) and human umbilical vein endothelial cells (HUVECs), we further confirmed that Blebbistatin (1 µmol/L) markedly ameliorated endothelial barrier dysfunction in MLECs and HUVECs by modulating NMMHC IIA/Wnt5a/ß-catenin pathway. Our data demonstrated that blebbistatin could inhibit the development of pulmonary endothelial barrier dysfunction and ALI via NMMHC IIA/Wnt5a/ß-catenin signaling pathway.

Microbial transformation and inhibitory effect assessment of uvaol derivates against LPS and HMGB1 induced NO production in RAW264.7 macrophages.

For the discovery of new pentacyclic triterpenes as a potential anti-inflammatory agent, microbial transformation of uvaol by Penicilium griseofulvum CICC 40293 and Streptomyces griseus ATCC 13273 was investigated. Stereoselective hydroxylation and epoxidation reactions were observed in the biotransformation. Moreover, six new metabolites were isolated and structurally elucidated by HR-ESI-MS and NMR spectrum. All the compounds were evaluated upon the inhibitory effects of nitric oxide (NO) release in RAW 264.7 cells induced by lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1). Among them, compound 3 (13, 28-epoxy-3ß, 7ß, 21ß-trihydroxy-urs-11-ene) with the unique epoxy structure and compound 5 (3ß, 21ß, 24, 28-tetrahydroxy-urs-12-en-30-oic acid), exhibited a considerable inhibitory effect on both models while compound 2 (urs-12-ene-3ß, 7ß, 21ß, 28-tetraol) showed a significant bias in the LPS-induced inflammatory response with IC50 value of 2.22 µM. Therefore, this study could provide some insights on the discovery of the pentacyclic triterpene leads for the treatment of either DAMPs or PAMPs triggered inflammation.

Aminoacylase-1 plays a key role in myocardial fibrosis and the therapeutic effects of 20(S)-ginsenoside Rg3 in mouse heart failure.

We previously found that the levels of metabolite N-acetylglutamine were significantly increased in urine samples of patients with heart failure (HF) and in coronary artery ligation (CAL)-induced HF mice, whereas the expression of its specific metabolic-degrading enzyme aminoacylase-1 (ACY1) was markedly decreased. In the current study, we investigated the role of ACY1 in the pathogenesis of HF and the therapeutic effects of 20(S)-ginsenoside Rg3 in HF experimental models in vivo and in vitro. HF was induced in mice by CAL. The mice were administered Rg3 (7.5, 15, 30 mg · kg-1· d-1, i.g.), or positive drug metoprolol (Met, 5.14 mg · kg-1· d-1, i.g.), or ACY1 inhibitor mono-tert-butyl malonate (MTBM, 5 mg · kg-1 · d-1, i.p.) for 14 days. We showed that administration of MTBM significantly exacerbated CAL-induced myocardial injury, aggravated cardiac dysfunction, and pathological damages, and promoted myocardial fibrosis in CAL mice. In Ang II-induced mouse cardiac fibroblasts (MCFs) model, overexpression of ACY1 suppressed the expression of COL3A1 and COL1A via inhibiting TGF-ß1/Smad3 pathway, whereas ACY1-siRNA promoted the cardiac fibrosis responses. We showed that a high dose of Rg3 (30 mg · kg-1· d-1) significantly decreased the content of N-acetylglutamine, increased the expression of ACY1, and inhibited TGF-ß1/Smad3 pathway in CAL mice; Rg3 (25 µM) exerted similar effects in Ang II-treated MCFs. Meanwhile, Rg3 treatment ameliorated cardiac function and pathological features, and it also attenuated myocardial fibrosis in vivo and in vitro. In Ang II-treated MCFs, the effects of Rg3 on collagen deposition and TGF-ß1/Smad3 pathway were slightly enhanced by overexpression of ACY1, whereas ACY1 siRNA partially weakened the beneficial effects of Rg3, suggesting that Rg3 might suppress myocardial fibrosis through ACY1. Our study demonstrates that N-acetylglutamine may be a potential biomarker of HF and its specific metabolic-degrading enzyme ACY1 could be a potential therapeutic target for the prevention and treatment of myocardial fibrosis during the development of HF. Rg3 attenuates myocardial fibrosis to ameliorate HF through increasing ACY1 expression and inhibiting TGF-ß1/Smad3 pathway, which provides some references for further development of anti-fibrotic drugs for HF.

Steroid hormone-inducible biosensor based on EGFP-tagged and environmental application.

Steroid hormones as a class of emerging organic pollutant and high concern, due to their potential risks for human and environmental. Accurate analytical methods of steroid hormones are necessary in quantifying and monitoring. Biosensor is a promising technique. In this study, though part of 3α-HSD DNA to construct a regulatory plasmid and with the EGFP reporter gene to generate a reporter plasmid. Separately transformed into Escherichia coli strain BL21 and extracted the cell lysates as novel biosensor reagents. Analyzed the total amounts of steroid hormones in water, sediment, and soil samples using biosensor reagents, and compared these results with those obtained by HPLC. In summary, detection method using an EGFP reporter that can detect trace amounts of steroid hormones to reached fg/L. The optimal reaction time range and temperature were 30 min and 30 °C, respectively, while the most suitable organic solvent for the steroid hormone was 100% ethanol, up to 96-well plate format. This method is very suitable for high-throughput detection of environmental steroid hormone pollutants.

Expression Analysis of AUX/IAA Family Genes in Apple Under Salt Stress.

Members of the auxin/indoleacetic acid (Aux/IAA) gene family in plants are primary auxin-responsive genes that play important roles in many aspects of plant development and in responses to abiotic stress. Recently, 33 Aux/IAA have been identified in the apple genome. The biological responses of MdIAAs to salt stress are still unknown. In this study, Malus zumi, Malus baccata, and Malus × domestica 'Fuji' plantlets were subjected to salt stress by supplementing hydroponic media with NaCl at various concentrations. M. zumi showed the strongest salt resistance, followed by 'Fuji', and M. baccata was the most sensitive to salt stress. Tissue-specific expression profiles of MdIAAs were determined by quantitative real-time polymerase chain reaction. When apple plantlets were subjected to salt stress, most of salt-responsive MdIAAs were up-regulated by 1 h, 3 h, and 6 h in roots, shoot tips, and leaves, respectively. Highly expressed MdIAAs in roots, especially for M. zumi, consisted with the salt tolerance of apple rootstocks. Transgenic apple calli were tolerant to salt stress when over-expressed salt-responsive genes, MdIAA8, -9, and -25. These results provide clues about salt resistance in these three Malus species, which helps apple breeding of salt tolerance by genetic transformation.

The Establishment of a Mouse Model of Recurrent Primary Dysmenorrhea.

Primary dysmenorrhea is one of the most common reasons for gynecologic visits, but due to the lack of suitable animal models, the pathologic mechanisms and related drug development are limited. Herein, we establish a new mouse model which can mimic the periodic occurrence of primary dysmenorrhea to solve this problem. Non-pregnant female mice were pretreated with estradiol benzoate for 3 consecutive days. After that, mice were injected with oxytocin to simulate menstrual pain on the 4th, 8th, 12th, and 16th days (four estrus cycles). Assessment of the cumulative writhing score, uterine tissue morphology, and uterine artery blood flow and biochemical analysis were performed at each time point. Oxytocin injection induced an equally severe writhing reaction and increased PGF2α accompanied with upregulated expression of COX-2 on the 4th and 8th days. In addition, decreased uterine artery blood flow but increased resistive index (RI) and pulsatility index (PI) were also observed. Furthermore, the metabolomics analysis results indicated that arachidonic acid metabolism; linoleic acid metabolism; glycerophospholipid metabolism; valine, leucine, and isoleucine biosynthesis; alpha-linolenic acid metabolism; and biosynthesis of unsaturated fatty acids might play important roles in the recurrence of primary dysmenorrhea. This new mouse model is able to mimic the clinical characteristics of primary dysmenorrhea for up to two estrous cycles.

Screening and Identification of Anti-Inflammatory Compounds from Erdong Gao via Multiple-Target-Cell Extraction Coupled with HPLC-Q-TOF-MS/MS and Their Structure-Activity Relationship.

Erdong Gao (EDG), consisting equally of roots of Asparagi Radix and Ophiopogonis Radix, is a well-known traditional Chinese formulation that has been used to treat cough and throat pain for centuries. However, the bioactive components in EDG remain to be elucidated. In this study, a rapid and effective method involving live cell bio-specific extraction and HPLC-Q-TOF-MS/MS was established to rapidly screen and identify the anti-inflammatory compounds of an EDG extract. One hundred and twenty-four components were identified in EDG extract using HPLC-Q-TOF-MS/MS analysis. After co-incubation with 16HBE, HPAEpiCs and HUVECs, which have been validated as the key target cells for pulmonary diseases, sixteen components were demonstrated to exhibit an affinity for binding to them. Furthermore, fifteen components were subsequently verified to exert anti-inflammatory effects on lipopolysaccharide (LPS)-induced 16HBE, HPAEpiCs and HUVECs via inhibiting the release of TNF-α and IL-6, indicating that nine steroidal saponins may possess potential for the treatment of lung-related diseases. Taken together, our study provides evidence that live cell biospecific extraction combined with the HPLC-Q-TOF-MS/MS technique was an efficient method for rapid screening potential bioactive components in traditional Chinese medicines and the structure activity relationship of steroidal saponins in EDG was summarized for the first time.

Endothelial Conditional Knockdown of NMMHC IIA (Nonmuscle Myosin Heavy Chain IIA) Attenuates Blood-Brain Barrier Damage During Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Endothelial detachment contributes to BBB disruption and neurovascular dysfunction, but its regulation in stroke has yet to be clarified. We investigated the function of NMMHC IIA (nonmuscle myosin heavy chain IIA) in the endothelium on BBB breakdown and its potential mechanisms. METHODS: Endothelial conditional knockdown NMMHC IIA (Myh9ECKD) was constructed in vivo and in vitro, and its role was explored in middle cerebral artery occlusion/reperfusion-injured mice and oxygen-glucose deprivation/reoxygenation-injured brain microvascular endothelial cells. The degree of brain injury was analyzed using staining (2,3,5-triphenyltetrazolium chloride, hematoxylin, and eosin) and electron microscopy. BBB breakdown was investigated with leakage of Evans Blue dye and expression of TJs (tight junctions) and MMP (matrix metallopeptidase)-2/9. Transcriptomics for enrichment analysis was adopted to explore the potential downstream signaling pathways of NMMHC IIA involved in middle cerebral artery occlusion/reperfusion-induced BBB dysfunction. RESULTS: NMMHC IIA expression was upregulated in endothelial cells after cerebral ischemia/reperfusion injury. Myh9ECKD mice exhibited improvement in endothelial barrier hyperpermeability and TJs integrity stimulated by cerebral ischemia/reperfusion. Blebbistatin (NMMHC II inhibitor) treatment exerted the same effect. Transcriptomics showed that NMMHC IIA was involved in regulating various BBB-related genomic changes in the middle cerebral artery occlusion/reperfusion model, and NMMHC IIA was confirmed to significantly modulate Hippo and peroxisome proliferator-activated receptor gamma/nuclear factor-kappa B signaling pathways, which are closely related to BBB damage. CONCLUSIONS: Our findings provide some new insights into how NMMHC IIA contributes to maintaining the integrity of the cerebral endothelial barrier. NMMHC IIA could be a potential therapeutic target for ischemic stroke.