Estrogen ameliorates sepsis-induced vascular hyporeactivity in thoracic aorta of female rats via permissive effect of GRα expression.

OBJECTIVE: Estrogen is correlated to the lower mortality and disease severity of female than that of male, which indicates the potential therapeutic role of estrogen supplement therapy in sepsis. The structure of Daidzein is similar to that of 17ß estradiol (E2), an estrogen in human body, causing the exogenous Daidzein can interact with estrogen receptor as well as E2 in the body. We aim to explore the therapeutic role of estrogen in sepsis-induced vascular dysfunction. Also, we wonder if estrogen regulates blood pressure via glucocorticoid-mediated vascular reactivity. METHODS: Female SD rats received ovariectomy (OVX) to induce estrogen deficiency. After 12 weeks of administration, cecal ligation and puncture (CLP) was used to establish the in vivo model of sepsis. Lipopolysaccharide (LPS) was used to construct the in vitro model of sepsis in vascular smooth muscle cells (VSMCs). E2 and Daidzein were used for estrogen supplement therapy. RESULTS: E2 and Daidzein significantly inhibited inflammation infiltration and histopathological injury in thoracic aorta in the rat model with CLP. E2 and Daidzein improved carotid pressure and vascular hyporeactivity in sepsis rats with OVX. Importantly, E2 and Daidzein promoted glucocorticoid permissive action and increased glucocorticoid receptor α (GRα) expression in thoracic aorta smooth muscle cells. E2 and Daidzein upregulated GRα, and inhibits cytokine production, proliferative phenotype and cell migration in LPS-induced VSMCs. CONCLUSION: Estrogen improved vascular hyporeactivity in thoracic aorta induced by sepsis via permissive effect of GRα expression.

Variable Control and Its Influence Before Urine Sample Analysis in a Field Environment.

Background and Objectives: The aim of the study was to store urine samples at different temperatures and humidity levels and analyze common biochemical test results and point-of-care testing (POCT) indicators according to different storage times and evaluate whether the samples should be centrifuged to study the best storage conditions for urine samples. Methods: Random midstream urine samples (100 mL) were collected from 10 healthy individuals. A portion of the samples was centrifuged. The remaining samples were not centrifuged and were stored under different temperature and humidity conditions for different periods. We measured urine indicators ([Na+], [K+], [Cl-], gamma-glutamyl transpeptidase [GGT], urea, and creatinine [Cr]) at 2, 4, 24, and 72 hours and 7 and 55 days, and we used POCT to measure myoglobin (Mb) and microalbumin (mAlb) concentrations. Results: Centrifugation of urine samples decreased the measured GGT and increased the measured Mb. In urine samples stored at 4°C and room temperature, electrolyte concentrations were scarcely affected by storage time. After storage at 50°C for 24 hours, the measured [Na+] and [Cl-] levels changed. Metabolites (urea and Cr) underwent no obvious change across temperatures. GGT did not change during long-term storage at 4°C. The mAlb level changed significantly only after storage at 4°C. When stored at 4°C, Mb changed little within 4 hours. Under humid conditions, [Na+] and [Cl-] increased significantly after 24 hours, and urea decreased significantly after 7 days of storage. Under dry storage conditions, urinary Cr and GGT decreased, and under humid conditions, these concentrations increased. At high humidity, mAlb increased significantly after 72 hours. Conclusions: Electrolyte and amino acid metabolite concentrations were less affected by storage time at 4°C and room temperature than at other temperatures. Some proteins are sensitive to environmental changes; samples collected for quantification of these proteins can be stored briefly at 4°C after centrifugation. Normal humidity conditions meet most physiological testing requirements.

The Influences of Cryopreservation Methods on RNA, Protein, Microstructure and Cell Viability of Skeletal Muscle Tissue.

Background: Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. Methods: We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN2) snap-freezing, LN2-cooled isopentane snap-freezing, RNAlater®-based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. Results: PCSF preserved RNA integrity better than LN2 and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN2. The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. Conclusion: PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.