The α1-adrenergic receptor is involved in hepcidin upregulation induced by adrenaline and norepinephrine via the STAT3 pathway.

Elevated body iron stores are associated with hypertension progression, while hypertension is associated with elevated plasma catecholamine levels in patients. However, there is a gap in our understanding of the connection between catecholamines and iron regulation. Hepcidin is a key iron-regulatory hormone, which maintains body iron balance. In the present study, we investigated the effects of adrenaline (AD) and norepinephrine (NE) on hepatic hepcidin regulation. Mice were treated with AD, NE, phenylephrine (PE, α1-adrenergic receptor agonist), prazosin (PZ, α1-adrenergic receptor antagonist), and/or propranolol (Pro, ß-adrenergic receptor antagonist). The levels of hepcidin, as well as signal transducer and activator of transcription 3 (STAT3), ferroportin 1 (FPN1), and ferritin-light (Ft-L) protein in the liver or spleen, were assessed. Six hours after AD, NE, or PE treatment, hepatic hepcidin mRNA levels increased. Pretreatment with PZ, but not Pro, abolished the effects of AD or NE on STAT3 phosphorylation and hepatic hepcidin expression. When mice were treated with AD or NE continuously for 7 days, an increase in hepatic hepcidin mRNA levels and serum hepcidin concentration was also observed. Meanwhile, the expected downstream effects of elevated hepcidin, namely decreased FPN1 expression and increased Ft-L protein and non-heme iron concentrations in the spleen, were observed after the continuous AD or NE treatments. Taken together, we found that AD or NE increase hepatic hepcidin expression via the α1-adrenergic receptor and STAT3 pathways in mice. The elevated hepatic hepcidin decreased FPN1 levels in the spleen, likely causing the increased iron accumulation in the spleen.

The Construction and Characterization of Mitochondrial Ferritin Overexpressing Mice.

Mitochondrial ferritin (FtMt) is a H-ferritin-like protein which localizes to mitochondria. Previous studies have shown that this protein can protect mitochondria from iron-induced oxidative damage, while FtMt overexpression in cultured cells decreases cytosolic iron availability and protects against oxidative damage. To investigate the in vivo role of FtMt, we established FtMt overexpressing mice by pro-nucleus microinjection and examined the characteristics of the animals. We first confirmed that the protein levels of FtMt in the transgenic mice were increased compared to wild-type mice. Interestingly, we found no significant differences in the body weights or organ to body weight ratios between wild type and transgenic mice. To determine the effects of FtMt overexpression on baseline murine iron metabolism and hematological indices, we measured serum, heart, liver, spleen, kidney, testis, and brain iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild type and transgenic mice. In conclusion, our results suggest that FtMt overexpressing mice have no significant defects and the overexpression of FtMt does not affect the regulation of iron metabolism significantly in transgenic mice.

[Research on Hemorheology in Rats with Acute Hyperuricemia].

Hyperuricemia is a risk factor for various diseases, but knowledge on acute hyperuricemia is still not sufficient. The present study was aimed to investigate the effect of acute hyperuricemia on red blood cells from hemorheological point of view, and to provide the reference for clinical treatment. The rats were gavaged with 500 mg/kg hypoxanthine and intraperitoneally injected with 100 mg/kg oxonate to induce the model of acute hyperuricemia. The same volume of blood samples were drawn within time period of 0, 1, 2, 3 and 6 h, respectively, from the inner canthus of rats to measure the serum uric acid, hemorheological parameters and the malondialdehyde level. It was found that in each period of 1, 2 and 3 h, the rats had significantly higher levels of uric acid. The integrated deformation index and relax index were increased. The hemolysis rate was significantly reduced. The plasma malondialdehyde level was obviously decreased at the end of 2 h. The results suggested that short-term elevated uric acid could improve the hemorheological parameters and the lipid oxidative level in red blood cells.

Urantide decreases hepatic steatosis in rats with experimental atherosclerosis via the MAPK/Erk/JNK pathway.

Hepatic steatosis, an indicator of atherosclerosis (AS), is always accompanied by inflammatory responses and disturbances in lipid metabolism. The present study investigated the protective effect of urantide, a urotensin II (UII) receptor antagonist, on the liver of rats with AS with hepatic steatosis by regulating the MAPK pathway. AS was induced in rats via an intraperitoneal injection of vitamin D3 and the administration of a high­fat diet. Urantide treatment was then administered to the rats. Pathology, liver index, lipid levels and liver function were measured to determine liver injury. The expression levels of UII and G protein­coupled receptor 14 (GPR14) were determined using immunohistochemistry, reverse transcription­quantitative PCR and western blotting. The expression levels of MAPK­related proteins in hepatocytes from each group were quantified using western blotting and immunofluorescence staining. Rats with AS had typical pathological changes associated with AS and hepatic steatosis, which were significantly improved by urantide treatment. Blood lipid levels, body weight, liver index and liver function were recovered in rats with AS after urantide treatment. Urantide downregulated the expression levels of UII and GPR14 in the livers of rats with AS; concurrently, the phosphorylation of Erk1/2 and JNK was significantly decreased. Moreover, no significant changes were observed in the phosphorylation of p38 MAPK in AS rat livers. In conclusion, urantide inhibits the activation of Erk1/2 and JNK by blocking the binding of UII and GPR14, thereby alleviating hepatic steatosis in rats with AS, ultimately restoring lipid metabolism in the liver and alleviating AS lesions.

Urantide alleviates the symptoms of atherosclerotic rats in vivo and in vitro models through the JAK2/STAT3 signaling pathway.

Atherosclerosis is the leading cause of human death, and its occurrence and development are related to the urotensin II (UII) and UII receptor (UT) system and the biological function of vascular smooth muscle cells (VSMCs). During atherosclerosis, impaired biological function VSMCs may promote atherosclerotic plaque formation. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway is an important mediator of signal transduction; however, the role of this signaling pathway in atherosclerosis and VSMCs remains unknown. This study aimed to investigate the effects of urantide on the JAK2/STAT3 signaling pathway in atherosclerosis. We examined the effect of urantide on the UII/UT system and the JAK2/STAT3 signaling pathway in a high fat diet induced atherosclerosis rat model and studied the effect and mechanism of urantide on the phenotypic transformation of VSMCs. We found that the UII/UT system and JAK2/STAT3 signaling pathway were highly activated in the thoracic aorta in atherosclerotic rats and in ox-LDL- and UII-induced VSMCs. After urantide treatment, the pathological changes in atherosclerotic rats were effectively improved, and the activities of the UII/UT system and JAK2/STAT3 signaling pathway were inhibited. Moreover, urantide effectively inhibited proliferation and migration and reversed the phenotypic transformation of VSMCs. These results demonstrated that urantide may control the JAK2/STAT3 signaling pathway by antagonizing the UII/UT system, thereby maintaining the biological function of VSMCs and potentially preventing and curing atherosclerosis.

Urantide attenuates myocardial damage in atherosclerotic rats by regulating the MAPK signalling pathway.

OBJECTIVE: To explore the effect of urantide on atherosclerotic myocardial injury by antagonizing the urotensin II/urotensin II receptor (UII/UT) system and regulating the mitogen-activated protein kinase (MAPK) signalling pathway. METHODS: Atherosclerosis (AS) was established in rats by administering a high-fat diet and an intraperitoneal injection of vitamin D3. The effect of treatment with urantide (30 µg/kg), a UII receptor antagonist, for 3, 7, or 14 days on AS-induced myocardial damage was evaluated. RESULTS: The heart of rats with AS exhibited pathological changes suggestive of myocardial injury, and the serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased. Additionally, significant increases in the levels of UII, its receptor (G protein-coupled receptor 14, GPR14), p-P38, p-extracellular signal-regulated kinase (ERK) and p-c-Jun N-terminal kinase (JNK) were observed in the heart. Urantide improved pathological changes in the heart of rats with AS and reduced the serum CK and LDH levels. Additionally, the UII antagonist decreased the increased levels of UII, GPR14, p-P38, p-ERK and p-JNK in the heart. CONCLUSIONS: Urantide alleviates atherosclerotic myocardial injury by inhibiting the UII-GPR14 interaction and regulating the MAPK signalling pathway. We hypothesized that myocardial injury may be associated with the regulation of the MAPK signalling pathway.

Quantitative proteomics reveals TMOD1-related proteins associated with water balance regulation.

The distal tubule and collecting duct in kidney regulate water homeostasis. TMOD1 is an actin capping protein that plays an important role in controlling the organization of actin filaments. In this study, we found TMOD1 was specifically expressed in distal tubules and collecting ducts. To investigate the role of TMOD1, we created Tmod1flox/flox mice and bred them with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice, Tmod1flox/flox/Ksp-Cre+ (designated as TFK). As compared with control mice, TFK mice showed oliguria, hyperosmolality urine, and high blood pressure. To determine the mechanisms underlying this phenotype, we performed label-free quantitative proteomics on kidneys of TFK and control mice. Total of 83 proteins were found differentially expressed. Bioinformatic analysis indicated that biological processes, including protein phosphorylation and metabolic process, were involved in TMOD1 regulatory network. Gene set enrichment analysis showed that multiple pathways, such as phosphatidylinositol signaling system and GnRH signaling pathway, were strongly associated with Tmod1 knockout. Western blot validated the down-regulation of three proteins, TGFBR2, SLC25A11, and MTFP1, in kidneys of TFK mice. Our study provides valuable information on the molecular functions and the regulatory network of Tmod1 gene in kidney, as well as the new mechanisms for the regulation of water balance.

Studies on the biomechanical properties of maturing reticulocytes.

We investigated the biomechanical properties of reticulocytes obtained from an animal model of hemolytic anemia induced by antibody injection. The hemorheological indices, membrane viscoelasticity, membrane fluidity, and the secondary structure of membrane proteins of the reticulocytes were monitored continuously during the course of their maturation into erythrocytes. The results indicate that reticulocytes had lower deformability, lower membrane fluidity, greater viscoelastic modulus and lesser proportions of alpha-helices and beta-sheets in protein secondary structures than mature erythrocytes. All these indices approached to the level of normal erythrocytes when reticulocytes transformed during maturation. The results help to enhance our understanding of the biomechanical properties of the reticulocytes in their maturing process with clinical diagnosis significances.

[On the biophysics characteristics of reticulocytes].

This paper reports an in vivo study on the biophysics characteristics of reticulocytes. Anemia was induced by injection of phenylhydrazine in rabbits. The measurements, including electrophoresis rate, hematolytic rate, fluorescent polarization and the changing anisotropic value, were performed in vivo for 72 hours in the process of reticulocytes growing into erythrocytes. It was shown that there were obvious changes in the biophysics characteristics of reticulocytes in this course. Therefore, the findings are of significance to basic, theoretical and clinical studies.

Influence of TRAIL gene on biomechanical properties of the human leukemic cell line Jurkat.

We cloned the cDNA fragment of human TNF-related apoptosis inducing ligand (TRAIL) into RevTet-On, a Tet-regulated and high-level gene expression system. Making use of the TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the biomechanics properties of Jurkat through measuring changes of cellular biomechanics properties before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the TRAIL gene expression led to significant changes in cellular biomechanics properties. The osmotic fragility increased and the cell stiffness increased after the expression of TRAIL gene. Thus, the apoptosis-inducing TRAIL gene caused significant changes in the biomechanics properties of Jurkat cells.

Microrheological characteristics of reticulocyte in vivo.

Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticulocytes, which synchronously grow, were got in vivo. The measurements of deformation index, orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of reticulocytes turning into red blood cells in vivo. There were obvious changes in the microrheological characteristics of reticulocytes in the course of turning into erythrocytes. The present study is significant in clinic for studying erythrocytes' microrheological characteristics when there are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes microrheological characteristics. It makes up a deficiency in the study on microrheological characteristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL.

Using the method of gene transfection with liposome, we obtained the mouse erythroleukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indicated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger osmotic fragility, lower cell surface charge density, increased elastic modulus K (1) which is inversely proportional to cells' maximal deformation ability, obviously diminished surface viscosity mu, with elastic modulus K (2)having no distinct changes. The above results provided some bases for recognizing the function of TFAR19 completely from the viewpoint of biorheology.

[FTIR spectroscopy studies on the apoptosis-promoting effect of TFAR19 on the erythroleukemia cell line MEL].

The changes in the cellular main components of the mouse erythroleukemia cell line MEL for TFAR19 gene transfection were studied by the technology of Fourier transform infrared spectroscopy (FTIR). Using the method of gene transfection with liposome, we obtained MEL-TF19 cell line, which stably carries TFAR19, a novel apoptosis-related gene. The expression of the gene on mRNA level was confirmed by RT-PCR. Then, FTIR spectra of the cells were measured in the course of apoptosis induced by serum deprivation. Our results indicated that after being transfected with TFAR19 gene, MEL-TF19 cells exhibited relatively higher protein content, higher transcriptional activity, and relatively lower phospholipid content as compared with those exhibited by MEL cells. All the above changes reflect the apoptosis-promoting effect of TFAR19 gene, and maybe account for the cellular rheological changes after TFAR19 gene transfection, which were discovered in our previous study.

The effects of non-ionic contrast medium on the hemorheology in vitro and in vivo.

Contrast media are the commonly used agents in radiology. However, because of their characteristics of high osmolality, high viscosity, and chemical toxicity, the administrations of contrast media have been shown to cause adverse effects especially on hemorheology in short time course. The present study is to find the effects of a non-ionic contrast medium, iopromide, on the hemorheology in long time course both in vitro and in vivo. For in vitro treatment, human peripheral blood samples were incubated with contrast medium at 37°C for 0.5, 1 and 2 h. For in vivo study, about 15 ml of contrast medium was injected into rabbits and blood samples were collected at 0.5, 2, 6, and 24 h after the bolus injection. Hemorheological parameters were examined. Results showed that hematocrit adjusted whole blood viscosity increased significantly at 1 h after in vitro treatment of contrast medium, while it decreased at 0.5 h and remained low till 6 h after bolus injection. Ektacytometer showed that erythrocyte deformability decreased to the lowest level at 2 h in vitro and it dropped at 0.5 h and resumed to normal after 2 h in vivo. Erythrocyte small deformation indices were reduced by contrast medium in both in vitro and in vivo studies. Erythrocyte orientation index was also reduced in in vivo study. Erythrocyte electrophoresis rates at all time points decreased but osmotic fragility did not change in both studies. These impaired hemorheological parameters may disturb the microcirculation and cause adverse effects in patients with kidney diseases.

Urantide improves atherosclerosis by controlling C-reactive protein, monocyte chemotactic protein-1 and transforming growth factor-ß expression in rats.

The aim of the present study was to investigate the effects of urantide on the expression status of C-reactive protein (CRP) and the inflammatory cytokines monocyte chemotactic protein (MCP)-1 and transforming growth factor (TGF)-ß in the aortas of rats with atherosclerosis (AS), and to identify its underlying mechanisms. The effects of urantide in a rat model of AS and in cultured rat vascular smooth muscle cells (VSMCs) were analyzed via hematoxylin and eosin staining, immunohistochemical staining and ELISA. The results in vivo demonstrated that urantide downregulated the expression of inflammatory mediators CRP and MCP-1 and upregulated the expression of TGF-ß. The results in vitro indicated that urantide inhibited the proliferation of VSMCs. In addition, urantide reduced the expression of CRP and downregulated the secretion of TGF-ß in the culture supernatant. In conclusion, urantide ameliorated the arterial inflammatory damage that was observed in the AS rat model at the cell and tissue levels by controlling the expression of CRP and the inflammatory cytokines MCP-1 and TGF-ß. Therefore, urantide may be a potential agent for the complementary treatment of AS.

The effects of long term aerobic exercise on the hemorheology in rats fed with high-fat diet.

Hypercholesterolemia is one of the cardiovascular risk factors sensitive to preventive and control interventions. Here we created a hypercholesterolemia model to investigate the effect of the long term aerobic exercise (swimming) on the hemorheology of rats fed with high-fat diet. We found that the rats fed with high-fat diet developed hypercholesterolmia and hepatic steatosis and their hemorheological and coagulative properties were all impaired as compared to those of the rats fed with standard diet. But after exercise, the total cholesterol and triglyceride in the plasma were significantly decreased and the severity of hepatic steatosis were reduced. Exercise greatly improved the erythrocytes' hemorheological properties, including deformability, electrophoretic mobility and osmotic fragility. Exercise also markedly lowered the activated partial thromboplastin time (APTT) but had moderate effects on other coagulative parameters. The high oxidative stress level, as indicated by plasma MDA concentration, in rats with high-fat diet was significantly attenuated to the normal level after exercise. The present study suggests that long term aerobic exercise could remarkably improve the abnormal hemorheological property and the oxidative stress in rats with hypercholesterolemia.

A microfluidics cytometer for mice anemia detection.

The design and fabrication of a microfluidic cytometer system and its application for reticulocyte detection are described. This chip can count the target cells, which are focused at the detection window without sheath flow. This cytometer system based on optimized epifluoresence has a competitive advantage in the signal-to-noise ratio. Induced fluorescence from the reticulocyte binded with antibody is detected by the optical module and then transformed into the electronic signal by a photo multiplier tube. After signal processing, the results are automatically read out by a digital module and displayed on the system. To evaluate this microfluidic cytometer system, experiments employing polystyrene (PS) micro beads and induced reticulocyte of mice anemia are carried out, respectively, and the results illustrate that the microfluidic cytometer system is effective in detecting the reticulocyte.

Biorheological changes of dendritic cells at the different differentiation stages.

The differentiation, maturation and functioning of Dendritic cells (DCs) are dynamic processes. This study investigated the changes of DCs' migration ability and biorheological properties during their differentiation. Transmigration assay showed that, DCs' migration rate was improved significantly as they differentiate (p < 0.05); NSC (Rac1 blocker) treatment could significantly decrease their migration rates (p < 0.05). Confocal images showed that, F-actin uniformly distributed in monocytes; with DC's differentiation, F-actin began to remodel and gather at the site of dendrites; the images presented surface ruffles and uneven sawtooth-like cytoskeletal structures. Fluorescence polarization analysis showed that, membrane fluidity was increased significantly with DC's differentiation (p < 0.05). CD62L was upregulated significantly (p < 0.05) on the third and ninth days. CD2 was upregulated significantly (p < 0.05) until the seventh day. DC's electrophoretic mobility was increased continuously, especially increased significantly from the third day to the fifth day and the final stage (p < 0.05). These results indicate that there are significant changes in the biorheological properties of DCs during their differentiation.

Biomechanical alterations of dendritic cells by co-culturing with K562 CML cells and their potential role in immune escape.

Dendritic cells (DCs), which are potent antigen presenting cells (APCs), are utilized to deliver the signals essential for the initiation of immune responses. In this study, we used an interdisciplinary approach to characterize the effect of K562 cells, a human chronic myeloid leukemia (CML) cell line, on the biomechanical characteristics and immune functions of DCs. When co-cultured with K562 cells, the biomechanical and immunological characteristics of immature DCs (imDCs) and mature DCs (mDCs) were severely impaired compared with controls. The changes include increased membrane viscoelasticity, reorganized cytoskeleton (F-actin), suppressed capability of antigen uptake, transendothelium migration, and activation of naïve T cells. In exploring the mechanisms of these changes, we identified several genes and proteins by microarray analysis and 2D gel electrophoresis. Changes were found in the cytoskeleton-related genes and proteins (such as cofilin1 and profilin1) and matrix-related genes and proteins (such as TIMP1 and MMP9). These findings provide a molecular basis for the biomechanical and immunological changes of DCs in response to K562 and may help to elucidate the mechanism for tumor immune escape.