[Practical exploration on the Online Four-step Teaching of Encouraging and Sharing during COVID-19 period].

The "Four-step Teaching of Encouraging and Sharing" is a learner-centered teaching method that advocates teamwork and gives full play to the role of the teacher in guiding learning. It is an innovative teaching approach to realize students' self-transcendence by stimulating students' internal motivation for independent learning, applying group task-driven learning, and giving teachers' feedback to students' sharing. It consists of four steps: teachers' guiding, students' self-regulated learning, team learning and practice, experience sharing. We have applied this method to the teaching practice of physiology and experimental physiological science with a significant impact on teaching effects. This teaching method has also been implemented to other courses in other majors. To solve the problems of reduced communication and interaction, low learning enthusiasm and motivation in online teaching course during COVID-19 pandemic, we recruited 21 undergraduates from different schools and majors. Using the "Tencent Meeting" platform, the authors tried to apply the whole process of the "Four-step Teaching of Encouraging and Sharing" to the online teaching of physiology. Group tests and questionnaires were used to evaluate teaching effects. The results showed that the implementation of the "Online Four-step Teaching of Encouraging and Sharing (OFST)" was feasible and effective, and to a certain extent alleviated the problems of loneliness and low learning motivation of students during online learning caused by home quarantine, which was particularly helpful for long-distance inter-school and inter-discipline team learning.

Preparedness of medical education in China: Lessons from the COVID-19 outbreak.

The COVID-19 outbreak can be seen as a 'big test' for China; a summative assessment of its preparedness on multiple fronts, including medical education. Being intimately involved in the coordinated response, the First Affiliated Hospital of Sun Yat-sen University has been a first-hand witness to the strengths and weaknesses of the current medical education system in China. On the one hand, we believe that the distinguished contributions in disease containment efforts by healthcare professionals indicated that our medical education system has achieved its intended outcomes and is socially accountable. On the other hand, we have also identified three major issues that need to be addressed from an educational standpoint: insufficient emphasis on public health emergency preparedness; unsophisticated mechanisms for interdisciplinary cooperation; and inadequate guidance in medical ethics. Whilst these reflections might be seen in its summative form, we would suggest changing it to that of a formative process, where we learn from our assessment through observation and feedback of the gaps, upon which improvement of our present situation can be made. We hope that these lessons may be helpful to our colleagues in the rest of China and around the world, who are engaged in medical educational reform.

Oestrogen Inhibits VEGF Expression And Angiogenesis In Triple-Negative Breast Cancer By Activating GPER-1.

Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer with ample vascularisation and high vascular endothelial growth factor (VEGF) expression. The sex steroid hormone oestrogen is involved in several cellular activities associated with TNBC regulation. However, the role of oestrogen in VEGF expression and angiogenesis in TNBC remains unclear. In this study, we found that treatment with 17ß-oestradiol (E2) inhibited VEGF mRNA and protein expression in the TNBC cell lines MDA-MB-468 and MDA-MB-436. To further elaborate on the phenomenon of E2-regulated angiogenesis, we showed that conditioned medium from the TNBC cell line MDA-MB-468 treated with E2 inhibits the tube formation ability of human umbilical vein endothelial cells (HUVECs). Additionally, the G-protein-coupled oestrogen receptor-1 (GPER-1)-specific agonist G-1 has a function similar to that of E2. While G-15, the selective antagonist of GPER-1, notably reversed the inhibitory effects of E2 and G-1 on VEGF expression and tube formation, suggesting that GPER-1 is involved in the E2-induced angiogenesis suppression in TNBC cells. Moreover, E2 inhibited in vivo tumour growth and angiogenesis and reduced the expression levels of VEGF, NF-κB/p65, STAT3, and the endothelial marker CD34 in MDA-MB-468 xenograft tumours. Our findings provide important evidence that E2 can inhibit VEGF expression and angiogenesis in TNBC by activating GPER-1, offering additional insight into tumour angiogenesis and targets for drug intervention in TNBC.

[Advances of the Role of Ezrin in Migration and Invasion of Breast Cancer Cells].

Ezrin, also known as cytovillin or vilin 2, is one of the members of ERM (Ezrin/Radixin/Moesin) protein family. Ezrin, which is a tyrosine kinase substrate, functions to bridge membrane proteins and the actin cytoskeleton. Recent studies have demonstrated that Ezrin regulates the proliferation, apoptosis, adhesion, invasion, metastasis and angiogenesis of breast cancer cells. These processes are not only associated with changes in expression level and subcellular localization of Ezrin itself, but also influenced by alteration in microenvironment of primary breast cancer cells. The regulation of Ezrin in mammary carcinoma cells involves interactions among signaling pathways mediated by adhesion molecules (CD44, ICAM, E-cadherin) and the tyrosine kinase growth factors, Epidermal Growth Factor (EGF), and Platelet-derived Growth Factor (PDGF) and their receptors. The determination of the functions and mechanism(s) of action of Ezrin in the migration and invasion of breast cancer cells will provide new information on the basic mechanisms of metastasis of breast cancer cells and has the potential to identify a novel drug target for the prevention and treatment of breast cancer. This article addresses the role of Ezrin in the migration and invasion of breast cancer cells.

Oestrogen exerts anti-inflammation via p38 MAPK/NF-κB cascade in adipocytes.

BACKGROUND: Oestrogen has anti-inflammatory property in obesity. However, the mechanism is still not defined. OBJECTIVE: To investigate the effect of oestrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1) production in adipocytes. METHODS: Lipopolysaccharides (LPS) was used to imitate inflammatory responses and monocyte chemotactic protein-1 (MCP-1) was selected as an inflammatory marker to observe. 17ß-Estradiol (E2), SB203580 (SB), pyrrolidine dithiocarbamate (PDTC), pertussis toxin (PTX), wortmannin (WM), p65 siRNA and p38 MAPK siRNA were pre-treated respectively or together in LPS-induced MCP-1. Then p38 MAPK and NF-κB cascade were silenced successively to observe the change of each other. Lastly, oestrogen receptor (ER) α agonist, ERß agonist and ER antagonist were utilised. RESULTS: LPS-induced MCP-1 largely impaired by pre-treatment with E2, SB, PDTC or silencing NF-κB subunit. E2 inhibited LPS-induced MCP-1 in a time- and dose-dependent manner, which was related to the suppression of p65 translocation to nucleus. Furthermore, LPS rapidly activated p38 MAPK, while E2 markedly inhibited this activation. It markedly attenuated LPS-stimulated p65 translocation to nucleus and MCP-1 production by transfecting with p38 MAPK siRNA or using p38 MAPK inhibitor. The oestrogen's inhibitory effect was mimicked by the ERα agonist, but not by the ERß agonist. The inhibition of E2 on p38 MAPK phosphorylation was prevented by ER antagonist. CONCLUSIONS: E2 inhibits LPS-stimulated MCP-1 in adipocytes. This effect is related to the inhibition of p38 MAPK/NF-κB cascade, and ERα appears to be the dominant ER subtype in these events.

Inhibitory Effects of Hydrogen on Proliferation and Migration of Vascular Smooth Muscle Cells via Down-Regulation of Mitogen/Activated Protein Kinase and Ezrin-Radixin-Moesin Signaling Pathways.

Molecular hydrogen (H2) has recently attracted considerable attention for the prevention of oxidative stress-related vascular diseases. The purpose of this study is to evaluate the effects of hydrogen on proliferation and migration of vascular smooth muscle cells (VSMCs) stimulated by angiotensin II (Ang II) in vitro, and on vascular hypertrophy induced by abdominal aortic coarctation (AAC) in vivo. Hydrogen-rich medium (0.6~0.9 ppm) was added 30 min before 10⁻7 M Ang II administration, then the proliferation and migration index were determined 24 h after Ang II stimulation. Hydrogen gas (99.999%) was given by intraperitoneal injection at the dose of 1 ml/100 g/day consecutively for one week before AAC and lasted for 6 weeks in vivo. Hydrogen inhibited proliferation and migration of VSMCs with Ang II stimulation in vitro, and improved the vascular hypertrophy induced by AAC in vivo. Treatment with hydrogen reduced Ang II- or AAC-induced oxidative stress, which was reflected by diminishing the induction of reactive oxygen species (ROS) in Ang II-stimulated VSMCs, inhibiting the levels of 3-nitrotyrosine (3-NT) in vascular and serum malondialdehyde (MDA). Hydrogen treatment also blocked Ang II-induced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK, c-Jun NH2-terminal kinase (JNK) and the ezrin/radixin/moesin (ERM) in vitro. Taken together, our studies indicate that hydrogen prevents AAC-induced vascular hypertrophy in vivo, and inhibits Ang II-induced proliferation and migration of VSMCs in vitro possibly by targeting ROS-dependent ERK1/2, p38 MAPK, JNK and ERM signaling. It provides the molecular basis of hydrogen on inhibiting the abnormal proliferation and migration of VSMCs and improving vascular remodeling diseases.

Caveolin-3 is involved in the protection of resveratrol against high-fat-diet-induced insulin resistance by promoting GLUT4 translocation to the plasma membrane in skeletal muscle of ovariectomized rats.

Insulin resistance is recognized as a common metabolic factor which predicts the future development of both type 2 diabetes and atherosclerotic disease. Resveratrol (RSV), an agonist of estrogen receptor (ER), is known to affect insulin sensitivity, but the mechanism is unclear. Evidence suggests that caveolin-3 (CAV-3), a member of the caveolin family, is involved in insulin-stimulated glucose uptake. Our recent work indicated that estrogen via ER improves glucose uptake by up-regulation of CAV-3 expression. Here, we investigated the role of CAV-3 in the effect of RSV on insulin resistance in skeletal muscle both in vivo and in vitro. The results demonstrated that RSV ameliorated high-fat-diet (HFD)-induced glucose intolerance and insulin resistance in ovariectomized rats. RSV elevated insulin-stimulated glucose uptake in isolated soleus muscle in vivo and in C2C12 myotubes in vitro by enhancing GLUT4 translocation to the plasma membrane rather than increasing GLUT4 protein expression. Through ERα-mediated transcription, RSV increased CAV-3 protein expression, which contributed to GLUT4 translocation. Moreover, after knockdown of CAV-3 gene, the effects of RSV on glucose uptake and the translocation of GLUT4 to the plasma membrane, as well as the association of CAV-3 and GLUT4 in the membrane, were significantly attenuated. Our findings demonstrated that RSV via ERα elevated CAV-3 expression and then enhanced GLUT4 translocation to the plasma membrane to promote glucose uptake in skeletal muscle, exerting its protective effects against HFD-induced insulin resistance. It suggests that this pathway could represent an effective therapeutic target to fight against insulin resistance syndrome induced by HFD.

E2-BSA activates caveolin-1 via PI3K/ERK1/2 and lysosomal degradation pathway and contributes to EPC proliferation.

BACKGROUND: The mechanism that estrogen (E(2)) increases the number of endothelial progenitor cells (EPC) is largely unknown. Here we used E(2)-conjugated bovine serum albumin (E(2)-BSA, membrane impermeable) to investigate whether the membrane estrogen receptor (mER) and its related protein caveolin-1 (CAV-1) are involved in these processes. METHODS AND RESULTS: E(2)-BSA promoted [(3)H]-thymidine incorporation of EPC through increasing CAV-1 expression via mER (ERα, but not ERß or GPR30). Both cholesterol depletion and CAV-1 knockdown with use of CAV-1 siRNA significantly attenuated E(2)-BSA-induced [(3)H]-thymidine incorporation. Western blot showed that E(2)-BSA increased membrane CAV-1 protein expression 12h after treatment, whereas mRNA levels of CAV-1 were augmented until 24h after E(2)-BSA treatment. Furthermore, pre-incubated EPC with ICI 182780 (a specific ER antagonist), LY 294002 (a selective PI(3)K inhibitor) or PD 98059 (a specific ERK1/2 inhibitor) before E(2)-BSA inhibited the late-stage effect of E(2)-BSA (≥24 h) on up-regulation of CAV-1 mRNA and protein expression. Pulse chase results demonstrated that E(2)-BSA inhibited lysosome-mediated degradation of CAV-1 protein at the early stage (≤12 h), and then resulted in the increased CAV-1 protein. CONCLUSION: In the present work we demonstrated that E(2)-BSA promotes EPC proliferation through mER (ERα) in CAV-1-dependent manner: prolonging the stability of CAV-1 protein through quick inhibition of the lysosomal degradation pathway at the early stage (≤12 h) and up-regulating CAV-1 at transcription levels through PI(3)K/ERK1/2 signaling pathway at the late stage (≥24 h). These data indicated that a there is a novel mechanism of E(2)-BSA in the regulation of EPC proliferation through CAV-1.

17 beta-estradiol attenuates pressure overload-induced myocardial hypertrophy through regulating caveolin-3 protein in ovariectomized female rats.

Our findings indicate that in ovariectomized female rats abdominal aortic constriction led to significant increases in left ventricular mass, myocyte diameter and heart weight/body weight (HW/BW) value, and decreases in interventricular septal thickness at diastole (IVSd), left ventricular percent fractional shortening (FS) and ejection fraction (EF). These pathophysiological alterations were largely reversed by administration with 17ß-estradiol for eight weeks. Furthermore, the enhanced expression of extracellular signal-regulated kinases 1/2 and decreased expression of caveolin-3 were found in left ventricle of AAC group. 17ß-estradiol (E(2)) administration increased the expression of caveolin-3 and reduced the level of ERK phosphorylation in these pressure-overloaded rats. Moreover, in cultured neonatal rat cardiomyocytes, E(2) inhibited the hypertrophic response to angiotensin II. This effect was reinforced by the addition of extracellular signal-regulated kinases 1/2 inhibitor PD98059, but was impaired when the cells were pretreated with caveolae disruptor, methyl-ß-cyclodextrin (M-ß-CD). In conclusion, our data indicate that estrogen attenuates the hypertrophic response induced by pressure overload through down-regulation of extracellular signal-regulated kinases 1/2 phosphorylation and up-regulation of caveolin-3 expression.

[Role of caveolin-1 on membrane estrogen receptor mediated proliferation of endothelial progenitor cells].

OBJECTIVE: To investigate the potential role of caveolin-1 (CAV-1) on membrane estrogen receptor (mER) mediated proliferation of endothelial progenitor cells (EPCs). METHODS: Bone marrow (BM)-derived EPCs were cultured. The proliferation of EPCs induced by estradiol (E2)-BSA in the absence or presence of ICI 182, 780 (a pure ER inhibitor), MßCD and CAV-1 siRNA was determined by [³H]-thymidine incorporation. The expression of CAV-1 was detected by Western blot. RESULTS: Proliferation of EPC peaked after 10(-8) mol/L E2-BSA culture for 24 h (87.5% increase vs. control), and this effect could be inhibited by estrogen receptor blocker ICI 182, 780, indicating that mER-initiated membrane signaling pathways was involved in the proliferation effect of estrogen on EPC. Both cholesterol depletion and CAV-1 siRNA significantly attenuated E2-BSA induced [³H]-thymidine incorporation. Western blot result confirmed that cholesterol depletion or CAV-1 siRNA significantly decreased CAV-1 protein expression (-18.6% or -41.2% vs. 10(-8) mol/L E2-BSA alone). CONCLUSION: Our results suggested that estradiol promoted EPC proliferation through activating CAV-1 pathway.

Effect of slow abdominal breathing combined with biofeedback on blood pressure and heart rate variability in prehypertension.

OBJECTIVE: Prehypertension is a new category designated by the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7) in 2003. Managing prehypertension with nonpharmacological intervention is possibly beneficial to the prevention of hypertension. In this study, we observed the effect of slow abdominal breathing combined with electromyographic (EMG) biofeedback training on blood pressure (BP) in prehypertensives and assessed the changes of heart rate variability (HRV) in order to find an optional intervention to prevent hypertension and acquire some experimental data to clarify the underlying neural mechanism. METHODS: Twenty-two (22) postmenopausal women with prehypertension were randomly assigned to either the experiment group or the control group. The experiment group performed 10 sessions of slow abdominal breathing (six cycles/min) combined with frontal electromyographic (EMG) biofeedback training and daily home practice, while the control group only performed slow abdominal breathing and daily home practice. BP and HRV (including R-R interval and standard deviation of the normal-normal intervals [SDNN]) were measured. RESULTS: Participants with prehypertension could lower their systolic blood pressure (SBP) 8.4 mm Hg (p < 0.001) and diastolic blood pressure (DBP) 3.9 mm Hg (p < 0.05) using slow abdominal breathing combined with EMG biofeedback. The slow abdominal breathing also significantly decreased the SBP 4.3 mm Hg (p < 0.05), while it had no effect on the DBP (p > 0.05). Repeated-measures analyses showed that the biofeedback group + abdominal respiratory group (AB+BF) training was more effective in lowering the BP than the slow breathing (p < 0.05). Compared with the control group, the R-R interval increased significantly during the training in the AB+BF group (p < 0.05). The SDNN increased remarkably in both groups during the training (p < 0.05). CONCLUSIONS: Slow abdominal breathing combined with EMG biofeedback is an effective intervention to manage prehypertension. The possible mechanism is that slow abdominal breathing combined with EMG biofeedback could reduce sympathetic activity and meanwhile could enhance vagal activity.

Modulatory role of estradiol in nicotinic antinociception in adult female rats.

AIMS: Differences in the response to nicotinic analgesia in males and females have been suggested by recent studies, and such differences are presumed to be due to the regulatory effects of gonadal hormones. The aim of this study was to investigate nicotinic antinociception and the effect of estradiol (E2) on this response in female rats. MAIN METHODS: Ovariectomized female rats were implanted with subcutaneous silastic tubes containing E2. On day 28 after implantation, epibatidine, a high-potency nicotinic acetylcholine receptor (nAChR) agonist, was administered intrathecally, and antinociception at the spinal level was assessed by the tail-flick test. In addition, immunohistochemical staining for nAChRalpha4 was performed in spinal cord sections. KEY FINDINGS: We found that female rats showed shorter nociceptive latencies than males, but there was no effect of ovarian status. However, OVX significantly increased epibatidine-induced antinociception compared to that in intact females, and this increase was attenuated by E2 treatment. In addition, OVX resulted in increased nAChRalpha4 immunostaining in the dorsal horn compared to that in intact females, and this increase was also attenuated by E2 treatment. SIGNIFICANCE: Results of this study provide new evidence that E2 modulates epibatidine-induced antinociception at the spinal level in female rats.

Non-genomic effects of 17beta-estradiol in activation of the ERK1/ERK2 pathway induces cell proliferation through upregulation of cyclin D1 expression in bovine artery endothelial cells.

OBJECTIVE: Growing evidence indicates that estrogen's non-genomic effects play important roles in cellular functions and backs up the hypothesis of the existence of a membrane estrogen receptor (mER) in a number of cell types, but little is known about the complementary effects between traditional genomic and novel non-genomic effects of estrogen. The aim of the present study was to explore the non-genomic activation of ERK1/2 mitogen-activated protein kinase (MAPK) by 17beta-estradiol (E(2)) through mER and its role in cell proliferation. METHODS: On cultured bovine artery endothelial cells (BAECs) we used the [(3)H]thymidine incorporation assay to evaluate the influence of E(2) on cell proliferation and fluorescence microscopy to show the presence of mER on the cell membrane. Scatchard analysis was performed to identify and characterize the mER on a purified membrane fraction of BAECs. RESULTS: E(2) upregulated cyclin D1 protein expression and enhanced cell proliferation. Inhibition of the MAPK cascade with PD98059 or of G protein with pertussis toxin (PTX) completely abolished the above effects, while the estrogen receptor antagonist tamoxifen attenuated E(2)-dependent upregulation of cyclin D1 and cell proliferation. Accordingly, E(2) rapidly led to ERK1/ERK2 activation, which was prevented by tamoxifen or PTX and was entirely reproduced by membrane-impermeable estradiol-bovine serum albumin conjugate (E(2)coBSA). Immunofluorescent staining with E(2)coBSA-fluorescein isothiocyanate resulted in a punctuate staining pattern of the plasma membrane and Scatchard analysis of the E(2)-binding protein in a purified membrane fraction of BAECs showed that E(2) binds to the membrane fraction with a dissociation constant of 0.2394 nmol/l. CONCLUSION: Our findings showed that E(2) induces cell proliferation through upregulation of cyclin D1 via non-genomic activation of the ERK1/ERK2 pathway mediated by mER and G protein.

Raloxifene plus 17beta-estradiol inhibits proliferation of primary cultured vascular smooth muscle cells and human mammary endothelial cells via the janus kinase/signal transducer and activator of transcription3 cascade.

Long-term use of estrogen replacement therapy increases the risk of breast cancer. Presently, we investigated the effects and mechanisms of Raloxifene, a second generation selective estrogen receptor modulator, plus 17beta-estradiol on the proliferation of primary cultured vascular smooth muscle cells (VSMC) and human mammary endothelial cells (HMEC). Raloxifene plus 17beta-estradiol inhibited angiotensin II-induced VSMC proliferation and rapid phosphorylation of STAT(3); these effects were blocked by AG490, the janus kinase/signal transducer and activator of transcription3 (JAK/STAT(3)) inhibitor. STAT(3) production was not affected. In primary cultured HMEC, immunofluorescence identified the ERbeta subtype, but not the ERalpha subtype, in the nucleus. Raloxifene plus 17beta-estradiol inhibited 17beta-estradiol-induced proliferation of HMEC. Western blot analysis established that Raloxifene attenuated the 17beta-estradiol-induced phosphorylation of STAT(3), and that this effect was blocked by AG490. We conclude that Raloxifene plus 17beta-estradiol inhibits the proliferation of VSMC and HMEC through the JAK/STAT(3) cascade, which in primary cultured HMEC may be implemented through ERbeta.

Effects of caveolin-1 on the 17beta-estradiol-mediated inhibition of VSMC proliferation induced by vascular injury.

Estrogen has a protective effect on the cardiovascular system. Yet the mechanism of how estrogen inhibits vascular smooth muscle cell (VSMC) proliferation after vascular injury and the role of caveolin-1 in this process are not clear. To understand the protection effect of estrogen and caveolin-1, we employed a vascular balloon-injury model. Sixteen New Zealand White rabbits with or without estrogen were tested. 17beta-estradiol is able to inhibit VSMC proliferation in a range from 10(-10)-10(-5) mol/L, with an optimal concentration of 10(-8) mol/L. Estrogen exerted its effect through suppressing the activity of p42/44 MAPK, which can be blocked by tamoxifen. Moreover, in estrogen pretreated cells as well as in common carotid arteries of the balloon injury model, expression of caveolin-1 is enhanced compared to the estrogen-deficient group, as assessed by both western blotting and RT-PCR and morphological studies. Our results showed that the inhibition effect of estrogen in VSMCs is mediated by p42/44 MAPK. Caveolin-1 plays an important role in this protective process.

[Role of NO signal pathway in the inhibitory of 17beta-estradiol on the production of endothelin-1 in vascular smooth muscle cells].

AIM: To investigate the mechanisms of 17beta-estradiol on the production of endothelin-1 in vascular smooth muscle cells. METHODS: After incubation VSMC with various concentrations of 17beta-estradiol (10(-9) - 10(-7) mol/L) or plus L-NAME(10(- 6) mol/L) for different times, the concentration of endothelin-1 was measured. At the same time, the activity of endothelin converting enzyme-1 was analyzed, and the expression of preproET-1mRNA was measured by RT-PCR. RESULTS: In basal conditions, 17beta-estradiol could inhibit the production of endothelin-1 in VSMC, and the action of 17beta-estradiol had nothing to do with the activity of endothelin converting enzyme-1. L-NAME inhibited the effect of 17-estradiol on the production of endothelin-1 in VSMC. RT-PCR results showed that 17-estradiol inhibited the preproET-1 mRNA expression, and whereas L-NAME reversed this action of 17beta-estradiol. CONCLUSION: In basal conditions, 17beta-estradiol decreases the preproET-1 mRNA expression through NO-pathway to inhibit the production of endothelin-1 in cultured VSMC.