Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.     

Effect of Yangjing-Zhongyu decoction on in vitro maturation and expression of PDGFA and PDGFRα of mouse oocytes

AIMTo investigate the effects of Yangjing-Zhongyu decoction on the in vitro maturation and expression of platelet-derived growth factor subunit A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) of mouse oocytes. METHODSThe SPF female KM mice were given Yangjing-Zhongyu decoction, and the blood was collected to prepare serum. The serum containing Yangjing-Zhongyu decoction was used to culture immature oocyte-granulosa cell complexes. After the in vitro culture, the oocyte maturation rate, fertilization rate and cleavage rate were observed and calculated, and the expression of PDGFA and PDGFRα in the oocytes at protein and mRNA levels was determined by West?ern blot and real-time PCR. RESULTSYangjing-Zhongyu decoction increased the in vitro maturation rate and fertil?ization rate of the oocytes (P<0.05 or P<0.01), and down-regulated the protein and mRNA expression of PDGFA and PDGFRα (P<0.01). CONCLUSION Yangjing-Zhongyu decoction may promote the in vitro maturation of mouse oocytes by down-regulating the expression of PDGFA and PDGFRα.     

Role of TGF-β1/Smad signaling in angiotensin Ⅱ mediated down-regulation of connexin 43

AIM: To analyze the alterations of angiotensin Ⅱ (Ang Ⅱ), connexin 43 (Cx43), angiotenisin Ⅱ receptor type 1 (AT1) and signaling molecules in the TGF-β1/Smad pathway in different regions of the left ventricular heart tissue for exploring whether Ang Ⅱ regulates Cx43 expression via the TGF-β1/Smad signaling pathway in myocardial infarction (MI) rats. METHODS: MI was induced in 20 male Sprague-Dawley rats by the left anterior descending coronary artery ligation. The rats were then randomized into 2 groups. In the losartan group, 20 mg·kg^-1·d^-1 of losartan were administered for 2 weeks. Heart functions were assessed after surgery and 2 weeks later again following the above treatments. All the rats were sacrificed and relevant molecules, including Ang Ⅱ, AT1, and Cx43 were determined thereafter in diffe-rent areas of the left ventricle. TGF-β1 and its downstream signaling molecules, including Smad 2, Smad 3 and Smad 7, were also detected. RESULTS: In losartan group, both left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs) were smaller, with diminished interventricular septal thickness (IVSd) and left ventricular posterior wall depth (LVPWd) and distinct improvement of left ventricular ejection fraction (LVEF) (P<0.05). Losartan therapy exhibited a reduction of Ang Ⅱ in the infarct zone and the border zone in the cardiac tissues. AT1 was obviously attenuated in the infarct zone with an enhanced expression of Cx43, which was also elevated in the border zone and none infarct zone. TGF-β1, Smad 2 and Smad 3 were decreased in different zones of the left ventricle, while Smad 7, in contrary to the above factors, presented a converse alteration.CONCLUSION: The activation of Ang Ⅱ provokes downregulation of Cx43 through TGF-β1/Smad signaling pathway in MI rats.     

Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

Effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

Effect of Ca2+/calmodulin-dependent protein kinaseⅡinhibitor KN-93 on late sodium current in rabbits model of heart failure

AIM: To investigate the change of late sodium current (I_NaL) and the effect of Ca²⁺/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on I_NaL in the cardiomyocytes after isoproterenol-induced heart fai-lure (HF) in rabbits. METHODS: The rabbit model of HF was induced by injecting isoproterenol (300 μg·kg^-1·d^-1) for 15 d. One month later, all rabbits received by echocardiography and HE staining to observe the morphological changes of myocardium for evaluating the HF model. The protein expression of Na_V1.5, CaMKⅡδ and phosphorylated CaMKⅡδ was determined by Western blot. The ventricular myocytes were isolated from the rabbits of normal saline (NS) group and HF group by Langendorff perfusion, and the whole-cell patch-clamp technique was used to record I_NaL. RESULTS: Compared with NS group, the heart rate in HF group was increased (P<0.01), the ventricular cavity was enlarged (P<0.05), and the cardiac function was decreased (P<0.01). Compared with NS group, the cardiomyocytes in HF group arranged in disorder, vacuolar degeneration and myocardial interstitial edema were observed, and fibrous tissue increased. The protein levels of Na_V1.5, CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group (P<0.01). I_NaL in HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of I_NaL in HF group and NS group was significantly increased, but that in HF group increased more obviously than that in NS group (P<0.01). After ATXⅡ had induced stable current, we added KN-93 into NS group and HF group, and we found that the ATXⅡ-increased I_NaL in NS group and HF group was significantly decreased (P<0.05).CONCLUSION: CaMKⅡ inhibitor KN-93 inhibits the increase in I_NaL in HF rabbits, which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on I_NaL.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

JAK-STAT pathway modulates NF-кB-mediated cytoprotection of H2O2 preconditioning

AIM: To investigate the role of nuclear factor-kappa B (NF-кB) in the adaptive cytoprotection of H₂O₂ preconditioning and modulation of NF-кB expression by JAK-STAT pathway. METHODS: In PC 12 cells, the experimental model of cytoprotection of H₂O₂ preconditioning against oxidative stress-induced injury was set up. The apoptotic cells were measured by propidium iodide staining and flow cytometry (FCM). The levels of NF-кB and STAT3 expression were detected by Western blotting. RESULTS: Preconditioning with 100 μmol/L H₂O₂ for 90 min significantly inhibited apoptosis induced by 300 μmol/L H₂O₂, and up-regulated expression of NF-кB and STAT3. Both MG-132 (10 μmol/L, an inhibitor of NF-кB) and AG-490 (an inhibitor of JAK2) obviously blocked the expression of NF-кB and cytoprotection induced by H₂O₂ preconditioning. CONCLUSION: JAK-STAT pathway modulates the cytoprotection of H₂O₂ preconditioning that is mediated by NF-кB.     

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

AIM:To investigate the mechanism of angiotensinⅡ (AngⅡ)/angiotensinⅡ type 1 receptor (AT₁R) pathway activating protein phosphatase 2A (PP2A) which leads to down-regulation endothelial nitric oxide synthase (eNOS) phosphorylation level in mesenteric arteries of rats. METHODS:The mesenteric arteries of adult male SD rats (weighing 160~180 g; n=90) were isolated under aseptic conditions. Firstly, to determine the effect of angiotensinⅡ down-regulated eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into normal control (control) group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1×10^-7 mol/L, 1×10^-6 mol/L and 1×10^-5 mol/L for 6 h, 12 h and 24 h, respectively. Secondly, to investigate the molecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan (CAN; a specific AT₁R blocker)+AngⅡ group. The mesenteric arteries were pretreated with 1×10^-5 mol/L CAN for 1 h, then incubated with 1×10^-7 mol/L AngⅡ for 12 h in CAN+AngⅡ group. The protein levels of eNOS, p-eNOS (Ser1177), PP2Ac, p-PP2Ac (Tyr307) and protein phosphatase 2A inhibitor 2 (I₂^PP2A) in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS:Compared with the control group, the protein level of p-eNOS (Ser1177) in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h, 12 h and 24 h (P<0.05). The decreasing tendency of p-eNOS (Ser1177) showed concentration-dependently, especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10^-7 mol/L for 12 h in vitro, the protein levels of p-eNOS (Ser1177) were down-regulated (P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS (Ser1177) (P<0.05); the protein levels of eNOS showed no significant difference in each group. Compared with the control group, the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A were decreased after the mesenteric arteries were treated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group, the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesarten pretreatment inhibited the activity of PP2A significantly (P<0.05). CONCLUSION:AngⅡ increases PP2A activity via AT₁R pathway, thus leading to down-regulation eNOS (Ser1177) phosphorylation level in mesenteric arteries. The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A.     

Average height of surrounding buildings and district age are the main predictors of tree failure on the streets of São Paulo/Brazil

Tree failure is an increasingly frequent issue in cities worldwide leading to the risk of property damage, financial loss, citizen injury, and death. Assessing tree failure is a challenging task since early signs are often not visible and require a detailed evaluation of each tree, which is limiting considering the management of trees across the whole city. We used Regression Trees and Bagging to assess tree failure on the streets of São Paulo / Brazil using parameters from the gray and green infrastructure that could be easily estimated in the field to support the proper preventive maintenance of street trees. We characterized the districts’ age, average building height, tree height, canopy cover, sidewalk width, sidewalk slope, and terrain slope of 26,616 fallen trees. The Regression Tree shows 82% accuracy and reveals that building height is the main predictor of tree failure, followed by district age, sidewalk width, and tree height. The proportion of tree failure in the most verticalized areas, with on average five stories buildings or taller, is twice that observed in the entire city. Tree failure also increases in districts older than 42 years. The proportion of tree failure is 37% lower than the city’s average in relatively newer districts with low building height, where trees taller than 9.58 m are more prone to failure. These results point to possible roles of wind tunneling, shading, pollution, canopy conflicts with service cables in the urban canyons, and the natural senescence of trees in the oldest districts. The present study establishes comprehensive guidelines for effective preventive maintenance of the street trees in São Paulo.     

Research progress of organic solute transporter α/β in bile acid metabolism

The organic solute transporter α/β （OSTα/β） is a recently discovered transporter that controls bile acid secretion into portal blood stream in the basal lateral membrane of intestinal epithelial cells. OSTα/β is a compound composed of 2 subunits, OSTα and OSTβ. Only when the 2 subunits are expressed at the same time, they exist stably and function properly. It is responsible for the transmembrane transport of organic solutes such as bile acids in a way of easy diffusion. OSTα/β is regulated by bile acid receptor, also named as farnesoid X receptor （FXR）. Studies showed that the bile acid synthesis in OSTα deficient mice is decreased, while the bile acid content in the urine is increased. It is worth mentioning that the single gene mutation leads to OSTβ deficiency in the patients with clinical symptoms such as chronic diarrhea and cholestatic liver disease. This paper reviews the structure, function and role of OSTα/β in enterohepatic circulation and the diseases caused by loss of OSTα/β.     

Effect of ERK1/2/c-Fos signal pathway on angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain induced by angiotensin Ⅱ

AIM: To investigate the effect of ERK1/2/c-Fos signal pathway during angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain (GMCS) induced by angiotensin Ⅱ. METHODS: Rat glomerular mesangial cells (GMC) were co-cultured with angiotensin Ⅱ and different doses of angiotensin-(1-7). The numbers of GMC were evaluated by crystal violet staining. The amounts of p-ERK1/2 and c-Fos expressions were detected by Western blotting. RESULTS: Angiotensin- (1-7) showed its inhibitory effects on GMC number increasing induced by angiotensin Ⅱ as well as the amounts of p-ERK1/2 and c-Fos expressions in a concentration dependent manner. CONCLUSION: ERK/c-Fos signal pathway is involved in the inhibitory effects of angiotensin-(1-7) on angiotensin Ⅱ -induced GMC proliferation.     

Effect of tebuconazole-tolerant and plant growth promoting Rhizobium isolate MRP1 on pea–Rhizobium symbiosis

Seed dressing with fungicides adversely affects the structure and function of beneficial soil microbial communities and consequently crop yield. This study was aimed to evaluate the impact of technical-grade fungicide tebuconazole on plant growth promoting potentials of tebuconazole-tolerant Rhizobium isolate MRP1. The performance of the isolate MRP1-inoculated pea plants grown in tebuconazole treated soils was also assessed. Generally, the three concentrations [100 (recommended dose), 200 and 300 μg kg⁻¹ soil] of tebuconazole when used alone, adversely affected the growth, symbiosis, grain yield and nutrient uptake by pea plants. Concentration dependent phytotoxicity of tebuconazole was observed for all the measured parameters. On the contrary, fungicide tolerant Rhizobium sp. MRP1 in the presence of fungicide increased the measured parameters at all tested concentrations. As an example, when inoculant MRP1 was also used with 300 μg tebuconazole kg⁻¹ soil, it substantially increased the root nitrogen, shoot nitrogen, root P, shoot P, seed yield and grain protein by 20, 19, 50, 31, 15 and 7%, respectively, when compared with uninoculated plants grown in fungicide-treated soils. The study suggests that the plant growth promoting Rhizobium sp. MRP1 can be used as bacterial inoculant to increase the production of pea in soils polluted with fungicides.