Effect of high glucose toxicity on JNK pathway,cell viability and apoptosis in pancreatic β-cell line INS-1

AIM: To investigate the effect of high glucose toxicity on JNK pathway and cell function of INS-1 cells.METHODS: Cultured INS-1 cells with or without IGF-1 exposure, were treated with glucose at 3 concentrations (5.6 mmol/L, 11.2 mmol/L and 33.3mmol/L), respectively. MTT was used to measure the cell viability. Apoptosis was determined by immuno-fluorescence and flow-cytometry analysis. The serine 270 phosphorylation of IRS and phosphorylation of JNK in INS-1 cells were detected in the presence or absence of SP600125 treatment.RESULTS: The cell viability decreased and apoptosis increased with elevated glucose concentrations. The percentage of apoptosis cells was 11.3% in 5.6 G group, 12.7% in 11.2 G group and 28.2% in 33.3 G group. There was remarkable increase in apoptosis in 33.3 G group with a 2.49-fold increase to the cells in the basal 5.6 mmol/L glucose. High glucose activated the serine 270 phosphorylation of IRS correlates with JNK phosphorylation in INS-1 cells. Using Western blotting analysis, the levels of JNK phosphorylation were 3.33 fold increased and serine 270 phosphorylation of IRS was 1.17 fold increased in 33.3 G group compared to 11.2 G group (P<0.01). IGF-1 treatment inhibited phosphorylation of JNK and IRS. SP600125 treatment completely blocked JNK phosphorylation in 11.2 G group and reduced JNK phosphorylation by 90% in 33.3 G group. In addition, SP600125 treatment partly reduced serine 270 phosphorylation of IRS by 88.3% in 11.2 G group and 80% in 33.3 G group, the viability of INS-1 cells increased and the apoptosis decreased.CONCLUSION: The toxicity of chronic high glucose, which inhibits the cells viability and induces the cell apoptosis, might be related to suppress IRS signal by activating the JNK pathway. Blocking the JNK pathway might relieve the effect of glucose toxicity to the β cell function by improving the IRS signal pathway.     

Role of ZIPK in the regulatory effects of PKCα and PKCε on calcium sensitivity during hemorrhagic shock in rats

AIM: To observe the role of zipper-interacting protein kinase (ZIPK) in the regulatory effects of protein kinase Cα (PKCα) and protein kinase Cε (PKCε) on calcium sensitivity during hemorrhagic shock(HS) in rats. METHODS: The skinned first class arborization of superior mesenteric artery (SMA) from HS rats were adopted to observe the influence of inhibitor of ZIPK on the effects of PKCα and PKCε agonists on calcium sensitivity after shock via measuring the contraction initiated by Ca2+ with isolated organ perfusion system, hypoxic vascualr smooth muscle cells (VSMCs) were adopted to measure the protein expression and activity of ZIPK after applying PKCα and PKCε agonists following hypoxia via Western blotting. RESULTS: (1) The calcium sensitivity of SMA was decreased after 2 h shock, and increased by agonists of PKCα and PKCε. Emax of Ca2+ was increased from 47.2％to 66.5％ (P<0.01) and 66.3％ (P<0.01) of normal control respectively as compared with 2 h shock group. The increasing effects of PKCα and PKCε agonists on calcium sensitivity of SMA after 2 h shock were weakened by the inhibitor of ZIPK. The cumulative dose-response curve of Ca2+ was shifted to the right, the Emax of Ca2+ was decreased to 42.6％ and 47.5％ of normal control (P<0.01), respectively. (2) The protein expression and activity of ZIPK in VSMCs were decreased after 2 h hypoxia, and were increased by the agonists of PKCα and PKCε following 2 h hypoxia. CONCLUSION: PKCα and PKCε regulate the calcium sensitization probably through changing the protein expression and activity of ZIPK following HS in rats.     

Inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue of mouse with asthma

AIM: To investigate the inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue with asthma. METHODS: The BALB/c mice were divided into four groups: control group (group A, n=20), asthmatic model group (group B, n=20), large-dose ground dragon treatment group (group C, n=20) and low-dose ground dragon treatment group (group D, n=20). To establish a mouse model of chronic asthma, we sensitized the mouse with 0.02% ovalbumin (OVA) by intraperitoneal injection, and stimulated the mice with 1% OVA by atomization. The treatment groups were given ground dragon before stimulation every time. After the last time of stimulation, the mice were subjected to laboratory tests. Inflammatory cells in bronchoalveolar lavage fluid were counted. Total level of IgE in serum was detected by ELISA. FN mRNA and α-SMA mRNA in the lung tissue were measured by RT-PCR and AlphaImager 2200 semi-quantitation analysis system. Expressions of FN and α-SMA were measured by the method of two-step immunohistochemistry and leica QWIN V3 analysis system. RESULTS: (1) Compared with those in group A, the expressions of α-SMA and FN in group B were significantly increased (P<0.01). Compared with group B, those in group C were significantly decreased (P<0.01), while those in group D were slightly decreased (P>0.05). (2) Compared with those in group A, the expression levels of α-SMA mRNA and FN mRNA in group B had a great increase (P<0.01). There was a notably decreases of α-SMA mRNA and FN mRNA levels in group C, compared with group B (P<0.01). However, α-SMA and FN mRNA level in group D was just a slightly decreased, compared with group B (P>0.05). CONCLUSION: The ground dragon inhibits α-SMA and FN expression in the lung tissue of mice with chronic asthma, indicating that ground dragon may inhibit airway remodeling in asthma through the inhibition of α-SMA and FN expressions.     

Effects of miR-92a and miR-19b on insulin gene expression in mouse pancreatic β-cells

AIM To investigate the effect of microRNA-92a （miR-92a） and microRNA-19b （miR-19b） on the insulin expression in mouse pancreatic β-cells. METHODS The relative expression levels of endogenous miR-92a and miR-19b in mouse insulinoma MIN6 cells were detected by qPCR. The MIN6 cells were divided into control group, and experimental groups I and II, with 3 samples in each group, and transfected with negative control miRNA （NC）, miR-92a and miR-19b, respectively. The over-expression of the miRNAs was detected by qPCR. The morphological changes and viability of the cells were detected by optical microscopy and CCK8 assay, respectively. The expression of insulin was detected by qPCR, Western blot and immunofluorescence. The possible mechanisms of miR-92a and miR-19b regulating insulin expression were analyzed by bioinformatics prediction, dual-luciferase reporter assay, and Western blot. RESULTS Compared with the adult pancreatic progenitor cells, the expression of endogenous miR-92a and miR-19b in the MIN6 cells was decreased significantly （P<0.05）. Over-expression of miR-92a and miR-19b had no effect on the viability of MIN6 cells, but inhibited the expression of insulin at mRNA and protein levels. miR-19b significantly inhibited the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1 （P<0.05）. miR-92a had a fine-tuning effect on the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1. CONCLUSION miR-92a and miR-19b inhibit the insulin expression in mouse pancreatic β-cells.     

Inhibitory effect of EGCG on proliferation and HIF-1α/VEGF expression in cell line HepG2

AIM: To study the molecular mechanism of EGCG on inhibiting the growth of hepatic carcinoma. METHODS: The proliferation of hepatic cell line HepG2 cultured with different doses of EGCG was studied by MTT and suspension/adherence methods. The effect of EGCG on the expression of HIF-1α/VEGF at mRNA and protein levels in vitro and in vivo was evaluated by RT-PCR and Western blotting, respectively. The inhibition of EGCG on the growth of tumor implanted into athymic nude mice was also observed. RESULTS: The proliferation of hepatic cell line HepG2 was inhibited by EGCG in a dose-dependent manner. The expression of HIF-1α/VEGF was suppressed markedly by EGCG at protein level. However, the inhibitory effect of EGCG on the mRNA expression was only observed on VEGF, not on HIF-1α. In the animal experiment, the implanted tumor growth was inhibited by 39.8%±5.1%. CONCLUSION: EGCG suppresses the hepatic carcinoma cell growth, and interrupts the HIF-1α/VEGF signaling pathway significantly, indicating a fundamental mechanism of EGCG for inhibiting tumor growth.     

Effects of HIF-1α silencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

AIM: To study the effect of hypoxia-inducible factor 1α(HIF-1α) silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919 under hypoxia. METHODS: Hypoxic condition was induced by CoCl₂ and the expression of HIF-1α was silenced by small interference RNA. HIF-1α-specific RNAi lentiviral vector was constructed. Real-time RT-PCR and Western blotting were used to detect the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF-1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS: Under hypoxic condition, the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells increased significantly (P<0.05). HIF-1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 (P<0.05) in CBRH-7919 cells. In transfected cells, the number of cells in G₀/G₁ phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION: Hypoxia induces the expression of HIF-1α. HIF-1α silencing can regulate the proliferation of hepatoma cells through reducing the expression of Ki67 and increasing the expression of p27.     

The effects of temperature and plant developmental stage on the occurrence of the curd quality defects “bracting” and “riciness” in cauliflower

Summary

Models to simulate the induction of the “bracting” and “riciness” defects in cauliflower were estimated from field experiments. Bracting, where small cauline leaves develop and penetrate the curd surface, is caused by high temperatures – a kind of de-vernalization. Riciness, on the other hand, where small flower buds develop on the curd surface, is caused by low temperatures, especially after a preceding period of high temperature – a kind of strong vernalization. Both bracting and riciness can be induced only at certain stages during plant development. Plots of cauliflower were given periods of different temperature treatments in the field by portable compartments with both cooling and heating units. The treatments were both constant high or low temperatures (24, 18, 13 and 8°C) in 10 d periods, and alternating high and low temperatures (23/23, 23/15, 23/10 and 23/5°C) in 7 d periods. The treatments were started both before and after curd induction. The incidences of bracting and riciness in harvested curds were recorded in 84 plots with different temperature regimes. These data were used to estimate the combined effect of plant development and temperature on quality defects. The responsive developmental stages in the plants were described by a parabolic function, which depends on the apex/curd diameter. The curd diameter with the highest risk of induction of bracting was estimated to be around 12 mm (range 1–23 mm). A combined model of the diameter function and the summation of daily average temperature with a base temperature of 15°C explained 66% of the variation in the data of observed bracting. The apex diameter with the highest risk of induction of riciness was estimated to be around 0.35 mm (range 0.2–0.5 mm). A combined model of sensitive apex diameter and the preceding temperatures (average daily temperature of 10 d), during a temperature drop (average minimum temperature of 10 d), and after (average daily temperature of 6 d) explained 71% of the variation in the data of observed riciness. These models may be used to set up test equipment and to test susceptibility of new cultivars in breeding programmes. On a farm level, the models may be used to forecast risk of quality defects in cauliflower production. Alternatively, the knowledge of how the combination of developmental stage and temperature scenario induce either bracting or riciness may be used to study flower induction by temperature.     

Role of danshensu on TGF-β signal transduction in rat’s hepatic stellate cells

AIM: To investigate the role of danshensu on Smad signal transduction in rat hepatic stellate cells (HSCs) stimulated with transforming growth factor (TGF-β1). METHODS: The rat HSCs was isolated with collagenase by in situ-liver recirculation perfusion and cultured in vitro. MTT colorimetric assay was used to detect proliferation of HSCs treated with different concentration of danshensu. The expressions of α-SMA and TβR I and II were observed by immunocytochemitry, indirect immunofluorescent staining and Western blotting when HSCs stimulated with TGF-β1 and with different concentrations of danshensu for 24 h. RESULTS: (1) Danshensu at the concentration from 0.0625 mmol/L to 1 mmol/L prevented the proliferation of HSCs in a dose-dependent manner (P<0.05). Danshensu also inhibited the proliferation of HSCs induced by TGF-β1 in a dose-dependent manner (P<0.05). (2) At concentration of 0.25 mmol/L, danshensu down-regulated α-SMA protein expression in HSCs with or without stimulation of TGF-β1 (P<0.05), and the activation of HSCs was inhibited also. (3) Danshensu down-regulated the protein expression of TβR I and II in HSCs stimulated with TGF-β1 (P<0.05, or P<0.01), these effects were correlated with the concentration. (4) TGF-β1 increased the mRNA level of Smad2, 3, and 7 in HSCs (P<0.01). Danshensu down-regulated the mRNA level of Smad2, 3 (P<0.05) and up-regulate the mRNA level of Smad7 in HSCs induced by TGF-β1 (P<0.05). CONCLUSION: Danshensu inhibits the activation and proliferation of HSCs through down-regulating the expression of TβR I and II located in cellular membrane of HSCs. Danshensu suppresses the activation of HSCs, and also inhibits the activation of HSCs stimulated by TGF-β1 through up-regulation of Smad7 mRNA and down-regulation of Smad2, Smad3 mRNA expression in HSCs.     

Effect of angiotensin Ⅱ on the remodeling of aorta in spontaneously hypertensive rats

AIM: To investigate the effects of a 10-weeks treatment with angiotensin Ⅱ (Ang Ⅱ) subtype I receptor antagonist losartan on vascular remodeling of thoracic aorta in male spontaneously hypertensive rats (SHR). METHODS: SHR were treated from 16 to 26 weeks of age with losartan at 15 mg/kg·d^-1 or 0.75 mg/kg·d^-1. RESULTS: Losartan (15 mg/kg·d^-1) treatment significantly decreased systolic blood pressure compared with the control group, while losartan (0.75 mg/kg·d^-1) had no the effect, losartan(15 mg) prevents the development of aortic hypertrophy by preventing hypertrophy of vascular smooth muscle cells (VSMC). In the losartan 0.75 group, these parameters were not changed. But in the losartan 15 and losartan 0.75 groups, the collagen content of the aortic media decreased significantly. CONCLUSION: It is inferred that the effect of Ang Ⅱ on stimulating VSMC growth of the aorta in SHR is dependent on arterial pressure, while the effect on collagen fibers is through pressure independent mechanism.     

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 proprotein convertases on transforming growth factor β1-induced HBV replication inhibition

AIM: To study the effect of proprotein convertases (PCs) on the transforming growth factor (TGF) β1-induced inhibition of HBV replication.METHODS: HepG2.2.15 cells cultured regularly were exposed to recombinant TGFβ1 at concentration of 2 μg/L or 5 μg/L and/or PC inhibitor at concentration of 20 μmol/L for 18 h. The total RNA and HBV core particle DNA were extracted from these cells, and PC mRNA and core-associated HBV DNA were detected by real-time PCR technique. RESULTS: The mRNA expression levels of 7 PCs in HepG2.2.15 cells were observed with various degrees. Recombinant TGFβ1 significantly up-regulated the mRNA expression of all PCs except for the down-regulation of PC5/6, though PC1/3 and PC2 were up-regulated most obviously. Furin and PACE4 were the predominant PCs before and after TGFβ1 exposure when the basic mRNA expression was taken into account. Further study showed that TGFβ1-induced the inhibition of HBV replication was abrogated by PC inhibitor in HepG2.2.15 cells. CONCLUSION: TGFβ1-induced the inhibition of HBV replication is mediated by the up-regulation of PCs, which might be of many implications in efficient interferences of TGFβ1 on HBV replication.     

Mini-review: The effects of apples on plasma cholesterol levels and cardiovascular risk – a review of the evidence

Summary

Evidence suggests that a high intake of fruits is associated with a reduced risk of cardiovascular disease (CVD) and lowered plasma cholesterol, but the specific effects of individual types of fruit, fruit fractions, and processed fruit are less well-studied. Apples are among the most frequently consumed fruits, and human and animal studies on apple may help to clarify the effect of this fruit on CVD risk markers. The aim of this mini-review is to summarise current evidence for a lowering effect of apple on the risk of CVD and plasma cholesterol levels, and to investigate whether such an effect is influenced by fruit processing or the form of intake. Possible mechanisms behind the cholesterol-lowering effect of apples are also considered. All relevant published experimental studies in humans and animals were identified within the open literature. Nine human studies were identified, of which four concerned the effects of whole apples, two the effects of dried apples, and three the effects of filtered apple juice. Additional studies considered specific apple components. In general, there was a cholesterol-lowering effect, in the range of 5 – 8%, after the intake of approx. three whole apples, whereas the consumption of apple juice (375 – 720 ml) had no effect on plasma cholesterol levels and may result in adverse effects on plasma triglyceride levels. Limitations in the study designs did not allow us to draw conclusions on the effect of the intake of whole, dried apples (15 – 52 g). We also identified a total of nine experimental studies in animal models. Feeding with apple products resulted in decreased levels of plasma (11 – 43%) and liver (23 – 67%) cholesterol in the majority of studies. There was an increased excretion of bile acids (3 – 56%) and cholesterol (5 – 41%) in rats fed with apple products. Based on the current evidence from human observational and intervention studies, it seems likely that a reduction in plasma total and LDL cholesterol occurs after a dietary intake of apples, which could lead to a decreased risk of CVD. On average, a daily intake of approx. three apples resulted in a decrease in total cholesterol of 5 – 8% (approx. 0.5 mmol l^?1). The consumption of filtered apple juice may result in adverse effects on plasma triglyceride levels. Evidence from animal studies suggests that the major mechanism behind the cholesterol-lowering effect of apples involves an increased clearance of plasma cholesterol due to enhanced faecal excretion of bile acids and cholesterol.     

Legacy effects of past land use on current biodiversity in a low-intensity farming landscape in Transylvania (Romania)

Context

Ecological impacts of past land use can persist for centuries. While present-day land use is relatively easy to quantify, characterizing historical land uses and their legacies on biodiversity remains challenging. Southern Transylvania in Romania is a biodiversity-rich area which has undergone major political and socio-economic changes, from the Austro-Hungarian Empire to two World Wars, communist dictatorship, capitalist democracy, and EU accession—all leading to widespread land-use changes.

Objectives

We investigated whether present-day community composition of birds, plants, and butterflies was associated with historical land use.

Methods

We surveyed birds, plants, and butterflies at 150 sites and classified those sites as forest, arable land, or managed grassland for six epochs using historical maps from the 1870s, 1930s, and 1970s, satellite imagery from 1985 to 2000, and field visits in 2012. Sites were labelled permanent if they had the same land use at all epochs and non-permanent otherwise. We used clustering and PERMANOVA based on community similarity to test for associations between community composition and land-use history.

Results

We found significant differences (p = 0.030) in bird communities between permanent and non-permanent forest sites, and permanent and non-permanent grassland sites (p = 0.051). No significant associations were found among plants or butterflies and land-use history.

Conclusions

Bird communities were associated with historical land use, though plants and butterflies were not. Historical land-use change in our study area was likely not sufficiently intense to cross relevant ecological thresholds that would lead to legacy effects in present-day plant and butterfly communities.