Effect of pioglitazone on glucose metabolism and PPAR-γ expression in lipid-infused rats

AIM: To investigate the effect of pioglitazone (Pio) on glucose metabolism and peroxisome proliferators-activated receptor (PPAR)-γ expression in free fatty acid (FFA) -induced insulin resistance in rats. METHODS: A hyperinsulinaemic-euglycaemic clamp and ［3-3H］-glucose tracing technique were used in awake rats. Glucose metabolism in vivo and PPAR-γ in adipose tissue expression were assessed with elevation FFA by lipid infusion over 4 h in rats pretreated with or without Pio.RESULTS: During steady-state of clamp, there was a significant increase in plasma FFA in two lipid-infused groups, compared to control rats (P<0.01). The glucose infusion rates (GIR) in Pio-treated rats (P/L group), compared with controls, were significantly reduced ［(20.6±0.4) mg·kg^-1·min^-1 vs (33.6±0.6)mg·kg^-1· min^-1, P<0.01］, whereas the GIR was lower in the lipid group (L group) than that in the P/L group［(12.6±0.8) mg·kg^-1·min^-1 vs (20.6±0.4) mg·kg^-1·min^-1, P<0.01］. The hepatic glucose production (HGP) was significantly suppressed (85%) ［(18.3±2.1)mg· kg^-1·min^-1 (basal) vs (2.7±2.4)mg· kg^-1·min^-1, and (17.5±2.6) mg· kg^-1·min^-1 vs (2.6±1.0)mg· kg^-1·min^-1］, all P<0.01 during clamp in control and P/L groups. The suppressive effect of insulin on HGP was significantly blunted in L group［(17.3±2.1)mg· kg^-1·min^-1 vs (15.8±1.5)mg· kg^-1·min^-1］. The rate of glucose disappearance (GRd) was significantly reduced in two lipid-infused rats compared with controls［(26.6±1.6)mg· kg^-1·min^-1 and (23.2±0.9)mg· kg^-1·min^-1 vs (37.7±2.6)mg·kg^-1·min^-1,P<0.01］. The PPAR-γ expression of adipose tissue in P/L group was significantly upregulated. CONCLUSION: Lipid-infusion induces an acute insulin-resistance in vivo. Pio treatment upregulates the PPAR-γ of adipose tissue and suppresses HGP. Pio can protect partly against lipid-induced insulin resistance.     

Protective effect of astragaloside Ⅳ on acute tubular injury induced by aristolochic acid I

AIM: To study the effect of astragaloside Ⅳ (AS Ⅳ) on acute aristolochic acid nephropathy (AAN). METHODS: MTT assay was used to observe the viability of human proximal tubule epithelial cell line HK-2 in vitro. In in vivoexperiments, Kunming mice were intra peritoneally injected with aristolochic acid I (AAⅠ) for 6 d to induce acute AAN model.AS Ⅳ at dose of 50 mg·kg^-1·d^-1 was gavaged for 6 d, and the levels of urine protein, urine γ-glutamyltransferase (γ-GT), serum creatinine (SCr) and blood urea nitrogen (BUN) were measured. The histological changes of the kidneys were observed under microscope by HE and periodic acid-silver methenamine (PASM) staining at the 7th day. RESULTS: The cell viability was significantly inhibited by AA I. However, the cell viability increased when AA I combined with AS Ⅳ was given as compared with control group, indicating that AS Ⅳ plays a dose-dependent protective role in HK-2 cells against the injury of AA I. The results of in vivo experiments showed that the levels of urine protein, urine γ-GT, SCr and BUN were decreased in AA I combined with AS Ⅳ group compared with AA I renal injury group. Histological study showed that AA I-induced kidney injury was improved with the decrease in the area of tubule necrosis and nude tubular basement membrane. CONCLUSION: AS Ⅳ has a protective effect on renal tubular damage induced by AA I.     

Protective effects of multi-enzyme Ⅱ on vascular endothelial cell injury induced by hyperlipidemia serum

AIM and METHODS: The protective effects of multi-enzyme Ⅱ was studied on cultured endothelial cells which was injuried by hyperlipidemia serum. RESULTS: Hyperlipidemia serum increased ICAM-1 expression on the surface of endothelial cells, and decreased NO₂^- release significantly (P＜0.01). ICAM-1 expression could be reduced and NO₂^- release could be enhanced markedly by multi-enzyme Ⅱ (P＜0.01). CONCLUSION: Multi-enzyme Ⅱ had an obvious protective effect on vascular endothelial cells which was injuried by hyperlipidemia seurm. Multi-enzyme Ⅱ could clean out oxide free radicals effectively because it had the acitive structure of both SOD and CAT.     

Protective effect and functional mechanism of curcumin on TNF-α induced neuronal damage in rat hippocampus

AIM: To observe the protective effect of curcumin on TNF-α induced neuronal damage in rat hippocampus and to explore the functional mechanism of curcumin. METHODS: The excitatory postsynaptic potential (EPSP) was recorded in CA1 pyramidal layer of rat hippocampal slices with in vitro brain slices recording techniques. High frequency stimulation was given on Schaffer branches to induce long-term potentiation (LTP). After treated with drugs, the initial slope of EPSP in each group was measured and calculated. RESULTS: Compared to control group, TNF-α and N-methyl-D-aspartate(NMDA) obviously inhibited the LTP in hippocampal slices of rat brain (P<0.05). Curcumin partly recovered the LTP, which was inhibited by TNF-α or NMDA, to near the control level (P>0.05). No effect of TNF-α, NMDA or curcumin on basal synaptic transmission in hippocampal slices was observed. CONCLUSION: Curcumin has protective effect on hippocampal neurons of rats. Curcumin can partly prevent the over-activation of NMDA receptor on neuronic membrane induced by TNF-α and maintain the long-term potentiation in neurons.     

Preventive effect of Sini decoction on expression of TGF-β1 in rat myocardiac fibrosis induced by isoproterenol

AIM: To explore the protective effects of Sini decoction (SD) on myocardial fibrosis induced by isoproterenol (Iso) in rats.METHODS: Nineteen Wistar rats were divided into Iso group, SD treatment group and control group. The rats in Iso group were injected with Iso and were then fed with saline. The rats in SD treatment group were injected with Iso and were then fed with SD. The rats in control group were injected with saline and were then fed with saline. The level of hydroxyproline (Hyp), the contents of angiotensin Ⅱ (AngⅡ) and transforming growth factor beta-1 (TGF-β1) in plasma were measured 4 weeks after administration. TGF-β1 at mRNA and protein levels were measured by the techniques of ELISA and RT-PCR. RESULTS: The plasma levels of TGF-β1 and AngⅡ were lower in control group than those in Iso group and SD treatment group (P<0.05). The plasma levels of TGF-β1 and AngⅡ in SD treatment group were lower than those in Iso group (P<0.05). Compared to Iso group, the cardiac diastolic function was significantly improved in SD treatment group (P<0.05). The results of immunohistochemistry and RT-PCR showed that the mRNA and protein expressions of TGF-β1 were lower in SD group than those in Iso group (P<0.05). CONCLUSION: SD alleviates myocardial fibrosis induced by Iso in rats by decreasing TGF-β1 expression at mRNA and protein levels.     

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.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

Effect of Notch1/Hes1 signaling pathway on proliferation and differentiation of AECⅡ by regulating expression of C/EBPα

AIM: To investigate whether Notch1/Hes1 signaling pathway regulates the expression of CCAAT/enhancer binding protein alpha (C/EBPα), thus affecting the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ). METHODS: Human AECⅡ were cultured in vitro, and randomly divided into control group, activator group (adding Notch pathway activator Jagged1 protein, 500 μg/L) and inhibitor group (adding Notch inhibitor DAPT, 10 μmol/L). AECⅡ in each group were collected after 24 h of intervention. The expression of Notch1, Hes1 and C/EBPα at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell proliferation ability of the AECⅡ was measured by living cell counting and CCK-8 assay. The cell cycle distribution and differentiation of the AECⅡ were analyzed by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly increased in activator group (P<0.05), AECⅡ entered G₂/M phase from S phase, the proliferation of AECⅡ was increased, and the differentiation of AECⅡ was reduced (P<0.05). The mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly reduced in inhibitor group (P<0.05), the cell cycle of AECⅡ cells was arrested in G₀/G₁ phase, the proliferation of AECⅡ cells was reduced, and the differentiation of AECⅡ cells was increased (P<0.05). CONCLUSION: Notch1/Hes1 signaling pathway regulates the expression of C/EBPα and affects the proliferation and differentiation of AECⅡ.     

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.     

Effect of Buyanghuanwu decoction on the expression of ERK2 and CaMKⅡβ in hippocampus tissue and on ability of learning and memory in vascular dementia rats

AIM: To investigate the effect of Buyanghuanwu decoction, a Chinese medicine, on the ability of learning and memory in the rats with vascular dementia (VD) and on the protein expression of extracellular signal-regulated kinase 2(ERK2) and calcium/calmodulin-dependent protein kinase Ⅱβ(CaMKⅡβ) in hippocampus CA1 area.METHODS: The rats were divided into 4 groups: sham group, VD group, VD+Buyanghuanwu decoction group and VD+nimodipine group. The VD rat model was prepared by Pulsinelli's four-vessel occlusion. At 7th day, 14th day or 28th day after operation, the behaviors of the rats were tested by Morris water maze. The morphological changes of the neurons in hippocampus CA1 area were observed by HE staining 30 d after operation. Western blotting was used to observe the protein expression of ERK2 and CaMKⅡβ in the brain tissues of hippocampal CA1 area of the VD rats. RESULTS: Compared with sham group, the pathological changes such as irregular arrangement, coagulation necrosis and obvious deletion in the neurons of hippocampus CA1 area in VD group appeared significantly. The obstacle of learning and memory ability was observed and the protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area was significantly decreased (P<0.05). Compared with VD group, the neurons in hippocampal CA1 area of VD+Buyanghuanwu decoction group and VD+nimodipine group were in eumorphism, lined up in order, and the structure was close to that in sham group. The ability of learning and memory also significantly improved (P<0.05). The protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area significantly increased (P<0.05). CONCLUSION: Buyanghuanwu decoction promotes the protein expression of ERK2 and CaMKⅡβ in hippocampus CA1 area to protect the neurons from injury, builds up the synapses and promotes the ability of learning and memory in VD rats.     

Effect of interfering TGF-β receptor Ⅱ expression on viability,differentiation and apoptosis of NB4 cells

AIM: To evaluate the effect of interfering TGF-β receptor Ⅱ (TβRⅡ) expression on the viability and differentiation of human acute promyelocytic leukemia NB4 cells induced by all-trans retinoic acid (ATRA) and their apoptosis induced by arsenic trioxide (ATO). METHODS: The technique of lentivirus-mediated RNA interference was used to obtain stable NB4 cells with TβRⅡ knockdown, named TβRⅡ-shRNA NB4 cells. CCK-8 assay was used to detect the viability of TβRⅡ-shRNA NB4 cells. The expression level of CD11b was analyzed by flow cytometry, and Wright-Giemsa staining was used to detect the effects of ATRA on the differentiation of TβRⅡ-shRNA NB4 cells. Double staining (Annexin V-FITC/PI) and AO/EB staining were used to detect the effects of ATO on the apoptosis of TβRⅡ-shRNA NB4 cells. RESULTS: The viability of TβRⅡ-shRNA NB4 cells was significantly higher than that of NB4 parental cells. The differentiation was induced in TβRⅡ-shRNA NB4 cells and NB4 parent cells by treatment with ATRA at different concentration (0.01, 0.02, 0.04, 0.08, 0.1 μmol/L) for 96 h. The differentiation rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells in a dose-dependent manner. ATO induced apoptosis of TβRⅡ-shRNA NB4 cells and NB4 parent cells at different concentrations (2, 4 and 8 μmol/L) for 24 h. The apoptotic rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells dose-dependently. At the concentration of 8 μmol/L for 24 h, the apoptotic rates in TβRⅡ-shRNA NB4 cells and NB4 cells were (49.15±2.05)% and (66.85±2.41)%, respectively (P<0.01). CONCLUSION: Down-regulation of TβRⅡ increases the viability of NB4 cells, inhibits NB4 cell differentiation induced by ATRA, and also inhibits apoptosis induced by ATO.     

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.     

The effect of nucleolin on interleukin-1β secretion induced by lipopolysaccharide

AIM: To explore the expression of nucleolin in lipopolysaccharide(LPS)-mediated inflammatory models, and further investigate the role of nucleolin in expression and secretion of LPS-induced interleukin-1β(IL-1β). METHODS: To establish inflammatory models, mice suffered intraperitoneal injection of LPS(15 mg/kg)and RAW264.7 cells were treated with LPS(500 μg/L).Western blotting were applied to identify the expression of nucleolin in these inflammatory models. After over-expression of nucleolin by transient pcDNA3.1-C23 transfection and down-regulation by transient transfection of nucleolin antisense oligonucleotides, the secretion of IL-1β were examined by enzyme-linked immunosorbent assay (ELISA) in LPS-stimulated RAW264.7 cells. RESULTS: Westem blotting assays showed that the 110 kD nucleolin increased in RAW264.7 cells treated with LPS (500 μg/L) and the lung tissues of the mice treated with LPS (15 mg/kg), while the 80 kD component of nucleolin decreased. ELISA showed that LPS-induced IL-1β release in RAW264.7 cells transfected with pcDNA3.1-C23 was higher than that in pcDNA3.1 empty vector transfected cells. LPS-induced IL-1β release in RAW264.7 cells transfected with C23 antisense oligonucleotide was lower than that in normal cells and scramble oligonucleotide transfected cells. CONCLUSION: In LPS-mediated mouse endotoxemia model and LPS-mediated RAW264.7 cell inflammatory model, the expression of 110 kD nucleolin was up-regulated, but 80 kD nucleolin fragment decreased. Nucleolin promoted secretion of LPS-induced IL-1β.     

Effects of aldosterone on expression of angiotensin Ⅱ receptors in cultured rat mesangial cells

AIM: To investigate the effect of aldosterone (ALD) on the mRNA expression of angiotensin Ⅱ (Ang II) type 1 (AT-1a R and AT-1bR) and 2 (AT-2R) receptors in cultured rat mesangial cells (RMCs) treated with high glucose. METHODS: Rat mesangial cells were cultured in high glucose medium containing different concentrations of ALD (10-8-10-6 mol/L). The antagonists of ALD and Ang II receptors including pironolactone (10-7 mol/L, aldosterone receptor antagonist, SPI), losartan (10-7 mol/L, Ang II type 1 receptor blocker, Los) or PD123319 (10-9 mol/L, Ang II type 2 receptor antagonist, PD) were added in the cell culture for 12 h. The control cells were only treated with high (30 mmol/L) or normal (5.6 mmol/L) glucose medium. The viability and proliferation of the RMCs were evaluated by MTT assay. The mRNA expression of AT-1aR, AT-1b R and AT-2R was detected by semi-quantitative RT- PCR. The expression of MCP-1 in cultured RMCs was detected by ELISA. RESULTS: The mRNA expression of AT-1aR, AT-1b R and AT-2R was increased significantly by treatment with ALD in a dose-dependent manner (1.62-1.77, 9.61-9.89 and 7.26-7.35 folds of high glucose control, respectively, P<0.01). SPI significantly reduced the mRNA expression of AT-1aR and AT-1b R (P<0.01) but not affected the mRNA expression of AT-2R. The ratio of AT-1aR/AT-1b R in cultured RMCs treated with high glucose decreased significantly after stimulated with ALD (P<0.01). However, the effect of ALD was inhibited by SPI (P<0.01). Aldosterone treatment induced a significant upregulation of MCP-1 expression in a dose-dependent manner, and previous treatment with spironolactone, losartan or PD123319 abolished this aldosterone-induced MCP-1 expression. CONCLUSION: The results suggest that aldosterone is involved in the inflammatory response by up-regulating the expression of AT-1aR, AT-1bR and AT-2R, changing the proportion of AT-1R subtype, and inducing MCP-1 overproduction in cultured RMCs treated with high glucose.     

Role of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in vascular adventitial fibroblasts

AIM:Effects of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in cultured vascular adventitial fibroblasts. METHODS:Vascular adventitial fibroblasts (VAF)were isolated, cultured from rat thoracic aorta by explant method. ELISA was used to study typeⅠcollagen synthesis and competitive RT-PCR was employed to detect its mRNA expression after angiotensin Ⅱ administration. RESULTS: Angiontensin Ⅱ caused a dose dependent increase of typeⅠcollagen synthesis and its mRNA expression in VAF. CONCLUSION:The results support that angiotensin Ⅱ is an important factor controlling collagen metabolism of VAF and VAF may play an important role in vascular remodelling of hypertension.     

Effects of exogenous cyanide on root physiology and metabolism of peach seedlings北大核心CSCD

【Objective】Peach (Prunus persica) is an economically important fruit crop worldwide. With the increasing demand and the reduced cultivated land acreage of peach, replant problem (also known as replant disease) has become increasingly prominent, and has been causing severe economic losses. Autotoxicity is a special kind of allelopathy, and is considered to be a major factor resulting in the prevalence of peach replant problem. Cyanide (CN-) is a major autotoxin that causes peach replant problem, but the information on physiological and metabolic responses of peach plants under CN- treatment is quite limited. Thus, the specific responsive mechanisms of peach plants to CN- are worthy of in-depth exploration. The study aimed to investigate the effects of CN- treatment on the morphological, physiological, and metabolic parameters in roots of peach seedlings, so as to provide new insights into the response mechanisms of peach plants to CN- treatment. 【Methods】The natural root environment of dead and living trees was investigated in the peach orchard of Huazhong Agricultural University. The effects of exogenous CN- treatment on root growth and seed germination were assessed on peach germinatedseeds (treated with 0, 0.25, 0.5, and 1.0 mmol•L-1 CN-) and lettuce seeds (treated with 0 and 0.5 mmol•L-1 CN- ). The peach root tips treated with 0 and 0.5 mmol • L-1 CN- were subjected to anatomical assessments using paraffin sectioning and staining (including transverse and longitudinal sections). The contents of H2O2 and MDA, as well as the activities of CAT, POD, and SOD from 0 and 0.5 mmol•L-1 CNstressed peach roots were detected. The expression levels of CAT, POD, and SOD-encoding genes were tested using qRT-PCR. To further understand the CN- -induced metabolic changes, peach roots treated with 0 and 0.5 mmol • L-1 CN- for 5 d were subjected to gas chromatography-mass spectrometry (GCMS) analysis. 【Results】The CN- was mainly distributed in 0-40 cm soil layer, and the content was significantly higher in the soil with dead trees than with living trees. The CN- content in soil was predominantly detected adjacent to peach roots, and gradually decreased with the distance from peach roots. The CN- contents showed an upward trend year by year in the bulk soils with the dead and living trees, where the dead trees contained more CN- than the living trees. The CN- contents varied in different size of peach roots, the fine roots (Φ < 5 mm) contained more CN- than the middle size (5 mm ≤ Φ ≤ 10 mm) and the large size roots (Φ > 10 mm). We evaluated the effect of different CN- concentrations (0, 0.25, 0.5, and 1.0 mmol•L-1) on the growth performance of peach germinated-seeds. The result showed that 0.25 mmol • L-1 CN- treatment boosted root growth, while 0.5 and 1.0 mmol • L-1 inhibited root growth with decreased root length and lateral root numbers. The allelopathy sensitivity index indicated that the effects of CN- treatment on peach growth in a concentration-dependent manner, showing low concentration promoted growth but high concentration inhibited it. Additionally, with 0.5 mmol•L-1 CNtreatment, the transverse and longitudinal sections of the root tip showed a severely wrinkled root epidermis, ruptured root cortex cells, and larger intercellular spaces. 0.5 mmol•L-1 CN- treatment also significantly inhibited lettuce seed germination and biomass. The contents of H2O2 and MDA in peach roots significantly increased, and the activities of SOD, POD, and CAT, as well as their respective encoding genes expression, significantly increased with 0.5 mmol•L-1 CN- treatment. The GC-MS analysis showed that 0.5 mmol • L-1CN- treatment dramatically increased contents of numerous amino acids, including proline, glycine, serine, asparagine, alanine, glutamate, GABA etc. Moreover, CN- treatment significantly affected carbohydrate levels in peach roots. 【Conclusion】The CN- contents were associated with the distribution and size of plant roots, and the decomposition of plant residuals. Exogenous CNsupply markedly retarded peach root growth. CN- feeding also gave rise to oxidative stress, reflecting by the increased ROS and MDA levels, and antioxidant enzyme activities. CN- supplementation also induced metabolic reprogramming, displaying a disorder of amino acid and carbohydrates metabolism. © 2022, Office of Journal of the Fruit Science. All right reserved.     

Killing effect and mIFN-γ expression of gene-viral therapeutic system CNHK300-mIFN-γ on malignant tumor cells in vitro

AIM: To investigate the cytotoxicity and mouse IFN-γ (mIFN-γ) expression of oncolytic adenovirus CNHK300-mIFN-γ (CNHK300-Mγ) containing mIFN-γ gene in malignant tumor cells in vitro . METHODS: Human lung cancer cell line A549, human liver cancer cell line SMMC-7721, human pancreatic cancer cell line PANC-1, and human normal fibroblast line BJ were cultured and treated with CNHK300-Mγ, CNHK300, ONYX-015 or AdEasy-mIFN-γ (AdEasy-Mγ). TCID₅₀ assay was used to evaluate the replication ability of CNHK300-Mγ, CNHK300 and ONYX-015 in carcinoma cell lines and normal cell line, and the cytotoxicity was evaluated by cytopathic effect assay and MTT assay. The mIFN-γ expression in the supernatant was detected by ELISA after CNHK300-Mγ or AdEasy-Mγ infection in carcinoma cell lines and normal cell line. RESULTS: The tumor-specific replication ability and cytotoxicity of CNHK300-Mγ were similar to those of CNHK300. The IC₅₀ was as low as MOI of 0.47 pfu/cell for A549 cells, 0.074 pfu/cell for SMMC-7721 cells, 0.532 pfu/cell for PANC-1 cells and was as high as MOI of 281.73 pfu/cell for BJ cells. CNHK300-Mγ was a more powerful killer of malignant tumor cells than ONYX-015 (P<0.01). The tumor cells infected with CNHK300-Mγ efficiently expressed mIFN-γ in vitro and mIFN-γ largely increased as the time prolonged in A549, SMMC-7721 and PANC-1 cells. The mIFN-γ expression in the carcinoma cell lines infected with CNHK300-Mγ was much higher than that in the cells infected with AdEasy-Mγ (P<0.01), but was similar to that in the normal cell line (P>0.05). CONCLUSION: CNHK300-Mγ selectively replicates and effectively promotes the expression of mIFN-γ in carcinoma cells, and specifically kills the tumor cells.     

The effect of rootstock on yield and quality of ‘Mutsu’ apples

Summary

Yield and fruit quality parameters were studied during three years in a rootstock trial with ‘Mutsu’ apple. M.9 provided the highest total yield per tree as well as the highest quantity of intermediate and yellow fruit as compared with J.9, M.26 and B.9. Within similar colour categories, significant differences among rootstocks were also recorded for fruit starch degradation pattern (SDP), firmness, titratable acidity (TA), and soluble solids concentrations (SSC). Generally, the lowest SDP and highest fruit firmness was found in fruits from trees on rootstocks M.26 and B.9. The highest TA was found in fruits from trees on rootstocks M.26 and J.9 and the highest SSC in fruits from trees on rootstocks J.9 and B.9. However, considerable variations among years were also recorded. Correlation coefficients between rootstock and several yield and quality parameters revealed high correlation to SSC for green fruit and TA for medium and yellow fruit and some correlation to yield and number of fruits per tree.     

Effects of immune-activated platelets and LDL on expression and activity of COX-2 and PPAR-α in HUVECs

AIM: To observe the effect of immune-activated platelets and low-density lipoprotein cholesterol (LDL) on the expression and activity of cyclooxygenase-2 (COX-2) and peroxisome proliferator activated receptor α (PPAR-α) in human umbilical vein endothelial cells (HUVECs) treated with activated platelets and LDL. METHODS: The platelets were activated by ADP. The co-culture system of HUVECs with immune activated platelets and/or LDL were established. The activity of COX-2 and expression of PPAR-α at mRNA and protein levels in HUVECs were detected by RT-PCR and Western blotting. The concentration of PGE2 was measured by ELISA for representing the COX-2 activity. The PPAR-α activity was determined by a nuclear factor assay kit. RESULTS: The COX-2 activity and mRNA expression of PPAR-α, the protein levels of COX-2 and PPAR-α and PGE2 concentration in activated platelets group were significant higher than those in un-activated platelets group (all P<0.01). No difference of PPAR-α binding activity was observed between two groups. LDL didnt affect the COX-2 activity and PPAR-α expression, but significantly promoted the stimulating effect of immune-activated platelets. CONCLUSION: Immune-activated platelets significantly promote COX-2 activity and PPAR-α expression in HUVECs, but dont change the PPAR-α binding activity. LDL at general concentration does not affect the expression and activity of COX-2 and PPAR-α, but promote the effect of activated platelets on HUVECs.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.