Effect of hypercholesterolemia on the ionic currents in cardiac ventricular myocytes of rats

AIM: To determine whether chronic hypercholesterolemia affects ionic currents on cardiac ventricular myocytes of rats. METHODS: Whole-cell patch-clamp technique was used to record the ionic currents in single cardiac myocytes isolated from normal cholesterolemia and hypercholesterolemia rats. RESULTS: In the hypercholesterol group (group Ⅱ), serum total-cholesterol level was significantly higher than that of normal group (group Ⅰ) ［(3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］. The serum triglyceride content of group II was remarkably higher than that of group Ⅰ ［(1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］. In ventricular myocytes of rats, 50% repolarization of action potential duration (APD50) prolonged from (70.86±8.12)ms (group Ⅰ) to (116.16±6.90)ms (group Ⅱ) (n=10 in each group, P<0.01); APD90 prolonged from (95.10±7.27)ms (group Ⅰ) to (144.04±7.39)ms (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of -120 mV, Ik1 increased from (-16.98±4.54) pA/pF(group Ⅰ) to (-19.92±4.08) pA/pF (group Ⅱ) (n=12 in each group, P<0.05); at the test potential of 0 mV, ICa-L decreased from (-8.56±1.29) pA/pF (group Ⅰ) to (-5.24±0.90) pA/pF (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of +60 mV, Ito decreased from (13.20±1.97) pA/pF (group Ⅰ) to (10.30±1.97) pA/pF (group Ⅱ) (n=8 in each group, P<0.05). CONCLUSION: Hypercholesterolemia affects the ionic currents on cardiomyocytes of rats greatly, which may be the ionic mechanism of cardiac toxicity induced by hypercholesterolemia.     

Reduction of inflammatory-related factor expression in experimental acute pancreatitis in Egr-1 knockout mice

AIM: To observe the effects of Egr-1 gene knockout on the expression of inflammatory-related factors in pancreatic tissue in a mouse acute pancreatitis model. METHODS: The experimental pancreatitis was induced by high-dose of cearulein in wildtype mice and Egr-1 knockout mice. The pancreatitis indexes, such as serum amylase, pancreata edema, and myeloperoxidase(MPO) levels in pancreata and lungs were recorded. The mRNA levels of tissue factor(TF), plasminogen activator inhibitor(PAI-1), monocyte chemoattractant protein(MCP-1), Gro-1, IL-6 and ICAM-1 were measured by quantitative PCR. RESULTS: Contrary to wildtype mice, typical pancreatitis was not induced by high-dose cearulein in the Egr-1 knockout mice, not only markedly reduced edema in pancreata and lungs, but decreased MPO levels in lungs as well were found. Furthermore, the mRNA of TF, PAI, MCAP, ICAM-1 and IL-6 in pancreata were significantly decreased in Egr-1 knockout mice. CONCLUSION: The severity of pancreatitis and lung damage is ameliorated in Egr-1 knockout mice stimulated by high-dosage of cearulein, which was probably mediated by decreasing expression of inflammatory-related factors in pancreata, such as TF, PAI, MCP-1, ICAM-1 and IL-6.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.     

Effect of acupuncture on mitophagy of skeletal muscle in rats with exercise-induced skeletal muscle damage

AIM: The effect of acupuncture on mitophagy-related protein expression in skeletal muscle of rats after heavy-load exercise was investigated to explore the role of acupuncture in the repairment of exercise-induced skeletal muscle damage. METHODS: Male adult Sprague-Dawley rats (n=128) were randomly divided into 4 groups:control (C, n=8) group, exercise (E, n=40) group, acupuncture (A, n=40) group, and exercise and acupuncture (EA, n=40) group. The rats in E group and EA group performed an eccentric exercise, and the rats in A group and EA group immediately after exercise received acupuncture treatment. The rats in the latter 3 groups were further divided into 0 h, 12 h, 24 h, 48 h and 72 h sub-groups (n=8), and soleus muscle was collected at each time point. The transmission electron microscopy was used to observe the ultrastructural changes of the mitochondria in skeletal muscle. The content of citrate synthase (CS) was measured by ELISA. The protein expression of skeletal muscle PTEN-induced putative kinase 1 (PINK1), parkin and microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot. RESULTS: After the heavy-load exercise, the mitochondria swelled and accumulated under cell membrane. The number of mitophagosomes was increased, and the content of CS was significantly decreased (P<0.05). The expression of PINK1, parkin and LC3 was significantly elevated (P<0.05). However, the acupuncture intervention after exercise promoted the recovery of mitochondrial ultrastructure, attenuated mitophagolysosome formation, maintained CS content and down-regulated the expression of PINK1, parkin and LC3 (P<0.05). CONCLUSION: Heavy-load exercise causes the damages of mitochondrial structure and function in the skeletal muscle and activates PINK1/parkin pathway to induce excessive occurrence of mitophagy. Acupuncture intervention after exercise is able to alleviate the damage of mitochondria in the skeletal muscle through decreasing the expression of mitochondrial outer membrane protein PINK1, reducing the recruitment of downstream cytoplasmic protein parkin, thereby affecting the combination of LC3 and mitochondria to inhibit the overactivation of mitophagy.     

Effect of Egr-1 gene transfection on renal inflammation in diabetic mice

AIM: To observe the effects of Egr-1 gene transfection on the expression of tumor necrosis factor-α(TNF-α) and intercellular adhesion molecule-1(ICAM-1), and to investigate the role of Egr-1 in the pathogenesis of diabetic nephropathy.METHODS: The diabetic mouse model was established. Ten mice were randomly selected as the diabetic group. The remaining 40 mice were injected with empty plasmid, Egr-1 expression plasmid or Egr-1 siRNA plasmid via the tail vein once a week. The normal control group was also set up. The animals were sacrificed at the end of the 4th week. The renal tissues were harvested. The expressions of Egr-1, TNF-α and ICAM-1 were detected by immunohistochemistry and Western blot. The pathological changes were observed under electron microscope.RESULTS: In diabetic mouse kidney, the expression of Egr-1, TNF-α and ICAM-1 was increased, and irregular thickening of glomerular basement membrane, mesangial expansion and fusion of foot were observed. The change trend was more significant in Egr-1 gene transfection group, and these changes in siRNA plasmid transfection group were obviously reduced compared with diabetes group.CONCLUSION: Egr-1 up-regulates the expression of TNF-α and ICAM-1, and induces mesangial cell proliferation and mesangial extracellular matrix accumulation, which is probably one of the mechanisms of accelerating glomerulosclerosis.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Effect of GLP-1 receptor agonist on lipolysis in adipose tissue of obese mice and its underlying mechanism

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) receptor agonist exendin-4 on white adipose tissue (WAT) and the underlying mechanisms. METHODS: Male C57BL/6J mice (8 weeks) were challenged by high-fat diet for 12 weeks, and were randomly divided into saline group and exendin-4 group. The mRNA expression of sirtuin 1(SIRT1), adipose triglyceride lipase (ATGL), TNF-α and adiponectin of WAT was detected by real-time PCR. 3T3-L1 adipocytes or mouse embryonic fibroblasts cells were treated with exendin-4 for 24 h. The protein levels of SIRT1, ATGL and hormone-sensitive lipase (HSL) were determined by Western blot.RESULTS: Exendin-4 significantly decreased epididymal fat weight, fasting blood glucose and serum triglyceride levels (P<0.05), and reduced body weight and serum TNF-α level. The mRNA expression of SIRT1, ATGL and adiponectin in WAT was all significantly up-regulated by exendin-4, which were contrary to the down-regulation of TNF-α mRNA expression (P<0.05). Exendin-4 promoted the protein expression of SIRT1, ATGL, and HSL in 3T3-L1 adipocytes in a dose-dependent manner. Less lipid droplets with up-regulation of lipolytic protein expression were observed when combined with SIRT1 agonist treatment, which were suppressed by SIRT1 inhibitor. Deletion of SIRT1 led to larger adipocytes with more lipid droplets, and the effect of exendin-4 on the lipolysis disappeared when SIRT1 was deficient.CONCLUSION: Exendin-4 promotes lipolysis in WAT of obese mice via activation of SIRT1.     

Effect of intermedin1-53 on aldosterone-induced cardiac fibroblast proliferation

AIM: To investigate the roles of intermedin_1-53 (IMD_1-53) and exteracellular signal-regulated kinase (ERK) signal pathway in the proliferation of rat cardiac fibroblasts (CFBs).METHODS: Isolated and cultured CFBs from new born SD rats were randomly divided into control group, aldosterone (ALD) groups (at different concentrations) and ALD+IMD groups (IMD at different concentrations). The viability of CFBs was determined by MTT assay. Western blotting was used to observe the effect of IMD on ALD-induced ERK phosphorylation.RESULTS: IMD_1-53 had no significant effect on the proliferation of CFBs in basal state, but inhibited the CFBs growth stimulated by ALD in a concentration (10^-9~10^-7 mol/L)-dependent manner. IMD_1-53 also inhibited ERK phosphorylation stimulated by ALD in a concentration (10^-9~10^-7 mol/L)-dependent manner.CONCLUSION: IMD_1-53 inhibits the proliferation of CFBs by ERK signal pathway.     

Effect of fluvastatin on migration of vascular smooth muscle cells from rats

AIM: To investigate the effect and mechanism of fluvastatin on the migration induced by platelet derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) in cultured vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: PDGF-BB and ET-1 significantly induced VSMCs migration, which was inhibited by pretreatment of VSMCs with fluvastatin (10-9-10-5 mol/L) in a dose-dependent manner, and the peak inhibition rate of migration induced by PDGF-BB and ET-1 was over 86.67%. Fluvastatin also attenuated the increase in ［Ca2+］i induced by PDGF-BB and ET-1, with a peak inhibition rate of 86.76% and 65.32%, respectively. CONCLUSION: PDGF-BB and ET-1 promote migration of VSMCs from SHR．Fluvastatin may have direct inhibitory effects on cell migration induced by PDGF-BB and ET-1. The increase in ［Ca2+］i may acts as intracellular signaling in the migration in response to PDGF-BB and ET-1 in VSMCs.     

钙营养对甜樱桃果实品质形成的影响

研究了生长期喷钙对甜樱桃果实品质形成的影响.结果表明:生长期喷钙能显著提高果实的可溶性固形物含量、极显著地提高果实硬度,对果实的花青素含量和色泽及多酚氧化酶含量均有影响,能有效提高果实品质.     

Effect of ligustrazine on expression of LFA-1 and ICAM-1 in bone marrow of radiation injured mice

AIM: To investigate the effect of ligustrazine on the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in bone marrow and on the mechanism of hematopoietic reconstitution in radiation injured mice.METHODS: The 24 mice (clean class) were randomly divided into 3 groups: normal group, radiation injured group and ligustrazine group. After irradiation by 6.0Gy ［⁶⁰Co］ γ-ray, the radiation injured animals were given normal saline (0.2 mL, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine through gastric tube (0.2 mL, twice a day). The mice in normal group received no treatment. At the 7th, 14th, 21st day after irradiation, the femur were taken and the bone marrow mononuclear cells (BMNCs) suspension were made to culture bone marrow stromal cells (BMSCs). The mRNA and protein expressions of ICAM in BMSCs were assayed by RT-PCR and Western blotting. The expression levels of LFA-1 in BMNCs were evaluated by flow cytometry analysis.RESULTS: In ligustrazine group the expression levels of LFA-1 at the 7th, 14th and 21st days after irradiation were higher than those in radiation injured group (P<0.01 or P<0.05). However, the expression level of ICAM-1 was lower than that in the compared group (P<0.01 or P<0.05).CONCLUSION: Ligustrazine can increase the LFA-1 expression level of BMNCs, decrease the ICAM-1 expression level in BMSCs, indicating that ligustrizine promotes the recovery of hematopoietic cells in bone marrow, then improves the bone marrow microenvironment and enhances hematopoietic reconstitution.     

Effect of OX-LDL and simvastatin on PKC activity and cytosolic free Ca2+ in cultured rat aortic smooth muscle cells

AMI:To clarify whether OX-LDL and simvastatin can induce the changes of PKC activity and cytosolic free Ca²⁺ in rat aortic smooth muscle cells (ASMC). METHODS:PKC activity and cytosolic free Ca²⁺ were measured by its ability to transfer phosphate from [³²P]ATP to lysine-rich histone and flow cytometric analysis after loading with the Ca²⁺dye fluo-3/Am, respectively. RESULTS:OX-LDL increased PKC total activity in a dose-dependent manner and induced translocation of PKC from the cytosolic to membrane, while OX-LDL induced biphasic [Ca²⁺]i responses including the rapid initial transient phase and the sustained phase. When simvastatin was added, the translocation of PKC was markedly decreased and simvastatin did not impair the initial peak response to OX-LDL but significantly reduced the subsequent plateau phase. CONCLUSSION:OX-LDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca²⁺ in ASMC and these two events are closely linked.     

硝酸钙处理对甜瓜幼苗抗冷性的影响

研究了硝酸钙溶液浇施和喷施对甜瓜幼苗生长及低温胁迫下抗冷性指标的影响。研究结果表明,硝酸钙浇施和喷施处理均有助于提高甜瓜幼苗在冷胁迫条件下叶绿素含量、根系活力、POD活性,降低电解质渗漏率和MDA含量,提高幼苗抗冷性,延缓冷害症状的发生。10～15mmol/L浇施和10mmol/L喷施处理优于其他处理。     

Effect of TRPV1 activation on lipopolysaccharide-induced lung inflammatory injury in mice

AIM:To investigate the effects of transient receptor potential cation channel subfamily V member 1 (TRPV1) activation by capsaicin on the inflammation and its underlying mechanisms in lipopolysaccharide (LPS)-induced lung injury in mice. METHODS:A total of 108 specific pathogen-free male ICR mice were randomly divided into 6 groups: normal control group, capsaicin (CAP) control group, capsazepine (CAPZ) control group, endotoxemia group, CAP treatment group and CAPZ treatment group. LPS was intraperitoneally injected 30 min after the subcutaneous injection of CAP or CAPZ. After modeling, the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-10, substance P (SP) and calcitonin gene-related peptide (CGRP) in the lung were measured by ELISA. The expression of Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) in the lung tissue was assessed by Western blotting. The pathological changes of the lung tissue were observed under light microscope. RESULTS:The expression of TNF-α, IL-6, IL-10 and NF-κB in the lung tissues at 3 h, 8 h and 16 h was dramatically higher in endotoxemia group than that in normal control group. Compared with endotoxemia group, the levels of TNF-α, IL-6 and nuclear NF-κB in CAP treatment group at 3 h, 8 h and 16 h were obviously decreased, but the level of IL-10 was increased. The changes of the factors mentioned above in CAPZ treatment group were absolutely adverse to those in CAP treatment group. The levels of SP and CGRP were significantly higher in endotoxemia group and CAP control group than those in normal control group, but those in CAPZ control group were lower. Compared with endotoxemia group, SP and CGRP were markedly increased in CAP treatment group and were obviously decreased in CAPZ treatment group. The level of TLR4 in endotoxemia group was distinctly higher than that in normal control group at 3 h, 8 h and 16 h. However, as compared with endotoxemia group, the expression of TLR4 in CAP treatment group and CAPZ treatment group didn’t change much. At 8 h and 16 h after modeling, the degree of lung damage was also decreased in CAP treatment group as compared with endotoxemia group, while that in CAPZ treatment group was aggravated. CONCLUSION: TRPV1 activation obviously inhibits the increase in TNF-α, IL-6 and NF-κB in the lung tissue of endotoxemia mice, and promotes the increase in the anti-inflammatory factor IL-10, as well as the levels of SP and CGRP, but has no effect on the expression of TLR4.     

苹果施用硝酸钙的效果

通过对苹果树叶面喷和土施试验证明:(1)叶面喷(包括盆栽和田间试验)0.3％Ca液2次,能促进树体生长、增加叶干重、提高树体N、K、Ca等营养元素水平,并明显提高果实产量和品质。在年生育期的前期或后期喷Ca,均能进入果实中,前期进入量高于后期。(2)土施Ca(NO_3)_2,同施用筹N量的尿素相比,叶片中N、P、K、Ca、Fe、Zn等元素含量,均有明显提高,并有增产和提高品质作用。在土施方式中,以Ca(NO_3)_2与有机肥混施为佳,但土施Ca(NO_3)_2对提高果实中Ca含量未显出明显效果。(3)总体效果,土施不如叶喷。     

叶面喷施硼酸对苹果果实硼和钙含量的影响

 以‘富士’苹果为试材，通过幼果期、果实膨大期和果实成熟期叶面喷施硼酸，研究硼对果实硼和钙含量的影响。结果表明：从叶面喷硼对果实吸收硼的短期效果来看，果实发育的不同时期叶面喷施硼酸均促进果实对硼、钙的吸收，并且处理的效果顺序是：幼果期>果实膨大期>果实成熟期；在果实采收时硼含量为：果实膨大期>果实成熟期>幼果期。不同时期叶面喷施硼酸均提高了果肉细胞中水溶态硼、半束缚态硼和束缚态硼的绝对含量，从3种形态硼所占的比例来看，随着果实不断发育，水溶态硼和半束缚态硼比例升高，而束缚态硼比例下降。     

硫酸钙对西瓜枯萎病的防治效果

以硫酸钙(生石膏粉)为材料,在西瓜上进行防治枯萎病试验,结果表明,与不施用硫酸钙的处理相比,施用硫酸钙后防病效果较好,且植株茎粗度、蔓长度、叶面积系数、单果质量、可溶性固形物含量等均有所提高,在试验设计范围内以667m2施用硫酸钙30kg的综合效果最好。     

Effects of cycloastragenol on cardiac fibrosis induced by isoproterenol in mice

AIM: To investigate the effects of cycloastragenol (CAG) on cardiac fibrosis in mice and the mechanism involved. METHODS: The mouse model of cardiac fibrosis induced by isoproterenol (ISO) was established and treated with high- and low-dose CAG. Cardiac function was measured by echocardiography, and heart sections were stained by Masson’s trichrome for fibrosis assessment. The expression of fibrosis-related factors was assayed using RT-qPCR, Western blot and immunohistochemistry. In addition, cardiac fibroblasts isolated from neonatal factors rat ventricles were cultured and administrated with ISO followed by CAG treatment, and then the expression profile of the factors above was assayed using RT-qPCR. RESULTS: Treatment with CAG significantly alleviated ISO-induced cardiac dysfunction and fibrosis, inhibited the mRNA expression of oxidative stress-related factors NADPH oxidase 4 and inducible nitric oxide synthase, and blocked the phosphorylation of proteins associated with nuclear factor-κB signaling pathway as well as the expression of transforming growth factor-β (TGF-β). In addition, it was demonstrated that CAG also inhibited the mRNA expression of the factors above in primary cardiac fibroblasts administrated with ISO. CONCLUSION: CAG markedly rescues ISO-induced cardiac dysfunction and fibrosis via inhibition of oxidative stress, inflammation and TGF-β levels.     

喷施醋酸钙对蓝莓果实发育过程中关键品质的影响

探讨不同浓度醋酸钙对蓝莓果实品质的影响,为蓝莓品质调控提供理论依据。以‘粉蓝’为试材,从盛花后10 d开始,并每隔10 d在选取的蓝莓树上喷施醋酸钙,醋酸钙溶液浓度设置为0.3%、0.6%、1.0%,以喷施蒸馏水为对照,研究喷施醋酸钙对蓝莓果实关键品质的影响。结果表明,喷施醋酸钙溶液可有效提高蓝莓果实发育过程中的可溶性糖、花色苷、总酚含量及发育后期类黄酮含量,降低可滴定酸含量,提升糖酸比,其中以0.6%醋酸钙处理效果最好,说明喷施适宜浓度醋酸钙可有效提高蓝莓果实品质。相关性分析表明,花色苷与可溶性糖、糖酸比呈极显著正相关,与可滴定酸呈显著或极显著负相关,其中以0.6%醋酸钙处理各指标相关性最显著,说明能提高细胞内糖含量的因素都可以不同程度地促进花色苷的合成。