Endogenous nitric oxide synthase inhibitor increase skeletal muscle contractility and mitochondria biosynthesis in 4-week running rats

AIM: To observe the effect of endogenous nitric oxide synthase(NOS) inhibitor asymmetric dimethylarginine(ADMA) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS: The 4 weeks running rat model was established. The twitch tension, tetanic tension and the fatigue test of soleus muscle induced by electrical stimulation ex vivo were detected. The ATP content, mitochondrial DNA levels and the mRNA expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), nuclear respiratory factor(NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle. Serum ADMA concentration was detected by high performance liquid chromatography. The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA metabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot. NOS activity and nitric oxide(NO) content were analyzed by colorimetric method. RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1α and NRF were significantly increased(P<0.01). In addition, the protein expression of constitute type NOS(cNOS) and NOS activity were significantly increased(P<0.01), but the increase in NO content was relatively smaller in soleus muscle in exercise group(P<0.05). Moreover, serum ADMA concentration in running group was increased, while the DDAH2 expression in skeletal muscle was decreased.CONCLUSION: Short-term endurance exercise enhances the twitch tension, tetanic tension and fatigue resistance of soleus muscle. The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochondria biosynthesis and mitochondrial function.     

Changes of diaphragmatic function and morphologic features in diabetic rats

AIM:To evaluate the changes of diaphragm contractile properties and morphologic features in experimental diabetic rats.METHODS:Diaphragm contractility was assessed by twitch kinetics,maximal tetanic force (P₀),tension-frequency relationship and fatigue index(FI)in vitro diaphragm strip preparations of 4th week diabetic Wistar rats,and compared with that of the control group.The structure of diaphragm was analysed in diabetic rats and control with Hematoxyline and eosin staining,Heidenhain staining and enzymatic histochemistry.RESULTS:The twitch (Pt),contraction time(CT),half relaxation time(RT_1/2)and FI decreased significantly in diabetic group as compared with the control group.There was no difference in Po between the two groups.The tension of diabetic diaphragm at frequencies of 25,50,75,100 and 125 Hz was significantly decreased compared with that of the normal rats.After diaphragm bundles were subjected to fatiguing stimulations and incubated with aminophylline at 250 mg/L for 30 minutes, the tension of diabetic diaphragm decreased remarkably at both high and low frequencies in comparison with that of the normal rats.In addition,the activity and optical density ofα-phosphoglycerol dehydrogenase in diaphragm from diabetic rats were significantly lower than that from control rats(P<0101).CONCLUSION:The reduction of contractile function,decreased diaphragm fatigue resistance,and damaged structure occurred in 4th week diabetic rat diaphragm.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Influence of metformin on LPIN1 expression and AMPK signaling in high-fat diet-induced insulin resistant rats

AIM: To explore the regulatory mechanism of LPIN1 in hepatic insulin resistance by investigating the influence of metformin on the expression of LPIN1 and AMP-activated protein kinase(AMPK) signaling in the rats with high-fat diet-induced insulin resistance. METHODS: Thirty-six 4-week-old male Wistar rats were randomly divided into 2 groups: control group and high-fat diet (HF) group. The rats in HF group were fed with high-fat diet for 8 weeks and then were randomly divided into 2 subgroups: HF group and metformin intervention group, and the animals were continuously raised for 8 months. The mRNA levels of α1 and α2 subunit of AMPK as well as LPIN1 were measured by real-time RT-PCR. Phospho-AMPKα (Thr-172) was detected by Western blotting to evaluate the activity of AMPK. RESULTS: After 4 months, the rats in HF group showed significant increase in the levels of body weight, fast plasma glucose and insulin, and the levels of triglyceride and total cholesterol significantly elevated.Significant decrease in LPIN1 and phospho-AMPKα (Thr-172) expression in the rat livers were also observed. After treated with metformin, the metabolic indexes of the HF rats were improved. The mRNA and protein expression of AMPKα1 and AMPKα2 had no significant difference among the 3 groups. Metformin treatment also increased the expression of LPIN1 in the liver tissues of HF rats. CONCLUSION: The decrease in LPIN1 expression and AMPK activity may contribute to hepatic insulin resistance in diet-induced obese rats. Metformin improves the LPIN1 expression and AMPK activity through the interaction between LPIN1 and AMPK signal pathways.     

Improving effects of ginsenoside Rb1 on glucose metabolism in cardiomyocytes under hypoxia by hypoxia-inducible factor 1α

AIM: To elucidate the effect of ginsenoside Rb1 (Gs-Rb1) on the glucose metabolism to improve the viability of the cardiomyocytes under hypoxia, and whether hypoxia-inducible factor 1α (HIF-1α) and/or AMPKα are involved in the process.METHODS: The neonatal rat cardiomyocytes were cultured, and randomly divided into control group, hypoxia (1% O₂, 94% N₂ and 5% CO₂) group, Gs-Rb1 (200 μmol/L) group, Ara-A (500 μmol/L) group, Gs-Rb1+Ara-A group, YC-1 (5 μmol/L) group, Gs-Rb1+YC-1 group, Ara-A+YC-1 group and Gs-Rb1+YC-1+Ara-A group. After the intervention for 8 h, the cell viability was analyzed by MTT assay. The protein levels of AMPK, HIF-1α and glucose transporter-4 (GLUT-4) were determined by Western blot. The activities of heterophosphatase (HK), phosphofructokinase (PFK) and lactic dehydrogenase (LDH) were measured by ELISA.RESULTS: Gs-Rb1 significantly improved the viability of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 and Ara-A. In addition, YC-1 and Ara-A had a synergistic effect. Gs-Rb1 increased the protein levels of AMPK and HIF-1α in the hypoxic cardiomyocytes, which was significantly inhibited by Ara-A and YC-1. Gs-Rb1 significantly increased the expression of GLUT-4 on the cytomembrane of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 or Ara-A, especially Ara-A+YC-1. Gs-Rb1 significantly increased the activities of HK, PFK and LDH, all those were significantly inhibited by YC-1 or Ara-A. Besides, YC-1 and Ara-A had a synergistic effect.CONCLUSION: Gs-Rb1 improves the viability of hypoxic cardiomyocytes, which may be related to the regulation of glucose uptake and enhancement of glycolysis by synergy of both HIF-1α and AMPK.     

Effect of crystallin βB2 on the aging of lens

AIM: To study the effect of crystallin βB₂ on the aging of lens. METHODS: SD rats were maintained routinely and killed at 6 different ages (1 d, 8 d, 2 weeks, 8 weeks, 8 months and 1.5 years). Water-soluble crystallins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (IEF/SDS-PAGE). After Comassize blue staining, the crystallin patterns were screened and analyzed. βB₂ crystallin and the main chaperone proteins (αA₂, αB₂) were identified and the relative quantity was measured. RESULTS: (1) The quantity of water-soluble crystalline βB₂ increased in close relation to the aging of the rat. (2) αA₂, αB₂ chaperone proteins increased with the aging of the rat too. (3) The change of the quantity of water-soluble crystalline βB₂ was closely related to the changes of αA₂ , αB₂ chaperone proteins. (4) Degraded and modified crystallins began to appear clearly in the lens after 8 months old. CONCLUSION: Based on our results, we infer that water-soluble crystalline βB₂ increases with the aging dof the rat, which is helpful to maintain the structure and transparency of the lens.     

Effects of prolonged heavy load swimming and the Panaxadiol saponins on expression of α-actin of quadriceps in rats

AIM: To study the effects of 7 weeks prolonged haevy load swimming and Panaxadiol Saponins (PDS) on the expression of α-actin in quadriceps muscle of thigh in rats. METHODS: After 7 weeks prolonged heavy load swimming training, the expression of α-actin in quadriceps muscle of thigh was quantitatively examined by Northern blotting analysis in the condition with or without PDS treatment. RESULTS: The expression of α-actin in rat quadriceps muscle of thigh in exercise+PDS group was significantly higher than that in sedentray control group and exercise+saline group at 24^th hour after intensive training. No statistical difference between exercise+androsan group and exercise+saline group was observed.CONCLUSION: PDS enhanced α-actin expression in quadriceps muscle of thigh in rats.     

Relationship between renal hypertension and cardiac α1-adrenoceptor autoantibodies in rats

AIM: To examine the autoantibody against α₁-adrenoceptor and its biologic activities during the development of renal hypertension. METHODS: Renal hypertension of rat was achieved by clipped renal artery, the titre of autoantibody to α₁-adrenoceptor was detected using ELISA immunoassay. Furthermore, the biological offects of these autoantibodies on cultured cardiomyocytes were also examined. RESULTS: After two weeks of clipping renal arteries, both the frequency of occurrence and the titre of autoantibodies to cardiac α₁-adrenergic receptor were significantly increased as compared with the control of pre-treatment. The increased autoantibodies lasted for several weeks and then automatically decreased gradually to the pre-clipping level at 12 weeks. The biological effects of these autoantibodies displayed an "agonistic-like" activities on the beating frequency of cultured neonatal cardiomyocytes. CONCLUSION: Autoantibodies against α₁-adrenoceptor may play a role in the elevation of peripheral vascular resistance and in the development of cardiac hypertrophy in rats with renal hypertension.     

Effects of cyclosporin on oxidative stress and mitochondrial energy metabolism in rat hippocampus after status epilepticus

AIM：To investigate the effects of cyclosporin on oxidative stress and mitochondrial energy metabolism in the rat hippocampus after status epilepticus. METHODS：Status epilepticus was induced by pilocarpine. The changes of malondialdehyde and superoxide dismutase in the rat hippocampus with or without cyclosporin injection were evaluated. Additionally, the mitochondrial permeability transition, the activity of mitochondrial respiratory chain complex I/III and ATP content in the rat hippocampus were detected. RESULTS：Cyclosporin significantly inhibited mitochondrial permeability transition in the rat hippocampus after status epilepticus. Decreased malondialdehyde and increased superoxide dismutase levels were detected in cyclosporin treatment group compared with status epilepticus group (P<0.05). More-over, the activity of mitochondria respiratory chain complex I, not III, in the mitochondrial fraction increased after cyclo-sporin treatment (P<0.05). In addition, cyclosporin significantly prevented the decrease of ATP content in rat hippocampus after status epilepticus (P<0.05). CONCLUSION：Cyclosporin suppresses oxidative stress in the rat hippocampus after status epilepticus. Cyclosporin alleviates the impairment of mitochondrial energy metabolism in rat hippocampus after status epilepticus.     

Early high-protein diet improves glucose metabolism in SGA rats via adiponectin-AMPK signaling

AIM: To investigate the effect of early high-protein diet on glucose metabolism in small-for-gestational-age (SGA) rats and the role of adiponectin-AMP-activated protein (AMPK) signaling in this process. METHODS: Forty-eight neonatal male SGA rats were established by maternal food restriction throughout the period of pregnancy. The animals were randomly divided into SGA control group (CS group, 24 rats) and high-protein intervention SGA group (HPS group, 24 rats) when born. Twenty-four normal neonatal male rats were used as normal control group (CN group). The rats in CN group and CS group were breastfed for 3 weeks and their mothers were provided free access to basic diet. After weaning, they were provided free access to basic diet until 12 weeks of age. The rats in HPS group were breastfed for 3 weeks and their mothers were provided free access to high-protein diet. After weaning, they were provided free access to high-protein diet until 4 weeks of age. At the 4th week of age, they were provided free access to basic diet until 12 weeks of age. From 4 to 12 weeks of age, fasting blood glucose and insulin were measured and homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated. The serum adiponectin level and the visceral fat mass (VFM) were detected. The percentage of VFM to body weight (VFM%) was calculated. The visceral fat was paraffin-embedded and the adipocyte area was determined. The expression of AMPKα,phosphorylated AMPKα (p-AMPKα) and glucose transporter 4 (GLUT4) in skeletal muscle was analyzed. At the 12th week of age, the oral glucose tolerance test (OGTT) was performed. RESULTS: At the 4th week of age, no significant difference of HOMA-IR among groups was observed. At the 12th week of age, HOMA-IR in CS group was significantly higher than that in CN group and HPS group. No significant difference of HOMA-IR between CN group and HPS group was found. From 4 to 12 weeks of age, VFM% and adipocyte area of visceral fat in CS group were significantly higher than those in CN group and HPS group. No significant difference of VFM% and adipocyte area of visceral fat between CN group and HPS group was observed. The level of serum adiponectin, and the expression of p-AMPKα and GLUT4 in skeletal muscle were significantly lower in CS group than those in CN group and HPS group. The protein levels of p-AMPKα and GLUT4 were not significantly different between CN group and HPS group. No significant difference in the expression of AMPKα in skeletal muscle among groups was observed. At the 12th week of age, the area under the curve (AUC) of glucose level in OGTT was higher in CS group than that in CN group and HPS group. No significant difference of AUC of glucose level between CN group and HPS group was found. CONCLUSION: Early high protein diet may improve glucose metabolism in SGA rats, partly by avoiding excessive accumulation of visceral fat and possibly by activating adiponectin-AMPK signal pathway to increase GLUT4 expression.     

Phasic change of water soluble major crystallins with aging in rats

AIM: To study the correlation between phasic change of the relative quantity of major crystallins with aging in rats. METHODS: Six groups of SD rats (age 1 d， 8 d，2 weeks，8 weeks，8 months and 1.5 years) were raised routinely. Water soluble crystallins were extracted and separated by two－dimensional polyacrylamide gelelectrophoresis. After comassize blue staining，the crystallins patterns were scanned and analyzed. RESULTS: (1) Out of the eighteen water soluble major rat crystallins tested in each group, seven showed gradual phasic changes in relative quantity of crystallins, but there were no significant changes in total quantity of water soluble crystallins. (2) Phasic changes in these crystallins presented four different patterns: increasing (βB₄、αB₂、αA₂、βA₁), decreasing (β₇、β₈、γ_2,3、γ_5,6),relatively stable(βA₃、βB₅), and irregular. (3) The ratio of βB₄ /αA₂ increased gradually with the rat aging process. CONCLUSION: The gradual phasic changes in relative quantity of crystallins reflect the aging status of rat crystalline.     

Inhibitory effect of ginkgo-dipyridamole injection on ischemia/reperfusion injury in rat hearts in vitro

AIM：To investigate the effect of ginkgo-dipyridamole injection (GD) on ischemia/reperfusion (I/R) injury in rat hearts in vitro and its possible mechanism. METHODS：Forty male Sprague-Dawley rats were randomly divided into 5 groups (n=8): normal control (NC) group, I/R group, ischemic preconditioning (IPC)+I/R group, GD+I/R group and GD+LaCl3+I/R group. Cardiac function indexes, including heart rate (HR), left ventricular systolic pressure (LVSP) and the maximal rise/fall rate of left ventricular pressure (±dp/dtmax), were detected at 5 time points, including stabilizing point, 30 min after ischemia, and 5, 30 and 60 min after reperfusion. The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent at the five time points was assayed. The concentration of Ca2+ and the content of α-ketoglutarate dehydrogenase (α-OGDH) in myocardial mitochondria were determined at the end of the whole experiment. RESULTS：Compared with I/R group, the cardiac function indexes in IPC+I/R and GD+I/R groups were improved at the reperfusion period (P<0.05), the activity of LDH and CK in coronary effluent and the concentration of Ca2+ in mitochondria were significant reduced (P<0.01), and the content of α-OGDH was increased (P<0.05). However, the protective effect of GD was inhibited by LaCl3 (P<0.05). CONCLUSION：GD protects rat hearts against I/R injury by inhibiting calcium overload and improving mitochondrial enzyme activity to stabilize mitochondrial energy metabolism.     

Excessive endoplasmic reticulum stress induces apoptotic cell death in chronic cyclosporine A nephrotoxicity

AIM：To investigate the impact of excessive endoplasmic reticulum stress on apoptotic cell death in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. METHODS：Male Sprague-Dawley rats on a low-salt diet were subcutaneously injected with vehicle (olive oil, 1 mL·kg-1·d-1) or CsA (15 mg/kg) daily for 1 or 4 weeks. Tubulointerstitial fibrosis and apoptotic cell death were estimated by trichrome staining and TUNEL staining. In addition, immunohistochemistry and immunoblotting were used to evaluate the expression of immunoglobulin-binding protein (BiP), eukaryotic initiation factor 2α (eIF2α), growth arrest and DNA damage-inducible protein 153 (GADD153), caspase-12 and caspase-3. RESULTS：The rats treated with CsA for 1 week did not develop tubulointerstitial fibrosis and TUNEL-positive cells, whereas 4-week treatment with CsA induced typical tubulointerstitial fibrosis and increased TUNEL-positive cells. CsA induced a significant increase in BiP and caspase-12 expression peaked at 1 week, and then returned to normal levels at 4 weeks. In contrast, the expression of eIF2α, GADD153 and caspase-3 in CsA-treated rat kidneys were significantly increased in a time-dependent manner. CONCLUSION：Excessive endoplasmic reticulum stress causes apoptotic cell death by depleting molecular chaperones and stimulating the proapoptotic pathway in chronic CsA nephrotoxicity.     

苹果乙烯响应因子基因MdERF11对脱落酸的敏感性分析

以‘嘎拉’苹果(Malus×domestica Borkh.)为试材,克隆了乙烯响应因子基因MdERF11(序列号MDP0000756341)。测序发现,该基因包含全长为483 bp的完整开放阅读框,编码161个氨基酸。系统进化树分析表明,这一乙烯响应因子与拟南芥AtERF11蛋白同源序列相似性最高。利用PlantCare数据库进行基因启动子顺式作用元件预测,MdERF11启动子序列中含有与脱落酸(ABA)、乙烯及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdERF11在苹果的各组织中均有表达,在叶柄和果实中表达量相对较高;并且MdERF11的表达明显受到ABA的诱导。在外源ABA的处理下,MdERF11过量表达的苹果愈伤组织的生长势明显比野生型强,表明MdERF11降低了苹果愈伤组织对ABA的敏感性。     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Effect of hyperbaric oxygen on HIF-1α expression in rat experimental periodontitis with psychological stress

AIM: To investigate the effect of hyperbaric oxygen (HBO) on hypoxia-inducible factor-1α (HIF-1α) expression in rat experimental periodontitis with psychological stress. METHODS: Male special pathogen-free Wistar rats (n=120) were randomly divided into 4 groups: normal control group; psychological stress stimulation group; experimental periodontitis group: the periodontitis model was induced by wrapping 3/0 silk ligature inoculated with Porphyromonas gingivalis around the left maxillary second molar of the rats; periodontitis model with stress stimulation group. Psychological stress was removed at the 9th weeks after ligature, 6 rats from each experiment group were randomly chosen to HBO treatment. The rats were sacrificed at the 2nd, 4th, 8th and 10th weeks after ligature. Gingival index (GI) and attachment loss (AL) were measured before sacrifice. The histological changes of periodontal tissues were observed under microscope with HE staining. The expression of HIF-1α was observed by the method of immunohistochemistry. RESULTS: The sites of gingival attachment were normal in control group and psychological stress stimulation group. Periodontal pocket, and periodontal attachment loss were observed in experimental periodontitis group. The tissue damage was much serious in periodontitis model with stress stimulation group. No significant difference of GI and AL among psychological stress stimulation group and normal control group during the experiment was observed. GI and AL in periodonitis model with stress stimulating group were significantly higher than those in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). The levels of GI and AL were significantly lower at the 10th weeks after HBO treatmnt than those in untreated groups (P < 0.05). No significant difference of HIF-1α expression scores among psychological stress stimulation group and normal control group was found. HIF-1α expression scores in periodonitis model with stress stimulating group was significantly higher than that in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). At the 10th weeks after HBO treatment the levels of HIF-1α were significantly lower than that in untreated groups (P < 0.01). CONCLUSION: Stress stimulation may aggravate periodontitis by decreasing tissue oxygenation in rats. HBO may represent a useful way in psychological stress periodontitis therapy.