Effect of Ganoderma lucidum spores on IL-1β and c-Fos in the brain tissue of epilepsy rats

AIM: To observe the influence of Ganoderma lucidum spores on the cytokine IL-1β and c-Fos in the brain tissue of epilepsy rats. METHODS: The IL-1β level was examined by radioimmunoassay and the c-Fos expression was measured by mmunohistochemical assay. RESULTS: Comparing the Ganoderma lucidum spores group with the epilepsy model group: c-Fos positive cell count in brain cortex and hippocampus in treatment group was obviously reduced. IL-1β level in brain tissue was also reduced obviously. CONCLUSION: Ganoderma lucidum spores effectively reduces cytokine IL-1β in brain tissue of epilepsy rats, improves the immunity dysfunction and plays a role in anti-epilepsy through suppressing c-Fos expression in epilepsy rats brain tissue and blocking of LRG.     

Effect of Ganoderma lucidum spores powder on the expression of IGF-1, NF-κB and apoptosis of nerve cells in the brain from epileptic rat

AIM: To investigate the effect of Ganoderma lucidum spores powder on the expression of insulin-like growth factor-1 (IGF-1), nuclear factor-κB (NF-κB) and apoptosis of nerve cells in rats with epilepsy established by pentetrazole. METHODS: The sub-eclampsia dosage of pentetrazole (PTZ) was used to make epilepsy model. Ganoderma lucidum spores powder group was given from stomach. The enduring time and latent period were recorded. The immune reactivity of IGF-1, NF-κB/P65 and apoptosis of nerve cells were measured with immunohistochemical staining and TUNEL method. RESULTS: In high power sight (×400), there were much more apoptosis cells in hippocampus and brain cortex of model group (18.80±2.13, 16.87±2.00) than those in control group (0.97±0.52, 0.58±0.25). The expressions of IGF-1, NF-κB in model group were higher than those in control group. Compared with model group, the latent period of Ganoderma lucidum spores powder group at the 17th, 21th, 25th days were longer (P<0.05, P<0.05, P<0.01, respectively).There were less apoptosis cells in hippocampus and brain cortex of Ganoderma lucidum spores powder group (12.30±2.46, 10.48±1.33) than those in model group. The expression of NF-κB/P65 in Ganoderma lucidum spores powder group was lower than that in model group, but the immune reactivity of IGF-1 increased more distinctly in Ganoderma lucidum spores powder group than that in model group. CONCLUSION: IGF-1, NF-κB and apoptosis of nerve cells may have play a role in occurrence and development of PTZ-induced epilepsy. Ganoderma lucidum spores powder can suppress expression of NF-κB strongly, and facilitate the immune reactivity of IGF-1, which may be one of the mechanisms by which Ganoderma lucidum spores powder restrains the apoptosis of nerve cells caused by epilepsy to prevent the damages of nerve cells.     

钙、铁和锰对两种灵芝液体培养菌丝体生物量的影响

探讨了水中不同含量的钙、铁、锰离子对赤芝(Ganoderma lucidum)和松杉灵芝(G.tsugae)液体培养下菌丝体生物量的影响.结果表明,在200～1200 mg/L时随着Ca浓度增加赤芝生物量逐渐减少,而松杉灵芝在Ca浓度为200 mg/mL时生物量最高,在600～1200 mg/mL低于不添加Ca的处理;在Fe浓度为200～600 mg/L及200～800 mg/L时,赤芝和松杉灵芝的生物量高于不添加的处理,但当浓度超过相应范围时,这两种灵芝的生物量显著降低;Mn浓度为200或400 mg/L时,两种灵芝的生物量较高,当浓度超过800 mg/L时低于不添加的处理.     

Effect of transplantation of hMSCs modified by BDNF and GDNF gene on injured brain in rat MCAO model

AIM：To study the therapeutic effect of human mesenchymal stem cells (hMSCs) modified by brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) gene transfer with liposome on middle cerebral artery occlusion (MCAO) model rats. METHODS：The nonviral expression vector was constructed and transfected into the hMSCs by liposomal method. The rat brain injury model was established by the method of MCAO. The gene-modified hMSCs, control hMSCs or PBS was transplanted into the rats 24 h after MCAO by femoral venous injection. The neurological function score, the change of the body weight and the behavior test were used to evaluate the damage of the brain in the rats. The degree of the damage and the migration of the cells 15 d after transplantation were analyzed by observing the histological changes of the brain tissues. RESULTS：The expression levels of BDNF and GDNF in gene-modified hMSCs were much higher than those in control hMSCs. The transplantation of BDNF and GDNF gene-modified hMSCs promoted the functional recovery and reduced the infarct size in the rats after MCAO. A few exogenesis cells only survived in the infarct area of the brain in the MCAO rats, and the cells showed no differentiation. CONCLUSION：Transplantation of BDNF and GDNF gene-modified hMSCs by nonviral expression vector is effective in treating cerebral ischemia. The effect may result from the action of the cytokines secreted by these cells, reducing the injuries induced by the brain ischemia and accelerating the nerve repair following the injury.     

Effect of zhiganning on the expressions of HSP60 and HSP70 in the hepatocytes of rats undergoing steatohepatitis

AIM: To observe the expressions of HPS60 and HPS70 in hepatocytes in rats under treatment with zhiganning on steatohepatitis. METHODS: Male SD rats were randomly divided into large dose zhiganning group, small dose zhiganning group, ursodeoxycholic acid (UDCA) group, model group, normal control group. Except the normal control group, all the other rats were fed with high fat (88% standard diet, 10% lard, 2% cholesterol) and 35% alcohol 10 mL/kg twice a day. Prophylactic drugs were used at the same time. All rats were sacrificed at the 9th week. Routine histologic features of hepatic sections were observed by HE staining and penetrated electron microscope. The expressions of HSP60 and HSP70 in the liver were detected by immunohistochemistry, respectively. RESULTS: ⑴ The degree of steatohepatitis in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly decreased compared with that in model group (P<0.05). ⑵ The expression of HSP70 in the large dose zhiganning group and ursodeoxycholic acid (UDCA) group were significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). The expression of HSP60 in the large dose zhiganning group was significantly higher than that in either model group or normal control group (P<0.05, P<0.01, respectively). ⑶ In the large dose zhiganning group and ursodeoxycholic acid group, ultramicroscopic structure of liver was nearly normal, which was significantly improved compared with model group. CONCLUSION: The results indicate that zhiganning and UDCA effectively prevente the steatohepatitis in rats induced by high fat diet and alcohol. The enhanced expressions of HSP60 and HSP70 may play an important role in the prevention of liver from injury.     

Effect of Jieyuwan on depression-like behaviors and expression of BDNF in hippocampus and prefrontal cortex of WKY rats

AIM:To investigate the mechanism of depression and its development, and to study the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex of Wistar-Kyoto (WKY) rats treated with Jieyuwan. METHODS:Adult male WKY rats were used as an animal model of endogenous depression. Wistar rats of the same strain were selected as control group. WKY rats were randomly divided into model group, citalopram group and Jieyuwan group. After intragastric administration for 21 d, the changes of depression-like behaviors were observed by sucrose preference test and forced swimming test. Immunofluorescence and Western blot were used to detect the expression of BDNF in the hippocampus and prefrontal cortex. RESULTS:WKY rats showed significant depression-like behaviors, and the expression of BDNF was significantly decreased in the hippocampus and prefrontal cortex (P<0.01). The reduction of neuronal axons in hippocampus was also observed. After drug treatment, the depression-like behaviors of WKY rats were significantly attenuated, and the expression of BDNF and the number of axons were increased (P<0.01). CONCLUSION:Jieyuwan effectively attenuates the depression-like behaviors of WKY rats, and BDNF is a key factor in its antidepressant effect. Our findings further confirm the involvement of BDNF in the development of depression.     

Effect of Ba2+ concentration on L-type Ca2+ channel in hypothalamic neurons

AIM:To investigate the effect of Ba²⁺ concentration on L-type of Ca²⁺ channel in hypothalamic neurons.METHODS:The cell acute isolation technique and cell-attached patch-clamp technique were used.RESULTS:The slope conductance of L-type Ca²⁺ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP₀) obviously different with different Ba²⁺ concentration as carrier. CONCLUSION:Ba²⁺ concentration had the obvious effect on the L-type Ca²⁺ channel.     

Change of L-type voltage-gated Ca2+ channel current during focal brain ischemia in diabetic rats

AIM: To observe the change of L-type voltage-gated Ca²⁺ channel current during focal brain ischemia in the normal rats and the rats with diabetes mellitus (DM). METHODS: Combination of high-fat diet with streptozotocin (STZ) was used to establish DM animal model. The operation of middle cerebral artery occlusion (MCAO) with monofilament on the rats was performed. The animals were divided into sham operation group, MCAO 1 h group, MCAO 3 h group, MCAO 6 h group, MCAO 24 h group, DM sham operation group, DM+MCAO 1 h group, DM+MCAO 3 h group, DM+MCAO 6 h group and DM+MCAO 24 h group. The score of neural function was determined to judge the degree of palsy in the rats in MCAO 24 h group and DM+MCAO 24 h group. The changes of L-type voltage-gated Ca²⁺ channel current of cortex neurons during ischemia were measured using the whole-cell patch clamp technique. RESULTS: The rats in DM+MCAO 24 h group awaked slowly, and the degree of semiplegia was more serious than that in the rats in MCAO 24 h group. The score of neural function in DM+MCAO group was higher than that in MCAO group (P<0.05). The longer the ischemic time was, the higher L-type voltage-gated Ca²⁺ channel current was observed in MCAO group and DM+MCAO group (P<0.05). L-type voltage-gated Ca²⁺ channel current in DM+MCAO group was higher than that in MCAO group at each time point(P<0.05). CONCLUSION: The aggratation of ischemic injury during DM+MCAO is probably associated with Ca²⁺ overload induced by calcium channel opening and current increasing.     

Effect of BDNF expression in cerebral cortex and hippocampus on ability of learning and memory in APP/PS1 transgenic mice

AIM: To investigate the expression changes of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus and their effects on the ability of learning and memory in the wild-type (WT) mice and APP/PS1 transgenic mice. METHODS: WT mice and APP/PS1 transgenic mice were selected as study subjects. Aβ plaques, apoptosis rate and BDNF expression in the cerebral cortex and hippocampus of WT mice and APP/PS1 transgenic mice were detected by the methods of Congo red staining, TUNEL, immunofluorescence and Western blot. The abilities of learning and memory were determined by Morris water maze test. RESULTS: The Aβ plaques appeared in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, and the number of Aβ plaques in 12-month-old mice was larger than that in 6-month-old mice (P<0.05). The number of apoptotic neurons in the cerebral cortex and hippocampus of 12-month-old APP/PS1 transgenic mice was larger than that of WT mice (P<0.01). The expression level of BDNF in the cerebral cortex and hippocampus of WT mice was higher than that of APP/PS1 transgenic mice (P<0.01). The Morris water maze test showed that the escape latency in APP/PS1 transgenic mice was longer than that in WT mice, and the times across the platform quadrant in 60 s was less than that in WT mice (P<0.01). The swim-tracking path of APP/PS1 transgenic mice was disordered and irregular. CONCLUSION: The expression of BDNF in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was lower than that of WT mice, accompanied by increased neuronal apoptosis and decreased spatial learning and memory ability. The decrease in learning and memory ability may be related to decreased BDNF expression in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, leading to increased neuronal apoptosis, which may be one of the pathological mechanisms of Alzheimer disease.     

ATPase activities and Ca2+/calmodulin alterations in hippocampi of rats with PTSD-like behaviors

AIM: To explore the pathophysiological bases in the pathogenesis of the lasting emotional behavioral disorders following posttraumatic stress disorder(PTSD). METHODS: 240 male Wistar rats were divided randomly into 3 groups. Group SE(n =96) for rats with PTSD-like behavior by constant pulsating current of 100 μA with intratrain frequencies of 16 Hz, pulsating duration of 1 ms, train duration of 10 s and interstimulus interval of 7 min for 5 days with 8 times per day. Group CE(n =96) for control with electrode implanted in hippocampus without stimulation, and Group NC(n =48) for normal control. The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase, levels of intracellular calcium and free calmodulin(CaM), and the total CaM expression were detected in hippocampi of experimental rats. RESULTS: The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase in mitochondria of hippocampal cells in Group SE rats were significantly decreased at 48 h and 72 h after the last stimulation, respectively. The intracellular free calcium levels were increased, and the mean channel fluorescence of intracellular free CaM decreased remarkably at 72 h poststimulation, while the expression of total CaM was significantly elevated at 48 h after the last stimulation in hippocampi of Group SE rats. CONCLUSION: The lasting increased levels of intracellular free calcium and expression of Ca²⁺ -CaM in hippocampus, as well as the dysfunction of Na⁺-K⁺ pump and Ca²⁺ -ATPase in mitochondria may play important roles in the long-term neuropsychological sequelae in PTSD.     

Influences of Ca2＋ antagonist on the mRNA levels of L-type Ca2＋ channel and Na＋-Ca2＋ exchanger in the atria of hypertensive rats

AIM: To investigate the mRNA changes of L-type Ca²⁺channel α_1Csubunit (CaL-α_1C) and Na⁺-Ca²⁺ exchanger (NCX) in the atria of renovascular hypertensive rats. METHODS: Two-kidney one-clip Goldblatt hypertensive Sprague-Dawley rats were divided into three groups 1 week after operation, HD group was treated with 250 mg/d diltiazem, LD group was treated with 50 mg/d diltiazem, control group (C group) was treated with vehicle. After 4 weeks treatment, semiquantitative RT-PCR was used to estimate the mRNA changes of CaL-α_1Cand NCX, and GAPDH was used as internal control. RESULTS: Systolic blood pressure in LD group was comparable with C group, and that in HD group was decreased to normal level after diltiazem treatment. In C group, CaL-α_1C mRNA level were 2.5, 2.4 and 2.1 times of S, HD and LD group, and NCX mRNA level were 1.9, 1.6 and 2.1 times of S, HD and LD group. There were no significant difference in the mRNA level of CaL-α_1Cand NCX among S, HD and LD group. CONCLUSION: The mRNA levels of CaL-α_1Cand NCX are upregulated in the atria of hypertensive rats. Ca²⁺ antagonist inhibites their upregulation independent of blood pressure.     

Antagonistic effects of cyproheptadine and anisodamine on [Ca2+]i elevation induced by TNFα in endothelial cell strains

AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca²⁺]_i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca²⁺]_i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca²⁺]_i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10^-5, 6×10^-5 mol/L) and Ani (2×10^-5, 4×10^-5 mol·L^-1) markedly inhibited TNFα (1.2×10^-9 mol·L^-1)-induced [Ca²⁺]_i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca²⁺]_i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca²⁺]_i elevation, which probably is one of the mechanisms of their antishock effects.     

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.     

Effect of chronic hypoxia on [Ca2+]iin pulmonary artery endothelial cells and smooth muscle cells under acute hypoxia

AIM AND METHODS: Using Ca²⁺-sensitive fluorescent probe Fura-2,we measured the changes of [Ca²⁺]_iin cultured rat pulmonary artery endothelial cells (PAEC) and porcine pulmonary artery smooth muscle cells (PASMC) from normoxic (NC group) or chronic hypoxic group (CH group) when they were exposed to acute hypoxia. RESULTS: The increase in [Ca²⁺]_iin 6th passage of PASMC caused by acute hypoxia in CH group was significantly lower than that in the same passage of NC group (P<0.05).On the contrary, in PAEC, the acute hypoxia induced increase in _i, which was significantly higher in 5th passage of CH group than that in NC group (P<0.05). CONCLUSION: The decrease of the elevation of [Ca²⁺]_icaused by acute hypoxia in PASMC of CH group indicated that it functioned to lower the constrictive response to hypoxia.The intensive increase in [Ca²⁺]_icaused by acute hypoxia in PAEC of CH group might lead to more relaxing factors derived from PAEC,which results in a decrease in HPV.     

Effects of fructose-1,6-diphosphate on adriamycin-induced calcium and sarcoplosnic reticulum Ca2+-ATPase activity in cardiomyocytes of rats

AIM: To study the effects of fructose-1,6-diphosphate (FDP) on adriamycin (ADR)-induced calcium and sarcoplasmic reticulum Ca²⁺-ATPase activity in cardiomyocytes of rats. METHODS: Rats were treated with ADR by intraperitoneal injection (2.5 mg·kg^-1 body weight) once every two days for 11 days, and then ADR-treated rats were intervened by FDP at different dosages (ip) once every other day for 41 days. Enzyme linked immune absorption assay (ELISA) was employed to detect froponin I (CTnI). CK-MB was examined by monoclonal antibody. Intracellular free calcium concentration was measured on fluorescent spectrophotometry and SRCa²⁺-ATPase activity was examined by inorganic phosphate. RESULTS: FDP (300, 600, 1 200 mg·kg^-1) significantly reduced the levels of CTnI and CK-MB in serum. Decreased calcium and increased SRCa²⁺-ATPase activity in cardiomyocytes were also observed when ADR-treated rats were intervened by FDP (P＜0.01). CONCLUSION: FDP reduced the injury of cardiotoxicity induced by ADR via decreasing intracellular free calcium and increasing SRCa²⁺-ATPase activity in cardiomyocytes.     

Effects of benazepril on cardiac function,free oxygen radicals,sarcoplasmic reticulum Ca2+-ATPase following cardiac ischemia-reperfusion in spontaneously hypertensive rats

AIM: To investigate the effects of angiotensin converting enzyme inhibitor (ACEI), benazepril (B), on cardiac function , free oxygen radicals, sarcoplasmic reticulum(SR) Ca²⁺-ATPase following ischemia-reperfusion in sportaneously hypertensive rats (SHRs). METHODS: Thirty 10-week-old female SHRs were randomly assigned into two groups: group SHR was control; The animal in group SHR+B was given with 10 mg/kg of benazepril per day. Another 15 Wistar rats with the same age and sex were normal control (group Wistar). After 12 weeks of pretreatment, all rats in each group were subjected to 30 min of left anterior descending coronary artery occlusion and 30 min of reperfusion. Hemodynamic parameters, left heart-to-body weight ratio (LVW/BW), myocardial malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and SR Ca²⁺-ATPase activity were measured. RESULTS: Compared to group Wistar, the rats in group SHR had higher blood pressure, LVW/BW and myocardial MDA concentration, more serious left cardiac function injury and lower myocardial SOD activity and SR Ca²⁺-ATPase activity; group SHR+B had lower myocardial MDA concentration, higher myocardial SOD activity, but no difference in blood pressure, LVW/BW, the degree of left cardiac function injury and myocardial SR Ca²⁺-ATPase activity. CONCLUSION: Benazepril can attenuate ischemia-reperfusion-induced cardiac function injury by regression of left ventricular hypertrophy (LVH), improving SR Ca²⁺-ATPase activity and decreasing oxygen free radicals injury in SHRs.     

Effect of Scutellaria barbata flavonoids on abnormal changes of Bcl-2, Bax,Bcl-xL and Bak protein expression in mitochondrial membrane induced by composite Aβ25-35

AIM：To study the abnormalities of Bcl-2, Bax, Bcl-xL and Bax in the cell mitochondrial membrane of cerebral cortex in the rats injected with β-amyloid beta protein 25-35 (Aβ25-35) in combination with aluminum trichloride (AlCl3) and recombinant human transforming growth factor β1 (rhTGF-β1) (composite Aβ25-35) into lateral cerebral ventricle, and to explore the intervention of Scutellaria Barbata flavonoids (SBF). METHODS：The male SD rats were used to establish the neuronal damaged model by receiving injection of RhTGF-β1 1 μL (10 ng) on day 1 of operation, and then from day 2 of operation, intracerebroventricular injection of Aβ25-35 (4 μg/d, consecutive 14 d) in the morning and 1% AlCl3 in the afternoon (3 μL/d, consecutive 5 d). On the day 49 of operation, the successful model rats were randomly divided into model control and 3 doses of SBF groupss. The rats in SBF groups were daily orally administered with SBF at doses of 35, 70 and 140 mg/kg for 36 d. All the rats were decapitated 60 min after the last administration. The protein expression of Bcl-2, Bax, Bcl-xL and Bak in rat cerebral cortex cell mitochondrial membrane was detected by Western blotting. RESULTS：The composite Aβ25-35 dramatically caused decreases in Bcl-2 and Bcl-xL (P<0.05), and increases in Bax and Bak (P<0.01) in rat cerebral cortex cell mitochondrial membrane. However, oral treatment with SBF for 36 d reversed the above disorders induced by composite Aβ25-35. CONCLUSION：The composite Aβ25-35 induces the expression abnormalities of Bcl-2, Bax, Bcl-xL and Bak in mitochondrial membrane and SBF reverses the above disturbances of apoptotic factors.