Healthy Adult Male SD Rats Research Articles

Emodin Regulates lncRNA XIST/miR-217 Axis to Protect Myocardial Ischemia-Reperfusion Injury.

This study is aimed at investigating the effect of emodin on myocardial ischemia-reperfusion injury (MIRI) and mechanism. Eighty healthy adult male SD rats (weighing 230-250 g) were utilized to establish I/R model, which were randomly divided into five groups (16 rats in each group): sham operation group, myocardial ischemia-reperfusion injury group (I/R group), emodin group, emodin +NC group, and emodin +XIST group. The contents of CK, CK-MB, LDH, and HBDH in serum were determined by ELISA kit. LDH was detected by ELISA assay, SOD was detected by the xanthine oxidase method, and MDA was detected by the thiobarbituric acid method. The relative expression of XIST and miR-217 was evaluated by RT-qPCR. Western blot was applied to detect the protein expression. Flow cytometry was applied to detect cardiomyocyte apoptosis. Myocardial infarction area was obviously increased in I/R model rats, while emodin decreased the myocardial infarction in I/R model rats. In addition, cardiac enzymes (CK, CK-MB, LDH, and HBDH) and apoptosis were obviously increased in MIRI model rats, while emodin obviously decreased cardiac enzymes and apoptosis. The ROS and MDA levels were raised while the activities of SOD were declined in the I/R model group. The ROS and MDA levels were decreased while the activities of SOD were raised in the emodin group. The XIST expression was markedly raised in the I/R model group while decreased in the emodin group, and the overexpression of XIST reversed the protective effect of emodin on myocardial infarction, oxidative stress, and cardiomyocyte apoptosis. In addition, XIST directly regulated the expression of miR-217, and si-XIST inhibited H/R-induced oxidative damage of cardiomyocytes via inhibiting miR-217. Emodin protected MIRI both in vitro and in vivo via inhibiting lncRNA XIST to upregulate miR-217.

Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7.

To investigate the protective effect of sevoflurane preconditioning on renal ischemia-reperfusion injury (renalischemiareperfusionmodel, RIRI) and its related mechanism. Eighty healthy adult male SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group) and TRPM7 inhibitor combined with sevoflurane pretreatment group (T + Sev group), 20 animals in each group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, and the levels of creatinine and urea nitrogen in each group were detected. Deoxyribonucleic acid terminal transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect renal cell apoptosis, and Western blottingwas used to detect the expression of apoptotic proteins cleaved-caspase-3, bax, Bcl-2, and TRPM7 in renal tissue; Detection of oxidative stress-related index levels in renal tissue and levels of inflammatory factors in renal tissue and serum. Compared with the Sham group, the renal tissue pathological damage was aggravated, the levels of creatinine and blood urea nitrogen were increased, and the apoptosis was increased in the RIR group and the Sev group. Death, malondialdehyde (MDA) levels and inflammatory factors were increased, and superoxide dismutase (SOD) levels were decreased (all p < .05); The scores, apoptosis rate, MDA level, and relative expression of inflammatory factor levels were decreased, and SOD levels were increased (all p < .05). Compared with the Sev group, the renal tissue pathological damage in the T + Sev group was aggravated, creatinine, blood urea nitrogen levels increased, apoptosis increased, apoptosis-related proteins cleaved-caspase-3, bax, Bcl-2 showed increased apoptosis, malondialdehyde (MDA) levels, inflammatory factor levels increased, ultrahigh The levels of oxide dismutase (SOD) were decreased (all p < .05). Therefore, we believe that sevoflurane is involved in the protection of rat renal ischemia-reperfusion injury by downregulating the expression of TRPM7.

Levosimendan in rats decreases acute kidney injury after cardiopulmonary resuscitation by improving mitochondrial dysfunction.

BackgroundAcute kidney injury (AKI), the most common complication after cardiac resuscitation, is highly prevalent and harmful. There is increasing evidence that levosimendan can improve cardiac output, increase renal blood flow, and prevent AKI. As a novel calcium sensitizer, levosimendan may exert its protective effect via mitochondria.MethodsRat models of asphyxia-induced cardiac arrest and cardiopulmonary resuscitation (CPR) were set up. Thirty healthy adult male SD rats were randomly divided into CPR group (CPR group, n=10), levosimendan-treated group (levo group, n=10), and sham-operated group (sham group, n=10). Twelve hours after CPR, serum renal function indicators were measured, the kidney injury and mitochondrial morphological changes were observed. Oxygen uptake of the mitochondria, mitochondrial adenosine triphosphate (ATP) and mitochondrial free Ca2+ concentration were measured. Oxidative stress-related indicator levels in rat kidney tissues were further detected to analyze the differences in apoptosis rates among these three groups. Mitochondrial optic atrophy 1 (Opa1), dynamin-related protein 1 (Drp1), and apoptosis-related proteins were detected using Western blotting.ResultsCompared with the sham group, the CPR group had a significant increase in renal tissue damage. PAS staining and HE stains confirmed that CPR led to renal histopathological damage and destruction of the mitochondrial structure. Levosimendan improved the histopathological and ultrastructural damages of kidneys. Further analysis revealed that mitochondrial ATP content, NADH dehydrogenase, succinate dehydrogenase/cytochrome C oxidase, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (CSH-Px) decreased. Free Ca2+ concentration and malondialdehyde (MDA) significantly increased (all P<0.05) in the kidney tissues of rats in the CPR group. However, mitochondrial ATP content, NADH dehydrogenase, succinate dehydrogenase/cytochrome C oxidase, SOD, CAT, and CSH-Px increased, whereas free Ca2+ concentration and MDA decreased (all P<0.05) in the levo group. The apoptosis rate increased in the CPR group. There were significantly increased levels of Drp1 protein levels, and significantly decreased Opa1 expression (all P<0.05). However, the levo group showed the opposite effects (all P<0.05).ConclusionsLevosimendan can alleviate AKI following CPR, which may be achieved by improving mitochondrial dysfunction and suppressing the mitochondrial apoptosis pathway.

Role of 5-Hydroxytryptamine and Intestinal Flora on Depressive-Like Behavior Induced by Lead Exposure in Rats.

Objective To investigate the effects of 5-hydroxytryptamine (5-HT) and intestinal flora on depression-like behavior induced by lead exposure in rats. Methods 30 healthy SPF adult male SD rats were randomly divided into control group and lead exposure group. The depression-like behavior of rats was detected. The blood, striatum, and intestinal tissue were collected. The lead content was detected by ICP-MS. The mRNA expressions of ChgA, TPH1, 5-HT, and 5-HT3R were tested by qRT-PCR. The content of 5HT was checked by HPLC-ECD. The content of 5-HT3R was detected by ELISA. The protein expressions of 5-HT, 5-HT3R, ChgA, and TPH were gauged by immunohistochemistry. Fecal samples were collected, and the composition of intestinal flora in experimental rats was analyzed by 16 s RNA metagene sequencing. Results Lead exposure can greatly cause depression. The content of 5-HT in blood and striatum in the lead exposure group decreased, and the expression levels of 5-HT, 5-HT3 R, ChgA, and TPH in the intestine decreased distinctly. Compared with the control group, the distribution of a-polymorphism related indexes Simpson, Chao1, Shannon, and ACE in rats with depressive-like behavior after lead exposure was significantly increased; in the lead exposure group, there were 61 different operational taxonomic units (OUTs) in intestinal flora at the family level. Based on linear discriminant analysis, it was found that the key bacteria were Lactobacillaceae and Bifidobacteriaceae, and their abundance decreased evidently in the lead exposure group. Conclusion Lead exposure improves depressive-like behavior by affecting intestinal flora and regulating neurotransmitter 5-HT through the intestinal-brain axis.

The Expression of ZnT3 and GFAP Is Potentiated in the Hippocampus of Drug-Resistant Epileptic Rats Induced by Amygdala Kindling

Objective: The first-line treatment for epilepsy, a chronic neurological disorder characterized by spontaneous seizures, includes the application of anticonvulsant drug therapy. Only one-third of patients are incapable of complete controlling of their seizures after the administration of ≥2 pharmaceuticals. Here, we aimed to observe the ultrastructure changes and the expression of ZnT3 and GFAP in the hippocampus of drug-resistant epileptic rats. Methods: A total of 50 healthy adult male SD rats were used to generate the model ofepilepsy by amygdala kindling. After the rats were successfully kindled, pharmacoresistant epileptic (PRE) rats were selected according to their response to phenobarbital and phenytoin. The ultrastructure as well as the expression of zinc transporter 3 (ZnT3, a member of a growing family of mammalian zinc transporters) and glial fibrillary acidic protein (GFAP) were compared among PRE, pharmacosensitive epileptic (PRE), and normal (NRC) rats. Results: The PRE rats displayed severe synapses, neuronal degeneration, and necrosis. Moreover, the expression of ZnT3 and GFAP was significantly increased in both PRE and PSE rats; compared with NRC rats, the promotion of this expression was more pronounced in the PRE rats. Conclusions: Taken together, obvious synapses, neuronal degeneration, necrosis, mossy fiber sprouting, and astrogliosis were found in the drug-resistant epileptic rat model induced by amygdala kindling.

Tongmai Yangxin pill reduces myocardial No-reflow via endothelium-dependent NO-cGMP signaling by activation of the cAMP/PKA pathway

Ethnopharmacological relevanceThe Tongmai Yangxin pill (TMYX) is derived from the Zhigancao decoction recorded in Shang han lun by Zhang Zhongjing during the Han dynasty. TMYX is used for the clinical treatment of chest pain, heartache, and qi-yin-deficiency coronary heart disease. Previous studies have confirmed that TMYX can improve vascular endothelial function in patients with coronary heart disease by upregulating nitric oxide activity and then regulating vascular tension. Whether TMYX can further improve myocardial NR by upregulating NO activity and then dilating blood vessels remains unclear. Aim of the studyThis study aimed to reveal whether TMYX can further improve myocardial NR by upregulating NO activity and then dilating blood vessels. The underlying cAMP/PKA and NO-cGMP signaling pathway-dependent mechanism is also explored. Materials and methodsThe left anterior descending coronary arteries of healthy adult male SD rats were ligated to establish the NR model. TMYX (4.0 g/kg) was orally administered throughout the experiment. Cardiac function was measured through echocardiography. Thioflavin S, Evans Blue, and TTC staining were used to evaluate the NR and ischemic areas. Pathological changes in the myocardium were assessed by hematoxylin–eosin staining. An automated biochemical analyzer and kit were used to detect the activities of myocardial enzymes and myocardial oxidants, including CK, CK-MB, LDH, reactive oxygen species, superoxide dismutase, malonaldehyde, and NO. The expression levels of genes and proteins related to the cAMP/PKA and NO/cGMP signaling pathways were detected via real-time fluorescence quantitative PCR and Western blot analysis, respectively. A microvascular tension sensor was used to detect coronary artery diastolic function in vitro. ResultsTMYX elevated the EF, FS, LVOT peak, LVPWd and LVPWs values, decreased the LVIDd, LVIDs, LV-mass, IVSd, and LV Vols values, demonstrating cardio-protective effects, and reduced the NR and ischemic areas. Pathological staining showed that TMYX could significantly reduce inflammatory cell number and interstitial edema. The activities of CK, LDH, and MDA were reduced, NO activity was increased, and oxidative stress was suppressed after treatment with TMYX. TMYX not only enhanced the expression of Gs-α, AC, PKA, and eNOS but also increased the expression of sGC and PKG. Furthermore, TMYX treatment significantly decreased ROCK expression. We further showed that TMYX (25–200 mg/mL) relaxed isolated coronary microvessels. ConclusionsTMYX attenuates myocardial NR after ischemia and reperfusion by activating the cAMP/PKA and NO/cGMP signaling pathways, further upregulating NO activity and relaxing coronary microvessels.

Basic Research on Neuroprotective Effect of Volatile Oil of Cang Ai after Cerebral Ischemia-Reperfusion Injury Based on Diffusion Tensor Imaging

To study the neuroprotective effect of inhalation of volatile oil of Cang Ai (VOCA) on cerebral ischemia-reperfusion injury model by MRI diffusion tensor imaging. Twenty-four healthy adult male SD rats were randomly divided into sham operation group, model (middle cerebral artery occlusion (MCAO) ) group and VOCA group. Evaluated the degree of neurological impairment of rats in each group immediately after successful establishment of model or 7 d later according to Zea Longa scoring. Coronal diffusion tensor imaging (DTI) scan was performed at 3 h, 3 d, and 7 d after the model successfully established by using 7.0 T magnetic resonance imaging. Measured the apparent diffusion coefficient (ADC) and anisotropy score (FA) of the DTI in the striatal region and the motion flat zone of the maximum infarct level and then calculate the relative apparent diffusion coefficient (rADC) and relative anisotropy score (rFA). TTC staining was used to evaluate the cerebral infarction volume of rats in each group at 7 d post model establishment, and the correlation analysis of rFA, rADC and neural score was performed. No neurological defect was detected in mice in the sham operation group. The MCAO group and the VOCA group showed neurological defect to different degrees. The neurological function score of the VOCA group was obviously lower than that of MCAO group at 7 th day (P<0.05). The DTI scan results showed that the rADC value of striatum of rats in VOCA group was higher than that in MCAO group at 3 h and 3 d after modeling (P<0.05), while there was no significant difference between the three groups at 7 th day. The rADC value of the motor cortex in the VOCA group was higher than that in the MCAO group at 3 h after modeling (P<0.01), and there was no significant difference at 3 rdday and 7 thday. The rFA value of striatum in VOCA group was higher than that in MCAO group at 3 rd day and 7 th day after modeling (P<0.05). There were no significant differences in rFA value between the MCAO and the VOCA group at three time points. TTC staining results showed that there was no infarcted area in the sham operation group, and the infarct volume in the VOCA group was smaller than that of the MCAO group (P<0.05). Correlation analysis showed that the striatum rFA value was highly correlated with neurological scores (r=－0.847, P<0.01). For the first time, we found that VOCA can effectively protect the neurological function of MCAO rats by reducing the toxic edema of cells in the ischemic area and accelerating the recovery of nerve fiber bundles after cerebral ischemia and reperfusion. rFA and rADC values can be used as effective indicators to evaluate the recovery of nerve function after cerebral ischemia and reperfusion.

Tongmai Yangxin pill reduces myocardial no-reflow by regulating apoptosis and activating PI3K/Akt/eNOS pathway

Ethnopharmacological relevanceTongmai Yangxin pill (TMYX) is derived from the Zhigancao decoction recorded in Shang han lun by Zhang Zhongjing during the Han dynasty and was further improved by Professor Ruan Shiyi, a cardiovascular expert at Tianjin University of Traditional Chinese Medicine. TMYX is used for the clinical treatment of chest pain, heartache, and qi-yin-deficiency coronary heart disease and can improve vascular endothelial function in patients with angina pectoris or coronary heart disease by up-regulating nitric oxide activity and then regulating vascular tension. Whether TMYX can further improve myocardial no-reflow by up-regulating NO activity and then dilating blood vessels remains unclear. Aim of the studyThis study aimed to reveal whether TMYX can further improve myocardial NR by up-regulating NO activity and then dilating blood vessels. The mechanism underlying PI3K/Akt/eNOS pathway activation and apoptosis regulation is also explored. Materials and methodsThe left anterior descending coronary arteries of healthy adult male SD rats were ligated to establish a NR model. The rats were assigned to 14 groups: control, sham, NR, TMYX (4.0 g/kg), sodium nitroprusside (SNP), Tongxinluo capsule (TXL), PI3K blocker (LY), TMYX + LY, SNP + LY, TXL + LY, eNOS blocker (L-NAME), TMYX + L-NAME, SNP + L-NAME, and TXL + L-NAME groups. Cardiac function was measured through echocardiography. Thioflavin S, Evans Blue, and TTC staining were adopted to evaluate NR and ischemic areas. Cell inflammation degree and edema were assessed by hematoxylin–eosin staining. Automated biochemical analyzer and kit were used to detect the activities of myocardial oxidants, including reactive oxygen species, super oxide dismutase, malonaldehyde, and NO. The expression levels of genes and proteins in the PI3K/Akt/eNOS signaling pathway and apoptosis were detected via real-time fluorescence quantitative PCR and Western blot analysis, respectively. A microvascular tension sensor was adopted to detect coronary artery diastolic function in vitro. ResultsTMYX reduced NR and ischemic areas; suppressed LV-mass; enhanced EF, FS, LVOT peak, and LVSV; and improved cardiac structure and function. Moreover, it decreased creatine kinase (CK), CK-MB, and lactic dehydrogenase activities. TMYX increased NO and super oxide dismutase activities; inhibited malonaldehyde activity; reduced muscle fiber swelling and inflammatory cell infiltration; and improved vasodilation in vitro. In the NR myocardium, TMYX stimulated myocardial PI3K activities and PI3K (Tyr458) phosphorylation and enhanced Akt activities and Akt phosphorylation at Tyr315. TMYX increased the activities of eNOS and the phosphorylation of eNOS at Ser1177 in the NR myocardium and attenuated cardiomyocyte apoptosis by increasing the expression of Bcl-2 and decreasing that of caspase-3 and Bax. All these effects of TMYX were abolished by the specific inhibitors of PI3K (LY) and eNOS (L-NAME). ConclusionsTMYX attenuates myocardial NR after ischemia and reperfusion by activating the PI3K/Akt/eNOS pathway and regulating apoptosis, further up-regulating NO activity and relaxing coronary microvessels.

Effect of astragaloside IV on cognitive dysfunction in rats with cerebrally infarcted via TGF-β / Smad signaling pathway

Cerebral infarction is an acute cerebrovascular disease caused by abnormal blood circulation in the brain. In the present study, we investigate the effect of astragaloside IV on cognitive dysfunction in cerebrally infarcted rats via transforming growth factor-β (TGF-β) / Smad signaling pathway. For this purpose, 45 rats were divided into three groups including astragaloside, model, and control. 30 of 45 healthy adult male SD rats were randomly selected to establish an acute cerebral infarction model. 15 modeled rats were enrolled as a model and astragaloside group, and another 15 rats as a blank control group. The rats in the astragaloside group were fed with astragaloside IV according to 1.08 g/kg body weight, and those in the blank group and model group were given matching normal saline. The levels of TGF-β, Smad1, Smad3 and Smad7 of TGF-β/Smad signaling transduction pathway at T0 (week 0), T1 (week 3) and T2 (week 6) were determined by enzyme-linked immunosorbent assay (ELISA). The modified neurological severity score (mNSS) was used to evaluate the improvement of cognitive dysfunction in rats. The mNSS of rats with cerebral infarction in the astragaloside group was lower than that in the control group and model group (P&lt; 0.05). While the levels of TGF-β, Smad1, Smad3 and Smad7 in the astragaloside group were higher than those in the control group and model group (P&lt; 0.05). Astragaloside IV plays an important role in improving cognitive dysfunction in rats with cerebral infarction while affecting the levels of TGF-β, Smad1, Smad3 and Smad7 and activating TGF-β / Smad signaling pathway.

DNA methylation in the medial prefrontal cortex regulates alcohol-related behavior in rats.

Alcohol abuse causes tissue and organ damage, and may participate neuropsychiatric diseases. Studies have shown that DNA methylation plays an important role in gene expression and behavioral changes induced by alcohol, however the causative neurobiological mechanisms have not been clarified. In this study, 32 healthy adult male SD rats were randomly divided into a drinking water control group (n=16) and a chronic alcohol exposure group (n=16). The alcohol preference and locomotor activity of rats were evaluated by two-bottle choice test (TBCT) and open-field test (OFT). DNA methylation in the medial prefrontal cortex (mPFC) tissue was detected by the reduced representative bisulfite sequencing (RRBS) technology. The methylation differential genes closely related to alcohol abuse were screened. qRT-PCR was used to verify the mRNA expression patterns of differential genes. qRT-PCR and Western blot were used to detect the expression of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MeCP2). Furthermore, the effect of short-term alcohol exposure (7 days) on DNMTs and MeCP2 in the mPFC of rats was tested (n=8/group). The results indicated that the methylation level of promoter region in the mPFC of rats exposed to chronic alcohol was significantly increased. In addition, the increased methylation levels in the promoter of Ntf3 and Ppm1G were accompanied by down-regulated mRNA levels in the chronic alcohol exposure group. The decreased methylation levels in the promoter of Hap1 and DUSP1 were accompanied by up-regulated mRNA levels. Furthermore, chronic alcohol exposure increased the mRNA and protein levels of DNMT3B and MeCP2. However, short term alcohol exposure did not affect their expression. This present study provides evidence that DNA methylation is associated with the development of alcohol abuse, which may be regulated by DNMT3B and MeCP2. The target genes Ntf3, Ppm1G, Hap1, and DUSP1 related to alcohol abuse were discovered as well, providing new insights into the neurobiological mechanism of alcohol abuse and the potential pharmacological targets for the treatment of alcohol abuse.

The changes of the expression of 2-type small conductance-Ca2+- activating-K+ (SK2) channel protein in hypertensive rat myocardium

To investigate the expression of 2-type small conductance-Ca2+-activating-K+ (SK2) channel protein in hypertensive rat myocardial cells. Twelve healthy adult male SD rats were randomly divided into control group (n=5) and experimental group (n=7). The rats of experimental group were injected intraperitoneally with N'-nitro-L-arginine (L-NNA 15 mg/(kg·d))while the rats of control group were injected intraperitoneally with isometrical normal saline(15 ml/(kg·d )). The body weight, blood pressure and electrocardiogram of the rats were measured every week. After 4 weeks, the rats were sacrificed to obtain hearts, and the expression of SK2 channel protein in myocardium was detected by Western blot. After 4 weeks of administration, compared with the control group, the blood pressure in the experimental group was significantly elevated (P＜0.05), QRS duration and R-R interval were prolonged, and the expressions of SK2 channel in the atrial and ventricular tissue of the experimental group were significantly higher than those in the control group (1.12±0.18,1.64±0.26, P ＜ 0.05). The expressions of atrial and ventricular SK2 pathway are increased in hypertensive model rats. It may be one of the mechanism leading to arrhythmias in hypertensive model rats and can provide new ideas and strategies for the treatment and prognosis of hypertensive diseases.

Effect of basic fibroblast growth factor on autophagy of nerve cells in rats after traumatic brain injury

Objective To investigate the effect of basic fibroblast growth factor (bFGF) on autophagy of nerve cells in rats after traumatic brain injury (TBI). Methods A total of 120 healthy adult male SD rats were randomly divided into sham group, TBI+ vehicle group, and TBI+ bFGF group by random number table method, with 40 rats in each group. PinPoint™ Precision Cortical Impactor was used to simulate the pathological damage after TBI. In the sham group, the dura was exposed without impact. In the TBI+ bFGF group, 250 μg/kg of human recombinant bFGF was given in the nasal cavity 1 hour before the modeling, while in the sham group and TBI+ Vehicle group, the same amount of saline was given in the nasal cavity 1 hour before the modeling. The necrotic cells were observed by propidium iodide(PI)staining 6 hours after injury. The effect of bFGF on the nerve function after TBI in rats was evaluated with modified neurological severity score (mNSS) 24 hours after injury. The water content of brain tissue was measured by dry and wet method 48 hours after injury. The ratio of Beclin-1, P62 protein and microtubule-associated protein 1 light 3 (LC3)-II/I protein was detected by western blot. The volume of brain injury was calculated by integral method of brain tissue section. The positive neuron specific nuclear protein (NeuN) cells were observed by immunofluorescence staining. The apoptotic cells were observed by TUNEL. Results Compared with the sham group [(4.0±1.2)%], the percentage of necrotic cells in TBI+ vehicle group [(54.3±10.1)%] and TBI+ bFGF group [(34.5±10.5)%] increased significantly (P<0.05), but the percentage of necrotic cells in TBI+ bFGF group increased less than that in TBI+ vehicle group (P<0.05). Compared with the sham group [(0.3±0.5)points], the mNSS in the TBI+ vehicle group [(5.8±0.8)points] and TBI+ bFGF group [(4.7±1.1)points] were significantly increased (P<0.05), but the mNSS of TBI+ bFGF group was lower than that of TBI+ vehicle group (P<0.05). Compared with the sham group [(76.7±0.7)%], the water content of brain tissue of TBI+ vehicle group [(79.2±0.5)%] and TBI+ bFGF group [(78.4±1.0)%]were significantly increased (P<0.05), but the water content of TBI+ bFGF group was significantly lower than that of TBI+ vehicle group (P<0.05). Compared with the sham group, protein expression of Beclin-1 and LC3-II/I in TBI+ vehicle group and TBI+ bFGF group were significantly improved (P<0.05), P62 protein expression was significantly decreased (P<0.05), the volume of brain tissue injury was significantly increased (P<0.05), the number of positive NeuN cells increased significantly (P<0.05), and the number of apoptotic cells and apoptotic cells were significantly increased (P<0.05). Compared with the TBI+ Vehicle group, the up-regulation of Beclin-1 protein and LC3-II/I protein ratio was obviously inhibited in the TBI+ bFGF group (P<0.05), the down-regulation of P62 protien was significantly suppressed, the volume of brain tissue injury was significantly decreased, and the number of positive NeuN cells and apoptotic cells was significantly reduced (P<0.05). Conclusion The bFGF can significantly inhibit excessive autophagy in rats after TBI, reduce brain edema, reduce cell apoptosis and necrosis, and improve neural function. Key words: Fibroblast growth factor 2; Autophagy; Brain injuries; Brain edema; Apoptosis

Effect of lead exposure on the accumulation of copper and iron in central nervous system of rats

Objective: To investigate the dynamic changes of copper and iron contents in brain tissue, body fluids and barriers of rats exposed to lead at different periods in order to provide a theoretical basis for the study of the mechanism of lead nerve injury. Methods: Sixty-four healthy adult SPF male SD rats were randomly divided into control group and lead exposure group, after one week of adaptive feeding, rats in the lead exposure group were treated with 250 mg/L lead acetate, and rats in control group were treated with ordinary drinking water, the experimental period was 12 weeks. After exposure for 3, 6, 9 and 12 weeks, the samples including blood, choroid plexus, cerebrospinal fluid, cortex, hippocampus, striatum, hypothalamus, amygdala, substantia nigra and cerebellum were obtained. Lead, copper and iron content in all kinds of samples were detected by Inductively Coupled Plasma Mass Spectrometry(ICP-MS). The measurement data were presented as Mean±SD, Comparison of metal contents in different tissues of rats at different time analyzed using repeated measurement analysis of variance, Two-variable correlation analysis using Spearman correlation test.The relationship between lead exposure experiod and copper and iron in samples was studied by using trend test. Results: After 12 weeks of lead exposure compared with the control group, lead contents in cortex, hippocampus, striatum, hypothalamus, amygdala, substantia nigra and cerebellum of rats were 2.21, 2.44, 2.95, 3.53, 4.01, 1.85 and 2.86 folds of control group, and the differences were statistically significant(P<0.05). At the same time, lead content in blood, cerebrospinal fluid,choroid plexus, brain microvessels and bones increased. The increase rate in the amygdala and cerebrospinal fluid ranked first among brain tissue or barrier,which were 4.01 and 3.0 folds respectively. Compared with the control group, Compared with the control group, copper content in cortex,hippocampus, striatum, hypothalamus,amygdala, cerebellum,blood,cerebrospinal fluid,choroid plexus and cerebral microvasculature showed an increasing trend among rats following 3,6,9,12 weeks of lead exposure. Copper content change in the striatum was highest among all brain tissue. The increase rate of copper content in the striatum was at the top among brain tissues. After 12 weeks of lead exposure,copper content in brain microvessels was 4.98 folds higher than that of the control group (P<0.05). After lead exposure at different periods,the iron content in the cortex, hippocampus, striatum,cerebrospinal fluid,choroid plexus and brain microvessels of experimental rats all increased(P<0.05). And the iron increase rate in the hypothalamus or cerebrospinal fluid increase ranked first among brain tissues or body fluid the most obviously. Conclusion: With the increase of exposure time, lead exposure can changes in the contents of copper and iron in different brain tissues,body fluids and barriers in rats,among which, the contents of copper and iron in the amygdala,cerebrospinal fluid and brain microvessels increase significantly. This may be related to nerve damage from lead exposure.

PO-243 Correlation between exercise performance and muscle electrical activity in Exercise-induced Fatigue Rats

Objective Assess the muscle contraction ability of rats before and after exercise fatigue quantitatively, and analyze the correlation between exercise performance and muscle electrical activity. This study intends to provide a theoretical principle for exercise fatigue.&#x0D; Methods 7 healthy adult male SD rats (300~350g) were used and trained for adaptive treadmill by running on the treadmill for 1 week and holding on a vertical rectangular railing (20×10cm, 10 iron bars) until it's mastered. After this training, we used rat grasping force tester( BioSEB GS3) to measure the maximum grasping force(MGF) of rat's limbs, each rat was carried out 3 times, bout interval is 2 minutes. In addition, the grasping bar time(GBT) was recorded 3 times , bout interval is 30 minutes. We let rats to grasp and hold on a vertical rectangular railing(20×10cm， 10 iron bars), evaluation of rat muscular endurance by grasping bar time(GBT), each rat was carried out 3 times, bout interval is 30 minutes; During the MGF and GBT test, motor unit recruitment and discharge frequency was predicted by measuring the EMG of extensor muscles of the right hindlimb and flexor elbow muscles of the right forelimb by wireless non-invasive miniature surface EMG tester (Italy, BTS FREEEMG), the max Root Mean Square (maxRMS) and Median Frequency (MF) parameter was used to evaluate motor unit recruitment and discharge frequency, respectively.&#x0D; After these, the rats were allowed to have a one-day rest, and then had a load motion program on the treadmill (three levels’ load: the first stage movement speed 8.2 m/min, exercise time 15 min; second stage speed 15 m/min, exercise time 15 min; third stage speed at 20 m/min, exercise to fatigue ) to build the rats EF model by monitoring the acceleration of the rat's sprint with a miniature wireless acceleration sensor (18g). 30 continuous sprint acceleration at the end of running was less than half of initial acceleration and the running posture of the rats changed to prostrate, and remained at the end of the runway for a long time. Later, quantitative correlational data analyses such as mean, Pearson correlation, analysis of one-way ANOVA and paired sample t test were performed in this study.&#x0D; Results (1) The rats’ sprint acceleration of treadmill exercise at the end stage (the final 1/5 of the total time) decreased by 56.9% (P &lt; 0.01) when compared with the early stage (the begining 1/5 of the total time). (2) The MGF and GBT of EF decreased by 68.1% (P &lt; 0.01) and 90.38% (P &lt; 0.01), respectively when compared with the beginning EF; in addition, the EMG maxRMS and MF of hindlimb and forelimb of EF rats had significantly reduced (P &lt; 0.01), and the rats’ MGF/GBT was positively correlated with EMG maxRMS/MF significantly (MGF: forelimb rmaxRMS = 0.901, P &lt; 0.01，rMF = 0.761, P &lt; 0.01; hindlimb rmaxRMS = 0.913, P &lt; 0.01，rMF = 0.783, P &lt; 0.01; GBT: forelimb rmaxRMS = 0.922, P &lt; 0.01，rMF = 0.806, P &lt; 0.01; hindlimb rmaxRMS = 0.908, P &lt; 0.01，rMF = 0.896, P &lt; 0.01).&#x0D; Conclusions Exercise fatigue reduced the muscle strength, muscular endurance and muscle power of rates significantly, which may be related to the decreased recruitment, rhythm synchronization and discharge frequency of muscle motor units of forelimb flexor and hindlimb extensor.

Role of p38MAPK signaling pathway in rats with phantom limb pain

To investigate the role of p38MAPK signal pathway in spinal cord and dorsal root ganglion (DRG) in rats with phantom limb pain and the effects of specific inhibitors. Methods: Healthy adult male SD rats (n=48) were cut off one side of the sciatic under anesthesia to establish a model of phantom limb pain. In addition, the healthy rats were taken as a sham group (group S, n=24). The animals were scored by observing the action of chewing (0=no chewing, 13=the worst chewing) after the operation and were sacrificed on the following day after the operation. The successful model of phantom limb pain were randomly divided into 2 groups: a phantom limb pain group (group P, n=24) and a phantom limb pain plus inhibitor group (group P+I, n=24). SB203580 was given to the rat at 0.8 mg/kg on every Monday until the rats were sacrificed, the rest of the rats received an equal amount of saline. Eight rats from each group were randomly taken for the determination of levels of P-p38MAPK in spinal cord and DRG before administration and on the 4th, 6th, 8th weekend following the administration, respectively. Results: In the sham group, no animal developed chewing. Meanwhile, rats in successful model of phantom limb pain group began chewing from the 2nd day after operation with scores at eight to eleven. The chewing scores in the P+I group were reduced after the treatment. Compared with group S, P-p38MAPK levels were elevated in groups of P and P+I (P<0.05 or P<0.01). Compared with group P, P-p38MAPK level was decreased in the group P+I (P<0.05 or P<0.01). Conclusion: P38MAPK signal pathway involves in the development of phantom limb pain.

Application and research of UW solution in replantation of rat limb

Objective To explore the application of UW solution in the replantation of the rat limb, and to explore a simple method for the ideal preservation of the amputated limbs. Methods From September, 2017 to Janurary, 2018, one hundred and twenty healthy adult male SD rats, weight 320-350 g, were randomly divided into 8 groups: A, B, C, D, E, F, G, H group. The isolated limbs of the control group A, C, E, G were cryopreserved in refrigerator at 4 °C, while those of the experiment group B, D, F, H were preserved under the same condition after being perfused with UW solution. All the severed limbs were replanted back to the rats after different time length of cry opreservation: A, B 12 hours, C, D 20 hours, E, F 28 hours and G, H 36 hours. During the operations the femoral artery and venous patency were detected immediately after anastomosis. And after the operations the survival rates of limb replantation were observed. On the third day after the limb replantation, the tibial posterior muscle tissue was examined for pathology. The cell areas in pathological photographs were compared with Image analysis software Image-pro Plus 6.0 and the ultra structural changes of skeletal muscle tissue were observed. Results The femoral artery and venous patency in experiment group A, C, E and control group B, D, F were all 100 percent. However, in the group G, H the severed limb vessels changed obviously, the patency dropped markedly and the survival rates of limb replantation turned out to be 0. The experiment groups perfused with UW solutions, had relatively higher survival rates compared with the control groups (12 h: 93.33% vs. 53.33%, 20 h: 80.00% vs. 40.00%,28 h: 53.33% vs. 13.33%) and the skeletal muscles edema was lighter than that of the control group, with a larger proportion of cell areas(12 h: 69.49±8.57 vs. 44.75±6.34, 20 h: 56.34±7.73 vs. 36.69±5.74, 28 h: 45.76±8.23 vs. 31.49±7.72). There was a significantly difference(P<0.05). Electron microscope observation showed that the experiment group bore a lighter damage of myofibril, interstitial tissue and mitochondria compared with the control group. Conclusion The combination of UW solution and cryopreservation of organs is an ideal and proper way to preserve the amputation limb, to extend the time limit for replantation, and to achieve a better recovery effect of postoperative limb function. Besides, UW solution is easy to be promoted in the treatment of isolated limb in clinical work. Key words: UW solution; Skeletal; Organ preservation; Replantation of severed limbs; Rat

Diosgenin Protects Rats from Myocardial Inflammatory Injury Induced by Ischemia-Reperfusion

BackgroundDiosgenin, a phytosteroid sapogenin, has anti-inflammatory properties shown to reduce myocardial ischemia-reperfusion injury (MIRI). However, the specific mechanism by which this is achieved is not clear. This study investigated the protective effects of diosgenin on myocardial ischemia/reperfusion (I/R) and the potential anti-inflammatory mechanisms.Material/MethodsHealthy adult male SD rats, body weight (b.w.) 250–280 g, were used to model ischemia-reperfusion injury (IRI) and were administered diosgenin (50 mg/kg and 100 mg/kg b.w.) intragastrically for 4 consecutive weeks before surgery. The left anterior descending artery (LAD) was ligated to induce myocardial ischemia for 30 min and reperfusion for 30 min, 60 min, and 120 min while relevant indicators were detected.ResultsBoth 50 mg and 100 mg diosgenin oral administration increased left ventricular developed pressure (LVDP) and maximum changing rate of ventricular pressure (±dp/dtmax), decreased left ventricular end-diastolic pressure (LVEDP), and myocardial enzyme markers. TTC staining suggested that diosgenin reduced myocardial infarct size in the rat model. Pathological results showed that myocardial ischemia and inflammation were alleviated by diosgenin. In addition, the increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) in serum, and myeloperoxidase (MPO) in myocardium were significantly suppressed by diosgenin administration. Diosgenin further inhibited the phosphorylation of transcription factor NF-κB and modulated the expression of downstream inflammatory cytokines by regulating the activation of p38-MAPK and JNK pathways.ConclusionsResults demonstrate diosgenin plays an anti-inflammatory role in the protection of MIRI through regulation of p38-MAPK and JNK pathways and phosphorylation of NF-κB.

Time-dependent Protein Expressions of MMP-2 and MMP-9 in Liver Contusion Rats after Impact

To observe the protein expression patterns of matrix metalloproteinase （MMP）-2 and MMP-9 in the liver tissue of liver contusion rats at different time after impact. Fifty healthy adult male SD rats were randomly and evenly divided into control group and experimental groups （1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 3 d, 5 d, 7 d after liver contusion）. A rat liver contusion model was established by a free-falling device. The rats were killed at corresponding time, and the contused hepatic lobes were extracted. The protein expressions of MMP-2 and MMP-9 in contused liver tissue of the rats in each group were observed by immunohistochemical staining （SP method） and Western blotting. After the liver contusion, the expression of positive cell and the protein semiquantitative result showed that the protein expression of MMP-2 enhanced at 6 h and peaked at 24 h, then decreased gradually at 3-5 d, and returned to normal levels at 7 d. The difference of expression between group and its previous adjacent group after 6 h （except 18 h） had statistical significance （P<0.05）. The protein expression of MMP-9 rose obviously at 1 h after liver contusion and peaked at 18 h, then decreased gradually at 3-7 d which still higher than control group. The expression difference between group and its previous adjacent group （except 12 h and 24 h） had statistical significance （P<0.05）. The protein expressions of MMP-2 and MMP-9 in contused liver tissue after impact show good time-dependent patterns, which may provide important reference indicators for the time estimation of liver contusion.

Effects of penehyclidine hydrochloride on severe acute pancreatitis-associated acute lung injury in rats

Penehyclidine hydrochloride (PHC) is a selective M1 and M3 receptor antagonist. This study was designed to investigate the effect of PHC on acute lung injury (ALI) induced by severe acute pancreatitis (SAP) and the expression of hypoxia-inducible factor-1α (HIF-1α) in rats. A total of 45 healthy adult male SD rats were randomly divided into 3 groups: an S group, sham operation; an ALI group, pancreatitis-associated acute lung injury (PALI); and a P group, PALI treated with PHC. Rats from the ALI and P groups were used to establish a model of acute lung injury associated with SAP by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct. Rats in the P group, reflecting acute lung injury caused by SAP, were treated with PHC immediately following SAP. Rats in the S and ALI groups were injected with the same amount of 0.9% sodium chloride solution. After modeling, the rats were sacrificed at 12h. The wet/dry weight (W/D) ratios of lung tissue were calculated. Pathological changes in pancreatic and lung tissues were scored. The expression levels of TLR4 and NF-κB p65 in lung tissue were detected by Western blot. RT-PCR was used to detect HIF-1α mRNA in lung tissue. The HIF-1α, IL-1β, and IL-6 expression levels in lung tissues and serum amylase levels were detected by ELISA. The results showed extensive infiltration of neutrophils, alveolar hemorrhage and necrosis and fat necrosis in the pancreatic tissue of rats in the PALI and P groups. Their pancreatic tissue injury scores were significantly higher than the score of the S group (P<0.01). However, no statistically significant difference was observed in the serum amylase levels of the P and ALI groups (P>0.05). The W/D ratios of lung tissue in the ALI and P group rats were significantly higher than those in the S group (P<0.05). Compared with those of the ALI group rats, the lung tissue pathological changes of the P group were significantly improved, and the lung W/D value was significantly lower than that of the ALI group (P<0.05). Compared with those of the S group, the TLR4, NF-κB p65, HIF-1α mRNA, and HIF-1α expression levels in the lung tissue of the ALI and P groups were significantly higher (P<0.01), and the TLR4, NF-κB p65, HIF-1α mRNA, HIF-1α, IL-1β and IL-6 expression levels in the P group were significantly lower than those in the ALI group (P<0.05). The current work indicates that PHC could not alleviate the damage to pancreatic tissue caused by SAP. However, PHC did suppress HIF-1α, IL-1β and IL-6 expression levels and reduced the acute lung injury induced by SAP in rats, which might depend on suppression of the expression of inflammatory factors, such as HIF-1α.

Effects of FTY720 pretreatment on ventilator-induced lung injury in rats

Objective To study the effects of FTY720 pretreatment on ventilator-induced lung injury (VILI) in rats in order to explore the role of sphingosine-1-phosphate receptor 1 (S1PR1). Methods Forty healthy adult male SD rats weighing 300-350 g were randomly (randlom number)divided into 4 groups (n=10 each): group CV (conventional tidal volume VT = 8 mL/kg), group HV (high tidal volume VT= 40 mL/kg), group HF and group HFW. The rats in group HF received intra-gastric administration of FTY720 10 mg·kg-1·d-1for 7 days, while additional dose of W146 (S1PR1 antagonist) 1 mg·kg-1·d-1 was administrated in group HFW before high tidal volume ventilation. After 4-hour mechanical ventilation, arterial blood samples were obtained for blood gas analysis before the animals were sacrificed. The lungs were harvested for histopathologic observation. Apoptosis rate in lung tissue was determined with flow cytometry. W/D lung weight ratio, pulmonary permeability index (PPI), total protein, and TNF-α, IL-1β in bronchoaveolar lavage fluid (BALF) were measured. All data were analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) test by using SPSS version 20.0 software. Differences were considered statistically significant if P<0.05. Results Compared with group CV, the level of PaO2 [(73.6± 8.9) vs.(50.5± 6.0)] was decreased, the levels of W/D [(3.12± 0.27)vs. (5.12± 0.56)], PPI [(0.08±0.03)vs.(0.30 ± 0.06)], apoptosis rate [(10.6±2.9)vs.(48.5±6.7)], total protein [(5.8±2.1)vs.(15.4±5.6)] and TNF-α [(24.3±5.7) vs. (108.4±16.0)] and IL-1β [(90.6±14.1) vs.(338.5±44.3)] were increased in group HV (P 0.05). Compare with group CV, The histopathologic damage of lung tissue was obvious in group HV and group HFW, it was attenuated by pretreatment with FTY720 in group HF. Conclusions FTY720 pretreatment provides protective effects against ventilation-induced lung injury in rats, and S1PR1 may mediate the protection through reducing apoptosis rate and inflammatory reaction. Key words: Receptions, Lysosphingolipid; Respiration, artificial; Respiratory distress syndrome, adult; Apoptosis; Inflammatory response; Rats