Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway / 中国当代儿科杂志

OBJECTIVES@#To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism.@*METHODS@#Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62).@*RESULTS@#Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05).@*CONCLUSIONS@#Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.

Effect of platelet-derived growth factor-BB on pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension and its mechanism / 中国当代儿科杂志

OBJECTIVES@#To study the effect of platelet-derived growth factor-BB (PDGF-BB) on pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension (HPH).@*METHODS@#A total of 128 neonatal rats were randomly divided into four groups: PDGF-BB+HPH, HPH, PDGF-BB+normal oxygen, and normal oxygen (n=32 each). The rats in the PDGF-BB+HPH and PDGF-BB+normal oxygen groups were given an injection of 13 μL 6×1010 PFU/mL adenovirus with PDGF-BB genevia the caudal vein. After 24 hours of adenovirus transfection, the rats in the HPH and PDGF-BB+HPH groups were used to establish a neonatal rat model of HPH. Right ventricular systolic pressure (RVSP) was measured on days 3, 7, 14, and 21 of hypoxia. Hematoxylin-eosin staining was used to observe pulmonary vascular morphological changes under an optical microscope, and vascular remodeling parameters (MA% and MT%) were also measured. Immunohistochemistry was used to measure the expression levels of PDGF-BB and proliferating cell nuclear antigen (PCNA) in lung tissue.@*RESULTS@#The rats in the PDGF-BB+HPH and HPH groups had a significantly higher RVSP than those of the same age in the normal oxygen group at each time point (P<0.05). The rats in the PDGF-BB+HPH group showed vascular remodeling on day 3 of hypoxia, while those in the HPH showed vascular remodeling on day 7 of hypoxia. On day 3 of hypoxia, the PDGF-BB+HPH group had significantly higher MA% and MT% than the HPH, PDGF-BB+normal oxygen, and normal oxygen groups (P<0.05). On days 7, 14, and 21 of hypoxia, the PDGF-BB+HPH and HPH groups had significantly higher MA% and MT% than the PDGF-BB+normal oxygen and normal oxygen groups (P<0.05). The PDGF-BB+HPH and HPH groups had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group at all time points (P<0.05). On days 3, 7, and 14 of hypoxia, the PDGF-BB+HPH group had significantly higher expression levels of PDGF-BB and PCNA than the HPH group (P<0.05), while the PDGF-BB+normal oxygen group had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group (P<0.05).@*CONCLUSIONS@#Exogenous administration of PDGF-BB in neonatal rats with HPH may upregulate the expression of PCNA, promote pulmonary vascular remodeling, and increase pulmonary artery pressure.

Expression of miR-155 and IFN-γ in lung injury model of neonatal rats with acute respiratory distress syndrome / 国际儿科学杂志

Objective:To study the expression of micro RNA-155(miR-155) and IFN-γ in lung tissue in a neonatal rat model of acute respiratory distress syndrome(ARDS)lung injury by intraperitoneal injection of lipopolysaccharide(LPS).Methods:Eighty neonatal SD rats on the 7th day after birth were assigned to the experimental group(LPS group)and control group(isotonic NaCl group), with 40 rats in each group.LPS solution(4 mg/kg)was injected into the abdominal cavity of neonatal SD rats in the experimental group to establish an animal model of neonatal acute respiratory distress syndrome(NARDS). The control group was established by isotonic NaCl solution(4 ml/kg)in the same way.The lung tissue samples were taken at 3 h, 6 h, 12 h and 24 h after drug administration to observe the surface changes.Then the lung sections were stained with HE to observe the pathological changes and score the lung tissue injury.Finally, the expression levels of miR-155 and IFN-γ in the lung tissue were tested by RT-PCR and ELISA techniques, respectively.Results:(1)At the beginning of the experiment, the neonatal rats in the experimental group gradually showed the clinical manifestations of ARDS, and the macroscopic observation, pathological changes and lung tissue injury scores of the lung tissues suggested the appearance of NARDS lung injury, indicating that the model was successful.(2)The expression levels of miR-155(1.33±0.12 vs 0.95±0.02、1.77±0.17 vs 0.96±0.01、2.18±0.09 vs 0.96±0.02 and 2.43±0.06 vs 0.96±0.02)and IFN-γ(370.79±13.89 vs 273.03±11.44、424.24±10.11vs270.70±13.05、466.63±6.57 vs 268.11±7.88 and 519.13±7.09 vs 272.97±12.54)ng/L in the lung tissue of rats between the experimental group and the control group were significantly different( P<0.01), and the difference was statistically significant among the groups in the experimental group( F values were 165.983 and 408.574, P<0.01). The expression levels of miR-155 and IFN-γ in the lung tissue of the experimental group increased gradually over time and showed an increasing trend. Conclusion:After the successful establishment of NARDS animal model, the expression levels of miR-155 and IFN-γ in the lung tissue of NARDS rats have significantly increased and showed a sequential pattern.MiR-155 is expected to become an early biomarker for the diagnosis of NARDS.

Panax quinquefolium saponin Optimizes Energy Homeostasis by Modulating AMPK-Activated Metabolic Pathways in Hypoxia-Reperfusion Induced Cardiomyocytes / 中国结合医学杂志

OBJECTIVE@#To investigate the effects and underlying mechanisms of Panax quinquefolium saponin (PQS) on energy deficiency in hypoxia-reperfusion (H/R) induced cardiomyocytes.@*METHODS@#The H/R injury involved hypoxia for 3 h and then reperfusion for 2 h. Cardiomyocytes recruited from neonatal rat ventricular myocytes (NRVMs) were randomly divided into control, H/R, H/R+compound C (C.C), H/R+PQS, and H/R+C. C+PQS groups. BrdU assay, lactase dehydrogenase (LDH) leakage and early apoptosis rate were evaluated to assess cell damages. Contents of high energy phosphate compounds were conducted to detect the energy production. Protein expression levels of adenosine monophosphate-activated protein kinase a (AMPKα), glucose transporter 4 (GLUT4), phosphate fructose kinase 2 (PFK2), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and acetyl CoA carboxylase 2 (ACC2) in the regulatory pathways were measured by Western blotting. Immunofluorescence staining of GLUT4 and FAT/CD36 was used to observe the mobilization of metabolic transporters.@*RESULTS@#PQS (50 mg/L) pretreatment significantly alleviated H/R-induced inhibition of NRVMs viability, up-regulation of LDH leakage, acceleration of early apoptosis, and reduction of energy production (P<0.05). Compared with the H/R group, up-regulated expression of AMPKα, GLUT4, PFK2, FAT/CD36 and ACC2 were observed, and more GLUT4 and FAT/CD36 expressions were detected on the membrane in the H/R+PQS group (P<0.05). These effects of PQS on H/R-induced NRVMs were eliminated in the H/R+C.C+PQS group (P<0.05).@*CONCLUSION@#PQS has prominent advantages in protecting NRVMs from H/R-induced cell damages and energy metabolic disorders, by activation of AMPKα-mediated GLUT4-PFK2 and FAT/CD36-ACC2 pathways.

Difference Analysis of Pharmacokinetic Behavior of Active Ingredients in Wujiwan Between Normal Rats and Rats with Irritable Bowel Syndrome / 中国实验方剂学杂志

Objective:To establish an ultra-performance liquid chromatography-mass spectrometry （UPLC-MS/MS） for determining the plasma concentrations of 10 active ingredients in Wujiwan at different time points after oral administration， and to compare the pharmacokinetic characteristics between normal rats and rats with chronic visceral hypersensitive irritable bowel syndrome （CVH-IBS）. Method:CVH-IBS rat model was prepared by the neonatal rat colon percutaneous transluminal coronary angioplasty （PTCA） balloon stimulation method. After intragastric administration of Wujiwan （0.245 g·kg^-1）， blood was collected from the jugular vein at different time points， and the plasma concentrations of 10 active ingredients （berberine hydrochloride， palmatine hydrochloride， coptisine hydrochloride， jatrorrhizine hydrochloride， epiberberine， dihydroberberine， evodiamine， evodine， paeoniflorin， albiflorin） in Wujiwan was detected simultaneously by UPLC-MS/MS， the pharmacokinetic parameters of each component in normal rats and CVH-IBS rats were calculated. Result:The established UPLC-MS/MS could sensitively and accurately detect the plasma concentrations of 10 active ingredients of Wujiwan in rats. Compared with the normal group， the absorption rates of these 10 active ingredients of Wujiwan in the blood of CVH-IBS rats all decreased to a certain extent， and the peak time （<italic>t</italic>_max） was prolonged. Among them， the <italic>t</italic>_max of berberine hydrochloride and jatrorrhizine hydrochloride were significantly prolonged from 54 minute and 39 minute to 90 minute， respectively （<italic>P</italic><0.05， <italic>P</italic><0.01）. Area under the plasma concentration-time curve （AUC_0-<italic>_t</italic>） of each component increased， and evodiamine and paeoniflorin were significantly different （<italic>P</italic><0.05，<italic> P</italic><0.01）. The clearance rates （CL/<italic>F</italic>） of these 10 active ingredients were all decreased， among which berberine hydrochloride， palmatine hydrochloride and evodiamine had significant differences （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:There are significant differences in the pharmacokinetic behavior of the active ingredients in Wujiwan between normal rats and CVH-IBS rats， which may be related to the destruction of microstructure of intestinal epithelial cells and the change of activity of liver enzymes under the pathological state of IBS.

Role of T-type Calcium Channels in Generating Hyperexcitatory Behaviors during Emergence from Sevoflurane Anesthesia in Neonatal Rats / 神经科学通报·英文版

In the current study, we sought to investigate whether T-type Ca channels (TCCs) in the brain are involved in generating post-anesthetic hyperexcitatory behaviors (PAHBs). We found that younger rat pups (postnatal days 9-11) had a higher incidence of PAHBs and higher PAHB scores than older pups (postnatal days 16-18) during emergence from sevoflurane anesthesia. The power spectrum of the theta oscillations (4 Hz-8 Hz) in the prefrontal cortex was significantly enhanced in younger pups when PAHBs occurred, while there were no significant changes in older pups. Both the power of theta oscillations and the level of PAHBs were significantly reduced by the administration of TCC inhibitors. Moreover, the sensitivity of TCCs in the medial dorsal thalamic nucleus to sevoflurane was found to increase with age by investigating the kinetic properties of TCCs in vitro. TCCs were activated by potentiated GABAergic depolarization with a sub-anesthetic dose of sevoflurane (1%). These data suggest that (1) TCCs in the brain contribute to the generation of PAHBs and the concomitant electroencephalographic changes; (2) the stronger inhibitory effect of sevoflurane contributes to the lack of PAHBs in older rats; and (3) the contribution of TCCs to PAHBs is not mediated by a direct effect of sevoflurane on TCCs.

Effects of Resveratrol on Cognitive Function and SIRT 1/NF-κB Signaling Pathway in Neonatal Rats with Hypoxic-ischemic Brain Injury / 中国药房

OBJECTIVE： To study the effects of resveratrol （Res） on cognitive function and SIRT1/NF-κB signaling pathway in neonatal rats with hypoxic-ischemic brain injury. METHODS： SD neonatal rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， Res low-dose and high-dose groups （30， 60 mg/kg）， with 12 rats in each group. Except that sham operation group received sham operation， hypoxic-ischemic brain injury model was established by Rice method in other groups. After modeling， the rats were given relevant medicine intraperitoneally each day， for consecutive 6 weeks. Water maze test was used to analyze spatial learning and memory function of rats in each group. The escape latency after 1， 3 and 6 weeks of administration and the times of crossing platform after 6 weeks of administration were recorded. TTC staining was used to detect cerebral infraction area of rats after 6 weeks of medication. Western blot was used to detect the expression of Bcl-2， Bax， Caspase-3， SIRT1， SIRT1/NF-κB pathway related protein SIRT1 and p-NF-κB in hippocampal CA1 region. RESULTS： Compared with sham operation group， escape latency of rats was prolonged significantly in model group after 1， 3， 6 weeks of medication （P＜0.05）， the times of crossing platform was decreased significantly after 6 weeks of medication （P＜0.05）； the area of cerebral infarction was increased significantly （P＜0.05）； the protein expression of Bax， Caspase-3 and p-NF-κB in hippocampus CA1 region were increased significantly， while the protein expression of Bcl-2 and SIRT1 were decreased significantly （P＜0.05）. Compared with model group， the escape latency of Res low-dose and high-dose groups were shortened significantly after 1， 3， 6 weeks of medication （P＜0.05）， while the times of crossing platform was increased significantly after 6 weeks of medication （P＜0.05）； the area of cerebral infarction was decreased significantly （P＜0.05）， and the protein expression of Bax， Caspase-3 and p-NF-κB protein in hippocampal CA1 area were decreased significantly， while the protein expression of Bcl-2 and SIRT1 were increased significantly （P＜0.05）. The improvement of above indexes in high-dose group were significantly better than low-dose group （P＜0.05）. CONCLUSIONS： Res can improve cognitive dysfunction in neonatal rats with hypoxic-ischemic brain injury， which is related with SIRT1/NF-κB signaling pathway.

Effects of Xenon on expression of CLIC4 mRNA in neonatal rats brain with white matter damage / 中华实用儿科临床杂志

Objective To investigate the mechanism of white matter damage (WMD) and the neuroprotective effect of Xenon on neonates with WMD.Methods Three-day-old SD rat pups (n =96) were randomly divided into the blank control group (n =24),the WMD control group (n =24),the Xenon intervention group A (n =24) and the Xenon intervention group B (n =24) by random number method according to their birth time.WMD rat models were successfully established by giving intraperitoneal injection of lipopolysaccharide(LPS) 0.05 mg/kg combined with carotid artery ligation and hypoxia for 1 hour in the WMD control group and the Xenon intervention groups.In the control group,only 9 g/L saline (0.05 mg/kg) was injected intraperitoneally,while carotid artery ligation and hypoxia were not administered.Rats in Xenon intervention group A and group B were given inhalation of 500 mL/L Xenon for 3 hours at 0 and 2 hours respectively after establishment of the models.Six rats in each group were randomly selected and decapitated at 0,24,48 and 72 hours after the intervention.The brain white matter on the right was analyzed by using HE staining and myelin basic protein(MBP) immunofluorescence staining,and real-time quantitative polymerase chain reaction was used to detect the expressions level of CLIC4 mRNA.Results (1) Brain tissue pathology:compared with the blank control group,the brain white matter on the right of the WMD control group and the Xenon intervention group A and group B had loose and disordered structure,nuclear pyknosis and cytoplasm loosening.However,the lesions in both Xenon intervention group A and group B were significantly less than those in the WMD control group,and there was no significant difference between the Xenon intervention group A and group B.(2) MBP measurement:the number of MBP-positive cells in the brain white matter on the right of WMD control group was significantly lower than that in the blank control group,while compared with WMD control group,they were significantly higher in Xenon intervention group A and group B.(3) CLIC4 mRNA expression level:compared with blank control group,the expressions levels of CLIC4 mRNA at most time point were higher both in the WMD control group and the Xenon intervention group A and group B (all P ＜ 0.05),except the time point 24 h in the Xenon intervention group A.The expressions of CLIC4 mRNA in group A and group B were significantly decreased compared with those in the WMD control group (all P ＜ 0.05).However,there were no significant differences between Xenon intervention group A and group B (P ＞ 0.05).Conclusions The expressions of CLIC4 mRNA in brain tissues on neonatal rats with WMD significantly increased,indicating that the mitochondrial pathway could be one of the pathological processes of WMD.Early Xenon intervention may reduce neonatal WMD by reducing the expression of CLIC4 mRNA,which plays a neuroprotective role.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts DNA protection against hypoxia / 中国药理学通报

Aim To investigate the protective roles of sonic hedgehog( Shh) signaling pathway in hypoxia-in-duced DNA damage with the neonatal rat cardiomyo-cytes. Methods The hypoxia model on neonatal car-diomyocytes was established with one to two days old Sprague Dawley rats by deprivation of oxygen and glu-cose ( OGD) . After pretreated with Shh pathway ago-nist SAG1.3 or antagonist GANT61, the survival rates of cardiomyocytes were assayed by MTT after OGD 6 hours or 12 hours. The protein levels of Shh pathway, phosphorylated histone H2AX at serine 139 (γH2AX), phosphorylated ATM (p-ATM), phospho-rylated p53 ( p-p53 ) , cleaved-caspase-3, Bcl-2 and Bax were detected by Western blot. The γH2AX foci was detected by immunofluorescence. Results Com-pared to control group, the protein expression of γH2AX, p-ATM, cleaved-caspase-3, p-p53 in OGD cardiomyocytes significantly increased, and Bcl-2/Bax ratio proportionally decreased. Particularly, the ex-pression of γH2AX, p-ATM was highest at OGD 6 h, and then gradually declined after OGD 12 h. After SAG1.3 pretreatment, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 dramatically de-creased and the Bcl2/Bax ratio increased in OGD 6 h or OGD 12 h cardiomyocytes. On the contrary, in GANT61 pretreatment group, the expression of γH2AX, p-ATM, cleaved-caspase-3 and p-p53 signifi-cantly increased and the Bcl-2/Bax ratio decreased compared to the OGD 6 h or OGD 12 h cardiomyo-cytes. Conclusion The activation of Shh pathway protects cardiomyocytes against hypoxia-induced apop-tosis through inhibition of DNA damage.

A model of transplanting neonatal cardiomycytes into the wall of rat inferior vena cava / 中国胸心血管外科临床杂志

@#Objective     To establish a model of transplanting neonatal cardiomycytes into the wall of rat inferior vena cava. Methods     Neonatal cardiomyocytes (n=6, 5×106cells each, A group) or medium (n=6, B group) only were transplanted into the wall of inferior vena cava in female Fisher rats. At 21 days after transplantation, the contraction of transplanted cardiomyocytes was assessed and the inferior vena cava was processed for histology. Results     Distinct rhythmic beating of the vena cava at the site of cell transplantation before and after the aorties were clamped (at a rate 141± 47 rpm and 88± 44 rpm which was dramaticly lower than aortic beating, with a statistical difference at P value of 0.03). Cardiomyocyte was seen in 6 rats who had neonatal cardiomyocyte transplantation, but not in 6 rats receiving media. Hematoxylin and eosin staining showed viable cardiomyocytes in the wall of the vena cava in 6 rats treated with neonatal cardiomyocytes, but not in 6 rats receiving media. Conclusion     This study shows that neonatal cardiomyocytes can survive, mature and spontaneously and rhythmically contract after they are transplanted in the wall of inferior vena cava.

Resveratrol reduces electrical remodeling in atrial fibrillation by down-regulating microRNA-21 in neonatal rat atrial myocytes / 中国病理生理杂志

AIM: To detect the effects of resveratrol (RSV) on the expression of microRNA-21 (miR-21) in primarily cultured neonatal rat atrial myocytes with electric remodeling induced by rapid electrical stimulation (RES).Furthermore, to find out the possible mechanism of miR-21 regulating electrical remodeling.METHODS: The neonatal rat atrial myocytes were isolated by double-enzyme (trypsin and collagenase I) digestion and differential adhesion method.The atrial fibrillation (AF) model was induced by RES.Atrial myocytes were randomly divided into 4 groups: control group, RSV group, RES group, and RSV+RES group.To further detect whether RSV regulated electric remodeling by miR-21, except the 4 groups, we add miR-21 over-expression group and miR-21 inhibitor group: RES+negative control (NC) group, RES+miR-21 mimics group, RES+miR-21 mimics+RSV group, RES+miR-21 inhibitor group, and RES+miR-21 inhibitor+RSV group.The optimal concentration and pretreatment time of resveratrol were determined by CCK-8 assay.The expression of miR-21 and the mRNA expression of L-type calcium channels CACNA1C and CACNB2 in atrial myocytes were detected by qPCR.The protein expression of L-type calcium channels Cav1.2 and Cavβ2 in the atrial myocytes was analyzed by Western blot.RESULTS: The expression of miR-21 in RES group was significantly increased compared with control group, while preconditioning with RSV decreased the expression of miR-21.Compared with RES+miR-21 mimics group, the expression of miR-21 in RES+miR-21 mimics+RSV group was significantly decreased.Meanwhile, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased (P<0.05).Compared with RES group, the expression of miR-21 in RES+miR-21 inhibitor group and RES+miR-21 inhibitor+RSV group was decreased, while the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased.However, no difference of the expression of miR-21, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 among RSV+RES, RES+miR-21 inhibitor and RES+miR-21 inhibitor+RSV groups was observed (P<0.05).CONCLUSION: In AF model induced by RES, RSV may reduce electric remodeling by inhibiting the expression of miR-21 and regulating the downstream target genes.

Expression of autophagy related proteins in neonatal rats after early infection induced celebral matter damage / 中国免疫学杂志

Objective:To explore the effect of infection on autophagy-related proteins,Beclin-1 and LC3,expression in cerebral white matter in newborn rats.Methods: A total of 64 two-day-old Sprague-Dawley(SD) rats were randomly divided into control and experimental groups(n=32 each).At day 2 to 6 after birth,the rats in experimental group were intraperitoneally injected with 0.6 mg/kg of lipopolysaccharide(LPS) once a day to establish a white matter injury induced by infection in neonatal rats while the rats in control group were injected with equal amounts of normal saline.Rats were sacrificed to collect brain tissues at 12 hour,1,3,5 d after model establishment.HE staining was performed to observe the pathological changes.Changes in the expression of Beclin-1 and LC3 in rat white matter were examined by Western blot and RT-PCR.Results: Growth and development of rat in experimental group was slow,cerebral white matter lesions were obvious.Compared with the control group,the experimental group Beclin1 and LC3 protein and mRNA levels in the model after 12 h began to express,1 d reached the peak,and then decreased,each time points were higher than the control group(P<0.05).Conclusion: Early infection in neonatal rats can cause white matter damage;the expression of autophagy-related proteins,Beclin1 and LC3,showed that autophagy may be involved in the pathological process of white matter damage induced by infection.

Cardiac fibroblasts of the rat transfected by lenti virus-stromal cell-derived factor-la-green fluorescent protein vector / 解剖学报

Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control C0N145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored. The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-la protein.

Role of miRNA-204 targeted LC3B in Ang Ⅱ induced myocardial hypertrophy / 重庆医学

Objective To explore the effects of miRNA-204 targeted LC3B expression on Ang Ⅱ induced cardiomyocytes hypertrophy.Methods The primary neonatal rat cardiomyocytes served as the research objects and divided into the control group,AngⅡ group,combination-treated group 1 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by negative control lentivirus vector),combination-treated group 2 (cardiomyocytes were given Ang Ⅱ stimulation,meanwhile infected by lentivirus carrying miRNA-204 overexpression vector) according to different treatments.About 48 h to 72 h after intervention treatment,the cardiomyocyte hypertrophy change was detected by confocal microscopy,the expression of miRNA-204 was analyzed by real time PCR,the protein expression of LC3B was measured by Western blot and targeted gene of miRNA-204 was demonstrated by dual-luciferase reporter assay system.Results Compared with the control group,the cardiomyocyte relative surface area in the Ang Ⅱ group was significantly enlarged,the protein expression of LC3B was significantly increased,the expression of miRNA-204 was upregulated,the differences were statistically significant (P＜0.05).Whereas comparing the combination-treated group 1 with combination-treated group 2,the protein expression of LC3B in the latter was down-regulated and the cell area was reduced (P＜0.05).The further luciferase activity report gene experiment results suggested that miRNA-204 was able to bind to LC3B 3'-UTR and decreased the luciferase activities (P＜0.05),but not to bind its mutated fragment for inactivating luciferase activity(P＞0.05).Conclusion miRNA-204 is able to inhibit Ang Ⅱ induced cardiomyocytes hypertrophy,its action is realized by targeting the expression of LC3B.

Protective effect of heat shock protein 70 on lungs in newborn rats with hypoxic pulmonary hypertension / 中华实用儿科临床杂志

Objective To investigate the protective effect of adenovirus mediated heat shock protein 70 (HSP70) on lungs in neonatal rats with hypoxic pulmonary hypertension (HPH).Methods One hundred and twenty-eight 7-10 d healthy Wistar neonatal rats were randomly divided into HPH model group and control group.HPH group was divided into saline group,empty virus group,and HSP70 group according to the transfection solution.HPH model was established in the hypoxia cabin of 80 mL/L nitrogen oxygen mixed gas after transfection.The mean pulmonary artery pressure(mPAP) was measured after 3,7,10 and 14 days of hypoxia in each group.The mRNA and protein expression of HSP70,hypoxia inducible factor-1 alpha(HIF-1 α),endothelin-1 (ET-1) and inducible nitric oxide synthase(iNOS) in the lung tissues of neonatal rats were detected by using reverse transcription-PCR and Western blot respectively.Results (1) The mPAP level was significantly higher in saline group (M,Q:12.00,2.50;15.00,2.00;18.00,1.75;20.00,2.25) than that in control group (M,Q:9.50,4.75;10.50,1.00;13.00,1.00;15.50,3.25),and the differences were significant (z =-3.28,-3.40,-3.34,-3.06,all P ＜ 0.01);and the differences were also significant between empty virus group (M,Q:13.50,2.00;15.50,1.75;18.00,1.00;22.00,4.25) and control group (z =-2.83,-3.42,-3.40,-2.97,all P ＜ 0.01) in 3,7,10,and 14 days;but there was no significant difference between HSP70 group (M,Q:8.50,4.00;10.50,1.00;13.00,1.00)and the control group in 3,7,and 10 days (z =-0.43-0.00,-3.06,all P ＞ 0.05).(2) The expressions of HSP70 mRNA among the groups were statistically significant(F =6.321,9.669,6.333,all P ＜ 0.01),and the expressions of HSP70 protein also had significant difference(F =16.463,3.637,17.749,all P ＜ 0.01).(3)The level of HIF-1α mRNA in saline group was significantly higher than that of the control group,and the differences were statistically significant (q =4.312,9.106,6.151,all P ＜ 0.01);and the level of HIF-1α mRNA in empty virus group was also significantly higher than that in the control group,and the differences were statistically significant (q =3.982,9.235,5.352,all P ＜ 0.01) in 3,7,and 10 days;hypoxia in HSP70 group was lower than that of the empty virus group in 3,7 days,and the differences were statistically significant (q =6.083,11.031,all P ＜ 0.05).The level of ET-1 mRNA in saline group was significantly higher than that in the control group(q =5.112,10.086,6.264,all P ＜ 0.01),in empty virus group was significantly higher than that in the control group,and the differences were statistically significant (q =4.182,12.238,5.864,all P＜0.01) in 3,7,and 10 days,but in HSP70 group it was lower than that in the empty virus group in 3,7,and 10 days,and the differences were statistically significant (q =6.912,10.235,7.021,all P ＜ 0.05).The level of iNOS mRNA in saline group was significantly higher than that of the control group,and the differences were statistically significant (q =4.998,8.056,5.369,all P ＜0.01),in empty virus group was significantly higher than that in the control group,and the differences were statistically significant (q =4.778,10.138,5.154,all P ＜0.01) in 3,7,and 10 days,but in HSP70 group it was lower than that in the empty virus group in 3,7,and 10 days,and the differences were statistically significant (q =7.819,9.838,6.156,all P ＜ 0.05).The level of HIF-1 α protein in saline group was significantly higher than that of the control group in 3,7,and 10 days,and the differences were statistically significant (q =3.146,3.012,4.106,all P ＜ 0.05),in empty virus group was significantly higher than that of the control group in 10 days,and the difference was statistically significant (q =3.468,P ＜ 0.05);but in HSP70 group it was lower than that in the empty virus group in 3,7,and 10 days,and the differences were statistically significant (q =3.876,4.108,4.021,all P＜ 0.05).The level of ET-1 protein of HSP70 group was lower than that of the saline group,the differences were statistically significant(q =3.367,2.983,3.246,all P ＜ 0.05),in HSP70 group was lower than that of the empty virus,and the differences were statistically significant (q =3.268,2.678,3.567,all P ＜0.05).The level of iNOS protein in saline group was significantly higher than that in the control group in 3,7,and 10 days,and the differences were statistically significant (q =3.360,3.567,3.567,all P ＜ 0.05),but in HSP70 group it was lower than that in the empty virus group,and the differences were statistically significant (q =3.126,3.908,3.087,all P ＜ 0.05).Conclusion Adenovirus mediated HSP70 can improve the HSP70 expression in HPH,down-regulate the expression of HIF-1 α,ET-1,iNOS,and reduce pulmonary arterial pressure.

Different effects of a single exposure or multiple exposures to sevoflurane on hippocampal structures in neonatal rats / 临床麻醉学杂志

Objective To investigate the effects of a single exposure or multiple exposures with equivalent total duration of exposure to sevoflurane on the histological morphology and neurons ultrastructure changes in neonatal rats hippocampus CA1.Methods A total of 45 male Sprague-Dawley rats on postnatal day 7,weighing 14-18 g,were randomly divided into three groups (n=15 each): Control group (group C),single exposure to sevoflurane group (group SS),multiple exposures to sevoflurane group (group TS).In group SS,the rats inhaled 3% sevoflurane for 6 h on postnatal day 7.In group TS,the rats inhaled 3% sevoflurane for 2 h on postnatal day 7,8 and 9.In group C,the rats inhaled 60% oxygen on the corresponding day age.Rats were sacrificed and brain were seperated on postnatal day 14.CA1 pyramidal neurons pathological morphology and quantity changes were observed by Hematoxylin-Eosin (HE) and Nissl staining.In the meantime,transmission electron microscopy was used for observing neurons ultrastructure and measuring the thickness of the postsynaptic density and the length of the postsynaptic active area.Results Nissl staining and HE indicated that multiple exposures and a single 6 h exposure to sevoflurane resulted in severer neurons loss and sparse arrangement relative to group C (P<0.05),Multiple exposures to sevoflurane resulted in greater neurons loss compared with a single 6-h exposure (P<0.05).Transmission electron microscope indicated that damage of CA1 neuronal subcellular organelle induced by multiple exposures and a single 6 h exposure was severer compared with group C.Both multiple exposures and a single exposure lead to decreased thickness of the postsynaptic density and shorter length of the postsynaptic active area (P<0.05).Multiple exposures to sevoflurane caused greater damaged than a single exposure (P<0.05).Conclusion Both a single and multiple exposure to sevoflurane induced CA1 neurons loss and ultrastructure changes in neonatal rats.Compared with a single exposure,multiple exposures to sevoflurane resulted in greater neurons morphology injury.

Effect of progesterone on the expression of O4 and O1 in the white matter of neonatal rat model with periventricular leukomalacia / 临床儿科杂志

Objective To explore the effect of progesterone on the expression of O4 and O1 in the white matter of neonatal rat model with periventricular leukomalacia (PVL). Methods 2-day-old neonatal SD rats were randomly divided into model group, experimental group, and sham operation group. Rats' left common carotid artery was ligated and exposed to hypoxia (8%O2+92%N2) for 0.5 h in both the model group and experimental group to build the PVL animal model. The rats in experimental group was injected intraperitoneally with progesterone 10 mg/(kg·d) immediately after cerebral hypoxia ischemia. In sham operation group, rats' left common carotid artery was only isolated without ligation and hypoxia. 1, 4, 7, and 14 days after operation, the pathological changes of brain tissue were compared among three groups. Immunohistochemical staining was used to detect the expression of O1 and O4 in the cerebral cortex of rats in three groups at different time points. Results There were no abnormal pathological changes in the white matter in the sham operation group at each time point. The left ventricular enlargement and periventricular leukomalacia were found in both model and experimental groups, while the pathological damages of white matter in experimental group were significantly lighter than those in model group at each time point. The integral optical density (IOD) of O1 and O4 positive cells in the cerebral cortex of the three groups was gradually increased at day 1, day 4, and day 7 after operation and reached the peak level at day 7 , then was decreased at day 14 after operation. There was statistically significant difference (P<0.01). At day 1, day 4, day 7, and day 14, the integral optical density (IOD) of O1 and O4 positive cells in the cerebral cortex of sham operation group was highest, followed by experimental group and model group, and there was significant difference (P<0.01). Conclusion Progesterone can reduce the pathological damage in the cerebral cortex in neonatal rats with PVL, and promote the expression of O1 and O4 in the periventricular white matter, which can promote the differentiation and maturation of oligodendrocytes.

Effects of isovitexin on transient outward potassium current of ventricular myocytes in rats / 中国中药杂志

To investigate the effects of isovitexin Ⅳ on transient outward potassium current in rat ventricular myocytes. In this study, MTT assay was used to investigate the safe range of isovitexin. The results showed that the IC₅₀ of the drug was in the range of 10-30 μmol•L⁻¹, and the drug concentration of 1-3 μmol•L⁻¹ for the patch clamp test was within the safe range. In addition, the single ventricular myocytes were obtained by single-enzymatic hydrolysis through aortic retrograde perfusion. The transient outward potassium current (Ito) of rat ventricular myocytes was guided and measured by whole-cell patch-clamp technique and the changes of current characteristics were recorded after isovite was applied. When the concentration of IV was less than 0.1 μmol•L⁻¹, there was no significant effect on Ito. However, with the increase in the concentration of IV (≥0.3 μmol•L⁻¹), the peak of Ito was decreased gradually, from (32.32±2.9) pA/pF to （25.83±4.3） pA/pF, 1 μmol•L⁻¹ IV and (19.51±3.5) pA/pF, 3 μmol•L⁻¹ IV respectively, with an inhibition effect in a concentration-dependent manner. In the range of 1-3 μmol•L⁻¹, IV down-regulated the I-V curve of Ito significantly. The activation curve showed that IV can enable the maximum half activation potential (V1/2) to move to the positive direction, and the V1/2 was increased from (19.59±1.6) mV to (22.81±1.7) mV and (28.86±1.4) mV at concentration of 1, 3 μmol•L⁻¹, meanwhile the activation curve moved to the right. However, the maximum half inactivating potential (V1/2) of the steady-state inactivation curve of Ito was significantly decreased from (－51.43±0.99) mV to (－61.81±1.3) mV with concentration of 1 μmol•L⁻¹ and (－71.50±1.4) mV with concentration of 3 μmol•L⁻¹. The inactivation time constant of recovery from inactivation (τ) was up-regulated significantly from (94.89±0.73) ms to (118.5±1.5) ms and (162.4±1.4) ms at concentration of 1, 3 μmol•L⁻¹ respectively. Meanwhile IV could enable the inactivation recovery curve to move to the right, which suggested that it can prolong the recovery time from inactivation of the transient outward potassium channel. In conclusion, isovitexin had a high inhibitory effect on Ito in rat ventricular myocytes.

Expression and significance of NLRP3 inflammatory body in neonatal rats with hypoxic-ischemic brain damage / 中华行为医学与脑科学杂志

Objective To investigate the expression of NLRP3 in different time point of HIBD neonatal rats and to search for critical time points and alleviate HIBD dysfunction.Methods 96 newborn rats of 7 days old were randomly divided into HIBD group(n=48) and Sham operation group(n=48).HIBD model was prepared by referring to Rice method.Brain tissue was taken after 6 h,24 h,72 h,7 d.Brain injury was detected by HE stain.The expression and distribution of NLRP3 and Caspase-1 were detected by immune fluorescence and Western blot,and IL-1β and IL-18 were detected by ELISA.Results HE staining and immunofluorescence showed that NLRP3 protein (HIBD group (0.63±0.07),Sham group(0.43±0.04)) was increased significantly since 6 h in HIBD group,and its downstream protein Caspase-1,IL-1β and IL-18 were successive activated.The results showed IL-1β (HIBD group(732.28± 108.42)pg/ml,Sham group(584.58± 36.35) pg/ml) was increased significantly since 6 h in HIBD group;Caspase-1 (HIBD group(0.67±0.09),Sham group(0.30±0.05)),IL-18 (HIBD group(683.84±31.83) pg/ml,Sham group(571.32±50.91) pg/ml) was increased significantly since 24 h in HIBD group(P＜0.05).Conclusion NLRP3 and its downstream inflammatory cytokines IL-1 β and IL-18 are up-regulated when HIBD occurs.The change of NLRP3protein expression in group HIBD is earlier than changes of neuron.NLRP3 signal may mediate and participate in the occurrence and development of HIBD.

Neuroprotective Effects of Amantadine on the Hypoxic-ischemic Brain Inj ury of Neonatal Rats / 华中科技大学学报(医学版)

Objective To examine the neuroprotective effects of amantadine(AMA),a non-competitive NMDA receptor an-tagonist,on the hypoxic-ischemic(HI)brain injury of neonatal rats and the possible mechanisms.Methods Hypoxic-ischemic encephalopathy(HIE)models were established in seven-day-old male and female Sprague-Dawley rats by ligating the right ce-phalic artery and then inhaling 8% oxygen for 2 h.All the rats were divided into 3 groups:control group(n=15),HIE group(n=15),and AMA group(n=45).Animals in AMA group were intranasally treated with AMA at 50 mg/kg 30 min before and 15 min after ligation and 30 min before inhalation(15 rats each used at the three time points).The right-to-left hemispheric weight ratio was calculated 7 days after the HI brain injury.The right hippocampus tissues of rats(n=45)were harvested 24 h after the HI brain injury and the concentrations of IL-1βand IL-6 were detected by ELISA.The outcomes of behavior tests(in-volving 45 rats)including Barnes maze test,motor coordination test and fear conditioning test,were evaluated 30 days after the HI brain injury.Results Intranasal AMA significantly increased the right-to-left hemispheric weight ratio,lowered the concen-trations of IL-1βand IL-6 in the right hippocampus of rats and promoted the behavior functions 15 min after ligation(P<0.05) . Conclusion Intranasal AMA can provide neuroprotection partially by reducing the hippocampal inflammation in the neonatal rats with HI brain injury.