1.Effect of minocycline on glutamate uptake in periventricular zone of neo-natal rats after hypoxia
Hongchun LI ; Xia LI ; Xuetao MA ; Jie XIAO ; Zhirui NIU ; Lei FENG ; Fan LI
Chinese Journal of Pathophysiology 2016;32(2):290-295
AIM:To investigate the role of minocycline on glutamate uptake in the periventricular zone and its putative mechanism after hypoxic exposure in neonatal rats.METHODS: A model of hypoxic-ischemic brain damage (HIBD) was developed by putting postnatal 1 d rat pups in 5%O2 for 3.5 h.The glutamate level in periventricular zone was measured by liquid chromatography coupled with tandem mass spectrometry assay ( LC-MS/MS) after hypoxic exposure for 4 h and 1 d.The dynamic changes of glutamate transporters EAAT1, and EAAT2 during developmental period in periventricular zone were determined by Western blot.Moreover, the expression of EAAT1, EAAT2, Iba-1, IL-1β, TNF-αand TGF-β1 was also detected by Western blot after hypoxic exposure for 4 h and 1 d in that region.The effects of mino-cycline on all parameters mentioned above were tested after minocycline treatment at the same time points and in the same region.RESULTS:After hypoxic exposure, glutamate level was increased, but it was decreased after minocycline treat-ment.EAAT1 and EAAT2 kept a low expression level at the first postnatal week, but a predominant elevation was found at the end of the second postnatal week.The expression of EAAT1, EAAT2, Iba-1, IL-1βand TGF-β1 was increased at 1 d after hypoxic exposure.EAAT1 and TNF-αexpression was significantly up-regulated, while EAAT2 was down-regulated af-ter minocycline treatment.CONCLUSION: Minocycline inhibits the increase in the glutamate level after hypoxia in periventricular region of the neonatal rats.The mechanism may relate to the selective regulation of glutamate transporters, rather than the inhibition of neuroinflammation in periventricular zone.
2.Neuroprotective effect of H2 S by inhibiting autophagy after restoration of spontaneous circulation in rats with cardiac arrest
Hongyan WEI ; Hengjie LI ; Fang LI ; Chunlin HU ; Xin LI ; Hui LI ; Ziran ZHAO ; Jie ZHANG ; Xiaoxing LIAO
Chinese Journal of Pathophysiology 2016;32(2):284-289
AIM:To investigate the neuroprotective effect of hydrogen sulfide ( H2 S) after cardiopulmonary re-suscitation in rats with cardiac arrest ( CA) , and to explore the effects of H2 S on neuron autophagy.METHODS:The CA model was established through asphyxia.Male Wistar rats were randomly divided into sham group, model group and NaHS group.The levels of beclin-1 and LC3 II/I were measured by Western blot at 2 h, 4 h, 12 h and 24 h after the restoration of spontaneous circulation (ROSC).At 12 h after ROSC, the formation of autophagic vacuole with LC3 dots was deter-mined by immunohistochemical ( IHC) method.The phenomenon of neuron autophagy was observed under transmission electron microscope.The numbers of apoptotic neurons were counted by TUNEL staining at 72 h after ROSC.The neurolo-gic deficit score ( NDS) was used to evaluate the neurologic function after ROSC.RESULTS: The level of beclin-1 was gradually increased in model group, but it was increased and then gradually recovered in NaHS group ( P<0.05 ) .The conversion of LC3 II in the cerebral cortex was the same as beclin-1.The results of IHC showed that LC3-positive nuclei in model group were more than those in NaHS group ( P<0.05) .The number of autophagic vacuole in model group was more than that in NaHS group (P<0.05).The number of the TUNEL-positive cells in model group was more than that in NaHS group (P<0.05).The NDS of the animals in NaHS group after ROSC was lower than that in model group(P<0.05). CONCLUSION:H2 S inhibits neuronal autophagy, decreases apoptosis and improves neurologic function in CA rats after ROSC.
3.Effects of imidapril on blood gas, oxidative stress and inflammatory fac-tors in rats with acute lung injury induced by ammonium chloride
Congfu HUANG ; Xueqin HAO ; Wen DENG ; Chuanfa LIU ; Tingting WANG ; Xiaoli WEN ; Zhongrong TAO
Chinese Journal of Pathophysiology 2016;32(2):278-283
AIM:In this study, the rat lung injury model was induced by ammonium chloride for studying the effect of imidapril on blood gas, serum TNF-α, IL-6 and MDA concentrations, and AngⅡand CD54 protein expression in rat lung tissue.METHODS:Male rats were randomly divided into 3 groups:control group, lung injury model group and drug group.The rats in control group were given saline (2 mL/kg), while the rats in lung injury model group were given 6% ammonium chloride (2 mL/kg).In drug group, imidapril (3 mg· kg-1· d-1) was given to the rats once daily for 1 week by intragastric gavage after given 6%ammonium chloride.On the 7th day, the rats were anesthetized with 2% so-dium pentobarbital.Abdominal aorta blood, venous blood and lung tissue were collected.The blood gas indexes and serum TNF-α, IL-6 and MDA concentrations were determined.The lung tissues were fixed and sliced, and the expression of AngⅡ and CD54 proteins was detected by immunohistochemistry.RESULTS: The PaCO2 increased in lung injury model group compared with control group and drug group (P<0.05).The expression of AngⅡand CD54, and the concentrations of TNF-α, IL-6 and MDA also increased significantly ( P<0.01) in model group.Pulmonary edema, inflammation, alveo-lus congestion, hemorrhage and hyperplasia in model group were obvious compared with control group and drug group. CONCLUSION:Imidapril improves blood gas indexes, and reduces lipid peroxidation and inflammatory responses in the rats with lung injury induced by ammonium chloride.
4.STAT3 signaling in pathogenesis of pancreatitis
Linfei ZONG ; Xiaohui XIANG ; Shihai XIA
Chinese Journal of Pathophysiology 2016;32(3):558-563
Signal transducer and activator of transcription 3 (STAT3), an acute-phase response protein, is ac-tivated to over-express by cytokines .STAT3 also acts as a transcriptional factor to regulate the expression of cytokines .O-ver-expression of cytokines is accompanied by STAT 3 activation and over-expression in acute pancreatitis .Meanwhile , the proliferation of pancreatic stellate cells in chronic pancreatitis is mediated by STAT 3.In this review, the research progress in STAT3 function is summarized to elaborate its potential role in the pathogenesis of pancreatitis .
5.Effect of Hedgehog signaling pathway inhibitor on biological behavior of intrahepatic cholangiocarcinoma cell line RBE
Yang ZHANG ; Jing XU ; Cuiming ZHANG ; Ling ZHANG ; Aixiu QIAO
Chinese Journal of Pathophysiology 2016;32(3):544-548
AIM:To investigate the effect of cyclopamine , a Hedgehog ( Hh) signaling pathway inhibitor , on the biological behavior of intrahepatic cholangiocarcinoma cell line RBE .METHODS:The proliferation of RBE cells was detected by cell counting with Typan blue staining and MTT assay , and the apoptosis was analyzed by the flow cytometry . The Transwell invasive cabin assay was used to detect the invasion ability , and Western blot was used to determine the pro-tein expression of Gli 1 and MMP-9 in the RBE cells before and after cyclopamine treatment .RESULTS:Cyclopamine in-hibited the growth of RBE cells in a time-and dose-dependent manner .After cyclopamine treatment for 24 h, 48 h and 72 h, the apoptotic rates were significantly higher than those in control group .In control group , the number of cells invading through the Matrigel of invasion chamber was 154.52 ±13.61, while in experimental group it was 62.00 ±12.17, indica-ting that the invasion ability of the cells declined significantly .Furthermore , Western blot showed that the protein levels of Glil and MMP-9 in the RBE cells were decreased after treatment with cyclopamine for 24 h and 48 h.CONCLUSION:Blockage of the Hh signaling pathway with cyclopamine suppresses the proliferation , promotes the apoptosis and inhibits the invasion ability of RBE cells .
6.Two de novo mutations including 1 novel mutation in FBN1 and genotype-phenotype correlation in 2 Chinese Marfan syndrome families
Qihao PAN ; Xiaobi LIANG ; Yong GUO ; Junwen ZHU ; Bin HU ; Yiming WANG
Chinese Journal of Pathophysiology 2016;32(3):527-533
[ ABSTRACT] AIM:To investigate the genetic cause of 2 Chinese families with Marfan syndrome .METHODS:The clinical and laboratory investigations were performed in the 2 unrelated Chinese families .Family 1 had 1 patient with cardiac problem.Family 2 had 2 patients:one died, and the other with respiratory and cardiac problems .Next generation sequencing and Sanger sequencing in the Marfan syndrome causal gene FBN1 were performed in the patient , his unaffected sister and the parents of family 1.Sanger sequencing covering all the exons and intron-exon boundaries were performed in the patient and the parents in family 2.Bioinformatic analysis was engaged in the variations unravelled .Fifty healthy indi-viduals were also investigated in the same manner .RESULTS:Both patients were diagnosed with Marfan syndrome .A no-vel mutation c.4685G>A (p.Cys1562Tyr) was detected in the patient of family 1 but was absent in his parents and the unaffected sister .This is a previously unreported novel mutation .In the mutation a conserved Cys was substituted by a Tyr in amino acid 1562 affecting a TGF-βbinding domain and the secondary structure in the encoded protein .We also detected the mutation c.3706T>C (p.Cys1236Arg) in the patient of family 2.It was absent in the unaffected parents , and there-fore was a de novo mutation too.This mutation has been previously reported and known to be associated with neonatal Marfan syndrome .Both mutations were absent in the 50 healthy controls .We also compared the genotype and phenotypes of the 2 families.CONCLUSION:We report 2 de novo mutations in 2 Chinese families with Marfan syndrome .One of the 2 mutations is novel.The phenotype of the mutation c.4685G>A(p.Cys1562Tyr) in family 1 is associated with classical Marfan syndrome, while that of c.3706T>C (p.Cys1236Arg) in family 2 is with neonatal type of Marfan syndrome .De novo mutations may be a cause for a proportion of mutations underlying the disease .The novel mutation also expends the mutational spectrum of the FBN1 gene.
7.Effects of different lighting on reproductive system in depressive female rats
Chinese Journal of Pathophysiology 2016;32(3):510-515
AIM:To investigate the effects of different lighting on the reproductive system in depressive female rats.METHODS:Healthy adult female rats were randomly chosen as control group , and the depressive adult female rats in SPF grade were randomly divided into 5 groups (7 rats each):depressive model group, sulfur lamp group, heat radia-tion lamp group , fluorescent lamp group and LED lamp group .After 45 d of continuous illumination , the estrous cycle was observed by the vaginal exfoliated cells , and the organ indexes of ovary and uterus were calculated .The concentrations of estiadrol (E2), prolactin (PRL), progesterone (PROG) and testosterone (T) in the serum were detected by ELISA, and the histopathological lesion of ovary was observed under microscope with HE staining .RESULTS:The estrous cycle exhib-ited serious disorder , the ovaries exhibited serious congestion , and the organ indexes of ovary and uterus and the concentra-tions of E2 , PRL, PROG and T decreased significantly in the rats in depressive model group compared with control group (P<0.05).The estrous cycle and histopathological damage of ovary were obviously improved , and the concentrations of E2 , PRL, PROG and T were significantly increased after the sulfur lamp lighting in the depressive female rats compared with depressive model group .No obvious change and improvement of the reproductive functions in the heat radiation lamp group, fluorescent lamp group and LED lamp group was observed .CONCLUSION:The reproductive functions of depres-sive female rats are improved by sulfur lamp lighting .
8.Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis
Yuefeng LIU ; Weimin LUO ; Yong ZHANG ; Xiaodong ZHONG
Chinese Journal of Pathophysiology 2016;32(3):504-509
AIM:To observe the effect of docosahexaenoic acid ( DHA) on H2 O2-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism .METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H2 O2 was used to mimic the oxidative stress condition .The cells were treated with 30~100μmol/L DHA for 4~24 h.The expression of heme oxygenase-1 (HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The enzymic activity of HO-1 was measured by colorimetry . Production of reactive oxygen species ( ROS) was determined by fluorescent probe .Activation of NF-E2-related factor 2 (Nrf2) was examined by immunofluorescence method .Apoptosis of ARPE-19 cells was analyzed by flow cytometry .RE-SULTS:The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment .Meanwhile , nuclear translocation of Nrf 2 was also observed .Apoptosis appeared and ROS was produced upon H2O2 incubation.In contrast, DHA at 100 μmol/L significantly abrogated H2O2-induced apoptosis and ROS production.Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H 2 O2-induced apoptosis and ROS production .CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase -1 expression after Nrf2 activation .
9.Effects of ClC-3 gene overexpression on bone mass and structure in mice
Yuan WANG ; Zhiqin DENG ; Haibo WANG ; Hong GAO ; Xiechou LIANG ; Qiuchan TAN ; Linyan ZHU ; Qingnan LI ; Liwei WANG ; Lixin CHEN
Chinese Journal of Pathophysiology 2016;32(3):499-503
[ ABSTRACT] AIM:To investigate the effect of the overexpression of voltage-gated chloride channel family protein 3 ( ClC-3) gene on bones of mice .METHODS: The tail gene detection assay was used to confirm the overexpression of ClC-3.The male FVB mice of three months old were divided into two groups , the wild type ( WT) group and the ClC-3 overexpressed (ClC-3 transgene) group.The body weight, length and weight of the right tibias were measured .The upper and middle parts of the tibias were dissected , decalcified, paraffin-imbed, sectioned and stained with HE staining .The bone morphology metrology was used to analyze the changes of bone structures .The percent trabecular area (%Tb.Ar), trabecular number ( Tb.N) , trabecular width ( Tb.Wi) and trabecular separation ( Tb.Sp) of cancellous bone in the upper part of the tibia were measured.The total tissue area (T.Ar), cortical area (Ct.Ar), percent cortical area (%Ct.Ar), marrow area ( Ma.Ar) and percent marrow area (%Ma.Ar) of the cortical bone in the middle part of the tibia were detec-ted .RESULTS:The wild type mice and the ClC-3-overexpressed mice were verified by the tail gene detection assay . Compared with WT group , the body weight and the length and weight of the tibia were decreased in ClC -3 transgene mice (P<0.05).In the cancellous bones of ClC-3 transgene mice, the%Tb.Ar and Tb.Wi were decreased (P<0.05), the Tb.Sp was increased (P<0.05) and the Tb.N was not significantly changed .In the cortical bones of ClC-3 transgene mice, the T.Ar, Ct.Ar and%Ct.Ar were decreased (P<0.05), the%Ma.Ar was increased (P<0.05), and the Ma. Ar was not significantly changed .CONCLUSION:ClC-3 overexpression may lead to the reduction of the bone mass and the destructure of the cancellous and cortical bones .The results suggest that ClC-3 may be involved in the regulation of bone resorption and/or formation.
10.Ischemia postconditioning induces tight junction protein expression and inhibits brain edema after thrombotic cerebral ischemia in tree shrews
Shuqing LI ; Fan LI ; Liang HE ; Bo HE
Chinese Journal of Pathophysiology 2016;32(3):477-484
AIM:To assess whether the expression of tight junction (TJ) proteins, occludin/zonula occludins (ZO)-1, and regional cerebral blood flow (rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning (PC), and to explore how TJ affects brain edema and cerebral infarction .METHODS:Tree shrews were randomly grouped into control , ischemia and cerebral ischemia +PC (n=23), and the remaining 3 ani-mals were used for magnetic resonance imaging ( MRI) .The local cerebral thrombosis were induced by photochemical reac-tion in the tree shrews , and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsi-lateral common carotid artery (5 min ×3).The changes of the neural ultrastructure were observed under electron micro-scope.The neuronal apoptosis was analyzed by the method of TUNEL .Laser Doppler brain flowmetry was used to monitor the rCBF.The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot .The cerebral in-farction volume was detected by MRI .The brain water content was measured by dry-wet weight method .RESULTS: In-duction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA 1 area, and conversely, the number of neurons with abnormal ultrastructure was increased .The TUNEL positive cells were increased significantly (P<0.01) in ischemia group.Moreover, the rCBF decreased significantly (P<0.01), and occludin/ZO-1 protein expression decreased ( P<0.01 ) .The brain water content and cerebral infarction volume were significantly in-creased (P<0.01).Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water con-tent, the TUNEL positive cells, and the infarction volume (P<0.01).CONCLUSION:Ischemic PC increases the rCBF but not the local water content , suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin /ZO-1 protein expression .