Tea polyphenols increase X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 expression in the hippocampus of rats during cerebral ischemia/reperfusion injury

Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apudnic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.

Xuebijing alters tumor necrosis factor-alpha, interleukin-1beta and p38 mitogen activated protein kinase content in a rat model of cardiac arrest following cardiopulmonary resuscitation

We established a rat model of cardiac arrest by clamping the endotracheal tube of adult rats at expiration. Twenty-four hours after cardiopulmonary resuscitation, nerve cell injury and expression of tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase content were increased. Rats injected with Xuebijing, a Chinese herb compound preparation, exhibited normal cellular structure and morphology, dense neuronal cytoplasm, and decreased tumor necrosis factor-α, interleukin-1β, and p38 mitogen activated protein kinase expression at 24 hours following cardiopulmonary resuscitation. These data suggest that Xuebijing can attenuate neuronal injury induced by hypoxia and reperfusion during cardiopulmonary resuscitation.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB

AIM:To study the effects of Helicobacter pylori(H. pylori)tumor necrosis factor-α(TNF)inducing protein (Tip-α)on cytokine expression and its mechanism. METHODS:We cloned Tip-αfrom the H.pylori strain 26695,transformed Escherichia coli with an expression plasmid,and then confirmed the expression product by Western blotting.Using different concentrations of Tip-αthat affected SGC7901 and GES-1 cells at different times,we assessed cytokine levels using enzyme-linked immunosorbent assay.We blocked SGC7901 cells with pyrrolidine dithiocarbamate(PDTC),a specific inhibitor of nuclear factorκB(NF-κB).We then detected interleukin(IL)-1βand TNF-αlevels in SGC7901 cells. RESULTS:Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein,which indicated that recombinant Tip-αprotein was recombined successfully.The levels of IL-1β,IL-8 and TNF-αwere sig-nificantly higher following Tip-αinterference,whether GES-1 cells or SGC-7901 cells were used(P<0.05).However,the levels of cytokines(including IL-1β,IL-8 and TNF-α)secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-αat the same concentration and for the same duration(P<0.05).After blocking NF-κB with PDTC, the cells(GES-1 cells and SGC-7901 cells)underwent interference with Tip-α.We found that IL-1βand TNF-αlevels were significantly decreased compared to cells that only underwent Tip-αinterference(P<0.05). CONCLUSION:Tip-αplays an important role in cyto-kine expression through NF-κB.

Expression and role of specificity protein 1 in the sclera remodeling of experimental myopia in guinea pigs

AIM：To study the expression of collagen I and transcription factor specificity protein 1（Sp1）,a transforming growth factor-β1（TGF-β1） downstream target,and reveal the impact of the TGF-β1-Sp1 signaling pathway on collagen remodeling in myopic sclera.METHODS：Seventy-five 1-week-old guinea pigs were randomly divided into normal control,form deprivation myopia（FDM）,and self-control groups.FDM was induced for different times using coverage with translucent latex balloons and FDM recovery was performed for 1wk after 4wk treatment;then,changes in refractive power and axial length were measured.Immunohistochemistry and reverse transcription-polymerase chain reaction were used to evaluate dynamic changes in collagen I and Sp1 expression in the sclera of guinea pigs with emmetropia and experimental myopia,and the relationship between collagen I and Sp1 levels was analyzed.RESULTS：In the FDM group,the refractive power was gradually changed（from 2.09±0.30 D at week 0 to-1.23±0.69 D,-4.17±0.59 D,-7.07±0.56 D,and-4.30±0.58 D at weeks 2,4,6,and 1wk after 4wk,respectively;P〈0.05）,indicating deepening of myopia.The axial length was increased（from 5.92±0.39 mm at week 0 to 6.62±0.36 mm,7.30±0.34 mm,7.99±0.32 mm,and 7.41±0.36 mm at weeks 2,4,6,and 1wk after 4wk;P〈0.05）.The m RNA and protein expression of Sp1 and collagen I in the sclera of the FDM group was lower than that of the control groups（P〈0.05）,and the reduction was eye-coverage time-dependent.Furthermore,correlation between Sp1 and collagen I down-regulation in the myopic sclera was observed.CONCLUSION：Our data indicate that transcription factor Sp1 may be involved in the regulation of type I collagensynthesis/degradation during myopic sclera remodeling,suggesting that TGF-β1 signaling plays a role in the development and progression of myopia.

Colonic expression of Runx3 protein and TGF-β_1 and their correlation in patients with irritable bowel syndrome

Objective:To investigate the role of Runx3 protein and TGF-β_1 in the pathogenesis of irritable bowel syndrome(IBS),as well as the correlation of these two proteins.Methods:Colonic tissue was collected from patients with IBS and normal persons.The colonic expression of Runx3 protein and TGF-β_1 was detected with immunohislochemistry method.Semi-quantitative analysis was used to evaluate the staining degree of these two proteins.Results:Compared with their counterparts,patients with IBS did not show any changes in the colonic expression of Runx3 protein and TGF-β_1(P>0.05).Interestingly,there was a significant correlation between Runx3 protein and TGF-β_1 in patients with IBS(P<0.05).Conclusions:The role of Runx3 protein and TGF-β_1 in the pathogenesis of IBS remains to be further studied.

Sphingosine kinase 1 dependent protein kinase C-δ activation plays an important role in acute liver failure in mice

AIM: To investigate the role of protein kinase C(PKC)-δ activation in the pathogenesis of acute liver failure(ALF) in a well-characterized mouse model of D-galactosamine(D-Gal N)/lipopolysaccharide(LPS)-induced ALF.METHODS: BALB/c mice were randomly assigned to five groups, and ALF was induced in mice by intraperitoneal injection of D-Ga IN(600 mg/kg) and LPS(10 μg/kg). Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels at different time points within one week were determined using a multiparameteric analyzer. Serum levels of high-mobility group box 1(HMGB1), tumor necrosis factor(TNF)-α, interleukin(IL)-1β, IL-6, and IL-10 as well as nuclear factor(NF)-κB activity were determined by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of PKC-δ in liver tissue and peripheral blood mononuclear cells(PBMCs) was analyzed by Western blot.RESULTS: The expression and activation of PKC-δ were up-regulated in liver tissue and PBMCs of mice with D-Gal N/LPS-induced ALF. Inhibition of PKC-δ activation with rottlerin significantly increased the survival rates and decreased serum ALT/AST levels at 6, 12 and 24 h compared with the control group(P < 0.001). Rottlerin treatment also significantly decreased serum levels of HMGB1 at 6, 12, and 24 h, TNF-α, IL-6 and IL-1 β at 12 h compared with the control group(P < 0.01). The inflammatory cell infiltration and necrosis in liver tissue were also decreased in the rottlerin treatment group. Furthermore, sphingosine kinase 1(Sph K1) dependent PKC-δ activation played an important role in promoting NF-κB activation and inflammatory cytokine production in ALF.CONCLUSION: Sph K1 dependent PKC-δ activation plays an important role in promoting NF-κB activation and inflammatory response in ALF, and inhibition of PKC-δ activation might be a potential therapeutic strategy for this disease.

Expression of c-erbB-2 oncogene protein, epidermal growth factor receptor, and TGF-β1 in human pancreatic ductal adenocarcinoma

Objective: To detect the relations of c-erbB-2 onco-gene protein, epidermal growth factor receptor (EG-FR) and transforming growth factor-β1 (TGF-β1)to the progression or metastasis of pancreatic carci-noma.Methods: Using streptavidinbiotin complex (SABC)method, c-erbB-2 oncongene protein, we examinedimmunohistochemically EGFR and TGF-β1 expres-sions in wax-tissue sections from 10 individuals withnormal pancreas (NP), 13 patients with chronic pan-creatitis (CP) and 36 patients with pancreatic ductaladenocarcinoma (PC).Results: The positive expression rates of c-cerbB-2oncogene protein, EGFR and TGF-β1 in the NP, CPand PC groups were 0, 0, 10%; 7.7%, 7.7%,7.7%; and 41.7%, 50.0%, 44.4%, respectively.The positive expression rates of the three specific pro-teins increased more significantly in the PC groupthan in the NP and CP groups (P【0.05). The indi-vidual expression of c-erbB-2, EGFR and TGF-β1was not related to the age and sex of the patients aswell as the site, size and histopathological grade oftumors (P】0.05), but to the clinical stage of tumors(P【0.01). The coexpression rate of the three pro-teins was 27.8 % (10/36). This coexpression in thePC group was correlated with the histopathologicalgrades and clinical stages of tumors (P【0.01).Conclusion: Detection of c-erbB-2 oncogene protein,EGFR, and TGF-β1 expressions in pancreatic tissueis helpful to judge the malignancy, progression, andmetastasis of PC.

Reactive oxygen species-induced activation of Yes-associated protein-1 through the c-Myc pathway is a therapeutic target in hepatocellular carcinoma

BACKGROUND The Hippo signaling pathway regulates organ size by regulating cell proliferation and apoptosis with terminal effectors including Yes-associated protein-1(YAP-1).Dysregulation in Hippo pathway has been proposed as one of the therapeutic targets in hepatocarcinogenesis.The levels of reactive oxygen species(ROS)increase during the progression from early to advanced hepatocellular carcinoma(HCC).AIM To study the activation of YAP-1 by ROS-induced damage in HCC and the involved signaling pathway.METHODS The expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761)was quantified using real-time polymerase chain reaction and immunoblotting.Human HCC cells were treated with H2O2,which is a major component of ROS in living organisms,and with either YAP-1 small interfering RNA(siRNA)or control siRNA.To investigate the role of YAP-1 in HCC cells under oxidative stress,MTS assays were performed.Immunoblotting was performed to evaluate the signaling pathway responsible for the activation of YAP-1.Eighty-eight surgically resected frozen HCC tissue samples and 88 nontumor liver tissue samples were used for gene expression analyses.RESULTS H2O2 treatment increased the mRNA and protein expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761).Suppression of YAP-1 using siRNA transfection resulted in a significant decrease in tumor proliferation during H2O2 treatment both in vitro and in vivo(both P<0.05).The oncogenic action of YAP-1 occurred via the activation of the c-Myc pathway,leading to the upregulation of components of the unfolded protein response(UPR),including 78-kDa glucoseregulated protein and activating transcription factor-6(ATF-6).The YAP-1 mRNA levels in human HCC tissues were upregulated by 2.6-fold compared with those in nontumor tissues(P<0.05)and were positively correlated with the ATF-6 Levels(Pearson’s coefficient=0.299;P<0.05).CONCLUSION This study shows a novel connection between YAP-1 and the UPR through the c-Myc pathway during oxidative stress in HCC.The ROS-induced activation of YAP-1 via the c-Myc pathway,which leads to the activation of the UPR pathway,might be a therapeutic target in HCC.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

细胞自噬与高迁移率族蛋白B-1(HMGB1)介导肿瘤耐药的研究进展

恶性肿瘤可通过多种细胞机制,产生对抗癌药物和放疗的抗性,即所谓耐药现象。细胞自噬是肿瘤细胞耐药的一个重要原因。高迁移率族蛋白B-1(HMGB1)是高度保守的非组蛋白DNA结合蛋白,在DNA结构、基因转录、基因重组、DNA损伤修复以及细胞存活等方面发挥重要的调控作用;HMGB1在细胞内的功能,与其氧化还原状态和细胞定位息息相关;在应激条件下,可从核内转位至胞质,启动细胞自噬,介导肿瘤细胞对抗癌药物和放疗的抗性。因此,HMGB1是克服肿瘤细胞耐药的潜在靶标。

缺氧诱导因子-1α和BCL-2/腺病毒E1B19 kDa相关蛋白3在中耳胆脂瘤表达及意义

目的探讨缺氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)和BCL-2/腺病毒E1B19KDa相关蛋白3(Bcl2/adenovirus E1B 19 kD interacting protein 3,BNIP3)在中耳胆脂瘤中的表达及胆脂瘤上皮的凋亡情况。方法采用免疫组织化学方法检测30例中耳胆脂瘤标本与18例外耳道皮肤标本中HIF-1α和BNIP3蛋白的表达情况,使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling,Tunel)检测20例中耳胆脂瘤标本和18例外耳道皮肤标本的凋亡情况。使用Pearson相关分析检验HIF-1α和BNIP3蛋白之间的相关性。结果 HIF-1α在胆脂瘤组和对照组的平均光密度分别为0.16±0.07和0.08±0.03,两组比较差异有统计学意义(t=4.279,P<0.01);BNIP3在胆脂瘤组和对照组的平均光密度分别为0.16±0.08和0.11±0.06,两组比较差异有统计学意义(t=2.463,P=0.0185);经pearson相关分析,在胆脂瘤上皮中,HIF-1α和BNIP3之间呈正相关(r=0.418,P=0.003);Tunel染色中,凋亡指数在胆脂瘤组和对照组分别为(52.8±12.5)%和(9.99±2.97)%,两组比较差异有统计学意义(t=14.166,P<0.01)。结论 HIF-1α和BNIP3在中耳胆脂瘤中的异常表达可能与胆脂瘤的高凋亡特性有关。

过氧化还原蛋白1血清水平变化与急性缺血性脑卒中患者疾病转归的关联性分析

目的:探讨过氧化还原蛋白1(PRDX1)血清水平变化与急性缺血性脑卒中(AIS)患者疾病转归的关联性。方法:选取2019年5月~2020年1月我院AIS患者128例作为观察组,同期健康体检者76例作为对照组。均检测血清PRDX1水平,对比两组及观察组不同神经功能损伤程度(NIHSS评分)患者血清PRDX1水平,并随访1个月,对比不同预后患者血清PRDX1水平,分析血清PRDX1水平变化与预后关联性。结果:观察组血清PRDX1水平高于对照组(P<0.05);不同神经功能损伤程度患者血清PRDX1水平,经单因素方差分析,差异有统计学意义(P<0.05);两两对比,随神经功能损伤程度加重,血清PRDX1水平呈升高趋势(P<0.05);经Spearman秩相关系模型分析,血清PRDX1水平与神经功能损伤程度呈正相关(P<0.05);随访1个月,预后良好106例,预后不良22例,预后良好患者血清PRDX1水平低于预后不良患者(P<0.05);偏相关性分析,血清PRDX1水平与预后相关(P<0.05)。结论:血清PRDX1水平变化与AIS发生发展及病情转归关系密切,临床可通过检测血清PRDX1水平,早期预测预后情况,并指导完善治疗方案以确保患者能最大化获益。

沙颍河铅污染对儿童GH-IGF-1轴和体格发育的影响

目的：探讨沙颍河铅污染现状及其对儿童生长激素-胰岛素样生长因子-1（ GH-IGF-1）轴和体格发育的影响。方法：随机选择淮河沙颍河流域S县两个村庄作为调查点（依距河岸距离远近分别定为对照区和污染区）；原子吸收分光光度法检测河水和调查点饮用水及土壤中的铅含量。整群抽取两村庄小学8～13岁在校学生作为研究对象，伏安极谱法测定儿童血清铅含量；ELISA法检测儿童血清胰岛素样生长因子-1（ IGF-1）、胰岛素样生长因子结合蛋白3（IGFBP3）、生长激素（GH）和生长激素结合蛋白（GHBP）水平；电子体重计、身高坐高计、皮褶厚度计及Gulick卷尺分别测量体重、身高、皮褶厚度和胸围。结果：河水3个采样断面铅浓度均超过国家地表水水质标准Ⅴ级。污染区饮用水及土壤铅含量均高于对照区（ t＝2．663和2．300，P＜0．05）。污染区儿童血清铅含量高于对照区儿童（t＝3．227，P＝0．002）。污染区男孩身高、体重、皮褶厚度、胸围与对照区比较差异均无统计学意义（P均＞0．05）。两区女孩身高、体重和胸围差异均无统计学意义（P均＞0．05），但污染区女孩皮褶厚度高于对照区（t＝3．036，P＝0．003）。结论：沙颍河流域的饮用水中铅含量明显升高，污染区儿童血清GH-IGF-1轴相关因子水平受到影响。

Effects of Hypoxia on Expression of P-gp and Mutltidrug Resistance Protein in Human Lung Adenocarcinoma A549 Cell Line

To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O_2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O_2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, or anti-TRIF antibodies(all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Hypoxia promotes pulmonary vascular remodeling via HIF-1α to regulate mitochondrial dynamics

Background Increasing research suggests that mitochondrial defect plays a major role in pulmonary hypertension(PH) pathogenesis. Mitochondrial dynamics and quality control have a central role in the maintenance of the cell proliferation and apoptosis balance. However, the molecular mechanism underlying of this balance is still unknown. Methods To clarify the biological effects of hypoxic air exposure and hypoxia-inducible factor-1α(HIF-1α) on pulmonary arterial smooth muscle cell(PASMC) and pulmonary arterial hypertension rats, the cells were cultured in a hypoxic chamber under oxygen concentrations. Cell viability, reactive oxygen species level, cell death, mitochondrial morphology, mitochondrial membrane potential, mitochondrial function and mitochondrial biosynthesis, as well as fission-and fusion-related proteins, were measured under hypoxic conditions. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure, right ventricular hypertrophy index and right ventricular weight/body weight ratio were examined and recorded. Further, we assessed the role of HIF-1α in the development and progression of PH using HIF-1α gene knockdown using small interfering RNA transfection. Mdivi-1 treatment was performed before hypoxia to inhibit dynamin-related protein 1(Drp1). Results We found that HIF-1α expression was increased during hypoxia, which was crucial for hypoxia-induced mitochondrial dysfunction and hypoxia-stimulated PASMCs proliferation and apoptosis. We also found that targeting mitochondrial fission Drp1 by mitochondrial division inhibitor Mdivi-1 was effective in PH model rats. The results showed that mitochondrial dynamics were involved in the pulmonary vascular remodeling under hypoxia in vivo and in vitro. Furthermore, HIF-1α also modulated mitochondrial dynamics in pulmonary vascular remodeling under hypoxia through directly regulating the expression of Drp1. Conclusions In conclusion, our data suggests that abnormal mitochondrial dynamics could be a marker for the early diagnosis of PH and monitoring disease progression. Further research is needed to study the signaling pathways that govern mitochondrial fission/fusion in PH.

Yes-associated protein promotes endothelial-tomesenchymal transition of endothelial cells in choroidal neovascularization fibrosis

AIM:To reveal whether and how Yes-associated protein(YAP)promotes the occurrence of subretinal fibrosis in agerelated macular degeneration(AMD).METHODS:Cobalt chloride(Co Cl2)was used in primary human umbilical vein endothelial cells(HUVECs)to induce hypoxia in vitro.Eight-week-old male C57 BL/6 J mice weighing 19-25 g were used for a choroidal neovascularization(CNV)model induced by laser photocoagulation in vivo.Expression levels of YAP,phosphorylated YAP,mesenchymal markers[αsmooth muscle actin(α-SMA),vimentin,and Snail],and endothelial cell markers(CD31 and zonula occludens 1)were measured by Western blotting,quantitative real-time PCR,and immunofluorescence microscopy.Small molecules YC-1(Lificiguat,a specific inhibitor of hypoxia-inducible factor 1α),CA3(CIL56,an inhibitor of YAP),and XMU-MP-1(an inhibitor of Hippo kinase MST1/2,which activates YAP)were used to explore the underlying mechanism.RESULTS:Co Cl2 increased expression of mesenchymal markers,decreased expression of endothelial cell markers,and enhanced the ability of primary HUVECs to proliferate and migrate.YC-1 suppressed hypoxia-induced endothelialto-mesenchymal transition(End MT).Moreover,hypoxia promoted total expression,inhibited phosphorylation,and enhanced the transcriptional activity of YAP.XMU-MP-1 enhanced hypoxia-induced End MT,whereas CA3 elicited the opposite effect.Expression of YAP,α-SMA,and vimentin were upregulated in the laser-induced CNV model.However,silencing of YAP by vitreous injection of small interfering RNA targeting YAP could reverse these changes.CONCLUSION:The findings reveal a critical role of the hypoxia-inducible factor-1α(HIF-1α)/YAP signaling axis in End MT and provide a new therapeutic target for treatment of subretinal fibrosis in AMD.