子宫内膜癌组织中缺氧诱导因子-1α和血管内皮生长因子表达的临床意义

目的探讨缺氧与子宫内膜癌发生、发展的关系。方法运用免疫组织化学技术检测缺氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)在正常子宫内膜、不典型增生子宫内膜及子宫内膜癌组织中的表达。结果 HIF-1α(73.21%,25.64%,4.88%)和VEGF(78.51%,30.77%,7.32%)在子宫内膜癌组织中、不典型增生子宫内膜和正常子宫内膜中的表达率差异有统计学意义(P<0.05);HIF-1α和VEGF的表达存在正相关性;HIF-1α和VEGF的表达与子宫内膜癌的浸润深度、淋巴结转移及临床分期密切相关,VEGF的表达还与子宫内膜癌的病理分级密切相关。结论 HIF-lα和VEGF的高表达对于子宫内膜癌的发生起着重要作用,并可能与其恶性进展密切相关。

Efficacy of Shouzhangshen(Rhizoma Gymnadeniae Crassinervidis)extract against acute high altitude hypoxia-induced brain injury in mice

OBJECTIVE:To evaluate the protective effect of Shouzhangshen(Rhizoma Gymnadeniae Crassinervidis)extract against acute high altitude hypoxia-induced brain injury in mice.METHODS:Sixty C57BL/6J mice were selected and assigned to six groups(n=10):normal control group,low-pressure hypoxia group,positive control group(dexamethasone 500 mg/kg),and three groups treated with Shouzhangshen extract(250,500,and 750 mg/kg,respectively).The Morris water maze test was performed to evaluate alterations in spatial learning and memory deficits.Nissl staining was performed to detect Nissl bodies and neuron damage.Hypoxia-inducible factor(HIF)-1α,interleukin(IL)-1β,tumor necrosis factor(TNF)-α,vascular endothelial growth factor(VEGF),and malondialdehyde(MDA)expression in brain tissue and serum,as well as superoxide dismutase(SOD)and glutathione(GSH)activity in brain tissues were measured by enzyme-linked immunosorbent assays,quantitative real-time-polymerase chain reaction and western blots.RESULTS:The Morris water maze test results showed that Shouzhangshen extract can significantly reduce the latency and swimming distance to escape onto a visible platform,increase neuron density and hierarchy and the number of pyramidal neurons,and decrease the expression of HIF-1α,IL-1β,TNF-α,and VEGF mRNAs and proteins in both brain tissue and serum(P<0.05).Furthermore,significantly lower MDA expression and higher GSH activity were detected in the three groups treated with Shouzhangshen compared with the low-pressure hypoxia group(P<0.05).However,no significant alteration was observed for SOD activity(P>0.05).CONCLUSION:Our findings suggest that Shouzhangshen extract may have a significant effect on acute high altitude hypoxia-induced brain injury in mice.

Transforming Growth Factor-beta I Involved in the Pathogenesis of Endometriosis through Regulating Expression of Vascular Endothelial Growth Factor under Hypoxia

Background： Endometriosis （EMs） is a common gynecological disorder characterized by endometrial-like tissue outside the uterus. Hypoxia induces the expression of many important downstream genes to regulate the implantation, survival, and maintenance ofectopic endometriotic lesions. Transtbrming growth factor-beta I （TGF-β1） plays a major role in the etiology of EMs. We aimed to determine whether TGF-β1 affects EMs development and progression and its related mechanisms in hypoxic conditions. Methods： Endometrial tissue was obtained from women with or without EMs undergoing surgery from October, 2015 to October, 2016. Endometrial cells were cultured and then exposed to hypoxia and TGF-β1 or TGF-β1 inhibitors. The messenger RNA （mRNA） and protein expression levels ofTGF-β1, vascular endothelial growth fhctor （VEGF）, and hypoxia-inducible fhctor-Ic~ （HIF-β1） were measured. A DuaI-Luciferase Reporter Assay was used to examine the effect ofTGF-[31 and hypoxia on a VEGF promoter construct. Student＇s t-test was pertbrmed/br comparison among groups （one-sided or two-sided） and a value ofP 〈 0.05 was considered statistically significant. Results： TGF-β1, VEGF, HIF-β1 mRNA, and protein expression were significantly higher in EMs tissue than that in normal endometrial tissue （t = 2.16, P = 0.042）. EMs primary cultured cells exposed to hypoxia expressed 43.8% higher VEGF mRNA and protein （t = 6.84, P - 0.023）. VEGF mRNA levels increased 12.5% in response to TGF-β1, whereas the combined treatment of hypoxia/TGF-β1 resulted in a much higher production （87.5% increases） of VEGF. The luciferase activity of the VEGF promoter construct was increased in the presence of either TGF-β1 （2.6-fold, t = 6.08, P = 0.032） or hypoxia （11.2-fold, t = 32.70, P 〈 0.001 ）, whereas the simultaneous presence of both stimuli resulted in a significant cooperative effect （ 18.5-fold, t = 33.50, P 〈 0.001 ）. Conclusions： The data support the hypothesis that TGF-β1 is involved in the pathogenesis of EMs through regulating VEGF expression. An additive effect of TGF-[31 and hypoxia is taking place at the transcriptional level.

External use of Ruyanneixiao cream efficiently blocks precancerous mammary lesions by interfering with glycolysis induced by inhibition of hypoxia inducible factor-1α, hexokinase 2, phosphofructokinase, and pyruvate kinase M2 expression

OBJECTIVE:To investigate the effect of Ruyanneixiao cream(RYNX) on the expression of hypoxia inducible factor-1α(HIF-1α), hexokinase 2(HK2),phosphofructokinase(PFK), and pyruvate kinase M2(PKM2) mRNA and protein in MCF-10 AT cells and in an animal model of precancerous mammary lesions.METHODS:Following treatment of MCF-10 AT cells with RYNX, tamoxifen(TAM) and YC-1 for 48 h,HIF-1α, HK2, PFK, PKM2 mRNA and protein expression was analyzed.Fifty female SD rats were randomly divided into control, model, TAM, and highand low-dose RYNX groups, with 10 rats in each group.A precancerous mammary lesion model was established for all groups except the control group.High-and low-dose RYNX cream containing TAM was coated on the breasts of animals in the corresponding groups.The rat mammary tissue was removed in the 10 th week and HIF-1α, HK2, PFK,PKM2 mRNA and protein was analyzed.RESULTS:In vitro analyses demonstrated that, compared with the matrix group, HIF-1α, HK2, PFK,PKM2 mRNA and protein expression was significantly decreased in the RYNX group(P < 0.05).Compared with the YC-1 + RYNX group, HK2, PFK,and PKM2 protein expression was significantly reduced in the RYNX group.HIF-1α, HK2, PFK, and PKM2 protein expression was increased significantly in the model group(P < 0.05) compared with the control group, while HIF-1α, HK2, PFK, and PKM2 mRNA and protein expression was significantly decreased in both the high-and low-dose RYNX groups(P < 0.05), with the effect being greater in the high-dose group.CONCLUSION:RYNX can block precancerous breast lesions by decreasing the expression of HK2,PFK, and PKM2 mRNA and protein via inhibition of HIF-1α mRNA and protein overexpression in a dose-dependent manner.

Efficacy of bioactive compounds of Chaihu(Radix Bupleuri Chinensis)on glaucomatous optic atrophy through interleukin-6/hypoxia inducible factor-1αsignal pathway

OBJECTIVE:To investigate the bioactive compounds of Chaihu(Radix Bupleuri Chinensis)(RB)on glaucomatous optic atrophy(GOA),and to study the pharmacological mechanism.METHODS:We collected information on the bioactive compounds of RB from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Targets related to bioactive compounds and GOA were also obtained.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and network analyses were performed to investigate the potential mechanism of RB against GOA.Subsequently,the main bioactive compounds of RB and targets of GOA were docked by Autodock software.Moreover,a GOA model of retinal ganglion cells(RGCs)induced by cobalt chloride was established to verify the effect of RB on GOA.RESULTS:There were 17 main bioactive compounds and 46 key targets were screened as potential players in GOA.The compound-target network mainly contained 17 compounds and 46 corresponding targets,and the key targets consisted of interleukin-6(IL-6),hypoxia inducible factor-1α(HIF1A),Caspase-3,estrogen receptor alpha(ESR1),MYC proto-oncogene(MYC),and vascular endothelial growth factor A(VEGFA).Forty-nine significantly enriched GO terms,and 134 KEGG signaling pathways were identified(P<0.05),including HIF-1,tumor necrosis factor,VEGF,prolaction,and other signaling pathways.Molecular docking results showed that the main bioactive compounds of RB exhibited the strongest binding activity with IL-6.Furthermore,experimental validation showed that the RB extract inhibited the activity and promoted apoptosis of RGCs in a dose-dependent manner.The RB extract also suppressed the expression of Bax,Caspase-3,and Caspase-9 and regulated malonaldehyde,superoxide dismutase,and glutathione peroxide by inhibiting the IL-6/HIF-1αsignaling pathway.CONCLUSIONS:The present study provided insights into the mechanism of RB on GOA.RB mainly reverses GOA by inhibiting the IL-6/HIF-1αsignaling pathway.

Forensic Significance of Messenger RNA and Protein Expression of Genes Downstream of Hypoxia Inducible Factor 2 in Myocardial Tissue for Death Discrimination

Background: As a heterodimeric transcription factor, hypoxia-inducible factor 2 alpha subunit (HIF2A), is an important member of the HIF family. It plays a significant role in the hypoxia adaptation process by regulating the different types of downstream transcription factors and auxiliary regulatory factors. HIF2A-related factors are believed to participate in the progression of myocardial injury or myocardial ischemia, support the protection of ischemic myocardium, and provide guiding significance for the diagnosis and discrimination of sudden cardiac death in forensic pathology. Aim and Objectives: This study aimed to explore the discriminability and applicability of HIF2A-related factors in myocardial infarction cases compared with other causes of death, provide further insights for the forensic diagnosis of heart failure (HF) cases with myocardial infarction, and support the clinical treatment of patients with HF after myocardial infarction. Materials and Methods: The relative expression levels of HIF2A, amphiregulin (AREG), potassium large conductance calcium-activated channel subfamily M β1 (KCNMB1), peroxisome proliferator-activated receptor α (PPARA), vascular endothelial growth factor (VEGF), and VEGFR2 messenger RNAs (mRNAs) in myocardial tissue samples were performed using quantitative reverse transcriptase-polymerase chain reaction. A partial least squares-discriminant analysis model was constructed to select the indicators with better identification effects for myocardial infarction cases. The protein levels of HIF2A, AREG, KCNMB1, and PPARA were further detected by immunohistochemistry. The forensic autopsy cases (27 cases in total, postmortem interval <72 h) included seven cases of acute myocardial infarction and ten cases of myocardial ischemia. There were ten cases in the control group, including four cases of traffic injury, one case of injury by fall from height, and five cases of blunt force injury. Results: Characteristic results were observed in the myocardial ischemia/infarction samples. Compared with the control group, the relative mRNA expression levels of AREG, KCNMB1, and PPARA were significantly increased during the progression of myocardial ischemia, but this was not observed for HIF2A, VEGF, or VEGFR2 mRNA. Immunohistochemistry assays further verified the expression levels of the related factors at the protein level, and H and E staining showed signs of angiogenesis and inflammation in the ischemia/infarction group. Conclusions: By controlling the expression of downstream target genes (AREG, KCNMB1, and PPARA) during myocardial cell hypoxia adaptation, HIF2A has a potential significance in the diagnosis of myocardial infarction in forensic medicine. We believe that HIF2A, AREG, KCNMB1, and PPARA can be used as molecular pathological biomarkers for the discrimination of causes of death in myocardial infarction cases.