The role of the HIF-1 signaling pathway in retinal ganglion cell injury caused by the OPTN (E50K) mutation

To investigate the correlation of changes in hypoxia-inducible factor-1α (HIF-1α) and p53 expressions with vitamin B3 deficiency in skin cancer patients. Twenty non-melanoma skin cancer patients with positive HIF-1α and p53 expressions were selected and randomly divided into two groups, the placebo group and the experimental group. After an appropriate number of cancer tissues were taken, the experimental group was treated with oral administration of 500 mg vitamin B3 every day, while the placebo group was treated with oral administration of the same amount of placebo; after 1 week, the skin cancer tissues in the same part were taken, and the skin tissues of healthy people were taken as the control group; the mRNA and protein expression levels of HIF-1α and p53 in tissues were detected. HIF-1α and p53 were mainly expressed in the nucleus in non-melanoma skin cancer. The protein and mRNA expression levels of HIF-1α and p53 in tissues of skin cancer patients were significantly increased compared with those in skin tissues of healthy people (P<0.05). The protein and mRNA expression levels of HIF-1α and p53 in tissues of skin cancer patients were significantly decreased at 1 week after the oral administration of vitamin B3 compared with those before the oral administration of vitamin B3 (P<0.05), but the protein and mRNA expression levels of HIF-1α and p53 in tissues of skin cancer patients in placebo group had no significant changes (P>0.05). The vitamin B3 deficiency in skin cancer patients was positively correlated with the expressions of HIF-1α and p53. The expression levels of HIF-1α and p53 in tissues of skin cancer patients are significantly increased compared with those in skin tissues of healthy people, and the changes in their expressions are positively correlated with the vitamin B3 deficiency. Supplementing vitamin B3 has a certain protective effect on skin cancer patients.

We aimed to explore the role of microRNA 195 (miR-195) in diabetic retinopathy (DR). From January 2019 to July 2020, 50 patients with DR undergoing vitrectomy and 40 patients with idiopathic macular holes undergoing vitrectomy were selected as the observation group (OG) and control group (CG), respectively. The mRNA and protein expression levels of miR-195, SIRT1, BAX, and BCL-2 were detected in the retinal tissues obtained from the two groups during surgery. In addition, human retinal endothelial cells and human dermal microvascular endothelial cells were cultured in a high-glucose environment to detect the targeted relationship between miR-195 and SIRT1; determine the mRNA and protein expression levels of SIRT1, BAX, and BCL-2 after miR-195 knockdown; and assess the levels of cell proliferation and apoptosis. In OG, the mRNA and protein expression levels of miR-195 and BAX were high, whereas those of BCL-2 and SIRT1 were low. Moreover, we detected a targeted relationship between miR-195 and SIRT1. Conversely, miR-195 knockdown led to the downregulation of the mRNA and protein expression levels of BAX and the upregulation of the mRNA and protein expression levels of SIRT1 and BCL-2 as well as improvement in cell growth and a decrease in the apoptosis rate. miR-195 is overexpressed in DR, and its targeted relationship with SIRT1 inhibits the growth of cells in the retina and accelerates apoptosis.

Effects and significance of formononetin on the expression levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in mouse cervical cancer tissue were investigated. The animal models of Balb/c nude mice with cervical cancer were established by the inoculation of HeLa cells, and randomly divided into positive control (n=10), cisplatin (n=15) and formononetin group (n=15). Mice were all sacrificed on the 31st day after administration, and their tumors were excised and weighed to calculate tumor inhibition rate. At the same time, their cancer tissues were obtained. RT-qPCR was used for detecting the mRNA expression levels of HIF-1α and VEGF, and western blotting for detecting the protein expression levels. During the medication intervention, mice in the formononetin group had no obvious adverse reactions, and were in good condition, whereas mice in the cisplatin group had poor appetite, drooping spirits and decreased activity. Mice in the cisplatin and the formononetin groups had significantly lower tumor mass and volume than those in the positive control group (P<0.05). The tumor inhibition rate of mice was 56.24% in the cisplatin group, and 50.17% in the formononetin group. Cervical cancer mice in the formononetin and the cisplatin groups had significantly lower mRNA and protein expression levels of HIF-1α and VEGF in tissues than those in the positive control group (P<0.05). Formononetin can inhibit the growth of cervical cancer and reduce the mRNA and protein expression levels of HIF-1α and VEGF in mouse cervical cancer. Formononetin has an inhibitory effect on cervical cancer tumors similar to that of cisplatin, but the former has smaller side effects, providing data for the clinical use in cervical cancer.

Fructose-1,6-bisphosphatase 1 (FBP1) is a rate-limiting enzyme in gluconeogenesis. Recently, the catalytic activity-independent function of FBP1, hypoxia-induced factor (HIF) repression in the nucleus, was identified. The aim of the present study was to investigate the association between FBP1 and hypoxia-related gene expression in clear cell renal cell carcinoma (ccRCC). The protein expression levels of FBP1, HIF-1α, HIF-2α, erythropoietin (EPO) and carbonic anhydrase IX (CA9) were assessed by immunohistochemical staining of ccRCC paraffin blocks from 123 patients using the tissue microarray technique. The expression level of FBP1 was then correlated with various clinicopathological factors, and the protein expression levels of HIF-1α, HIF-2α, EPO and CA9. Clinicopathological factors, including age, gender, T stage and Fuhrman grade, were not significantly different between patients with low and high FBP1 expression in ccRCC (P>0.05). FBP1 protein expression level was significantly correlated with the expression levels of HIF-1α (P=0.005) and EPO (P=0.010), but not significantly correlated with the expression levels of HIF-2α (P=0.123) and CA9 (P=0.513) in ccRCC tissues. The current findings confirm the association between FBP1 and hypoxia-related gene expression, and may facilitate understanding of the mechanisms of ccRCC tumorigenesis.

To observe the effect of "nape seven needles" on the expressions of hypoxia inducible factor-1α (HIF-1α) and Notch1 in cervical intervertebral disc of cervical disc degeneration (CDD) rats, so as to reveal its underlying mecha-nisms in improving CDD. SD male rats were randomly divided into normal, model, non-acupoint and nape seven needles groups, with 10 rats in each group. Staticdynamic imbalance method was used to establish CDD model. Rats in the nape se-ven needles group were treated with acupuncture at "Fengfu"(GV16), and bilateral "Fengchi"(GB20), "Wangu"(GB12) and "Tianzhu"(BL10), and rats in the non-acupoint group received acupuncture at the sham acupoints at the caudal tip and armpit, both for 20 min, 6 days a week for 4 weeks. After intervention, tilted plane test and spiral CT were used to assess the neck movement function and cervical degeneration degree of rats; immunohistochemistry was used to observe the protein expression levels of HIF-1α and Notch1 in cervical discs tissue; Western blot and real-time quantitative PCR were used to detect the protein and mRNA expression levels of HIF-1α and Notch1 in cervical discs tissue, respectively. After modeling, the cervical curvature was straightened, with narrowed intervertebral space, rough and hardened articular surface, osteophytes, and blurred articular space and articular process, which was relatively milder in the nape seven needles group. Compared with the normal group, the angle of tilted plane was significantly reduced (P<0.05), while cervical scores, HIF-1α mRNA expression level in cervical intervertebral disc tissue were significantly increased (P<0.05) in the model group. Compared with the model and the non-acupoint groups, cervical scores were significantly reduced (P<0.05), while the angle of tilted plane, HIF-1α and Notch1 positive expressions, HIF-1α mRNA expression level, Notch1 protein and mRNA expression levels in cervical intervertebral disc tissue were significantly increased (P<0.05) in the nape seven needles group. Acupuncture of "nape seven needles" can reduce the degree of cervical degeneration in rats, which was possibly associated with its effects in up-regulating the expressions of HIF-1α and Notch1 in cervical intervertebral disc tissue, promoting the proliferation and recovery of endogenous cells in nucleus pulposus.

The aim of the present study was to investigate the cell proliferation-inhibiting and anti-rheumatic activities of chemical components from Aconitum soongoricum Stapf. Chemical constituents of Aconitum soongoricum Stapf. were separated and purified by silica gel and Sephadex LH-20 chromatography. Structure was identified by spectroscopic technique, and physical/chemical properties were analyzed. The following four compounds were identified: i) Aconitine, ii) songorine, iii) 16, 17-dihydro-12β, 16β-epoxynapelline, and iv) 12-epi-napelline. Cell Counting kit-8 assay was performed to assess cell proliferation. ELISA was conducted to determine the cytokine contents, and reverse transcription-quantitative polymerase chain reaction and Western blot analysis were performed to detect the mRNA and protein expression levels. Compared with the lipopolysaccharide (LPS) group, the contents of IL-6, IL-1β, TNF-α and PGE-2 in the culture supernatant were significantly declined in the leflunomide + LPS and intervention+LPS groups, as well as the mRNA expression levels of HIF-1α, VEGFA and TLR4. Treatments with songorine, benzoylaconine and aconitine (at different concentrations) significantly inhibited the proliferation of HFLS-RA cells. Compared with the LPS group, the contents of PGE-2, IL-6, IL-1β and TNF-α in the culture supernatant were significantly decreased in the intervention groups, and the mRNA expression levels of TLR4, HIF-1α and VEGFA in the cells in the intervention groups. Songorine, benzoylaconine and aconitine from Aconitum soongoricum Stapf. have anti-rheumatic activities in vitro, which may inhibit the proliferation of HFLS-RA cells, and the underlying mechanisms may be associated with inhibiting the inflammatory cytokine production and downregulating the expression levels of HIF-1α, VEGF and TLR4.

Objective: To investigate the expressions of vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1α), and epidermal growth factor receptor (EGFR) in different morphological regions of Marjolin ulcer and their clinical relationship with angiogenesis. Methods: From January 2012 to December 2017, the patients admitted to our hospital who met the inclusion criteria were selected, including 92 patients with Marjolin ulcer [56 males and 36 females, aged (55±15) years], 100 patients with chronic non-cancerous skin ulcer [59 males and 41 females, aged (51±16) years], and 100 patients performed with other skin-related surgery [58 males and 42 females, aged (52±15) years], and they were enrolled into Marjolin ulcer group (MU), chronic non-cancerous ulcer group (CNU), and other skin surgery group (OSS) respectively. The etiology, pathogenic site, ulcer diameter, and course of patients in group MU were retrospectively analyzed. Ulcer tissue specimens from patients of group MU and group CNU and specimens of normal skin tissue attached to the tissue resected during operation from patients of group OSS were collected. The expressions of VEGF, HIF-1α, EGFR, and CD34 in the above-mentioned tissue and the surrounding normal skin, ulcer, epitheliomatous hyperplasia, and canceration areas in Marjolin ulcer tissue were detected by immunohistochemical method, and the positive expression rate and protein expression level were calculated. Data were processed with Pearson chi-square test, Mann-Whitney U test, Bonferroni method, and Bonferroni correction, and Spearman correlation analysis was used to analyze the relationship among the total protein expression levels. Results: In group MU, burns accounted for 91.3% (84/92) of the causes of patients, 44.6% (41/92) of the patients had tumors in the lower extremities, 62.0% (57/92) of the patients had skin ulcer diameter of 2.1-5.0 cm, and 75.0% (69/92) of the patients had a course of disease of more than 20 years. The positive rates of VEGF, HIF-1α, and EGFR in ulcer tissue of patients in group CNU were 41.0% (41/100), 77.0% (77/100), and 83.0% (83/100), respectively, significantly higher than those of normal skin tissue of patients in group OSS [12.0% (12/100), 45.0% (45/100), and 67.0% (67/100), χ(2)=21.589, 21.522, 6.827, P<0.01]. The positive rates of VEGF, HIF-1α, and EGFR in ulcer tissue of patients in group MU were 91.3% (84/92), 100.0% (92/92), and 100.0% (92/92), respectively, which were significantly higher than those in corresponding tissue of patients in group CNU and group OSS (χ(2)=53.372, 24.772, 17.159; 120.543, 72.777, 36.661, P<0.01). In ulcer tissue of patients in group MU, the positive expression rates of VEGF in ulcer, epitheliomatous hyperplasia, and canceration areas were significantly higher than the rate in surrounding normal skin area (χ(2)=87.120, 42.368, 89.624, P<0.01); the positive expression rates of VEGF in canceration and ulcer areas were significantly higher than the rate in epitheliomatous hyperplasia area (χ(2)=22.586, 16.060, P<0.01). In ulcer tissue of patients in group MU, the positive expression rates of EGFR in ulcer, epitheliomatous hyperplasia, and canceration areas were significantly higher than the rate in surrounding normal skin area (χ(2)=21.679, 27.600, 27.600, P<0.01), but the positive expression rates of HIF-1α in four morphological areas were similar (χ(2)=3.008, P>0.05). In ulcer tissue of patients in group MU, the protein expression levels of VEGF and CD34 in ulcer, epitheliomatous hyperplasia, and canceration areas were significantly higher than those in surrounding normal skin area (Z=-6.765, -6.819; -6.765, -6.640; -6.765, -6.819, P<0.01), the protein expression levels of VEGF and CD34 in epitheliomatous hyperplasia area were significantly lower than those in ulcer area (Z=-4.484, -5.266, P<0.01), and the protein expression levels of VEGF and CD34 in canceration area were significantly higher than those in ulcer area (Z=-6.427, -6.723, P<0.01) and epitheliomatous hyperplasia area (Z=-6.427, -6.462, P<0.01). In ulcer tissue of patients in group MU, the protein expression levels of HIF-1α and EGFR in ulcer, epitheliomatous hyperplasia, and canceration areas were significantly higher than those in surrounding normal skin area (Z=-6.819, -6.393; -6.819, -6.393; -6.819, -6.393, P<0.01), the protein expression levels of HIF-1α and EGFR in ulcer area were significantly lower than those in epitheliomatous hyperplasia and canceration areas (Z=-6.118, -5.638; -6.640, -6.393, P<0.01), and the protein expression levels of HIF-1α and EGFR in canceration area were significantly higher than those in epitheliomatous hyperplasia area (Z=-6.558, -6.819, P<0.01). In ulcer tissue of patients in group MU, the total protein expression levels of VEGF, HIF-1α, and EGFR were significantly positively correlated with the total protein expression level of CD34 (r=0.772, 0.415, 0.502, P<0.01) respectively; the total protein expression level of EGFR was significantly positively correlated with that of HIF-1α (r=0.839, P<0.01), both of which were significantly positively correlated with the total protein expression level of VEGF (r=0.531, 0.440, P<0.01) respectively. Conclusions: The expressions of VEGF, HIF-1α, and EGFR are the highest in Marjolin ulcer canceration area, and EGFR may promote angiogenesis through HIF-1α or directly increasing the expression of VEGF.

Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism

Objective: To investigate the expression and clinicopathological significance of hypoxia-inducible factor 1 alpha (HIF-1α), glucose transporter 1(GLUT-1) and lactate dehydrogenase(LDH)-5 in colorectal cancer. Methods: The expression levels of HIF-1α, GLUT-1 and LDH-5 were detected by immunohistochemical staining in 142 specimens of human carcinoma in comparison with adjacent normal tissues. Results: The expression levels of HIF-1α(78.2%, 111/142), GLUT-1(75.4%, 107/142) and LDH-5(68.3%, 97/142) were higher in tumor tissues than in adjacent normal tissues(14.8%, 21/142; 11.3%, 16/142; 7.0%, 10/142; P<0.01 for all three proteins), and such over-expression was significantly associated with lymphovascular invasion, tumor grade and pathological stages(all P<0.01). Additional studies showed that HIF-1α, GLUT-1 and LDH-5 were positively associated with each other(r<0.3, P<0.05 for all three proteins). Conclusion: The data suggest that HIF-1α, GLUT-1 and LDH-5 expression may serve as prognostic indicators for colorectal cancer patients.

The aim of the present study was to investigate the effects of microRNA-18a (miR-18a) on the invasiveness and metastasis of invasive meningiomas and the underlying mechanism. A total of 69 patients with meningiomas (30 patients in the invasive meningioma group and 39 patients in the non-invasive meningioma group) and 48 cases in the control group were enrolled. Samples of meningioma tissues, serum and cerebrospinal fluid were collected. Reverse transcription-quantitative polymerase chain reaction was performed to quantify the expression levels of hypoxia-inducible factor-1α (HIF-1α) mRNA and miR-18a. Western blot analysis was used to determine protein expression levels of HIF-1α. The expression levels of HIF-1α mRNA and protein in all three types of sample from the invasive meningioma group were significantly higher compared with those in the control and non-invasive meningioma groups (P<0.05), and the expression levels of HIF-1α mRNA in the serum and cerebrospinal fluid of the non-invasive meningioma group were significantly higher compared with those in the control group (P<0.05). The expression levels of miR-18a in the invasive meningioma group were significantly reduced compared with those in the control and non-invasive meningioma groups (P<0.05), whereas the levels of miR-18a in the non-invasive meningioma group were significantly lower compared with those in the control group (P<0.05). The expression of HIF-1α is significantly upregulated in patients with invasive meningiomas, possibly due to the downregulation of miR-18a expression. Therefore, miR-18a may regulate invasive meningiomas via HIF-1α.

BackgroundMicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs.MethodsHSC-T6 cells were treated with cobalt chloride (CoCl2), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1α (HIF-1α), collagen type I, transforming growth factor-β1 (TGF-β1), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1α, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 µmol/L CoCl2 medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ResultsWith the increase of CoCl2 concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1α, collagen I, TGF-β1, and Smad7, and the protein expressions levels of HIF-1α, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 µmol/L CoCl2 medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing.ConclusionsOur results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.

The aim of this study was to investigate whether abnormal expression of matrix metalloproteinase (MMP)-9/tissue inhibitors of MMPs (TIMP)-1 and B cell lymphoma 2 (BCL-2)/BCL-2-associated X protein (BAX) are correlated with the characteristic accelerated fibrosis and apoptosis during ageing and in atrial fibrillation (AF). Four groups of dogs were studied: adult dogs in sinus rhythm (SR), aged dogs in SR, adult dogs with AF induced by rapid atrial pacing and aged dogs with AF induced by rapid atrial pacing. The mRNA and protein expression levels of the target gene in the left atrium were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Pathohistological and ultrastructural changes were assessed by light and electron microscopy. The apoptotic indices of myocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The mRNA and protein expression levels of MMP-9 and BAX and those of TIMP-1 and BCL-2 were significantly upregulated and down-regulated, respectively, in the aged groups compared with the adult groups. Compared with the control groups, the adult and aged groups with AF exhibited significantly increased mRNA and protein expression levels of MMP-9 and BAX and decreased expression levels of TIMP-1 and BCL-2. Samples of atrial tissue demonstrated abnormal pathohistological and ultrastructural changes, accelerated fibrosis and apoptosis. MMP-9/TIMP-1 and BCL-2/BAX hold potential for use as substrates conducive to AF and their abnormal expression plays a major role in structural remodeling of the atrium.

BackgroundTo examine changes of mRNA and protein expressions of MMP-2, Bcl-2, and BAX in atrial fibrillation (AF) patients, and investigate the correlations among these 3 biomarkers.Material/MethodsRheumatic heart disease patients (n=158) undergoing cardiac surgical procedures for mitral valve repair or replacement were included as the AF group (n=123), containing paroxysmal AF (n=42), persistent AF (n=36), and permanent AF (n=45). Rheumatic heart disease patients with sinus rhythm (SR) (n=35) were enrolled as the SR group (control group). Immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR) were applied to detect the protein and mRNA expression levels of MMP-2, Bcl-2, and BAX. Apoptosis was observed with light and electron microscopes and detected by TdT-mediated dUTP nick-end labeling (TUNEL).ResultsCompared with the SR group, the left atrial diameters (LADs), protein and mRNA expression levels of MMP-2 and BAX, apoptotic index (AI), and Bcl-2/BAX ratio were evidently increased in the 3 AF groups, but protein and mRNA expression levels of Bcl-2 decreased in the AF groups (all P<0.05). Correlation analysis found that MMP-2 protein expression levels was positively correlated with BAX expression, but negatively correlated with Bcl-2 expression levels.ConclusionsOur study results suggest that elevated MMP-2 expression and disturbance balance of Bcl-2/BAX expressions may be associated with the development and maintenance of AF. MMP-2 may be involved in the development of AF through promoting BAX expressions and inhibiting Bcl-2.

Protective effects and mechanisms of lactoferrin and HIF-1α on dry eye syndrome in mice.

SummaryBackgroundTo investigate the expression levels of importin13 (IPO13), c-kit, CD146, telomerase, caspase-3, bcl-2 and bax in endometrial polyps (EPs).Material/MethodsWe detected the mRNA expression levels of IPO13, c-kit, bcl-2 and bax in endometrial polyps (EPs) using real-time PCR. We detected the protein expression levels of IPO13, telomerase, CD146, caspase-3, bcl-2 and bax in EPs using S-P (Streptavidin-Peroxidase) immunohistochemistry. Western blotting was performed to determine the levels of importin13 and bcl-2 proteins in EPs.ResultsThe expression levels of IPO13, c-kit, telomerase, caspase3, and bax were lower in the EP tissue compared to normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05). The expression of CD146 was decreased in the EP tissue compared to the normal endometrial tissue during the proliferation phase of the menstrual cycle (p<0.05). The expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05).ConclusionsThe expression levels of IPO13, c-kit, telomerase, caspase3, and bax were decreased; however, the expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue. These findings suggest that the development of EPs is associated with the deregulated activities of the endometrial stem/progenitor cells and the decreased apoptosis of endometrial cells, with the latter being the major factor involved in the development of EPs.