HO-1 nuclear accumulation and interaction with NPM1 protect against stress-induced endothelial senescence independent of its enzymatic activity.

Heme oxygenase-1 (HO-1) has attracted accumulating attention for its antioxidant enzymatic activity. However, the exact regulatory role of its non-enzymatic activity in the cardiovascular system remains unaddressed. Here, we show that HO-1 was accumulated in the nuclei of stress-induced senescent endothelial cells, and conferred protection against endothelial senescence independent of its enzymatic activity. Overexpression of ΔHO-1, a truncated HO-1 without transmembrane segment (TMS), inhibited H2O2-induced endothelial senescence. Overexpression of ΔHO-1H25A, the catalytically inactive form of ΔHO-1, also exhibited anti-senescent effect. In addition, infection of recombinant adenovirus encoding ΔHO-1 with three nuclear localization sequences (NLS), alleviated endothelial senescence induced by knockdown of endogenous HO-1 by CRISPR/Cas9. Moreover, repression of HO-1 nuclear translocation by silencing of signal peptide peptidase (SPP), which is responsible for enzymatic cleavage of the TMS of HO-1, exacerbated endothelial senescence. Mechanistically, nuclear HO-1 interacted with NPM1 N-terminal portion, prevented NPM1 translocation from nucleolus to nucleoplasm, thus disrupted NPM1/p53/MDM2 interactions and inhibited p53 activation by NPM1, finally resisted endothelial senescence. This study provides a novel understanding of HO-1 as a promising therapeutic strategy for vascular senescence-related cardiovascular diseases.

Pterostilbene and its nicotinate derivative ameliorated vascular endothelial senescence and elicited endothelium-dependent relaxations via activation of sirtuin 1.

Vascular endothelial cell senescence is a leading cause of age-associated diseases and cardiovascular diseases. Interventions and therapies targeting endothelial cell senescence and dysfunction would have important clinical implications. This study evaluated the effect of 10 resveratrol analogues, including pterostilbene (Pts) and its derivatives, against endothelial senescence and dysfunction. All the tested compounds at the concentrations from 10-9 M to 10-6 M did not show cytotoxicity in endothelial cells by MTT assay. Among the 10 resveratrol analogues, Pts and Pts nicotinate attenuated the expression of senescence-associated ß-galactosidase, downregulated p21 and p53, and increased the production of nitric oxide (NO) in both angiotensin II - and hydrogen peroxide - induced endothelial senescence models. In addition, Pts and Pts nicotinate elicited endothelium-dependent relaxations, which were attenuated in the presence of endothelial NO synthase (eNOS) inhibitor L-NAME or sirtuin 1 (SIRT1) inhibitor sirtinol. Pts and Pts nicotinate did not alter SIRT1 expression but enhanced its activity. Both Pts and Pts nicotinate have high binding activities with SIRT1, according to surface plasmon resonance results and the molecular docking analysis. Inhibition of SIRT1 by sirtinol reversed the anti-senescent effects of Pts and Pts nicotinate. Moreover, Pts and Pts nicotinate shared similar ADME (absorption, distribution, metabolism, excretion) profiles and physiochemical properties. This study suggests that the Pts and Pts nicotinate ameliorate vascular endothelial senescence and elicit endothelium-dependent relaxations via activation of SIRT1. These two compounds may be potential drugs for the treatment of cardiovascular diseases related to endothelial senescence and dysfunction.

Rhamnocitrin extracted from Nervilia fordii inhibited vascular endothelial activation via miR-185/STIM-1/SOCE/NFATc3.

BACKGROUND: Vascular endothelial activation is pivotal for the pathological development of various infectious and inflammatory diseases. Therapeutic interventions to prevent endothelial activation are of great clinical significance to achieve anti-inflammatory strategy. Previous studies indicate that the total flavonoids from the endemic herbal medicine Nervilia fordii (Hance) Schltr exerts potent anti-inflammatory effect and protective effect against endotoxin lipopolysaccharide (LPS)-induced acute lung injury, and shows clinical benefit in severe acute respiratory syndromes (SARS). However, the exact effective component of Nervilia fordii and its potential mechanism remain unknown. PURPOSE: The aim of this study was to investigate the effect and mechanism of rhamnocitrin (RH), a flavonoid extracted from Nervilia fordii, on LPS-induced endothelial activation. METHODS: The in vitro endothelial cell activation model was induced by LPS in human umbilical vein endothelial cells (HUVECs). Cell viability was measured to determine the cytotoxicity of RH. RT-PCR, Western blot, fluorescent probe and immunofluorescence were conducted to evaluate the effect and mechanism of RH against endothelial activation. RESULTS: RH was extracted and isolated from Nervilia fordii. RH at the concentration from 10-7 M-10-5 M inhibited the expressions of interlukin-6 (IL-6) and -8 (IL-8), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell-adhesion molecule-1 (VCAM-1), and plasminogen activator inhibitor-1 (PAI-1) in response to LPS challenge. Mechanistically, RH repressed calcium store-operated Ca2+ entry (SOCE) induced by LPS, which is due to downregulation of stromal interaction molecule-1 (STIM-1) following upregulating microRNA-185 (miR-185). Ultimately, RH abrogated LPS-induced activation of SOCE-mediated calcineurin/NFATc3 (nuclear factor of activated T cells, cytoplasmic 3) signaling pathway. CONCLUSION: The present study identifies RH as a potent inhibitor of endothelial activation. Since vascular endothelial activation is a pivotal cause of excessive cytokine production, leading to cytokine storm and severe pathology in infectious diseases such as SARS and the ongoing COVID-19 pneumonia disease, RH might suggest promising therapeutic potential in the management of cytokine storm in these diseases.

[The inhibiting role of recombinant plasmid PGCsi-AQP1 on laryngeal carcinoma in vivo].

OBJECTIVE: To construct a kind of recombinant plasmid PGCsi-AQP1 delivery with DOPC and explore the inhibit effect of laryngeal carcinoma by RNAi targeting AQP1 in vivo. METHOD: Male BALB/c mice, 6 weeks of age transplanted with laryngeal carcinoma cell line Hep-2, four groups were divided randomly: Tail vein injection group (TVIG), Carcinoma around injection group (CAIG), negative control group (NCG) and blank control group (BCG). The recombinant plasmid PGCsi-AQP1 delivery with DOPC were inject into tail vein or surrounding tumor. HE pathological slides and tumor size were observed and inhibitory rate was figured up. The level of AQP1 protein expression and high microvessel density were detected by Immunohistochemical staining (IHC). RESULT: We constructed BALB/c mice models of laryngeal carcinoma successfully (1) HE staining: cell putrescence, nuclear pyknosis and apoptotic bodies were more in the tumor tissues of experimental groups than two control groups. (2) The total volumes of tumor in experimental group were both smaller than in two control groups (P < 0.01). The inhibition rate of TVIG and CAIG were 52.4% and 53.5% respectively and there was no significant difference (P > 0.05). (3) IHC: the AQP1 positive cells and microvessel density in TVIG and CAIG were both less than in two control groups (P < 0.01). CONCLUSION: Neutral lipsomes DOPC could help carriaging the recombinant plasmid PGCsi-AQP1 to tumor and then play an inhibit role in laryngeal carcinoma tissue by RNAi targeting AQP1 in vivo.

[Efficacy and safety analysis with standardized mite allergen subcutaneous immunotherapy in 90 patients with allergic rhinitis].

OBJECTIVE: To evaluate the three-year efficacy and safety with standardized dust mite subcutaneous immunotherapy in patients with allergic rhinitis. METHODS: Ninety patients who were diagnosed as allergic to mite by skin prick test and serum IgE were include in the standardized allergen-specific dose-escalation regimen. Nasal symptom score (0-3) were collected before treatment and three years after treatment; VAS (visual analogue scale, 0-10) of all nasal symptoms and drug use score were collected every four months; frequency of local and systemic reactions were recorded in the duration of dose escalation and maintenance. RESULTS: Nasal blocking, sneeze, rhinorrhea and nasal itch were significantly improved after 3 years treatment (before treatment: 2[2;3], 2[2;3], 2[2;3], 2[1;2] ; after treatment: all were 0[0;0]; Z value were -8.310, -8.408, -8.377, -8.287, all P were 0.000). VAS of all nasal symptoms and drug use score decreased dramatically after escalation period (before treatment: 8.00[7.00;8.85], 2.00[1.50;2.00]; after treatment: 1.00[1.00;1.50], 0 [0;0]; Z value were -8.287, -8.248, P value 0.086, 0.744), and maintained afterwards (F value were 2.483, 0.296; P value were 0.086, 0.744). Ninety-eight case times (64.47%) local reactions mainly happened in maintenance period; the frequency of systemic reactions was 2.54%. CONCLUSION: The standardized specific allergen immunotherapy for allergic rhinitis is safe and effective.

[Correlation between the expression of aquaporin 1 and the micro-angiogenesis in laryngeal carcinoma].

OBJECTIVE: To confirm the expression and distribution of aquaporin 1 in laryngeal carcinoma, and analyze the correlation to micro angiogenesis in the development of laryngeal carcinoma. METHOD: The expression and distribution of aquaporin 1 were examined by immunohistochemical technique in laryngeal carcinoma. New vessels were labelled using CD105 monoclonal antibody. Systematic analysis had been done on IMVD and data of clinical pathology. RESULT: Positive AQP1 cells and IMVD in primary carcinoma were higher than that in normal laryngeal mucosa. The expression of AQP1 was gradually increased with the decrease of differentiation grade of laryngeal carcinoma. The expression of AQP1 in lymphatic metastasis groups was higher than that of controls. CONCLUSION: The expression of AQP1 in tumor cells and IMVD of primary laryngeal carcinoma are higher than normal. Not only is increased expression of AQP1 positive relevant to IMVD, but to lymphatic metastasis and malignant cell grading from laryngeal carcinoma. The increased AQP1 expression in epithelial cells of carcinoma and vascular endothelial cells may play a considerable role in growing, infiltrating, metastasis of malignant tumor by promoting vascular permeability.

[Role of aquaporin 1 in the migration of eosinophils from asthmatic guinea pigs].

OBJECTIVE: To investigate the role of aquaporin 1 (AQP1) in the migration of eosinophils (EOS) and to determine if AQP-1 can be viewed as the chemotactic activity marker of EOS. METHODS: Asthma model of guinea pigs were developed and EOS were purified from both peripheral blood and bronchoalveolar lavage fluid (BALF). The smears of EOS were studied by in situ hybridization for determining AQP1 mRNA and immunofluorescence under laser scanning confocal microscope for determining AQP1 protein. RESULTS: AQP1 was found expressed in EOS both from peripheral blood and from BALF. Compared with the expression of AQP1 mRNA (mean grey value 109.200 +/- 5.756, x +/- s) and protein (average fluorescence intensity 279.926 +/- 11.293) in EOS from BALF, there was stronger expression of AQP1 mRNA (92.904 +/- 3.290) and protein (425.081 +/- 17.474) in EOS from peripheral blood. The difference both of AQP1 mRNA (t = 9.519, P < 0.05) and protein(t = 27.020, P < 0.05) were considered statistically significant respectively. CONCLUSIONS: AQP1 plays a crucial role in EOS movement. It is possible that EOS produce more AQP1 protein to accelerate its migration to inflammatory tissues under allergic disease and EOS with AQP1 highly expressed are activated. AQP1 can be viewed as the chemotactic activity marker of EOS.

[Expression of monocyte chemotactic protein 1 and vascular endothelial growth factor in nasal polyps].

OBJECTIVE: To evaluate the relationship between the expression of monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) in nasal polyp (NP) tissues and the role of MCP-1 in the formation of nasal polyps. METHODS: Forty nasal polyp tissues obtained from NP patients were used to detect the expression of MCP-1 and VEGF at the levels of protein and mRNA by immunohistochemical and in situ hybridization assay. Twenty-five inferior turbinate (IT) samples obtained from chronic rhinitis patients were used as control. RESULTS: The expression of MCP-1 and VEGF was higher in NP tissue than in IT at protein and mRNA levels (P <0.01). The expression of these two cytokines was positively correlated(r = 0.871, P <0.05). CONCLUSIONS: MCP-1 and VEGF were involved in the formation of nasal polyps.

[Effect of inhibiting aquaporin-1 on proliferation and apoptosis of the Hep-2 cell].

OBJECTIVE: To explore the effect of inhibiting aquaporin-1 (AQP-1) on proliferation and apoptosis of the Hep-2 cell. METHOD: The expression of AQP-1 in Hep-2 cell was identified through immunohistochemistry. After using acetazolamide, an antagonist of AQP-1, the assay of cell proliferation, apoptosis and the expression of AQP-1 were performed by microculture tetrazolium salt(MTT) and flow cytometry, TUNEL respectively. RESULT: The expression of AQP-1 is mainly found on membrane of Hep-2 cell and the expression is significantly reduced when the concentration of acetazolamide is 5 x 10(-5) mol/L. Noteworthily, inhibiting AQP-1 by acetazolamide, the rate of cell growth and cell apoptosis significantly increases with the time prolong. It showed time-dependent phenomena. There is positive relationship obviously between cell apoptosis rate and inhibition rate of the AQP-1 protein expression. CONCLUSION: The inhibiting AQP-1 with acetazolamide may significantly induce apoptosis of Hep-2 cell. It suggest that AQP-1 may be as new target of therapy of laryngeal cancer.

[Expression of prolactin in macrophage of nasal polyps and its significance].

OBJECTIVE: To detect the expression level and distribution of prolactin (PRL) in nasal polyp and to find out the significance of the mechanism of PRL in the invasion of nasal polyp. METHODS: Twenty-five polyp tissues were obtained from the patients who were subjected to nasal polypectomy in our Department. Inferior turbinate mucosa was used as control obtained from 12 patients with rhinogenous snoring. HE staining was performed for routine histopathologic examination. The expression of PRL in nasal polyps was observed by immunohistochemical staining, and six polyp tissues were estimated through double staining for determining cells which expressed PRL. RESULTS: (1) Positive expression of PRL was significantly stronger (t =4.004, P < 0.01) in 25 nasal polyp tissues (2.05 +/- 0.88) than that in 12 normal inferior turbinate mucosa (0.96 +/- 0.50). Positive expression of macrophage (CD68) was significantly stronger (t = 3.519, P < 0.01) in 25 nasal polyp tissues (1.85 +/- 0.83) than that in 12 normal inferior turbinate mucosa (0.93 +/- 0.52). (2) The PRL expressing cell mainly was the macrophage as demonstrated by double immunohistochemical method. CONCLUSION: PRL derived from macrophages of nasal mucosa may participate in the formation of nasal polyp through its local immune modulation.

[Expression of aquaporin-1 and Bcl-2 mRNA in eosinophils of the nasal polyps and its significance].

OBJECTIVE: To study the significance of aquaporin-1 (AQP-1) expression in the eosinophils of nasal polyps. The expression and location of AQP-1 mRNA and apoptosis associated gene Bcl-2 mRNA in nasal polyps were explored. METHODS: Sixteen nasal polyp samples were collected from 11 women and 5 men aged 20-65 years during routine endonasal surgery. Nasal mucosa specimens from the inferior turbinates of 10 patients with allergic rhinitis (7 women and 3 men, aged 16-58 years), collected during septoplasty, were used as controls. The expression of AQP-1 mRNA and Bcl-2 mRNA was detected in serial adjacent sections by in situ hybridization and eosinophils were examined by stain MGG. RESULTS: AQP-1 mRNA expression was found in all 16 nasal polyps and in 4 of 10 inferior turbinate tissues, the mean expression rates were (93.16 +/- 13.25)% and (19.54 +/- 4.98)%, respectively. All 16 nasal polyps and 10 control nasal tissues expressed Bcl-2 mRNA, by the average rates of (84.74 +/- 12.10)% and (16.45 +/- 3.12)%, respectively. The expression of AQP-1 mRNA was positively correlated with Bcl-2 mRNA expression in nasal polyps (r = 0.875, P < 0.01). CONCLUSIONS: AQP-1 contributes to the survival of eosinophils in nasal polyps by keeping the permeation balance of eosinophils.

[Expression of inducible nitric oxide synthase mRNA in epithelial cell of nasal mucosa is upregulated through Toll-like receptor-4].

OBJECTIVE: To investigate the upregulation of inducible nitric oxide synthase (iNOS) in epithelial cell of nasal mucosa through Toll-like receptor-4 (TLR-4). METHOD: Specimens from 30 patients of chronic sinusitis and 21 healthy adults were examined by in situ hybridization for TLR-4 and iNOS mRNA. RESULT: All 30 samples of chronic sinusitis showed a stronger expression of TLR-4 and iNOS than in controls (P<0.01). iNOS upregulated in nasal epithelium correlated with TLR-4 (r=0.435, P<0.01). CONCLUSION: It suggested that TLR-4 may play a role in enhancing local host defence and inflammation via upregulating the expression of iNOS mRNA and the subsequent increased production of NO.

[Study on molecular mechanism of anti-aging effect of rhidosin on olfactory bulb in senile rats].

OBJECTIVE: To research the molecular mechanism of anti-aging effect of rhidosin on olfactory bulb in senile rats. METHOD: Ten 3 month-old rats and twenty 26-month-old rats were randomed in rhidosin and saline groups. After decapitated the rats, the olfactory bulb were cut and immediately fixed with neutral formation, followed with paraffin-embedding, serial sectioning, immunohistochemical staining and light microscopic observation. RESULT: The proteins of FGF, Bcl-2 and Bax were mainly expressed in mitral cells in the olfactory bulb of the rats. The positive expression rate of FGF and Bcl-2 in young rats group was significantly higher than that in old rats group(P < 0.01). The FGF and Bcl-2 were positive relationship(r = 0.8971; P < 0.01). The expression of Bax in young control group was slightly lower than that in old control group, but there was no significant difference (P > 0.05). The positive expression rate of FGF and Bcl-2 in rhidosin group was significantly higher than that in control group in old rate group(P < 0.01). CONCLUSION: FGF is a factor of ascending the anti-apoptosis gene Bcl-2 in mitral cells; rhidosin can increase the expression of FGF and Bcl-2 proteins. We guess that the mechanism of rhidosin anti-aging effect on the olfactory system may be through the effect of increasing the expression of FGF to inhibit the apoptosis of mitral cells.

[Immunoregulatory effect of Bacillus Calmette Guerin aerosol to allergic airway diseases].

OBJECTIVE: To investigate whether bacillus calmette guerin (BCG) is effective used transairway and its preventive mechanisms to allergic airway diseases. METHODS: Animal experiment was finished. Adult rats were devided into four groups: control group, ovalbumin-sensitized group, BCG used transairway group, BCG used transairway + ovalbumin-sensitized group. Then these animals symptoms were studied and the pathology change were studied under microscope about nasal and bronchi mucosa and bronchoalveolar lavege fluid cells. IL-4 mRNA and IFN-gamma mRNA in lung tissue were detected through RT-PCR, the protein production of IL-4 and IFN-gamma about bronchoalveolar lavege fluid and serum were detected through ELISA. RESULTS: In ovalbumin-sensitized group, allergic animal model were made successfully. In only BCG used transairway group, the symptoms of animals were normal, few inflammation cells infiltrated into the mucosa of nasal and bronchi, the numbers of macrophage were greatly increased in smear of bronchoalveolar lavege fluid, IFN-gamma mRNA and protein production were greatly increased. In BCG used transairway + ovalbumin-sensitized group, the allergic symptoms and inflammation were greatly reduced, not only IFN-gamma mRNA and protein production were increased but also IL-4 mRNA and protein production were greatly decreased. CONCLUSION: It is a good pathway that BCG used transairway. The immunoloregulation mechanisms of BCG to allergic airway diseases are to enhance Th1 response, in the meantime, to suppress Th2 response.