Endothelial cell ferroptosis mediates monocrotaline-induced pulmonary hypertension in rats by modulating NLRP3 inflammasome activation.

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

Interferon regulatory factor 7 inhibits rat vascular smooth muscle cell proliferation and inflammation in monocrotaline-induced pulmonary hypertension.

AIMS: Although interferon regulatory factor 7 (IRF7) has known roles in regulating the inflammatory response, vascular smooth muscle cell proliferation, and apoptosis, its role in the pathogenesis of pulmonary hypertension (PH) is unclear. We hypothesized that IRF7 overexpression could inhibit pulmonary vascular remodeling and slow the progression of PH. MAIN METHODS: IRF7 mRNA and protein levels in the lung samples and pulmonary artery smooth muscle cells (PASMCs) isolated from monocrotaline (MCT)-induced PH rats were assessed. We evaluated the effects of IRF7 on inflammation, proliferation, and apoptosis using an in vivo MCT-induced PH rat model and in vitro methods. KEY FINDINGS: We noted decreased IRF7 mRNA and protein levels in the pulmonary vasculature of MCT-induced PH rats. IRF7 upregulation attenuated pulmonary vascular remodeling, decreased the pulmonary artery systolic pressure, and improved the right ventricular (RV) structure and function. Our findings suggest that nuclear factor kappa-Bp65 (NF-κBp65) deactivation could confer pulmonary vasculature protection, reduce proinflammatory cytokine (tumor necrosis factor-α, interleukin 6) release, and decrease PASMC proliferation and resistance to apoptosis via deactivating transcription factor 3 (ATF3) signaling. ATF3 deactivation induced the downregulation of the proliferation-dependent genes proliferating cell nuclear antigen (PCNA), cyclin D1, and survivin, coupled with increased levels of B cell lymphoma-2-associated X protein (Bax)/B cell lymphoma-2 (Bcl2) ratio, and cleaved caspase-3 in PASMCs. SIGNIFICANCE: Our findings showed that IRF7 downregulation could initiate inflammation via NF-κBp65 signaling, causing PASMC proliferation via ATF3 signaling pathway activation. Therefore, IRF7 could be a potential molecular target for PH therapy.

Activation of Nicotinic Acetylcholine α7 Receptor Attenuates Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Downregulating the NLRP3 Inflammasome.

Background: Inflammation and altered immunity contribute to the development of pulmonary arterial hypertension (PH). The alpha 7 nicotinic acetylcholine receptor (α7nAChR) possesses anti-inflammatory activities. The current study was performed to investigate the effects of a selective α7nAChR agonist, PNU-282987, on controlling a monocrotaline (MCT)-induced rat model of PH and explored the underlying mechanisms. Methods: Sprague-Dawley rats were injected with MCT and treated with PNU-282987 at the prevention (starting 1 week before MCT) and treatment (starting 2 weeks after MCT) settings. Four weeks after MCT injection, hemodynamic changes, right ventricular structure, and lung morphological features were assessed. Enzyme-linked immunosorbent assay, Western blot and qRT-PCR were performed to assess levels of inflammatory cytokines and NLRP3 (Nod-like receptor family pyrin domain-containing 3) inflammasome pathway in the rat lung tissues. In addition, the lung macrophage line NR8383 was used to confirm the in vivo data. Results: Monocrotaline injection produced PH in rats and downregulated α7nAChR mRNA and protein expression in rat lung tissues compared to sham controls. Pharmacological activation of α7nAChR by PNU-282987 therapy improved the rat survival rate, attenuated the development of PH as assessed by remodeling of pulmonary arterioles, reduced the right ventricular (RV) systolic pressure, and ameliorated the hypertrophy and fibrosis of the RV in rats with MCT-induced PH. The expression of TNF-α, IL-6, IL-1ß, and IL-18 were downregulated in rat lung tissues, which implied that PNU-282987 therapy may help regulate inflammation. These protective effects involved the inhibition of the NLRP3 inflammasome. In vitro assays of cultured rat lung macrophages confirmed that the anti-inflammation effect of PNU-282987 therapy may contribute to the disturbance of NLRP3 inflammasome activation. Conclusion: Targeting α7nAChR with PNU-282987 could effectively prevent and treat PH with benefits for preventing ongoing inflammation in the lungs of rats with MCT-induced PH by inhibiting NLRP3 inflammasome activation.

Protein expression of nucleophosmin, annexin A3 and nm23-H1 correlates with human nasopharyngeal carcinoma radioresistance in vivo.

Radioresistance is a significant obstacle in the treatment of endemic nasopharyngeal carcinoma (NPC). The present study aimed to identify proteins associated with radioresistance in NPC in vitro and in vivo. Proteomics analyses were conducted to screen for differentially-expressed proteins (DEPs) in parental CNE-2 cells and CNE-2R cells. Using proteomics approaches, 16 DEPs were identified. Of these DEPs, nucleophosmin (NPM1), annexin A3 and nm23-H1, were verified using western blot analyses. The tumorigenicity was investigated using mouse xenograft tumorigenicity assays, and tumor growth curves were generated. The protein expression of NPM1, annexin A3 and nm23-H1 was examined by immunohistochemically staining tumor tissues. NPM1 and annexin A3 protein levels were downregulated in the CNE-2R cells, whereas nm23-H1 expression was upregulated. In vivo tests showed that compared with the CNE-2 tumors, CNE-2R tumor growth was significantly retarded (P<0.05). CNE-2 tumor progression was inhibited by irradiation, but CNE-2R tumor progression was not, indicating that the CNE-2R cells were also radioresistant in vivo. NPM1 and annexin A3 expression was significantly lower in non-irradiated (NIR)-CNE-2R tumors compared with NIR-CNE-2 tumors (P<0.01). However, Nm23-H1 protein levels were significantly higher (P<0.05). Overall, the present study established comparable radioresistant and radiosensitive tumor models of human NPC, and identified candidate biomarkers that may correlate with radioresistance. The data showed that dysregulation of NPM1, annexin A3 and nm23-H1 expression correlated with the cellular and tumor radioresponse. These proteins are involved in the regulation of intracellular functions, including stress responses, cell proliferation and DNA repair. However, further clinical evaluations are required.

[Malaria Incidence and Control in Baoshan City during the Twelfth Five-Year Plan Period].

A retrospective analysis was made on malaria incidence in Baoshan City of Yunnan Province during the 2011-2015 Twelfth Five-Year Plan Period. The epidemiological characteristics and endemic situation of malaria were analyzed. A total of 1 301 malaria cases were reported in Baoshan City during 2011-2015, with an average incidence rate of 10.2 per 100 000 individuals, showing a relatively low prevelence of malaria. The cases were mostly imported（98.5%, 1 282/1 301）. No local malaria cases were found in Baoshan City since 2014. The cases were mostly in Tengchong City（65.6%, 853/1 301）, and mainly in the age range of 20-50 years （84.4%, 1 098/1 301）.

[Analysis of Time Distribution of Malaria Incidence in Baoshan City During 1990-2014].

The malaria epidemic data in Baoshan city from 1990 to 2014 were analyzed with concentration ratio and circular distribution. Results showed that the incidence of malaria in Baoshan city displayed a trend of decrease from 1990 to 2014. The total concentration ration M was 0.36, the circular distribution r was 0.30, and the average angle α was 148.78°. The malaria incidence peaked on May 31, with the epidemic period being from March 2 to August 29, which is inconsistent with the seasonal ebb and flow of local malaria vector.

[Expression and significance of Th17 cells and related cytokines in a murine model of systemic sclerosis].

OBJECTIVE: To study the expression and significance of Th17 cells and related cytokines in the peripheral blood, skin and lung in a murine model of systemic sclerosis (SSc). METHODS: Twenty female BALB/c mice were randomly divided into 2 groups, including a control group and a bleomycin(BLM) -injected-4-week group (SSc group). Pathological changes of the skin and lung were detected. The proportion of CD4(+)IL-17(+)Th17 cells in the peripheral blood, skin and lung of the mice was determined by flow cytometry. The mRNA expressions of RORγt, IL-17A, and IL-6 of the skin and lung were evaluated by real-time PCR. Enzyme linked immunosorbent assay was used to measure the levels of IL-17 and IL-6 in the serum. RESULTS: Dermal inflammation and the score of PF were significantly increased in the SSc group as compared with the control group (2.60±0.84 vs. 0.40±0.52, 2.80± 1.81 vs.0.60±0.70). Hydroxyproline(HYP) contents of the skin and lung were obviously increased in the SSc group than in the control group [(3.17±1.74) mg/g vs. (1.45±0.40) mg/g,(0.53±0.14) mg/g vs. (0.38±0.16) mg/g], all P<0.05. The percentage of Th17 cells in the peripheral blood, skin and lung of the SSc group were significantly increased as compared with the control group [(2.07±0.89)% vs. (1.02±0.32)%,(5.80±2.02)% vs. (1.64±0.58)%,(5.24±2.43)% vs. (1.92±0.98)%,P <0.01]. Compared with the control group, the mRNA levels of IL-17A, RORγt, IL-6 in the skin and lung of the SSc group were higher. The levels of IL-17, IL-6 of the SSc group in the serum were significantly increased, all P<0.05. The frequency of Th17 cells, and the levels of IL-17 and IL-6 in the blood had a positive correlation with dermal and pulmonary inflammation, fibrosis and HYP contents of the skin and lung, The frequency of Th17, IL-17 and IL-6 in the skin and lung had, respectively, a positive correlation with dermal and pulmonary inflammation, HYP contents of the skin and lung, all P<0.01. CONCLUSION: Th17 cells were significantly increased in the peripheral blood, skin and lung of a murine model of SSc, and had an intimate relationship with inflammation and fibrosis of the skin and lung, and involved the pathogenesis of SSc through producing IL-17, IL-6.

[In vivo short hairpin RNA interference of vascular endothelial growth factor on drug-resistant tongue cancer cells].

OBJECTIVE: To study the in vivo interference effects of vascular endothelial growth factor (VEGF) short hairpin RNA (shRNA) on xenografts of drug-resistant tongue cancer cells. METHODS: Drug-resistant tongue caner cells Tca/Cisplatin (DDP) were injected subcutaneously into nude mice to establish xenograft models, which were randomly divided into non-transfected group, mock control group, control group transfected with scrambled sequence plasmid, interference group transfected with VEGF-shRNA expression plasmid. Liposome-mediated plasmid transfection was done in the latter three groups every three days. Xenografts were observed and tumor growth curve was measured. After the 10th transfection, tumors were anatomized and weigh. Microvessel density was detected by immunohistochemical staining. In situ hybridization assay was used to test VEGF mRNA, and immunohistochemistry to test VEGF, P-glycoprotein (P-gp), B cell lymphoma/leukemia-2 (bcl-2) and extracellular signal-regultaed kinase 2 (ERK-2) protein. RESULTS: Tumor growth in VEGF-shRNA interference group was significantly slow. Tumor weight was (0.4781 ± 0.0860) g, microvessel density (7.35 ± 1.31)/view, VEGF mRNA (0.0767 ± 0.0234), VEGF protein (0.1301 ± 0.0433), P-gp (0.1517 ± 0.0184), bcl-2 (0.1218 ± 0.0251) and ERK-2 protein (0.1178 ± 0.0291) in VEGF-shRNA interference group; all of them were less than those in the other three groups (P < 0.05). CONCLUSIONS: Inhibition targeting VEGF may become a potential therapy for drug-resistant tongue cancer.

[Tyrosine kinase A and vascular endothelial growth factor receptor 2 in the invasion and metastasis of salivary adenoid cystic carcinoma].

OBJECTIVE: To determine the effect of tyrosine kinase A (TrkA) and vascular endothelial growth factor receptor 2 (VEGFR2) in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC). METHODS: The expression of TrkA and VEGFR2 were detected by immunohistochemical staining in 47 cases of SACC of salivary glands. Clinical data were reviewed by multivariate prognostic analysis. RESULTS: The positive rate of TrkA and VEGFR2 in SACC was 87.23% (41/47) and 85.11% (40/47) respectively. Express of TrkA and VEGFR2 in perineural invasion and recurrence group were higher than non-perineural invasion and non-recurrence group. Significant difference was found in microvessel density (MVD) and VEGFR2 expression within different groups (P < 0.05). MVD in perineural invasion group (25.14 +/- 2.83) was significantly higher than that in none perineural invasion group (18.81 +/- 1.33) (P < 0.05). MVD in recurrence or metastasis group (26.58 +/- 2.38) was significantly higher than that (19.06 +/- 1.39) in none recurrence nor metastasis group (P < 0.05). CONCLUSION: Positive correlation between expression of TrkA, VEGFR2 and nerve invasion and vessel metastasis of SACC indicate that TrkA and VEGFR2 play important roles in the invasion and metastasis of SACC. It is possible that TrkA and VEGFR2 could be an aid for evaluating the prognosis of SACC patients.

Study on expression of laminin in patients with intractable epilepsy.

In this study, we examined differences in serum laminin expression in patients with intractable epilepsy. Our results suggest that elevated laminin may contribute to the pathogenesis of intractable epilepsy. ELISA and western blots were used to measure laminin in the serum of 30 intractable epilepsy patients, 46 nonintractable epilepsy patients, and 20 normal subjects. By ELISA, serum laminin levels were greater in intractable epilepsy patients (177.396 +/- 30.602) and nonintractable epilepsy patients (121.915 +/- 35.215) than in normal control subjects (67.474 +/- 7.197); laminin was significantly greater in the intractable epilepsy group than in the nonintractable epilepsy group. In western blots, the optical density ratio of laminin to ss-actin was 0.871 +/- 0.032 for the intractable epilepsy group, 0.686 +/- 0.017 for the nonintractable epilepsy group, and 0.385 +/- 0.024 for the normal control group. The optical density ratios of the intractable and nonintractable epilepsy groups were higher than those for the normal control group, and the intractable epilepsy group was even greater than the nonintractable epilepsy group. Thus, laminin is significantly increased in epilepsy patients, and this increase is more profound in intractable epilepsy patients.

[Vasoactive intestinal peptide expression and its clinical significance in gastric adenocarcinoma].

OBJECTIVE: To investigate the expression of vasoactive intestinal peptide (VIP) in gastric adenocarcinoma, and to evaluate the correlation of VIP level with clinical pathologic parameters. METHODS: The level of VIP in sera from gastric adenocarcinoma patients and healthy people was investigated by ELISA. Moreover, the differential gene expression between gastric adenocarcinoma, gastric dysplasia, and the corresponding normal gastric mucosa were determined by RT-PCR. Western Blot was also used to measure the expression of VIP in the gastric adenocarcinoma and the normal gastric mucosa. RESULTS: The serum level of VIP was (5.794 +/- 0.014) ng/ ml in normal control and was (14.437 +/- 0.825) ng/ml in gastric adenocarcinoma patients, showing significant difference (P < 0.05). Meanwhile,the V/B of gastric adenocarcinoma tissues was greater than that of gastric dysplasia and the corresponding normal gastric mucosa (P <0.01), the values of V/B were 1.5261 +/- 0.3028, 0.9334 +/- 0.2872,and 0.9051 +/- 0.2794, respectively. The values of V/B between normal gastric mucosa and gastric dysplasia were not different significantly (P > 0.05). There were significantly negative correlation between the VIP mRNA expression of the differentiation degree of tumor (P < 0.05). The VIP mRNA expression was higher in gastric adenocarcinoma with lymph node metastasis than that without lymph node matastsis (P < 0.05). The VIP protein expression of the gastric adenocarcinoma tissues was greater than that of normal control. CONCLUSION: This findings provide a direct evidence to support the possibility that VIP play a cofactor role in the pathogenesis of gastric adenocarcinoma.

[Combining of TNP-470 and 5-Fu in inhibition of adenoid cystic carcinoma in nude mice model].

OBJECTIVE: To study the effect of angiogenesis inhibitor and its combine with chemical drug in suppressing the growth of adenoid cystic carcinoma (ACC). METHODS: Acc-M cells were inoculated subcutaneous into BABL/C nu/nu mice. The mice were divided into control, different dose of TNP-470 treatment groups, 5-Fu treatment group and TNP-470 plus 5-Fu treatment group. Treatments were given 48 hours after inoculation. The mice were sacrificed on the 22nd day and excised tumors were weighted. Tumors were also investigated by immunohistochemistry and ultrastructural observations. RESULTS: TNP-470 100 mg/kg/qod efficiently inhibited the growth of Acc-M tumors. TNP-470 30 mg/kg/qod combined with 50 mg/kg/week 5-Fu also resulted in significant growth inhibit of the tumors. TNP-470 suppressed tumor growth by inhibiting neovascularization, therefore inducing apoptosis of Acc-M cells. All experimental groups had different degrees of VEGF and bFGF express. CONCLUSION: Since ACC is a slow developing tumor, blood supply is not so sufficient as sarcomas. Angiogensis inhibitor may inhibit its growth in high dosage. Combining medium dosage of angiogensis inhibitor with chemical drug may have synergistic result in inhibiting ACC growth.