[Isolation and identification of Arbovirus in Hainan province, 2017-2018].

Objective: To understand the types and distribution of Arboviruses in Hainan province. Methods: Blood-sucking insects were collected in Hainan province from 2017 to 2018. After laboratory treatment, BHK-21 cells and C6/36 cells were inoculated with grinding supernatant of all blood-sucking insects to isolate all of involving virus. Arbovirus genes in blood-sucking insects were detected in parallel by RT-PCR method. Results: A total of 15 062 mosquitoes were classified into four genera (Culex, Armigeres, Aedes, Anopheles) and 11 360 midges were collected. Culex tritaeniorhynchus was in the majority and accounted for 92.88% (13 990/15 062) of all the mosquitoes collected. Four strains of virus isolates were notified by tissue culture method. Three strains of viruses belonged to Japanese encephalitis virus (JEV), with the other one as Getah virus (GETV). Five pools of JEV gene amplification were positive, from Culex tritaeniorhynchus. Results from the phylogenetic analysis showed that they belonged to genotype JEV-Ⅰ. The minimum infection rate of JEV was 0.57‰ (8/13 990). A total of 5 pools of Akabane virus (AKV) gene amplification were positive. The minimum infection rate of AKV was 0.44‰ (5/11 360). Based on the S gene and M gene sequences of the virus, data from the phylogenetic analysis showed that the five AKV strains carried by midges in Hainan province were in a separate evolutionary branch and with formed unique geographical distribution. Conclusions: JEV and GETV had been isolated again from the mosquito specimens in this survey, since the 1980s. AKV was detected from the midge specimens in Hainan province. These results showed the needs of strengthening the programs on detection and monitor of JEV, GETV and AKV that were related to animal and human diseases in order to reduce the risks of related diseases in this area.

Effects of hydroxycamptothecin on the expression of matrix metalloproteinase-1 (MMP-1), tissue inhibitor of MMP-1, and type I collagen in rats with pulmonary fibrosis.

The aim of this study was to investigate the effects of hydroxycamptothecin (HCPT) on the expression of matrix metalloproteinase-1 (MMP-1), tissue inhibitor of MMP-1 (TIMP-1), and type I collagen in the lung tissue of rats with pulmonary fibrosis induced by bleomycin A5. We used hematoxylin eosin staining to observe the degree of pulmonary fibrosis in rats; Masson staining, reverse transcription polymerase chain reaction, and immunohistochemistry were used to observe the expression of collagen, MMP-1, and TIMP-1, and type I collagen. The expression of MMP-1 in the model group decreased significantly, while the expression of TIMP-1 and type I collagen significantly increased. After treatment with HCPT, the degree of pulmonary fibrosis and the expression of TIMP-1 and type I collagen decreased in all treatment groups. However, the expression of MMP-1 increased in a dose-dependent manner. Our results showed that HCPT decreased the pulmonary fibrosis induced by bleomycin A5 in rats, and an increase in MMP-1 expression and decrease in the TIMP-1 and type I collagen expression may be the mechanism that regulates the metabolism of the extracellular matrix.

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells.

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.

[Protective effect of ONO-1078, a leukotriene antagonist, on focal cerebral ischemia in mice].

AIM: To determine whether ONO-1078 [pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy) benzoyl-amino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate], a potent leukotriene antagonist, has protective effect on focal cerebral ischemia in mice. METHODS: Focal cerebral ischemia was induced by permanent middle cerebral artery (MCA) occlusion in mice. ONO-1078 (0.01, 0.05, 0.10 mg.kg-1), dexamethasone (0.5 mg.kg-1), nimodipine (0.2 mg.kg-1) or saline (control) were injected i.p. once daily for 3 days, and 30 min before MCA occlusion. Twenty-four hours after cerebral ischemia, the neurological scores were evaluated, infarct volumes and areas of the right and left cerebral hemispheres were measured by computer imaging analysis. RESULTS: ONO-1078, dexamethasone and nimodipine reduced the neurological scores. ONO-1078 and dexamethasone reduced the ratio of right/left hemisphere area, indicating inhibition of brain edema, while nimodipine showed no effect. ONO-1078 dose-dependently reduced infarct size, and dexamethasone and nimodipine showed the same effect. CONCLUSION: ONO-1078 showed protective effect on focal cerebral ischemia. This may represent a novel approach to the treatment of acute cerebral ischemia.

MCI-186: further histochemical and biochemical evidence of neuroprotection.

The bioactivity of 3-methyl-1-phenyl-pyrazolin-5-one (MCI-186) was examined based on histochemical changes in drastic global ischemic rat brains. Rats with mean arterial blood pressure reduction were subjected to 60 min cerebral ischemia/80 min reperfusion. Infusion of MCI-186 at 3.0 mg/Kg reduced brain infarction from 21 +/- 4% (saline control, n= 15) to 11 +/- 3% (n=16, p<0.05). By comparison, infusion of up to 20 mg/Kg propyl galalate (PG)--a well documented antioxidant--produced an infarct percentage of 14 +/- 5% (n=8), close to the saline control. Biochemically, the neuroprotective effect of MCI-186 was demonstrated by diminishing the release of creatine kinase (CK) in serum from 3363 +/- 608 U/L (n=14) in saline control to 1989 +/- 293 U/L (n= 15) in MCI group (p<0.05), while PG did not lower the activity of CK significantly. MCI-186 behaves as a free radical scavenger by suppressing the formation of superoxide anion in xanthine oxidase (XO)-hypoxanthine (HP) system (p<0.05). Our data supported our contention that MCI-186 has potent anti-stroke effect with antioxidant activities.

Aortic endothelial cells damaged by a nitric oxide donor and protected by flavonoids.

Cultured porcine aortic endothelial cells (PAEC) were exposed to four concentrations (0.00 mM - 5.00 mM) of 3-Morpholino-sydnonimine-hydrochloride (SIN-1, a nitric oxide donor). SIN-1 demonstrated a dose dependent cytotoxicity against PAEC as indicated by the thiobarbituric acid (TBA) assay. Morphologically and biochemically, the presence of selected flavonoids (morin, quercetin, or catechin) was shown to protect the PAEC from SIN-1 toxicity. Protection levels determined from the TBA assay were significant (p<0.05) for all flavonoids, with morin at 72+/-8%. Quercetin and catechin had comparable protective activities of 54+/-6% and 43+/-3%, respectively. This study supports the contention that SIN-1 is cytotoxic to PAEC and that antioxidants such as flavonoids may attenuate such toxicity.

Bronchodilating effects of bambuterol on bronchoconstriction in guinea pigs.

AIM: To study the effects of bambuterol (Bam) on bronchoconstriction in guinea pigs. METHODS: Bronchospasm induced by histamine aerosol, lung resistance (RL) and dynamic lung compliance (Cdyn) changes induced by ovalbumin aerosol in vivo, isolated resting lung parenchyma strips, and carbamylcholine-induced tracheal constriction in vitro in guinea pig were investigated. RESULTS: Bam dose-dependently prolonged the time to histamine-induced collapse, ED50 values (95% confidence limits) of Bam intragastric gavage (i.g.) after 1 h, 4 h, and 24 h were 0.74 (0.60-0.91), 0.75 (0.61-0.91) and 1.00 (0.77-1.30) mg.kg-1, respectively. Bam 2 or 10 mg.kg-1 i.g. 2 h before ovalbumin aerosol partly or almost completely inhibited bronchial challenge of ovalbumin-induced change of RL and Cdyn. Bam 0.1-1.0 mumol.L-1 gave a weak relaxation on isolated tracheal strips induced by carbamylcholine and failed to relax the isolated resting lung parenchyma strips in guinea pig. CONCLUSION: Bam showed a long-acting bronchodilation by its slow metabolism in vivo.

Morin hydrate: a better protector than purpurogallin of corneal endothelial cell damage induced by xanthine oxidase and SIN-1.

PURPOSE: Free radicals are responsible for tissue injury in corneal preservation and transplantation. Morin hydrate, a flavonoid from Brazil wood, has been shown to be cytoprotective in several types of cells. The aim of this study was to investigate the effectiveness of morin hydrate on rabbit corneal endothelial cells against damage induced by oxyradicals and nitric oxide. METHODS: Corneal endothelial cell cultures were prepared from New Zealand white rabbits, using standard microcarrier technique. Two free-radical-generating systems were used-17 IU/L xanthine oxidase/1 mM hypoxanthine and 5 mM 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1, a nitric oxide-donating agent). RESULTS: Over 95% of cultured corneal endothelial cells necrosed within 3.6 +/- 1.5 min after exposure to xanthine oxidase/hypoxanthine. Adding morin hydrate delayed cell necrosis to 5.8 +/- 0.3 min (0.25 mM morin hydrate), 13.3 +/- 5.0 min (0.5 mM), and 41.5 +/- 8.6 min (1.0 mM). Exposed to nitric oxide generated by SIN-1, cells necrosed by 9.5 +/- 2.5 min, versus 14.1 +/- 1.3 min (0.25 mM morin hydrate), 27.2 +/- 2.0 min (0.5 mM), and 43.3 +/- 5.4 min (1.0 mM). Morin hydrate significantly prolonged survival of cells compared to equimolar concentrations of purpurogallin, Trolox, or ascorbate (P < 0.01). CONCLUSION: This study demonstrates that morin hydrate behaves as a broad-spectrum antioxidant: it scavenges not only xanthine oxidase/hypoxanthine-generated oxyradicals, but also nonenzymatic, nitrogen-derived radicals, better than those above mentioned antioxidants. This property of morin hydrate may help prevent free radical damage in corneal preservation solutions.

Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals.

The peroxidation of human erythrocytes induced by peroxyl radical initiator and its inhibition by several gallate esters (e.g., propyl, methyl, ethyl) and Trolox (a more polar analogue of vitamin E) have been studied. The antioxidant activity was determined on erythrocytes against hemolysis generated by a thermal activator, 2,2'-azobis-(2-amidinopropane)dihydrogenchloride. It was found that propyl gallate and its two analogues were more effective than Trolox in preventing cell lysis. However, the combination of gallate esters and Trolox produced a protective effect exceeding the arithmetic sum of their individual contributions. These perceived synergisms occur at more than one level of Trolox at a given level of a gallate ester.

Elevated plasma levels of tumor necrosis factor in chronic heart failure with cachexia.

To study the potential role of tumor necrosis factor (TNF) in chronic heart failure, we measured the plasma levels of TNF by enzyme linked immunoabsorbent assay in 109 patients with various heart diseases grouped as 'non-heart failure' (n = 36), 'heart failure' (n = 36) and 'cachectic' (n = 37). The daily food intake was also investigated. The results showed that there was no obvious difference of daily caloric intake among the three groups of patients. Plasma levels of TNF were significantly elevated in the patients with 'heart failure' (0.51 +/- 0.26 ng/ml, mean +/- S.E.M.), and even higher in the patients with 'cachexia' (6.19 +/- 2.76 ng/ml), as compared with the patients with 'non-heart failure' (0.09 +/- 0.03 ng/ml). Twenty-five patients with 'cachexia' and 11 patients with 'heart failure' had plasma levels of TNF > or = 100 pg/ml, whereas only 5 patients with 'non-heart failure' had plasma levels of TNF above that level. The patients with high levels of TNF were more cachectic than those with normal levels of TNF (body mass index 19.5 +/- 3.4 vs. 22.3 +/- 3.6, P < 0.05). In multivariate analysis, elevated levels of TNF were associated with the level of serum total protein, presence of heart failure and cachexia. These findings indicate that plasma levels of TNF are increased in patients with heart failure, and high levels of TNF may play an important role in the pathogenesis of cardiac cachexia.

Comparative protection against oxyradicals by three flavonoids on cultured endothelial cells.

Oxygen-derived free radicals are known to injure the endothelium of aorta in diverse disorders. In this study we compared the cytoprotective effects of three flavonoids against oxyradical damage to porcine aortic endothelial cells in vitro. Cultured porcine aortic endothelial cells were exposed to oxyradicals generated by xanthine oxidase--hypoxanthine (XO-HP). The cytoprotective activities of morin, quercetin, and catechin on these systems were compared using established morphologic criteria. The results in the XO-HP system showed that morin at 0.125, 0.25, and 0.5 mM delayed cell necrosis to 27.4 +/- 1.3, 46.8 +/- 1.8, and longer than 70 min, respectively, compared with 12.0 +/- 1.3 min in the control group. These degrees of protection were significantly stronger than those provided by quercetin and catechin at corresponding concentrations (p < 0.01). Morin and quercetin were moderate inhibitors of xanthine oxidase on the basis of the oxygen consumption rate, whereas catechin at the same concentrations had little inhibitory effect. The data from uric acid formation and cytochrome c reduction were consistent with the oxygen consumption measurement for the three flavonoids.

Molecular structure and antioxidant specificity of purpurogallin in three types of human cardiovascular cells.

Purpurogallin (PPG) in an active cytoprotector found in certain oak barks. We have shown that PPG prolongs the survival of cultured cardiocytes from rats and rabbits against different oxidants better than do antioxidants such as Trolox (a hydrophilic analogue of vitamin E) in a morphometric assay system. First, we verified by X-ray crystallography that PPG is a bicyclic molecule comprising a phenolic ring fused with a seven-membered ring in a highly planar conformation. In analogues of PPG wherein the two double bonds in the seven membered ring of the parent molecule are saturated or where the four OH groups of the parent compound are substituted by four OCH3 groups, the derivatives are less planar and less protective of the human cells than native PPG. Second, PPG in a concentration-dependent manner protected myocytes and endothelial cells of humans against oxyradicals generated with any one of the following oxyradical generators: (a) xanthine oxidase plus hypoxanthine, (b) menadione, or (c) paraquat. In each case, PPG was more cytoprotective than comparative antioxidants. Also, PPG protected erythrocytes against peroxyl radicals better than the two PPG derivatives mentioned. Third, the cytoprotective action of PPG detected in vitro was accompanied by declines of malondialdehyde. Finally, we observed that PPG chelated ferrous ions and, therefore, can suppress the formation of radicals in the Fenton reaction. Thus, PPG with its molecular architecture and presumably its affinity for ferrous ions protects multiple types of cardiovascular cells against oxyradicals.

Comparative cytoprotection of cultured corneal endothelial cells by water-soluble antioxidants against free-radical damage.

We reported previously that purpurogallin (PPG) markedly protects the cultured rabbit corneal endothelial cells (RCEC) against oxyradical damage generated with hypoxanthine (HX) and xanthine oxidase (XO)(1). In this study, we further compared the cytoprotective activities of PPG versus Trolox (TX, alpha-tocopherol, a water-soluble analogue of vitamin E) and ascorbate (Asc) in confluent cultured RCEC with phase contrast microscopy and confirmed by transmission electron microscopy. PPG prolonged survival of the oxyradical damaged cells longer than those without PPG present (18.6 +/- 1.4 min at 1.0 mM and 11.2 +/- 1.0 at 0.25 mM respectively vs. 7.3 +/- 0.8 min in control). At levels equimolar to PPG, TX, and Asc were less effective in delaying cell necrosis caused by HX and XO (p < 0.01). When exposed to superoxide radicals generated by menadione, RCEC necrosed at 29.8 +/- 1.5 min compared to PPG 47.2 +/- 1.0 min at 1.0 mM and 38.9 +/- 1.0 min at 0.25 mM. This was significantly different from TX and Asc at corresponding concentrations (p < 0.01). PPG scavenges not only HX-XO-generated oxyradicals, but also nonenzymatically produced superoxide radicals, more actively than two well known antioxidants--TX and Asc.

Molecular properties and myocardial salvage effects of morin hydrate.

Morin hydrate is a bioactive pigment found in yellow Brazil wood. Recently, we reported that morin hydrate prolongs the survival of three types of cells from the human circulatory system against oxyradicals generated in vitro. The protection excels that given by equimolar concentrations of ascorbate, mannitol, and Trolox. Here, we demonstrate that, in vivo, morin hydrate at 5 mumol/kg actually reduced by > 50% the tissue necrosis in post-ischemic and reperfused rabbit hearts. Mechanistically, morin hydrate not only scavenges oxyradicals, but also moderately inhibits xanthine oxidase, a free-radical generating enzyme from the ischemic endothelium. Among other possibilities, morin hydrate appears to chelate some metal ions (e.g. Fe2+) in oxyradical formation, although this needs to be examined further. Nuclear magnetic resonance (at 500 mHz) and electron-impact mass spectrometry also supported a molecular formula of C15H10O7 for morin hydrate. Only by X-ray crystallography was it clearly revealed that there are two water molecules attached by intermolecular hydrogen bonds to a morin molecule. Also, the three rings of morin hydrate approach coplanarity. This conformation favours a delocalization of electrons after oxyradical reduction, making morin an effective antioxidant. Thus, we have documented some of the molecular properties and myocardial salvage effects of morin hydrate.

Comparative antioxidant protection of cultured rabbit corneal epithelium.

Oxygen-free radicals may injure the epithelium of corneas in storage awaiting transplantation. This study compared the cytoprotective effects of several antioxidants against oxygen-free radical injury to rabbit corneal epithelial cells in vitro. Cultured rabbit corneal epithelial cells were exposed to oxygen-free radicals generated by xanthine oxidase and hypoxanthine. The cytoprotective activities of various antioxidants on this system were compared using established morphologic criteria. The results demonstrated that purpurogallin at 1.0 mM delayed cell necrosis to 9.98 +/- 1.16 min compared with 2.96 +/- 0.67 min without antioxidant protection. This degree of protection was significantly different from that provided by ascorbate (1.0 mM), trolox (1.0 mM), superoxide dismutase + catalase, catalase (92,000 IU/L), mannitol (1.0 mM), and superoxide dismutase (24,200 IU/L) (p < 0.01). We concluded that purpurogallin effectively protects corneal epithelium from oxygen-free radical injury and may help prevent such injury in corneal preservation solutions.

Purpurogallin as a cytoprotector of cultured rabbit corneal endothelium.

We examined the protective properties of purpurogallin, a naturally occurring phenol, in delaying necrosis of cultured corneal endothelial cells caused by oxygen free radicals. Endothelial cell cultures were prepared from New Zealand white rabbits using microcarrier cell culture techniques. Corneal endothelial cells were treated with hypoxanthine (2 mM) and xanthine oxidase (67 IU/L) to generate free radicals. The criteria for cell necrosis were cytoplasmic shrinkage, dissolution of plasma membranes and presence of "haloes" around the cells on phase contrast microscopy, confirmed by transmission electron microscopy. More than 95% of second-generation cells exhibited morphologic evidence of necrosis within 4.62 +/- 0.82 minutes after exposure to oxyradicals. The addition of purpurogallin (0.25 or 1.0 mM) significantly increased time to cell necrosis to 8.18 +/- 0.83 and 11.59 +/- 1.71 minutes respectively (p < 0.05). Further studies are under way to determine whether purpurogallin may be useful in preventing endothelial cell damage in corneas preserved for corneal transplantation.

Propyl gallate as a hepatoprotector in vitro and in vivo.

Recently, there has been renewed interest in propyl gallate, a preservative in foods and fuels. This compound, which exhibits antimicrobial activity, has been found to be toxicologically safe after almost 30 years of evaluation. In the present study, we examined whether propyl gallate is a hepatoprotective antioxidant, and investigated some of its bases of action vis-à-vis Trolox, a vitamin E analogue. In isolated rat hepatocytes, propyl gallate prolonged substantially cell survival against oxyradicals generated with xanthine oxidase-hypoxanthine. The protection was dose dependent and excelled that of Trolox, mannitol, or ascorbate, each at or near its optimum level in the same system. In rats undergoing an 80-min partial hepatic ischemia, infusion of propyl gallate at 20 mumol/kg body weight just before a 24-hr reperfusion salvaged the organ by 80.0 +/- 11.5%, an extent comparable to that with Trolox. Mechanistically, we found that propyl gallate (a) protected hepatocytes against the cascade of oxyradicals produced by xanthine oxidase-hypoxanthine; (b) protected hepatocytes against superoxide radicals generated specifically by menadione; (c) protected the functionally important hepatic vascular endothelial cells more effectively than Trolox against xanthine oxidase-hypoxanthine, and (d) approximately halved the amount of lipid conjugated dienes (a more specific marker of oxyradical damage than malondialdehyde) formed in tissues after oxidant damage. Therefore, there are fundamental reasons why propyl gallate is an effective antioxidant-based hepatoprotector, both in vitro and in vivo.

[Pars plana lensectomy and anterior vitrectomy for the treatment of congenital subluxated lenses].

The conventional surgical management of congenital subluxated lenses is frequently associated with a high incidence of complications leading to poor visual prognosis. Lensectomy through the pars plana with modern techniques of microsurgery and automated suction-cutting devices was performed on 11 patients (18 eyes) whose pre-operative visual acuities with the best optical correction ranged from 0.05-0.3, which were improved postoperatively to 0.5-1.0 during a follow-up of 3 to 60 months with an average of 36 months. The complications were accidental small cuts at the pupillary borders of 3 eyes, indicating that pars plana lensectomy is a safe procedure for the treatment of congenital subluxated lenses.

Morin: a wood pigment that protects three types of human cells in the cardiovascular system against oxyradical damage.

Morin is a yellowish pigment extractable from the wood of Chlorophora tinctoria. In the present study, we have determined that morin protects three types of human cells--ventricular myocytes, saphenous vein endothelial cells, and erythrocytes--against damage by oxyradicals generated in situ. In myocytes and endothelial cells, morin prolonged substantially and in a concentration-dependent manner the survival of cells exposed to either xanthine oxidase-generated oxyradicals or superoxide radicals produced with menadione. Morin protected erythrocytes from lytic attack by peroxyl radicals generated with 2,2'-azo-bis (2-amidinopropane) dihydrochloride. In all three types of human cells, the protective effect of morin clearly excelled that displayed by Trolox (a vitamin E analog), ascorbate, or mannitol, which are water-soluble antioxidants of similar molecular size. Chemically, we verified that morin behaves as an antioxidant by diminishing markedly the amount of malondialdehyde (lipid peroxidation product) found in human cardiocytes despite their exposure to oxyradicals. In agreement with related reports, we also observed that morin is non-toxic in rats even when used at concentrations 2-3 orders of magnitude higher than those in our in vitro studies. Thus, morin acts as a broad-spectrum and non-toxic antioxidant.