Carbon Monoxide Alleviates Salt-Induced Oxidative Damage in Wheat Seedling Leaves

Carbon monoxide （CO）, a by-product released during the degradation of heme by heme oxygenases （EC 1.14.99.3） In animals, is regarded as an important physiological messenger or bioactive molecule involved in many biological events that has been recently reported as playing a major role in mediating the cytoprotectlon against oxidant-induced lung Injury. In the present study, we first determined the protective effect of exogenous CO against salt-induced oxidative damage in wheat seedling leaves. Wheat seedlings treated with 0.01μmol/L hematin as the CO donor demonstrated significant reversal of chlorophyll decay, dry weight, and water loss induced by 300 mmol/L NaCl stress. Interestingly, the increase in lipid peroxidation observed in salt-treated leaves was reversed by 0.01μmol/L hematin treatment. Time-couree analyses showed that application of 0.01μmol/L hematln enhanced gualacol peroxidase, superoxide dismutase, ascorbate peroxidase and catalase activities in wheat seedling leaves subjected to salt stress. These effects are specific for CO because the CO scavenger hemoglobin （1.2 mg/L） blocked the actions of the CO donor hematln. However, higher concentration of the CO donor （1.0μmol/L） did not alleviate dry weight and water loss of salt-stressed wheat seedlings. These results suggest that exogenous application of low levels of a CO donor may be advantageous against salinity toxicity.

Endogenous Hydrogen Peroxide Plays a Positive Role in the Upregulation of Heme Oxygenase and Acclimation to Oxidative Stress in Wheat Seedling Leaves

Pretreatment of lower H2O2 doses （0.05, 0.5 and 5 mM） for 24 h was able to dose-dependently attenuate lipid peroxidation in wheat seedling leaves mediated by further oxidative damage elicited by higher dose of H2O2 （150 mM） for 6 h, with 0.5 mM H2O2 being the most effective concentrations. Further results illustrated that 0.5 mM H2O2 pretreatment triggered the biphasic production of H2O2 during a 24 h period. We also noticed that only peak Ⅰ （0.25 h） rather than peak Ⅱ （4 h） was approximately consistent with the enhancement of heme oxygenase （HO） activity, HO-1 gene expression. Meanwhile, enhanced superoxide dismutase （SOD） activity, Mn-SOD and Cu, Zn-SOD transcripts might be a potential source of peak Ⅰ of endogenous H2O2. Further results confirmed that 0.5 mM H2O2 treatment for 0.5 h was able to upregulate HO gene expression, which was detected by enzyme activity determination, semi-quantitative reverse transcription-polymerase chain reaction and western blotting. Meanwhile, the application of N,N＇-dimethylthiourea, a trap for endogenous H2O2, not only blocked the upregulation of HO, but also reversed the corresponding oxidation attenuation. Together, the above results suggest that endogenous H2O2 production （peak Ⅰ） plays a positive role in the induction of HO by enhancing its mRNA level and protein expression, thus leading to the acclimation to oxidative stress.

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide （CO）, an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signal molecule in mammalian cells, with its effects mediated mainly by nitric oxide （NO）. In the present report, we show that exogenous CO induces lateral root （LR） formation, an NO-dependent process. Administration of the CO donor hematin to rapeseed （Brassica napus L. Yangyou 6） seedlings for 3 days, dose-dependently promoted the total length and number of LRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturated concentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin （Hb） or the CO-specific synthetic inhibitor zinc protoporphyrin-IX（ZnPPIX） were added. Interestingly, depletion of endogenous NO using its specific scavenger 2-（4-carboxyphenyl）-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt （cPTIO） or the nitric oxide synthase （NOS） inhibitor N^G-nitro-L-arginine methyl ester （L-NAME）, led to the complete abolition of LR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, the induction of LR development by 200 μmol/L sodium nitroprusside （SNP）, an NO donor, was not affected by the presence or absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopy with the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NO release compared with control samples and that the NO signal was mainly distributed in the LR primordia （LRP）, especially after 36 h treatment. The LRP were found to have similar morphology in control, SNP- and hematin-treated seedlings. Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME, ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore, we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstream of CO signaling, which has also been shown to occur in animals.

Two new terpenoids from Reduning Injection

Objective:To study the antipyretic and anti-inflammatory constituents from the active fraction of Reduning(RDN)Injection.Methods:In this study,the active fraction of RDN Injection was screened by the LPS-induced mouse endotoxin shock model.The chemical constituents were isolated by chromatography on HP-20 macroporous adsorptive resins,silica gel,ODS columns and reverse phase MPLC and HPLC repeatedly,and their structures were elucidated based on spectroscopic analysis(HR-ESI-MS,NMR,ECD)and chemical methods.Meanwhile,we evaluated the anti-inflammatory activities of the isolates by measuring their inhibitory effects on TNF-αproduction in LPS stimulated RAW 264.7 macrophages.Results:The 95%ethanol eluate of RDN Injection by the macroporous adsorption resin column was proved to be the antipyretic and anti-inflammatory active fraction of this injection.A novel iridoid,named jasminoide A(1),and a new guaiane sesquiterpenoid,named(1 R,7 R,8 S,10 R)-7,8,11-trihydroxy-4-guaien-3-one(2),were isolated from Reduning injection,and compound 2 can inhibit TNF-αproduction with IC50 values of 72.24μmol/L.Conclusion:In this study,two new terpenoids were isolated from Reduning Injection,and compound 2 showed inhibitory activity against LPS-activated TNF-αproduction in RAW 264.7 cells in vitro.