Objective To observe the effects of substance P (SP), NK1 receptor antagonist and nitric oxide synthase (NOS) inhibitors on the secretion of nitric oxide (NO) and expression of inducible NOS (iNOS) by immortalized human keratinocyte line HaCaT. Methods The NO level in supernatant of cultured HaCaT cells was measured by nitrate reductase assay after treatment with different concentrations (10-9 to 10-6 mol/L) of SP, or the combination of SP (10-8 mol/L) and spantide (3×10-7 mol/L), aminoguanidine (10-7 mol/L), 7-nitroindazole (10-6 mol/L) or L-NAME (10-5 mol/L) for various durations. Reverse transcription-PCR was performed to measure the expression of iNOS mRNA in HaCaT cells incubated with SP of 10-8 mol/L for 1, 24 and 48 hours. Results The SP of 10-9 to 10-6 mol/L significantly induced the production of NO by HaCaT cells, and the highest level of NO was observed in HaCaT cells treated with SP of 10-8 mol/L. The synthesis of NO by HaCaT cells induced by SP was inhibited by Spantide of 3 × 10~7 mol/L at all time points (30 minutes, 1, 3, 6, 12, 24 hours, all P< 0.01), by L-NAME of 10-5 mol/L at 3 time points (30 minutes, 1, 24 hours) and by 7-nitroin-dazole of 10-6 mol/L at 2 time points (30 minutes, 1 hour, both P< 0.05), but not by aminoguanidine of 10-7 mol/L at any time point (all P > 0.05). After treatment with SP of 10-8 mol/L, the relative mRNA expression of iNOS was 0.199 ± 0.018 and 0.516 ± 0.030 at 24 and 48 hours, respectively, and there was a statistical difference between the two time points (P < 0.01). Conclusions SP can reinforce the secretion of NO by HaCaT cells via NK1 receptor activation, but iNOS is unlikely to be the primary origin of NO secreted by HaCaT cells induced by SP.

Phytoremediation plays an important role in the treatment of heavy metal pollution in soil. In order to elucidate the mechanism of salicylic acid (SA) on copper absorption, seedlings from Xuzhou (with strong Cu-tolerance) and Weifang Helianthus tuberosus cultivars (with weak Cu-tolerance) were selected for pot culture experiments. 1 mmol/L SA was sprayed upon 300 mg/kg soil copper stress, and the photosynthesis, leaf antioxidant system, several essential mineral nutrients and the changes of root upon copper stress were analyzed to explore the mechanism of copper resistance. The results showed that Pn, Tr, Gs and Ci upon copper stress decreased significantly compared to the control group. Meanwhile, chlorophyll a, chlorophyll b and carotenoid decreased with significant increase in initial fluorescence (F₀), maximum photochemical quantum yield of PSⅡ (Fv/Fm), electron transfer rate (ETR) and photochemical quenching coefficient (qP) content all decreased. The ascorbic acid (AsA) content was decreased, the glutathione (GSH) value was increased, the superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity in the leaves were decreased, and the peroxidase (POD) activity was significantly increased. SA increased the Cu content in the ground and root system, and weakened the nutrient uptake capacity of K, Ca, Mg, and Zn in the root stem and leaves. Spray of exogenous SA can maintain the opening of leaf stomata, improve the adverse effect of copper on photosynthetic pigment and PSⅡ reaction center. Mediating the SOD and APX activity started the AsA-GSH cycle process, effectively regulated the antioxidant enzyme system in chrysanthemum taro, significantly reduced the copper content of all parts of the plant, and improved the ion exchange capacity in the body. External SA increased the content of the negative electric group on the root by changing the proportion of components in the root, promoted the absorption of mineral nutrient elements and the accumulation of osmoregulatory substances, strengthened the fixation effect of the root on metal copper, and avoided its massive accumulation in the H. tuberosus body, so as to alleviate the inhibitory effect of copper on plant growth. The study revealed the physiological regulation of SA upon copper stress, and provided a theoretical basis for planting H. tuberosus to repair soil copper pollution.
Antioxidants ; Copper ; Helianthus/metabolism* ; Salicylic Acid/pharmacology* ; Chlorophyll A/pharmacology* ; Spectroscopy, Fourier Transform Infrared ; Chlorophyll/pharmacology* ; Ascorbic Acid ; Superoxide Dismutase/metabolism* ; Photosynthesis ; Glutathione ; Plant Leaves ; Stress, Physiological ; Seedlings