Inflammatory mechanism of cerebral ischemia-reperfusion injury with treatment of stepharine in rats.

BACKGROUND: Increasing evidence has shown that microglia-induced neuroinflammation is involved in the pathogenesis of ischemic stroke. Stepharine, one of the alkaloids extracted from Stephania japonica (Thunb.) Miers, exhibited strong inhibitory effect on microglial overactivation. However, it is not known whether it has the potential to prevent ischemic stroke. METHODS: The neuroprotective and anti-neuroinflammatory effects of stepharine were investigated in vivo and in vitro, using a rat model of middle cerebral artery occlusion (MCAO) and lipopolysaccharide (LPS)-stimulated BV-2 cells, respectively. RESULTS: In vivo, stepharine (500 µg/kg) suppressed neurological deficits scores, brain water content and cerebral infarct volume induced by MCAO. Moreover, stepharine (500 µg/kg) inhibited NeuN+ cells loss and Iba-1+ cells increase in the MCAO ischemic cortex. In vitro, stepharine (10, 30 µM) substantially inhibited nitric oxide release as well as the mRNA and protein expression of pro-inflammatory mediators [inducible nitric oxide synthase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1ß] in LPS-activated BV-2 cells. LPS-induced increase of TLR4 expression, IκBα phosphorylation, and NF-κB p65 nuclear translocation was inhibited by stepharine (10, 30 µM). Molecular docking analysis showed that stepharine directly interacted with TLR4. SPR assay further confirmed that stepharine could bind to the TLR4/MD2 complex. Meanwhile, stepharine exhibited neuroprotective effects on SH-SY5Y cells cultured with LPS-treated conditioned medium. CONCLUSION: Our study demonstrated for the first time that stepharine improved the outcomes in MCAO rats, reduced neuronal loss, and suppressed microglial overactivation via the inhibition of TLR4/NF-κB pathway. These results suggest that stepharine might be a potential therapeutic agent for the treatment of ischemic stroke.

Amide-Iminoate Isomerism in Antineuroinflammatory Isoquinoline Alkaloids from Stephania cepharantha.

Six new (1-6) and two known (7, 8) alkaloids that were chemically inseparable geometrical isomers (two isomers present in a 1:1 ratio for 1-4 and 6 and a 1:3 ratio for 5, 7, and 8) were identified from Stephania cepharantha. Their structures and absolute configurations were determined by spectroscopic data analyses and comparison of their experimental and calculated ECD spectra. Moreover, using NOE correlations and DFT-based calculations, the NMR data of each geometrical isomer of 1-6 were assigned. The biological evaluation of 1-8 showed that 5 and 6 have stronger inhibitory effects (IC50 values, 12.0 and 12.6 µM, respectively) than minocycline (IC50 value, 17.5 µM) against NO production in overactivated BV2 cells, suggesting they have great potential in the development of neuroinflammatory therapeutics for treating neurodegenerative diseases.

Mechanisms underlying enhanced Cd translocation and tolerance in roots of Populus euramericana in response to nitrogen fertilization.

A pot experiment was conducted to evaluate how nitrogen (N) availability influences cadmium (Cd) absorption, translocation and stress tolerance in roots of Populus euramericana. Seedling growth was sensitive to N deficiency, but it was unaltered by Cd exposure. Cadmium absorption by roots was promoted by N deficiency, resulting in a higher root Cd concentration compared to the N-sufficient condition. Fine-root length was tightly correlated (R2 = 0.73) with Cd concentration in roots, indicating that vigorous fine-root proliferation under N deficiency contributed to active absorption and accumulation of Cd in roots. Despite accumulation in roots, Cd translocation from roots to shoots was less active under N deficiency compared to N sufficiency. This was related to elevated glutathione reductase (GR) activity and glutathione (GSH) levels in roots after N application, which may not only promote antioxidant defence, but also facilitate the formation of GSH-Cd complexes that are uploaded into root cylinders. Nitrogen application also promoted antioxidant defense in roots via increased production of phytohormones and the level of enzymatic and non-enzymatic antioxidants. Transcript levels for genes responsible for antioxidant defense, Cd detoxification and Cd uploading were increased in roots by N application. The N-stimulated Cd tolerance, detoxification and uploading in roots are factors likely to promote Cd translocation from roots to shoots, which may enhance the biological capacity of this poplar species for phytoremediation of Cd pollution.

Natural neuroprotective alkaloids from Stephania japonica (Thunb.) Miers.

Modulating inflammatory responses after stroke can prevent brain injury and, therefore, improve neurological outcome. Stephania japonica (Thunb.) Miers is a Chinese folk medicine with the function of dispelling the "wind and blockage" in the human body according to the Chinese medicine theory, in which the symptoms of stroke are caused by the "wind and blockage" in the body. In this paper, we for the first time linked S. japonica to stroke by clarifying fifteen alkaloidal constituents including five undescribed (1-5) ones and screening out six hasubanan type alkaloids (1-4, 7, 15) that elicited stronger anti-neuroinflammatory activities than the positive drug. Moreover, the total alkaloid fraction (ASJ) with previously undescribed 3 as the main component was subject to the in vivo evaluation of the protective effect in the MCAO-induced brain injury. The results showed that ASJ exhibited potent protective effect against brain injury in the MCAO rat model. The results reported in this paper suggested that the hasubanan alkaloids from S. japonica would be an important molecular source for discovering novel therapeutic agents for neuroinflammation-related diseases, such as stroke diseases.

The influence of nitrogen availability on anatomical and physiological responses of Populus alba × P. glandulosa to drought stress.

BACKGROUND: Drought and nitrogen (N) deficiency are two major limiting factors for forest productivity in many ecosystems. Elucidating the mechanisms underlying the influence of soil N availability on drought responses of tree species is crucial to improve tree growth under drought. RESULTS: The root proliferation under drought was enhanced by adequate N application. Vessel frequency in xylem increased upon drought, with more significant increase under adequate N conditions compared with that under low N conditions, possibly leading to increased hydraulic safety. Nitrogen application under drought increased indole acetic acid (IAA), which contributed to the adaptive changes of xylem. Nitrogen application increased leaf abscisic acid (ABA) concentration, therefore regulated stomata adjustment, and promoted intrinsic water use efficiency (WUEi). Moreover, N application promoted antioxidant defense in leaves by showing increased level of free proline and carotenoid, which improved drought tolerance and growth performance of poplars. CONCLUSIONS: Anatomical and physiological responses of Populus to drought were suppressed by N deficiency. Adequate N application promoted adaptive changes of root and xylem under drought and increased hydraulic safety. Nitrogen addition under drought also increased leaf ABA level which may regulate stomata adjustment and promote WUEi. Moreover, nitrogen application improved antioxidant defense in leaves with increased levels of antioxidants. These positive regulations improved drought tolerance and growth performance of poplars.

Rapid characterization of TCM Qianjinteng by UPLC-QTOF-MS and its application in the evaluation of three species of Stephania.

A method for rapid identification of bioactive components in herbals from Stephania species was developed by utilizing UPLC-QTOF-MS/MS in this study. Sixty-three alkaloids including the types of proaporphine, aporphine, protoberberine, morphine, hasubanan, benzylisoquinoline and bisbenzylisoquinoline were characterized. Among them, 29 components were selected for semi-quantitative analyses to characterize the difference of chemical compositions among three Stephania species. The results revealed that bisbenzylisoquinoline-type of alkaloids are the representative components of SB (Subgen. Botryodiscia). The content of aporphine-type of alkaloids was much higher in ST (Subgen. Tuberiphania) than that in SS (Subgen. Stephania), while the content of protoberberine-type of alkaloids was higher than that in ST. The present study offered an efficient approach for quality control of Qianjinteng based on the rapid identification and semi-quantification of alkaloids in Stephania species by using UPLC-QTOF-MS.

Physiological and transcriptional responses of Catalpa bungei to drought stress under sufficient- and deficient-nitrogen conditions.

Many semi-arid ecosystems are simultaneously limited by soil water and nitrogen (N). We conducted a greenhouse experiment to address how N availability impacts drought-resistant traits of Catalpa bungei C. A. Mey at the physiological and molecular level. A factorial design was used, consisting of sufficient-N and deficient-N combined with moderate drought and well-watered conditions. Seedling biomass and major root parameters were significantly suppressed by drought under the deficient-N condition, whereas N application mitigated the inhibiting effects of drought on root growth, particularly that of fine roots with a diameter <0.2 mm. Intrinsic water-use efficiency was promoted by N addition under both water conditions, whereas stable carbon isotope compositions (δ13C) was promoted by N addition only under the well-watered condition. Nitrogen application positively impacted drought adaptive responses including osmotic adjustment and homeostasis of reactive oxygen species, the content of free proline, soluble sugar and superoxide dismutase activity: all were increased upon drought under sufficient-N conditions but not under deficient-N conditions. The extent of abscisic acid (ABA) inducement upon drought was elevated by N application. Furthermore, an N-dependent crosstalk between ABA, jasmonic acid and indole acetic acid at the biosynthesis level contributed to better drought acclimation. Moreover, the transcriptional level of most genes responsible for the ABA signal transduction pathway, and genes encoding the antioxidant enzymes and plasma membrane intrinsic proteins, are elevated upon drought only under sufficient-N addition. These observations confirmed at the molecular level that major adaptive responses to drought are dependent on sufficient N nutrition. Although N uptake was decreased under drought, N-use efficiency and transcription of most genes encoding N metabolism enzymes were elevated, demonstrating that active N metabolism positively contributed drought resistance and growth of C. bungei under sufficient-N conditions.