The dopamine D1-D2DR complex in the rat spinal cord promotes neuropathic pain by increasing neuronal excitability after chronic constriction injury.

Dopamine D1 receptor (D1DR) and D2 receptor (D2DR) are closely associated with pain modulation, but their exact effects on neuropathic pain and the underlying mechanisms remain to be identified. Our research revealed that intrathecal administration of D1DR and D2DR antagonists inhibited D1-D2DR complex formation and ameliorated mechanical and thermal hypersensitivity in chronic constriction injury (CCI) rats. The D1-D2DR complex was formed in the rat spinal cord, and the antinociceptive effects of D1DR and D2DR antagonists could be reversed by D1DR, D2DR, and D1-D2DR agonists. Gαq, PLC, and IP3 inhibitors also alleviated CCI-induced neuropathic pain. D1DR, D2DR, and D1-D2DR complex agonists all increased the intracellular calcium concentration in primary cultured spinal neurons, and this increase could be reversed by D1DR, D2DR antagonists and Gαq, IP3, PLC inhibitors. D1DR and D2DR antagonists significantly reduced the expression of p-PKC γ, p-CaMKII, p-CREB, and p-MAPKs. Levo-corydalmine (l-CDL), a monomeric compound in Corydalis yanhusuo W.T. Wang, was found to obviously suppress the formation of the spinal D1-D2DR complex to alleviate neuropathic pain in CCI rats and to decrease the intracellular calcium concentration in spinal neurons. l-CDL-induced inhibition of p-PKC γ, p-MAPKs, p-CREB, and p-CaMKII was also reversed by D1DR, D2DR, and D1-D2DR complex agonists. In conclusion, these results indicate that D1DR and D2DR form a complex and in turn couple with the Gαq protein to increase neuronal excitability via PKC γ, CaMKII, MAPK, and CREB signaling in the spinal cords of CCI rats; thus, they may serve as potential drug targets for neuropathic pain therapy.

Blockade of spinal dopamine D1/D2 receptor suppresses activation of NMDA receptor through Gαq and Src kinase to attenuate chronic bone cancer pain.

INTRODUCTION: Spinal N-methyl-D-aspartate receptor (NMDAR) is vital in chronic pain, while NMDAR antagonists have severe side effects. NMDAR has been reported to be controlled by G protein coupled receptors (GPCRs), which might present new therapeutic targets to attenuate chronic pain. Dopamine receptors which belong to GPCRs have been reported could modulate the NMDA-mediated currents, while their exact effects on NMDAR in chronic bone cancer pain have not been elucidated. OBJECTIVES: This study was aim to explore the effects and mechanisms of dopamine D1 receptor (D1DR) and D2 receptor (D2DR) on NMDAR in chronic bone cancer pain. METHODS: A model for bone cancer pain was established using intra-tibia bone cavity tumor cell implantation (TCI) of Walker 256 in rats. The nociception was assessed by Von Frey assay. A range of techniques including the fluorescent imaging plate reader, western blotting, and immunofluorescence were used to detect cell signaling pathways. Primary cultures of spinal neurons were used for in vitro evaluation. RESULTS: Both D1DR and D2DR antagonists decreased NMDA-induced upregulation of Ca2+ oscillations in primary culture spinal neurons. Additionally, D1DR/D2DR antagonists inhibited spinal Calcitonin Gene-Related Peptide (CGRP) and c-Fos expression and alleviated bone cancer pain induced by TCI which could both be reversed by NMDA. And D1DR/D2DR antagonists decreased p-NR1, p-NR2B, and Gαq protein, p-Src expression. Both Gαq protein and Src inhibitors attenuated TCI-induced bone cancer pain, which also be reversed by NMDA. The Gαq protein inhibitor decreased p-Src expression. In addition, D1DR/D2DR antagonists, Src, and Gαq inhibitors inhibited spinal mitogen-activated protein kinase (MAPK) expression in TCI rats, which could be reversed by NMDA. CONCLUSIONS: Spinal D1DR/D2DR inhibition eliminated NMDAR-mediated spinal neuron activation through Src kinase in a Gαq-protein-dependent manner to attenuate TCI-induced bone cancer pain, which might present a new therapeutic strategy for bone cancer pain.

Global convergence in the balance between leaf water supply and demand across vascular land plants.

Coordination between the density of veins (water supply) and stomata (demand for water) has been found in the leaves of modern angiosperms and also in ferns. This suggests that this coordinated development is not a unique adaptation of derived angiosperms that enables their high productivity. To test this, we compiled leaf vein and stomatal density data from 520 land vascular plant species including derived angiosperms, basal angiosperms, gymnosperms and ferns. We found global coordination across vascular land plants, although the relationships were not significant in gymnosperms and vessel-less angiosperms. By comparing the evolution of xylem conduit elements with variation in the density of veins and stomata and theoretical stomatal conductance among plant lineages, we found that the physiological advantage of modern angiosperms is associated with the emergence of xylem with low intraconduit resistance and leaves with high vein and stomata densities. Thus our results indicate two major events associated with surges in xylem hydraulic capacity in angiosperms: (1) the origin of vessels and (2) the emergence of vessels with simple perforation plates, which diminished physical limitations on stomatal conductance. These evolutionary innovations may have enabled derived angiosperms to be more productive and adaptive to the changing climate.

Levo-corydalmine attenuates microglia activation and neuropathic pain by suppressing ASK1-p38 MAPK/NF-κB signaling pathways in rat spinal cord.

BACKGROUND AND OBJECTIVES: Neuropathic pain is partially refractory to currently available treatments. Although some studies have reported that apoptosis signal-regulating kinase 1 (ASK1) may inhibit chronic pain, the mechanisms underlying this process have not been fully elucidated. METHODS: Chronic constriction injury (CCI) of the rat sciatic nerve was used to establish a neuropathic pain model. Nociception was assessed using von Frey hair and Hargreaves' methods. Western blot and immunofluorescence were used to detect the cell signaling pathway. BV2 cell line was cultured for in vitro evaluation. RESULTS: Our results indicated that spinal ASK1 was co-expressed with the microglia marker ionized calcium binding adaptor 1. Additionally, intrathecal administration of ASK1 inhibitor suppressed the activation of spinal microglia and attenuated CCI-induced neuropathic pain. The ASK1 inhibitor also decreased the levels of phosphorylated ASK1 (p-ASK1), p65, p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) messenger RNA in lipopolysaccharide-stimulated BV2 microglia cells. Intragastric administration of levo-corydalmine (l-CDL) significantly attenuated CCI-induced neuropathic pain and inhibited the expression of p-ASK1 in the spinal cord. l-CDL conspicuously suppressed the activation of spinal microglia in vitro and in vivo. Translocation of nuclearfactor-kappa B (NF-κB) and upregulation of p-p65, TNF-α, IL-1ß were inhibited by l-CDL. Further, the analgesic effects of l-CDL were associated with reduced levels of phosphorylated protein kinase C (PKC γ), c-JunNH2-terminal kinase, and extracellular signal-regulated kinase. CONCLUSIONS: This study showed that the expression of ASK1 in spinal microglia and ASK1 inhibitor suppressed microglia activation via suppression of p38 MAPK/NF-κB, which ultimately attenuated CCI-induced neuropathic pain. l-CDL also inhibited the ASK1-P38 MAPK/NF-κB axis to attenuate CCI-induced neuropathic pain.

Diuretic Activity of Compatible Triterpene Components of Alismatis rhizoma.

Alismatis rhizoma (AR), the dried rhizoma of Alisma orientale Juzepzuk (Alismataceae), is a traditional Chinese medicine. AR is an important part of many prescriptions and is commonly used as a diuretic agent in Asia. This study aimed to evaluate the diuretic effects of total triterpene extract (TTE) and triterpene component compatibility (TCC, the mixture of alisol B 23-acetate, alisol B, alisol A 24-acetate, alisol A, and alisol C 23-acetate) of AR in saline-loaded rats. The optimal diuretic TCC of AR was optimized using a uniform design. Different doses (5, 20, and 40 mg/kg) of TTE and TCC groups (N1-N8) were orally administered to rats. Urinary excretion rate, pH, and electrolyte excretion were measured in the urine of saline-loaded rats. Results showed that TTE doses increased urine volume and electrolyte excretion compared with the control group. All uniformly designed groups of TCC also increased urine excretion. In addition, optimal diuretic TCC was calculated (alisol B 23-acetate: alisol B: alisol A 24-acetate: alisol A: alisol C 23-acetate 7.2:0.6:2.8:3.0:6.4) and further validated by saline-loaded rats. This study demonstrated that TTE presented a notable diuretic effect by increasing Na⁺, K⁺, and Cl − displacements. The most suitable TTC compatible proportion of alisol B 23-acetate: alisol B: alisol A 24-acetate: alisol A: alisol C 23-acetate for diuretic activity was validated, and triterpenes were the material basis for the diuretic activity of AR.

Stable stomatal number per minor vein length indicates the coordination between leaf water supply and demand in three leguminous species.

The coordination between minor vein density (MVD) and stomatal density (SD) has been found in many plants. However, we still know little about the influence of leaf node on this correlation relationship. Here, we devised the new functional trait 'stomatal number per minor vein length' (SV). By measuring leaflet area (LA), MVD, SD, and SV, we demonstrated the significance of this functional trait in Arachis hypogaea (peanut) grown under different light regimes and in sun leaves of Dalbergia odorifera and Desmodium renifolium. We found that SV did not change significantly with leaflet node or with LA within each light treatment, while shading caused a significant decrease in SV. The positive correlation between SD and MVD was found in peanut under each light regime. Sun leaves of D. odorifera and D. renifolium also had stable SV along the leaflet node, with a positive correlation between MVD and SD. We conclude that under a certain light regime, a stable SV similar to the positive correlation between MVD and SD can also indicate the coordination between leaf water supply and demand. Our findings highlight the significance of SV and provide new insight into the coordination between stomatal number and minor vein length.

The effects of anticancer drugs TSA and GSK on spermatogenesis in male mice.

OBJECTIVE: The effect of anticancer drugs Trichostation A (TSA) and GSK2126458 (GSK) on genetic recombination of sperm meiosis in mice was investigated, and their clinical feasibility of fertility preservation in cancer patients was also assessed. METHODS: Eighteen Kunming mice were randomly given TSA or GSK at the concentrations of 0, 0.1 and 0.2 umol/L for three months. Immunofluorescence was used to evaluate the genetic recombination of homologous chromosomes and fidelity of chromosome synapsis. Sperm density, motility and viability were also examined to investigate the spermatogenic function. RESULTS: The average number of MLH1 foci in each spermatocyte was greatly higher in TSA (0.1) group than that in control (P<0.05), but no difference with the TSA (0.2) group (P>0.05). The frequency of SC with no MLH1 foci was lower while the frequency of SC with one MLH1 foci was higher in spermatocyte of mice with different doses of TSA compared with controls (P<0.05). The weight of left testis in TSA (0.1) group was significant decreased compared with that in control (P<0.05). However, no significant differences were observed in average number of MLH1, frequency of SC with 0-3 MLH1 foci, spermatocyte percentage of XY chromosomes containing MLH1 foci and percentages of cells containing gaps and splits among groups with or without the treatment of GSK. Furthermore, there were no statistical differences in body weight, testicular weight, sperm density, sperm motility and sperm viability among the three groups. CONCLUSION: TSA increased genetic recombination frequency of spermatocyte meiosis. GSK had no significant effect on genetic recombination frequency of spermatocyte meiosis and spermatogenic function.

Weak co-ordination between vein and stomatal densities in 105 angiosperm tree species along altitudinal gradients in Southwest China.

Leaf-level water balance, as revealed by a correlation between stomatal density (SD) and vein density (VD), has been reported in some plants. However, the generality of this correlation and how it may be affected by altitude changes are unclear. Here, we investigated whether this balance is maintained across tree species of diverse families along a large altitudinal gradient. We measured leaf area (LA), SD, stomata length (SL), and VD in 105 angiosperm species across two altitudinal ranges, 800-1400m above sea level (a.s.l.) in tropical montane forests (TMF) and 2000-2600m a.s.l. in subtropical montane forests (SMF) in Yunnan, South-west China. The average SD was independent of altitude in both regions. Similarly, the average VD within either SMF or TMF was also not significantly different. However, overall, TMF had significantly larger VD and LA but smaller SL than SMF. Vein density was positively correlated with SD across SMF species, with a weaker correlation for TMF species and all species combined. Stomatal length was negatively correlated with SD and VD across all species. Our results extend the leaf water balance theory to diverse angiosperm tree species, and indicate decoupled adaptation of SD and VD in these species along a large altitudinal gradient.