Co-Administration of nalbuphine to improve morphine tolerance in mice with bone cancer pain.

BACKGROUND: Kappa-opioid receptor (KOR) agonists are known for having opposite and/or different effects compared with Mu-opioid receptor (MOR) agonists. This study is aimed at clarifying the analgesic effect and tolerance of nalbuphine combined with morphine, and quantifying the mRNA and protein expression of spinal MOR and KOR in a mouse bone cancer pain (BCP) model treated with nalbuphine and morphine. METHOD: BCP model was prepared in C3H/HeNCrlVr Mice by implanting the sarcoma cells into the intramedullary space of the femur. The paw withdrawal thermal latency (PWL) measured by thermal radiometer was used to assess thermal hyperalgesia. PWL testing was performed after implantation and drug administration according to the protocol. Hematoxylin-eosin staining in the spinal cord and x-ray in the femoral intramedullary canal was detected. Real-time PCR and western blot analysis played a role in detecting spinal MOR and KOR expression changes. RESULTS: In tumor-implanted mice, the spinal MOR and KOR protein and mRNA expression was down-regulated when compared to that in sham-implanted mice (p < 0.05). Morphine therapy can lead to a decrease in spinal µ receptor expression. Similarly, the nalbuphine therapy can lead to a decrease in the expression of κ receptor protein and mRNA at the spinal cord level (p < 0.05). Morphine, nalbuphine, or nalbuphine co-administration with morphine all can extend the paw withdrawal thermal latency (PWL) to radiant thermal stimulation in tumor-implanted mice (p < 0.05). Compared with the morphine treatment group, nalbuphine co-administration with morphine delayed the reduction of PWL value again (p < 0.05). DISCUSSION: BCP itself may induce down-regulation of the spinal MOR and KOR expression. A low dose of nalbuphine co-administration with morphine led to the delayed emergence of morphine tolerance. The part of the mechanism may be due to the regulation of spinal opioid receptors expression.

[Intrathecal injection of AG-490 reduces bone-cancer-induced spinal cord astrocyte reaction and thermal hyperalgesia in a mouse model].

OBJECTIVE: To investigate the role of spinal interleukin-6-Janus kinase 2 (IL-6-JAK2) signaling transduction pathway in regulating astrocytes activation during the maintenance of bone cancer pain (BCP).
 Methods: NCTC 2472 fibrosarcoma cells were injected into the femur marrow cavity in C3H/HeNCrlVr male mice to establish BCP model and they were replaced by the equal volume of α-MEM in the sham model. The paw withdrawal latency (PWL) was measured after inoculation of tumor cells. The lumbar enlargement of spinal cord (L3-L5) was isolated, and Real-time RT-PCR and Western blot were used to detect the expression of spinal glial fibrillary acidic protein (GFAP) and JAK2 mRNA and protein, respectively. The expression level of spinal GFAP mRNA indirectly reflect astrocytes activation level. Pain behaviors and spinal cord GFAP mRNA and protein expression were observed at the given time points after intrathecal administration of JAK2 antagonist AG-490.
 Results: The PWL at 10, 14, 21 d after operation in BCP model group were significantly shorter than that in the sham group (P<0.05); the spinal GFAP and JAK2 mRNA and protein levels were higher in the BCP model group in comparison to mice in the sham group (P<0.05); intrathecal injection of JAK2 agonist AG-490 (30 or 90 nmol) significantly alleviated PWL, and downregulated the expression of spinal GFAP mRNA and protein (P<0.05).
 Conclusion: The IL-6-JAK2 signaling pathway plays an important role in maintaining the BCP by regulating the expression of GFAP in the spinal cord. Intrathecal injection of AG-490 can reduce the BCP, and inhibit the activation of IL-6-JAK2 signaling pathway, which may be one of the mechanisms for spinal astrocyte activation.

Molecular mechanism underlying miR-204-5p regulation of adipose-derived stem cells differentiation into cells from three germ layers.

The limited differentiation ability of adipose-derived stem cells (ADSCs) limits their application in stem cell therapy and regenerative medicine. Here, we explore the molecular mechanism by which miR-204-5p regulates ADSCs differentiation into cells derived from the three germ layers (i.e., adipocytes, neurocytes, and hepatocytes). Although miR-204-5p overexpression inhibited ADSCs differentiation into adipocytes, neurocyte and hepatocyte differentiation were promoted. Mechanistically, miR-204-5p inhibited the expression of PPARG by regulating the AMPK signaling pathway, thereby inhibiting ADSCs differentiation into adipocytes. Further, miR-204-5p regulated JAG1/NOTCH3 axis for the inhibition of differentiation into adipocytes and promotion of differentiation into neurocytes. miR-204-5p might also promote ADSCs differentiation into hepatocytes by upregulating E2F8. The findings of this study provide novel insights into the regulatory mechanisms underlying early embryonic development and will help to facilitate the application of ADSCs in stem cell therapy and regenerative medicine.

Intrathecal injection of metabotropic glutamate receptor subtype 3 and 5 agonist/antagonist attenuates bone cancer pain by inhibition of spinal astrocyte activation in a mouse model.

BACKGROUND: Astrocytes and metabotropic glutamate receptors play important roles in nociceptive processing. However, their roles in bone cancer pain were not well understood. This study sought to investigate whether selective mGluR3 and mGluR5 agonist or antagonist develop antinociceptive effects on bone cancer pain by inhibition of spinal astrocyte activation. METHODS: C3H/HeNCrlVr mice were inoculated into the intramedullary space of the femur with sarcoma NCTC 2472 cells to induce bone cancer pain. Quantitative real-time reverse transcription-polymerase chain reaction and Western blot experiments examined messenger RNA and protein expression of spinal glial fibrillary acidic protein, mGluR3, and mGluR5. The authors further investigated effects of intrathecal treatment with the mGluR3 agonist (APDC), the mGluR3 antagonist (LY341495), the mGluR5 agonist (CHPG), or the mGluR5 antagonist (MTEP) on nociceptive behaviors and spinal astrocyte activation associated with bone cancer pain. RESULTS: Inoculation of sarcoma cells, but not α-MEM solution, induced progressive bone cancer pain and resulted in up-regulation of glial fibrillary acidic protein, mGluR3, and mGluR5 expression on days 10, 14, and 21 postinoculation. Intrathecal administration of APDC and MTEP attenuated bone cancer-evoked spontaneous pain, mechanical allodynia, thermal hyperalgesia, and reduced spinal glial fibrillary acidic protein expression. However, treatment with LY341495 and CHPG induced thermal hyperalgesia and spinal glial fibrillary acidic protein expression in control mice. CONCLUSIONS: Spinal mGluR3 activation or mGluR5 inhibition reduced bone cancer pain. Inhibition of spinal astrocyte activation may contribute to the analgesic effects. These findings may lead to novel strategies for the treatment of bone cancer pain.

[Role of cannabinoid 2 receptor in the development of bone cancer pain].

OBJECTIVE: To explore the effects of cannabinoid 2 receptor (CB2) in the development of bone cancer pain in mice. METHODS: A total of 84 mice (C3H/HeJ) were randomly divided into 4 groups:tumor group (Group T, n = 30), medication administration group (Group J, n = 12), vehicle group (Group D, n = 12) and sham group (Group S, n = 30). And 2 × 10(5) osteolytic NCTC2472 cells in α-MEM were injected into medullary cavity of right distal femur to induce bone cancer pain in a murine model while sham mice received an injection of only α-MEM. All mice were tested for pain-related behaviors at pre-inoculation and at Days 5, 7, 10 and 14 post-inoculation. The tests included paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). Group J and Group D were injected intrathecally with 2 µg JWH015 dissolved in 4% DMSO and only 4% DMSO respectively in a 5 µl. volume. Pain behavior tests were performed before and at 1, 6, 24, 48 and 72 h after an intrathecal injection. Lumbar intumescentia of mice in each group were harvested to examine the expression level of CB2 by Western blot after pain behavior tests at Days 5, 7, 10 and 14 post-inoculation and 12 h after an intrathecal injection. RESULTS: (1) Pain behavior tests:Mechanical allodynia appeared at Day 7 post-inoculation. The value of PWMT was (1.27 ± 0.28) g (P < 0.05) and it declined gradually to (0.53 ± 0.20) g at Day 14. The threshold of mechanical hyperalgesia increased to (1.00 ± 0.20) g at 6 h after an intrathecal injection of JWH015, peaked at (1.40 ± 0.39) g at 12 h, became alleviated after 48 h and recovered to the pre-dosing levels at 72 h. Thermal hyperalgesia appeared at Day 10 post-inoculation. The value of PWTL was (16.9 ± 0.4) s (P < 0.05) at Day 10 and declined to (11.5 ± 0.7) s at Day 14 post-inoculation. The threshold of thermal hyperalgesia increased to (15.7 ± 1.9) g at 6 h after an intrathecal injection of JWH015, peaked at (18.6 ± 2.3) g at 12 h, became alleviated after 48 h and recovered to the pre-dosing levels at 72 h. (2) Western blot: From Day 5 post-inoculation, the ratio of CB2/ß-actin increased gradually. Compared with the ratio of 0.190 ± 0.010 at Day 5 post-inoculation, the ratio of CB2/ß-actin increased to 0.660 ± 0.010 at Day 14 post-inoculation (P < 0.05); compared with the ratio of 0.903 ± 0.006 in group D at 12 h after an intrathecal injection of JWH015, the ratio of CB2/ß-actin 0.510 ± 0.010 significantly decreased (P < 0.05). CONCLUSION: The cannabinoid 2 receptor plays an important role in the formation of bone cancer pain.

Integration of single-cell transcriptome and chromatin accessibility of early gonads development among goats, pigs, macaques, and humans.

The early gonads of mammals contain primordial germ cells (PGCs) and somatic cell precursors that are essential for sex determination and gametogenesis. Although it is extensively documented in mice, the development of early gonads in non-rodents remains to be delineated. Because molecular differences between mouse and human gonadal cells have been reported, it warrants the study of the key markers and regulatory features that are conserved or divergent between non-rodent species and human. Here, we integrate single-cell transcriptome and chromatin accessibility analysis to identify regulatory signatures of PGCs and somatic cells in the early gonads of goats, pigs, macaques, and humans. We identify the evolutionarily conserved and species-specific events, including genes expression, signaling pathways, and cell-cell interactions. We also uncover potential cis-regulatory elements and key transcription factors in PGCs and somatic cells. Our datasets provide important resources for better understanding the evolutionary programs of PGCs and gonadal somatic cell development in mammals.

Glutamate receptors and signal transduction in learning and memory.

The plasticity of the central nervous system helps form the basis for the neurobiology of learning and memory. Long-term potentiation (LTP) is the main form of synaptic plasticity, reflecting the activity level of the synaptic information storage process, and provides a good model to study the underlying mechanisms of learning and memory. The glutamate receptor-mediated signal pathway plays a key role in the induction and maintenance of LTP, and hence the regulation of learning and memory. The progress in the understanding of the glutamate receptors and related signal transduction systems in learning and memory research are reviewed in this article.

Effect of ketamine administration on memory consolidation, p-CREB and c-fos expression in the hippocampal slices of minor rats.

To investigate the effect of ketamine administration on memory consolidation, p-CREB and c-fos expression in hippocampus of infant rats and the possible mechanism. Ninety-six SD rats of 21 days old were divided into seven groups, Y-maze was used to test the ability of learning and memory for minor rats. p-CREB and c-fos protein expression was tested by immunhistochemistry. The results showed that the memory consolidation rate in normal saline group mice was higher than that in k1a and k7a group (P<0.05). However, normal saline group between k1b and k7b group was not significant difference. The p-CREB and c-fos protein expressing cells of normal saline group were higher than those in passive control group, pseudotraining group, k1a and k7a group (P<0.05). However, there was not significant difference between normal saline group and k1b or k7b group. The c-fos protein expressing cells of pseudotraining group was higher than those in passive control group, however, the p-CREB protein expressing cells was not significant difference. Therefore, administration of ketamine may temporally affect the ability of memory consolidation rate of minor rats through suppressing the expression of p-CREB and c-fos protein in hippocampus.

Anesthetic ketamine counteracts repetitive mechanical stress-induced learning and memory impairment in developing mice.

The aim of this study is to investigate whether ketamine, a noncompetitive N-methyl-D: -aspartate receptor (NMDAR) antagonist, had an influence on learning and memory in developing mice. Fifty Kunming mice aged 21 days were randomly divided into 5 subgroups (n = 10 for each) to receive intraperitoneal injection of equal volume of saline (S group) or ketamine (25, 50 or 100 mg/kg of body weight/day) for 7 consecutive days, or to be left untreated (C group). A step-down passive avoidance test was performed to evaluate learning and memory in these mice on days 8 and 9. Additionally, the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus was determined. Rats receiving saline or sub-anesthetic dose of ketamine (25 mg/kg) showed significantly decreased abilities of learning and memory and reduced expression of BDNF, compared to the normal controls (P < 0.05). In contrast, comparable abilities of learning and memory and expression of BDNF were found for anesthetic doses of ketamine (50 or 100 mg/kg)-treated rats and controls (P > 0.05). Repetitive mechanical stress impairs learning and memory performance in developing mice, which may be associated with decreased BDNF expression. The stress-induced learning and memory impairment can be prevented by anesthetic doses of ketamine.

[Effect of shentong zhuyu decoction on pain behavior and spinal cord astrocytes model of osteocarcinoma pain].

OBJECTIVE: To study the analgesic effect of shentong zhuyu decoction (SZD) and its effect on the expression of the spinal cord glial fibrillary acidic protein (GFAP). METHODS: One hundred C3H/HeNCrlVr male mice were randomly divided into the normal group (n=8), the sham operation group (n=30), the model group (n=30), the Chinese medicine (CM) group 1 (n=8), the CM group 2 (n=8), the CM group 3 (n=8), and the vehicle group (n=8). 0.1 g crude drug of SZD/0.4 mL, 0.3 g crude drug of SZD/0.4 mL, 0.9 g crude drug of SZD/0.4 mL, and 0.4 mL normal saline were respectively given by gastrogavage to mice in CM 1, 2, 3 groups and the vehicle group, once daily for seven days starting from Day 14. The paw withdrawal thermal latency (PWTL), as the behavior indicator, was assessed in mice using radiant thermal stimulator. The lumbar enlargement of the spinal cord was taken after the behavioral test on Day 21. GFAP mRNA and protein expressions were detected using real-time quantitative RT-PCR and Western blot. RESULTS: Compared with the normal group (Day 0) (PWTL: 15.91 +/- 1.65 s) and the sham operation group (PWTL: Day 4: 13.33 +/- 1.44 s; Day 7: 11.28 +/- 0.61 s; Day 10: 15.47 +/- 2.46 s; Day 14: 15.69 +/- 1.98 s; Day 21: 15.69 +/- 1.68 s), the PWTL value in the model group (Day 4: 13.24 +/- 1.02 s; Day 7: 11.30 +/- 1.09 s; Day 10: 9.12 +/- 0.54 s; Day 14: 7.79 +/- 0.77 s; Day 21: 6.36 +/- 0.59 s) progressively decreased (P < 0.05) as time went by, while the spinal cord GFAP mRNA and protein expressions gradually increased. Compared with the normal group (Day 0) and the sham operation group (Day 14), the PWTL value in the CM groups and the vehicle group obviously decreased on Day 14 (P < 0.05). The PWTL value was not significantly different among the model group, CM groups, and the vehicle group on Day 14 (P > 0.05). On Day 21 the PWTL value of CM group 2 and 3 increased and the spinal cord GFAP mRNA and protein expression levels decreased when compared with the model group and the vehicle group (P < 0.05). But no significant difference in the PWTL value or GFAP expression levels was shown among the CM 1 group, the vehicle group, and the model group (P > 0.05). CONCLUSION: SZD had analgesic effect. Inhibition of the proliferation and activation of the spinal cord astrocytes might be one of its mechanisms.

Generation of VEGF knock-in Cashmere goat via the CRISPR/Cas9 system.

Cashmere is a rare and specialised animal fibre, which grows on the outer skin of goats. Owing its low yield and soft, light, and warm properties, it has a high economic value. Here, we attempted to improve existing cashmere goat breeds by simultaneously increasing their fibre length and cashmere yield. We attempted this by knocking in the vascular endothelial growth factor (VEGF) at the fibroblast growth factor 5(FGF5) site using a gene editing technology and then studying its hair growth-promoting mechanisms. We show that a combination of RS-1 and NU7441 significantly improve the efficiency of CRISPR/Cas9-mediated, homologous-directed repair without affecting the embryo cleavage rate or the percentages of embryos at different stages. In addition, we obtained a cashmere goat, which integrated the VEGF gene at the FGF5 site, and the cashmere yield and fibre length of this gene-edited goat were improved. Through next-generation sequencing, we found that the up-regulation of VEGF and the down-regulation of FGF5 affected the cell cycle, proliferation, and vascular tone through the PI3K-AKT signalling pathway and at extracellular matrix-receptor interactions. Owing to this, the gene-edited cashmere goat showed impressive cashmere performance. Overall, in this study, we generated a gene-edited cashmere goat by integrating VEGF at the FGF5 site and provided an animal model for follow-up research on hair growth mechanisms.

Intraperitoneal injection of thalidomide attenuates bone cancer pain and decreases spinal tumor necrosis factor-α expression in a mouse model.

BACKGROUND: Tumor necrosis factor α (TNF-α) may have a pivotal role in the genesis of mechanical allodynia and thermal hyperalgesia during inflammatory and neuropathic pain. Thalidomide has been shown to selectively inhibit TNF-α production. Previous studies have suggested that thalidomide exerts anti-nociceptive effects in various pain models, but its effects on bone cancer pain have not previously been studied. Therefore, in the present study, we investigated the effect of thalidomide on bone cancer-induced hyperalgesia and up-regulated expression of spinal TNF-α in a mouse model. RESULTS: Osteosarcoma NCTC 2472 cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce ongoing bone cancer related pain behaviors. At day 5, 7, 10 and 14 after operation, the expression of TNF-α in the spinal cord was higher in tumor-bearing mice compared to the sham mice. Intraperitoneal injection of thalidomide (50 mg/kg), started at day 1 after surgery and once daily thereafter until day 7, attenuated bone cancer-evoked mechanical allodynia and thermal hyperalgesia as well as the up-regulation of TNF-α in the spinal cord. CONCLUSIONS: These results suggest that thalidomide can efficiently alleviate bone cancer pain and it may be a useful alternative or adjunct therapy for bone cancer pain. Our data also suggest a role of spinal TNF-α in the development of bone cancer pain.

Effect of ketamine on ERK expression in hippocampal neural cell and the ability of learning behavior in minor rats.

To study the effects of ketamine on ERK expression in hippocampal neural cell and the ability of learning behavior in minor rats. Seventy-two Sprague-Dawley rats of 21 days old were randomly divided into nine groups. The Y-maze was used to test the ability of learning and spatial localization. At the end of training, all rats were killed and the expression levels of ERK1, ERK2 and p-ERK1/2 were tested by immunohistochemistry. The learning times and total reaction time (TRT) of group K2a, K2b, K2c and K3 have significant differences compared with T group (P < 0.05). Immunohistochemical staining showed that the level of ERK1, ERK2 and p-ERK1/2 in all rats which received light-electricity integrated training increased remarkably relative to the C group (P < 0.01). The expression levels of ERK1, ERK2 and p-ERK1/2 in hippocampal neural cell of group K2a, K2b and K3 significantly decreased when compared with T group (P < 0.05). Therefore, the results demonstrate that administration of over-anesthetic ketamine may impair learning ability of 21 days old rats within 24 h. ERK signal transduction pathway may be involved in the ability of learning and spatial localization. The inhibition of ERK signal transduction pathway may be one of the mechanisms of the impairment of learning and memory ability by ketamine.

ERK in learning and memory: a review of recent research.

The extracellular signal-regulated kinase (ERK) pathway is a member of the mitogen-activated protein kinase (MAPK) superfamily, which is an important, highly conserved family of enzymes associated with cell membrane receptors and regulative targets. In the central nervous system, there is almost no mature neuronal proliferation and differentiation, but the regulation of MAPK and its upstream and downstream molecular pathways is still widespread, with the ERK signaling pathway being one of the most actively studied signal transduction pathways. It is activated by a variety of cell growth factors and substances which promote mitotic activity, and transmits extracellular signals from the cell surface to the nucleus, which transmission plays an important role in the process of cell proliferation and differentiation. In recent years, accumulating evidence has shown that the ERK signaling pathway has an important link with the higher functions of learning and memory.

The antidepressant effects of rosiglitazone on rats with depression induced by neuropathic pain.

A growing number of studies reported that rosiglitazone (a PPARgamma agonist) could ameliorate the painful state and prevent stress-induced depression. However, whether rosiglitazone can prevent pain-induced depression is unclear. This study aimed to explore the antidepressant effects of rosiglitazone in L5 spinal nerve transection (SNT) induced neuropathic pain rats. In addition, AMPK inhibitor (Compound C) and autophagic antagonist (3-methyladenine, 3-MA) were applied to investigate the underlying therapeutic mechanisms. L5 SNT-induced neuropathic pain symptoms and depressive like-behaviors were detected by paw pressure threshold test (PPT), open-field test (OFT), forced swimming test (FST), tail suspension test (TST), sucrose preference test (SPT). Rosiglitazone could ameliorate L5 SNT-induced neuropathic pain symptoms and depressive like-behaviors and the effect could be reversed by Compound C or 3-MA. Compared with the sham group, the levels of BDNF, AMPK, Beclin-1 and LC3B in rats hippocampus significantly decreased in L5 SNT group. On the contrary, rosiglitazone administration significantly up-regulated the levels of AMPK, BDNF, Beclin-1 and LC3B in rats hippocampus. Compared with sham group, the levels of TNF-α, IL-1ß, superoxide dismutase (SOD) and malondialdehyde (MDA) in rat hippocampus significantly increased in L5 SNT group. Besides, rosiglitazone administration significantly decreased the levels of TNF-α, IL-1ß, SOD and MDA in hippocampus. Compared with rosiglitazone group, 3-MA administration, but not Compound C administration, significantly increased the levels of TNF-α, IL-1ß, SOD and MDA in hippocampus. In conclusion, rosiglitazone can counteract down-regulation of AMPK and BDNF induced by L5 SNT rats in hippocampus, and activate autophagic pathway. These effects may contribute to the antidepressant effect of rosiglitazone on the rats with depression induced by L5 SNT.