Comorbid anxiety and depressive symptoms in chronic pain are a common health problem, but the underlying mechanisms remain unclear. Previously, we have demonstrated that sensitization of the CeA neurons via decreased GABAergic inhibition contributes to anxiety-like behaviors in neuropathic pain rats. In this study, by using male Sprague Dawley rats, we reported that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain. Bilateral electrolytic lesions of CeA, but not lateral/basolateral nucleus of the amygdala (LA/BLA), abrogated both pain hypersensitivity and aversive and depressive symptoms of neuropathic rats induced by spinal nerve ligation (SNL). Moreover, SNL rats showed structural and functional neuroplasticity manifested as reduced dendritic spines on the CeA neurons and enhanced LTD at the LA/BLA-CeA synapse. Disruption of GluA2-containing AMPAR trafficking and endocytosis from synapses using synthetic peptides, either pep2-EVKI or Tat-GluA2(3Y), restored the enhanced LTD at the LA/BLA-CeA synapse, and alleviated the mechanical allodynia and comorbid aversive and depressive symptoms in neuropathic rats, indicating that the endocytosis of GluA2-containing AMPARs from synapses is probably involved in the LTD at the LA/BLA-CeA synapse and the comorbid aversive and depressive symptoms in neuropathic pain in SNL-operated rats. These data provide a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlight that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain. SIGNIFICANCE STATEMENT Several studies have demonstrated the high comorbidity of negative affective disorders in patients with chronic pain. Understanding the affective aspects related to chronic pain may facilitate the development of novel therapies for more effective management. Here, we unravel that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain, and LTD at the amygdaloid LA/BLA-CeA synapse mediated by GluA2-containing AMPAR endocytosis underlies the comorbid aversive and depressive symptoms in neuropathic pain. This study provides a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlights that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.
Objective To explore the expressions and functions of miR-155, miR-125b-5p, miR-210, and their target proteins, in vascular endothelial cells in oxidative stress. Methods Human umbilical vein endothelial cells (HUVECs) were isolated and cultured; the expression of endothelial cell factor VIII related antigen was detected by immunofluorescence staining; and the expression of CD31 was detected and identified by flow cytometry. The oxidative stress model of vascular endothelial cells was established by preeclampsia serum and hydrogen peroxide (H2O2) treatment. The treated HUVECs were divided into 4 groups: normal pregnancy serum group, preeclampsia serum group, 0 μmol/L H2O2 group and 100 μmol/L H2O2 group. The levels of miR-155, miR-125b-5p, and miR-210 were tested by real-time quantitative PCR within 24 hours after modeling. The expression of Smad4 protein was detected by Western blotting and immunofluorescence staining. Cell apoptosis was detected by flow cytometry combined with annexin V-FITC/PI staining. Results Compared with 0 μmol/L H2O2 group, miR-125b-5p significantly decreased in 100 μmol/L H2O2 group, while Smad4 protein significantly increased. In addition, 100 μmol/L H2O2 treatment promoted cell apoptosis and necrosis. Compared with normal pregnancy serum group, the apoptosis and necrosis were significantly enhanced in preeclampsia serum group, but there were no significant differences in miR-125b-5p and Smad4 between the groups. Conclusion High level of H2O2 up-regulates Smad4 protein expression and inhibits miR-125b-5p expression.
Ginseng, the roots and rhizomes of Panax ginseng C. A. Meyer, is used not only as a herbal medicine but also as a functional food to support body functions. Ginsenoside Rg3 (GRg3) is a major bioactive component in ginseng. In this study, the beneficial effects of GRg3 on rats with Alzheimer's disease (AD) were evaluated via the behavioral experiment and antioxidant capacity. Moreover, metabolomic analysis based on UPLC-QTOF-MS/MS and apoptosis analysis was used to obtain the change between AD and GRg3-administrated rats to assess the underlying mechanisms on improving mitochondrial dysfunction. Results showed that GRg3 could prevent the cognitive impairment of AD rats by improving the mitochondrial dysfunction. The potential mechanisms were related to regulate the abnormality of energy metabolism, electron transport chain, amino acid metabolism, purine metabolism, and antiapoptosis. These findings support the exploitation of GRg3 as an effective complementary and functional food to prevent and delay AD.
Background: Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease. Growing evidence indicated that IPAH is a chronic immune disease. This study explored the molecular mechanisms and T cell infiltration of IPAH using integrated bioinformatics methods. Methods: Gene expression profiles of dataset GSE113439 were downloaded from the Gene Expression Omnibus and analyzed using R. Protein-protein interaction (PPI) network and gene set enrichment analysis (GSEA) were established by NetworkAnalyst. Gene Ontology enrichment analysis was performed using ClueGO. Transcription factors of differentially expressed genes (DEGs) were estimated using iRegulon. Transcription factors and selected hub genes were verified by real-time polymerase chain reaction (qPCR) in the lung tissues of rats with pulmonary artery hypertension. The least absolute shrinkage and selection operator regression model and the area under the receiver operating characteristic curve (AUC) were applied jointly to identify the crucial hub genes. Moreover, immune infiltration in IPAH was calculated using ImmuCellAI, and the correlation between key hub genes and immune cells was analyzed using R. Results: A total of 512 DEGs were screened, and ten hub genes and three transcription factors were filtered by the DEG PPI network. The DEGs were mainly enriched in mitotic nuclear division, chromosome organization, and nucleocytoplasmic transport. The ten hub genes and three transcription factors were confirmed by qPCR. Moreover, MAPK6 was identified as the most potent biomarker with an AUC of 100%, and ImmuCellAI immune infiltration analysis showed that a higher proportion of CD4-naive T cells and central memory T cells (Tcm) was apparent in the IPAH group, whereas the proportions of cytotoxic T cells (Tc), exhausted T cells (Tex), type 17 T helper cells, effector memory T cells, natural killer T cells (NKT), natural killer cells, gamma-delta T cells, and CD8 T cells were lower. Finally, MAPK6 was positively correlated with Tex and Tcm, and negatively correlated with Tc and NKT. Conclusion:MAPK6 was identified as a crucial hub gene to discriminate IPAH from the normal group. Dysregulated immune reactions were identified in the lung tissue of patients with IPAH.
To investigate the expression of peptidylarginine deiminase 4 (PADI4) during the process of differentiation into granulocyte of NB4 cells induced by all-trans-retinoic acid (ATRA) and whether PADI4 is involved in the inflammatory cytokines expression.Granulocyte differentiation model of NB4 cells induced by ATRA was established. The cell morphology changes were observed by Wright-Giemsa staining. The expression of cell differentiation marker CD11b was analyzed by flow cytometry. The mRNA and protein expression of PADI4 was detected by RT-PCR and Western blot, respectively. The expression of tumor necrosis factor (TNF) α and interleukin (IL) 1β was analyzed by ELISA, and also examined with the knockdown of PADI4 expression by siRNA.After NB4 cells induced by ATRA, the cytoplasm increased and the ratio of nuclear to cytoplasmic was reduced. Nuclear dented, and rod-shaped nucleus, lobulated phenomenon increased (P<0.05). Flow cytometry analysis results showed that the cell surface molecule CD11b expression increased (P<0.01). RT-PCR and Western blot showed the expression of PADI4 increased at both transcriptional and translational levels during the process of the differentiation. ELISA showed TNF-α and IL-1β secretion increased in differentiated macrophages, while they could be inhibited by PADI4-specific siRNA.During the differentiation into granulocyte of NB4 cells induced by ATRA, PADI4 expression increased. Furthermore, PADI4 appeared to play a critical role in inflammatory cytokines secretion.PADI4对NB4细胞分化过程中炎性因子表达水平的影响.探讨全反式维甲酸（ATRA）诱导人急性早幼粒细胞白血病细胞株NB4向粒细胞分化过程中肽酰基精氨酸脱亚胺酶4（PADI4）的表达变化及其是否参与炎性因子的表达.建立ATRA诱导的NB4细胞分化模型，采用Wright-Giemsa染色观察细胞形态学变化；采用流式细胞术检测细胞表面分化抗原CD11b的表达；RT-PCR方法检测PADI4基因转录水平的表达；Western bolt方法检测PADI4翻译水平的表达；ELISA检测培养液中肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β的表达；siRNA技术干扰PADI4表达后ELISA检测TNF-α和IL-1β的表达.ATRA诱导后NB4细胞胞浆增多，核浆比例降低，核有凹陷，杆状和分叶现象增多。流式细胞术检测结果显示细胞表面分子CD11b表达明显升高(P<0.01)。在分化模型中，RT-PCR及Western blot检测结果显示PADI4转录水平和翻译水平表达均明显升高(P<0.05)。ELISA方法检测培养液中TNF-α和IL-1β的表达水平明显升高(P<0.05)，而siRNA技术干扰PADI4表达后TNF-α和IL-1β的表达水平明显下降(P<0.05).ATRA诱导NB4细胞向粒细胞分化过程中PADI4的表达升高，同时PADI4参与了NB4细胞分化过程中炎性因子的表达.
In this study, the protective effects of 3,4-dihydroxyphenyl lactic acid (DLA) on blood-brain barrier (BBB) injury following subarachnoid hemorrhage (SAH) has been explored.Male Sprague-Dawley rats (weight 300-350 g) were used to establish the SAH model using the endovascular perforation method. The animals were randomly divided into four groups: sham (n = 40), SAH (n = 46), SAH + vehicle (n = 44), and SAH + DLA (n = 40) treatment groups. At 1 h after SAH, either DLA (10 mg/kg) or normal saline (vehicle) was administered by femoral vein injection. The effects of DLA on mortality, neurological function, brain water content, and BBB were observed. Additionally, immunohistochemistry and western blot techniques were applied to investigate the mechanism of action of DLA.We found that the administration of DLA (10 mg/kg) following SAH could improve neurological functions, reduce brain water content, and maintain BBB integrity. The expression of pro-inflammatory and pro-apoptotic factors such as toll-like receptor 4 (TLR4), NF-κB (p-p65), tumor necrosis factor-α, p-p38 MAPK, p-p53, and caspase-3 were significantly increased after SAH. These same factors were markedly attenuated following treatment with DLA.These findings showed that DLA can alleviate BBB injury following SAH through its anti-inflammatory and anti-apoptotic effects via suppression of TLR4 and its downstream NF-κB and p38 MAPK pathways.
As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor (MC3R) is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals, and its pharmacological effect could be directly influenced by the presence of melanocortin receptor accessory proteins (MRAPs), MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not yet been thoroughly evaluated. In this work, we performed sequence alignment, phylogenetic tree, and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH-stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.
Objective
To develop a new method of immune isolation for transplanting pig islets to rat to observe the effects on diabetic rats.
Method
Diabetes of rat was induced by streptozotocin. Pancreas of pig was digested with type V collagenase. The pig islets were purified by density gradient centrifugation. Insulin stimulation index of the purified pig islets was evaluated. Liver of the 18 diabetic rats and 6 normal rats was exposed by operation. Capsule at one edge of the liver lobe was cut apart and separated from liver parenchyma on both sides of the lobe. An artificial cyst was constructed by a cellulose ester (CE) dialysis bag with a ball of hollow fibers in the bag, and each end of the bag was closed by a thread. Each end of the bag was stuffed into the subcapsule of each side of the liver lobe. The thread at each end of the bag was passed through a hole of the capsule at other edge of the lobe and linked to each other around the lobe to hold the artificial cyst on the liver parenchyma. Middle part of the cyst (with the ball of hollow fibers in) was put between the cuts of capsule and under the skin. The collagen solution (pH 7.4) with pig islets (4000 IEQ) was injected into the subcapsule artificial cyst of 12 diabetic rats as experiment group. The collagen solution without pig islets was injected into the subcapsule artificial cyst of 6 diabetic rats and 6 normal rats as diabetes control group and normal control group respectively. The cut was sutured. The collagen solution became a collagen gel in the artificial cysts. At 8th week of the initial transplantation, the solution with pig islets (2000 IEQ) was injected percutaneously again into the subcapsule artificial cyst of each rat of the experiment group. Rats of the three groups were raised as usual, and the levels of fasting blood glucose were measured regularly.
Result
The insulin stimulation index of the purified pig islets was 2.2±0.2. At 1st to 8th week of the initial transplantation, the levels of fasting blood glucose in the experiment group at every time point were lower significantly than those in the diabetes control group (P<0.01). At 13th week of the initial transplantation, there was no significant fibroplasia around the subcasule artificial cysts in the rats by visual observation.
Conclusion
CE dialysis bag in the liver capsule at 13th week did not show significant stimulation to the surrounding tissues. Pig islets isolated in the subcapsule artificial cyst of rats might live normally and perform hypoglycemic action. Pig islets could be percutaneously injected into the subcapsule artificial cyst.
Key words:
Pig; Islet; Subcapsule; Liver; Transplant; Immune isolation; Rat
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