Effect of transcutaneous electrical nerve stimulation based on wrist-ankle acupuncture theory for pain relief during colonoscopy without anesthesia: a randomized controlled trial.

BACKGROUND: Colonoscopy is essential for diagnosing colorectal diseases but can cause pain during the procedure. This study explored the analgesic effects of transcutaneous electrical nerve stimulation based on wrist-ankle acupuncture theory (TENS-WAA) in colonoscopy without anesthesia. METHODS: This prospective study included 120 participants undergoing colonoscopies without anesthesia. The trial group received low-frequency, high-intensity TENS-WAA adjusted to the maximum tolerable current, while the control group received minimal current. The primary outcome was the retrospective pain score on a visual analog scale (VAS). Secondary outcomes included time, heart rate, and credibility/expectancy questionnaire (CEQ) scores. RESULTS: Participants who received TENS-WAA reported significantly lower pain VAS scores than the control group (estimated median difference -1.1, 95%CI -2 to -0.4; P = 0.002). Male participants in the trial group experienced significantly lower pain scores than the control group (mean difference -1.4, 95%CI -2.41 to -0.39; P = 0.008). The trial group showed significantly less variation in heart rates (P<0.001) and higher CEQ scores (P = 0.001) than the control group. No adverse events were reported. CONCLUSION: TENS-WAA effectively reduced pain during colonoscopy without anesthesia, especially in male participants, providing a promising noninvasive analgesic method.

Arginine vasotocin (AVT)/mesotocin (MT) receptors in chickens: Evidence for the possible involvement of AVT-AVPR1 signaling in the regulation of oviposition and pituitary prolactin expression.

Two structurally related peptides, arginine vasotocin (AVT) and mesotocin (MT), are reported to regulate many physiological processes, such as anti-diuresis and oviposition in birds, and their actions are likely mediated by four AVT/MT receptors (AVPR1A, AVPR1B, MTR and AVPR2b), which are orthologous/paralogous to human AVPR1A, AVPR1B, OXTR and AVPR2 respectively. However, our knowledge regarding the functions of these avian AVT/MT receptors has been limited. Here, we examined the functionality and expression of these receptors in chickens and investigated the roles of AVT in the anterior pituitary. Our results showed that 1) AVPR1A, AVPR1B and AVPR2b could be preferentially activated by AVT, monitored by cell-based luciferase reporter assays and/or Western blot, indicating that they are AVT-specific receptors (AVPR1A; AVPR1B) or AVT-preferring receptor (AVPR2b) functionally coupled to intracellular calcium, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, MTR could be activated by AVT and MT with similar potencies, indicating that MTR is a receptor common for both peptides; 2) Using qPCR, differential expression of the four receptors was found in chicken tissues including the oviduct and anterior pituitary. In particular, only AVPR1A is abundantly expressed in the uterus, suggesting its involvement in mediating AVT-induced oviposition. 3) In cultured chick pituitary cells, AVT could stimulate ACTH and PRL expression and secretion, an action likely mediated by AVPR1B and/or AVPR1A abundantly expressed in anterior pituitary. Collectively, our data helps to elucidate the roles of AVT/MT in birds, such as the 'oxytocic action' of AVT, which induces uterine muscle contraction during oviposition.

Effect of Aerobic Exercise on Serum Metabolites in Mice with Hepatocellular Carcinoma After Surgery.

BACKGROUND Modern medicine has suggested exercise therapy is one of the main treatments for postoperative rehabilitation of tumors. It can influence the recovery of cancer patients by changing the body's material metabolism and energy metabolism. However, studies on metabolic changes of exercise therapy on hepatocellular carcinoma (HCC) patients after surgery are limited. The aim of this study was to explore the effect of aerobic exercise on mice after orthotopic HCC surgery by serum metabolomics test and explore the related mechanism. MATERIAL AND METHODS A total of 60 C57Bl/6 mice were used to establish an orthotopic xenograft model of H22 mouse hepatoma cells. Mice were randomly divided into 6 groups and it was found that the metabolic products of the early postoperative exercise group and sedentary group mainly included L-tryptophan, citric acid, and other energy-related metabolites. RESULTS Energy metabolites, such as succinic acid of the high-intensity exercise group were increased after surgery, whereas phospholipid metabolites, including phosphatidylethanolamine (18: 0/0: 0), were decreased. In the moderate-intensity exercise group, the change tendency was consistent, and the level of various metabolites decreased. CONCLUSIONS Thus, it is likely that aerobic exercise reduced the degree of postoperative stress responses and improved energy metabolism in mice. The underlying mechanism involves improving the tricarboxylic acid cycle, intervening in energy metabolism, reorganization caused by the tumor, reducing the abnormal increase of phospholipase activity caused by the stress of liver cancer, reducing the level of hemolytic phospholipids, thereby inhibiting mitochondrial pathway-initiated apoptosis.

Bibliometric analysis of breast cancer-related lymphedema research trends over the last 2 decades.

Objective: As breast cancer cases rise globally, post-mastectomy lymphedema garners increasing scholarly attention. This study aims to conduct a comprehensive bibliometric analysis of Breast Cancer-Related Lymphedema (BCRL) research from 2003 to 2022, identifying trends and providing global research insights for future studies. Method: The literature for this analysis was extracted from the Web of Science (WoS) Core Collection, encompassing 1199 publications, including 702 articles and 101 reviews, totaling 803. Using advanced bibliometric tools such as VOSviewer and CiteSpace, quantitative and visual analyses were performed to map collaboration networks, research clusters, and emerging trends. The search strategy included specific terms related to lymphedema, breast cancer, and BCRL, ensuring a comprehensive representation of the research landscape. Results: The bibliometric analysis revealed a steady increase in BCRL publications over the studied period, reaching a peak in 2018. The United States emerged as the leading contributor to BCRL literature, with China also demonstrating a significant presence. Collaboration networks were visualized, showcasing the interconnectedness of institutions and researchers globally. Key research hotspots identified include preventive strategies, complex decongestive therapy, and reconstructive interventions. Conclusion: In conclusion, this pioneering bibliometric analysis provides a comprehensive overview of BCRL research trends and collaborations globally. The findings contribute valuable insights into the evolution of the field, highlighting areas of focus and emerging research themes. This study serves as a foundational resource for researchers, clinicians, and policymakers, fostering evidence-based practices and interventions for BCRL in the future.

Melittin inhibits the proliferation migration and invasion of HCC cells by regulating ADAMTS9-AS2 demethylation.

BACKGROUND: Melittin (MEL) has been reported to exhibit anti-cancer effects in vitro against several types of cancer. Long non-coding RNA (lncRNA) ADAMTS9-AS2 can be used as a tumor suppressor. However, there is insufficient data on the potential link between MEL and ADAMTS9-AS2 in hepatocellular carcinoma (HCC). METHODS: RT-qPCR, CCK-8, colony formation, scratch wound healing and transwell assays were used to detect the function of MEL or ADAMTS9-AS2 on HCC cells. Furthermore, Western blot analysis was applied to determine that whether an association existed in MEL or ADAMTS9-AS2 with the PI3K/AKT/mTOR signal pathway. In addition, RT-qPCR and Western blot analysis validated that whether MEL has a demethylation effect. RESULTS: All the experimental data showed that MEL or ADAMTS9-AS2 inhibited the proliferation, migration and invasion of MHCC97-H and HepG2 cells, which may relate to PI3K/AKT/mTOR signal pathway. Moreover, the result showed that MEL treatment inhibited the expression of DNA methyltransferase protein-1 (DNMT1), which acted as the role of demethylation, and then up-regulated the expression of ADAMTS9-AS2, affecting the development of HCC. CONCLUSIONS: ADAMTS9-AS2 played a role in MEL-induced HCC inhibition. This study provided an interesting theoretical basis and further evidence for the potential application of MEL in the treatment of HCC.

Transferrin-Targeted Cascade Nanoplatform for Inhibiting Transcription Factor Nuclear Factor Erythroid 2-Related Factor 2 and Enhancing Ferroptosis Anticancer Therapy.

Ferroptosis, an iron-dependent cell death driven by the lethal levels of lipid peroxidation (LPO), becomes a promising anticancer strategy. However, the anticancer efficacy of ferroptosis is often hindered by the activation of nuclear factor erythrocyte 2-associated factor 2 (Nrf2), which is an indispensable regulator of the cellular antioxidant balance by preventing the accumulation of intracellular reactive oxygen species (ROS). Herein, we present a rational design of a Tf-targeted cascade nanoplatform TPM@AM based on mesoporous polydopamine (MPDA) co-encapsulating a ferroptosis inducer (artesunate, ART) and an Nrf2-specific inhibitor (ML385) to enhance intracellular ROS and therefore amplify ferrotherapy. Transferrin (Tf) can specifically recognize the transferrin receptor (TfR) on the surface of the cell membrane, which binds and transports iron into cells. When TPM@AM is endocytosed, the high-acid tumor microenvironment and laser irradiation trigger the collapse of MPDA to release ART and ML385. Furthermore, MPDA endows the nanoplatform with photothermal capability. The nanoplatform exhibits high efficiency for synergistic tumor suppression, representing a spatiotemporal controllable therapeutic strategy for precise synergistic cancer therapy.

Serum metabonomics as a diagnostic approach for cancer-related fatigue.

In the present study, differences in metabolic pathways between patients with and without cancer-related fatigue (CRF) were examined to identify metabolic serum biomarkers of CRF. In this preliminary study, metabolic profiling was applied to analyze the serum samples from 14 patients with CRF and 11 non-CRF individuals (non-fatigue cancer survivors) by ultra-performance liquid chromatography coupled with mass spectrometry. Orthogonal partial least-squares discriminant analysis was adopted to evaluate the differences between the CRF and non-CRF groups. The CRF group was characterized by increases in phosphatidylethanolamine (PE; 18:0/0:0), LysoPE (0:0/20:4 and 0:0/16:0), lysophosphatidylcholine (LysoPC; 20:4, 22:4 and 16:0) and LysoPC/PC, phosphatidylserine (21:0/0:0), glycerophosphocholine and N-docosahexaenoyl γ-aminobutyric acid. Furthermore, decreases in anandamide, uric acid, dihydrouracil, LysoPE (0:0/22:5), 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman, 19(R)-hydroxy-prostaglandin F1α, N-(3α,12α-dihydroxy-5ß-cholan-24-oyl)-glycine, ketoleucine, indoxyl sulfate, α-N-phenylacetyl-L-glutamine and 1-linoleoyl-glycerophosphocholine were detected. These data indicate a possible disturbance in the metabolism of phospholipids and adjustments in the endocannabinoid system. The metabonomic approach may be helpful to determine the pathophysiological mechanisms of CRF and the identification of potential biomarkers for the accurate diagnosis of CRF. All clinical data were obtained from the 'Research on the efficacy of traditional Chinese medicine comprehensive intervention in cancer-related fatigue' (TCM-CRF) project. Medical Ethical Approval for TCM-CRF was approved by the Chinese Ethics Committee of Registering Clinical Trials. The approval number for the TCM-CRF study was ChiECRCT-2013038, and the TCM-CRF study was completed.

Characterization of relaxin 3 and its receptors in chicken: Evidence for relaxin 3 acting as a novel pituitary hormone.

Mammalian relaxin (RLN) family peptides binding their receptors (RXFPs) play a variety of roles in many physiological processes, such as reproduction, stress, appetite regulation, and energy balance. In birds, although two relaxin family peptides (RLN3 and INSL5) and four receptors (RXFP1, RXFP2, RXFP2-like, and RXFP3) were predicated, their sequence features, signal properties, tissue distribution, and physiological functions remain largely unknown. In this study, using chickens as the experimental model, we cloned the cDNA of the cRLN3 gene and two receptor (cRXFP1 and cRXFP3) genes. Using cell-based luciferase reporter assays, we demonstrate that cRLN3 is able to activate both cRXFP1 and cRXFP3 for downstream signaling. cRXFP1, rather than cRXFP3, is a cognate receptor for cRLN3, which is different from the mammals. Tissue distribution analyses reveal that cRLN3 is highly expressed in the pituitary with lower abundance in the hypothalamus and ovary of female chicken, together with the detection that cRLN3 co-localizes with pituitary hormone genes LHB/FSHB/GRP/CART and its expression is tightly regulated by hypothalamic factors (GnRH and CRH) and sex steroid hormone (E2). The present study supports that cRLN3 may function as a novel pituitary hormone involving female reproduction.

Effect of Ultrasonic Penetration with Volatile Oil of Olibanum and Chuanxiong Rhizoma on Acute Knee Synovitis Induced by Sports Training: An Open-Label Randomized Controlled Study.

Background: Knee synovitis is a common sports injury. We proposed the use of UTVOR, which is a combination of the use of volatile oil of Olibanum (VOO) and volatile oil of Chuanxiong Rhizoma (VOCR) and conventional ultrasound (US) therapy, to treat knee synovitis. Design, Setting, Participants, and Interventions. Participants were randomly assigned into a control group (conventional US therapy group) and a test group (UTVOR group). The control group received conventional US therapy with a coupling agent as the medium. The test group received a revised US therapy with VOO and VOCR as media. Both groups were treated once per day for three consecutive days. Main Outcome Measures. The subjects' Visual Analogue Scale (VAS) pain score, Lysholm knee score, knee swelling degree, circumference, and range of motion of the knee joint were evaluated before the first treatment and 24 h after the third treatment. The VAS pain score was considered the primary outcome, while the three other measurements were regarded as the secondary outcomes. An adverse event was reported subjectively and recorded. Results: A total of 116 participants were included in the analysis (test group: n = 64; control group: n = 52). The evaluation results showed that the VAS pain scores of the male and female participants in both groups decreased after treatment (P < 0.001), but only the difference among the male sub-group had significant between-group difference (P < 0.001). After treatment, the Lysholm scores in both groups increased significantly (all P < 0.001), the range of motion and the circumference of the injured knee decreased significantly (P < 0.001), while no between-group difference was observed in general or in the gender sub-groups (all P > 0.025). No side effect or complication was reported during the treatment. Conclusion: UTVOR had a superior analgesic effect to conventional US therapy in the male population, but its effects on alleviating joint function, swelling, and range of motion were comparable to that of conventional US therapy. Our study found that UTVOR can be an effective method to reduce pain and treat knee synovitis, and it is subjectively safe. Trial registration. This study was registered under the Chinese Clinical Trial Registry (Trial Registration Number: ChiCTR2000035671).

Characterization of Chicken α2A-Adrenoceptor: Molecular Cloning, Functional Analysis, and Its Involvement in Ovarian Follicular Development.

Adrenoceptors are suggested to mediate the functions of norepinephrine (NE) and epinephrine (EPI) in the central nervous system (CNS) and peripheral tissues in vertebrates. Compared to mammals, the functionality and expression of adrenoceptors have not been well characterized in birds. Here, we reported the structure, expression, and functionality of chicken functional α2A-adrenoceptor, named ADRA2A. The cloned chicken ADRA2A cDNA is 1335 bp in length, encoding the receptor with 444 amino acids (a.a.), which shows high amino acid sequence identity (63.4%) with its corresponding ortholog in humans. Using cell-based luciferase reporter assays and Western blot, we demonstrated that the ADRA2A could be activated by both NE and EPI through multiple signaling pathways, including MAPK/ERK signaling cascade. In addition, the mRNA expression of ADRA2A is found to be expressed abundantly in adult chicken tissues including thyroid, lung, ovary and adipose from the reported RNA-Seq data sets. Moreover, the mRNA expression of ADRA2A is also found to be highly expressed in the granulosa cells of 6-8 mm and F5 chicken ovarian follicles, which thus supports that ADRA2A signaling may play a role in ovarian follicular growth and differentiation. Taken together, our data provide the first proof that the α2A-adrenoceptor is functional in birds involving avian ovarian follicular development.

Characterization of four urotensin II receptors (UTS2Rs) in chickens.

Urotensin II receptor (UTS2R) is suggested to mediate the actions of urotensin II (UTS2) and UTS2-related peptide (URP, also called UTS2B) in mammals. However, the information regarding the gene structure, functionality and tissue expression of UTS2/URP receptor remains largely unknown in non-mammalian vertebrates including birds. In this study, using RACE-PCR, we cloned the full-length cDNAs of four chicken UTS2/URP receptors and designated them as cUTS2R1, cUTS2R2, cUTS2R3 and cUTS2R5 respectively, according to their evolutionary origin. The cloned cUTS2R1, cUTS2R2, cUTS2R3 and cUTS2R5 are predicted to encode 7-transmembrane receptors of 382, 343, 331 and 363 amino acids respectively, which show 50-66 % amino acid sequence identity with human UTS2R. Using cell-based luciferase reporter assays and Western blot, we demonstrated that chicken UTS2Rs expressed in HEK293 cells could be effectively activated by synthetic chicken UTS2-12, UTS2-17 and URP peptides, and their activation can elevate intracellular calcium concentration and activate MAPK/ERK signaling cascade, indicating that the four UTS2Rs are functional and capable of mediating UTS2/URP actions in chickens. Quantitative real-time PCR revealed that the four receptors are widely, but differentially, expressed in adult chicken tissues, while cUTS2 and cURP are highly expressed in the hindbrain and spinal cord, and moderately/weakly expressed in other tissues examined including the spleen and gonads. Taken together, our data provide first piece of evidence that all four UTS2Rs are functional in an avian species and help to reveal the conserved roles of UTS2R signaling across vertebrates.

Single-Cell RNA Sequencing Analysis of Chicken Anterior Pituitary: A Bird's-Eye View on Vertebrate Pituitary.

It is well-established that anterior pituitary contains multiple endocrine cell populations, and each of them can secrete one/two hormone(s) to regulate vital physiological processes of vertebrates. However, the gene expression profiles of each pituitary cell population remains poorly characterized in most vertebrate groups. Here we analyzed the transcriptome of each cell population in adult chicken anterior pituitaries using single-cell RNA sequencing technology. The results showed that: (1) four out of five known endocrine cell clusters have been identified and designated as the lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs, respectively. Somatotrophs were not analyzed in the current study. Each cell cluster can express at least one known endocrine hormone, and novel marker genes (e.g., CD24 and HSPB1 in lactotrophs, NPBWR2 and NDRG1 in corticotrophs; DIO2 and SOUL in thyrotrophs, C5H11ORF96 and HPGDS in gonadotrophs) are identified. Interestingly, gonadotrophs were shown to abundantly express five peptide hormones: FSH, LH, GRP, CART and RLN3; (2) four non-endocrine/secretory cell types, including endothelial cells (expressing IGFBP7 and CFD) and folliculo-stellate cells (FS-cells, expressing S100A6 and S100A10), were identified in chicken anterior pituitaries. Among them, FS-cells can express many growth factors, peptides (e.g., WNT5A, HBEGF, Activins, VEGFC, NPY, and BMP4), and progenitor/stem cell-associated genes (e.g., Notch signaling components, CDH1), implying that the FS-cell cluster may act as a paracrine/autocrine signaling center and enrich pituitary progenitor/stem cells; (3) sexually dimorphic expression of many genes were identified in most cell clusters, including gonadotrophs and lactotrophs. Taken together, our data provides a bird's-eye view on the diverse aspects of anterior pituitaries, including cell composition, heterogeneity, cell-to-cell communication, and gene expression profiles, which facilitates our comprehensive understanding of vertebrate pituitary biology.

DNA methylation is involved in sexual differentiation and sex chromosome evolution in the dioecious plant garden asparagus.

DNA methylation is a crucial regulatory mechanism in many biological processes. However, limited studies have dissected the contribution of DNA methylation to sexual differentiation in dioecious plants. In this study, we investigated the variances in methylation and transcriptional patterns of male and female flowers of garden asparagus. Compared with male flowers, female flowers at the same stages showed higher levels of DNA methylation. Both male and female flowers gained DNA methylation globally from the premeiotic to meiotic stages. Detailed analysis revealed that the increased DNA methylation was largely due to increased CHH methylation. Correlation analysis of differentially expressed genes and differentially methylated regions suggested that DNA methylation might not have contributed to the expression variation of the sex-determining genes SOFF and TDF1 but probably played important roles in sexual differentiation and flower development of garden asparagus. The upregulated genes AoMS1, AoLAP3, AoAMS, and AoLAP5 with varied methylated CHH regions might have been involved in sexual differentiation and flower development of garden asparagus. Plant hormone signaling genes and transcription factor genes also participated in sexual differentiation and flower development with potential epigenetic regulation. In addition, the CG and CHG methylation levels in the Y chromosome were notably higher than those in the X chromosome, implying that DNA methylation might have been involved in Y chromosome evolution. These data provide insights into the epigenetic modification of sexual differentiation and flower development and improve our understanding of sex chromosome evolution in garden asparagus.

Opioid Peptides and Their Receptors in Chickens: Structure, Functionality, and Tissue Distribution.

Opioid peptides, derived from PENK, POMC, PDYN and PNOC precursors, together with their receptors (DOR, MOR, KOR and ORL1), constitute the opioid system and are suggested to participate in multiple physiological/pathological processes in vertebrates. However, the question whether an opioid system exists and functions in non-mammalian vertebrates including birds remains largely unknown. Here, we cloned genes encoding opioid system from the chicken brain and examined their functionality and tissue expression. As in mammals, 6 opioid peptides encoded by PENK (Met-enkephalin and Leu-enkephalin), POMC (ß-endorphin), PDYN (dynorphin-A and dynorphin-B) and PNOC (nociceptin) precursors and four opioid receptors were found to be highly conserved in chickens. Using pGL3-CRE-luciferase and pGL4-SRE-luciferase reporter systems, we demonstrated that chicken opioid receptors (cDOR, cMOR, cKOR and cORL1) expressed in CHO cells, could be differentially activated by chicken opioid peptides, and resulted in the inhibition of cAMP/PKA and activation of MAPK/ERK signaling pathways. cDOR is potently activated by Met-enkephalin and Leu-enkephalin, and cKOR is potently activated by dynorphin-A, dynorphin-B and nociceptin, whereas cORL1 is specifically activated by nociceptin. Unlike cDOR, cKOR and cORL1, cMOR is moderately/weakly activated by enkephalins and other opioid peptides. These findings suggest the ligand-receptor pair in chicken opioid system is similar, but not identical to, that in mammals. Quantitative real-time PCR revealed that the opioid system is mainly expressed in chicken central nervous system including the hypothalamus. Collectively, our data will help to facilitate the better understanding of the conserved roles of opioid system across vertebrates.

Regulation of Pituitary Cocaine- and Amphetamine-Regulated Transcript Expression and Secretion by Hypothalamic Gonadotropin-Releasing Hormone in Chickens.

There is increasing evidence that cocaine- and amphetamine-regulated transcript (CART) peptide is abundantly expressed in the anterior pituitary of birds and mammals, suggesting that CART peptide may be a novel pituitary hormone and its expression and secretion is likely controlled by the hypothalamic factor(s). To substantiate this hypothesis, using chicken as an animal model, we examined the effects of gonadotropin-releasing hormone (GnRH) on pituitary CART secretion and expression and investigated whether GnRH could modulate plasma CART levels. The results showed that: (1) chicken GnRH (GnRH1 and GnRH2) could potently stimulate CART peptide secretion in intact pituitaries incubated in vitro, as detected by Western blot; (2) GnRH could also stimulate CART mRNA expression in cultured pituitary cells, as revealed by quantitative real-time polymerase chain reaction (qPCR) assay; (3) GnRH actions on pituitary CART expression and secretion are likely mediated by GnRH receptor coupled to the intracellular Ca2+, MEK/ERK, and cAMP/PKA signaling pathways; and (4) plasma CART levels are high in chickens at various developmental stages (1.2-3.5 ng/ml) and show an increasing trend towards sexual maturity, as detected by enzyme-linked immunosorbent assay (ELISA). Moreover, plasma CART levels could be significantly induced by intraperitoneal administration of GnRH in chicks. Taken together, our data provide the first collective evidence that CART peptide is a novel pituitary hormone and its expression and secretion are tightly controlled by hypothalamic GnRH, thus likely being an active player in the hypothalamic-pituitary-gonadal (HPG) axis.

The anti-tumour effect of Mel and its role in autophagy in human hepatocellular carcinoma cells.

Melittin (Mel), a major component of venom of honey bee (Apismellifera), has various biological effects. Recent researches have reported the anti-tumor activity of Mel in various human cancers, including hepatocellular carcinoma (HCC). In this study, we aimed to further discuss the role of Mel in HCC and investigate the correlation of autophagy with the effect of Mel in HCC cells. Methyl thiazolyl tetrazolium (MTT) assay and flow cytometry were used to detect the viability and apoptosis of HCC cells, respectively. To examine the changes of autophagy in HCC cells treated with Mel, transmission electronmicroscope (TEM) and immunofluorescence detection were adopted. Finally, we used western blot method to detect the changes of pivotal proteins in autophagy and mitochondrial apoptotic pathways. The results of MTT assay and flow cytometry revealed that Mel could suppress the cell viability and promote the apoptosis of HCC cells. Autophagy could be induced by the treatment with Mel in HCC cells. The inhibition of autophagy by chloroquine (CQ) contributed to the enhanced anti-tumor effect of Mel, but autophagy induction by RAPA decreased Mel effect in HCC cells. Mel was closely associated with the expression of proteins in mitochondrial apoptotic pathway. In summary, Mel could induce the autophagy of HCC cells, and the autophagy might offer protection against apoptosis in HCC. Mel might suppress the tumor through activating mitochondrial apoptotic pathway.

Identification of a Novel Functional Corticotropin-Releasing Hormone (CRH2) in Chickens and Its Roles in Stimulating Pituitary TSHß Expression and ACTH Secretion.

Corticotropin-releasing hormone (CRH), together with its structurally and functionally related neuropeptides, constitute the CRH family and play critical roles in multiple physiological processes. Recently, a novel member of this family, namely CRH2, was identified in vertebrates, however, its functionality and physiological roles remain an open question. In this study, using chicken (c-) as the animal model, we characterized the expression and functionality of CRH2 and investigated its roles in anterior pituitary. Our results showed that (1) cCRH2 cDNA is predicted to encode a 40-aa mature peptide, which shares a higher amino acid sequence identity to cCRH (63%) than to other CRH family peptides (23-38%); (2) Using pGL3-CRE-luciferase reporter system, we demonstrated that cCRH2 is ~15 fold more potent in activating cCRH receptor 2 (CRHR2) than cCRHR1 when expressed in CHO cells, indicating that cCRH2 is bioactive and its action is mainly mediated by CRHR2; (3) Quantitative real-time PCR revealed that cCRH2 is widely expressed in chicken tissues including the hypothalamus and anterior pituitary, and its transcription is likely controlled by promoters near exon 1, which display strong promoter activity in cultured DF-1 and HEK293 cells; (4) In cultured chick pituitary cells, cCRH2 potently stimulates TSHß expression and shows a lower potency in inducing ACTH secretion, indicating that pituitary/hypothalamic CRH2 can regulate pituitary functions. Collectively, our data provides the first piece of evidence to suggest that CRH2 play roles similar, but non-identical, to those of CRH, such as its differential actions on pituitary, and this helps to elucidate the roles of CRH2 in vertebrates.

Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution, functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expression.

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary.

Characterization of NMB, GRP and their receptors (BRS3, NMBR and GRPR) in chickens.

The two structurally and functionally related peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) play critical roles in many physiological/pathological processes in mammals. However, the information regarding the expression and functionality of avian NMB, GRP and their receptors is limited. Here, we characterized cNMB, cGRP and their receptors (cNMBR, cGRPR and cBRS3) in chickens. Our results showed that: (1) cNMBR and cGRPR expressed in CHO cells could be potently activated by cNMB and cGRP, respectively, as monitored by cell-based luciferase reporter assays, indicating that cNMBR and cGRPR are cNMB- and cGRP-specific receptors; strikingly, BRS3 of chickens (/spotted gars), which is orthologous to mouse bombesin receptor subtype-3 (BRS3), could be potently activated by GRP and NMB, demonstrating that both peptides are the endogenous ligands for chicken (/spotted gar) BRS3; (2) quantitative real-time PCR (qPCR) revealed that cGRPR is widely expressed in chicken tissues with abundant expression in the ovary, pancreas, proventriculus, spinal cord and brain, whereas cNMB, cNMBR and cBRS3 are mainly expressed in the brain and testes; (3) interestingly, qPCR, Western blot and immunostaining revealed that cGRP is predominantly expressed in the anterior pituitary and mainly localized to LH-cells, suggesting that cGRP is likely a novel pituitary hormone in chickens. In summary, our data help to uncover the roles of GRP, NMB and their receptors in birds, and provide the first persuasive evidence from an evolutionary prospective that in vertebrates, GRP and NMB are the endogenous ligands for BRS3, an orphan receptor that has puzzled endocrinologists for more than two decades.

Characterization of melanin-concentrating hormone (MCH) and its receptor in chickens: Tissue expression, functional analysis, and fasting-induced up-regulation of hypothalamic MCH expression.

Melanin-concentrating hormone (MCH) is a neuropeptide expressed in the brain and exerts its actions through interaction with the two known G protein-coupled receptors, namely melanin-concentrating hormone receptor 1 and 2 (MCHR1 and MCHR2) in mammals. However, the information regarding the expression and functionality of MCH and MCHR(s) remains largely unknown in birds. In this study, using RT-PCR and RACE PCR, we amplified and cloned a MCHR1-like receptor, which is named cMCHR4 according to its evolutionary origin, and a MCHR2 from chicken brain. The cloned cMCHR4 was predicted to encode a receptor of 367 amino acids, which shares high amino acid identities with MCHR4 of ducks (90%), western painted turtles (85%), and coelacanths (77%), and a comparatively low identity to human MCHR1 (58%) and MCHR2 (38%), whereas chicken MCHR2 encodes a putative C-terminally truncated receptor and is likely a pseudogene. Using cell-based luciferase reporter assays or Western blot, we further demonstrated that chicken (and duck) MCHR4 could be potently activated by chicken MCH1-19, and its activation can elevate calcium concentration and activate MAPK/ERK and cAMP/PKA signaling pathways, indicating an important role of MCHR4 in mediating MCH actions in birds. Quantitative real-time PCR revealed that both cMCH and cMCHR4 mRNA are expressed in various brain regions including the hypothalamus, and cMCH expression in the hypothalamus of 3-week-old chicks could be induced by 36-h fasting, indicating that cMCH expression is correlated with energy balance. Taken together, characterization of chicken MCH and MCHR4 will aid to uncover the conserved roles of MCH across vertebrates.