Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway.

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS: EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

Environmental enrichment accentuates glucose-induced feeding suppression and glial cell line-derived neurotrophic factor gene expression in the hypothalamus of mice.

The hypothalamus controls food intake by integrating nutrient signals, of which one of the most important is glucose. Consequently, impairments in hypothalamic glucose-sensing mechanisms are associated with hyperphagia and obesity. Environmental enrichment (EE) is an animal housing protocol that provides complex sensory, motor, and social stimulations and has been proven to reduce adiposity in laboratory mice. However, the mechanism by which EE promotes adiposity-suppressing effect remains incompletely understood. Neurotrophic factors play an important role in the development and maintenance of the nervous system, but they are also involved in the hypothalamic regulation of feeding. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are expressed in the hypothalamus and their expression is stimulated by glucose. EE is associated with increased expression of Bdnf mRNA in the hypothalamus. Therefore, we hypothesized that EE potentiates the anorectic action of glucose by altering the expression of neurotrophic factor genes in the hypothalamus. Male C57BL/6 mice were maintained under standard or EE conditions to investigate the feeding response to glucose and the associated expression of feeding-related neurotrophic factor genes in the hypothalamus. Intraperitoneal glucose injection reduced food intake in both control and EE mice with a significantly greater reduction in the EE group compared to the control group. EE caused a significantly enhanced response of Gdnf mRNA expression to glucose without altering basal Gdnf mRNA expression and Bdnf mRNA response to glucose. These findings suggest that EE enhances glucose-induced feeding suppression, at least partly, by enhancing hypothalamic glucose-sensing ability that involves GDNF.

Intravenously engrafted human multilineage-differentiating stress-enduring (Muse) cells rescue erectile function after rat cavernous nerve injury.

OBJECTIVE: To evaluate the effect of intravenous administration of human multilineage-differentiating stress-enduring (Muse) cells on rat postoperative erectile dysfunction (ED) with cavernous nerve (CN) injury without an immunosuppressant. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomised into three groups after CN crush injury. Either human-Muse cells, non-Muse mesenchymal stem cells (MSCs) (both 1.0 × 105 cells), or vehicle was infused intravenously at 3 h after CN injury without immunosuppressant. Erectile function was assessed by measuring intracavernous pressure (ICP) and arterial pressure (AP) during pelvic nerve electrostimulation 28 days after surgery. At 48 h and 28 days after intravenous infusion of Muse cells, the homing of Muse cells and non-Muse MSCs was evaluated in the major pelvic ganglion (MPG) after CN injury. In addition, expressions of C-X-C motif chemokine ligand (Cxcl12) and glial cell line-derived neurotrophic factor (Gdnf) in the MPG were examined by real-time polymerase chain reaction. Statistical analyses and comparisons among groups were performed using one-way analysis of variance followed by the Tukey test for parametric data and Kruskal-Wallis test followed by the Dunn-Bonferroni test for non-parametric data. RESULTS: The mean (SEM) ICP/AP values at 28 days were 0.51 (0.02) in the Muse cell group, 0.37 (0.03) in the non-Muse MSC group, and 0.36 (0.04) in the vehicle group, showing a significant positive response in the Muse cell group compared with the non-Muse and vehicle groups (P = 0.013 and P = 0.010, respectively). In the MPG, Muse cells were observed to be engrafted at 48 h and expressed Schwann cell markers S100 (~46%) and glial fibrillary acidic protein (~24%) at 28 days, while non-Muse MSCs were basically not engrafted at 48 h. Higher gene expression of Cxcl12 (P = 0.048) and Gdnf (P = 0.040) was found in the MPG of the Muse group than in the vehicle group 48 h after infusion. CONCLUSION: Intravenously engrafted human Muse cells recovered rat erectile function after CN injury in a rat model possibly by upregulating Cxcl12 and Gdnf.

Modulating Effects of Zingiberaceae Phenolic Compounds on Neurotrophic Factors and Their Potential as Neuroprotectants in Brain Disorders and Age-Associated Neurodegenerative Disorders: A Review.

The Zingiberaceae family possess various phenolic compounds that have significant systemic bioactivities in the brain, including in age-related neurodegenerative diseases. Neurotrophins are growth factors that protect neurons from oxidative stress, and dysregulation of the neurotrophic system may result in neurocognitive disease. Phenolic compounds from the Zingiberaceae family have been used in traditional and complementary medicine (TCM) to improve cognitive functions. These compounds may affect the expression of neurotrophic agents, but their underlying molecular mechanisms require further investigation. Therefore, the goal of this review is to determine the expression and functional roles of phenolic compounds from the Zingiberaceae family in brain disorders and age-related neurodegenerative disorders. While previous studies have proposed various mechanisms for the neuroprotective activity of these compounds, their precise mechanism of action remains complex and poorly understood. Despite some promising findings, there are still shortcomings in the therapeutic use of these herbs, and current interventions involving the Zingiberaceae family appear to be clinically insufficient. This article aims to summarize recent discoveries of phenolic compounds from several Zingiberaceae family members and their use as neuroprotectants and provide the first review of evidence-linked neuroprotective activity of bioactive ingredients from prominent members of the Zingiberaceae family.

Age-dependent effects of resveratrol in hypothalamic astrocyte cultures.

OBJECTIVES: The hypothalamus plays critical roles in maintaining brain homeostasis and increasing evidence has highlighted astrocytes orchestrating several of hypothalamic functions. However, it remains unclear how hypothalamic astrocytes participate in neurochemical mechanisms associated with aging process, as well as whether these cells can be a target for antiaging strategies. In this sense, the aim of this study is to evaluate the age-dependent effects of resveratrol, a well-characterized neuroprotective compound, in primary astrocyte cultures derived from the hypothalamus of newborn, adult, and aged rats. METHODS: Male Wistar rats (2, 90, 180, and 365 days old) were used in this study. Cultured astrocytes from different ages were treated with 10 and 100 µM resveratrol and cellular viability, metabolic activity, astrocyte morphology, release of glial cell line-derived neurotrophic factor (GDNF), transforming growth factor ß (TGF-ß), tumor necrosis factor α (TNF-α), interleukins (IL-1ß, IL-6, and IL-10), as well as the protein levels of Nrf2 and HO-1 were evaluated. RESULTS: In vitro astrocytes derived from neonatal, adults, and aged animals changed metabolic activity and the release of trophic factors (GDNF and TGF-ß), as well as the inflammatory mediators (TNF-α, IL-1ß, IL-6, and IL-10). Resveratrol prevented these alterations. In addition, resveratrol changed the immunocontent of Nrf2 and HO-1. The results indicated that the effects of resveratrol seem to have a dose- and age-associated glioprotective role. CONCLUSION: These findings demonstrate for the first time that resveratrol prevents the age-dependent underlying functional reprogramming of in vitro hypothalamic astrocytes, reinforcing its antiaging activity, and consequently, its glioprotective role.

[Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats].

OBJECTIVE: To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. METHODS: Thirty male SD rats were randomly divided into sham operation, model, and acupuncture groups, with 10 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery (MCAO) for 1 h, followed by reperfusion. After modeling, rats in the acupuncture group received manual acupuncture stimulation of "Dazhui" (GV14), "Baihui"(GV20), "Shuigou" (GV26), bilateral "Zusanli" (ST36) and "Fengchi" (GB20) by twirling the needles rapidly for 10 s/acupoint every 10 min, with the needles retained for 20 min. The treatment was conducted once daily for successive 7 days. The neurological function was evaluated according to Longa's method. The state of CIRI was observed after Nissl staining, and the expression levels of Iba-1, iNOS, Arg1, BDNF, GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining. The contents of TNF-α, IL-6 and IL-10 in the ischemic tissue were assayed by ELISA. The protein expression levels of BDNF, GDNF, TLR4, MyD88 and NF-κB in the ischemic tissues were detected by Western blot. RESULTS: The neurological deficit score on the 24 h and 7th day was considerably higher in the model group than in the sham operation group (P<0.01), and evidently lower on the 7th day in the acupuncture group than in the model group (P<0.01). The number of NeuN positive cells，the area of immunofluorescence dual labelling of Arg1, BDNF and GDNF positive staining, IL-10 content, BDNF and GDNF protein expressions were significantly decreased (P<0.01), and the immunofluorescence dual labelling area of Iba-1 and iNOS, TNF-α and IL-6 contents, the pretein expression levels of TLR4, MyD88 and NF-κB considerably increased (P<0.01) in the model group relevant to the sham operation group. In contrast to the model group, the acupuncture group had a significant increase in the number of NeuN positive cells, the immunofluorescence dual labelling area of Arg1, BDNF and GDNF positive staining, IL-10 content, and BDNF and GDNF protein expressions (P<0.05, P<0.01), and an evident decrease in Iba-1 and iNOS positive staining, contents of TNF-α and IL-6, and the protein expression levels of TLR4, MyD88 and NF-κB (P<0.01, P<0.05). Nissl staining showed a marked reduction in the number of neurons, the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group, which was relatively milder in the acupuncture group. CONCLUSION: Acupuncture intervention can improve neurological function in CIRI rats, which may be related to its effects in regulating the polarization of microglia, reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain, inhibiting TLR4/MyD88/NF-κB signaling pathway.

The effects of acrylamide-mediated dorsal root ganglion neurons injury on ferroptosis.

Acrylamide (ACR) is a water-soluble chemical applied in industrial and laboratory processes. The neurotoxicity induced by acrylamide involves both peripheral and central nervous system. Hence, there is a growing urgency to investigate the mechanisms of acrylamide-induced neurotoxicity and search novel therapeutic target for the nerve repair. The effects of ACR on the proliferation, reactive oxygen species (ROS) and iron production of dorsal root ganglia (DRG) neurons and Schwann cells were determined. 5-Ethynyl-2'-deoxyuridine (EDU) staining and transwell assay were applied to detect the proliferation and migration capacity of DRG cells. Ferrostatin-1 (Fer-1) was used to suppress ferroptosis induced by ACR. RT-PCR analysis was performed to examine the expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF). Moreover, Iron, ROS, malondialdehyde (MDA) and glutathione (GSH) contents were measured to reveal the regulation of ferroptosis in ACR-related nerve injury. ACR inhibited the proliferation and migration of DRG neurons and the supplementation of Fer-1 reversed the effects induced by ACR. Besides, the treatment of Fer-1 effectively increased the expression of NGF, BDNF, VEGF and GDNF. Furthermore, ACR increased the iron level, MDA and ROS contents while inhibited the level of GSH. It was unveiled that ACR attenuated the proliferation, migration and neuron repair of DRG neurons through regulating ferroptosis. The modulation of ferroptosis might be a promising therapeutic strategy and provide references for future treatment of acrylamide-induced nerve damage.

[Effect of electroacupuncture on gastrointestinal motility in rats with slow transit constipation based on GDNF methylation modification].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Dachangshu"(BL25) on the intestinal transit function， expression level of glial cell-derived neurotrophic factor (GDNF) and methylation level of GDNF gene promoter region in colon tissue of rats with slow transit constipation (STC)， so as to explore its mechanisms underlying improvement of STC. METHODS: Male Sprague-Dawley (SD) rats were randomized into control， saline， model and EA groups (n=16 in each group). The STC model was replicated by gavage of compound diphenoxylate suspension (10 mL· kg-1· d-1) for 28 days. Rats of the saline group received the same dose of normal saline via gavage. EA (2 Hz/15 Hz， 0.1-1 mA) was applied to bila-teral ST25 and BL25 for 15 min， once daily for 14 days. The intestinal transmission function (the intestinal propulsion rate) was assessed by recording the first black grain stool discharge time and the number and weight of the discharged stool grains in 30 min after gavage of the activated carbon suspension (1 mL/100 g， 150 g/L). The score of fecal trait and the weight of stool within 24 h were recorded. The ultrastructural changes of Cajal interstitial cells in the colon tissue were observed by transmission electron microscope. The expression levels of GDNF protein and mRNA in the colon tissue were detected by using Western blot and real-time fluorescent quantitative PCR， separately， and changes of methylation level in the promoter region of GDNF gene detected by using Bisulfite sequencing method. RESULTS: Compared with the control group， the time of the 1st black stool grain discharging was obviously prolonged， and the number and weight of the discharged black stool grains were significantly decreased in the mo-del group (P<0.05)， suggesting a success of STC. The weight and trait score of stool in 24 h， intestinal propulsive rate， and the expression levels of GDNF protein and mRNA were significantly lower in the model group than in the control group (P<0.01， P<0.05). After EA， the weight and trait score of stool within 24 h， intestinal propulsive rate，and the expression levels of GDNF protein and mRNA were significantly increased in the EA group in contrast to the model group (P<0.01，P<0.05). The total CpGs methylation level of GDNF gene in colon tissue was considerably higher in the model group than in the control group (P<0.05)， and markedly lower in the EA group than in the model group (P<0.05). No significant differences were found between the control and saline groups in all the above-mentioned indexes (P>0.05). CONCLUSION: EA of back-shu and front-mu acupoints can effectively improve symptoms of constipation and intestinal transport function in STC rats， which may be related to its function in up-regulating the expression of GDNF and down-regulating the methylation level in the promoter region of GDNF gene in colon tissue.

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

DHI Increases the Proliferation and Migration of Schwann Cells Through the PI3K/AKT Pathway and the Expression of CXCL12 and GDNF to Promote Facial Nerve Function Repair.

The facial nerve is one of the vulnerable nerves in otolaryngology. Repair and recovery of facial nerve injury have a high priority in clinical practice. The proliferation and migration of Schwann cells are considered of great significance in the process of nerve injury repair. Danhong injection (DHI), as a common drug for cardiovascular and cerebrovascular diseases, has been fully certified in neuroprotection research, but its role in facial nerve injury is still not clear. Our study found that DHI can promote the proliferation and migration of RSC96 cells, a Schwann cell line, and this effect is related to the activation of the PI3K/AKT pathway. LY294002, an inhibitor of PI3K, inhibits the proliferation and migration of RSC96 cells. Further studies have found that DHI can also promote the expression of CXCL12 and GDNF at gene and protein levels, and CXCL12 is, while GDNF is not, PI3K/AKT pathway-dependent. Animal experiments also confirmed that DHI could promote CXCL12 and GDNF expression and promote facial nerve function recovery and myelin regeneration. In conclusion, our in vitro and in vivo experiments demonstrated that DHI could promote the proliferation and migration of Schwann cells through the PI3K/AKT pathway and increase the expression of CXCL12 and GDNF to promote facial nerve function repair.

Genetic Background Influences Severity of Colonic Aganglionosis and Response to GDNF Enemas in the Holstein Mouse Model of Hirschsprung Disease.

Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.

Mechanistic Insight from Preclinical Models of Parkinson's Disease Could Help Redirect Clinical Trial Efforts in GDNF Therapy.

Parkinson's disease (PD) is characterized by four pathognomonic hallmarks: (1) motor and non-motor deficits; (2) neuroinflammation and oxidative stress; (3) pathological aggregates of the α-synuclein (α-syn) protein; (4) neurodegeneration of the nigrostriatal system. Recent evidence sustains that the aggregation of pathological α-syn occurs in the early stages of the disease, becoming the first trigger of neuroinflammation and subsequent neurodegeneration. Thus, a therapeutic line aims at striking back α-synucleinopathy and neuroinflammation to impede neurodegeneration. Another therapeutic line is restoring the compromised dopaminergic system using neurotrophic factors, particularly the glial cell-derived neurotrophic factor (GDNF). Preclinical studies with GDNF have provided encouraging results but often lack evaluation of anti-α-syn and anti-inflammatory effects. In contrast, clinical trials have yielded imprecise results and have reported the emergence of severe side effects. Here, we analyze the discrepancy between preclinical and clinical outcomes, review the mechanisms of the aggregation of pathological α-syn, including neuroinflammation, and evaluate the neurorestorative properties of GDNF, emphasizing its anti-α-syn and anti-inflammatory effects in preclinical and clinical trials.

Differentiation of the 50B11 dorsal ganglion cells into NGF and GDNF responsive nociceptor subtypes.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.

Crocin inhibits the activation of mouse hepatic stellate cells via the lnc-LFAR1/MTF-1/GDNF pathway.

Crocin is the main monomer of saffron, which is a momentous component of traditional Chinese medicine Lang Qing A Ta. Here, we tried to probe into the role of crocin in liver fibrosis. Hematoxylin-eosin staining and Sirius Red staining were used to observe the pathological changes of liver tissues. After hepatic stellate cells (HSCs) were isolated from liver tissues, lnc-LFAR1, MTF-1, GDNF, and α-SMA expressions were detected by qRT-PCR and western blot. Immunohistochemistry and immunofluorescence were used to detect α-SMA expression. Chromatin immunoprecipitation was used to analyze the binding of MTF-1 to the GDNF promoter. Moreover, the dual-luciferase reporter gene, RNA pull-down, and RNA immunoprecipitation were used to clarify the interaction between MTF-1 and GDNF, lnc-LFAR1 and MTF-1. The degree of liver fibrosis was more severe in the mice from the liver fibrosis model, while the liver fibrosis was alleviated by the injection of crocin. lnc-LFAR1, GDNF, and α-SMA were up-regulated, and MTF-1 was down-regulated in liver fibrosis tissues and cells, while these trends were reversed after the injection of crocin. Besides, lnc-LFAR1 negatively regulated MTF-1 expression, and positively regulated GDNF and α-SMA expressions, and MTF-1 was enriched in the promoter region of GDNF. Furthermore, the cellular direct interactions between MTF-1 and GDNF, lnc-LFAR1 and MTF-1 were verified. In vivo experiments confirmed the relief of crocin on liver fibrosis. Our research expounded that crocin restrained the activation of HSCs through the lnc-LFAR1/MTF-1/GDNF axis, thereby ameliorating liver fibrosis.

Epidural Stimulation Combined with Triple Gene Therapy for Spinal Cord Injury Treatment.

The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.

Review on significance of GDNF, PTCH1 and RNF213 in chromophobe renal cell carcinoma illustrated by the case of 71-years-old man.

Here we review the role of GDNF, PTCH1, RNF213 illustrated by a case of renal cell carcinoma, chromophobe type (pT2a 8th pTNM edition) of the left kidney of 71-year-old man. Status of potential hotspots in 409 tumor genes were studied by means of next generation sequencing (NGS) technology (IonTorrent - Thermo Fisher Scientific, USA) using Ion AmpliSeq™ Comprehensive Cancer Panel. Next-generation sequencing (NGS) revealed mutations of GDNF (NM_001190468: c. 328C>T, p.R110W, allelic frequency 46%), PTCH1 (NM_001083607:c. 2969C

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Berberine ameliorates non-steroidal anti-inflammatory drugs-induced intestinal injury by the repair of enteric nervous system.

The study was to detect the role of GDNF, PGP9.5 (a neuronal marker), and GFAP (EGCs' marker) in the mechanism of non-steroidal anti-inflammatory drugs (NSAIDs) related to intestinal injury and to clarify the protective effect of berberine in the treatment of NSAID-induced small intestinal disease. Forty male SD rats were divided randomly into five groups (A-E): Group A: control group; Group B: model group received diclofenac sodium 7.5 mg/(kg*day) for 5 days; Group C-E: berberine low, medium and high dose groups were treated by 7.5 mg/(kg*day) diclofenac sodium for 5 days then received berberine 25 mg/(kg*day), 50 mg/(kg*day), and 75 mg/(kg*day), respectively, between the sixth and eighth day. Intestinal mucosa was taken on the ninth day to observe the general, histological injuries, and to measure the intestinal epithelial thickness. Then, immunohistochemistry was performed to detect the expression of PGP9.5 and GFAP, and Western blot was performed to detect GDNF expression. The histological score and the general score in the model group were, respectively, 5.75 ± 1.04 and 4.83 ± 0.92. Scores in berberine medium and high berberine group were lower compared with the model group (P < 0.05). The intestinal epithelial thickness in the model group was lower than in the control group and the berberine groups (P < 0.05). PGP9.5, GFAP, and GDNF content in the model group and the three berberine groups were significantly lower than in the control groups (P < 0.05). PGP9.5, GFAP, and GDNF content in the control group and the three berberine groups were higher compared with the model groups (P < 0.05). Berberine can protect the intestinal mucosa of NSAID users, and the mechanism is associated with the reparation of the enteric nervous system via upregulating the expression of PGP9.5, GFAP, and GDNF.

Electroacupuncture Therapy Ameliorates Motor Dysfunction via Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic neuron loss in the substantia nigra. However, specific sensory stimulation via electroacupuncture (EA) therapy may attenuate this loss by promoting the expression of endogenous neurotrophic factors in a manner similar to physical therapy. We investigated the potential protective effects of EA on dopaminergic neurons in a mouse model of PD and whether these effects are associated with the promotion of endogenous brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Mouse models of PD were generated using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine. Motor performance was assessed using behavioral tests, and Western blot experiments, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemical assays were performed. In both mouse models, EA treatment ameliorated motor impairments and dopaminergic neuron loss; these changes were accompanied by increases in BDNF and GDNF. In the MPTP group, EA treatment improved motor dysfunction by attenuating dopaminergic neuron loss in the substantia nigra, similar to the effects of levodopa. EA treatment significantly upregulated BDNF and GDNF expression in both the substantia nigra and striatum. Moreover, EA treatment induced the expression of cAMP response element binding protein (CREB) as well as Akt and Pitx3 in dopaminergic neurons in the substantia nigra. However, levodopa treatment did not induce BDNF/GDNF activation or related signaling factors. Thus, EA therapy may exert protective effects on dopaminergic neurons by upregulating the expression of BDNF, GDNF, and related signaling factors, thereby improving motor function. Hence, EA may represent an effective adjuvant therapy for motor deficits in patients with PD.

Effects of lithium and valproate on behavioral parameters and neurotrophic factor levels in an animal model of mania induced by paradoxical sleep deprivation.

The present study aimed to evaluate the effects of treatment with lithium (Li) and valproate (VPA) on behaviors and brain BDNF, NGF, NT-3, NT-4 and GDNF levels in mice submitted to paradoxical sleep deprivation (PSD), which induces an animal model of mania. Male C57BL/6J mice received an intraperitoneal (i.p.) injection of saline solution (NaCl 0.09%, 1 ml/kg), Li (47.3 mg/kg, 1 ml/kg) or VPA (200 mg/kg, 1 ml/kg) once a day for seven days. Animals were randomly distributed into six groups (n = 10 per group): (1) Control + Sal; (2) Control + Li; (3) Control + VPA; (4) PSD + Sal; (5) PSD + Li; or (6) PSD + VPA. Animals were submitted to 36 h of PSD, and then, they were submitted to the open field test. The frontal cortex and hippocampus were dissected from the brain. The manic-like behaviors in the mice were analyzed. Treatment with Li and VPA reversed the behavioral alterations induced by PSD. PSD decreased BDNF, NGF, and GDNF levels in the frontal cortex and hippocampus of mice. The administration of Li and VPA protected the brain against the damage induced by PSD. However, PSD and the administration of Li and VPA did not affect the levels of NT-3 and NT-4 in either brain structure evaluated. In conclusion, the PSD protocol induced manic-like behavior in rats and induced alterations in neurotrophic factor levels. It seems that neurotrophic factors and sleep are essential targets to treat BD.