In vitro drug screening models derived from different PC12 cell lines for exploring Parkinson's disease based on electrochemical signals of catecholamine neurotransmitters.

Gold nanostructures and a Nafion modified screen-printed carbon electrode (Nafion/AuNS/SPCE) were developed to assess the cell viability of Parkinson's disease (PD) cell models. The electrochemical measurement of cell viability was reflected by catecholamine neurotransmitter (represented by dopamine) secretion capacity, followed by a traditional tetrazolium-based colorimetric assay for confirmation. Due to the  capacity to synthesize, store, and release catecholamines as well as their unlimited homogeneous proliferation, and ease of manipulation, pheochromocytoma (PC12) cells were used for PD cell modeling. Commercial low-differentiated and highly-differentiated PC12 cells, and home-made nerve growth factor (NGF) induced low-differentiated PC12 cells (NGF-differentiated PC12 cells) were included in the modeling. This approach achieved sensitive and rapid determination of cellular modeling and intervention states. Notably, among the three cell lines, NGF-differentiated PC12 cells displayed the enhanced neurotransmitter secretion level accompanied with attenuated growth rate, incremental dendrites in number and length that were highly resemble with neurons. Therefore, it was selected as the PD-tailorable modeling cell line. In short, the electrochemical sensor can be used to sensitively determine the biological function of neuron-like PC12 cells with negligible destruction and to explore the protective and regenerative impact of various substances on nerve cell model.

Effects of electroacupuncture on repairing neurological damage following ischemic stroke based on miR-381-mediated SDF-1/CXCR4 signaling pathway. / 基于miR-381调控SDF-1/CXCR4信号通路探讨电针对缺血性脑卒中后神经损伤修复的影响.

OBJECTIVES: To investigate the effects of electroacupuncture (EA) on the miR-381, leucine-rich repeat C4 protein (LRRC4), and downstream stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling pathway in rat model of ischemic stroke, and to explore the mechanism by which EA improves neurological damage following ischemic stroke. METHODS: Among 50 SPF male SD rats, 10 rats were randomly selected into a sham surgery group, and the remaining rats were used to establish the middle cerebral artery occlusion (MCAO) model. The 30 successfully modeled rats were randomly divided into a model group, an EA group, and an agonist group, with 10 rats in each group. The rats in the EA group received EA at "Baihui" (GV 20) and "Dazhui" (GV 14), with disperse-dense wave, a frequency of 2 Hz/10 Hz, and a current intensity of 1 mA, 30 min per session, once daily for a total of 14 days. The rats in the agonist group received miR-381 agonist injections into the lateral ventricle, with 10 µL per injection, every 7 days for a total of 2 injections. After intervention, ZeaLonga neurobehavioral deficit score was observed in each group. HE staining was performed to observe the morphological changes in the ischemic brain tissue of rats in each group. ELISA was used to measure the levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and nerve growth factor (NGF) in serum. Western blot was employed to detect the protein expression of LRRC4, SDF-1, CXCR4, and extracellular regulated protein kinase 1 (ERK1) in the ischemic brain tissue. Real-time PCR was utilized to assess the expression of miR-381 and LRRC4, SDF-1, CXCR4, ERK1 mRNA in the ischemic brain tissue. RESULTS: After intervention, the brain tissue showed disordered cell arrangement, reduced quantity, and significant interstitial edema, with numerous vacuoles in the model group. The pathological changes mentioned above were alleviated in the brain tissue of rats in the EA group and the agonist group. Compared with the sham surgery group, the rats in the model group exhibited increased ZeaLonga neurobehavioral deficit scores, elevated levels of serum TNF-α and IL-6 (P<0.01), and decreased serum NGF level (P<0.01)；the protein expression of SDF-1, CXCR4 and ERK1 in ischemic brain tissue was reduced (P<0.01), while LRRC4 protein expression was increased (P<0.01)；the expression of miR-381, as well as SDF-1, CXCR4 and ERK1 mRNA in ischemic brain tissue was decreased (P<0.01), while LRRC4 mRNA expression was increased (P<0.01). Compared with the model group, the rats in the EA group and the agonist group showed decreased ZeaLonga neurobehavioral deficit scores and reduced levels of serum TNF-α and IL-6 (P<0.05, P<0.01), and increased serum NGF levels (P<0.05, P<0.01); the protein expression of SDF-1, CXCR4 and ERK1 in ischemic brain tissue was increased (P<0.01), while LRRC4 protein expression was decreased (P<0.01)；the expression of miR-381, as well as SDF-1, CXCR4 and ERK1 mRNA in ischemic brain tissue was increased (P<0.05, P<0.01), while LRRC4 mRNA expression was decreased (P<0.01). CONCLUSIONS: EA at "Baihui" (GV 20) and "Dazhui" (GV 14) may promote the repair of neurological damage following ischemic stroke by up-regulating miR-381 to selectively inhibit LRRC4 expression, thereby activating the SDF-1/CXCR4 signaling pathway.

Effect of electroacupuncture at Neiguan (PC6) at different time points on myocardial ischemia reperfusion arrhythmia in rats.

OBJECTIVE: To observe the effects of electroacupuncture at Neiguan (PC6) at different time points on reperfusion arrhythmia (RA) after myocardial ischemia and reperfusion in rats, and to investigate the correlation of this protective effect with nerve growth factor (NGF), tyrosine kinase A (TrkA), tyrosine hydroxylase (TH), and norepinephrine (NE). METHODS：A total of 72 Sprague-Dawley male rats were randomly divided into six groups (n = 12 rats/group): normal group (Norm), sham operation group (Sham), ischemia reperfusion group (I/R), pre-ischemic electroacupuncture group (EAI), pre-reperfusion electroacupuncture group (EAII), post-reperfusion electroacupuncture group (EAIII). The myocardial ischemia-reperfusion injury (MIRI) model was induced by occlusion of left anterior descending coronary artery for 20 min followed by reperfusion for 40 min in rats. With no intervention in the Norm group and only threading without ligation in the Sham group. Electroacupuncture pre-treatment at 20 min/d for 7 d before ligation in the EAⅠ group, 20 min of electroacupuncture before reperfusion in the EAII group and 20 min of electroacupuncture after reperfusion in the EAIII group. The electrocardiogram (ECG) of each group was recorded throughout the whole process, and the success of the MIRI model was determined based on the changs of J-point and T-wave in the ECG. The arrhythmia score was used to record premature ventricular contractions, ventricular tachycardia and ventricular fibrillation during the reperfusion period to assess the reperfusion induced arrhythmias. The expression levels of NGF, TrkA, TH protein were measured by Western blot. Moreover, the expression levels of plasma and myocardial NE levels were detected by enzyme linked immunosorbent assay. RESULTS: The differences between Norm group and Sham group were not statistically significant in all indexes. Arrhythmia score, myocardial NGF, TrkA, TH, and NE expression were significantly higher in the I/R group compared with the Sham group. Arrhythmia score, myocardial NGF, TrkA, TH, and NE expression were significantly lower in each EA group compared with the I/R group. CONCLUSION: Electroacupuncture at Neiguan (PC6) at different time points can reduce the incidence and severity of reperfusion arrhythmias in rats. This protective effect is related to electroacupuncture regulating NGF, TrkA, TH, NE expression and reducing sympathetic hyperactivation.

Prevalence and Related Factors of Attention Deficit Hyperactivity Disorder in School-age Children With Atopic Dermatitis.

Context: Atopic dermatitis (AD) is a chronic inflammatory skin disease and commonly affects children. AD is associated with a high incidence of ADHD, the most common psychological and neurobehavioral disorder in children and adolescents. If clinicians don't identify ADHD and intervene early, preschool children can experience adverse effects. Objective: The study intended to investigate the prevalence of attention deficit hyperactivity disorder (ADHD) in preschool children with AD, analyze the associated factors, and provide insights for early identification of risk factors and the development of interventions to reduce the likelihood of ADHD occurrence. Design: The research team performed a prospective, observational, case-control study. Setting: The study took place at the Zhoushan branch of Ruijin Hospital at the Shanghai Jiaotong University School of Medicine in Zhoushan, Zhejiang, China. Participants: Participants were 80 school-aged children diagnosed with AD and admitted to the hospital between May 2019 and May 2023. Groups: Based on the presence or absence of ADHD, the research team divided the children into two groups: (1) the Simple AD group with 71 participants with AD only, and the AD + ADHD group, with 9 participants with AD and ADHD. Outcome Measures: The research team: (1) collected and analyzed participants' demographic and clinical data, including an assessment of the AD severity using the SCORing Atopic Dermatitis (SCORAD) scale and the presence of sleep disorders using the Children's Sleep Habits Questionnaire (CSHQ); (2) assessed the presence of ADHD using the Swanson, Nolan, and Pelham-IV rating scales (SNAP-IV); (3) analyzed the factors influencing the occurrence of ADHD in AD children, using univariate and multivariate logistic regression analysis. Results: Among the 80 school-age children with AD, 9 participants (11.25%) had received a diagnosis of ADHD. The AD + ADHD group's age (P < .001); body mass index (BMI), with P < .001; AD severity (P = .013); rate of sleep disorders (P = .001); and levels of serum interleukin 6 (IL-6), with (P < .001), interleukin 4 (IL-4), with (P < .001), and nerve growth factor (NGF), with (P < .001) were all significantly greater than those of the Simple AD group. The univariate logistic regression analysis indicated that age (P = .014), BMI (P = .024), AD severity (P = .022), sleep disorders (P = .042), and levels of IL-6 (P = .044), IL-4 (P = .045), and NGF (P = .046) were all significantly related to the development of ADHD in school-age children with AD. The multivariate logistic regression analysis revealed that sleep disorders (P = .018) and elevated levels of serum IL-6 (P = .032), IL-4 (P = .021), and NGF (P = .016 ) were independent risk factors for ADHD (OR = 2.651, 3.074, 2.686, 3.340). Conclusions: School-aged children with AD are more likely to develop ADHD, which is mainly associated with sleep disorders and elevated levels of serum IL-6, IL-4, and NGF. Clinicians should give attention to these risk factors and implement early interventions to reduce the risk of children with AD developing ADHD.

Efficacy and safety of short-term edaravone or nerve growth factor add-on therapy for alcohol-related brain damage: A multi-centre randomised control trial.

AIMS: To measure the therapeutic effect of an anti-oxidant, edaravone (EDV), or neurotrophic treatment with nerve growth factor (NGF) as an add-on treatment for alcohol-related brain damage (ARBD). DESIGN: Multi-centre, randomised, single-blinded, comparative clinical trial. SETTING AND PARTICIPANTS: One hundred and twenty-two inpatients recruited from seven hospitals in different regions of China, all diagnosed with ARBD and aged 18 to 65 years old; among them, only two were female. INTERVENTION AND COMPARATOR: Patients were randomly assigned to receive one of three treatments for 2 weeks: 40 patients, treatment as usual (TAU: a combination of intramuscular injections of thiamine, intravenous infusions of other B vitamins with vitamin C and oral medication with vitamin E per day); 40, EDV add-on treatment to TAU (intravenous infusion with 30 mg of EDV twice per day); and 42, NGF add-on treatment to TAU (intramuscular injection of 20 µg of NGF per day). The patients underwent follow-up for 24 weeks. MEASUREMENTS: The primary outcome was the composite score of executive cognitive function in the 2nd week after treatment, which was measured as the mean of the Z scores of the assessments, including the digit symbol substitute test (DSST), digit span memory test-forward (DST-F), digit span memory test-reverse (DST-R) and space span memory test (SSMT). The secondary outcomes were the composite scores at later follow-ups, the score for each component of cognitive function, global cognitive function measured by the Montreal Cognitive Assessment (MoCA), craving for alcohol and the safety of the therapies. FINDINGS: EDV add-on treatment improved the composite score of executive cognitive function better than TAU in the 2nd week (adjusted mean difference: 0.24, 95% confidence interval 0.06 to 0.41; P = 0.008), but NGF add-on treatment did not (adjusted mean difference: 0.07, 95% confidence interval -0.09 to 0.24; P = 0.502). During the follow-up to 24 weeks, EDV add-on treatment improved the composite score of executive cognitive function and DST-R score better than TAU (both P < 0.01). Craving for alcohol was relieved in all three groups. No severe adverse events were observed. CONCLUSION: The short-term addition of edaravone to supplementary therapy treatment for alcohol-related brain damage (ARBD) improved executive cognitive function in patients with ARBD.

NGF/PI3K/TRPV1 pathway mediates the regulation of visceral pain by acupuncture at "Shangjuxu" (ST37) in irritable bowel syndrome rats with chronic visceral hyperalgesia. / NGF/PI3K/TRPV1通路介导针刺"上巨虚"è°ƒèŠ‚è‚ æ˜“æ¿€ç»¼åˆå¾æ ¢æ€§å† è„ç—›æ•æ¨¡åž‹å¤§é¼ å† è„ç—›.

OBJECTIVES: To investigate the effect of manual acupuncture at "Shangjuxu"(ST37) on nerve growth factor(NGF)/phosphatidylinositol 3-kinase(PI3K)/transient receptor potential vanilloid 1(TRPV1) signaling pathway in rats with chronic visceral hyperalgesia of irritable bowel syndrome (IBS), so as to explore its underlying mechanism in treating IBS chronic visceral hyperalgesia. METHODS: IBS chronic visceral hyperalgesia model was established by colorectal dilation stimulation for 2 weeks for SD pups at 8 d after birth, which were fed until 8-week age after the stimulation. Then the verified successfully modeled adult rats were randomly divided into model, Shangjuxu, and non-acupoint groups, with 6 rats in each group, and 6 unmodeled rats were selected as normal group. On the next day of model evaluation, rats in the Shangjuxu group received acupuncture at right ST37 while rats in the non-acupoint group received acupuncture at the non-meridian and non-acupoint point in the right hypochondrium, both for 15 min, with manual twisting of mild reinforcing and reducing performed for 30 s at an interval of 5 min, once a day, for a total of 7 d. Abdominal withdrawal reflex(AWR) score was used to evaluate the degree of chronic visceral pain in rats. Western blot and real-time fluorescence quantitative PCR were used to detect the colonic protein and mRNA expressions of NGF, tropomyosin receptor kinase A (TrkA), PI3K and TRPV1. The positive expressions of PI3K and TRPV1 proteins in the colon of rats were detected by immunohistochemistry method. RESULTS: Compared with the normal group, AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1 in colon tissue, and positive expressions of PI3K and TRPV1 in colon tissue were significantly increased(P<0.05) in the model group. After intervention, compared with the model group, rats in the Shangjuxu group had reduced AWR scores corresponding to 4 pressure levels of 20, 40, 60 and 80 mm Hg, lower colonic mRNA and protein expressions of NGF, TrkA, PI3K and TRPV1, and decreased positive expressions of PI3K and TRPV1 in colon tissue(P<0.05), while there were no significant differences in the above indexes of the non-acupoint group. CONCLUSIONS: Manual acupuncture at ST37 can alleviate IBS chronic visceral hyperalgesia in rat and its analgesic effect may be related to regulating NGF/PI3K/TRPV1 signaling pathway.

Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

Qinghua decoction improves chronic nonbacterial prostatitis possibly regulating the chromogranin A/nerve growth factor/tyrosine kinase A signaling pathway mediated by inflammatory factors.

OBJECTIVE: To explore the mechanism by which Qinghua decoction regulates neuroendocrine inflammation in chronic nonbacterial prostatitis (CNP) model rats and provide an experimental basis for clinical treatment. METHODS: The rats were randomly divided into six groups: normal control, model, Qianlie Tongyu capsule, low-dose Qinghua decoction, medium-dose Qinghua decoction, and high-dose Qinghua decoction group with six rats in each group. Rats in each group were sacrificed on the 29th day of treatment, and blood and prostate tissues were collected. Serum levels of tumor necrosis factor-alpha and interleukins 1-beta, 6, 8, and 10 (TNF-α and IL-1ß, -6, -8, and -10, respectively) were measured using enzyme-linked immunosorbent assay. The pathological changes in the rat prostate tissue in each group were observed under a light microscope. The expression levels of chromogranin A (CgA), nerve growth factor (NGF), and tyrosine kinase A (TrkA) were detected using reverse transcription quantitative polymerase chain reaction. Western blotting was used to detect protein expression of CgA, NGF, and TrkA. RESULTS: In the model group, the prostate capsule membrane and stroma were significantly dilated with more inflammatory cells infiltrating the stroma and perivessels. TNF-α, IL-1ß, -6, and -8, CgA, NGF, and TrkA levels increased, whereas the content of IL-10 decreased, which was statistically significant compared to that in the normal control group ( < 0.05). Prostate tissue cells in the high-dose group were neatly arranged with no obvious inflammatory cell infiltration. When compared with the model group, the high-dose Qinghua decoction group showed a significant improvement in these indices ( < 0.05). CONCLUSION: Qinghua decoction led to inhibition of pathological changes in the prostate tissue of rats with CNP, regulation of inflammatory cytokine expression, and inhibition in the expression of CgA, NGF, and TrkA. This mechanism may be primarily related to regulation of the CgA/NGF/TrkA signaling pathway mediated by various inflammatory factors.

[Antidepressant mechanism of Shenling Kaixin Granules based on BDNF/TrkB/CREB pathway].

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1ß), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), B-cell lymphoma-2(Bcl-2)/Bcl-2 associated X protein(Bax) and caspase-3 in hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.

[Lianmei Qiwu Decoction relieves diabetic cardiac autonomic neuropathy by regulating AMPK/TrkA/TRPM7 signaling pathway].

This study investigated the effect of Lianmei Qiwu Decoction(LMQWD) on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient receptor potential melastatin 7(TRPM7) signaling pathway. The diabetic rats were randomly divided into a model group, an LMQWD group, an AMPK agonist group, an unloaded TRPM7 adenovirus group(TRPM7-N), an overexpressed TRPM7 adenovirus group(TRPM7), an LMQWD + unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N), an LMQWD + overexpressed TRPM7 adenovirus group(LMQWD+TRPM7), and a TRPM7 channel inhibitor group(TRPM7 inhibitor). After four weeks of treatment, programmed electrical stimulation(PES) was employed to detect the arrhythmia susceptibility of rats. The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE) staining and Masson staining. The immunohistochemistry, immunofluorescence, real-time quantitative polymerase chain reaction(RT-PCR), and Western blot were adopted to detect the distribution and expression of TRPM7, tyrosine hydroxylase(TH), choline acetyltransferase(ChAT), growth associated protein-43(GAP-43), nerve growth factor(NGF), p-AMPK/AMPK, and other genes and related neural markers. The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues, decrease the levels of TH, ChAT, and GAP-43 in the myocardium and ganglion, increase NGF, inhibit the expression of TRPM7, and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels. This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state, and its mechanism was associated with the activation of AMPK, further phosphorylation of TrkA, and inhibition of TRPM7 expression.

Omega-3 Polyunsaturated Fatty Acids Protect Neurological Function After Traumatic Brain Injury by Suppressing Microglial Transformation to the Proinflammatory Phenotype and Activating Exosomal NGF/TrkA Signaling.

The transformation of microglia to a pro-inflammatory phenotype at the site of traumatic brain injury (TBI) drives the progression of secondary neurodegeneration and irreversible neurological impairment. Omega-3 polyunsaturated fatty acids (PUFA) have been shown to suppress this phenotype transformation, thereby reducing neuroinflammation following TBI, but the molecular mechanisms are unknown. We found that Omega-3 PUFA suppressed the expression of disintegrin metalloproteinase (ADAM17), the enzyme required to convert tumor necrosis factor-α (TNF-α) to the soluble form, thereby inhibiting the TNF-α/NF-κB pathway both in vitro and in a mouse model of TBI. Omega-3 PUFA also prevented the reactive transformation of microglia and promoted the secretion of microglial exosomes containing nerve growth factor (NGF), activating the neuroprotective NGF/TrkA pathway both in culture and TBI model mice. Moreover, Omega-3 PUFA suppressed the pro-apoptotic NGF/P75NTR pathway at the TBI site and reduced apoptotic neuronal death, brain edema, and disruption of the blood-brain barrier. Finally, Omega-3 PUFA preserved sensory and motor function as assessed by two broad-spectrum test batteries. The beneficial effects of Omega-3 PUFA were blocked by an ADAM17 promotor and by a NGF inhibitor, confirming the pathogenic function of ADAM17 and the central neuroprotective role of NGF. Collectively, these findings provide a strong experimental basis for Omega-3 PUFA as a potential clinical treatment for TBI.

Electroacupuncture Increases the Expression of Gas7 and NGF in the Prefrontal Cortex of Male Rats with Focal Cerebral Ischemia.

OBJECTIVE: To investigate the potential mechanisms underlying the migration of endogenous neural stem cells (eNSCs) to the frontal cortex to differentiate into neurons, and to monitor the effect of electroacupuncture (EA) regulation of focal cerebral ischemia (FCI) in rats on the expression of growth arrest-specific protein 7 (Gas7) and nerve growth factor (NGF) in the prefrontal cortex (PFC). METHODS: Randomly, forty-eight male Sprague-Dawley rats were divided into four groups: Normal, Sham operation, Model, and EA. The right middle cerebral artery was embolized utilizing the thread-embolism technique. In the EA group, "Baihui" and "Zusanli" points were treated with electroacupuncture for 30 minutes, once a day, for 21 days. Nissl staining revealed the neuronal morphology of the PFC. Using immunohistochemistry and Western blot, the expression of Gas7 and NGF in the right PFC was observed. RESULTS: Nissl staining showed clear PFC neurons with centered nuclei and distinct nucleoli in the Normal and Sham groups. In the Model group, the PFC nuclei were distinctively smaller. The neuronal morphology in the EA group resembled that of the Normal group. Results from Western blot and immunohistochemistry were comparable. The expression of Gas7 and NGF in the Sham surgery group did not differ significantly from the Normal group. However, the expression of Gas7 and NGF in the Model group was significantly lower than in the Normal group. The expression of Gas7 and NGF was significantly higher in the EA group than in the Model group. CONCLUSIONS: EA can increase the expressions of Gas7 and NGF in the ischemic prefrontal cortex, which may be one of the mechanisms by which EA promotes the differentiation of eNSCs into neurons in the injured area.

[Protective mechanism of electroacupuncture combined with acellular nerve allografts on spinal ganglia in rats with sciatic nerve injury].

OBJECTIVE: To observe the effects of electroacupuncture (EA) combined with acellular nerve allograft (ANA) on the morphological structure of spinal ganglion cells and the protein expressions of nerve growth factor (NGF) and phosphorylated protein kinase B (p-Akt) in rats with sciatic nerve injury (SNI), so as to explore the protective mechanism of EA combined with ANA on spinal ganglia. METHODS: SPF male SD rats were randomly divided into normal, model, single ANA bridging (bridging) and EA + ANA (combination) groups， with 10 rats in each group. The SNI rat model was established by right sciatic nerve transection. Rats in the bridging group were bridged with ANA to the two broken ends of injured sciatic nerves. Rats in the combination group were treated with EA at "Yanglingquan" (GB34) and "Huantiao" (GB30) 2 d after ANA bridging, with dilatational wave, frequency of 1 Hz/20 Hz, intensity of 1 mA, 15 min/d, 7 d as a course of treatment for 4 consecutive courses. Sciatic function index (SFI) was observed by footprint test. Wet weight ratio of tibialis anterior muscle was calculated after weighing. Morphology of rat spinal ganglion cells was observed after Nissl staining. The protein expressions of NGF and p-Akt were detected by immunofluorescence and Western blot. RESULTS: Compared with the normal group, the SFI and wet weight ratio of tibialis anterior muscle were significantly decreased (P<0.05), the number of Nissl bodies in spinal ganglion cells was significantly reduced (P<0.05) with dissolution and incomplete structure， the protein expressions of NGF and p-Akt in ganglion cells were significantly decreased (P<0.05) in the model group. Following the interventions and in comparison with the model group, the SFI and the wet weight ratio of tibialis anterior muscle were significantly increased (P<0.05), the damage of Nissl bodies in ganglion cells was reduced and the number was obviously increased (P<0.05), and the protein expressions of NGF and p-Akt in ganglion cells were significantly increased (P<0.05) in the bridging and combination groups. Compared with the bridging group, the SFI and the wet weight ratio of tibialis anterior muscle were increased (P<0.05), the morphology of Nissl bodies in ganglion cells was more regular and the number was increased (P<0.05), the protein expressions of NGF and p-Akt in spinal ganglion cells were significantly increased (P<0.05) in the combination group. CONCLUSION: EA combined with ANA can improve the SFI and the wet weight ratio of tibialis anterior muscle in SNI rats, improve the morphology and structure of Nissl bodies in spinal ganglion cells, and increase the protein expressions of NGF and p-Akt in spinal ganglion, so as to play a protective role on spinal ganglia.

3, 3'-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway.

BACKGROUND: 3, 3'-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PURPOSE: This study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. METHODS: Cytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. RESULTS: DIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. CONCLUSION: DIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via "AhR-Nrf2/Arg-1" signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

Efficacy of acupoint application in patients with overactive bladder: A randomized, placebo-controlled pilot trial.

OBJECTIVE: Conventional treatments for alleviating the symptoms of Overactive bladder (OAB) have been reported to have limited efficacy and a high rate of side effects. Traditional Chinese medicine (TCM) has been used in Asia countries because of its low side effects and being easy to operate. To confirm the efficacy of acupoint application treatment for alleviating OAB symptoms, a randomized and placebo-controlled pilot trial was conducted in this study. METHODS: All participants were randomly allocated into a treatment group or control group, receiving either a "Dinggui" acupoint application or placebo treatment for 4 weeks. The outcome measures were OAB symptom scores (OABSS), OAB questionnaire (OAB-q) scores, and TCM syndrome scores. Urine nerve growth factor (NGF) level, NGF normalized to urine creatinine (NGF/Cr), and maximum flow rate (Qmax) were also measured to evaluate the OAB symptoms. RESULTS: In total, 69 participants were included with 34 in the treatment group and 35 in the placebo-treated group. Treatment with "Dinggui" acupoint application showed a statistically significant decrease in OABSS scores (8.10±1.54 to 3.67±1.77), OAB-q scores (61.43±13.93 to 38.13±15.42), and TCM syndrome scores (15.60±5.98 to 9.20±4.82). The NGF and NGF/Cr were also observed meaningful changes in a decrease from 379.68 to 136.17 pg/ml and from 0.30 to 0.16 pg/mg, respectively. The Qmax value showed a significant increase from 14.40 to 24.05 ml/s. CONCLUSIONS: Treatment with "Dinggui" acupoint application could be considered an effective and alternative therapy for OAB management. Further studies with larger sample sizes and longer treatment periods are needed to investigate.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury.

AIMS: Nerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. MAIN METHODS: We used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 µg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. KEY FINDINGS: In capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SIGNIFICANCE: P38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

Wogonin, a Bioactive Ingredient from Huangqi Guizhi Formula, Alleviates Discogenic Low Back Pain via Suppressing the Overexpressed NGF in Intervertebral Discs.

Purpose: To investigate whether wogonin, a key bioactive ingredient of Huangqi Guizhi formula (HQGZ formula; a Traditional Chinese Medicine herbal formula) according to network pharmacology analysis, has analgesic effects on discogenic low back pain (LBP) via regulating the nerve growth factor (NGF) in intervertebral discs (IVDs). Methods: The lumbar IVDs of rats were punctured to discogenic LBP, and the therapeutic effect of orally administrated HQGZ for discogenic LBP was investigated by measuring mechanical and cold allodynia and histological analysis. A network pharmacology analysis was conducted to search for bioactive ingredients from the HQGZ formula, and wogonin was suggested to be the most possible bioactive ingredient for LBP treatment. Subsequently, the analgesic effect of wogonin was investigated in the LBP model, and the gene expression of propain peptides in the bilateral dorsal root ganglia was analyzed using RT-PCR. Finally, immunohistochemical staining was performed for NGF expression of NGF in the IVDs to determine whether wogonin treatment would ameliorate NGF-induced LBP. Results: Oral administration of HQGZ for two weeks significantly ameliorated puncture-induced IVD degeneration (IDD) and LBP. In addition, the network pharmacology analysis revealed that wogonin, quercetin, and kaempferol were the potential candidate components of HQGZ for LBP treatment. Furthermore, we proved that wogonin had significant analgesic effects in the LBP model. Finally, wogonin was demonstrated to suppress the upregulated NGF in the IVD and ameliorate NGF-induced LBP in rats. Conclusions: The HQGZ formula has significant analgesic effects for LBP. In addition, the bioactive ingredient of wogonin was extracted from HQGZ and ameliorated LBP by suppressing the overexpressed NGF in degenerated IVDs. Therefore, wogonin has potential to be alternative treatment for LBP in clinical.

Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

In brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

Celastrus paniculatus oil ameliorates NF-KB mediated neuroinflammation and synaptic plasticity in the scopolamine-induced cognitive impairment rat model.

BACKGROUND AND AIM: Traditionally, Celastrus paniculatus Willd. (CP) oil has been utilized as a tranquilizer and memory enhancer. The present study investigated the neuropharmacological activity and efficacy of CP oil in ameliorating scopolamine-induced cognitive impairment in rats. EXPERIMENTAL PROCEDURE: Cognitive deficiency was induced in rats by administration of scopolamine (2 mg/kg intraperitoneal injection) for a period of 15 days. Donepezil served as a reference drug and CP oil was tested as both preventive and curative treatments. Animals' behaviour was assessed through the Morris water maze (MWM), novel object preference (NOR), and conditioned avoidance (CA) tests. Oxidative stress parameters, bioamine concentration (dopamine, noradrenaline, and 5-hydroxytryptamine), nerve growth factor (NGF), interleukin-6 (IL-6), nuclear factor kappa B (NF-кB), and tumor necrosis factor-alpha (TNFα) were estimated. Synaptophysin immunohistochemistry was performed. RESULTS: Our results showed that CP oil ameliorated behavioural deficits. It reduced latency to find a hidden platform in MWM. Reduced novel object exploration time and discrimination index (p < 0.05) in the NOR. Reduced step-down latency and normalized conditioned avoidance response (p < 0.001) in the CA test. CP oil increased dopamine, serotonin, norepinephrine, superoxide dismutase (SOD), glutathione, and catalase levels. It decreased malondialdehyde (MDA), acetylcholinesterase activity, IL-6, NF-кB (P < 0.001), TNFα, and NGF levels. Treatment showed approximate typical reactivity to synaptophysin. CONCLUSION: Our data is suggestive that CP oil treatment improves behavioural test outcomes, increases biogenic amine concentration, and decreases acetylcholinesterase activity, and neuroinflammatory biomarkers. It also restores synaptic plasticity. It thus improves cognitive functions against scopolamine-induced amnesia in rats by improving cholinergic function.

Stimulation of functional recovery via neurorepair mechanisms by the traditional Japanese Kampo medicine, Ninjin'yoeito, and physical exercise in a rat ischemic stroke model.

ETHNOPHARMACOLOGICAL RELEVANCE: Ninjin'yoeito (NYT), a traditional Japanese Kampo medicine consisting of 12 herbs, has been reported to improve cognitive dysfunction, depression, and neurological recovery in patients with neurovascular diseases such as Alzheimer's disease and stroke. Several studies have reported that the NYT components exert neurotrophic, neurogenic, and neuroprotective effects. In addition, exercise enhances neuroprotection and functional recovery after stroke. Rehabilitative exercises and pharmacological agents induce neurophysiological plasticity, leading to functional recovery in stroke patients. These reports indicate that NYT treatment and exercise may promote functional recovery following stroke through their beneficial effects. However, no study has determined the effects of NYT and the possible mechanisms of neurorepair and functional recovery after stroke. AIM OF THE STUDY: This study aimed to investigate the combined effects of NYT and exercise on neuroprotection and functional recovery and the underlying mechanisms in a rat ischemic stroke model. MATERIALS AND METHODS: Stroke was induced with 60-min middle cerebral artery occlusion (MCAO) followed by reperfusion in adult male Sprague-Dawley rats. After stroke, the rats were assigned to four groups: ischemia reperfusion (IR), NYT, exercise (Ex), and NYT + Ex. NYT-treated rats were fed a diet containing 1% NYT one day after stroke. Exercise was performed using a motorized treadmill for 5 days a week (8-15 m/min, 20 min/day), starting 3 days after stroke. The NYT treatment and exercise were continued for 4 weeks after the stroke. Infarct volume, neurological deficits, sensorimotor functions, expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase A (TrkA) and B (TrkB), caspase-3 activity, and the p-Akt/Akt ratio were examined by immunohistochemistry and western blotting. RESULTS: Compared to the IR group, all treated groups indicated reduced infarct volumes. The NYT + Ex group showed significantly improved waking time and beam walking score compared with the IR group. The expression of NGF/TrkA/p-TrkA and BDNF/TrkB was significantly increased in the NYT + Ex group compared with those in the IR group, whereas the number of caspase-3 positive cells around the lesion was significantly lower in the NYT + Ex group than in the IR group. In addition, the ratio of p-Akt/Akt was significantly higher in the NYT + Ex group than in the IR group. CONCLUSIONS: This study suggests that NYT in combination with exercise provides neuroprotective effects and improves sensorimotor function by stimulating NGF/TrkA and BDNF/TrkB, and by activating the Akt pathway in ischemic stroke of rats. NYT may be an effective adjunctive agent in post-stroke rehabilitation.