Glutamate overactivity in basal ganglia critically contributes to the exacerbation of dopaminergic neuron degeneration in Parkinson's disease (PD). Activation of group II metabotropic glutamate receptors (mGlu2/3 receptors), which can decrease excitatory glutamate neurotransmission, provides an opportunity to slow down the degeneration of the dopaminergic system. However, the roles of mGlu2/3 receptors in relation to PD pathology were partially recognized. By using mGlu2/3 receptors agonist (LY354740) and mGlu2/3 receptors antagonist (LY341495) in mice challenged with different cumulative doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we demonstrated that systemic injection of LY354740 reduced the level of extracellular glutamate and the extent of nigro-striatal degeneration in both acute and sub-acute MPTP mice, while LY341495 amplified the lesions in sub-acute MPTP mice only. LY354740 treatment improved behavioral dysfunctions mainly in acute MPTP mice and LY341495 treatment seemed to aggravate motor deficits in sub-acute MPTP mice. In addition, ligands of mGlu2/3 receptors also influenced the total amount of glutamate and dopamine in brain tissue. Interestingly, compared with normal mice, MPTP-treated mice abnormally up-regulated the expression of polo-like kinase 2 (PLK2)/pS129 α-synuclein and phosphorylation of Fyn/N-methyl-D-aspartate receptor subunit 2A/2B (GluN2A/2B). Both acute and sub-acute MPTP mice treated with LY354740 dose-dependently reduced all the above abnormal expression. Compared with MPTP mice treated with vehicle, mice pretreated with LY341495 exhibited much higher expression of p-Fyn Tyr416/p-GluN2B Tyr1472 and PLK2/pS129 α-synuclein in sub-acute MPTP mice models. Thus, our current data indicated that mGlu2/3 receptors ligands could influence MPTP-induced toxicity, which supported a role for mGlu2/3 receptors in PD pathogenesis.

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

The lignan-rich fraction (SWR) of Sambucus Williamsii Ramulus, a folk herbal medicine in China for treatment of bone diseases, has previously reported to exert protective effects on bone without exerting uterotrophic effects in ovariectomized (OVX) mice. The aim of the present study was to identify the potential metabolites and the associated metabolic pathways that contribute to the beneficial effects of SWR on bone in vivo. Aged female Sprague Dawley rats (9 months old) were either sham-operated or ovariectomized for 12 weeks, before receiving treatment for another 12 weeks with the following treatment groups (n = 12 each): vehicle (Sham), vehicle (OVX), Premarin (130 μg/kg) or low (57 mg/kg), medium (114 mg/kg), and high (228 mg/kg) doses of SWR. The results showed that SWRH significantly suppressed bone loss, improved bone micro-architecture and increased bone strength on tibia without stimulating uterus weight gain in OVX rats. Premarin exerted similar bone protective effects as SWRH but elicited uterotrophic effects in OVX rats. The metabolic profiles of serum samples were analyzed by using ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry and gas chromatography time-of flight mass spectrometry, and the metabolites that were significantly altered were identified by multivariate statistical analysis. Our study indicated that SWRH effectively restored the changes of 26 metabolites induced by estrogen-deficiency in OVX rats, which related to lipids, amino acids, tryptophan metabolisms, and anti-oxidative system. A subsequent validation showed that the serum level of superoxide dismutase and catalase were indeed up-regulated, while the serotonin level in a tryptophan hydroxylase 1 (TPH1) high expressing cells (rats RBL-2H3 cells) was down regulated after treatment with SWR. The results also suggested that the gut-microbiota may play an important role on the bone protective effects of SWR. The current study provides insight for understanding the unique mechanism of actions of SWR that might be involved in achieving bone protective effects in vivo.

Acetylglutamine (NAG) is the derivative of glutamine, which is the richest free amino acid in the human body. In this work, a novel reliable method of the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and microdialysis (MD) technique for the evaluation of NAG and its metabolites γ-aminobutyric acid (GABA) and glutamic acid (Glu) in rat blood and brain was proposed. A Zorbax SB-C18 column (2.1 × 100 mm, 3.5 μM) was applied to separate the analytes. The mobile phase was acetonitrile-water (70:30, v/v) containing 5 mM ammonium acetate and the flow rate was 0.3 ml/min. Based on the multiple reaction monitoring (MRM) mode of positive ion, the precursors of product ions chosen for NAG, Glu, GABA, and N-carbamyl-L-glutamic (NCG, IS) were (m/z) 189.1→130.0, 148.0→84.1, 104→87.1, and 191.0→130.1, respectively. All the validation data, including precision, accuracy, inter-day repeatability, matrix effect, and stability, were within the acceptable ranges according to the reference of Bioanalytical Method Validation Guidance for Industry (2018). Rats with microdialysis probes inserted into jugular vein and hippocampus were administered the low (75 mg/kg, NAG-L), medium (150 mg/kg, NAG-M), and high (300 mg/kg, NAG-H) doses of NAG and 10 ml/kg Guhong injection (GHI) by tail vein, respectively. In the blood test, the Cmax values of NAG-L group were markedly lower (P < 0.01) than those of NAG-M, NAG-H, and GHI groups, respectively. No differences were observed between NAG-M and GHI groups, while the Cmax values in GHI group were significantly upgraded compared with NAG-H group. There were notable differences in the Cmax values of NAG in brain dialysate after administration of NAG and GHI. The drug distribution coefficients of NAG, Glu, GABA in brain and blood at low, medium, high doses of NAG and GHI groups were 13.99, 27.43, 34.81, 31.37; 11.04, 59.07, 21.69, 2.69%; 212.88, 234.92, 157.59, and 102.65%, respectively. Our investigation demonstrates that NAG and its related metabolites in rat blood and brain can be simultaneously measured according to the above proposed method. Meanwhile, NAG has easy and dose-dependently access to the blood-brain barrier and exhibits a medium retention time in rat.

Morchella conica (M. conica) Pers. is one of six wild edible mushrooms that are widely used by Asian and European countries for their nutritional value. The present study assessed the anti-diabetic potential of M. conica methanolic extract (100 mg/kg body weight) on streptozotocin (STZ)-induced diabetic mice. STZ was used in a single dose of 65 mg/kg to establish diabetic models. Body weights, water/food intake and fasting blood glucose levels were measured. Histopathological analysis of the pancreas and liver were performed to evaluate STZ-induced tissue injuries. In addition, in vitro assays such as α-amylase and protein tyrosine phosphatase 1B (PTP1B) inhibitory, antiglycation, antioxidant and cytotoxicity were performed. The in vitro study indicated potent PTP1B inhibitory potential of M. conica with an IC50 value of 26.5 μg/ml as compared to the positive control, oleanolic acid (IC50 36.2 μg/ml). In vivo investigation showed a gradual decrease in blood sugar level in M. conica-treated mice (132 mg/dl) at a concentration of 100 mg/kg as compared to diabetic mice (346 mg/dl). The extract positively improved liver and kidney damages as were shown by their serum glutamic pyruvic transaminase, serum glutamic oxaloacetate, alkaline phosphatase, serum creatinine and urea levels. Histopathological analysis revealed slight liver and pancreas improvement of mice treated with extract. Cytotoxicity assays displayed lower IC50 values. Based on the present results of the study, it may be inferred that M. conica are rich in bioactive compounds responsible for antidiabetic activity and this mushroom may be a potential source of antidiabetic drug. However, further studies are required in terms of isolation of bioactive compounds to validate the observed results.

Insufficient transport of therapeutic cargo into tumor bed is a bottleneck in cancer nanomedicine. Block copolymers are promising carriers with smaller particle size by ratio modification. Here, we constructed cisplatin nanoparticles with sizes ranging from 8 to 40 nm to study the permeability and therapy of Lewis lung carcinoma. We synthesized methoxypoly(ethylene glycol)2000-block poly(L-glutamic acid sodium salt)1979 loading cisplatin through complexation reaction. The cisplatin nanomedicine has high drug loading and encapsulation efficiency. In vitro data demonstrated that cisplatin nanoparticles had equivalent growth-inhibiting effects on Lewis lung carcinoma cells compared to free cisplatin. In vivo evidences showed cisplatin nanoparticles had superior antitumor effects on the Lewis lung carcinoma mouse model with no obvious side effects. All results indicated that optimizing the ratio of block copolymers to obtain smaller sized nanomedicine could act as a promising strategy for overcoming the inadequate accumulation in poorly vascularized tumors.

Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the health of the elderly. At present, no drugs have been proven to cure or delay the progression of the disease. Due to the multifactorial aetiology of this disease, the multi-target-directed ligand (MTDL) approach provides an innovative and promising idea in search for new drugs against AD. In order to find potential multi-target anti-AD drugs from traditional Chinese medicine (TCM) formulae, a compound database derived from anti-AD Chinese herbal formulae was constructed and predicted by the anti-AD multi-target drug prediction platform established in our laboratory. By analyzing the results of virtual screening, 226 chemical constituents with 3 or more potential AD-related targets were collected, from which 16 compounds that were predicted to combat AD through various mechanisms were chosen for biological validation. Several cell models were established to validate the anti-AD effects of these compounds, including KCl, Aβ, okadaic acid (OA), SNP and H2O2 induced SH-SY5Y cell model and LPS induced BV2 microglia model. The experimental results showed that 12 compounds including Nonivamide, Bavachromene and 3,4-Dimethoxycinnamic acid could protect model cells from AD-related damages and showed potential anti-AD activity. Furthermore, the potential targets of Nonivamide were investigated by molecular docking study and analysis with CDOCKER revealed the possible binding mode of Nonivamide with its predicted targets. In summary, 12 potential multi-target anti-AD compounds have been found from anti-AD TCM formulae by comprehensive application of computational prediction, molecular docking method and biological validation, which laid a theoretical and experimental foundation for in-depth study, also providing important information and new research ideas for the discovery of anti-AD compounds from traditional Chinese medicine.

4-(methylthio)butyl isothiocyanate (4-MTBITC) is a hydrolytic product from the plant Eruca sativa Thell. In the present study, we explored the anti-cancer effect of 4-MTBITC against 7,12-dimethylbenz [a] anthracene (DMBA) induced breast cancer. Hypoxic conditions were developed using a single dose of 60 mg/kg DMBA. Hepatic and renal parameters were increased along with antioxidants in cancer-bearing rats which were lowered with the treatment of 4-MTBITC. Further, it inhibited the up-regulation of glycolytic enzymes caused by DMBA. The hypoxia pathway was evaluated using RT-PCR and it was found that the 40 mg/kg doses of 4-MTBITC statistically lowered the expression of HIF-1α. Akt/mTOR signaling pathway was one of the major pathways involved in 4-MTBITC-induced cell growth arrest by western blotting. Amino acid profiling serum-free plasma revealed the downregulation of specific amino acids required for vital components of fast-growing cancer cells. 4-MTBITC reduced the levels of serine, arginine, alanine, asparagines, and glutamic acid. Histological examination also showed neoplastic growth following DMBA doses. 4-MTBITC treated rats showed less infiltration and normal physiology. Our findings for the first time demonstrated the potential therapeutic significance of 4-MTBITC on modulation of glycolytic enzymes and hypoxia pathway in female rats.

Epilepsy is a common neurological disease with recurrent seizures and neurobehavioral comorbidities, including cognitive impairment and psychiatric disorders. Recent studies suggest that L-3-n-butylphthalide (NBP), an extract from the seeds of Apium graveolens Linn. (Chinese celery), ameliorates cognitive dysfunction in ischemia and/or Alzheimer's disease animal models. However, little is known about the role of NBP in epilepsy and the associated comorbidities. Here, using a pilocarpine-induced chronic epileptic mouse model, we found that NBP supplement not only alleviated seizure severity and abnormal electroencephalogram, but also rescued cognitive and emotional impairments in these epileptic mice. The possible underlying mechanisms may be associated with the protective role of NBP in reducing neuronal loss and in restoring the expression of neural synaptic proteins such as postsynaptic density protein 95 (PSD95) and glutamic acid decarboxylase 65/67 (GAD65/67). In addition, NBP treatment increased the transcription of neuroprotective factors, brain-derived neurotrophic factor and Klotho. These findings suggest that NBP treatment may be a potential strategy for ameliorating epileptogenesis and the comorbidities of cognitive and psychological impairments.

Accumulating evidence suggests that natural medicines have notable curative effects on neurological conditions, such as migraine, that are mediated by regulating the gut microbial flora. A natural medicine pair used in traditional Chinese medicine, Gastrodia elata Blume and Uncaria rhynchophylla (Miq.) Miq. ex Havil. (GU), have shown excellent effect in treating migraine, yet the role of gut microbes in the therapeutic effect of GU in chronic migraine (CMG) is unknown. Here, we performed a 16S rRNA gene sequencing and metabolomics study of the effects of GU in a nitroglycerin (NTG)-induced rat model of CMG. Our results showed that the gut microbial community structure changed significantly and was similar to that of control rats after GU administration in CMG rats. Specifically, GU increased the relative abundance of Bacteroides and Coprococcus and reduced the abundance of Prevotella_1 and Escherichia-Shigella in CMG rats. The metabolomics profiles of the plasma and ileum contents of CMG rats obtained with an ultra-performance liquid chromatography-mass spectrometer (UPLC-MS) revealed similar biomarkers in both samples, and GU treatment reduced 3-indoxyl sulfate, glutamic acid, L-tyrosine, and L-arginine levels, and increased 5-HIAA, L-tryptophan, and linoleic acid levels in plasma. Correlation analysis showed that the affected bacteria were closely related to amino acid metabolism. Most importantly, GU treatment hardly affected biomarkers in feces samples after inhibiting the activity of gut microbes. Collectively, these findings indicate that structural changes in gut flora are closely related to host metabolism and that regulating the gut microbial community structure and function may be one of the important mechanisms underlying the therapeutic effects of GU in migraine.

Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F.Chow [Rhamnaceae; Ziziphi Spinosae Semen (ZSS)] has attracted extensive attention as the first choice of traditional Chinese medicine in the treatment of insomnia. However, recent studies on the sleep-improving mechanism of ZSS have mainly focused on the role of single components. Thus, to further reveal the potential mechanism of ZSS, an assessment of its multiple constituents is necessary. In this study, ZSS extract (ZSSE) was obtained from ZSS via detailed modern extraction, separation, and purification technologies. The chemical constituents of ZSSE were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). For in vivo experiments, a rat model of insomnia induced by p-chlorophenylalanine (PCPA) was established to investigate the potential effect and corresponding mechanism of ZSSE on improving sleep. Hematoxylin-eosin staining (HE) results revealed that the drug group showed prominent advantages over the model group in improving sleep. Moreover, the brain levels of γ-aminobutyric acid (GABA), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), and dopamine (DA) were monitored via enzyme-linked immunosorbent assay (ELISA) to further study the sleep-improving mechanism of ZSSE. We found that sleep was effectively improved via upregulation of GABA and 5-HT and downregulation of Glu and DA. In addition, molecular mechanisms of ZSSE in improving sleep were studied by immunohistochemical analysis. The results showed that sleep was improved by regulating the expression levels of GABA receptor subunit alpha-1 (GABAARα1) and GABA acid receptor subunit gamma-2 (GABAARγ2) receptors in the hypothalamus and hippocampus tissue sections. Therefore, this work not only identified the active ingredients of ZSSE but also revealed the potential pharmacological mechanism of ZSSE for improving sleep, which may greatly stimulate the prospective development and application of ZSSE.

Two kinds of tumor microenvironment-responsive polypeptide nanogels were developed for intracellular delivery of cytotoxics to enhance the antitumor efficacies and reduce the side effects in the chemotherapy of lung carcinoma. The sizes of both doxorubicin (DOX)-loaded nanogels methoxy poly(ethylene glycol)-poly(L-phenylalanine-co-L-cystine) [mPEG-P(LP-co-LC)] and methoxy poly(ethylene glycol)-poly(L-glutamic acid-co-L-cystine) [mPEG-P(LG-co-LC)] (NGP/DOX and NGG/DOX) were less than 100 nm, which was appropriate for the enhanced permeability and retention (EPR) effect. The bigger and smaller scale of nanoparticle could induce the elimination of reticuloendothelial system (RES) and decrease the in vivo circulating half-life, respectively. The loading nanogels were stable in the neutral environment while quickly degraded in the mimic intracellular microenvironment. Furthermore, the DOX-loaded reduction-responsive nanogels showed significantly higher tumor cell uptake than free DOX⋅HCl as time went on from 2 to 6 h. In addition, these DOX-loaded nanogels showed efficient antitumor effects in vivo, which was verified by the obviously increased necrosis areas in the tumor tissues. Furthermore, these DOX-loaded nanogels efficiently reduced the side effects of DOX. In conclusion, these reduction-responsive polypeptides based nanogels are suitable for the efficient therapy of lung carcinoma.

Crocetin (apo carotenoid dicarboxylic acid) is a common constituent of saffron. Its importance is well documented in Chinese medicine. Some studies have reported the inhibitory effect on inflammation in rats. The aim of the current experimental investigation to scrutinize the anti-inflammatory effect of Crocetin using the lipo polysaccharide (LPS) induced mouse macrophages (RAW 264.7) in vitro and complete Freund's adjuvant-induced arthritis model and to explore in vivo possible mechanism of action. RAW 264.7 macrophages were used for estimation of the effect of crocetin on the cyclooxygenase (COX-2), nitric oxide (NO)production, anti-inflammatory and along with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). Single intraperitoneal injection of complete freund's adjuvant (CFA) was used to induce arthritis. The rats were divided into different group and received the oral administration of crocetin in a dose-dependent manner with indomethacin till 28 days. The paw edema and body weight was estimated at regular interval of time. The biochemical parameters, hematological and pro-inflammatory cytokines such as tumor necrosis factor receptor 1 (TNF-R1), IL-6, and IL-1β, Vascular endothelial growth factor (VEGF); heme oxygenase-1/nuclear factor erythroid 2-related factor 2 (HO-1/Nrf-2) expression were estimated at end of the experimental study. Crocetin inhibited the COX-2 catalyzed prostaglandin (PGE2) and inducible nitric oxide synthase catalyzed NO production on RAW 264.7. The paw edema and body weight was significantly (P < 0.001) modulated by the Crocetin in a dose-dependent manner. Crocetin treatment increased the level of red blood cells (RBC), hemoglobin (Hb) and decreased level of white blood cells (WBC), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase (SGPT), and serum glutamic-oxaloacetic transaminase (SGOT) parameters, with reduction of TNF-α, IL-6, and IL-1β.The protective effect of crocetin was substantiated with a reduction in expression of IL-6, IL-1β, VEGF, and TNF-R1, respectively. Crocetin also increased the HO-1/Nrf-2 and decreased the nuclear factor kappa-B (NF-κB) mRNA, protein expression. On the basis of the result, we can conclude that the reduction of HO-1/Nrf-2 expression, as well as inflammatory mediators, may be involved in the protective effect of Crocetin in the CFA model.

The analogous β-carboline alkaloids, harmaline (HAL) and harmine (HAR), possess a variety of biological properties, including acetylcholinesterase (AChE) inhibitory activity, antioxidant, anti-inflammatory, and many others, and have great potential for treating Alzheimer's disease (AD). However, studies have showed that the two compounds have similar structures and in vitro AChE inhibitory activities but with significant difference in bioavailability. The objective of this study was to comparatively investigate the effects of HAL and HAR in memory deficits of scopolamine-induced mice. In the present study, mice were pretreated with HAL (2, 5, and 10 mg/kg), HAR (10, 20, and 30 mg/kg) and donepezil (5 mg/kg) by intragastrically for 7 days, and were daily intraperitoneal injected with scopolamine (1 mg/kg) to induce memory deficits and then subjected to behavioral evaluation by Morris water maze. To further elucidate the underlying mechanisms of HAL and HAR in improving learning and memory, the levels of various biochemical factors and protein expressions related to cholinergic function, oxidative stress, and inflammation were examined. The results showed that HAL and HAR could effectively ameliorate memory deficits in scopolamine-induced mice. Both of them exhibited an enhancement in cholinergic function by inhibiting AChE and inducing choline acetyltransferase (ChAT) activities, and antioxidant defense via increasing the antioxidant enzymes activities of superoxide dismutase and glutathione peroxidase, and reducing maleic diadehyde production, and anti-inflammatory effects through suppressing myeloperoxidase, tumor necrosis factor α, and nitric oxide as well as modulation of critical neurotransmitters such as acetylcholine (ACh), choline (Ch), L-tryptophan (L-Trp), 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (γ-GABA), and L-glutamic acid (L-Glu). Furthermore, the regulations of HAL on cholinergic function, inflammation, and neurotransmitters were more striking than those of HAR, and HAL manifested a comparable antioxidant capacity to HAR. Remarkably, the effective dosage of HAL (2 mg/kg) was far lower than that of HAR (20 mg/kg), which probably due to the evidently differences in the bioavailability and metabolic stability of the two analogs. Taken together, all these results revealed that HAL may be a promising candidate compound with better anti-amnesic effects and pharmacokinetic characteristics for the treatments of AD and related diseases.

Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP) cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with L-arginine (YRFK) and D-arginine (YrFK) were synthesized and coupled with carboxyethyl-TPP (TPP-3) and carboxypentyl-TPP (TPP-6) units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, L-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.

The alleviation of oxidative stress is considered an effective treatment for acetaminophen (APAP)-induced acute liver injury (AILI). However, it remains unknow whether the potential antioxidant Smilax china L. polysaccharide (SCLP) protects against AILI. In this study, in vitro and in vivo experiments were conducted to verify the hepatoprotective effect of SCLP against AILI and explore the potential mechanism. We found that SCLP relieved liver histopathological changes; reversed the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and reactive oxygen species (ROS); reversed the change in liver myeloperoxidase (MPO) activity; and enhanced liver antioxidant (GSH, GSH-Px, and t-SOD) levels in APAP-treated mice, thereby significantly reducing APAP-induced liver toxicity. SCLP rescued the cell viability and alleviated oxidative stress in H2O2-treated mouse AML12 (Alpha mouse liver 12) hepatocytes. The results of the mechanistic studies showed that SCLP upregulated nuclear factor E2 related factor (Nrf2) expression, promoted Nrf2 nuclear translocation, and enhanced the ability of Nrf2 to bind antioxidant response elements (AREs). Furthermore, SCLP activated Nrf2-ARE pathway, thus upregulating the expression of oxidative stress-related proteins heme oxygenase 1(HO-1), NAD(P)H quinone dehydrogenase 1(NQO-1) and glutamic acid cysteine ligase catalytic subunit (GCLC). In conclusion, this study confirmed the close correlation between liver protection by SCLP upon exposure to APAP and activated of the Nrf2-ARE pathway. These findings suggest that SCLP is an attractive therapeutic candidate drug for the treatment of AILI.

Apelin is the endogenous ligand for APJ, a G-protein-coupled receptor. Apelin gene and protein are widely distributed in the central nervous system and peripheral tissues. The role of apelin in chronic inflammatory pain is still unclear. In the present study, a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain was utilized, and the paw withdrawal latency/threshold in response to thermal stimulation and Von Frey filament stimulation were recorded after intrathecal (i.t.) injection of apelin-13 (0.1, 1, and 10 nmol/mouse). The mRNA and protein expression, concentration of glutamic acid (Glu), and number of c-Fos immunol staining in lumbar spinal cord (L4/5) were determined. The results demonstrated that Apln gene expression in the lumbar spinal cord was down-regulated in the CFA pain model. Apelin-13 (10 nmol/mouse, i.t.) alleviated CFA-induced inflammatory pain, and it exhibited a more potent antinociceptive effect than apelin-36 and (pyr)apelin-13. The antinociception of apelin-13 could be blocked by APJ antagonist apelin-13(F13A). I.T. apelin-13 attenuated the increased levels of Aplnr, Grin2b, Camk2d, and c-Fos genes expression, Glu concentration, and NMDA receptor 2B (GluN2B) protein expression caused by CFA. Apelin-13 significantly reduced the number of Fos-positive cells in laminae III and IV/V of the dorsal horn. This study indicated that i.t. apelin-13 exerted an analgesic effect against inflammatory pain, which was mediated by activation of APJ, and inhibition of Glu/GluN2B function and neural activity of the spinal dorsal horn.

Zhi zhu xiang (ZZX) is the root and rhizome of Valeriana jatamansi Jones ex Roxb. Recent studies have shown that ZZX can exert antianxiety, antidepressant, and sedative effects. Because post-traumatic stress disorder (PTSD) is similar to depression and anxiety in terms of its etiology, pathogenesis, and clinical manifestations, it is possible that ZZX may also be useful for the prevention and treatment of PTSD. In this study, a mouse model of PTSD was established and used to study the pharmacological action of a 95% ethanol extract of ZZX on PTSD via a series of classic behavioral tests. We found that a 95% ethanol extract of ZZX was indeed effective for relieving the symptoms of PTSD in mice. Moreover, network pharmacology analysis was used to predict the potential active ingredients, targets, and possible pathways of ZZX in the treatment of PTSD. The neurotransmitter system, the hypothalamic-pituitary-adrenal (HPA) axis, and the endocannabinoid (eCB) system were identified to be the most likely pathways for anti-PTSD action in ZZX. Due to the lack of a falsification mechanism in network pharmacology, in vivo tests were carried out in mice, and the expression levels of neurotransmitters, hormones, and genes of key targets were detected by enzyme-linked immunosorbent assay and real-time PCR to further verify this inference. Analysis showed that the levels of norepinephrine, 5-hydroxytryptamine, and glutamic acid were increased in the hippocampus, prefrontal cortex, and amygdala of PTSD mice, while the levels of dopamine and γ-aminobutyric acid were decreased in these brain regions; furthermore, ZZX could restore the expression of these factors, at least to a certain extent. The levels of adrenocorticotropic hormone, corticosterone, and corticotropin-releasing hormone were increased in these different brain regions and the serum of PTSD mice; these effects could be reversed by ZZX to a certain extent. The expression levels of cannabinoid receptor 1 and diacylglycerol lipase α mRNA were decreased in PTSD mice, while the levels of fatty acid amide hydrolase and monoacylglycerol lipase mRNA were increased; these effects were restored by ZZX to a certain extent. In conclusion, our findings suggest that ZZX may provide new therapeutic pathways for treating PTSD by the regulation of neurotransmitters, the HPA, and expression levels of eCB-related genes in the brain.

Background: Central fatigue (CF) is a subjective sense of tiredness associated with cognitive and memory disorders, accompanied by reduced physical endurance and negative emotions, such as anxiety and depression. Disease progression and prognosis with regards to CF have been unfavorable and possibly contribute to dementia, schizophrenia, and other diseases. Additionally, effective treatments for CF are lacking. KangPiLao decoction (KPLD) has been widely applied in clinical treatment and is composed of six Chinese herbal medicines, some of which have confirmed anti-fatigue effects. While glutamic acid (Glu) is the main excitatory transmitter in the central nervous system (CNS), gamma-aminobutyric acid (GABA) is the major inhibitory transmitter. Both are involved in emotional, cognitive, and memory functions. This research was designed to explore how KPLD regulates cognitive and emotional disorders in rats with CF and to identify the relationship between the regulatory effect and the GABA/Glu pathway. Methods: The compounds comprising KPLD were analyzed using high-performance liquid chromatography-mass spectrometry. Sixty Wistar rats were randomly divided into six groups. The modified multiple platform method was used to induce CF. Cognitive, emotional, and fatigue states were evaluated by performing behavioral tests (Morris water maze [MWM], open-field test [OFT], and grip strength test). Histomorphology, western blotting, immunohistochemistry, and RT-qPCR were performed to investigate protein and mRNA expression levels in the hippocampus and prefrontal cortexes involved in the GABA/Glu pathway. Results: Rats with CF exhibited impaired spatial cognition and increased negative emotions in the MWM and OFT. KPLD enabled the improvement of these symptoms, especially in the high-concentration group. Western blotting and RT-qPCR demonstrated that the expression of GABAARα1, GABAARγ2, GABABR1, and GAD67 in rats with CF was higher, whereas GAT-1 and NMDAR2B were lower in the hippocampus and prefrontal cortex. KPLD decreased the expression of GABAARα1, GABABR1, GABAARγ2, and GAD67 in the hippocampus and prefrontal cortex and enhanced the expression of NR2B in the prefrontal cortex. Conclusion: KPLD significantly improved cognitive and emotional disorders in rats with CF by regulating the GABA/Glu pathway. Overall, KPLD may be a promising candidate for developing a drug for treating CF.

Yokukansan (YKS) and yokukansankachimpihange (YKSCH) are traditional Japanese Kampo medicines. The latter comprises YKS along with the medicinal herbs Citrus unshiu peel and Pinellia tuber. Both of these Kampo medicines are indicated for the treatment of night crying and irritability in children and for neurosis and insomnia in adults. In recent clinical trials, YKS exhibited ameliorative effects on the behavioral and psychological symptoms of dementia, such as aggressiveness, excitement, and irritability. In the present study, we aimed to clarify the involvement of cholinergic degeneration in the nucleus basalis of Meynert (NBM) in the development of aggressiveness in rats. Subsequently, using this animal model, the effects of YKS and YKSCH on aggressiveness were compared and the mechanisms underlying these effects were investigated. L-Glutamic acid (Glu) was injected into the right NBM of rats to induce deterioration of cholinergic neurons. On day 8 after Glu injection, aggressive behaviors were evaluated using resident-intruder tests. After the evaluation, YKS or YKSCH was administered to rats with aggressive behaviors daily for 7 days. In some groups, the 5-HT1A receptor antagonist WAY-100635 was coadministered with YKS or YKSCH over the same period. In other groups, locomotor activity was measured on days 12-14 after Glu injection. On day 15, immunohistochemistry was then performed to examine choline acetyltransferase (ChAT) activities in the NBM. Aggressive behaviors had developed on day 8 after Glu injection and were maintained until day 15. YKS and YKSCH significantly ameliorated the aggressive behaviors. These suppressive effects were entirely abolished following coadministration of WAY-100635. Finally, the number of ChAT-positive cells in the right NBM was significantly reduced on day 15 after Glu injection, and treatment with YKS or YKSCH did not ameliorate these reduced cell numbers. Our results show that unilateral Glu injections into the NBM of rats leads to the development of aggressive behaviors, which is thought to reflect cholinergic degeneration. YKS and YKSCH treatments ameliorated Glu-induced aggressive behaviors, and these effects were suggested to be mediated by 5-HT1A receptor stimulation, but not by improvement of cholinergic degeneration.