[Non-targeted metabolomics of spleen and liver metabolism in mice treated with Pruni Semen processed with different methods].

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.

Changes in chemical components and hepatoprotective effect of red Panax notoginseng processed by aspartic acid impregnation treatment.

BACKGROUND: Red Panax notoginseng (RPN) is one of the major processed products of P. notoginseng (PN), with more effective biological activities. However, the traditional processing method of RPN has some disadvantages, such as low conversion rate of ginsenosides and long processing time. RESULTS: In this work, we developed a green, safe, and efficient approach for RPN processing by aspartic acid impregnation pretreatment. Our results showed that the optimized temperature, steaming time, and concentration of aspartic acid were 120 °C, 1 h, and 3% respectively. The original ginsenosides in PN treated by aspartic acid (Asp-PN) were completely converted to rare saponins at 120 °C within just 1 h. The concentration of the rare ginsenosides in Asp-PN was two times higher than that in untreated RPN. In addition, we examined the protective effect of RPN and Asp-PN on acetaminophen-induced liver injury in a mouse model. The results showed that Asp-PN has significantly more potent hepatoprotective action than the RPN. The hepatoprotection of Asp-PN in acetaminophen-induced hepatotoxicity may be due to its anti-oxidative stress, anti-apoptotic, and anti-inflammatory activities. CONCLUSION: These results indicated that aspartic acid impregnation pretreatment may provide an effective method to shorten the steaming time, improve the conversion rate of ginsenosides, and enhance hepatoprotective activity of RPN. © 2024 Society of Chemical Industry.

Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m/z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

[UPLC-Q-TOF-MS-based metabolomics reveals mechanism of Morinda officinalis iridoid glycosides in treating rheumatoid arthritis and bone loss].

This study aimed to investigate the therapeutic effects of Morinda officinalis iridoid glycosides(MOIG) on paw edema and bone loss of rheumatoid arthritis(RA) rats, and analyze its potential mechanism based on ultra-high performance liguid chromatography-guadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS) serum metabolomics. RA rats were established by injecting bovin type Ⅱ collagen. The collagen-induced arthritis(CIA) rats were administered drug by gavage for 8 weeks, the arthritic score were used to evaluate the severity of paw edem, serum bone metabolism biochemical parameters were measured by ELISA kits, Masson staining was used to observe the bone microstructure of the femur in CIA rats. UPLC-Q-TOF-MS was used to analyze the alteration of serum metabolite of CIA rats, principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were used to screen the potential biomarkers, KEGG database analysis were used to construct related metabolic pathways. The results demonstrated that the arthritic score, serum levels of IL-6 and parameters related with bone metabolism including OCN, CTX-Ⅰ, DPD and TRAP were significantly increased, and the ratio of OPG and RANKL was significantly decreased, the microstructure of bone tissue and cartilage were destructed in CIA rats, while MOIG treatments could significantly reduce arthritis score, mitigate the paw edema, reverse the changes of serum biochemical indicators related with bone metabolism, and improve the microstructure of bone tissue and cartilage of CIA rats. The non-targeted metabolomics results showed that 24 altered metabolites were identified in serum of CIA rats; compared with normal group, 13 significantly altered metabolites related to RA were identified in serum of CIA rats, mainly involving alanine, aspartate and glutamate metabolism; compared with CIA model group, MOIG treatment reversed the alteration of 15 differential metabolites, mainly involving into alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism, valine, leucine and isoleucine biosynthesis. Therefore, MOIG significantly alleviated paw edema, improved the destruction of microstructure of bone and cartilage in CIA rats maybe through involving into the regulation of amino acid metabolism.

Guhan Yangsheng Jing mitigates hippocampal neuronal pyroptotic injury and manifies learning and memory capabilities in sleep deprived mice via the NLRP3/Caspase1/GSDMD signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guhan Yangsheng Jing (GHYSJ) is a traditional Chinese patent medicine, that has the function of nourishing the kidney and replenishing the essence, invigorating the brain and calming the mind. It is often used to treat dizziness, memory loss, sleep disorders, fatigue, and weakness, etc. However, its mechanism for improving sleep has not yet been determined. AIM OF THE STUDY: This study aims to explore the effects of GHYSJ on Sleep Deprivation (SD)-induced hippocampal neuronal pyroptotic injury, learning and cognitive abilities, and sleep quality in mice. METHODS: In this study, a PCPA-induced SD mouse model was established. We assessed the influence of GHYSJ on sleep quality and mood by using the pentobarbital-induced sleep test (PIST) and sucrose preference test (SPT). The pharmacological effects of GHYSJ on learning and memory impairment were evaluated by the Morris Water Maze (MWM) and Open Field Test (OFT). Pathological changes in the hippocampal tissue of the SD rats were observed via HE staining and Nissl staining. The severity of neuronal damage was evaluated by detecting the expression of the neuronal marker Microtubule-associated protein 2 (MAP2), via immunohistochemistry and immunofluorescence. Furthermore, the levels of neurotransmitter 5-hydroxytryptophan (5-HTP), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), and Glutamic acid (Glu) in hippocampal tissues, as well as the expression of inflammatory factors Interleukin-1ß (IL-1ß) and Interleukin-18 (IL-18) in serum, were determined by ELISA. The expressions of mRNA and protein NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Gasdermin D (GSDMD), Cysteinyl aspartate specific proteinase1 (Caspase1), High mobility group box-1 protein (HMGB1) and Apoptosis-associated speck-like protein containing CARD (ASC) related to the cellular ferroptosis pathway were tested and analyzed by RT-PCR and WB respectively. RESULTS: PCPA significantly diminishes the sleep span of experimental animals by expediting the expenditure of 5-HT, consequently establishing an essentially direct SD model. The intervention of GHYSJ displays remarkable efficacy in mitigating insomnia symptoms, encompassing difficulties in initiating sleep and insufficient sleep duration. Likewise, it ameliorates memory function impairments induced by sleep deprivation, along with symptoms such as fatigue and depletion of vitality. GHYSJ exerts a protective influence on hippocampal neurons facilitated by inhibiting the down regulation of MAP2 and maintaining the equilibrium of neurotransmitters (5-HTP, 5-HT, GABA, and Glu). It diminishes the expression of intracellular pyroptosis-associated inflammatory factors (IL-1ß and IL-18) and curbs the activation of the NLRP3/Caspase1/GSDMD pyroptosis-related signaling pathways, thereby alleviating the damage caused by hippocampal neuronal pyroptosis.

Effects of Supplementing Zinc Magnesium Aspartate on Sleep Quality and Submaximal Weightlifting Performance, following Two Consecutive Nights of Partial Sleep Deprivation.

Purpose: We examined whether supplementation of zinc magnesium aspartate (ZMA), while partially sleep deprived, was beneficial to sleep quality and subsequent morning (07:00 h) submaximal weightlifting. Methods: Using a double-blinded, randomized counterbalanced design, sixteen trained males were recruited and completed six sessions: (i) one repetition max (1 RM) for bench press and back squat; (ii) two familiarisation sessions; (iii) three conditions with 4 h sleep and either: ZMA, placebo (PLA), or NoPill control (NoPill). Submaximal exercise session consisted of three repetitions at 40, 60 and 80% of 1 RM for bench press and back squat. Average power (AP), average velocity (AV), peak velocity (PV), displacement (D) and time-to-peak velocity (tPV) were recorded using MuscleLab linear encoders. Data were analysed using a general linear model with repeated measures and linear correlation. Results: No significant main effect for condition was found for performance values or subjective ratings of fatigue. Main effect for "load" on the bar was found, where AP and tPV values increased with load (p < 0.05). No significant relationship between dose of zinc or magnesium ingested and change in performance for 80% 1 RM power-outputs was found. Conclusion: Supplementation of ZMA for two nights of partial sleep deprivation had no effect on sleep or subsequent morning performance.

Neurometabolic alterations in children and adolescents with functional neurological disorder.

OBJECTIVES: In vivo magnetic resonance spectroscopy (MRS) was used to investigate neurometabolic homeostasis in children with functional neurological disorder (FND) in three regions of interest: supplementary motor area (SMA), anterior default mode network (aDMN), and posterior default mode network (dDMN). Metabolites assessed included N-acetyl aspartate (NAA), a marker of neuron function; myo-inositol (mI), a glial-cell marker; choline (Cho), a membrane marker; glutamate plus glutamine (Glx), a marker of excitatory neurotransmission; γ-aminobutyric acid (GABA), a marker of inhibitor neurotransmission; and creatine (Cr), an energy marker. The relationship between excitatory (glutamate and glutamine) and inhibitory (GABA) neurotransmitter (E/I) balance was also examined. METHODS: MRS data were acquired for 32 children with mixed FND (25 girls, 7 boys, aged 10.00 to 16.08 years) and 41 healthy controls of similar age using both short echo point-resolved spectroscopy (PRESS) and Mescher-Garwood point-resolved spectroscopy (MEGAPRESS) sequences in the three regions of interest. RESULTS: In the SMA, children with FND had lower NAA/Cr, mI/Cr (trend level), and GABA/Cr ratios. In the aDMN, no group differences in metabolite ratios were found. In the pDMN, children with FND had lower NAA/Cr and mI/Cr (trend level) ratios. While no group differences in E/I balance were found (FND vs. controls), E/I balance in the aDMN was lower in children with functional seizures-a subgroup within the FND group. Pearson correlations found that increased arousal (indexed by higher heart rate) was associated with lower mI/Cr in the SMA and pDMN. CONCLUSIONS: Our findings of multiple differences in neurometabolites in children with FND suggest dysfunction on multiple levels of the biological system: the neuron (lower NAA), the glial cell (lower mI), and inhibitory neurotransmission (lower GABA), as well as dysfunction in energy regulation in the subgroup with functional seizures.

Effect of Nutraceutical Factors on Hepatic Intermediary Metabolism in Wistar Rats with Induced Tendinopathy.

Tendinopathy (TP) is a complex clinical syndrome characterized by local inflammation, pain in the affected area, and loss of performance, preceded by tendon injury. The disease develops in three phases: Inflammatory phase, proliferative phase, and remodeling phase. There are currently no proven treatments for early reversal of this type of injury. However, the metabolic pathways of the transition metabolism, which are necessary for the proper functioning of the organism, are known. These metabolic pathways can be modified by a number of external factors, such as nutritional supplements. In this study, the modulatory effect of four dietary supplements, maslinic acid (MA), hydroxytyrosol (HT), glycine, and aspartate (AA), on hepatic intermediary metabolism was observed in Wistar rats with induced tendinopathy at different stages of the disease. Induced tendinopathy in rats produces alterations in the liver intermediary metabolism. Nutraceutical treatments modify the intermediary metabolism in the different phases of tendinopathy, so AA treatment produced a decrease in carbohydrate metabolism. In lipid metabolism, MA and AA caused a decrease in lipogenesis at the tendinopathy and increased fatty acid oxidation. In protein metabolism, MA treatment increased GDH and AST activity; HT decreased ALT activity; and the AA treatment does not cause any alteration. Use of nutritional supplements of diet could help to regulate the intermediary metabolism in the TP.

Thalamic neurometabolite alterations in chronic low back pain: a common phenomenon across musculoskeletal pain conditions?

ABSTRACT: Recently, we showed that patients with knee osteoarthritis (KOA) demonstrate alterations in the thalamic concentrations of several metabolites compared with healthy controls: higher myo-inositol (mIns), lower N-acetylaspartate (NAA), and lower choline (Cho). Here, we evaluated whether these metabolite alterations are specific to KOA or could also be observed in patients with a different musculoskeletal condition, such as chronic low back pain (cLBP). Thirty-six patients with cLBP and 20 healthy controls were scanned using 1 H-magnetic resonance spectroscopy (MRS) and a PRESS (Point RESolved Spectroscopy) sequence with voxel placement in the left thalamus. Compared with healthy controls, patients with cLBP demonstrated lower absolute concentrations of NAA ( P = 0.0005) and Cho ( P < 0.05) and higher absolute concentrations of mIns ( P = 0.01) when controlling for age, as predicted by our previous work in KOA. In contrast to our KOA study, mIns levels in this population did not significantly correlate with pain measures (eg, pain severity or duration). However, exploratory analyses revealed that NAA levels in patients were negatively correlated with the severity of sleep disturbance ( P < 0.01), which was higher in patients compared with healthy controls ( P < 0.001). Additionally, also in patients, both Cho and mIns levels were positively correlated with age ( P < 0.01 and P < 0.05, respectively). Altogether, these results suggest that thalamic metabolite changes may be common across etiologically different musculoskeletal chronic pain conditions, including cLBP and KOA, and may relate to symptoms often comorbid with chronic pain, such as sleep disturbance. The functional and clinical significance of these brain changes remains to be fully understood.

Self-Sorting Collagen Heterotrimers.

Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into three─out of 512 possible─triple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.

Wu-zhu-yu Decoction reduces early brain injury following subarachnoid hemorrhage in vivo and in vitro by activating the Nrf2 antioxidant system via SIRT6 targeting.

ETHNOPHARMACOLOGICAL RELEVANCE: Early brain damage (EBI) following subarachnoid hemorrhage (SAH) is a long-lasting condition with a high occurrence. However, treatment options are restricted. Wu-zhu-yu Decoction (WZYD) can treat headaches and vomiting, which are similar to the early symptoms of subarachnoid hemorrhage (SAH). However, it is yet unknown if WZYD can reduce EBI following SAH and its underlying mechanisms. AIM OF THE STUDY: This study aimed to investigate whether WZYD protects against EBI following SAH by inhibiting oxidative stress through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling via Sirtuin 6 (SIRT6)-mediated histone H3 lysine 56 (H3K56) deacetylation. MATERIALS AND METHODS: In the current investigation, the principal components of WZYD were identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). The SAH model in rats using the internal carotid artery plug puncture approach and the SAH model in primary neurons using hemoglobin incubation were developed. WZYD with different doses (137 mg kg-1, 274 mg kg-1, 548 mg kg-1) and the positive drug-Nimodipine (40 mg kg-1) were intragastrically administered in SAH model rats, respectively. The PC12 cells were cultured with corresponding medicated for 24h. In our investigation, neurological scores, brain water content, Evans blue leakage, Nissl staining, TUNEL staining, oxidative stress, expression of apoptosis-related proteins, and Nrf2/HO-1 signaling were evaluated. The interaction between SIRT6 and Nrf2 was detected by co-immunoprecipitation. SIRT6 knockdown was used to confirm its role in WZYD's neuroprotection. RESULTS: The WZYD treatment dramatically reduced cerebral hemorrhage and edema, and enhanced neurological results in EBI following SAH rats. WZYD administration inhibited neuronal apoptosis via reducing the expression levels of Cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3), cysteinyl aspartate specific proteinase-3(caspase-3), and Bcl-2, Associated X Protein (Bax) and increasing the expression of B-cell lymphoma-2(Bal2). It also decreased reactive oxygen species and malondialdehyde levels and increased Nrf2 and HO-1 expression in the rat brain after SAH. In vitro, WZYD attenuated hemoglobin-induced cytotoxicity, oxidative stress and apoptosis in primary neurons. Mechanistically, WZYD enhanced SIRT6 expression and H3K56 deacetylation, activated Nrf2/HO-1 signaling, and promoted the interaction between SIRT6 and Nrf2. Knockdown of SIRT6 abolished WZYD-induced neuroprotection. CONCLUSIONS: WZYD attenuates EBI after SAH by activating Nrf2/HO-1 signaling through SIRT6-mediated H3K56 deacetylation, suggesting its therapeutic potential for SAH treatment.

Sodium L-Aspartate Supplementation Improves Repeated-Sprint Performance.

Aspartate supplementation has been reported to improve endurance performance by facilitating the tricarboxylic acid cycle flux. The present study was performed to investigate the effects of aspartate supplementation on repeated-sprint performance and blood pH. Following an overnight fast, fourteen healthy males completed three sets of 10 × 6 s maximal sprints after consuming sodium L-aspartate (ASP) or placebo (PLA), in a double-blind manner. Both supplements were taken twice on each test day (2 × 4.5 g). Exercise performance (e.g., cadence and power output) and blood variables (e.g., pH and plasma amino acid levels) were measured. The ASP trial evidenced significantly higher plasma aspartate concentration during the first (ASP, 45.3 ± 9.2 µM; PLA, 6.1 ± 0.8 µM) and the second exercise sets (ASP, 24.2 ± 4.5 µM; PLA, 6.6 ± 0.9 µM) and peak cadence during the second set (ASP, 153 ± 3 rpm; PLA, 152 ± 3 rpm) compared with the PLA trial (all p < 0.05). The peak power output during the second exercise set (ASP, 743 ± 32 W; PLA, 734 ± 31 W; p = 0.060) and the blood pH immediately before (ASP, 7.280 ± 0.020; PLA, 7.248 ± 0.016; p = 0.087) and after the third exercise set (ASP, 7.274 ± 0.019; PLA, 7.242 ± 0.018; p = 0.093) tended to be higher in the ASP than in the PLA trial. In conclusion, ASP supplementation partially improved repeated-sprint performance (peak cadence during the second exercise set). However, it did not affect the mean power output.

A window into the human immune system: comprehensive characterization of the complexity of antibody complementary-determining regions in functional antibodies.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

[Effect of aqueous extract of Corni Fructus on Aß_(25-35)-induced brain injury and neuroinflammation in mice with Alzheimer's disease].

The purpose of this study was to investigate the effect of aqueous extract of Corni Fructus on ß-amyloid protein 25-35(Aß_(25-35))-induced brain injury and neuroinflammation in Alzheimer's disease(AD) mice to provide an experimental basis for the treatment of AD by aqueous extract of Corni Fructus. Sixty C57BL/6J male mice were randomly divided into a sham group, a model group, a positive control group(huperizine A, 0.2 mg·kg~(-1)), a low-dose aqueous extract of Corni Fructus group(1.3 g·kg~(-1)), a medium-dose aqueous extract of Corni Fructus group(2.6 g·kg~(-1)), and a high-dose aqueous extract of Corni Fructus group(5.2 g·kg~(-1)). The AD model was induced by lateral ventricular injection of Aß_(25-35) in mice except for those in the sham group, and AD model mice were treated with corresponding drugs by gavage for 24 days. The behavioral test was performed one week before animal dissection. Hematoxylin-eosin(HE) staining was performed to observe the morphology of neurons in the hippocampal region. Flow cytometry was used to detect the apoptosis level of primary hippocampal cells in mice. ELISA kits were used to detect the levels of ß-amyloid protein 1-42(Aß_(1-42)) and phosphorylated microtubule-associated protein Tau(p-Tau) in mouse brain tissues. Immunofluorescence and Western blot were used to detect the expression of related proteins in mouse brain tissues. MTT assay was used to detect the effect of compounds in aqueous extract of Corni Fructus on Aß_(25-35)-induced N9 cell injury. Molecular docking was employed to analyze the interactions of caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-ß-D-glucopyranoside, esculetin, and(+)-lyoniresinol with ß-amyloid precursor protein(APP), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α). Aqueous extract of Corni Fructus could improve the learning and memory abilities of Aß_(25-35)-induced mice by increasing the duration of the autonomous activity, the rate of autonomous alternation, the preference coefficient, and the discrimination coefficient, and reduce Aß_(25-35)-induced brain injury and neuroinflammation in mice by increasing the expression levels of interleukin-10(IL-10) and B-cell lymphoma-2(Bcl-2) in brain tissues, decreasing the expression levels of Aß_(1-42), p-Tau, IL-6, TNF-α, cysteine aspartate-specific protease 3(caspase-3), cysteine aspartate-specific protease 9(caspase-9), and Bcl-2-associated X protein(Bax), and decreasing the number of activated glial cells in brain tissues. The results of cell experiments showed that esculetin and(+)-lyoniresinol could improve Aß_(25-35)-induced N9 cell injury. Molecular docking results showed that caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-ß-D-glucopyranoside, esculetin, and(+)-lyoniresinol had good binding affinity with APP and weak binding affinity with IL-6 and TNF-α. Aqueous extract of Corni Fructus could ameliorate cognitive dysfunction and brain damage in Aß_(25-35)-induced mice by reducing the number of apoptotic cells and activated glial cells in the brain and decreasing the expression level of inflammatory factors. Caffeic acid, trans-p-hydroxy cinnamic acid, isolariciresinol-9'-O-ß-D-glucopyranoside, esculetin, and(+)-lyoniresinol may be the material basis for the anti-AD effect of aqueous extract of Corni Fructus.

[Moxibustion with seed-size moxa cone enhances anti-tumor effect of cyclophosphamide on Hepa1-6 hepatoma-bearing mice by regulating apoptosis factor cysteine family].

OBJECTIVE: To observe the anti-tumor effect of moxibustion with seed-sized moxa cones on Hepa1-6 liver cancer bearing (HLCB) mice and its regulatory mechanism on cell apoptosis. METHODS: A total of 40 male C57BL/6 mice were randomly divided into control, moxibustion, cyclophosphamide (CTX) and moxibustion+CTX groups, with 10 mice in each group. The HLCB model was established by subcutaneous inoculation of Hepa1-6 cancer cells into the right armpit. Mice of the CTX and moxibustion+CTX groups were given intraperitoneal injection of CTX (30 mg/kg), once daily for 3 days. Moxibustion with seed-sized moxa cones were applied to "Dazhui" (GV14), bilateral"Zusanli" (ST36) and "Sanyinjiao" (SP6), with 5 moxa cones for each acupoint, once daily for 10 consecutive days. The survival status scores and body weight of HLCB mice were observed, and the tumor weight and tumor inhibition rate were detected. HE staining was used to observe the morphological changes of tumor tissue. ELISA was used to detect the levels of serum interleukin (IL)-2, IL-4 and tumor necrosis factor-α (TNF-α). Western blot and fluorescent quantitative real-time PCR were used to detect the protein and mRNA expressions of cysteine aspartate protease (Caspase) -3 and Caspase-9 in tumor tissues, separately. RESULTS: Compared with the control group, the survival status scores, body weight, serum IL-2 and TNF-α levels were significantly increased (P<0.05, P<0.01), the tumor weight and serum IL-4 levels were significantly decreased (P<0.05) in the moxibustion group；while the survival status, body weight, tumor weight, serum IL-2 and IL-4 levels were significantly decreased (P<0.01, P<0.05), the content of TNF-α was significantly increased (P<0.01) in the CTX group. The protein and mRNA expressions of Caspase-3 and Caspase-9 in the 3 trentment groups were significantly increased (P<0.05, P<0.01). In comparison with the moxibustion group, the survival status scores, body weight and tumor weight, serum content of IL-2 were significantly decreased (P<0.01, P<0.05). In contrast to the CTX group, the survival status scores, body weight, serum IL-2 and TNF-α content, and the expressions of Caspase-3 and Caspase-9 in tumor tissue were significantly increased (P<0.01, P<0.05), and the tumor weight and serum IL-4 content were significantly decreased (P<0.05) in the moxibustion+CTX group. Results of HE staining showed that the tumor cells in the control group had clear nuclear membranes and nucleoli, with more dividing cells； while less nuclear division and an increase in tumor necrosis areas were found in the 3 treatment groups. CONCLUSION: Moxibustion with seed-size moxa cone can enhance the anti-tumor effect of CTX and improve the quality of life of HLCB mice, which may be related with its effect in activating the expressions of Caspase-3 and Caspase-9 in tumor tissue.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

The malate-aspartate shuttle is important for de novo serine biosynthesis.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD+. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-13C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD+/NADH ratio), consistent with the NAD+ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD+-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders.

[Effect of "Tiaoshen Tongluo" acupuncture on nerve function injury, muscle tension and neurotransmitters through Nrf2/ROS pathway in spastic rats after stroke].

OBJECTIVE: To observe the effect of "Tiaoshen Tongluo" acupuncture (TTA) at "Dingzhongxian" (MS5) and right "Dingpangxian" (MS8) on neurological injury, muscle tension and neurotransmitters through nuclear transcription factor E2 related factor 2 (Nrf2)/reactive oxygen species (ROS) signaling pathway in spastic rats after stroke, so as to explore its mechanisms underlying relief of post-stroke spasm (PSS). METHODS: A total of 90 male SD rats were randomly divided into 6 groups, i.e. sham operation, PSS model, medication, non-acupoint acupuncture, TTA, TTA+ML385 groups, with 15 rats in each group. The PSS model was established by middle cerebral artery occlusion. After modeling, rats of the medication group were treated by gavage of baclofen (0.4 mg/kg), once daily for 7 days. For rats of the non-acupoint acupuncture group, the spot about 10 mm above the iliac crest and below the armpit of the affected side was needled, and for those of the TTA group and TTA+ML385 group, EA stimulation (1 mA, 2 Hz/15 Hz) was applied to MS5 and right MS8 for 10 min, once daily for 7 consecutive days. Intraperitoneal injection of ML385 ［ a specific nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg］ was given to rats of the TTA+ML385 group before TTA was performed. The rats' neurological deficit score (0-4 points) was evaluated by referring to Zea Longa's methods and the muscular spasm degree of the quadriceps femoris of the left hindlimb (0-4 points) assessed by using Ashworth scale (MAS). The muscular tension of the left quadriceps femoris was measured by using a tension sensor, and Hoffman (H)-reflex response and M and H waves of electromyogram of the muscle between the metatarsals of the left foot were measured using an electrophysiological recorder. The cerebral infarction volume was measured after 2,3,5-triphenyltetrazolium chloride (TTC) staining. The contents of γ-aminobutyric acid (GABA), glycine (Gly), glutamic acid (Glu) and aspartic acid (Asp) of the right cortical infarct area were detected by using high performance capillary electrophoresis, and the contents of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) were detected by fluorescence spectrophoto-metry, as well as the level of ROS in the right cerebral cortical infarction tissues was detected by dihydroethidium staining. The expression levels of Nrf2 and heme oxygenase-1 (HO-1) proteins in the infarcted cerebral area were detected using Western blot. RESULTS: Compared with the sham operation group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu and Asp and ROS level were significantly increased (P<0.001), whereas the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, cerebral Nrf2 and HO-1 protein expression levels were apparently decreased (P<0.001) in the model group. In comparison with the model group, the neurological deficit score, MAS score, percentage of cerebral infarction volume, Hmax/Mmax ratio, contents of Glu, Asp and ROS levels were decreased (P<0.001), and the muscle tone, stimulation threshold for inducing H-reflex, GABA, Gly, 5-HT, DA and NE contents, Nrf2 and HO-1 protein expressions were increased (P<0.001, P<0.01) in both the medication and TTA groups. No significant differences were found between the non-acupoint group and model group, and between the medication and TTA groups in all the indexes mentioned above (P>0.05). After administration of ML385, the effects of TTA in reducing neurological deficit score, MAS score, Hmax/Mmax, percentage of cerebral infarct volume, Glu, Asp, ROS, and up-regulating H-reflex threshold, GABA, Gly, 5-HT, DA, NE, Nrf2 and HO-1 levels were eliminated (P<0.001，P<0.05,P<0.01). CONCLUSION: TTA can improve neurological behavior and muscle spasm in rats with PSS, which may be associated with its functions in regulating the levels of neurotransmitters in the cortical infarcted area by activating the Nrf2/ROS signaling pathway.

N-carbamoyl aspartate reduced body weight by stimulating the thermogenesis of iBAT.

Uridine has formerly been shown to alleviate obesity and hepatic lipid accumulation. N-carbamoyl aspartate (NCA) provides carbon atoms to uridine in de novo pyrimidine biosynthesis pathway. However, whether NCA is involved in the lipid metabolism remains elusive. Here we showed that NCA supplementation significantly decreased (P < 0.05) serum cholesterol (CHOL), high-density lipoprotein (HDL), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) levels of mice, and significantly increased (P < 0.05) relative mRNA expression of genes related to the synthesis of pyrimidine nucleotides and polyunsaturated fatty acids. Besides, supplemented with NCA significantly decreased body weight and area under the curve (AUC), and increased body temperature in the high-fat diet fed mice. For further, relative protein expression of uridine monophosphate synthase (UMPS), sterol regulatory element-binding protein 1(SREBP-1) and phosphorylated hormone-sensitive triglyceride lipase (P-HSL) in the liver, and uncoupling protein 1 (UCP-1) in interscapular brown adipose tissue (iBAT) also showed upregulated in the high-fat diet fed mice. Thus, NCA promoted de novo synthesis of pyrimidine and polyunsaturated fatty acid, and reduced body weight by stimulating high-fat diet-induced thermogenesis of iBAT.

Effects of Scutellaria strigillosa Hemsl. extract on HepG2 cell proliferation and apoptosis through binding to aspartate ß-hydroxylase.

This study aims to examine the impacts of Scutellaria strigillosa Hemsl. (SSH) on the proliferation, apoptosis of human hepatoma cell HepG2 and screen the bioactive components. We found that SSH extract inhibited HepG2 proliferation, arrested cell division prior to S phase. Additionally, SSH extract exposure induced apoptosis, and increased the proportions of late apoptotic cells. Specifically, we focus on the inhibitory effect of SSH extract on aspartate ß-hydroxylase, a key therapeutic target of hepatocellular carcinoma closely related with the proliferation and apoptosis of HepG2. We found SSH extract with notable inhibitory activity against aspartate ß-hydroxylase, elucidated the main bioactive constituents by HPLC-Q-TOF/MS and Molecular docking analysis. In conclusion, these results provided the antiproliferative and proapoptotic effects of SSH on HepG2 cell, elucidated the main bioactive constituents based on aspartate ß-hydroxylase inhibition. These data revealed the potential value of SSH and its bioactive components for the prevention and treatment of liver cancer for the first time.