Enzyme-Driven LC-HRMS Approach for Specific Recognition of 12α-Hydroxy Bile Acids.

The synthesis of 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical and alternative pathways, respectively. The composition of these BAs is a crucial index for pathophysiologic assessment. However, accurately differentiating 12HBAs and non-12HBAs is highly challenging due to the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro under mild conditions. Coupled to the conversion rate determined by liquid chromatography-high resolution mass spectrometry (LC-HRMS), this enzymatic probe allows for the straightforward distinguishing of 210 12HBAs and 312 non-12HBAs from complex biological matrices, resulting in a BAs profile with a well-defined hydroxyl feature at the C12 site. Notably, this enzyme-driven LC-HRMS approach can be extended to any molecule with explicit knowledge of enzymatic transformation. We demonstrate the practicality of this BAs profile in terms of both revealing cross-species BAs heterogeneity and monitoring the alterations of 12HBAs and non-12HBAs under asthma disease. We envisage that this work will provide a novel pattern to recognize the shift of BA metabolism from classical to alternative synthesis pathways in different pathophysiological states, thereby offering valuable insights into the management of related diseases.

Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.

Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.

Mitochondrial complex activity controls a multitude of physiological processes by regulating the cellular metabolism. Current methods for evaluating mitochondrial complex activity mainly focus on single metabolic reactions within mitochondria. These methods often require fresh samples in large quantities for mitochondria purification or intact mitochondrial membranes for real-time monitoring. Confronting these limitations, we shifted the analytical perspective toward interactive metabolic networks at the whole-cell level to reflect mitochondrial complex activity. To this end, we compiled a panel of mitochondrial respiratory chain-mapped metabolites (MRCMs), whose perturbations theoretically provide an overall reflection on mitochondrial complex activity. By introducing N-dimethyl-p-phenylenediamine and N-methyl-p-phenylenediamine as a pair of mass spectrometry probes, an ultraperformance liquid chromatography-tandem mass spectrometry method with high sensitivity (LLOQ as low as 0.2 fmol) was developed to obtain accurate quantitative data of MRCMs. Machine learning was then combined to capture the relationship between MRCMs and mitochondrial complex activity. Using Complex I as a proof-of-concept, we identified NADH, alanine, and phosphoenolpyruvate as metabolites associated with Complex I activity based on the whole-cell level. The effectiveness of using their concentrations to reflect Complex I activity was further validated in external data sets. Hence, by capturing the relationship between metabolites and mitochondrial complex activity at the whole-cell level, this study explores a novel analytical paradigm for the interrogation of mitochondrial complex activity, offering a favorable complement to existing methods particularly when sample quantities, type, and treatment timeliness pose challenges. More importantly, it shifts the focus from individual metabolic reactions within mitochondria to a more comprehensive view of an interactive metabolic network, which should serve as a promising direction for future research into the functional architecture between mitochondrial complexes and metabolites.

Pre-electroacupuncture Ameliorates Cerebral Ischemia-reperfusion Injury by Inhibiting Microglial RhoA/pyrin/GSDMD Signaling Pathway.

Cerebral ischemia reperfusion injury is a severe neurological impairment that occurs after blood flow reconstruction in stroke, and microglia cell pyroptosis is one of its important mechanisms. Electroacupuncture has been shown to be effective in mitigating and alleviating cerebral ischemia reperfusion injury by inhibiting neuroinflammation, reducing cellular pyroptosis, and improving neurological function. In this experiment, we divided the rats into three groups, including the sham operation (Sham) group, the middle cerebral artery occlusion/reperfusion (MCAO/R) group, and the pre-electroacupuncture (EAC) group. Pre-electroacupuncture group was stimulated with electroacupuncture of a certain intensity on the Baihui (GV 20) and Dazhui (GV 14) of the rat once a day from the 7th day to the 1st day before the MCAO/R operation. The extent of cerebral infarction was detected by TTC staining. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. The number of neurons and morphological changes were accessed by Nissl staining and HE staining. The cellular damage was detected by TUNEL staining. In addition, the expression levels of RhoA, pyrin, GSDMD, Caspase1, cleaved-Caspase1, Iba-1, CD206, and ROCK2 were examined by western blotting and immunofluorescence. The results found that pre-electroacupuncture significantly attenuated neurological impairment and cerebral infarction compared to the post-MCAO/R rats. In addition, pre-electroacupuncture therapy promoted polarization of microglia to the neuroprotective (M2) phenotype. In addition, pre-electroacupuncture inhibited microglia pyroptosis by inhibiting RhoA/pyrin/GSDMD signaling pathway, thereby reducing neuronal injury and increasing neuronal survival in the MCAO/R rats. Taken together, these results demonstrated that pre-acupuncture could attenuate cerebral ischemia-reperfusion injury by inhibiting microglial pyroptosis. Therefore, pre-electroacupuncture might be a potential preventive strategy for ischemic stroke patients.

Elevated neutrophil extracellular trap levels in periodontitis: Implications for keratinization and barrier function in gingival epithelium.

AIM: To explore the levels of neutrophil extracellular traps (NETs) in patients with periodontitis and examine their effects on keratinization, barrier function of human gingival keratinocytes (HGKs) and the associated mechanisms. MATERIALS AND METHODS: Saliva, gingival crevicular fluid (GCF), clinical periodontal parameters and gingival specimens were collected from 10 healthy control subjects and 10 patients with stage II-IV periodontitis to measure the NET levels. Subsequently, mRNA and protein levels of keratinization and barrier indicators, as well as intracellular calcium and epithelial barrier permeability, were analysed in HGKs after NET stimulation. RESULTS: The study showed that NET levels significantly elevated in patients with periodontitis, across multiple specimens including saliva, GCF and gingival tissues. Stimulation of HGKs with NETs resulted in a decrease in the expressions of involucrin, cytokeratin 10, zonula occludens 1 and E-cadherin, along with decreased intracellular calcium levels and increased epithelial barrier permeability. Furthermore, the inhibition of keratinization by NETs is ERK-KLF4-dependent. CONCLUSIONS: This study indicates that NETs impair the barrier function of HGKs and suppress keratinization through ERK/KLF4 axis. These findings provide potential targets for therapeutic approaches in periodontitis to address impaired gingival keratinization.

[Effect of superfine powder and aqueous extract of Polygonati Rhizoma on rats with natural perimenopausal syndrome].

This study aims to examine the effect of superfine powder and aqueous extract of Polygonati Rhizomaon on natural perimenopausal syndrome in rats and explore the underlying mechanism. To be specific, a total of 60 female SD rats(14-15 months old) with estrous cycle disorder were screened by the vaginal smear and randomized into model control group, ß-estradiol 3-benzoate group(0.1 mg·kg~(-1)), superfine powder of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)) and aqueous extract of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)), and another 10 female SD rats(14-15 months old) were selected as the youth control group. The administration lasted 6 weeks. Then the perimenopausal syndrome-related indexes such as body temperature, microcirculatory blood flow of face and ear, vertigo period, salivary secretion, grip force, and bone strength were determined and open field test was conducted. The immune system-related indexes such as the wet weight and index of thymus and spleen, percentage of T lymphocytes and subgroups in peripheral blood, and hematological indexes were measured. In addition, the ovary-related indexes such as estrous cycle, the wet weight and index of uterus and ovary, ovarian tissue morphology, and cell apoptosis were determined. Moreover, hypothalamus-pituitary-ovary axis(HPO)-related indexes such as serum sex hormone levels, cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and cytochrome P450 family 17 subfamily A member 1(P450 17A1) in ovarian tissue were measured. The results showed that the superfine powder and aqueous extract of Polygonati Rhizoma significantly decreased body temperature(anal, facial and dorsal temperature), microcirculatory blood flow in the ear, and vertigo period, increased salivary secretion, grip force, bone strength, total distance and total speed in the open field test, wet weight and index of thymus and spleen, lymphocyte ratio, CD3~+ level, and CD4~+/CD8~+ ratio, reduced neutrophil number and ratio, estrous cycle disorder ratio, and number of ovarian apoptotic cells, raised wet weight and index of uterus, wet weight of ovary, levels of inhibin B(INHB), estradiol(E_2), anti-müllerian hormone(AMH), and ovarian CYP11A1 and CYP19A1, decreased follicle-stimulating hormone(FSH) and luteinizing hormone(LH) content, and improved ovarian tissue morphology. It is suggested that the superfine powder and aqueous extract of Polygonati Rhizoma can improve the symptoms associated with natural perimenopausal syndrome in rats and enhance ovarian function and immune function. The mechanism is that they regulate HPO axis function by increasing estrogen synthesis.

Least absolute shrinkage and selection operator-based prediction of collision cross section values for ion mobility mass spectrometric analysis of lipids.

Collision cross section (CCS) values generated from ion mobility mass spectrometry (IM-MS) have commonly been employed to facilitate lipid identification. However, this is hindered by the limited available lipid standards. Recently, CCS values were predicted by means of computational calculations, though the prediction precision was generally not good and the predicted CCS values of the lipid isomers were almost identical. To address this challenge, a least absolute shrinkage and selection operator (LASSO)-based prediction method was developed for the prediction of lipids' CCS values in this study. In this method, an array of molecular descriptors were screened and optimized to reflect the subtle differences in structures among the different lipid isomers. The use of molecular descriptors together with a wealth of standard CCS values for the lipids (365 in total) significantly improved the accuracy and precision of the LASSO model. Its accuracy was externally validated with median relative errors (MREs) of <1.1% using an independent data set. This approach was demonstrated to allow differentiation of cis/trans and sn-positional isomers. The results also indicated that the LASSO-based prediction method could practically reduce false-positive identifications in IM-MS-based lipidomics.

In silico production of relative correction factor for the quantitative analysis of multi-components by single marker method.

INTRODUCTION: Traditional methods to derive experimentally-generated relative correction factors (RCFs) for the quantitative analysis of herbal multi-components by single marker (QAMS) method require reference standards and multiple validations with different instruments and columns, which hampers high throughput implementation. OBJECTIVES: To effectively reduce the application amounts of raw material and provide higher and more stable accuracy, this study aimed to develop a method to computationally generate RCFs of herbal components. MATERIALS AND METHODS: This strategy included the published data collection, calibration curves screening, computer algorithm-based RCFs generation and accuracy validation. RESULTS: Using the in silico approach, we have successfully produced 133 RCFs for the multi-component quantitative analysis of 63 widely used herbs. CONCLUSION: Compared with conventional RCFs, this in silico method would be a low cost and highly efficient way to produce practical RCFs for the QAMS method.

Saponins from Clematis mandshurica Rupr. regulates gut microbiota and its metabolites during alleviation of collagen-induced arthritis in rats.

Gut microbiota and their metabolites (short-chain fatty acids, SCFAs) are associated with the pathogenesis of rheumatoid arthritis (RA). Total Clematis triterpenoid saponins (CTSs) prepared from Clematis mandshurica Rupr. possess therapeutic benefits for arthritic diseases. However, the poor pharmacokinetic properties of CTSs have obstructed the translation of these natural agents to drugs. Here, we examined the effects of CTSs on arthritis symptoms, gut microbiota and SCFAs in rats with collagen-induced arthritis (CIA). Our results showed that the arthritis index scores of CIA rats treated with CTSs were significantly lower than those of the model group. Most importantly, CTSs moderated gut microbial dysbiosis and significantly downregulated the total SCFA concentration in CIA rats. Compared to the control group, CTSs treatment have no significant side effects on the gut microbiota and SCFA metabolism in normal rats. Two differential analyses (LEfSe and DESeq2) were combined to study the details of the changes in gut microbiome, and twenty-four marker taxa at the genus level were identified via a comparison among control, model and CIA rats treated with high doses of CTSs. In particular, the mostly significantly increased gram-negative (G-) and decreased gram-positive (G+) genera in CIA rats were well restored by CTSs. The observed SCFA concentrations demonstrated that CTSs tend to maintain the balance of the gut microbiota. The data presented herein suggest that CTSs could ameliorate arthritis-associated gut microbial dysbiosis and may be potential adjuvant drugs that could provide relief from the gastrointestinal damage caused as a side effect of commonly used drugs.

Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4+CD25+ Regulatory T Cells Mainly through Axl Receptor.

Background. Growth arrest-specific (Gas) 6 is one of the endogenous ligands of TAM receptors (Tyro3, Axl, and Mertk), and its role as an immune modulator has been recently emphasized. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. The present study was designed to investigate whether Tregs express TAM receptors and the potential role of Gas6-TAM signal in regulating the suppressive function of Tregs. Methods. The protein and mRNA levels of TAM receptors were determined by using Western blot, immunofluorescence, flow cytometry, and RT-PCR. Then, TAM receptors were silenced using targeted siRNA or blocked with specific antibody. The suppressive function of Tregs was assessed by using a CFSE-based T cell proliferation assay. Flow cytometry was used to determine the expression of Foxp3 and CTLA4 whereas cytokines secretion levels were measured by ELISA assay. Results. Tregs express both Axl and Mertk receptors. Gas6 increases the suppressive function of Tregs in vitro and in mice. Both Foxp3 and CTLA-4 expression on Tregs are enhanced after Gas6 stimulation. Gas6 enhances the suppressive activity of Tregs mainly through Axl receptor. Conclusion. Gas6 has a direct effect on the functions of CD4+CD25+Tregs mainly through its interaction with Axl receptor.

Role of mitofusin-2 in high mobility group box-1 protein-mediated apoptosis of T cells in vitro.

BACKGROUND: High mobility group box-1 protein (HMGB1), a ubiquitous nuclear protein, which is recognized as a danger-associated molecular pattern (DAMP) triggering activation of the innate immune system. Previous studies have shown that HMGB1 also plays a role in T cell-mediated immunity, but the effect of HMGB1 on apoptosis of T cells and its precise mechanism remain to be determined. METHODS: Two kinds of apoptosis assay techniques were used, i.e., Annexin V-FITC conjunction with PI to identify early apoptotic cells, Hoechst 33342 staining for double-stranded DNA to observe nuclear fragmentation or apoptotic body. The activation status of caspase-3, caspase-8, as well as caspase-9 was examined by colorimetric assay. The dynamic changes in intracellular calcium concentration ([Ca(2+)]i) was monitored by flow cytometry. Overexpression of Mfn2 was preformed by lentiviral vector transfection. The mRNA and protein levels of Mfn2 were determined by RT-PCR and Western-blotting. RESULTS: Treatment of Jurkat T cells with recombinant human HMGB1 (rhHMGB1) causes a significant dose-dependent increase in percentage of apoptotic cells. When T cells are incubated with HMGB1 they express decreased mitochondria fusion-related protein mitofusin-2 (Mfn2) and activate mitochondrial apoptotic pathway via elevation of [Ca(2+)]i, Bax insertion, and activation of caspase. Furthermore, overexpression of Mfn2 ameliorates the apoptosis of T cells induced by HMGB1. This occurs at least partly through Mfn2 keeps Ca(2+) homeostasis in T cells evidenced by monitoring [Ca(2+)]i dynamics. CONCLUSION: HMGB1 can trigger apoptosis of T lymphocytes through mitochondrial death pathway associated with [Ca(2+)]i elevation. Mfn2 plays a pivotal role in this process, and it might be a novel therapeutic target in T cell apoptosis related disorders.

Thinking Induced by Acute Kidney Injury of Diquat Poisoning: Cases Report.

Diquat poisoning is a fatal condition that is becoming increasingly common. The mortality risk of patients taking lethal doses of diquat is extremely high. It typically leads to rapid dysfunction of multiple organs, including the kidneys, heart, lungs, and brain. Acute kidney injury is usually the first manifestation of this poisoning. However, the optimal treatment strategy for diquat poisoning remains uncertain. Additionally, the mechanism of multiple organ dysfunction syndrome caused by diquat poisoning may resemble the progression of sepsis. In this report, we present 3 cases of diquat poisoning, all of which resulted in death. We emphasize that acute kidney injury is the primary cause of death, and suggest that some strategies used in the treatment of sepsis could be beneficial in managing diquat poisoning-induced acute kidney injury.

Differential cytokine expression in gastric tissues highlights helicobacter pylori's role in gastritis.

Helicobacter pylori (H. pylori), known for causing gastric inflammation, gastritis and gastric cancer, prompted our study to investigate the differential expression of cytokines in gastric tissues, which is crucial for understanding H. pylori infection and its potential progression to gastric cancer. Focusing on Il-1ß, IL-6, IL-8, IL-12, IL-18, and TNF-α, we analysed gene and protein levels to differentiate between H. pylori-infected and non-infected gastritis. We utilised real-time quantitative polymerase chain reaction (RT-qPCR) for gene quantification, immunohistochemical staining, and ELISA for protein measurement. Gastric samples from patients with gastritis were divided into three groups: (1) non-gastritis (N-group) group, (2) gastritis without H. pylori infection (G-group), and (3) gastritis with H. pylori infection (GH-group), each consisting of 8 samples. Our findings revealed a statistically significant variation in cytokine expression. Generally, cytokine levels were higher in gastritis, but in H. pylori-infected gastritis, IL-1ß, IL-6, and IL-8 levels were lower compared to H. pylori-independent gastritis, while IL-12, IL-18, and TNF-α levels were higher. This distinct cytokine expression pattern in H. pylori-infected gastritis underscores a unique inflammatory response, providing deeper insights into its pathogenesis.

Huang Lian Jie Du decoction attenuated colitis via suppressing the macrophage Csf1r/Src pathway and modulating gut microbiota.

Introduction: Ulcerative colitis, a subtype of chronic inflammatory bowel disease (IBD), is characterized by relapsing colonic inflammation and ulcers. The traditional Chinese herbal formulation Huang Lian Jie Du (HLJD) decoction is used clinically to treat diarrhea and colitis. However, the mechanisms associated with the effects of treatment remain unclear. This study aims to elucidate the molecular mechanistic effects of HLJD formulation on colitis. Methods: Chronic colitis in mice was induced by adding 1% dextran sulfate sodium (DSS) to their drinking water continuously for 8 weeks, and HLJD decoction at the doses of 2 and 4 g/kg was administered orally to mice daily from the second week until experimental endpoint. Stool consistency scores, blood stool scores, and body weights were recorded weekly. Disease activity index (DAI) was determined before necropsy, where colon tissues were collected for biochemical analyses. In addition, the fecal microbiome of treated mice was characterized using 16S rRNA amplicon sequencing. Results: HLJD decoction at doses of 2 and 4 g/kg relieved DSS-induced chronic colitis in mice by suppressing inflammation through compromised macrophage activity in colonic tissues associated with the colony-stimulating factor 1 receptor (Csf1r)/Src pathway. Furthermore, the HLJD formula could modify the gut microbiota profile by decreasing the abundance of Bacteroides, Odoribacter, Clostridium_sensu_stricto_1, and Parasutterella. In addition, close correlations between DAI, colon length, spleen weight, and gut microbiota were identified. Discussion: Our findings revealed that the HLJD formula attenuated DSS-induced chronic colitis by reducing inflammation via Csf1r/Src-mediated macrophage infiltration, as well as modulating the gut microbiota profile.

Metformin improves cognitive dysfunction through SIRT1/NLRP3 pathway-mediated neuroinflammation in db/db mice.

Diabetes mellitus (DM), an important public health problem, aggravates the global economic burden. Diabetic encephalopathy (DE) is a serious complication of DM in the central nervous system. Metformin has been proven to improve DE. However, the mechanism is still unclear. In this study, the db/db mice, a common model used for DE, were employed to explore and study the neuroprotective effect of metformin and related mechanisms. Behavioral tests indicated that metformin (100 or 200 mg/kg/day) could significantly improve the learning and memory abilities of db/db mice. The outcomes from the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) demonstrate that metformin effectively modulates glucose and insulin signaling pathways in db/db mice. The results of body weight and blood lipid panel (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol) show that metformin promotes the level of lipid metabolism in db/db mice. Furthermore, data from oxidative stress assays, which measured levels of malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase, suggest that metformin suppresses oxidative stress-induced brain damage in db/db mice. In addition, western blot, Nissl staining, and immunofluorescence results showed that metformin increased the expressions of nerve growth factor and postsynaptic density 95 and repaired neuronal structural damage. For the mechanism study, metformin activated SIRT1 and inhibited the expression of NLRP3 inflammasome (NLRP3, ASC, caspase-1, IL-1ß, and IL-18) and inflammatory cytokines (TNFα and IL-6). In conclusion, metformin could ameliorate cognitive dysfunction through the SIRT1/NLRP3 pathway, which might be a promising mechanism for DE treatment.

FEN1-assisted DNA logic amplifier circuit for fast and compact DNA computing.

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

Taohong Siwu decoction alleviates cognitive impairment by suppressing endoplasmic reticulum stress and apoptosis signaling pathway in vascular dementia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (TSD), a classic traditional Chinese medicine formula, is used for the treatment of vascular diseases, including vascular dementia (VD). However, the mechanisms remain unclear. AIM OF STUDY: This study aimed to investigate whether TSD has a positive effect on cognitive impairment in VD rats and to confirm that the mechanism of action is related to the Endoplasmic Reticulum stress (ERs) and cell apoptosis signaling pathway. MATERIALS AND METHODS: A total of 40 male adult Sprague-Dawley rats were divided into four groups: sham-operated group (Sham), the two-vessel occlusion group (2VO), the 2VO treated with 4.5 g/kg/d TSD group (2VO + TSD-L), the 2VO treated with 13.5 g/kg/d TSD group (2VO + TSD-H). The rats underwent either 2VO surgery or sham surgery. Postoperative TSD treatment was given for 4 consecutive weeks. Behavioral tests were initiated at the end of gastrulation. Open-field test (OFT) was used to detect the activity level. The New Object Recognition test (NOR) was used to test long-term memory. The Morris water maze (MWM) test was used to examine the foundation of spatial learning and memory. As a final step, the hippocampus was taken for molecular testing. The protein levels of GRP78 (Bip), p-PERK, PERK, IRE1α, p-IRE1α, ATF6, eIF2α, p-eIF2α, ATF4, XBP1, Bcl-2 and Bax were determined by Western blot. Immunofluorescence visualizes molecular expression. RESULTS: In the OFT, residence time in the central area was significantly longer in both TSD treatment groups compared to the 2VO group. In the NOR, the recognition index was obviously elevated in both TSD treatment groups. The 2VO group had a significantly longer escape latency and fewer times in crossing the location of the platform compared with the Sham group in MWM. TSD treatment reversed this notion. Pathologically, staining observations confirmed that TSD inhibited hippocampal neuronal loss and alleviated the abnormal reduction of the Nissl body. In parallel, TUNEL staining illustrated that TSD decelerated neuronal apoptosis. Western Blot demonstrated that TSD reduces the expression of ERs and apoptotic proteins. CONCLUSION: In this study, the significant ameliorative effect on cognitive impairment of TSD has been determined by comparing the behavioral data of the 4 groups of rats. Furthermore, it was confirmed that this effect of TSD was achieved by suppressing the ERs-mediated apoptosis signaling pathway.

Dengzhan Shengmai Capsule Ameliorates Cognitive Impairment via Inhibiting ER stress in APP/PS1 Mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD) is a common type of neurodegenerative disease with the ß-amyloid plaques (Aß) deposition. Previously, Dengzhan Shengmai capsule (DZSM) has been shown to reduce the pathology associated with AD, but the underlying mechanism is unclear. AIM OF STUDY: This study investigated the potential mechanisms of DZSM against AD. MATERIALS AND METHODS: The six-month-old wild-type male mice and APP/PS1 double transgenic male mice were administered 0.9 % saline or DZSM for 8 weeks by gavage. Open field test, new object recognition test, and Morris Water maze test were used to assess spatial learning and memory. Aß plaques in brains were visualized using ThT staining. Nissl staining, TUNEL staining, and western blot analyses were used to detect the neuronal function and apoptosis level. The superoxide dismutase (SOD), glutathione peroxidase assay kit (GSH-Px), and malondialdehyde (MDA) kits were performed to assess oxidative stress levels. Then, immunofluorescence and western blot analysis were applied to evaluate ER stress pathway protein levels. Finally, HT22 cells were treated by Aß1-42 with or without DZSM medicated serum. Cell viability was assessed using the CCK-8 assay, and western blot analysis was applied to evaluate ER stress pathway protein levels. RESULTS: Open filed test, new object recognition test and Morris Water maze test showed that DZSM restored cognitive disorders in APP/PS1 mice. Immunohistochemistry and Thioflavin T staining results indicated that DZSM reduced Aß plaques in the brain. Deeper and denser Nissl bodies were found in APP/PS1 mice after DZSM administration. Besides, APP/PS1 mice treated with DZSM showed a lower level of TUNEL and Bax/Bcl-2 ratio. DZSM improved the acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity while reducing acetylcholinesterase (AChE) and malondialdehyde (MDA) activity. In addition, the levels of ER stress pathway containing Phospho-PKR-like ER kinase (P-PERK), phosphorylate eukaryotic initiation factor 2 (P-eIF2α), activating transcription factor 4 (ATF4), glutamine-rich protein 1 (QRICH1), phosphorylate inositol-requiring protein 1α (P-IRE1α), the spliced form of X-box binding protein 1 (XBP1s), activating transcription factor-6 (ATF6) and C/EBP homologous binding protein (CHOP) were decreased by DZSM. CCK-8 results indicated that DZSM medicated serum played cytoprotective effects on Aß1-42-induced HT22 cells. Western blot results suggested DZSM possibly inhibited ER stress pathways in Aß1-42-induced HT22 cells. CONCLUSION: The potential protective mechanism of DZSM on cognitive impairment in AD might be related to ER stress pathways.

Melatonin attenuates scopolamine-induced cognitive dysfunction through SIRT1/IRE1α/XBP1 pathway.

BACKGROUND: The prevalence of dementia around the world is increasing, and these patients are more likely to have cognitive impairments, mood and anxiety disorders (depression, anxiety, and panic disorder), and attention deficit disorders over their lifetime. Previous studies have proven that melatonin could improve memory loss, but its specific mechanism is still confused. METHODS: In this study, we used in vivo and in vitro models to examine the neuroprotective effect of melatonin on scopolamine (SCOP)-induced cognitive dysfunction. The behavioral tests were performed. 18F-FDG PET imaging was used to assess the metabolism of the brain. Protein expressions were determined through kit detection, Western blot, and immunofluorescence. Nissl staining was conducted to reflect neurodegeneration. MTT assay and RNAi transfection were applied to perform the in vitro experiments. RESULTS: We found that melatonin could ameliorate SCOP-induced cognitive dysfunction and relieve anxious-like behaviors or HT22 cell damage. 18F-FDG PET-CT results showed that melatonin could improve cerebral glucose uptake in SCOP-treated mice. Melatonin restored the cholinergic function, increased the expressions of neurotrophic factors, and ameliorated oxidative stress in the brain of SCOP-treated mice. In addition, melatonin upregulated the expression of silent information regulator 1 (SIRT1), which further relieved endoplasmic reticulum (ER) stress by decreasing the expression of phosphorylate inositol-requiring enzyme (p-IRE1α) and its downstream, X-box binding protein 1 (XBP1). CONCLUSIONS: These results indicated that melatonin could ameliorate SCOP-induced cognitive dysfunction through the SIRT1/IRE1α/XBP1 pathway. SIRT1 might be the critical target of melatonin in the treatment of dementia.

Transcriptomic decoding of regional cortical vulnerability to major depressive disorder.

Previous studies in small samples have identified inconsistent cortical abnormalities in major depressive disorder (MDD). Despite genetic influences on MDD and the brain, it is unclear how genetic risk for MDD is translated into spatially patterned cortical vulnerability. Here, we initially examined voxel-wise differences in cortical function and structure using the largest multi-modal MRI data from 1660 MDD patients and 1341 controls. Combined with the Allen Human Brain Atlas, we then adopted transcription-neuroimaging spatial correlation and the newly developed ensemble-based gene category enrichment analysis to identify gene categories with expression related to cortical changes in MDD. Results showed that patients had relatively circumscribed impairments in local functional properties and broadly distributed disruptions in global functional connectivity, consistently characterized by hyper-function in associative areas and hypo-function in primary regions. Moreover, the local functional alterations were correlated with genes enriched for biological functions related to MDD in general (e.g., endoplasmic reticulum stress, mitogen-activated protein kinase, histone acetylation, and DNA methylation); and the global functional connectivity changes were associated with not only MDD-general, but also brain-relevant genes (e.g., neuron, synapse, axon, glial cell, and neurotransmitters). Our findings may provide important insights into the transcriptomic signatures of regional cortical vulnerability to MDD.