Phosphoproteomic analysis of APP/PS1 mice of Alzheimer's disease by DIA based mass spectrometry analysis with PRM verification.

Traditional Chinese medicine has been utilized in China for approximately thousands of years in clinical settings to prevent Alzheimer's disease (AD) and enhance memory, despite the lack of a systematic exploration of its biological underpinnings. Exciting research has corroborated the beneficial effects of tetrahydroxy stilbene glycoside (TSG), an extract derived from Polygonum multiflorum, in delaying learning and memory impairment in a model that mimics AD. Therefore, the primary objective of this study is to investigate the major function of TSG upon protein regulation in AD. Herein, a novel approach, encompassing data independent acquisition (DIA), DIA phosphorylated proteomics, and parallel reaction monitoring (PRM), was utilized to integrate quantitative proteomic data collected from APP/PS1 mouse model exhibiting toxic intracellular aggregation of Aß. Initially, we deliberated upon both single and multi-dimensional data pertaining to AD model mice. Furthermore, we authenticated disparities in protein phosphorylation quantity and expression, phosphorylation function, and ultimately phosphorylation kinase analysis. In order to validate the results, we utilized PRM ion monitoring technology to identify potential protein or peptide biomarkers. In the mixed samples, targeted detection of 50 target proteins revealed that 26 to 33 target proteins were stably detected by PRM. In summary, our findings provide new candidates for AD biomarker, which have been identified and validated through protein researches conducted on mouse brains. This offers a wealth of potential resources for extensive biomarker validation in neurodegenerative diseases. SIGNIFICANCE: DIA phosphorylated proteomics technique was used to detect and analyze phosphorylated proteins in brain tissues of mice with AD. Data were analyzed by various bioinformatics tools to explore the phosphorylation events and characterize them related to TSG. The results of DIA were further verified by PRM. Besides, we mapped the major metabolite classes emerging from the analyses to key biological pathways implicated in AD to understand the potential roles of the molecules and the interactions in triggering symptom onset and progression of AD. Meanwhile, we clarified that in the context of AD onset and TSG intervention, the changes in proteins, protein phosphorylation, phosphorylation kinases, and the internal connections.

Ginsenoside Rg1 treats ischemic stroke by regulating CKLF1/CCR5 axis-induced neuronal cell pyroptosis.

BACKGROUND: Ischemic stroke, a severe and life-threatening neurodegenerative condition, currently relies on thrombolytic therapy with limited therapeutic window and potential risks of hemorrhagic transformation. Thus, there is a crucial need to explore novel therapeutic agents for ischemic stroke. Ginsenoside Rg1 (Rg1), a potential neuroprotective agent, exhibits anti-ischemic effects attributed to its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. Nevertheless, the precise underlying mechanism of action remains to be fully elucidated. PURPOSE: This study aimed to explore whether Rg1 exerts anti-ischemic stroke effects by inhibiting pyroptotic neuronal cell death through modulation of the chemokine like factor 1 (CKLF1)/ C-C chemokine receptor type 5 (CCR5) axis. METHODS: In this study, the MCAO model was used as an ischemic stroke model, and experimental tests were performed after 6 hours of ischemia. The anti-ischemic effect of Rg1 was examined by TTC staining, nissl-staining and neurobehavioral tests. In the in vitro experiments, PC12 cells were subjected to stimulation with CKLF1's mimetic peptide C27 to assess the potential of CKLF1 to induce focal neuronal cell death. Additionally, the impact of CKLF1 mimetic peptide C27, antagonistic peptide C19, and CCR5 inhibitor MVC on PC12 cells subjected to oxygen-glucose deprivation (OGD) and subsequently treated with Rg1 was investigated. In vivo, Rg1 treatment was examined by quantitative real-time PCR (qPCR), ELISA, immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and co-immunoprecipitate (Co-IP) assays to perspective whether Rg1 treatment reduces CKLF1/CCR5 axis-induced pyroptotic neuronal cell death. In addition, to further explore the biological significance of CKLF1 in ischemic stroke, CKLF1-/- rats were used as the observation subjects in this study. RESULTS: The in vitro results suggested that CKLF1 was able to induce neuronal cells to undergo pyroptosis. In vivo pharmacodynamic results showed that Rg1 treatment was able to significantly improve symptoms in ischemic stroke rats. In addition, Rg1 treatment was able to inhibit the interaction between CKLF1 and CCR5 after ischemic stroke and inhibited CKLF1/CCR5 axis-induced pyroptosis. The results of related experiments in CKLF1-/- rats showed that Rg1 lost its therapeutic effect after CKLF1 knockdown. CONCLUSION: Our findings indicate that the activation of the NLRP3 inflammasome is initiated by the CKLF1/CCR5 axis, facilitated through the activation of the NF-κB pathway, ultimately resulting in the pyroptosis of neuronal cells. Conversely, Rg1 demonstrates the capability to mitigate neuronal cell damage following CKLF1-induced effects by suppressing the expression of CKLF1. Thus, CKLF1 represents a crucial target for Rg1 in the context of cerebral ischemia treatment, and it also holds promise as a potential target for drug screening in the management of ischemic stroke.

Mechanisms of dihydromyricetin against hepatocellular carcinoma elucidated by network pharmacology combined with experimental validation.

CONTEXT: Dihydromyricetin (DMY) is extracted from vine tea, a traditional Chinese herbal medicine with anti-cancer, liver protection, and cholesterol-lowering effects. OBJECTIVE: This study investigated the mechanism of DMY against hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Potential DMY, HCC, and cholesterol targets were collected from relevant databases. PPI networks were created by STRING. Then, the hub genes of co-targets, screened using CytoHubba. GO and KEGG pathway enrichment, were performed by Metascape. Based on the above results, a series of in vitro experiments were conducted by using 40-160 µM DMY for 24 h, including transwell migration/invasion assay, western blotting, and Bodipy stain assay. RESULTS: Network pharmacology identified 98 common targets and 10 hub genes of DMY, HCC, and cholesterol, and revealed that the anti-HCC effect of DMY may be related to the positive regulation of lipid rafts. Further experiments confirmed that DMY inhibits the proliferation, migration, and invasion of HCC cells and reduces their cholesterol levels in vitro. The IC50 is 894.4, 814.4, 467.8, 1,878.8, 151.8, and 156.9 µM for 97H, Hep3B, Sk-Hep1, SMMC-7721, HepG2, and Huh7 cells, respectively. In addition, DMY downregulates the expression of lipid raft markers (CAV1, FLOT1), as well as EGFR, PI3K, Akt, STAT3, and Erk. DISCUSSION AND CONCLUSION: The present study reveals that DMY suppresses EGFR and its downstream pathways by reducing cholesterol to disrupt lipid rafts, thereby inhibiting HCC, which provides a promising candidate drug with low toxicity for the treatment of HCC.

Paeoniae Radix Rubra extract attenuates cerebral ischemia injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. AIM OF THE STUDY: The present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. MATERIALS AND METHODS: The neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. RESULTS: The in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. CONCLUSION: PRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

Oleanolic acid and ursolic acid: therapeutic potential in neurodegenerative diseases, neuropsychiatric diseases and other brain disorders.

Brain disorders such as neurodegenerative diseases and neuropsychiatric diseases have become serious threatens to human health and quality of life. Oleanolic acid (OA) and ursolic acid (UA) are pentacyclic triterpenoid isomers widely distributed in various plant foods and Chinese herbal medicines. Accumulating evidence indicates that OA and UA exhibit neuroprotective effects on multiple brain disorders. Therefore, this paper reviews researches of OA and UA on neurodegenerative diseases, neuropsychiatric diseases and other brain disorders including ischemic stroke, epilepsy, etc, as well as the potential underlying molecular mechanisms.

New Therapeutic Approaches to and Mechanisms of Ginsenoside Rg1 against Neurological Diseases.

Neurological diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), stroke, cerebral infarction, ischemia-reperfusion injury, depression and, stress, have high incidence and morbidity and often lead to disability. However, there is no particularly effective medication against them. Therefore, finding drugs with a suitable efficacy, low toxicity and manageable effects to improve the quality of life of patients is an urgent problem. Ginsenoside Rg1 (Rg1) is the main active component of ginseng and has a variety of pharmacological effects. In this review, we focused on the therapeutic potential of Rg1 for improving neurological diseases. We introduce the mechanisms of Ginsenoside Rg1 in neurological diseases, including apoptosis, neuroinflammation, the microRNA (miRNA) family, the mitogen-activated protein kinase (MAPK) family, oxidative stress, nuclear factor-κB (NF-κB), and learning and memory of Rg1 in neurological diseases. In addition, Rg1 can also improve neurological diseases through the interaction of different signal pathways. The purpose of this review is to explore more in-depth ideas for the clinical treatment of neurological diseases (including PD, AD, HD, stroke, cerebral infarction, ischemia-reperfusion injury, depression, and stress). Therefore, Rg1 is expected to become a new therapeutic method for the clinical treatment of neurological diseases.

Metabonomics Study on Naotaifang Extract Alleviating Neuronal Apoptosis after Cerebral Ischemia-Reperfusion Injury.

Naotaifang extract (NTE) is a clinically effective traditional Chinese medicine compound for cerebral ischemia-reperfusion injury. Although NTE can achieve neuroprotective function through different mechanisms, the pharmacodynamic substances of NTE corresponding to these mechanisms have rarely been reported. Alleviating or inhibiting neuronal apoptosis is an important way to achieve neuroprotection. Accordingly, this study has evaluated the effects of NTE on alleviating neuronal apoptosis after cerebral ischemia-reperfusion injury from two levels of cells and tissues. Meanwhile, the serum pharmacochemistry of NTE was analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with the guidance of Chinmedomics. The results included three aspects: (1) NTE could significantly alleviate neuronal apoptosis caused by in vitro cellular models and in vivo animal models; (2) a total of 21 serum differential metabolites was discovered, including adenosine, inosine, ferulic acid, calycosin, salidroside, 6-gingerol, 2-methoxycinnamaldehyde, and so on; (3) the metabolic pathway regulated by NTE was mainly purine metabolism. From these results, it can be concluded that alleviating neuronal apoptosis by NTE after cerebral ischemia-reperfusion injury is one of the important mechanisms to achieve neuroprotection. The pharmacodynamic substances of NTE for alleviating neuronal apoptosis on the one hand are related to components directly absorbed into blood, such as ferulic acid, calycosin, salidroside, 6-gingerol, and 2-methoxycinnamaldehyde and on the other hand are also closely linked to its indirect regulation of purine metabolism in the body to produce adenosine and inosine. Therefore, our research not only identified the main pharmacodynamic substances of NTE that alleviated neuronal apoptosis but also provided a methodological reference for studying other neuroprotective effects of NTE.

Novel antidepressant mechanism of ginsenoside Rg1: Regulating biosynthesis and degradation of connexin43.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Meyer is a valuable medicinal herb and "alternative" remedy for the prevention and treatment of depression. Dysfunction of connexin43 (Cx43)-gap junction in astrocytes is predisposed to the precipitation of depression. Ginsenoside Rg1 (Rg1), the main bioactive constituent extracted from ginseng, is efficacious in the management of depression by upregulating the content of Cx43. Our previous results indicated that pretreatment with Rg1 significantly improved Cx43-gap junction in corticosterone (CORT)-treated astrocytes. However, the antidepressant mechanism underlying how Rg1 upregulates Cx43-gap junction in astrocytes hasn't been proposed. AIM OF THE STUDY: To dissect the mechanisms of Rg1 controlling Cx43 levels in primary astrocytes. METHODS: We examined the changes of the level of Cx43 mRNA, the degradation of Cx43, as well as the ubiquitin-proteasomal and autophagy-lysosomal degradation pathways of Cx43 followed by Rg1 prior to CORT in rat primary astrocytes isolated from prefrontal cortex and hippocampus. Furthermore, the recognized method of scrape loading/dye transfer was performed to detect Cx43-gap junctional function, an essencial indicator of the antidepressant effect. RESULTS: Pretreatment with Rg1 could reverse CORT-induced downregulation of Cx43 biosynthesis, acceleration of Cx43 degradation, and upregulation of two Cx43 degradation pathways in primary astrocytes. CONCLUSION: The findings in the present study provide the first evidence highlighting that Rg1 increases Cx43 protein levels through the upregulation of Cx43 mRNA and downregulation of Cx43 degradation, which may be attributed to the effect of Rg1 on the ubiquitin-proteasomal and autophagy-lysosomal degradation pathways of Cx43.

The Anti-Neuroinflammatory Effect of Fuzi and Ganjiang Extraction on LPS-Induced BV2 Microglia and Its Intervention Function on Depression-Like Behavior of Cancer-Related Fatigue Model Mice.

The Chinese herb couple Fuzi and Ganjiang (FG) has been a classic combination of traditional Chinese medicine that is commonly used clinically in China for nearly 2000 years. Traditional Chinese medicine suggests that FG can treat various ailments, including heart failure, fatigue, gastrointestinal upset, and depression. Neuroinflammation is one of the main pathogenesis of many neurodegenerative diseases in which microglia cells play a critical role in the occurrence and development of neuroinflammation. FG has been clinically proven to have an efficient therapeutic effect on depression and other neurological disorders, but its mechanism remains unknown. Cancer-related fatigue (CRF) is a serious threat to the quality of life of cancer patients and is characterized by both physical and psychological fatigue. Recent studies have found that neuroinflammation is a key inducement leading to the occurrence and development of CRF. Traditional Chinese medicine theory believes that extreme fatigue and depressive symptoms of CRF are related to Yang deficiency, and the application of Yang tonic drugs such as Fuzi and Ganjiang can relieve CRF symptoms, but the underlying mechanisms remain unknown. In order to define whether FG can inhibit CRF depression-like behavior by suppressing neuroinflammation, we conducted a series of experimental studies in vitro and in vivo. According to the UPLC-Q-TOF/MSE results, we speculated that there were 49 compounds in the FG extraction, among which 30 compounds were derived from Fuzi and 19 compounds were derived from Ganjiang. Our research data showed that FG can effectively reduce the production of pro-inflammatory mediators IL-6, TNF-α, ROS, NO, and PGE2 and suppress the expression of iNOS and COX2, which were related to the inhibition of NF-κB/activation of Nrf2/HO-1 signaling pathways. In addition, our research results revealed that FG can improve the depression-like behavior performance of CRF model mice in the tail suspension test, open field test, elevated plus maze test, and forced swimming test, which were associated with the inhibition of the expression of inflammatory mediators iNOS and COX2 in the prefrontal cortex and hippocampus of CRF model mice. Those research results suggested that FG has a satisfactory effect on depression-like behavior of CRF, which was related to the inhibition of neuroinflammation.

Kanglaite enhances the efficacy of cisplatin in suppression of hepatocellular carcinoma via inhibiting CKLF1 mediated NF-κB pathway and regulating transporter mediated drug efflux.

ETHNOPHARMACOLOGICAL RELEVANCE: Kanglaite (KLT) is an active extract of the Coix lacryma-jobi seed, which can benefit Qi and nourish Yin, and disperse the accumulation of evils. It is used as a biphasic broad-spectrum anti-cancer drug, and shows synergistic effects with radiotherapy and chemotherapy. However, the mechanism of KLT combined with cisplatin (CDDP) against hepatocellular carcinoma (HCC) has not been elucidated. AIM OF THE STUDY: The aim of present study was to investigate the potential synergistic effects of KLT and CDDP on HepG2 cells, discussing the possible mechanisms from the perspective of CKLF1 and NF-κB mediated inflammatory response and chemoresistance, and the involvement of drug efflux transporters. MATERIALS AND METHODS: CDDP injured HepG2 cells were used to investigate the effects of KLT on chemotherapeutics treated HCC. Effects of KLT pretreatment on CDDP injured HepG2 cells were determined by MTT, wound healing assay, and transwell assay. Expression of chemokine-like factor 1 (CKLF1) and activation of nuclear factor κB (NF-κB) were examined by qPCR, western blot, and immunofluorescence staining. Furthermore, to study the role of CKLF1 in KLT mediated effects on this CDDP injured HCC cell model, HepG2 cells overexpressed with CKLF1 gene were used. Cell viability and NF-κB activation were investigated. Moreover, TNF-α and IL-1ß levels were measured by Elisa analysis and western blot to evaluate the inflammatory response. Additionally, ATP-binding cassette (ABC) drug efflux transporters, MDR1, MRP2, and BCRP were also determined in present study. RESULTS: KLT pretreatment followed by CDDP treatment was found to show synergistic effects, which showed by decreased cell viability, migration and invasion ability of HepG2 cells. Expression of CKLF1 enhanced significantly in CDDP treated HepG2 cells, and KLT decreased this elevation obviously. Furthermore, CDDP activated NF-κΒ and promoted translocation of NF-κB toward the nucleus. KLT inhibited the activation of NF-κΒ, which sensitized cancer cells. Overexpression of CKLF1 reversed the effects of KLT on CDDP injured HepG2 cells, which exhibited by increased cell viability and enhanced activation of NF-κΒ. CDDP induced NF-κΒ activation could also lead to excessive inflammatory response, and KLT can suppress the aggravating inflammation which may be beneficial for tumor progression. Furthermore, we found that ABC drug efflux transporters MDR1, MRP2, and BCRP in CDDP treated HepG2 cells were decreased when pretreated with KLT. CONCLUSIONS: KLT pretreatment may increase the effects of CDDP on HepG2 cells, by exhibiting cooperative effects on suppression of HepG2 cells. The mechanisms may partly by inhibiting CKLF1 mediated NF-κB pathway, which may contribute to inflammation of tumor microenvironment and chemoresistance of CDDP. Inhibition of transporter-mediated drug efflux is also involved in KLT mediated sensitization effects of CDDP.

SIRT1/AMPK and Akt/eNOS signaling pathways are involved in endothelial protection of total aralosides of Aralia elata (Miq) Seem against high-fat diet-induced atherosclerosis in ApoE-/- mice.

Total aralosides of Aralia elata (Miq) Seem (TASAESs) possess multiple pharmacological activity, such as anti-inflammation, antioxidation, and antiapoptosis. However, there is no literature reporting the antiatherosclerotic effect and mechanism of TASAES so far. The aim of this study was to investigate the antiatherosclerotic effects in high-fat diet-induced ApoE-/- mice and potential mechanism of TASAES in ox-LDL-injured endothelial cells. In vivo assay, our data demonstrated that TASAES significantly reduced the atherosclerotic plaque size and caspase-3 expression level in aortic valve. In vitro, we found that TASAES could increase endothelial cell viability, attenuated mitochondrial membrane potential depolarization, and endothelial cells apoptosis. In addition, we found that TASAES could activate SIRT1/AMPK and Akt/eNOS signaling pathways. Importantly, EX527, SIRT1 siRNA, and LY294002, Akt siRNA, remarkably abolished the antiapoptotic effects of TASAES. In conclusion, this study demonstrated that SIRT1/AMPK and Akt/eNOS signaling pathways are involved in endothelial protection of TASAES against atherosclerotic mice, suggesting that TASAES is a candidate drug for atherosclerosis treatment.

Ginsenoside RK3 Prevents Hypoxia-Reoxygenation Induced Apoptosis in H9c2 Cardiomyocytes via AKT and MAPK Pathway.

Reperfusion therapy is widely utilized for acute myocardial infarction (AMI), but further injury induced by rapidly initiating reperfusion of the heart is often encountered in clinical practice. Ginsenoside RK3 (RK3) is reportedly present in the processed Radix notoginseng that is often used as a major ingredient of the compound preparation for ischemic heart diseases. This study aimed to investigate the possible protective effect of RK3 against hypoxia-reoxygenation (H/R) induced H9c2 cardiomyocytes damage and its underlying mechanisms. Our results showed that RK3 pretreatment caused increased cell viability and decreased levels of LDH leakage compared with the H/R group. Moreover, RK3 pretreatment inhibited cell apoptosis, as evidenced by decreased caspase-3 activity, TUNEL-positive cells, and Bax expression, as well as increased Bcl-2 level. Further mechanism investigation revealed that RK3 prevented H9c2 cardiomyocytes injury and apoptosis induced by H/R via AKT/Nrf-2/HO-1 and MAPK pathways. These observations indicate that RK3 has the potential to exert cardioprotective effects against H/R injury, which might be of great importance to clinical efficacy for AMI treatment.