Investigating the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles in atherosclerosis treatment.

BACKGROUND: Carthamus tinctorius L., a traditional herbal medicine used for atherosclerosis (AS), lacks a clear understanding of its therapeutic mechanisms. This study aimed to investigate the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles (CDNVs) in AS treatment. METHODS: CDNVs were isolated and characterized using improved isolation methods. Transmission electron microscopy, nanoparticle tracking analysis, and protein analysis confirmed their morphology, size, and protein composition. Small RNA sequencing was performed to identify the miRNA profile of CDNVs, and bioinformatics analysis was used to determine their potential biological roles. In vivo biodistribution and toxicity studies were conducted in mice to assess the stability and safety of orally administered CDNVs. The anti-atherosclerotic effects of CDNVs were evaluated in ApoE-/- mice through plaque burden analysis. The protective effects of CDNVs on ox-LDL-treated endothelial cells were assessed through proliferation, apoptosis, reactive oxygen species activation, and monocyte adhesion assays. miRNA and mRNA sequencing of CDNV-treated endothelial cells were performed to explore their regulatory effects and potential target genes. RESULTS: CDNVs were successfully isolated and purified from Carthamus tinctorius L. tissue lysates. They exhibited a saucer-shaped or cup-shaped morphology, with an average particle size of 142.6 ± 0.7 nm, and expressed EV markers CD63 and TSG101. CDNVs contained proteins, small RNAs, and metabolites, including the therapeutic compound HSYA. Small RNA sequencing identified 95 miRNAs, with 10 common miRNAs accounting for 72.63% of the total miRNAs. These miRNAs targeted genes involved in cell adhesion, apoptosis, and cell proliferation, suggesting their relevance in cardiovascular disease. Orally administered CDNVs were stable in the gastrointestinal tract, absorbed into the bloodstream, and accumulated in the liver, lungs, heart, and aorta. They significantly reduced the burden of atherosclerotic plaques in ApoE-/- mice and exhibited superior effects compared to HSYA. In vitro studies demonstrated that CDNVs were taken up by HUVECs, promoted proliferation, attenuated ox-LDL-induced apoptosis and ROS activation, and reduced monocyte adhesion. CDNV treatment resulted in significant changes in miRNA and mRNA expression profiles of HUVECs, with enrichment in inflammation-related genes. CXCL12 was identified as a potential direct target of miR166a-3p. CONCLUSION: CDNVs isolated from Carthamus tinctorius L. tissue lysates represent a promising oral therapeutic option for cardiovascular diseases. The delivery of miRNAs by CDNVs regulates inflammation-related genes, including CXCL12, in HUVECs, suggesting their potential role in modulating endothelial inflammation. These findings provide valuable insights into the therapeutic potential of CDNVs and their miRNAs in cardiovascular disease.

Saikosaponin B2 ameliorates depression-induced microglia activation by inhibiting ferroptosis-mediated neuroinflammation and ER stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Saikosaponins B2 (SSB2) is one of the main active components isolated from Radix Bupleuri (Bupleurum chinense DC.), a herb widely used of traditional Chinese medicine. It has been used for the treatment of depression for more than two thousand years. However, the molecular mechanisms remain to be determined. AIM OF THE STUDY: In this study, we evaluated the anti-inflammatory effect and elucidated underlying molecular mechanisms of SSB2 in LPS-induced primary microglia and CUMS-induced mice model of depression. METHOD: The effects of SSB2 treatment were investigated both in vitro and in vivo. The chronic unpredictable mild stimulation (CUMS) procedure was applied to establish the animal model of depression. Behavioural tests were used to evaluate the depressive-like behaviors in CUMS-exposed mice, including sucrose preference test, open field test, tail suspension test, and forced swimming test. The GPX4 gene of microglia was silenced using shRNA, and inflammatory cytokines were determined by Western Blot and immunofluorescence analysis. Endoplasmic reticulum stress and ferroptosis-related markers were detected by qPCR, flow cytometry and confocal microscopy. RESULT: SSB2 reversed depressive-like behaviours in CUMS-exposed mice and relieved central neuroinflammation and ameliorated hippocampal neural damage. SSB2 alleviated LPS-induced activation of microglia through the TLR4/NF-κB pathway. LPS-induced ferroptosis, with increased levels of ROS, intracellular Fe2+, mitochondrial membrane potential, lipid peroxidation, GSH, SLC7A11, FTH, GPX4 and Nrf2, and decreased transcription levels of ACSL4 and TFR1, was attenuated with SSB2 treatment in primary microglia cells. GPX4 knockdown activated ferroptosis, induced endoplasmic reticulum (ER) stress, and abrogated the protective effects of SSB2. Further, SSB2 attenuated ER stress, balanced calcium homeostasis, reduced lipid peroxidation and intracellular Fe2+ content by regulating the level of intracellular Ca2+. CONCLUSIONS: Our study suggested that SSB2 treatment can inhibit ferroptosis, maintain calcium homeostasis, relieve endoplasmic reticulum stress and attenuate central neuroinflammation. SSB2 exhibited anti-ferroptosis and anti-neuroinflammatory effects through the TLR4/NF-κB pathway in a GPX4-dependent manner.

Zanthoxylum armatum DC. extract induces liver injury via autophagy suppression and oxidative damage by activation of mTOR/ULK1 pathway.

Zanthoxylum armatum DC. (ZADC) has anti-inflammatory, antioxidative, and antibacterial effects. The cytotoxicity of methanol extract of Zanthoxylum armatum DC. (MZADC) has been reported for BRL 3 A cell lines. However, whether MZADC can induce liver damage in vivo remains unclear. Therefore, it is essential to explore whether ZADC causes liver injury and, if the results confirm hepatotoxicity, to further study the potential mechanisms for the in-vitro cytotoxicity of the BRL 3 A cell lines. In vivo, different doses (0.346, 0.519, and 1.038 g/kg/day) of MZADC treatment were given by intragastric administration among male Sprague Dawley rats for 28 days. Levels of serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in the high dose group increased. Steatosis and focal necrosis were found in liver cells in rats in the high dose group. In vitro, BRL 3 A cells were cultivated with MZADC at different concentrations (30, 50, and 70 µg/mL) for 24 h. The cell viability, the number of autophagosomes, and the expression levels of LC3 and Beclin-1 were on a decreasing trend. Besides, proportions of p-mTOR/mTOR and p-ULK1/ULK1 increased. Meanwhile, reactive oxygen species (ROS) accumulation and the content of malondialdehyde (MDA) were on the rise while the activity of superoxide dismutase (SOD) and the content of glutathione (GSH) was on the decline. This research suggests that MZADC may cause rats liver injury and inhibit autophagy in BRL 3 A cells by the mTOR/ULK1 pathway, and further induce intracellular oxidative damage.

Aucubin Exerts Anticancer Activity in Breast Cancer and Regulates Intestinal Microbiota.

Aucubin, a natural compound isolated from herbal medicine, has been reported to possess multiple beneficial properties. In this study, we aimed to verify the anticancer effect of aucubin on breast cancer and investigate the effect of cancer on the intestinal flora and whether aucubin has a therapeutic effect on intestinal problems caused by cancer. We established the breast cancer model with mouse 4T1 cell line and BALB/c mice. Aucubin was given once a day by gavage for 14 days. The results showed that aucubin suppress the growth of tumor in vivo by inducing tumor cell apoptosis. The tumor suppression rate of aucubin could reach 51.31 ± 4.07%. Organ histopathology was evaluated by tissue staining, which demonstrated that aucubin could alleviate the organ inflammatory damage caused by breast cancer without visible side effects. Moreover, aucubin could increase the expression of colonic tight junction protein occluding and adjust the gut microbiome to normal level according to 16S rDNA high-throughput sequencing. Herein, our results provide evidence for developing aucubin as an alternative and safe therapeutic for breast cancer treatment.

Aconitine induces autophagy via activating oxidative DNA damage-mediated AMPK/ULK1 signaling pathway in H9c2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum species, with a medicinal history of 2000 years, was traditionally used in the treatment of rheumatism, arthritis, bruises, and pains. However, many studies have reported that Aconitum species can cause arrhythmia in experimental animals, resulting in myocardial fibrosis and cardiomyocyte damage. Cardiotoxicity is the main toxic effect of aconitine, but the detailed mechanism remains unclear. AIM OF THE STUDY: This study aimed to explore the effects and underlying mechanism of autophagy in H9c2 cardiomyocytes induced by aconitine. MATERIALS AND METHODS: H9c2 cells were incubated with different concentrations of aconitine for 24 h, and the intervention sections were pretreated with various inhibitors for 1 h. The effects of aconitine on the oxidative DNA damage, autophagy and viability of H9c2 cells were evaluated by flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and Western blot. RESULTS: In H9c2 cells, the cell viability declined, LDH release rate, the number of autophagosomes, protein expression levels of LC3 and Beclin-1 increased significantly after 24 h of aconitine incubation. The pretreatment of autophagy inhibitor 3-MA decreased markedly autophagosomes and protein expression levels of LC3 and Beclin-1, which suggested that aconitine could induce cell autophagy. The significant increase of ROS and 8-OHdG showed that aconitine could cause oxidative DNA damage through ROS accumulation. Meanwhile, treatment of aconitine dramatically increased AMPKThr172 and ULK1Ser317 phosphorylation, and Compound C inhibited AMPKThr172 and ULK1Ser317 phosphorylation, which proved that aconitine induced autophagy via AMPK activation mediated ULK1 phosphorylation. Antioxidant NAC significantly reduced LDH, ROS and 8-OHdG, inhibited the phosphorylation of AMPKThr172 and ULK1Ser317, and down-regulated autophagosomes and proteins expression levels of LC3 and Beclin-1. Consequently, the inhibition of oxidative DNA damage and AMPK/ULK1 signaling pathway alleviated the aconitine-induced autophagic death of H9c2 cells. CONCLUSIONS: These results showed that aconitine induces autophagy of H9c2 cardiomyocytes by activating AMPK/ULK1 signaling pathway mediated by oxidative DNA damage. The autophagy induced by aconitine in cardiomyocytes is dependent on the activation of the AMPK pathway, which may provide novel insights into the prevention of aconitine-related toxicity.

Activation of ATM/Chk2 by Zanthoxylum armatum DC extract induces DNA damage and G1/S phase arrest in BRL 3A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC is a traditional medicinal plant. It is widely used in clinical treatment and disease prevention in China, India and other regions. Modern studies have reported the phytotoxicity, cytotoxicity and the animal toxicity of Zanthoxylum armatum DC, and the damage of genetic material has been observed in plants, but the detailed mechanism has not been explored. Besides, the toxicity of normal mammalian cells has not been evaluated. AIM OF THE STUDY: To evaluate the effects and underlying mechanism of genetic material damage in BRL 3A cells induced by Zanthoxylum armatum DC. MATERIALS AND METHODS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry was used for identification of compounds in methanol extract of Zanthoxylum armatum DC. BRL 3A cells were incubated with different concentrations of methanol extract of Zanthoxylum armatum DC (24 h). The cytotoxicity of extract was assessed with cell viability, LDH release rate, and ROS production. The damage of genetic material was assessed with OTM value of comet cells, cell cycle and the expression levels of p-ATM, p- Chk2, Cdc25A, and CDK2. RESULTS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry investigation revealed the presence of compounds belonging to flavonoid, fatty acid and alkaloid groups. The viability of BRL 3A cells was reduced in a time-dose dependent manner treated by methanol extract of Zanthoxylum armatum DC. It increased LDH release rate and ROS production, activated the DNA double strand damage marker of γH2AX and produced comet cells. In addition, methanol extract of Zanthoxylum armatum DC caused ATM-mediated DNA damage, further phosphorylated Chk2, inhibited cell cycle related proteins, and arrested the G1/S cycle. CONCLUSIONS: Methanol extract of Zanthoxylum armatum DC induces DNA damage and further leads G1/S cell cycle arrest by triggering oxidative stress in the BRL 3A cells. This study provides some useful evidences for its development as an antitumor drug via activation of ATM/Chk2.

Banxia Xiexin Decoction in the treatment of chronic atrophic gastritis: A protocol for systematic review and meta-analysis.

BACKGROUND: Chronic atrophic gastritis (CAG) is a common digestive disease. Without active treatment, it may induce gastric cancer. Western medicine has a certain effect on chronic atrophic gastritis, but there are many adverse reactions after long-term medication, and the disease is prone to relapse after treatment, which will affect the health and life of patients. Traditional Chinese medicine has obvious advantages in the treatment of chronic stomach diseases with reliable effect. A number of clinical data have also confirmed that Banxia Xiexin decoction has significant effect in the treatment of chronic atrophic gastritis, but there is no evidence of evidence-based medicine. Therefore, this study aims to explore the clinical efficacy and safety of Banxia Xiexin decoction in the treatment of chronic atrophic gastritis by means of systematic evaluation. METHOD: Databases including PubMed, The Cochrance Library, Embase, Web of Science, CNKI, VIP, and Wanfang were searched by computer. Besides, Baidu Scholar and Google Scholar were manually searched, and all randomized controlled trials of Banxia xiexin decoction for the treatment of chronic atrophic gastritis were collected. The retrieval time was from the establishment of the database to July 31, 2020. After 2 reviewers independently screened the literature, extracted the data and evaluated the bias risk of the included study, RevMan5.3 software (developed by the UK's International Cochrane Collaboration) was used to analyze the data. RESULTS: In this study, the effectiveness and safety of Banxia Xiexin decoction for the treatment of chronic atrophic gastritis were evaluated by the clinical efficiency, traditional Chinese medicine syndrome score (traditional Chinese medicine syndrome score), quality of life score, gastrin level, epidermal growth factor, eradication rate of helicobacter pylori and incidence of adverse reactions. CONCLUSION: This study will provide reliable evidence for the clinical application of Banxia Xiexin decoction in the treatment of chronic atrophic gastritis. ETHICS AND DISSEMINATION: The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/7K6QW.

[Science of Meridians, Collaterals and Acupoints--Exploration on teaching method of meridian syndromes].

Meridian syndromes are the required basic knowledge for mastering Science of Meridians, Collaterals and Acupoints but have not brought the adequate attention on the teaching program. The writers discovered' that the content of this section occupied a decisive role for developing the students' clinical thinking ability and, stimulating their interests to learn classical TCM theories. It's necessary to enhance the importance on meridian syndromes during teaching program. The teaching program was discussed in three aspects, named workshop pattern, competitive pattern and multimedia pattern. This teaching method may improve students' interests in the study on classical TCM theories, deepen the understanding on knowledge and motivate students' learning autonomy so that the teaching quality can be improved.