Disulfiram/Copper Induce Ferroptosis in Triple-Negative Breast Cancer Cell Line MDA-MB-231.

BACKGROUND: The complex formed by disulfiram (DSF) and copper (Cu) is safe and effective for the prevention and treatment of triple-negative breast cancer (TNBC). Although previous studies have shown that DSF/Cu induces ferroptosis, the mechanism remains unclear. METHODS: The mitochondrial morphology of TNBC treated with DSF/Cu was observed by transmission microscopy, and intracellular levels of iron, lipid reactive oxygen species (ROS), malondialdehyde, and glutathione were evaluated to detect the presence of ferroptosis. Target genes for the DSF/Cu-activated ferroptosis signaling pathway were examined by transcriptome sequencing analysis. Expression of the target gene, HOMX1, was detected by qRT-PCR, immunofluorescence and western blot. RESULTS: The mitochondria of TNBC cells were significantly atrophied following treatment with DSF/Cu for 24 h. Addition of DSF/Cu supplement resulted in significant up-regulation of intracellular iron, lipid ROS and malondialdehyde levels, and significant down-regulation of glutathione levels, all of which are important markers of ferroptosis. Transcriptome analysis confirmed that DSF/Cu activated the ferroptosis signaling pathway and up-regulated several ferroptosis target genes associated with redox regulation, especially heme oxygenase-1 (HMOX-1). Inhibition of ferroptosis by addition of the ROS scavenger N-acetyl-L-cysteine (NAC) significantly increased the viability of DSF/Cu-treated TNBC cells. CONCLUSIONS: These results show that DSF/Cu increases lipid peroxidation and causes a sharp increase in HMOX1 activity, thereby inducing TNBC cell death through ferroptosis. DSF/Cu is a promising therapeutic drug for TNBC and could lead to ferroptosis-mediated therapeutic strategies for human cancer.

A Mechanistic Exploratory Study on the Therapeutic Efficacy of Astragaloside IV Against Diabetic Retinopathy Revealed by Network Pharmacology.

Purpose: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus, which nearly happens to all the diabetic sufferers. This study aims to identify the preliminary molecular regulation involved in the therapeutic efficacy of astragaloside IV (AS- IV) for DR. Methods: Diabetic rat models were established and treated with AS-IV. Optical coherence tomography (OCT) and Hematoxylin-eosin (HE) staining was employed to demonstrate the histopathological changes. The main targets of AS-IV were identified by searching from public databases of traditional Chinese medicine (GeneCards, PharmMapper and Swiss Target Prediction). Besides, disease targets of DR were also obtained by integrated data from GEO datasets and predicted from public databases. Protein-protein interaction (PPI) network was constructed by Cytoscape with overlapping genes and 10 core targets were selected, on which Gene Ontology (GO) along with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted. The interaction between AS-IV and these crucial genes were analyzed using molecular docking. RT-qPCR and western blot were used to verify the expression variation of core targets. Results: OCT imaging and HE staining demonstrated that AS-IV administration significantly increased retinal thickness in diabetic rats, obviously alleviating DR induced histopathological changes as well as elevated blood glucose levels. 107 common targets of AS-IV and DR were determined after intersection. PPI network analysis filtered 10 hub genes potentially targeted by AS-IV, including VEGFA, CASP3, HIF1α, STAT3, CTNNB1, SRC, AKT1, EGFR, IL1ß and IL6. Enrichment analysis indicated that these genes were mainly enriched in biological processes like T cell activation, epithelial cell proliferation and protein kinase B signaling, and involved in oxidative stress, apoptosis and inflammation-related pathways. The molecular docking prediction suggested that AS-IV exhibited stable binding to these core targets. In addition, mRNA levels of core targets in diabetic rats were differentially expressed before and after AS-IV treatment. Western blot further revealed that AS-IV treatment elevated DR-depressed protein levels of PI3K and AKT. Conclusion: Our study elucidated the effect of AS-IV in attenuating retinopathy induced by diabetes in rats and preliminarily unveiled the therapeutic efficacy of AS-IV in the treatment of DR might be attributed to activation of PI3K-AKT signaling pathway.

Impact of Acupuncture on Sleep and Comorbid Symptoms for Chronic Insomnia: A Randomized Clinical Trial.

STUDY OBJECTIVES: To evaluate the efficacy and safety of acupuncture at HT 7 (Shenmen) and KI 7 (Fuliu) on sleep and comorbid symptoms for chronic insomnia. METHODS AND DESIGN: A randomized, single-blind, parallel and sham-controlled trial consisted of an acupuncture group (n = 41) and a sham acupuncture group (n = 41). Setting: a tertiary hospital of integrated Chinese and Western medicine. Participants: 82 subjects with chronic insomnia based on the International Classification of Sleep Disorders, Third Edition (ICSD-3). Interventions: a 10-session acupuncture treatment at bilateral HT 7 and KI 7 or sham acupoints with shallow needling was performed over 3 weeks. Measurements: the Pittsburgh sleep quality index (PSQI) and insomnia severity index (ISI) were evaluated at baseline, posttreatment, and at two follow-ups as the primary outcome measures. Polysomnography (PSG) on two consecutive nights, the Beck anxiety inventory (BAI), the Beck depression inventory (BDI) fatigue severity scale (FSS) and the Epworth sleepiness scale (ESS) were evaluated at baseline and posttreatment as the secondary outcome measures. RESULTS: After the treatments, PSQI scores decreased by 5.04 in the acupuncture group and 2.92 in the sham acupuncture group. ISI scores decreased by 7.65 in the acupuncture group and 5.05 in the sham acupuncture group. The between-group differences in the primary outcome measures posttreatment were statistically significant. However, no differences were found between the two groups during the two follow-ups. Regarding the PSG data, there were significantly lower levels of sleep onset latency (SOL), a lower percentage of sleep stage N1 and a higher percentage of sleep stage N3 in the acupuncture group than in the sham acupuncture group. After treatment, there were lower levels of comorbid symptoms (BAI, BDI, FSS and ESS) in both groups. However, no significant differences were observed between the groups. CONCLUSION: Acupuncture at HT 7 and KI 7 is an effective and safe nonpharmacologic intervention option for chronic insomnia. CLINICAL TRIAL REGISTRATION: The study was registered at the Chinese Clinical Trial Registry, registration ID: ChiCTR1900023787, China.

Effects of Electroacupuncture on Sleep via the Dopamine System of the HPA Axis in Rats after Cage Change.

BACKGROUND: Insomnia is often related to stressful events. The hypothalamus-pituitary-adrenal (HPA) axis is related to stress, and dopamine (DA) and DA receptors are involved in the regulation of HPA axis. Electroacupuncture (EA) can improve sleep in individuals with insomnia, but the mechanism is unclear. We demonstrated that EA can improve sleep in rats after cage change through DA and the DA receptors in the HPA axis. METHODS: A rat model of insomnia was established by cage change to a dirty cage. The rats in treatment groups were intervened by EA and D1R (or D2R) antagonists. Electroencephalography (EEG) and electromyogram (EMG) were recorded to compare the changes in sleep. The DA, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and cortisol (CORT) levels in the plasma and hypothalamus were measured by ELISAs, and the D1R and D2R levels were measured by RT-PCR and immunohistochemistry. RESULTS: The dirty group showed a significant increase in the amount of wakefulness and decrease in the amount of NREM sleep, with decreased numbers of long NREM sleep bouts and REM sleep bouts and increased mean duration of wakefulness during the light period. EA and D1R (or D2R) antagonists intervention could improve sleep disturbance by decreasing wakefulness in the light period after cage change, EA and D1R (or D2R) antagonists could increase the hypothalamus DA, CRH, ACTH, CORT level, and the D1R and D2R mRNA levels in the HPA axis, and the effect of EA plus D1R (or D2R) antagonist was not superior to that of EA or D1R (or D2R) antagonists alone. CONCLUSIONS: EA can improve the sleep of rats after cage change, and the mechanism may be related to the regulation of DA and D1R or D2R in the HPA axis.

Acupuncture for insomnia with short sleep duration: protocol for a randomised controlled trial.

INTRODUCTION: Insomnia with short sleep duration has a more serious negative impact on patient health. The existing literature suggests that medication therapy is more effective for this phenotype of insomnia compared with cognitive-behavioural therapy. However, the potential side effects of hypnotic medications hinder their clinical application. Acupuncture has been widely used in the treatment of insomnia, but it remains unclear whether it has therapeutic efficacy for insomnia with short sleep duration. The purpose of this trial is to evaluate the efficacy and safety of acupuncture for insomnia with short sleep duration. METHODS AND ANALYSIS: This study is designed as a randomised, single-centre, single-blinded, placebo acupuncture controlled trial involving 152 participants. Eligible patients will be divided into two groups according to the objective total sleep time: insomnia with normal sleep duration group and insomnia with short sleep duration group. Then, patients in each group will be randomly assigned to two subgroups, the treatment group (acupuncture) and the control group (placebo acupuncture), in a 1:1 ratio with 38 subjects in each subgroup. The primary outcome is the Pittsburgh Sleep Quality Index and the Insomnia Severity Index. Secondary outcomes are actigraphy, the Beck Anxiety Inventory, the Beck Depression Inventory and the Fatigue Severity Scale. All adverse effects will be assessed by the Treatment Emergent Symptom Scale. Outcomes will be evaluated at baseline, post treatment, as well as at 1-week and 1-month follow-up. ETHICS AND DISSEMINATION: This protocol has been approved by the ethics committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (no. 2019-17). Written informed consent will be obtained from all participants. The results will be disseminated through peer-reviewed journals for publications. TRIAL REGISTRATION NUMBER: ChiCTR1900023473; Pre-results.

Acupuncture Improves Peri-menopausal Insomnia: A Randomized Controlled Trial.

Study Objectives: To evaluate the short-term efficacy of acupuncture for the treatment of peri-menopausal insomnia (PMI). Methods: Design: A randomized, participant-blind, placebo-controlled trial consisted of the acupuncture group (n = 38) and placebo-acupuncture group (n = 38). Setting: A tertiary teaching and general hospital. Participants: 76 peri-menopausal women with insomnia disorder based on the International Classification of Sleep Disorders, Third Edition. Interventions: A 10-session of acupuncture at bilateral Shenshu (BL 23) and Ganshu (BL 18) with unilateral Qimen (LR 14) and Jingmen (GB 25) or Streitberger needles at the same acupoints was performed for over 3 weeks. Measurements: Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI) with over-night polysomnography (PSG) exam were completed at baseline and post-treatment. Results: After the treatments, the decrease from baseline in PSQI score was 8.03 points in acupuncture group and 1.29 points in placebo-acupuncture group. The change from baseline in ISI score was 11.35 points in acupuncture group and 2.87 points in placebo-acupuncture group. In PSG data, acupuncture significantly improved the sleep efficiency and total sleep time, associated with less wake after sleep onset and lower percent stage 1 after the treatment. No significant differences from baseline to post-treatment were found in placebo-acupuncture group. Conclusions: Acupuncture can contribute to a clinically relevant improvement in the short-term treatment of PMI, both subjectively and objectively. Clinical Trial Registration: Acupuncture for peri-menopause insomnia: a randomized controlled trial, http://www.chictr.org.cn/showproj.aspx?proj=12118 ChiCTR-IPR-15007199, China.

Electroacupuncture attenuates collagen-induced arthritis in rats through vasoactive intestinal peptide signalling-dependent re-establishment of the regulatory T cell/T-helper 17 cell balance.

OBJECTIVE: Imbalance between T-helper 17 (Th17) cells and regulatory T (Treg) cells is causally linked to the development of rheumatoid arthritis (RA). In this study, we tested the hypothesis that electroacupuncture (EA) confers therapeutic benefits in RA through activation of vasoactive intestinal peptide (VIP)-dependent signalling and restoration of the Th17/Treg cell balance. MATERIALS AND METHODS: A collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by injection of bovine type II collagen in incomplete Freund's adjuvant on day 0 and day 7. Three days after the second injection, EA was given at acupuncture points GB39 and ST36 three times per week for 4 weeks. To block VIP signalling, [D-P-Cl-Phe(6)-Leu(17)]-VIP, a VIP receptor antagonist, was administered intraperitoneally 30 min before EA. Inflammatory and pathological responses in the joint were assessed. Synovial VIP receptor mRNA levels and Treg and Th17 cell frequencies in the spleen were determined. RESULTS: EA significantly reduced the severity of CIA, as evidenced by reduced paw volumes, arthritis scores and inflammation scores. EA significantly increased mRNA expression of the VIP receptor VPAC1 and led to an elevation in CD4(+)FOXP3(+) Treg cell frequency and a reduction in CD4(+)IL17(+) Th17 cell frequency. Pre-injection of a VIP receptor antagonist significantly reversed EA-induced expansion of Treg cells, but did not alter the frequencies of Th17 cells. CONCLUSIONS: EA exerts anti-inflammatory effects in a collagen-induced rat model of arthritis. These effects appear to be mediated through activation of VIP signalling and re-establishment of the Th17/Treg cell balance.

Radiographic healing after a root canal treatment performed in single-rooted teeth with and without ultrasonic activation of the irrigant: a randomized controlled trial.

INTRODUCTION: The aim of this study was to compare the outcome of a root canal treatment with and without additional ultrasonic activation of the irrigant. METHODS: Single-rooted teeth with radiographic evidence of periapical bone loss were randomly assigned to 2 treatment groups. In both groups syringe irrigation was performed, and in one group the irrigant was also activated by ultrasound. Ten to 19 months after treatment, the teeth were examined by using periapical radiography (PA) and cone-beam computed tomography (CBCT). Area and volume of the periapical lesions were measured, and the outcome was presented in 4 categories: absence, reduction or enlargement of the radiolucency, or uncertain. Lesions were classified as reduced or enlarged when the change in size of the radiolucency was 20% or more. RESULTS: The recall rate was 82%, and 84 teeth were analyzed. CBCT detected significantly more post-treatment lesions than PA (P = .038), but the percentages of absence and reduction of the radiolucency together revealed by CBCT and PA were similar (P = .383). The CBCT results showed that absence of the radiolucency was observed in 16 of 84 teeth (19%) and reduction of the radiolucency in 61 of 84 teeth (72.6%), but there was no significant difference between the results of the 2 groups (P = .470). Absence and reduction of the radiolucency together were observed in the ultrasonic group in 39 of 41 teeth (95.1%) and in the syringe group in 38 of 43 teeth (88.4%). CONCLUSIONS: Root canal treatments with and without additional ultrasonic activation of the irrigant contributed equally to periapical healing.