Berberine Inhibits Ferroptosis and Stabilizes Atherosclerotic Plaque through NRF2/SLC7A11/GPX4 Pathway.

OBJECTIVE: To investigate potential mechanisms of anti-atherosclerosis by berberine (BBR) using ApoE-/- mice. METHODS: Eight 8-week-old C57BL/6J mice were used as a blank control group (normal), and 56 8-week-old AopE-/- mice were fed a high-fat diet for 12 weeks, according to a completely random method, and were divided into the model group, BBR low-dose group (50 mg/kg, BBRL), BBR medium-dose group (100 mg/kg, BBRM), BBR high-dose group (150 mg/kg, BBRH), BBR+nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor group (100 mg/kg BBR+30 mg/kg ML385, BBRM+ML385), NRF2 inhibitor group (30 mg/kg, ML385), and positive control group (2.5 mg/kg, atorvastatin), 8 in each group. After 4 weeks of intragastric administration, samples were collected and serum, aorta, heart and liver tissues were isolated. Biochemical kits were used to detect serum lipid content and the expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in all experimental groups. The pathological changes of atherosclerosis (AS) were observed by aorta gross Oil Red O, aortic sinus hematoxylin-eosin (HE) and Masson staining. Liver lipopathy was observed in mice by HE staining. The morphology of mitochondria in aorta cells was observed under transmission electron microscope. Flow cytometry was used to detect reactive oxygen species (ROS) expression in aorta of mice in each group. The content of ferrous ion Fe2+ in serum of mice was detected by biochemical kit. The mRNA and protein relative expression levels of NRF2, glutathione peroxidase 4 (GPX4) and recombinant solute carrier family 7 member 11 (SLC7A11) were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: BBRM and BBRH groups delayed the progression of AS and reduced the plaque area (P<0.01). The characteristic morphological changes of ferroptosis were rarely observed in BBR-treated AS mice, and the content of Fe2+ in BBR group was significantly lower than that in the model group (P<0.01). BBR decreased ROS and MDA levels in mouse aorta, increased SOD activity (P<0.01), significantly up-regulated NRF2/SLC7A11/GPX4 protein and mRNA expression levels (P<0.01), and inhibited lipid peroxidation. Compared with the model group, the body weight, blood lipid level and aortic plaque area of ML385 group increased (P<0.01); the morphology of mitochondria showed significant ferroptosis characteristics; the serum Fe2+, MDA and ROS levels increased (P<0.05 or P<0.01), and the activity of SOD decreased (P<0.01). Compared with BBRM group, the iron inhibition effect of BBRM+ML385 group was significantly weakened, and the plaque area significantly increased (P<0.01). CONCLUSION: Through NRF2/SLC7A11/GPX4 pathway, BBR can resist oxidative stress, inhibit ferroptosis, reduce plaque area, stabilize plaque, and exert anti-AS effects.

Inhibition of hexokinase 2 with 3-BrPA promotes MDSCs differentiation and immunosuppressive function.

The adoptive transfer of ex vivo generated myeloid-derived suppressor cells (MDSCs) may be a promising therapeutic strategy for preventing allograft rejection after solid organ transplantation. Currently, the precise role of immune-metabolic pathways in the differentiation and function of MDSCs is not fully understood. Hexokinase 2 (HK2) is an isoform of hexokinase and is a key enzyme involved in the increased aerobic glycolysis of different immune cells during their activation and function. Here, we demonstrate that the addition of HK2 inhibitor 3-Bromopyruvic acid (3-BrPA) into traditional MDSCs induction system in vitro significantly promoted MDSCs production and enhanced their immunosuppressive function. Treatment with 3-BrPA increased the expression of MDSC-related immunosuppressive molecules, such as iNOS, Arg1, and CXCR2. Moreover, the adoptive transfer of 3-BrPA-treated MDSCs significantly prolonged the survival time of mouse heart allografts. This study provides a novel strategy to solve the problems of harvesting enough autologous cells for MDSC production from sick patients, and producing functionally enhanced MDSCs for preventing graft rejection and inducing tolerance.

Ketogenic Diet Suppressed T-Regulatory Cells and Promoted Cardiac Fibrosis via Reducing Mitochondria-Associated Membranes and Inhibiting Mitochondrial Function.

Ketogenic diet (KD) is popular in diabetic patients but its cardiac safety and efficiency on the heart are unknown. The aim of the present study is to determine the effects and the underlined mechanisms of KD on cardiac function in diabetic cardiomyopathy (DCM). We used db/db mice to model DCM, and different diets (regular or KD) were used. Cardiac function and interstitial fibrosis were determined. T-regulatory cell (Treg) number and functions were evaluated. The effects of ketone body (KB) on fatty acid (FA) and glucose metabolism, mitochondria-associated endoplasmic reticulum membranes (MAMs), and mitochondrial respiration were assessed. The mechanisms via which KB regulated MAMs and Tregs were addressed. KD improved metabolic indices in db/db mice. However, KD impaired cardiac diastolic function and exacerbated ventricular fibrosis. Proportions of circulatory CD4+CD25+Foxp3+ cells in whole blood cells and serum levels of IL-4 and IL-10 were reduced in mice fed with KD. KB suppressed the differentiation to Tregs from naive CD4+ T cells. Cultured medium from KB-treated Tregs synergically activated cardiac fibroblasts. Meanwhile, KB inhibited Treg proliferation and productions of IL-4 and IL-10. Treg MAMs, mitochondrial respiration and respiratory complexes, and FA synthesis and oxidation were all suppressed by KB while glycolytic levels were increased. L-carnitine reversed Treg proliferation and function inhibited by KB. Proportions of ST2L+ cells in Tregs were reduced by KB, as well as the production of ST2L ligand, IL-33. Reinforcement expressions of ST2L in Tregs counteracted the reductions in MAMs, mitochondrial respiration, and Treg proliferations and productions of Treg cytokines IL-4 and IL-10. Therefore, despite the improvement of metabolic indices, KD impaired Treg expansion and function and promoted cardiac fibroblast activation and interstitial fibrosis. This could be mainly mediated by the suppression of MAMs and fatty acid metabolism inhibition via blunting IL-33/ST2L signaling.

Metformin Attenuates Cyclosporine A-induced Renal Fibrosis in Rats.

BACKGROUND: The aim of the present study was to investigate the therapeutic potential of metformin in preventing cyclosporine A (CsA)-induced nephrotoxicity. METHODS: Three groups of adult male Sprague-Dawley rats were treated with vehicle, CsA, and CsA + metformin for 4 weeks following 1 week on low sodium diet, respectively. At the end of treatment, all animals were euthanized, and the samples of kidney, urine, and blood were collected for functional, morphological, and molecular biological evaluation. RESULTS: Metformin effectively prevented CsA-induced renal dysfunction with increased creatinine clearance rate and reduced blood urea nitrogen and serum creatinine, as well as less proteinuria in comparison to the CsA group. Morphologically, metformin ameliorated CsA-induced renal fibrosis and tissue collapse in the areas of arteries, glomeruli, and proximal tubules. We further demonstrated that the antifibrotic effects of metformin in kidneys treated with CsA were associated with decreased phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2). CONCLUSIONS: In conclusion, our study revealed new therapeutic potential of metformin to attenuate calcineurin inhibitor-induced renal fibrosis, which was closely related to the suppression of MEK/ERK1/2 pathway.

Personalized management of anastomotic leak after surgery for esophageal carcinoma.

OBJECTIVE: To summarize the management of anastomotic leak following surgery for esophageal carcinoma. METHODS: The medical records of the patients developing digestive tract leak after surgery for esophageal carcinoma in our hospital from January 2003 to March 2011 were retrospectively analyzed. RESULTS: A total of 36 patients were included, in whom 13 developed cervical anastomotic leak, 18 had intra-thoracic anastomotic leak, and 5 had intra-thoracic gastric necrosis. Of these patients, 7 were treated with resurgery, 6 with esophageal stent implantation, and 23 with conservative treatment. Treatment lasted for 5 to 181 days, averagely 47.0 +/- 31.9 days. After management, 9 patients died (25.0%). Among seven patients with resurgery, four had deceased, two were cured, and one developed leak again and was switched to conservative treatment until discharged. All the 6 patients treated with stent implantation were cured. Of the 24 patients receiving conservative treatment (including one switched from resurgery), 18 (75.0%) were cured and 1 was not cured but survived. CONCLUSIONS: Anastomotic leak following surgery for esophageal carcinoma should be treated individually based on the onset time, location, size, and extent of the leakage. Conservative treatment is still a safe and effective method. The efficacy of stent implantation needs further investigation to confirm.

[bcl-2 expression in small cell lung cancer: a mechanism for apoptosis antagonism and immune evasion].

OBJECTIVE: To observe bcl-2 expression and its correlation with apoptosis antagonism and immune evasion of small cell lung cancer cell line and explore the application of bcl-2 antisense thio-oligonucleotide (bcl-2 SON) in gene therapy for lung cancer. METHODS: Western blotting was performed to detect bcl-2 expression in NCI-H69 cell line expressing Bcl-2 protein treated with bcl-2 SON. The treated NCI-H69 cells, along with the cells without bcl-2 SON treatment and NCI-H82 cells that did not express Bcl-2 protein, were respectively co-cultured with tumor-infiltrating lymphocytes (TILs) isolated from fresh tumor samples, and the apoptosis of the lung cancer cells was assessed by JAM assay. RESULTS: Western blotting revealed obvious inhibition of bcl-2 expression in NCI-H69 cells in response to bcl-2 SON treatment. JAM assay showed that the apoptosis of bcl-2 SON-treated H69 cells and NCI-H82 cells increased with the elevation of TIL ratio in the co-culture, while such changes were not observed in NCI-H69 cells without bcl-2 SON treatment. CONCLUSION: Small cell lung cancer cells expressing bcl-2 may antagonize the antitumor immune attack in the host, and bcl-2 SON may provide an effective alternative in gene therapy for small cell lung cancer.