Identification of a PANoptosis-related prognostic model in triple-negative breast cancer, from risk assessment, immunotherapy, to personalized treatment.

Background: Triple-negative breast cancer is a breast cancer subtype characterized by its challenging prognosis, and establishing prognostic models aids its clinical treatment. PANoptosis, a recently identified type of programmed cell death, influences tumor growth and patient outcomes. Nonetheless, the precise impact of PANoptosis-related genes on the prognosis of triple-negative breast cancer has yet to be determined. Methods: Clinical information for the triple-negative breast cancer samples was collected from the Gene Expression Omnibus and The Cancer Genome Atlas databases, while 19 PANoptosis-related genes were sourced from previous studies. We first categorized PANoptosis-related subtypes and determined the differentially expressed genes between them. Subsequently, we developed and validated a PANoptosis-associated predictive model using LASSO and Cox multivariate regression analyses. Statistical evaluations were conducted using R software, and the mRNA expression levels of the genes were quantified using real-time PCR. Results: Using consensus clustering analysis, we divided triple-negative breast cancer patients into two clusters based on PANoptosis-related genes and identified 1054 differentially expressed genes between these clusters. Prognostic-related genes were subsequently selected to re-cluster patients, validating their predictive ability. A prognostic model was then constructed based on four genes: BTN2A2, CACNA1H, PIGR, and S100B. The expression and enriched cell types of these genes were examined and the expression levels were validated in vitro. Furthermore, the model was validated, and a nomogram was created to enhance personalized risk assessment. The risk score, proven to be an independent prognostic indicator for triple-negative breast cancer, showed a positive correlation with both age and disease stage. Immune infiltration and drug sensitivity analyses suggested appropriate therapies for different risk groups. Mutation profiles and pathway enrichment were analyzed, providing insights into potential therapeutic targets. Conclusion: A PANoptosis-related prognostic model was successfully developed for triple-negative breast cancer, offering a novel approach for predicting patient prognosis and guiding treatment strategies.

ß,ß-Dimethylacrylalkannin, a key component of Zicao, induces cell cycle arrest and necrosis in hepatocellular carcinoma cells.

BACKGROUND: ß,ß-Dimethylacrylalkannin (DMAKN), a natural naphthoquinone found in Zicao, a traditional Chinese medicine (TCM), serves as the designated quantitative marker in the Chinese Pharmacopoeia. Despite its established role in assessing Zicao quality, DMAKN's biological potential remains underexplored in research. METHODS: We investigated DMAKN's involvement in Zicao's anti-hepatocellular carcinoma (HCC) properties using a combination of HPLC content analysis and comprehensive bioinformatics. Subsequently, both in vitro and in vivo experiments were conducted to evaluate DMAKN's efficacy against HCC. Mechanistic investigations focused on elucidating DMAKN's impact on cell cycle regulation and induction of cell death. RESULTS: Integrated HPLC analysis and bioinformatics identified DMAKN as the primary active compound responsible for Zicao's anti-HCC activity. In vitro and in vivo studies confirmed DMAKN's potent efficacy against HCC. Notably, DMAKN demonstrated dual effects on HCC cells: inhibiting proliferation at lower doses and inducing rapid cell death at higher doses. Mechanistic insights revealed that low-dose DMAKN induced G2/M phase cell cycle arrest through modulation of CDK1 and Cdc25C phosphorylation, while high-dose DMAKN triggered necrosis. Importantly, high-dose DMAKN caused a sharp increase in intracellular ROS levels in a short time, while low-dose DMAKN gradually increased ROS levels over a long period. Additionally, low-dose DMAKN-induced ROS activated the JNK pathway, crucial for cell cycle arrest, whereas high-dose DMAKN-induced necrosis was ROS-dependent but JNK-independent. CONCLUSION: This study underscores DMAKN's pivotal role as the principal anti-HCC compound in Zicao, delineating its differential effects and underlying mechanisms. These results demonstrate the potential of DMAKN as a therapeutic agent for the treatment of HCC, providing important information for further study and advancement in cancer therapy.

ß,ß-Dimethylacrylalkannin, a Natural Naphthoquinone, Inhibits the Growth of Hepatocellular Carcinoma Cells by Modulating Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) are pivotal in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC), influencing various stages from initiation to metastasis. Understanding the role of TAMs in HCC is crucial for developing novel therapeutic strategies. Macrophages exhibit plasticity, resulting in M1 and M2 phenotypes, with M1 macrophages displaying antitumor properties and M2 macrophages promoting tumor progression. Targeting TAMs to alter their polarization could offer new avenues for HCC treatment. ß,ß-dimethylacrylalkannin (DMAKN), a natural naphthoquinone, has gained attention for its antitumor properties. However, its impact on TAMs modulation remains unclear. This study investigates DMAKN's modulation of TAMs and its anti-HCC activity. Using an in vitro model with THP-1 cells, we induced M1 macrophages with LPS/IFN-γ and M2 macrophages with IL-4/IL-13, confirming polarization with specific markers. Co-culturing these macrophages with HCC cells showed that M1 cells inhibited HCC growth, while M2 cells promoted it. Screening for non-toxic DMAKN concentrations revealed its ability to induce M1 polarization and enhance LPS/IFN-γ-induced M1 macrophages, both showing anti-HCC effects. Conversely, DMAKN suppressed IL-4/IL-13-induced M2 polarization, inhibiting M2 macrophages' promotion of HCC cell viability. In summary, DMAKN induces and enhances M1 polarization while inhibiting M2 polarization of macrophages, thereby inhibiting HCC cell growth. These findings suggest that DMAKN has the potential to regulate TAMs in HCC, offering promise for future therapeutic development.

Saturated long-chain fatty acid-producing bacteria contribute to enhanced colonic motility in rats.

BACKGROUND: The gut microbiota is closely associated with gastrointestinal (GI) motility disorder, but the mechanism(s) by which bacteria interact with and affect host GI motility remains unclear. In this study, through using metabolomic and metagenomic analyses, an animal model of neonatal maternal separation (NMS) characterized by accelerated colonic motility and gut dysbiosis was used to investigate the mechanism underlying microbiota-driven motility dysfunction. RESULTS: An excess of intracolonic saturated long-chain fatty acids (SLCFAs) was associated with enhanced bowel motility in NMS rats. Heptadecanoic acid (C17:0) and stearic acid (C18:0), as the most abundant odd- and even-numbered carbon SLCFAs in the colon lumen, can promote rat colonic muscle contraction and increase stool frequency. Increase of SLCFAs was positively correlated with elevated abundances of Prevotella, Lactobacillus, and Alistipes. Functional annotation found that the level of bacterial LCFA biosynthesis was highly enriched in NMS group. Essential synthetic genes Fabs were largely identified from the genera Prevotella, Lactobacillus, and Alistipes. Pseudo germ-free (GF) rats receiving fecal microbiota from NMS donors exhibited increased defecation frequency and upregulated bacterial production of intracolonic SLCFAs. Modulation of gut dysbiosis by neomycin effectively attenuated GI motility and reduced bacterial SLCFA generation in the colon lumen of NMS rats. CONCLUSIONS: These findings reveal a previously unknown relationship between gut bacteria, intracolonic SLCFAs, and host GI motility, suggesting the importance of SLCFA-producing bacteria in GI motility disorders. Further exploration of this relationship could lead to a precise medication targeting the gut microbiota for treating GI motility disorders.

Magnolol, a Natural Polyphenol, Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice.

Magnolol is a lignan with anti-inflammatory activity identified in Magnolia officinalis. Ulcerative colitis (UC), one of the types of inflammatory bowel disease (IBD), is a disease that causes inflammation and ulcers in the colon. To investigate the effect of magnolol in dextran sulfate sodium (DSS)-induced experimental UC model, male C57 mice were treated with 2% DSS drinking water for 5 consecutive days followed by intragastric administration with magnolol (5, 10 and 15 mg/kg) daily for 7 days. The results showed that magnolol significantly attenuated disease activity index, inhibited colonic shortening, reduced colonic lesions and suppressed myeloperoxidase (MPO) activity. Moreover, colonic pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) induced by colitis were dramatically decreased by magnolol. To further unveil the metabolic signatures upon magnolol treatment, mass spectrometry-based metabolomic analysis of the small molecular metabolites in mice serum were performed. Compared with controls, abnormality of serum metabolic phenotypes in DSS-treated mice were effectively reversed by different doses of magnolol. In particular, magnolol treatment effectively elevated the serum levels of tryptophan metabolites including kynurenic acid (KA), 5-hydroxyindoleacetic acid, indoleacetic acid (IAA), indolelactic acid and indoxylsulfuric acid, which are potential aryl hydrocarbon receptor (AHR) ligands to impact colitis. These findings suggest that magnolol exerts anti-inflammatory effect on DSS-induced colitis and its underlying mechanisms are associated with the restoring of tryptophan metabolites that inhibit the colonic inflammation.

Dihydro-Resveratrol Ameliorates Lung Injury in Rats with Cerulein-Induced Acute Pancreatitis.

Acute pancreatitis is an inflammatory process originated in the pancreas; however, it often leads to systemic complications that affect distant organs. Acute respiratory distress syndrome is indeed the predominant cause of death in patients with severe acute pancreatitis. In this study, we aimed to delineate the ameliorative effect of dihydro-resveratrol, a prominent analog of trans-resveratrol, against acute pancreatitis-associated lung injury and the underlying molecular actions. Acute pancreatitis was induced in rats with repetitive injections of cerulein (50 µg/kg/h) and a shot of lipopolysaccharide (7.5 mg/kg). By means of histological examination and biochemical assays, the severity of lung injury was assessed in the aspects of tissue damages, myeloperoxidase activity, and levels of pro-inflammatory cytokines. When treated with dihydro-resveratrol, pulmonary architectural distortion, hemorrhage, interstitial edema, and alveolar thickening were significantly reduced in rats with acute pancreatitis. In addition, the production of pro-inflammatory cytokines and the activity of myeloperoxidase in pulmonary tissues were notably repressed. Importantly, nuclear factor-kappaB (NF-κB) activation was attenuated. This study is the first to report the oral administration of dihydro-resveratrol ameliorated acute pancreatitis-associated lung injury via an inhibitory modulation of pro-inflammatory response, which was associated with a suppression of the NF-κB signaling pathway.

5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing ß-catenin.

5-Hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/ß-catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/ß-catenin signalling by 5-HT. The expression of various 5-HT receptors was studied by quantitative real-time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non-tumour tissues. Receptors 5-HT1D (21/33, 63.6%), 5-HT2B (12/33, 36.4%) and 5-HT7 (15/33, 45.4%) were overexpressed whereas receptors 5-HT2A (17/33, 51.5%) and 5-HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5-HT increased total ß-catenin, active ß-catenin and decreased phosphorylated ß-catenin protein levels in serum deprived HuH-7 and HepG2 cells compared to control cells under serum free medium without 5-HT. Activation of Wnt/ß-catenin signalling was evidenced by increased expression of ß-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, biochemical analysis revealed 5-HT disrupted Axin1/ß-catenin interaction, a critical step in ß-catenin phosphorylation. Increased Wnt/ß-catenin activity was attenuated by antagonist of receptor 5-HT7 (SB-258719) in HCC cell lines and patient-derived primary tumour tissues in the presence of 5-HT. SB-258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/ß-catenin signalling activation by 5-HT and may represent a potential therapeutic target for hepatocarcinogenesis.

Qing-dai powder promotes recovery of colitis by inhibiting inflammatory responses of colonic macrophages in dextran sulfate sodium-treated mice.

BACKGROUND: Qing-dai powder (QDP), comprising Indigo naturalis (Qing-dai) and dried alum (Ku-fan), was used in Chinese medicine to treat the conditions associated with mucosal hemorrhage, such as ulcerative colitis (UC). This study aims to investigate the effects and potential mechanism of QDP on dextran sulfate sodium (DSS)-induced acute colitis in mice and to examine the regulatory effects of QDP on macrophages. METHODS: Seven- to eight-week-old male C57BL/6 mice were challenged with 2.0 % DSS in drinking water for 5 days and then the colitic mice were arbitrarily allocated into five groups (n = 10 for each group). QDP (0.77, 1.54 and 3.08 g/kg) and sulfasalazine (SASP) (0.20 g/kg) were orally administered for 7 days. The disease activity index was determined by scores of body weight loss, diarrhea and rectal bleeding; histological signs of damage was analyzed by H&E staining; myeloperoxidase activity was measured by colorimetric method, levels of proinflammatory cytokines were determined by ELISA; changes in macrophages in the colon were analyzed by immunohistochemistry (IHC) and flow cytometry. Lipopolysaccharide (LPS)-induced RAW264.7 cells were treated with or without QDP, then the production of TNF-α and IL-6 were measured by ELISA; and protein molecules such as COX-2, iNOS, IкB-α were determined by Western blot. RESULTS: Oral administration of QDP at dosages of 1.54 and 3.08 g/kg significantly reduced disease activity index on day 12 (P < 0.001 for 1.54 g/kg and P < 0.0008 for 3.08 g/kg), colon shortening (P = 0.012 for 1.54 g/kg, P = 0.001 for 3.08 g/kg), histological damage (P < 0.001 for 1.54 g/kg, P < 0.001 for 3.08 g/kg) and colonic myeloperoxidase activity (P = 0.002 for 1.54 g/kg, P < 0.001 for 3.08 g/kg) of DSS-treated mice. Moreover, QDP treatment (1.54 and 3.08 g/kg) significantly decreased DSS-induced infiltration of macrophages, and production of TNF-α (P = 0.005 for 1.54 g/kg, P = 0.002 for 3.08 g/kg), IL-1ß (P = 0.008 for 1.54 g/kg, P = 0.002 for 3.08 g/kg) and IL-6 (P = 0.011 for 1.54 g/kg, P = 0.004 for 3.08 g/kg) in colonic tissues, and also reduced serum MCP-1 levels (P = 0.001 for 1.54 g/kg, P < 0.001 for 3.08 g/kg). In RAW264.7 cells, QDP significantly suppressed LPS-induced production of TNF-α and IL-6 (Both P < 0.001 for 1.0 µg/mL QDP treatment) and expression levels of COX-2 (P = 0.002 and P = 0.001 for 1 and 3 µg/mL QDP treatment, respectively) and iNOS (P < 0.001 for 3 µg/mL QDP treatment) by inhibiting IкB-α degradation (P = 0.007 and P = 0.004 for 1 and 3 µg/mL QDP treatment, respectively) and NF-кB p65 nuclear translocation. CONCLUSION: QDP suppressed the inflammatory responses of colonic macrophages in DSS-induced UC in mice and LPS-induced RAW264.7 cells.

Anti-fibrotic effects of phenolic compounds on pancreatic stellate cells.

BACKGROUND: Pancreatic fibrosis is a prominent histopathological characteristic of chronic pancreatitis and plausibly a dynamic process of transition to the development of pancreatic ductal adenocarcinoma. Conversely, the activation of pancreatic stellate cells (PSCs) has been recently suggested as the key initiating step in pancreatic fibrosis. As natural polyphenols had been largely applied in complementary therapies in the past decade, in this study, we aimed to investigate which groups of phenolic compounds exert promising inhibitory actions on fibrogenesis as there are few effective strategies for the treatment of pancreatic fibrosis to date. METHODS: We examined the anti-fibrotic effects of a variety of herbal constituents using a cellular platform, the LTC-14 cells, which retained essential characteristics and morphologies of primary PSCs, by means of various biochemical assays including cell viability test, real-time polymerase chain reaction and Western blotting analysis. RESULTS: Among a number of commonly used herbal constituents, we found that the application of rhein, emodin, curcumin and resveratrol significantly suppressed the mRNA and protein levels of several fibrotic mediators namely alpha-smooth muscle actin, type I collagen and fibronectin in LTC-14 cells against transforming growth factor-beta stimulation. Though the values of cytotoxicity varied, the mechanism of the anti-fibrotic action of these four phenolic compounds was principally associated with a decrease in the activation of the nuclear factor-kappaB signaling pathway. CONCLUSIONS: Our findings suggest that the mentioned phenolic compounds may serve as anti-fibrotic agents in PSC-relating disorders and pathologies, particularly pancreatic fibrosis.

Anti-fibrotic effect of trans-resveratrol on pancreatic stellate cells.

Trans-resveratrol, also known as 3,5,4'-trihydroxy-trans-stilbene, is a natural stilbenoid found at high concentration in skins of red grapes and berries. Over the recent years, it has been reported with a variety of beneficial effects such as antioxidant, anti-aging and anti-inflammatory bioactivities; thus often utilized as an active substance in human and veterinary therapeutics. In the current study, we aimed to delineate the mechanism of its anti-fibrotic action by means of various biochemical assays, such as immunofluorescent staining, real-time polymerase chain reaction and Western blotting analyses in a cellular model, the LTC-14 cells, which retain essential characteristics and morphological features of primary pancreatic stellate cells (PSCs). Our results demonstrated that the application of trans-resveratrol as low as 10 µM notably suppressed the mRNA and protein levels of different fibrotic mediators namely alpha-smooth muscle actin, type I collagen and fibronectin in the LTC-14 cells stimulated with transforming growth factor-beta, a well recognized pro-fibrotic inducer. Importantly, the mechanism of the anti-fibrotic action of trans-resveratrol was associated with a decrease in nuclear factor-kappaB activation and protein kinase B phosphorylation. In conclusion, our finding suggests that trans-resveratrol may serve as a therapeutic or an adjuvant agent in anti-fibrotic approaches and/or PSC-relating pathologies.

Traditional Chinese medicine formulas for irritable bowel syndrome: from ancient wisdoms to scientific understandings.

Traditional Chinese Medicine (TCM) serves as the most common alternative therapeutic approach for Western medicine and benefits IBS patients globally. Due to the lack of scientific evidence in the past, TCM formulas were not internationally well recognized as promising IBS remedies. In this review, firstly, we present the etiology and therapy of IBS in terms of traditional Chinese medical theory. Secondly, we summarize the clinical randomized controlled trials (RCTs) of TCM formulas for IBS patients that are available in the literature (from 1998 to September 2013), in which 14 RCTs conducted of high quality were discussed in detail. Of the 14 selected trials, 12 of those concluded that TCM formulas provided superior improvement in the global symptoms of IBS patients over the placebo or conventional medicines. As well, all 14 RCTs suggested that TCM formulas have good safety and tolerability. Last but not least, we explore the pharmacological mechanisms of the anti-IBS TCM formulas available in the literature (from 1994 to September, 2013). Collectively, in combating IBS symptoms, most TCM formulas exert multi-targeting actions including the regulation of neurotransmitters and hormones in the enteric nervous system (ENS), modulation of smooth muscle motility in the gastrointestinal (GI) tract, modulation of the hypothalamic-pituitary-adrenal (HPA) axis, attenuation of intestinal inflammation and restoration of intestinal flora, etc. In conclusion, TCM formulas appear to be promising for IBS treatment. This review provides a useful reference for the public in furthering a better understanding and acceptance of TCM formulas as IBS remedies.