DHPS-Mediated Hypusination Regulates METTL3 Self-m6A-Methylation Modification to Promote Melanoma Proliferation and the Development of Novel Inhibitors.

Discovering new treatments for melanoma will benefit human health. The mechanism by which deoxyhypusine synthase (DHPS) promotes melanoma development remains elucidated. Multi-omics studies have revealed that DHPS regulates m6A modification and maintains mRNA stability in melanoma cells. Mechanistically, DHPS activates the hypusination of eukaryotic translation initiation factor 5A (eIF5A) to assist METTL3 localizing on its mRNA for m6A modification, then promoting METTL3 expression. Structure-based design, synthesis, and activity screening yielded the hit compound GL-1 as a DHPS inhibitor. Notably, GL-1 directly inhibits DHPS binding to eIF5A, whereas GC-7 cannot. Based on the clarification of the mode of action of GL-1 on DHPS, it is found that GL-1 can promote the accumulation of intracellular Cu2+ to induce apoptosis, and antibody microarray analysis shows that GL-1 inhibits the expression of several cytokines. GL-1 shows promising antitumor activity with good bioavailability in a xenograft tumor model. These findings clarify the molecular mechanisms by which DHPS regulates melanoma proliferation and demonstrate the potential of GL-1 for clinical melanoma therapy.

Photothermal therapy improves the efficacy of topical immunotherapy against melanoma.

BACKGROUND: Melanoma is an aggressive cancer with poor response to traditional therapies. A combination of photothermal therapy and topical immunotherapy is expected to eliminate melanoma effectively. MATERIALS AND METHODS: C57BL/6 mice with early stage and metastatic melanoma were treated with laser immunotherapy (LIT), combining near-infrared laser-based photothermal therapy (PTT) and topical imiquimod (IMQ)-based immunotherapy. The volume of primary and abscopal melanoma, animal survival, tissue temperature, transcriptome, and immune cell response were investigated to evaluate the effect of LIT. RESULTS: LIT could eliminate primary tumors, inhibited abscopal tumors, and prolonged animal survival. The tumor tissues were selectively destroyed under a photothermal gradient between 38.2 ± 3.7°C and 73.0 ± 2.3°C. Gene expression analysis showed a significant increase in the expression of damage associated molecular patterns. Additionally, the expression of mature dendritic cells, CD4+ T cells, and CD8+ T cells were increased, while myeloid-derived suppressor cells were downregulated after LIT. CONCLUSION: The study showed that LIT inhibited the growth of both primary and abscopal melanoma by activating systemic antitumor immune responses and reversing the immunosuppressive tumor microenvironment, making LIT a potential method for advanced melanoma treatment.

Capsaicin pretreatment attenuates salt-sensitive hypertension by alleviating AMPK/Akt/Nrf2 pathway in hypothalamic paraventricular nucleus.

Background: Long term hypertension seriously promotes target organ damage in the brain and heart, and has increasingly become serious public health problem worldwide. The anti-hypertensive effects of capsaicin has been reported, however, the role and mechanism of capsaicin within the brain on salt-induced hypertension have yet to be elucidated. This study aimed to verify the hypothesis that capsaicin attenuates salt-induced hypertension via the AMPK/Akt/Nrf2 pathway in hypothalamic paraventricular nucleus (PVN). Methods: Dahl salt-sensitive (Dahl S) rats were used as animal model for the present study. Rats were randomly divided into four groups based on their dietary regimen (0.3% normal salt diet and 8% high salt diet) and treatment methods (infusion of vehicle or capsaicin in the PVN). Capsaicin was chronically administered in the PVN throughout the animal experiment phase of the study that lasted 6 weeks. Results: Our results demonstrated that PVN pretreatment with capsaicin can slow down raise of the blood pressure elevation and heart rate (HR) of Dahl S hypertensive rats given high salt diet. Interestingly, the cardiac hypertrophy was significantly improved. Furthermore, PVN pretreatment with capsaicin induced decrease in the expression of mRNA expression of NADPH oxidase-2 (NOX2), inducible nitric oxide synthase (iNOS), NOX4, p-IKKß and proinflammatory cytokines and increase in number of positive cell level for Nrf2 and HO-1 in the PVN of Dahl S hypertensive rats. Additionally, the protein expressions of phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase-B (p-AKT) were decreased, phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were increased after the PVN pretreatment with capsaicin. Conclusion: Capsaicin pretreatment attenuates salt-sensitive hypertension by alleviating AMPK/Akt/iNOS pathway in the PVN.

The mechanism of Xihuang pills' intervention in the tumour immune microenvironment for the treatment of liver cancer based on the STAT3-PDL1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xihuang pills, a time-honored Chinese compound formula with a history spanning thousands of years, have demonstrated remarkable efficacy in treating various cancers, such as breast cancer, colon cancer, and liver cancer. Clinical applications over the years have established their effectiveness. Several scholars conducting experimental studies have elucidated the potent tumor-suppressing effects of Xihuang pills. While the inhibition of tumor vascular development and prevention of tumor cell invasion and metastasis have been well-explored mechanisms, the impact on the tumor immune microenvironment has received less attention. This study focuses on investigating the immune microenvironment adjustments induced by Xihuang pills in hepatocellular carcinoma. AIM OF THE STUDY: Tumour cells will find an escape phenomenon during tumour immunotherapy, which will affect immunotherapy results. We will research the regulation of the tumour immune microenvironment, to provide a more complete and precise basis for the elucidation of the mechanism of Xihuang pills in treating cancers. It provides new research ideas for people to treat liver cancer. MATERIALS AND METHODS: Through in vivo and in vitro assessments confirming the intervention effects of Xihuang pills, we observed alterations in T cell typing, macrophage polarization, and tumor-associated cytokine levels. The primary active ingredients of Xihuang pills were identified using UPLC-MS/GC-MS, and relevant pathways in the treatment of hepatocellular carcinoma were predicted through network pharmacology. Combining the network pharmacology approach, we predicted the pathways relevant to Xihuang pills in treating hepatocellular carcinoma and experimentally validated the involvement of PD-1/PD-L1, a key immunity-related axis. RESULTS: Xihuang Pill has a regulatory effect on the tumor immune microenvironment. CONCLUSIONS: The results indicated that Xihuang pills could impact splenic lymphocyte phenotyping, macrophage polarization, and IL-6 cytokine expression in liver cancer mice. The mechanism of action was associated with the regulation of the PD-1/PD-L1 signaling pathway by the STAT3 protein.

Luteolin, apigenin, and chrysin inhibit lipotoxicity-induced NLRP3 inflammasome activation and autophagy damage in macrophages by suppressing endoplasmic reticulum stress.

Lipotoxicity leads to numerous metabolic disorders such as nonalcoholic steatohepatitis. Luteolin, apigenin, and chrysin are three flavones with known antioxidant and anti-inflammatory properties, but whether they inhibit lipotoxicity-mediated NLRP3 inflammasome activation was unclear. To address this question, we used J774A.1 macrophages and Kupffer cells stimulated with 100 µM palmitate (PA) in the presence or absence of 20 µM of each flavone. PA increased p-PERK, p-IRE1α, p-JNK1/2, CHOP, and TXNIP as well as p62 and LC3-II expression and induced autophagic flux damage. Caspase-1 activation and IL-1ß release were also noted after 24 h of exposure to PA. In the presence of the PERK inhibitor GSK2656157, PA-induced CHOP and TXNIP expression and caspase-1 activation were mitigated. Compared with PA treatment alone, Bcl-2 coupled to beclin-1 was elevated and autophagy was reversed by the JNK inhibitor SP600125. With luteolin, apigenin, and chrysin treatment, PA-induced ROS production, ER stress, TXNIP expression, autophagic flux damage, and apoptosis were ameliorated. Moreover, TXNIP binding to NLRP3 and IL-1ß release in response to LPS/PA challenge were reduced. These results suggest that luteolin, apigenin, and chrysin protect hepatic macrophages against PA-induced NLRP3 inflammasome activation and autophagy damage by attenuating endoplasmic reticulum stress.

Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2 times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cells in nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Anaplastic thyroid cancer spheroids as preclinical models to test therapeutics.

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer. Despite advances in tissue culture techniques, a robust model for ATC spheroid culture is yet to be developed. In this study, we created an efficient and cost-effective 3D tumor spheroids culture system from human ATC cells and existing cell lines that better mimic patient tumors and that can enhance our understanding of in vivo treatment response. We found that patient-derived ATC cells and cell lines can readily form spheroids in culture with a unique morphology, size, and cytoskeletal organization. We observed both cohesive (dense and solid structures) and discohesive (irregularly shaped structures) spheroids within the same culture condition across different cell lines. BRAFWT ATC spheroids grew in a cohesive pattern, while BRAFV600E-mutant ATC spheroids had a discohesive organization. In the patient-derived BRAFV600E-mutant ATC spheroids, we observed both growth patterns, but mostly the discohesive type. Histologically, ATC spheroids had a similar morphology to the patient's tumor through H&E staining and proliferation marker staining. Moreover, RNA sequencing analysis revealed that the gene expression profile of tumor cells derived from the spheroids closely matched parental patient tumor-derived cells in comparison to monolayer cultures. In addition, treatment response to combined BRAF and MEK inhibition in BRAFV600E-mutant ATC spheroids exhibited a similar sensitivity to the patient clinical response. Our study provides a robust and novel ex vivo spheroid model system that can be used in both established ATC cell lines and patient-derived tumor samples to better understand the biology of ATC and to test therapeutics.

Central administration of AICAR attenuates hypertension via AMPK/Nrf2 pathway in the hypothalamic paraventricular nucleus of hypertensive rats.

BACKGROUND: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity. METHODS: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks. RESULTS: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1ß expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes. CONCLUSION: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension.

Multiparametric MRI radiomics improves preoperative diagnostic performance for local staging in patients with endometrial cancer.

PURPOSE: To determine whether multiparametric magnetic resonance imaging (MRI) radiomics-based machine learning methods can improve preoperative local staging in patients with endometrial cancer (EC). METHODS: Data of patients with histologically confirmed EC who underwent preoperative MRI were retrospectively analyzed and divided into a training or test set. Radiomic features extracted from multiparametric MR images were used to train and test the prediction of deep myometrial invasion (DMI) and cervical stromal invasion (CSI). Two radiologists assessed the presence of DMI and CSI on conventional MR images. A combined model incorporating a radiomic signature and conventional MR images was constructed and presented as a nomogram. Performance of the predictive models was assessed using the area under curve (AUC) in the receiver operating curve analysis and pairwise comparison using DeLong's test with Bonferroni correction. RESULTS: This study included 198 women (training set = 138, test set = 60). Conventional MRI achieved AUCs of 0.837 and 0.799 for detecting DMI and 0.825 and 0.858 for detecting CSI in the training and test sets, respectively. The nomogram achieved AUCs of 0.928 and 0.869 for detecting DMI and 0.913 and 0.937 for detecting CSI in the training and test sets, respectively. The ability of the nomogram to detect DMI and CSI in the two sets was superior to that of conventional MRI (adjusted p < 0.05), except for the ability to detect CSI in the test set (adjusted p > 0.05). CONCLUSION: A nomogram incorporating radiomics signature into conventional MRI improved the efficacy of preoperative local staging of EC.

Extracellular vesicles from Lacticaseibacillus paracasei PC-H1 inhibit HIF-1α-mediated glycolysis of colon cancer.

Aims: Extracellular vesicles from Lacticaseibacillus paracasei PC-H1 have antiproliferative activity of colon cells, but the effect on glycolytic metabolism of cancer cell remains enigmatic. The authors investigated how Lacticaseibacillus paracasei extracellular vesicles (LpEVs) inhibit the growth of colon cancer cells by affecting tumor metabolism. Materials & methods: HCT116 cells were treated with LpEVs and then differentially expressed genes were analyzed by transcriptome sequencing, the sequencing results were confirmed in vivo and in vitro. Results: LpEVs entered colon cancer cells and inhibited their growth. Transcriptome sequencing revealed differentially expressed genes were related to glycolysis. Lactate production, glucose uptake and lactate dehydrogenase activity were significantly reduced after treatment. LpEVs also reduced HIF-1α, GLUT1 and LDHA expression. Conclusion: LpEVs exert their antiproliferative activity of colon cancer cells by decreasing HIF-1α-mediated glycolysis.

Benzyl isothiocyanate inhibits TNFα-driven lipolysis via suppression of the ERK/PKA/HSL signaling pathway in 3T3-L1 adipocytes.

Tumor necrosis factor α (TNFα), an inflammatory cytokine, induces lipolysis and increases circulating concentrations of free fatty acids. In addition, TNFα is the first adipokine produced by adipose tissue in obesity, contributing to obesity-associated metabolic disease. Given that benzyl isothiocyanate (BITC) is a well-known anti-inflammatory agent, we hypothesized that BITC can ameliorate TNFα-induced lipolysis and investigated the working mechanisms involved. We first challenged 3T3-L1 adipocytes with TNFα to induce lipolysis, which was confirmed by increased glycerol release, decreased protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and perilipin 1 (PLIN1), and increased phosphorylation of ERK, protein kinase A (PKA), and hormone-sensitive lipase (HSL). However, inhibition of ERK or PKA significantly attenuated the lipolytic activity of TNFα. Meanwhile, pretreatment with BITC significantly ameliorated the lipolytic activity of TNFα; the TNFα-induced phosphorylation of ERK, PKA, and HSL; the TNFα-induced ubiquitination of PPARγ; the TNFα-induced decrease in PPARγ nuclear protein binding to PPAR response element; and the TNFα-induced decrease in PLIN1 protein expression. Our results indicate that BITC ameliorates TNFα-induced lipolysis by inhibiting the ERK/PKA/HSL signaling pathway, preventing PPARγ proteasomal degradation, and maintaining PLIN1 protein expression.

Discovery of (quinazolin-6-yl)benzamide derivatives containing a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety as potent reversal agents against P-glycoprotein-mediated multidrug resistance.

P-glycoprotein (P-gp) is an important factor leading to multidrug resistance (MDR) in cancer treatment. The co-administration of anticancer drugs and P-gp inhibitors has been a treatment strategy to overcome MDR. In recent years, tyrosine kinase inhibitor Lapatinib has been reported to reverse MDR through directly interacting with ABC transporters. In this work, a series of P-gp inhibitors (1-26) was designed and synthesized by integrating the quinazoline core of Lapatinib into the molecule framework of the third-generation P-gp inhibitor Tariquidar. Among them, compound 14 exhibited better MDR reversal activity than Tariquidar. The docking results showed compound 14 displayed the L-shaped molecular conformation. Importantly, compound 14 increased the accumulation of Adriamycin (ADM) and rhodamine 123 (Rh123) in MCF7/ADM cells. Besides, compound 14 significantly increased ADM-induced apoptosis and inhibited the proliferation, migration and invasion of MCF7/ADM cells. It was also demonstrated that compound 14 significantly inhibited the growth of MCF7/ADM xenograft tumors by increasing the sensitivity of ADM. In summary, compound 14 has the potential to overcome MDR caused by P-gp.

Protective Effect of Alpha-Linolenic Acid on Human Oral Squamous Cell Carcinoma Metastasis and Apoptotic Cell Death.

Oral cancer ranks sixth among Taiwan's top 10 cancers and most patients with poor prognosis acquire metastases. The essential fatty acid alpha-linolenic acid (ALA) has been found to diminish many cancer properties. However, the anti-cancer activity of ALA in oral cancer has yet to be determined. We examined the mechanisms underlying ALA inhibition of metastasis and induction of apoptotic cell death in oral squamous cell carcinoma (OSCC). Migration and invasion assays confirmed the cancer cells' EMT capabilities, whereas flow cytometry and Western blotting identified molecular pathways in OSCC. ALA dramatically reduced cell growth in a concentration-dependent manner according to the findings. Low concentrations of ALA (100 or 200 µM) inhibit colony formation, the expression of Twist and EMT-related proteins, the expression of MMP2/-9 proteins, and enzyme activity, as well as cell migration and invasion. Treatment with high concentrations of ALA (200 or 400 µM) greatly increases JNK phosphorylation and c-jun nuclear accumulation and then upregulates the FasL/caspase8/caspase3 and Bid/cytochrome c/caspase9/caspase3 pathways, leading to cell death. Low concentrations of ALA inhibit SAS and GNM cell migration and invasion by suppressing Twist and downregulating EMT-related proteins or by decreasing the protein expression and enzyme activity of MMP-2/-9, whereas high concentrations of ALA promote apoptosis by activating the JNK/FasL/caspase 8/caspase 3-extrinsic pathway and the Bid/cytochrome c/caspase 9 pathway. ALA demonstrates potential as a treatment for OSCC patients.

Andrographolide Attenuates Oxidized LDL-Induced Activation of the NLRP3 Inflammasome in Bone Marrow-Derived Macrophages and Mitigates HFCCD-Induced Atherosclerosis in Mice.

Andrographolide (AND) is a bioactive component of the herb Andrographis paniculata and a well-known anti-inflammatory agent. Atherosclerosis is a chronic inflammatory disease of the vasculature, and oxidized LDL (oxLDL) is thought to contribute heavily to atherosclerosis-associated inflammation. The aim of this study was to investigate whether AND mitigates oxLDL-mediated foam cell formation and diet-induced atherosclerosis (in mice fed a high-fat, high-cholesterol, high-cholic acid [HFCCD] diet) and the underlying mechanisms involved. AND attenuated LPS/oxLDL-mediated foam cell formation, IL-1[Formula: see text] mRNA and protein (p37) expression, NLR family pyrin domain containing 3 (NLRP3) mRNA and protein expression, caspase-1 (p20) protein expression, and IL-1[Formula: see text] release in BMDMs. Treatment with oxLDL significantly induced protein and mRNA expression of CD36, lectin-like oxLDL receptor-1 (LOX-1), and scavenger receptor type A (SR-A), whereas pretreatment with AND significantly inhibited protein and mRNA expression of SR-A only. Treatment with oxLDL significantly induced ROS generation and Dil-oxLDL uptake; however, pretreatment with AND alleviated oxLDL-induced ROS generation and Dil-oxLDL uptake. HFCCD feeding significantly increased aortic lipid accumulation, ICAM-1 expression, and IL-1[Formula: see text] mRNA expression, as well as blood levels of glutamic pyruvic transaminase (GPT), total cholesterol, and LDL-C. AND co-administration mitigated aortic lipid accumulation, the protein expression of ICAM-1, mRNA expression of IL-1[Formula: see text] and ICAM-1, and blood levels of GPT. These results suggest that the working mechanisms by which AND mitigates atherosclerosis involve the inhibition of foam cell formation and NLRP3 inflammasome-dependent vascular inflammation as evidenced by decreased SR-A expression and IL-1[Formula: see text] release, respectively.

Leg disorders in broiler chickens: a review of current knowledge.

Ensuring improved leg health is an important prerequisite for broilers to achieve optimal production performance and welfare status. Broiler leg disease is characterized by leg muscle weakness, leg bone deformation, joint cysts, arthritis, femoral head necrosis, and other symptoms that result in lameness or paralysis. These conditions significantly affect movement, feeding and broiler growth performance. Nowadays, the high incidence of leg abnormalities in broiler chickens has become an important issue that hampers the development of broiler farming. Therefore, it is imperative to prevent leg diseases and improve the health of broiler legs. This review mainly discusses the current prevalence of broiler leg diseases and describes the risk factors, diagnosis, and prevention of leg diseases to provide a scientific basis for addressing broiler leg health problems.

Hypusination-induced DHPS/eIF5A pathway as a new therapeutic strategy for human diseases: A mechanistic review and structural classification of DHPS inhibitors.

The discovery of new therapeutic strategies for diseases is essential for drug research. Deoxyhypusine synthase (DHPS) is a critical enzyme that modifies the conversion of the eukaryotic translation initiation factor 5A (eIF5A) precursor into physiologically active eIF5A (eIF5A-Hyp). Recent studies have revealed that the hypusine modifying of DHPS on eIF5A has an essential regulatory role in human diseases. The hypusination-induced DHPS/eIF5A pathway has been shown to play an essential role in various cancers, and it could regulate immune-related diseases, glucose metabolism-related diseases, neurological-related diseases, and aging. In addition, DHPS has a more defined substrate and a well-defined structure within the active pocket than eIF5A. More and more researchers are focusing on the prospect of advanced development of DHPS inhibitors. This review summarizes the regulatory mechanisms of the hypusination-induced DHPS/eIF5A pathway in a variety of diseases in addition to the inhibitors related to this pathway; it highlights and analyzes the structural features and mechanisms of action of DHPS inhibitors and expands the prospects of future drug development using DHPS as an anticancer target.

Single-cell transcriptomics reveals that tumor-infiltrating natural killer cells are activated by localized ablative immunotherapy and share anti-tumor signatures induced by immune checkpoint inhibitors.

Rationale: Natural killer (NK) cells provide protective anti-cancer immunity. However, the cancer therapy induced activation gene signatures and pathways in NK cells remain unclear. Methods: We applied a novel localized ablative immunotherapy (LAIT) by synergizing photothermal therapy (PTT) with intra-tumor delivering of the immunostimulant N-dihydrogalactochitosan (GC), to treat breast cancer using a mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mouse model. We performed single-cell RNA sequencing (scRNAseq) analysis to unveil the cellular heterogeneity and compare the transcriptional alterations induced by PTT, GC, and LAIT in NK cells within the tumor microenvironment (TME). Results: ScRNAseq showed that NK subtypes, including cycling, activated, interferon-stimulated, and cytotoxic NK cells. Trajectory analysis revealed a route toward activation and cytotoxicity following pseudotime progression. Both GC and LAIT elevated gene expression associated with NK cell activation, cytolytic effectors, activating receptors, IFN pathway components, and cytokines/chemokines in NK subtypes. Single-cell transcriptomics analysis using immune checkpoint inhibitor (ICI)-treated animal and human samples revealed that ICI-induced NK activation and cytotoxicity across several cancer types. Furthermore, ICI-induced NK gene signatures were also induced by LAIT treatment. We also discovered that several types of cancer patients had significantly longer overall survival when they had higher expression of genes in NK cells that were also specifically upregulated by LAIT. Conclusion: Our findings show for the first time that LAIT activates cytotoxicity in NK cells and the upregulated genes positively correlate with beneficial clinical outcomes for cancer patients. More importantly, our results further establish the correlation between the effects of LAIT and ICI on NK cells, hence expanding our understanding of mechanism of LAIT in remodeling TME and shedding light on the potentials of NK cell activation and anti-tumor cytotoxic functions in clinical applications.

Hypothalamic Paraventricular Nucleus Hydrogen Sulfide Exerts Antihypertensive Effects in Spontaneously Hypertensive Rats via the Nrf2 Pathway.

BACKGROUND: Hydrogen sulfide (H2S) is widely distributed throughout the nervous system with various antioxidant and anti-inflammatory properties. Hypertension involves an increase in reactive oxygen species (ROS) and inflammation in the hypothalamic paraventricular nucleus (PVN). However, it is unclear how H2S in PVN affects hypertension. METHODS: Our study used spontaneously hypertensive rats (SHR) and control Wistar Kyoto (WKY) rats, microinjected with adenovirus-associated virus (AAV)-CBS (cystathionine beta-synthase overexpression) or AAV-ZsGreen in bilateral PVN, or simultaneously injected with virus-carrying nuclear factor erythroid 2-related factor 2 (Nrf2)-shRNA for 4 weeks. Blood pressure (BP) and plasma noradrenaline level were detected, and the PVN was collected. Finally, levels of CBS, H2S, Nrf2, Fra-LI, ROS, gp91phox, p47phox, superoxide dismutase 1, interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor-α, tyrosine hydroxylase, and glutamate decarboxylase 67 were measured. RESULTS: We found that AAV-CBS increased H2S in the PVN, and BP, neuronal activation, oxidative stress, and inflammation of PVN were substantially reduced. Furthermore, endogenous H2S in the PVN activated Nrf2 and corrected the PVN's imbalance of excitatory and inhibitory neurotransmitters. However, Nrf2 knockdown in the PVN was similarly observed to abolish the beneficial effect of H2S on hypertension. CONCLUSIONS: The findings imply that endogenous H2S in SHR PVN is reduced, and PVN endogenous H2S can alleviate hypertension via Nrf2-mediated antioxidant and anti-inflammatory effects.

Luteolin Attenuates Hypertension via Inhibiting NF-κB-Mediated Inflammation and PI3K/Akt Signaling Pathway in the Hypothalamic Paraventricular Nucleus.

BACKGROUND: Luteolin is widely distributed among a number of vegetal species worldwide. The pharmacological effects of luteolin are diverse and amongst antioxidant, free radical scavenging, and anti-inflammatory activities. Preliminary study showed that luteolin can ameliorate hypertension. However, the precise mechanism needs further investigation. There is no evidence that luteolin affects the paraventricular nucleus of the hypothalamus (PVN), a brain nucleus associated with a critical neural regulator of blood pressure. Our main aim was to explore the effect of luteolin on the PI3K/Akt/NF-κB signaling pathway within the PVN of hypertensive rats. METHODS: spontaneously hypertensive rats (SHRs) and corresponding normotensive control rats, the Wistar Kyoto (WKY) rats were divided into four groups and subsequently treated for 4 weeks with bilateral PVN injections of either luteolin (20 µg/0.11 µL, volume: 0.11 µL/h) or vehicle (artificial cerebrospinal fluid). RESULTS: luteolin infusion to the PVN significantly decreased some hemodynamic parameters including the mean arterial pressure (MAP), heart rate (HR), circulating plasma norepinephrine (NE) and epinephrine (EPI). Additionally, there was a decrease in the expressions of the phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase-B (p-AKT), levels of reactive oxygen species (ROS), NAD(P)H oxidase subunit (NOX2, NOX4) in the PVN of SHRs. Meanwhile, the expression of inflammatory cytokines and the activity of nuclear factor κB (NF-κB) p65 in the PVN of SHRs were lowered. Furthermore, immunofluorescence results showed that injection of luteolin in the PVN reduced the expression of tyrosine hydroxylase (TH), and increased that of superoxide dismutase (SOD1) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN of SHRs. CONCLUSION: Our novel findings revealed that luteolin lowered hypertension via inhibiting NF-κB-mediated inflammation and PI3K/Akt signaling pathway in the PVN.

Gut microbiome dysregulation drives bone damage in broiler tibial dyschondroplasia by disrupting glucose homeostasis.

Tibial dyschondroplasia (TD) with multiple incentives is a metabolic skeletal disease that occurs in fast-growing broilers. Perturbations in the gut microbiota (GM) have been shown to affect bone homoeostasis, but the mechanisms by which GM modulates bone metabolism in TD broilers remain unknown. Here, using a broiler model of TD, we noted elevated blood glucose (GLU) levels in TD broilers, accompanied by alterations in the pancreatic structure and secretory function and damaged intestinal barrier function. Importantly, faecal microbiota transplantation (FMT) of gut microbes from normal donors rehabilitated the GM and decreased the elevated GLU levels in TD broilers. A high GLU level is a predisposing factor to bone disease, suggesting that GM dysbiosis-mediated hyperglycaemia might be involved in bone regulation. 16S rRNA gene sequencing and short-chain fatty acid analysis revealed that the significantly increased level of the metabolite butyric acid derived from the genera Blautia and Coprococcus regulated GLU levels in TD broilers by binding to GPR109A in the pancreas. Tibial studies showed reduced expression of vascular regulatory factors (including PI3K, AKT and VEFGA) based on transcriptomics analysis and reduced vascular distribution, contributing to nonvascularization of cartilage in the proximal tibial growth plate of TD broilers with elevated GLU levels. Additionally, treatment with the total flavonoids from Rhizoma drynariae further validated the improvement in bone homoeostasis in TD broilers by regulating GLU levels through the regulation of GM to subsequently improve intestinal and pancreatic function. These findings clarify the critical role of GM-mediated changes in GLU levels via the gut-pancreas axis in bone homoeostasis in TD chickens.