BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

One of the basic questions in the aging field is whether there is a fundamental difference between the aging of lower invertebrates and mammals. A major difference between the lower invertebrates and mammals is the abundancy of noncoding RNAs, most of which are not conserved. We have previously identified a noncoding RNA Terc-53 that is derived from the RNA component of telomerase Terc. To study its physiological functions, we generated two transgenic mouse models overexpressing the RNA in wild-type and early-aging Terc-/- backgrounds. Terc-53 mice showed age-related cognition decline and shortened life span, even though no developmental defects or physiological abnormality at an early age was observed, indicating its involvement in normal aging of mammals. Subsequent mechanistic study identified hyaluronan-mediated motility receptor (Hmmr) as the main effector of Terc-53. Terc-53 mediates the degradation of Hmmr, leading to an increase of inflammation in the affected tissues, accelerating organismal aging. adeno-associated virus delivered supplementation of Hmmr in the hippocampus reversed the cognition decline in Terc-53 transgenic mice. Neither Terc-53 nor Hmmr has homologs in C. elegans. Neither do arthropods express hyaluronan. These findings demonstrate the complexity of aging in mammals and open new paths for exploring noncoding RNA and Hmmr as means of treating age-related physical debilities and improving healthspan.
Animals ; Mice ; RNA, Untranslated/metabolism* ; Aging/genetics* ; Mice, Transgenic ; Telomerase/metabolism* ; RNA/genetics* ; Hippocampus/metabolism* ; Humans ; Mice, Inbred C57BL

The incomplete degradation of tumour cells by macrophages (Mϕ) is a contributing factor to tumour progression and metastasis, and the degradation function of Mϕ is mediated through phagosomes and lysosomes. In our preliminary experiments, we found that overactivation of NADPH oxidase 2 (NOX2) reduced the ability of Mϕ to degrade engulfed tumour cells. Above this, we screened out liquiritin from Glycyrrhiza uralensis Fisch, which can significantly inhibit NOX2 activity and inhibit tumours, to elucidate that suppressing NOX2 can enhance the ability of Mϕ to degrade tumour cells. We found that the tumour environment could activate the NOX2 activity in Mϕ phagosomes, causing Mϕ to produce excessive reactive oxygen species (ROS), thus prohibiting the formation of phagolysosomes before degradation. Conversely, inhibiting NOX2 in Mϕ by liquiritin can reduce ROS and promote phagosome-lysosome fusion, therefore improving the enzymatic degradation of tumour cells after phagocytosis, and subsequently promote T cell activity by presenting antigens. We further confirmed that liquiritin down-regulated the expression of the NOX2 specific membrane component protein gp91 phox, blocking its binding to the NOX2 cytoplasmic component proteins p67 phox and p47 phox, thereby inhibiting the activity of NOX2. This study elucidates the specific mechanism by which Mϕ cannot degrade tumour cells after phagocytosis, and indicates that liquiritin can promote the ability of Mϕ to degrade tumour cells by suppressing NOX2.

The disorder of group 3 innate lymphoid cells(ILC3)subgroup,such as the predominance of NCR-ILC3 but the deple-tion of NCR+ILC3,is unfavorable to damaged intestinal barrier repair,which leads to the prolongations and obstinacy of ulcerative colitis(UC).Our previous studies had shown that luteolin promoted NCRILC3 differentitating into NCR+ILC3 to improving the de-pletion of NCR+ILC3 in UC mice,while the mechanism is unclear.This article aimed to explore the underlying mechanism of luteolin enhancing the proportion NCR+ILC3.UC mice model was established with 2％DSS and Notch signaling was blocked,then luteolin was used to intervene.The results showed that the effect of luteolin on ameliorating disease symptoms in UC mice,including inhibit-ing the weight loss,reducing the pathological damage of colon mucosa,etc.,was diminished with blocking Notch signaling pathway.In addition,luteolin increased the proportion of NCR+ILC3,NCR+MNK3 and IL-22+ILC3,decreased intestinal permeability,pro-moted mucin secretion,and promoted ZO-1 and Occludin expression,the above effect of luteolin was neutralized by Notch inhibitor LY-411575.Luteolin activated the abnormally blocked Notch signaling pathway in UC mice.And molecular docking predicted the af-finity of luteolin for RBPJ to be-7.5 kcal·mol-1 in mouse,respectively;the affinity of luteolin for Notchl and RBPJ was respectively scored to be-6.4 kcal·mol-1 and-7.7 kcal·mol-1 homo sapiens.These results proved that luteolin is positive for enhancing the propor-tion of NCR+ILC3 via Notch signaling,and it provides a basis for targeting NCR+ILC3 for restoring intestinal barrier function to alle-viating ulcerative colitis.

Objective To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer.Methods Tumor associated fibroblasts(CAFs)co-cultured with breast cancer cells were treated with metformin,and the changes in expressions of hypoxia-inducible factor-1α(HIF-1α),p-AMPK,stroma-derived factor-1(SDF-1)and interleukin-8(IL-8)in the CAFs were detected using ELISA,RT-qPCR or Western blotting;Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1,IL-8 and TGF-β1.The effects of HIF-1α shRNA or overexpression plasmid,AMPK shRNA,and treatment with OG(a proline hydroxylase inhibitor)or 2-OXO(a proline hydroxylase activator)were examined on p-AMPK,HIF-1α,SDF-1 and IL-8 expressions and invasiveness of the CAFs.Results Metformin treatment significantly increased the expression levels of p-AMPK,SDF-1 and IL-8(P<0.05)and decreased HIF-1α expression(P<0.05)without affecting AMPK expression level(P>0.05)in the CAFs.The invasion ability of metformin-treated breast cancer cells was significantly decreased(P<0.05).Exogenous SDF-1 and IL-8,HIF-1α overexpression,and OG-induced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion(P<0.05)and HIF-1α,SDF-1 and IL-8 expressions in CAFs(P<0.05).Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells(P<0.05).P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells(P<0.05).Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells(P<0.05).Conclusion Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Long non-coding RNAs(lncRNAs),a group of non-coding RNAs with lengths exceeding 200 nucleotides,play a crucial role in the human transcriptome,particularly in various types of cancers,where they are pivotal in regulating the tumor immune microenvironment and tumor cell immune evasion.However,there is currently a lack of systematic literature review.Myeloid-derived suppressor cells(MDSCs),a subset of myeloid cells with immunosuppressive functions,play a significant role in cancer by directly and indirectly suppressing T cell activity,promoting the expansion of regulatory T cells,modulating the tumor microenvironment,and facilitating tumor metastasis.This review emphasizes the importance of in-depth investigation into the regulatory mechanisms of MDSCs for the discovery of effective tumor therapeutic targets and the development of novel treatment strategies.With further understanding and research into the relationship between lncRNAs and MDSCs,it is anticipated that more targets and new therapeutic approaches will be identified for cancer treatment,thereby improving clinical outcomes for patients.

Objective To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer.Methods Tumor associated fibroblasts(CAFs)co-cultured with breast cancer cells were treated with metformin,and the changes in expressions of hypoxia-inducible factor-1α(HIF-1α),p-AMPK,stroma-derived factor-1(SDF-1)and interleukin-8(IL-8)in the CAFs were detected using ELISA,RT-qPCR or Western blotting;Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1,IL-8 and TGF-β1.The effects of HIF-1α shRNA or overexpression plasmid,AMPK shRNA,and treatment with OG(a proline hydroxylase inhibitor)or 2-OXO(a proline hydroxylase activator)were examined on p-AMPK,HIF-1α,SDF-1 and IL-8 expressions and invasiveness of the CAFs.Results Metformin treatment significantly increased the expression levels of p-AMPK,SDF-1 and IL-8(P<0.05)and decreased HIF-1α expression(P<0.05)without affecting AMPK expression level(P>0.05)in the CAFs.The invasion ability of metformin-treated breast cancer cells was significantly decreased(P<0.05).Exogenous SDF-1 and IL-8,HIF-1α overexpression,and OG-induced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion(P<0.05)and HIF-1α,SDF-1 and IL-8 expressions in CAFs(P<0.05).Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells(P<0.05).P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells(P<0.05).Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells(P<0.05).Conclusion Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

OBJECTIVE: Our study aimed to conduct genomic characterization of Salmonella strains carrying the bla _NDM-1 gene in the intestinal tract of healthy individuals. The objectives were to underscore the importance of genomic surveillance for drug resistance in both commensal and pathogenic bacteria among healthy populations, and to establish protocols for regulating drug resistance plasmids based on the completion of a comprehensive map of drug resistance plasmid genomes. METHODS: We performed antimicrobial susceptibility testing and employed second- and third-generation sequencing techniques to analyze Salmonella strains harboring the bla _NDM-1 gene, to surveil drug-resistant bacteria in the intestines of healthy subjects. Sequence comparison was conducted using both core- and pan-genome approaches. Concurrently, conjugation experiments were carried out to assess the efficiency of plasmid transfer. RESULTS: We isolated a carbapenem-resistant Salmonella enterica serovar Typhimurium strain from a healthy food worker in China. This strain harbored an IncHI2/IncHI2A plasmid carrying bla _NDM-1 along with multiple antibiotic resistance genes (ARGs). Our findings highlight the potential for asymptomatic carriers to facilitate the transmission of ARGs. Pan-genomic analysis revealed that bla _NDM-1-positive plasmids could traverse bacterial species barriers, facilitating cross-host transmission. CONCLUSION This study marks the first detection of bla _NDM-1 in Salmonella strains isolated from healthy individuals. We underscore the risk associated with the transmission of conjugative hybrid plasmids carrying bla _NDM-1, which have the potential to be harbored and transmitted among healthy individuals. Enhanced surveillance of drug-resistant pathogens and plasmids in the intestinal microbiota of healthy individuals could provide insights into the risk of ARG transmission and pathways for population-wide dissemination via ARG transfer factors.
beta-Lactamases/genetics* ; Plasmids/genetics* ; Humans ; Anti-Bacterial Agents/pharmacology* ; China ; Microbial Sensitivity Tests ; Salmonella typhimurium/isolation & purification* ; Salmonella/isolation & purification* ; Salmonella Infections/microbiology*

Objective To evaluate the effect of hypothermic machine perfusion (HMP) on the expression levels of inflammatory cytokines in rat kidney. Methods Thirty male rats were randomly divided into the control (Control group), static cold storage group (SCS group) and HMP group, with 10 rats in each group. The velocity, intrarenal resistance and pH value of perfusion effluent were recorded during HMP. The expression levels of CXC chemokine ligand (CXCL)1, CXCL2, interferon (IFN)-β1, IFN-α4, CC chemokine ligand (CCL)2, CCL20, interleukin (IL)-17α, IL-17C and tumor necrosis factor (TNF)-α messenger RNA (mRNA) in renal tissues were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Pathological changes of the kidney were observed by hematoxylin-eosin (HE) staining. Results During HMP, the velocity and intrarenal resistance remained stable, and the pH value of perfusion effluent was decreased slowly. RT-PCR showed that the relative expression levels of CXCL1, CXCL2, CCL2, CCL20, IL-17α, IL-17C and TNF-α mRNA in the SCS and HMP groups were higher compared with those in the Control group. Compared with the SCS group, the relative expression levels of CXCL1, CXCL2, CCL2, CCL20, IL-17α and TNF-α mRNA were up-regulated in the HMP group (all P<0.05). HE staining revealed that the morphology of renal cells was normal in the Control group, whereas evident epithelial necrosis, cytoplasmic vacuolation, brush border loss and epithelial shedding were observed in the SCS group. Compared with the SCS group, pathological changes in the HMP group were alleviated. Conclusions HMP may activate renal inflammation, and inhibiting the activation of inflammation during HMP is expected to further improve the effect of allograft preservation.

Objective:To explore the effects of different concentrations of 2-hydroxybenzylamine(2-HOBA)on atherosclerosis and vascular smooth muscle cell senescence and the underlying mechanisms.Methods:Fourteen apolipoprotein E-deficient(ApoE-/-)mice were used to establish an atherosclerosis model and were divided into two groups(n=7)using the random number method: a high-fat diet(HD)group and a high-fat diet plus 2-HOBA(1 mg/ml)(HD+ HOBA)group.Pulse wave velocity was used to assess vascular stiffness and a treadmill was used to assess exercise endurance.Oil Red O staining was used to detect the size and number of atherosclerotic plaques.Masson staining was used to detect the morphology of collagen fibers and elastic fibers in the plaque, the size of the necrotic core area of the plaque, and the thickness of the fibrous cap.Mouse smooth muscle cells were treated with different concentrations of 2-HOBA(100 μmol/L, 250 μmol/L and 500 μmol/L)to establish an H 2O 2-induced senescence model.Senescence-associated β-galactosidase staining was used to detect cell senescence.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression levels of senescence-related secretory phenotype factors, and Western blot was used to detect the expression levels of senescence-related signaling proteins. Results:Compared with the HD group, the HD+ HOBA group showed that the area and number of aortic atherosclerotic plaques were decreased, and the atherosclerotic plaques were stabilized.In addition, compared with the HD group, vascular stiffness in the HD+ 2-HOBA group decreased by 26%(2.59±0.32 mm/ms vs.3.50±0.28 mm/ms), with a statistically significant difference( P<0.01), and exercise endurance increased by 62%[(143.74±24.25)m vs.(233.50±30.21)m, P<0.01], suggesting that 2-HOBA was able to improve aortic vascular stiffness and exercise endurance in mice.2-HOBA ameliorated H 2O 2-induced vascular smooth muscle cell senescence and decreased the mRNA levels of H 2O 2-induced senescence-associated secretory phenotype factors such as interleukin-1β, interleukin-6, tumor necrosis factor-α and monocyte chemoattractant protein-1.Meanwhile, 2-HOBA also inhibited the expression of p53 and p21, the key signaling factors of senescence. Conclusions:2-HOBA suppresses the development and progression of atherosclerosis through inhibiting oxidative stress-related p53/p21 signaling activation and ameliorating vascular smooth muscle cell senescence and the aging-related inflammatory phenotype.