Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

Understanding the Manufacturing Process of Lipid Nanoparticles for mRNA Delivery Using Machine Learning.

Lipid nanoparticles (LNPs), used for mRNA vaccines against severe acute respiratory syndrome coronavirus 2, protect mRNA and deliver it into cells, making them an essential delivery technology for RNA medicine. The LNPs manufacturing process consists of two steps, the upstream process of preparing LNPs and the downstream process of removing ethyl alcohol (EtOH) and exchanging buffers. Generally, a microfluidic device is used in the upstream process, and a dialysis membrane is used in the downstream process. However, there are many parameters in the upstream and downstream processes, and it is difficult to determine the effects of variations in the manufacturing parameters on the quality of the LNPs and establish a manufacturing process to obtain high-quality LNPs. This study focused on manufacturing mRNA-LNPs using a microfluidic device. Extreme gradient boosting (XGBoost), which is a machine learning technique, identified EtOH concentration (flow rate ratio), buffer pH, and total flow rate as the process parameters that significantly affected the particle size and encapsulation efficiency. Based on these results, we derived the manufacturing conditions for different particle sizes (approximately 80 and 200 nm) of LNPs using Bayesian optimization. In addition, the particle size of the LNPs significantly affected the protein expression level of mRNA in cells. The findings of this study are expected to provide useful information that will enable the rapid and efficient development of mRNA-LNPs manufacturing processes using microfluidic devices.

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Growth behavior and mRNA expression profiling during growth of IPEC-J2 cells.

OBJECTIVE: The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. RESULTS: Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.

The m6A reader HNRNPC promotes glioma progression by enhancing the stability of IRAK1 mRNA through the MAPK pathway.

Glioma is the most common and aggressive type of primary malignant brain tumor. The N6-methyladenosine (m6A) modification widely exists in eukaryotic cells and plays an important role in the occurrence and development of human tumors. However, the function and mechanism of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an RNA-binding protein and m6A reader in gliomas remains to be comprehensively and extensively explored. Herein, we found that HNRNPC mRNA and protein overexpression were associated with a poor prognosis for patients with gliomas, based on the data from TCGA, the CGGA, and the TMAs. Biologically, HNRNPC knockdown markedly repressed malignant phenotypes of glioma in vitro and in vivo, whereas ectopic HNRNPC expression had the opposite effect. Integrative RNA sequencing and MeRIP sequencing analyses identified interleukin-1 receptor-associated kinase 1 (IRAK1) as a downstream target of HNRNPC. The glioma public datasets and tissue microarrays (TMAs) data indicated that IRAK1 overexpression was associated with poor prognosis, and IRAK1 knockdown significantly repressed malignant biological behavior in vitro. Mechanistically, HNRNPC maintains the mRNA stability of IRAK1 in an m6A-dependent manner, resulting in activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which was necessary for the malignant behavior of glioma. Our findings demonstrate the HNRNPC-IRAK1-MAPK axis as a crucial carcinogenic factor for glioma and the novel underlying mechanism of IRAK1 upregulation, which provides a rationale for therapeutically targeting epitranscriptomic modulators in glioma.

PPIH acts as a potential predictive biomarker for patients with common solid tumors.

BACKGROUND: Our previous studies have indicated that mRNA and protein levels of PPIH are significantly upregulated in Hepatocellular Carcinoma (LIHC) and could act as predictive biomarkers for patients with LIHC. Nonetheless, the expression and implications of PPIH in the etiology and progression of common solid tumors have yet to be explored, including its potential as a serum tumor marker. METHODS: We employed bioinformatics analyses, augmented with clinical sample evaluations, to investigate the mRNA and protein expression and gene regulation networks of PPIH in various solid tumors. We also assessed the association between PPIH expression and overall survival (OS) in cancer patients using Kaplan-Meier analysis with TCGA database information. Furthermore, we evaluated the feasibility and diagnostic efficacy of PPIH as a serum marker by integrating serological studies with established clinical tumor markers. RESULTS: Through pan-cancer analysis, we found that the expression levels of PPIH mRNA in multiple tumors were significantly different from those in normal tissues. This study is the first to report that PPIH mRNA and protein levels are markedly elevated in LIHC, Colon adenocarcinoma (COAD), and Breast cancer (BC), and are associated with a worse prognosis in these cancer patients. Conversely, serum PPIH levels are decreased in patients with these tumors (LIHC, COAD, BC, gastric cancer), and when combined with traditional tumor markers, offer enhanced sensitivity and specificity for diagnosis. CONCLUSION: Our findings propose that PPIH may serve as a valuable predictive biomarker in tumor patients, and its secreted protein could be a potential serum marker, providing insights into the role of PPIH in cancer development and progression.

Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides.

Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.

In vivo and in vitro studies on the role of regulating Smac expression in the occurrence and development of colon cancer.

We aimed to explore the role of regulating Smac expression levels in the occurrence and development of colon cancer through in vitro and in vivo experiments. Colon cancer cells HT-29 were cultured and transfected into different groups. qRT-PCR was used to detect the expression level of Smac in cells; Flow cytometry was used to detect the apoptotic ability of each group of cells; Western blot was used to detect the protein expression of Smac and apoptosis-related factors Survivin and Caspase-3; The nude mouse tumorigenesis experiment was conducted to detect the regulatory effect of regulating Smac expression levels on the growth of colon cancer transplanted tumors in vivo. In comparison to the FHC group, the HT-29 group exhibited a decrease in Smac expression. The si-Smac group, when compared with the si-NC group, showed significant reductions in Smac mRNA and protein levels, weaker cell apoptosis, increased Survivin, and decreased Caspase-3 expression. Contrarily, the oe-Smac group, against the oe-NC group, displayed increased Smac mRNA and protein levels, enhanced apoptosis, reduced Survivin, and elevated Caspase-3 expression. In nude mice tumor transplantation experiments, the LV-sh-Smac group, as opposed to the LV-sh-NC group, had tumors with greater volume and weight, reduced Smac and Caspase-3, and increased Survivin expression. In contrast, the LV-oe-Smac group, compared with the LV-oe-NC group, showed tumors with decreased volume and mass, increased expressions of Smac and Caspase-3, and decreased Survivin. Smac is lowly expressed in colon cancer. Upregulation of Smac expression can inhibit the occurrence and development of colon cancer, possibly by inhibiting Survivin expression and promoting Caspase-3 expression, thereby enhancing the pro-apoptotic function.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

Differential expression of circRNAs during osteogenic/odontogenic differentiation of stem cells from apical papilla promoted by blue light-emitting diode.

BACKGROUND: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED. MATERIALS AND METHODS: SCAPs were divided into the irradiation group (4 J/cm2) and the control group (0 J/cm2), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed. RESULTS: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed. CONCLUSION: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.

Improving dermal fibroblast-to-epidermis communications and aging wound repair through extracellular vesicle-mediated delivery of Gstm2 mRNA.

Skin aging is characterized by the disruption of skin homeostasis and impaired skin injury repair. Treatment of aging skin has long been limited by the unclear intervention targets and delivery techniques. Engineering extracellular vesicles (EVs) as an upgraded version of natural EVs holds great potential in regenerative medicine. In this study, we found that the expression of the critical antioxidant and detoxification gene Gstm2 was significantly reduced in aging skin. Thus, we constructed the skin primary fibroblasts-derived EVs encapsulating Gstm2 mRNA (EVsGstm2), and found that EVsGstm2 could significantly improve skin homeostasis and accelerate wound healing in aged mice. Mechanistically, we found that EVsGstm2 alleviated oxidative stress damage of aging dermal fibroblasts by modulating mitochondrial oxidative phosphorylation, and promoted dermal fibroblasts to regulate skin epidermal cell function by paracrine secretion of Nascent Polypeptide-Associated Complex Alpha subunit (NACA). Furthermore, we confirmed that NACA is a novel skin epidermal cell protective molecule that regulates skin epidermal cell turnover through the ROS-ERK-ETS-Cyclin D pathway. Our findings demonstrate the feasibility and efficacy of EVs-mediated delivery of Gstm2 for aged skin treatment and unveil novel roles of GSTM2 and NACA for improving aging skin.

Adjuvants for cancer mRNA vaccines in the era of nanotechnology: strategies, applications, and future directions.

Research into mRNA vaccines is advancing rapidly, with proven efficacy against coronavirus disease 2019 and promising therapeutic potential against a variety of solid tumors. Adjuvants, critical components of mRNA vaccines, significantly enhance vaccine effectiveness and are integral to numerous mRNA vaccine formulations. However, the development and selection of adjuvant platforms are still in their nascent stages, and the mechanisms of many adjuvants remain poorly understood. Additionally, the immunostimulatory capabilities of certain novel drug delivery systems (DDS) challenge the traditional definition of adjuvants, suggesting that a revision of this concept is necessary. This review offers a comprehensive exploration of the mechanisms and applications of adjuvants and self-adjuvant DDS. It thoroughly addresses existing issues mentioned above and details three main challenges of immune-related adverse event, unclear mechanisms, and unsatisfactory outcomes in old age group in the design and practical application of cancer mRNA vaccine adjuvants. Ultimately, this review proposes three optimization strategies which consists of exploring the mechanisms of adjuvant, optimizing DDS, and improving route of administration to improve effectiveness and application of adjuvants and self-adjuvant DDS.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

ZDHHC20-mediated S-palmitoylation of YTHDF3 stabilizes MYC mRNA to promote pancreatic cancer progression.

Post-translational modifications of proteins in malignant transformation and tumor maintenance of pancreatic ductal adenocarcinoma (PDAC) in the context of KRAS signaling remain poorly understood. Here, we use the KPC mouse model to examine the effect of palmitoylation on pancreatic cancer progression. ZDHHC20, upregulated by KRAS, is abnormally overexpressed and associated with poor prognosis in patients with pancreatic cancer. Dysregulation of ZDHHC20 promotes pancreatic cancer progression in a palmitoylation-dependent manner. ZDHHC20 inhibits the chaperone-mediated autophagic degradation of YTHDF3 through S-palmitoylation of Cys474, which can result in abnormal accumulation of the oncogenic product MYC and thereby promote the malignant phenotypes of cancer cells. Further, we design a biologically active YTHDF3-derived peptide to competitively inhibit YTHDF3 palmitoylation mediated by ZDHHC20, which in turn downregulates MYC expression and inhibits the progression of KRAS mutant pancreatic cancer. Thus, these findings highlight the therapeutic potential of targeting the ZDHHC20-YTHDF3-MYC signaling axis in pancreatic cancer.

Senolytic effects of exercise in human muscles require acute inflammation.

Higher intensity exercise, despite causing more tissue damage, improved aging conditions. We previously observed decreased p16INK4a mRNA in human skeletal muscle after high-intensity interval exercise (HIIE), with no change following equivalent work in moderate-intensity continuous exercise. This raises the question of whether the observed senolytic effect of exercise is mediated by inflammation, an immune response induced by muscle damage. In this study, inflammation was blocked using a multiple dose of ibuprofen (total dose: 1200 mg), a commonly consumed nonsteroidal anti-inflammatory drug (NSAID), in a placebo-controlled, counterbalanced crossover trial. Twelve men aged 20-26 consumed ibuprofen or placebo before and after HIIE at 120% maximum aerobic power. Multiple muscle biopsies were taken for tissue analysis before and after HIIE. p16INK4a+ cells were located surrounding myofibers in muscle tissues. The maximum decrease in p16INK4a mRNA levels within muscle tissues occurred at 3 h post-exercise (-82%, p < 0.01), gradually recovering over the next 3-24 h. A concurrent reduction pattern in CD11b mRNA (-87%, p < 0.01) was also found within the same time frame. Ibuprofen treatment attenuated the post-exercise reduction in both p16INK4a mRNA and CD11b mRNA. The strong correlation (r = 0.88, p < 0.01) between p16INK4a mRNA and CD11b mRNA in muscle tissues suggests a connection between the markers of tissue aging and pro-inflammatory myeloid differentiation. In conclusion, our results suggest that the senolytic effect of high-intensity exercise on human skeletal muscle is mediated by acute inflammation.

Serine racemase expression profile in the prefrontal cortex and hippocampal subregions during aging in male and female rats.

Aging is associated with a decrease in N-methyl-D-aspartate (NMDA) receptor function, which is critical for maintaining synaptic plasticity, learning, and memory. Activation of the NMDA receptor requires binding of the neurotransmitter glutamate and also the presence of co-agonist D-serine at the glycine site. The enzymatic conversion of L-serine to D-serine is facilitated by the enzyme serine racemase (SR). Subsequently, SR plays a pivotal role in regulating NMDA receptor activity, thereby impacting synaptic plasticity and memory processes in the central nervous system. As such, age-related changes in the expression of SR could contribute to decreased NMDA receptor function. However, age-associated changes in SR expression levels in the medial and lateral prefrontal cortex (mPFC, lPFC), and in the dorsal hippocampal subfields, CA1, CA3, and dentate gyrus (DG), have not been thoroughly elucidated. Therefore, the current studies were designed to determine the SR expression profile, including protein levels and mRNA, for these regions in aged and young male and female Fischer-344 rats. Our results demonstrate a significant reduction in SR expression levels in the mPFC and all hippocampal subfields of aged rats compared to young rats. No sex differences were observed in the expression of SR. These findings suggest that the decrease in SR levels may play a role in the age-associated reduction of NMDA receptor function in brain regions crucial for cognitive function and synaptic plasticity.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Identifying competing endogenous RNA regulatory networks and hub genes in alcoholic liver disease for early diagnosis and potential therapeutic target insights.

Alcoholic liver disease (ALD) has a complex pathogenesis. Although early-stage ALD can be reversed by ceasing alcohol consumption, early symptoms are difficult to detect, and several factors contribute to making alcohol difficult to quit. Continued alcohol abuse worsens the condition, meaning it may gradually progress into alcoholic hepatitis and cirrhosis, ultimately, resulting in irreversible consequences. Therefore, effective treatments are urgently needed for early-stage ALD. Current research mainly focuses on preventing the progression of alcoholic fatty liver to alcoholic hepatitis and cirrhosis. However, challenges remain in identifying key therapeutic targets and understanding the molecular mechanisms that underlie the treatment of alcoholic hepatitis and cirrhosis, such as the limited discovery of effective therapeutic targets and treatments. Here, we downloaded ALD microarray data from Gene Expression Omnibus and used bioinformatics to compare and identify the hub genes involved in the progression of alcoholic fatty liver to alcoholic hepatitis and cirrhosis. We also predicted target miRNAs and long non-coding RNAs (lncRNAs) to elucidate the regulatory mechanisms (the mRNA-miRNA-lncRNA axis) underlying this progression, thereby building a competitive endogenous RNA (ceRNA) mechanism for lncRNA, miRNA, and mRNA. This study provides a theoretical basis for the early treatment of alcoholic hepatitis and cirrhosis and identifies potential therapeutic targets.

Effect of ADSCs on Th17/Treg and T-bet/GATA-3 in model mice with primary immune thrombocytopenia.

We aimed to observe the effects of adipose-derived mesenchymal stem cells (ADSCs) on T helper 17 (Th17)/regulatory T cells (Treg) and T-box transcription factor (T-bet)/GATA-binding protein 3 (GATA-3) in model mice with primary immune thrombocytopenia (ITP). 32 BALB/C mice were selected. ADSCs were isolated from 2 mice and cultured. The other 30 mice were randomly divided into the normal control group, the ITP model control group, and the ITP experimental group. Platelet count (PLT), Th17/Treg cells, related serum cytokines [interleukin-6 (IL-6), IL-17A, IL-10, and transforming growth factor ß1 (TGF-ß1)], T-bet and GATA-3 mRNA levels in peripheral blood mononuclear cells (PBMCs) in the 3 groups were detected. PLT and Treg in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). Th17 and Th17/Treg in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-6 and IL-17A levels, and T-bet mRNA levels in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-10 and TGF-ß levels, and GATA-3 mRNA levels in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). ADSCs can effectively regulate Th17/Treg balance and improve T-bet/GATA-3 mRNA expression levels in ITP model mice.

Trimethylamine N-oxide ameliorates hepatic damage including reduction of hepatic bile acids and cholesterol in Fxr-null mice.

There are conflicting animal experiments on the effect of trimethylamine N-oxide (TMAO), the dietary metabolite, on non-alcoholic fatty liver disease (NAFLD). This study aims to determine the effect of TMAO on NAFLD. A diet containing 0.3% TMAO was fed to farnesoid X receptor (Fxr)-null mice, a model of NAFLD, for 13 weeks. Fxr-null mice fed TMAO showed significant reductions in liver damage markers but not wild-type mice. Hepatic bile acid and cholesterol levels were significantly decreased, and triacylglycerol levels tended to decrease in TMAO-fed Fxr-null mice. Changes in mRNA levels of hepatic bile acid and cholesterol transporters and synthetic enzymes were observed, which could explain the decreased hepatic bile acid and cholesterol levels in Fxr-null mice given the TMAO diet but not in the wild-type mice. These results suggest that TMAO intake ameliorates liver damage in Fxr-null mice, further altering bile acid/cholesterol metabolism in an FXR-independent manner.