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SUMMARYThe messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccine platform is the newest tool in the vaccination arsenal for combating infectious diseases, rare genetic diseases, and cancer. Since 2020, major advances in preclinical and clinical studies have demonstrated the efficacy of mRNA-LNP vaccines against viral pathogens and in cancer. However, studies investigating the efficacy of mRNA-LNP vaccines against bacterial pathogens remain sparse due to substantial bacterial vaccine-specific challenges. Here, we highlight key challenges impeding the development of mRNA-LNP vaccines against human bacterial pathogens, the advantages that can be gained by utilizing the mRNA-LNP platform for bacterial vaccines, and progress toward the development of mRNA-LNP vaccines against bacterial infections.
With the rapid development of the mRNA pharmaceutical industry, Oligo(dT) chromatography, as a core step in mRNA separation and purification, has garnered increasing attention from both the industry and researchers. This article begins with the fundamental principles of mRNA affinity chromatography, analyzes the advantages and disadvantages of two typical affinity techniques, and highlights the sufficiency of Oligo(dT) chromatography as a platform technology. Aiming to the poor performance of the Oligo(dT) chromatography, the article reviews the main methods and mechanisms for enhancing its performance from three perspectives, resin structure, ligand structure, and process intensification. It also highlights the factors influencing the performance of Oligo(dT) chromatography and the challenges associated with these methods. The article provides an outlook on the future development directions of Oligo(dT) chromatography, offering insights for the advancement of next-generation Oligo(dT) chromatography resins and processes.
The response to lifestyle modification (LSM) in children with obesity is variable and difficult to predict. A systematic search for molecular markers to predict outcomes of LSM in pediatric obesity management. Out of 240 children with obesity (BMI>97%) recruited to a prospective 'multi-OMICS' study granted by ESPE Research Unit, 159 subjects (age 8-17 yrs, median 12.8 yrs; 45% females) finished twelve-months (between visits V0-V12) of LSM at three clinical centers (Poland, Turkey, Italy). Four of >180 baseline (V0, before LSM) clinical and laboratory features (sex, age, severity of obesity at V0, steroid metabolome) were used to select 50 representatives for RNA-seq analysis. We searched for circulating mRNA & miRNA capable of establishing the link with positive response to LSM defined by a decrease in z-score BMI (delta z-score BMI V12-V0<0, Responders/RS). First, the candidate gene approach with previously documented STAT3, CORO1C, SERPINH1, MVP, ITGB5 (upregulated in obesity), PCM1, SIRT1, EEF1G, PTEN and RPS2 (down-regulated in obesity) hub genes was applied. The pairwise ratios of transcripts per million (TPM)s of these genes were tested for statistical significance between RS (n=31) and non-RS (n=19) groups. Secondly, the TPM expression values of 2,129 miRNAs and 33,246 RPL13A-normalized mRNAs were compared between RS and non-RS to get a list of putative significant genes (p<0.05; the Welch t-test). Enrichr, a web-based tool (http://amp.pharm.mssm.edu/Enrichr), was used for analysing gene sets. Expression values for 909 mRNA and 24 miRNA genes were significantly different between RS vs non-RS. For example, STAT3/PTEN, CORO1C/PTEN, MVP/PTEN; miRNA-10a, miRNA-122, miRNA-127, miRNA-502, miRNA-210 were significantly higher and SIRT1/PTEN, EEF1G/PTEN, RPS2/PTEN, miRNA-27a were lower comparing RS vs. non-RS (p= 0.009, 0.022, 0.030, 0.048, 0.029, 0.045, 0.035, 0.020, 0.047, 0.04, 0.039, 0.029, respectively). Several pathways, including "Vitamin B6 metabolism" and "Insulin receptor signaling", were enriched in our differentially expressed mRNA (p<0.001): PNPO, PHOSPHO2, PDXP, PSAT1 were up-regulated in RS vs. non-RS. "Leptin signaling pathway WP2034" were enriched in STAT3 and PTEN (p<0.0001). The role of the balance between STAT3 and PTEN in LSM was discovered. The importance of previously described miRNA markers (miRNA-122, miRNA-127, miRNA-27a) related to adipogenesis risk, insulin resistance, hepatic fat reduction, PPAR-γ expression were confirmed. A combination of the previously reported V0 Leptin feature together with STAT3/PTEN and miRNA-27a levels showed promising predictive results. Our findings not only showed the predictive molecular markers but also pointed to their role in personalization of therapeutic management.
The Epstein-Barr virus (EBV) infects over 95% of adults, establishing lifelong latency and contributing to the development of various malignancies, including Burkitt lymphoma and nasopharyngeal carcinoma. However, the RNA structures regulating the splicing of the critical EBV gene, latent membrane protein 1 (LMP1), remain uncharacterized. To identify these regulatory elements, we applied spliceosome inhibition with RNA probing and sequencing (SIRP-seq) to the BJAB-B1 cell line. By utilizing the spliceosome inhibitor pladienolide B, we enriched pre-mRNA species, enabling the detection of structural features within both the full-length pre-mRNA (LMP1-FL) and an alternatively spliced isoform retaining intron 2 (LMP1-AS). The resulting chemical probing datasets informed the RNA folding algorithms RNAfold and ScanFold to generate the first high-resolution secondary structure models for the LMP1 pre-mRNA, encompassing both exonic and intronic regions. Our results identify 11 novel, thermodynamically stable RNA structures, with several key elements positioned near splice junctions. Notably, three structures (Structures 8, 9, and 10) were identified near the 3' splice site of intron 2, appearing in alternative conformations that may influence splicing accessibility. Furthermore, these structures map to regions containing disease-relevant mutations associated with patient survival in Burkitt lymphoma. This structural framework provides new insights into how LMP1 splicing may be regulated by RNA structure and identifies potential novel therapeutic targets for mitigating EBV-associated diseases.
This is a review of modified mRNA (mmRNA) science and a description of a novel application of mmRNA for the creation of personalized cosmetic products.
Plants activate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) to combat pathogens. However, how these systems coordinate immune activation while preventing autoimmunity remains poorly understood. In this study, we uncovered a regulatory mechanism in which surface immune signaling unlocks nucleotide-binding leucine-rich repeat (NLR) immune receptor activation through mRNA splicing. We identified an N-terminal prodomain in the potato late blight resistance protein Rpi-vnt1.1 that inhibits resistosome formation, preventing potential autoactivation of this NLR. Upon pathogen perception, PTI signaling induced alternative splicing of Rpi-vnt1.1 mRNA, removing this inhibitory element. This primed Rpi-vnt1.1 for activation by the Phytophthora infestans effector AVRvnt1, enabling resistosome assembly and immune signaling. The widespread conservation of N-terminal extensions in coiled coil-type NLRs points to a common regulatory mechanism in preventing potential autoactivation while preserving pathogen sensitivity.
Polymer-based delivery vehicles are a promising nonviral alternative for nucleic acid therapeutics; however, current formulations often have low transfection efficiency or if effective, have high toxicity. Herein, we investigate the use of blended polymeric micelles, an ordered assembly of amphiphilic block copolymers in solution, for the delivery of mRNA. Three cationic amphiphiles consisting of a primary amine (C1), dimethylamine (C2), or diethyleneimine (C3) were synthesized. Neutral amphiphiles containing morpholino (M), hydroxy (H), dihydroxy (DH), brush PEG (PB), or two linear PEGs (P5K, P10K) were also created. Seventy-five micelles were formed by blending each cationic and neutral amphiphile at 5 compositions. In the absence of serum, C2 micelles with PEG incorporations above 10% had the highest transfection efficiency; however, C3 formulations were more optimal, having more moderate transfection efficiencies and improved viability. In the presence of serum, C3 formulations with 5 and 10% neutral group incorporations were the most effective, maintaining cell viabilities on par with untreated controls and transfection efficiencies superior to purely cationic micelles. Higher neutral loadings of 20 and 40% reduced transfection efficiency. IL-1β release from macrophages during transfection was also measured as an indicator for potential inflammatory responses. Neutral group loadings of 10% with C1 and C2 significantly reduced cytokine release. In contrast, 10% neutral loadings for C3 micelles slightly increased cytokine release; however, these levels were still below those measured for C2 formulations, indicative of improved biocompatibility of C3 relative to C2. One notable exception was 10% M in C3 micelles, which produced levels on par with Nigericin, a microbial toxin known to include inflammasome activation. Neutral loadings of 40% reduced cytokine release across the formulation space, indicative of improved biocompatibility. Overall, this work demonstrates that changes in formulation chemistry can produce polymeric systems of improved transfection efficiency, without a coinciding increase in toxicity and immunogenicity, to yield more desirable vehicles for nucleic acid delivery.
[This retracts the article DOI: 10.1155/2022/3472745.].
To investigate the regulatory role of epigenetic regulator disruptor of telomeric silencing 1-like (DOT1L) and its mediated histone H3 lysine 79 (H3K79) methylation in modulating neuronal amyloid precursor protein (APP) expression, and to elucidate the underlying mechanisms involving mitochondrial homeostasis and the upstream p38 kinase. Alzheimer's disease (AD) models were established using APP/presenilin-1 (APP/PS1) double-transgenic mice and N2a cells overexpressing the human Swedish mutant APP (N2a-APPswe). Immunofluorescence staining was employed to assess DOT1L expression and localization in mouse brain tissues. N2a-APPswe cells were treated with the DOT1L-specific inhibitor EPZ5676 and divided into four groups: blank control, solvent control, DOT1L inhibitor, and DOT1L inhibitor plus p38 agonist (Gynostemma pentaphyllum extract). Western blotting was performed to measure the phosphorylation levels of DRP1 at Ser616 and Ser637 (key mitochondrial fission regulators), the levels of autophagy-related proteins p62 and the LC3-Ⅱ/LC3-Ⅰ ratio, the phosphorylation level of p38, as well as the expression of APP and APP-processing proteins BACE1 and PS1. Real-time quantitative polymerase chain reaction was used to detect mRNA levels of APP and genes involved in mitochondrial fission and fusion. Proteomics data were systematically analyzed through Gene Ontology analysis, WikiPathways enrichment analysis, and STRING protein-protein interaction network analysis to identify key signaling pathways. Mitochondrial morphology was evaluated by Mito-Tracker fluore-scence staining to measure average branch length. DOT1L expression was signifi-cantly reduced in neurons of APP/PS1 mice compared to wild-type controls. DOT1L inhibition led to decreased H3K79me2 levels (P<0.01), accompanied by a marked increase in APP protein expression (P<0.01), although APP mRNA levels were reduced (P<0.01). Proteomics analysis revealed that differentially expressed proteins were highly enriched in the mitochondrial electron transport chain. Compared with the solvent control, the DOT1L inhibitor group showed inhibited mitochondrial fission, as evidenced by decreased p-DRP1 (Ser616), increased p-DRP1 (Ser637), downregulated MIEF1 mRNA, upregulated MFN1 mRNA (all P<0.05), and increased average mitochondrial branch length (P<0.05), along with reduced p-p38 levels (P<0.05). Co-administration of the p38 agonist significantly reversed these mitochondrial dynamics abnormalities (all P<0.05) and attenuated the abnormally elevated protein levels of APP, BACE1, and PS1 (P<0.05) compared to the DOT1L inhibitor group. DOT1L maintains normal mito-chondrial fission and functional homeostasis through regulation of the p38 signaling pathway, thereby modulating APP expression. 目的: 明确表观遗传调节因子类端粒沉默干扰体1（DOT1L）及其介导的组蛋白H3第79位赖氨酸（H3K79）甲基化修饰对神经元淀粉样前体蛋白（APP）表达的调控作用，并阐明线粒体稳态及其上游p38激酶在该调控过程中的核心机制。方法: 采用APP/早老蛋白1（PS1）双转基因小鼠模型及过表达人源瑞典突变型APP的N2a（N2a-APPswe）细胞模拟AD。通过免疫荧光染色法检测小鼠脑组织中DOT1L的表达和定位。利用DOT1L特异性抑制剂EPZ5676处理N2a-APPswe细胞，分别设置空白对照组、溶剂对照组、DOT1L抑制剂组及DOT1L抑制剂+p38激动剂（绞股蓝提取物）组。采用蛋白质印迹法检测线粒体分裂关键蛋白质DRP1的Ser616和Ser637位点磷酸化水平、自噬相关蛋白质p62水平和LC3-Ⅱ/LC3-Ⅰ比值、信号分子p38磷酸化水平以及APP代谢相关蛋白质APP、BACE1和PS1的表达水平;采用实时定量聚合酶链反应检测APP、线粒体分裂及融合相关基因的表达;基于蛋白质组学数据，通过基因本体分析、WikiPathways富集分析及STRING蛋白质相互作用网络系统筛选关键信号通路;采用Mito-Tracker荧光染色法检测线粒体分支长度。结果: 与野生型小鼠比较，APP/PS1小鼠神经元中DOT1L表达减少。抑制DOT1L后，H3K79二甲基化水平降低（P<0.01），APP表达水平升高但其mRNA表达水平下降（均P<0.01）。差异蛋白高度富集于线粒体电子传递链。与溶剂对照组比较，DOT1L抑制剂组线粒体分裂受到抑制，表现为磷酸化DRP1（Ser616）水平下降、磷酸化DRP1（Ser637）水平升高、MIEF1 mRNA表达水平下降、MFN1 mRNA表达水平升高（均P<0.05），线粒体平均分支长度增加（P<0.05），并伴随磷酸化p38水平下降（P<0.05）。与DOT1L抑制剂组比较，加用p38激动剂可显著逆转上述线粒体动力学异常（均P<0.05），并降低APP、BACE1及PS1的异常高表达（均P<0.05）。结论: DOT1L通过调控p38介导的信号通路维持线粒体正常分裂及功能稳态，进而调控APP表达。.
Based on the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling pathway, this study investigated the effects of Zhuyu Pills on the immune balance of helper T cell(Th) 1/Th2 and Th17/regulatory T cell(Treg) populations in a mouse model of acne. The acne model was established by applying oleic acid to the right auricle combined with subcutaneous injection of Propionibacterium acnes, resulting in auricle lesions of 1 cm~2 over a 14-day modeling period. Fifty successfully modeled ICR mice were randomly assigned to five groups: model, low-, medium-, and high-dose Zhuyu Pills, and isotretinoin(n=10 per group). An additional 10 age-matched healthy ICR mice were included as a control group without modeling. The control and model groups received equal volumes of sterile distilled water via gavage, while the low-, medium-, and high-dose Zhuyu Pills groups received Zhuyu Pills at 130.54, 261.08, and 522.16 mg·kg~(-1), respectively. The isotretinoin group received isotretinoin at 4.55 mg·kg~(-1), once daily. All treatments were administered for four weeks. After administration, auricle tissue pathology was evaluated by hematoxylin and eosin(HE) staining. Serum levels of interleukin(IL)-17, interferon-γ(IFN-γ), IL-10, transforming growth factor-β(TGF-β), IL-12, IL-22, IL-4, and IL-5 were measured by ELISA. Protein expression of p-JAK2/JAK2 and p-STAT3/STAT3 in auricle tissue was assessed by Western blot. Th1, Th2, Th17, and Treg cell populations in the spleen were analyzed by flow cytometry. Immunohistochemistry was used to detect T-box expressed in T cell(T-bet) and GATA binding protein 3(GATA3) expression, and immunofluorescence was used to detect retineic-acid-receptor-related orphan nuclear receptor gamma(RORγt) and forkhead box P3(Foxp3) expression in auricle tissue. Western blot and real-time PCR were used to measure protein and mRNA levels of T-bet, GATA3, RORγt, and Foxp3. Compared with the control group, the model group exhibited significantly thickened auricle epidermis and extensive inflammatory cell infiltration(P<0.01); increased serum IL-17, IFN-γ, IL-12, and IL-22(P<0.01); decreased TGF-β, IL-10, IL-4, and IL-5(P<0.01); elevated p-JAK2/JAK2 and p-STAT3/STAT3 levels in auricle tissue(P<0.01); increased Th1 and Th17(P<0.01) and decreased Th2 and Treg cell populations in the spleen(P<0.01); and upregulated protein and mRNA levels of T-bet and RORγt(P<0.01) but downregulated GATA3 and Foxp3 protein and mRNA levels in auricle tissue(P<0.01). Compared with the model group, all Zhuyu Pills groups and the isotretinoin group showed reduced epidermal thickness and inflammatory infiltration(P<0.01); decreased serum IL-17, IFN-γ, IL-12, and IL-22(P<0.05 or P<0.01) and increased TGF-β, IL-10, IL-4, and IL-5(P<0.05 or P<0.01); lower p-JAK2/JAK2 and p-STAT3/STAT3 levels in auricle tissue(P<0.01); decreased Th1 and Th17(P<0.01) and increased Th2 and Treg cell populations in the spleen(P<0.01); and decreased protein and mRNA levels of T-bet and RORγt(P<0.01) but increased GATA3 and Foxp3 protein and mRNA levels in auricle tissue(P<0.05 or P<0.01). In conclusion, Zhuyu Pills may regulate inflammatory cytokines through the JAK2/STAT3 signaling pathway, restore Th1/Th2 and Th17/Treg polarization balance, maintain immune cell homeostasis, and thereby exert therapeutic effects on inflammatory skin lesions in acne.
This study aims to investigate the effect of modified Xiangsha Liujunzi Decoction on adipose tissue browning-neuregulin 4(Nrg4)-liver fatty acid synthesis pathway in the rat model of both spleen deficiency and hyperlipidemia. Seventy SPF-grade male SD rats were randomly assigned into blank control(CON), high-fat diet(HFD), spleen deficiency and high-fat diet(SD-HFD), rosuvastatin(RSF), low-, medium-, and high-dose modified Xiangsha Liujunzi Decoction(XS-L, XS-M, and XS-H, respectively) groups, with 10 rats in each group. The rats in the SD-HFD, RSF, XS-L, XS-M, and XS-H groups were modeled for spleen deficiency by an improper diet combined with swimming exhaustion for a total of 15 days. The CON group was fed with a normal diet while the other groups with a high-fat diet for 10 weeks after successful modeling of spleen deficiency. The RSF, XS-L, XS-M, and XS-H groups were administrated with corresponding drugs by gavage for 8 weeks and the other groups were administrated with an equal volume of distilled water. The feeding method of each group was kept unchanged during the period of gavage. Four items of serum lipids were measured by an automatic biochemical analyzer. The pathological changes in the interscapular brown adipose tissue(BAT), abdominal white adipose tissue(WAT), and liver were observed by hematoxylin-eosin(HE) staining. The liver lipid deposition was observed by oil red O staining and the serum Nrg4 level was measured by enzyme-linked immunosorbent assay(ELISA). RT-qPCR was employed to determine the mRNA levels of peroxisome proliferator-activated receptor gamma(PPARγ), peroxisome proliferator-activated receptor gamma cofactor 1α(PGC1α), uncoupling protein 1(UCP1), and Nrg4 in the adipose tissue and liver X receptor alpha(LXRα), sterol regulatory element-binding protein-1c(SREBP-1c), acetyl-CoA carboxylase(ACC), and stearoyl-CoA desaturase 1(SCD1) in the liver tissue of rats in each group. Western blot was employed to quantify the protein levels of PPARγ, PGC1α, UCP1, and Nrg4 in the adipose tissue and Erb-B2 receptor tyrosine kinase 3(ErbB3), phosphorylated ErbB3(p-ErbB3), Erb-B2 receptor tyrosine kinase 4(ErbB4), phosphorylated ErbB4(p-ErbB4), signal transducer and activator of transcription 5(STAT5), phosphorylated STAT5(p-STAT5), LXRα, SREBP-1c, ACC, and SCD1 in the liver tissue. Compared with the CON group, the HFD and SD-HFD groups showed significantly elevated serum levels of triglycerides(TG), total cholesterol(TC), high-density lipoprotein-cholesterol(HDL-C), and low-density lipoprotein-cholesterol(LDL-C) and a significantly declined level of Nrg4. HE staining revealed enlarged BAT and WAT cells and increased lipid vacuoles in hepatocytes in the HFD and SD-HFD groups. The oil red O staining showed that the HFD and SD-HFD groups had more orange lipid droplets in hepatocytes than the CON group. Compared with the CON group, the SD-HFD group showed significantly down-regulated mRNA and protein levels of PPARγ, PGC1α, and Nrg4, significantly down-regulated protein levels of UCP1 in both BAT and WAT cells, and a significantly down-regulated mRNA level of UCP1 in BAT cells. In addition, the SD-HFD group showed significantly decreased p-ErbB3/ErbB3, p-ErbB4/ErbB4, and p-STAT5/STAT5 ratios and significantly up-regulated mRNA and protein levels of LXRα, SREBP-1c, ACC, and SCD1 in the liver tissue. Compared with the SD-HFD group, the RSF, XS-M, and XS-H groups showed significantly declined serum TG, TC, and LDL-C levels, and the XS-M and XS-H groups presented significantly elevated serum Nrg4 levels. In addition, these groups showed reductions in volumes of BAT and WAT cells, alleviated hepatocyte swelling, and decreased lipid droplets in hepatocytes. The mRNA and protein levels of related factors were improved in XS-M and XS-H groups compared with those in the SD-HFD group. This study indicates that modified Xiangsha Liujunzi Decoction could promote the browning of adipose tissue, increase the expression of Nrg4 in the adipose tissue, raise the level of circulating Nrg4, and reduce liver fatty acid synthesis in the rat model of both spleen deficiency and hyperlipidemia.
Tissue fibrosis is described as the excessive accumulation of extracellular matrix (ECM) components. This process is associated with inflammation, and the interaction between fibroblasts and immune cells plays a key role, driving the progression of fibrosis. Results of recent study suggest that interleukin (IL)-12 may play a role in the processes associated with the development of fibrosis. This proinflammatory cytokine is synthesized and released by macrophages, dendritic cells, and B cells. The objective of this study was to determine: (i) the expression of IL-12 and its receptor in different mare endometrial categories; (ii) the effects of IL-12 on the expression of ECM-related factors; and (iii) endometrial fibroblast functional characteristics. The mRNA expression of IL-12 subunits and IL-12 receptor was determined using quantiative polymerase chain reaction (qPCR). The expression of ECM-related factors and functional characteristics in endometrial fibroblasts were determined using qPCR, Western blotting, zymography with bromodeoxyuridine and scratch assays. IL-12Rβ2 mRNA expression was upregulated in category IIB endometrium during the mid-luteal phase compared with the follicular phase of the estrous cycle. The IL-12 treatment of endometrial fibroblasts increased mRNA expression of COL1A1, COL3A1, ACTA2, and LOXL2, as well as matrix metalloproteinase (MMP)-9 and MMP3, with elevated pro-MMP2 and MMP9 gelatinolytic activity. Moreover, IL-12 reduced fibroblast proliferation after 96 h, without effect on migration. The findings indicate that IL-12 has a direct impact on the mRNA expression of fibrotic markers, MMP-2 and MMP-9 gelatinolytic activity and fibroblast proliferation. This suggests a potential role for IL-12 in the processes associated with ECM remodeling.
Dysregulation of the phosphoinositide 3-kinase/protein kinase B (AKT)/mechanistic target of rapamycin pathway is a hallmark of breast cancer, making AKT a focus of therapeutic interest. AKT inhibitor-IV is a benzimidazolium compound that inhibits AKT phosphorylation, but its activity and binding mechanism in breast cancer cells have not been characterized in detail. We compared AKT inhibitor-IV with tamoxifen in MCF-7 cells. Cell viability after 24-h treatment was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay (AKT inhibitor-IV: 0.1-5 μM; tamoxifen: 5-50 μM); apoptotic death was quantified by nucleosome ELISA; and AKT1, BCL-2, BAX, and CASP3 mRNA levels were measured by quantitative real-time PCR. Both compounds were docked into the ATP-binding pockets of 3-phosphoinositide-dependent protein kinase-1 (PDK1) (PDB 1H1W) and AKT1 (PDB 3MVH) using AutoDock Vina v1.2.6. AKT inhibitor-IV reduced viability with an IC50 of 1.1 μM, compared with 5.2 μM for tamoxifen, and induced 5.9-fold apoptosis enrichment versus 3.65-fold for tamoxifen. AKT inhibitor-IV downregulated AKT1 (0.32-fold) and BCL-2 (0.55-fold) and upregulated BAX (5.97-fold) and CASP3 (6.43-fold) mRNA; tamoxifen showed qualitatively similar but quantitatively weaker transcriptional changes. Docking returned affinities of -9.91 kcal/mol for PDK1 and -9.66 kcal/mol for AKT1, with AKT inhibitor-IV contacting catalytic-core residues of both kinases. AKT inhibitor-IV was more potent than tamoxifen in this in-vitro setting and warrants further preclinical evaluation. Because MCF-7 cells carry the CASP3 exon 3 deletion and lack functional caspase-3 protein, the observed CASP3 mRNA upregulation reflects transcriptional reprogramming rather than restored executioner activity. The modest docking difference requires biochemical confirmation before any PDK1-versus-AKT1 selectivity claim can be established.
This study explored the mechanism of Sangpi Zhike Formula in regulating the "oxidation-reduction" balance, reducing the accumulation of reactive oxygen species(ROS), thereby alleviating inflammatory injury and interfering with the process of ferroptosis, and relieving post-infection cough(PIC). The potential pharmacological mechanism was investigated using ultrahigh performance liquid chromatography-Q Exactive-high resolution mass spectrometry(UPLC-QE-MS) and network pharmacology. A total of 70 SPF-grade SD rats(half male and half female) were randomly divided into a normal control group, a model group, a montelukast sodium(western medicine) group, a Ferrostatin-1(ferroptosis inhibitor) group, and low-, medium-, and high-dose Sangpi Zhike Formula(TCM) groups, with 10 rats in each group. The dosages of Sangpi Zhike Formula for the low-, medium-, and high-dose TCM groups were 2.43, 4.85, and 9.70 g·kg~(-1)·d~(-1), respectively. Montelukast sodium was administered at 0.5 mg·kg~(-1)·d~(-1), while Ferrostatin-1 was injected intraperitoneally at 5 mg·kg~(-1). The treatment period lasted 14 d. Pathological damages in lung lobes was assessed by HE staining, ROS accumulation was observed by immunofluorescence, target protein expression was examined by immunohistochemistry, and lung index was calculated to evaluate pulmonary infiltration and congestion. Biochemical assays were performed to measure the levels of total superoxide dismutase(T-SOD), catalase(CAT), malondialdehyde(MDA), total antioxidant capacity(T-AOC), reduced glutathione(GSH), glutathione peroxidase(GSH-PX), oxidized glutathione(GSSG), and total iron in lung homogenates. ELISA was used to quantify tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6). Western blot and qRT-PCR were employed to determine protein and mRNA levels of tumor protein p53(p53), cystine/glutamate transporter(SLC7A11), glutathione peroxidase 4(GPX4), long-chain acyl-CoA synthetase 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), heme oxygenase-1(HO-1), NFE2 related factor 2(Nrf2), NLR family pyrin domain containing 3(NLRP3), Toll-like receptor 4(TLR4), and nuclear factor-kappa light chain enhancer of activated B cells p65(NF-κB p65) in lung tissue. Network pharmacology analysis indicated that the Sangpi Zhike Formula might exert therapeutic effects by targeting TNF, AKT1, IL-6, and TP53, thereby modulating ferroptosis-and pyroptosis-related signaling pathways. Experimental verification showed that compared with the normal group, the model group had obvious inflammatory cell infiltration, structural disorders, significant ROS accumulation in lung tissue, with a significantly increased fluorescence intensity(P<0.01), significantly increased levels of MDA, CAT, GSSG, T-AOC, and total iron, decreased GSH level, and significantly upregulated expressions of p53, ACSL4, LPCAT3, HO-1, NLRP3, TLR4, NF-κB proteins and mRNAs(P<0.01), and significantly downregulated expressions of SLC7A11, GPX4, Nrf2 proteins and mRNAs(P<0.01). Compared with the model group, the medium-dose TCM group and the ferroptosis inhibitor group showed significant differences in the above indicators(P<0.01); other drug intervention groups also showed significant differences in the above indicators compared with the model group(P<0.05). These results suggest that the Sangpi Zhike Formula may alleviate PIC symptoms by adjusting the "oxidation-reduction" balance in lung tissue, reducing ROS accumulation, and thereby alleviating inflammatory injury and interfering with the ferroptosis process.
This study aimed to explore the mechanism of Qingxue Xiaozhi Formula in ameliorating endothelial inflammation in glucose and lipid metabolism disorders through the visceral adipose tissue exosome miR-27b-3p-mediated peroxisome proliferator-activated receptor α(PPARα)/nuclear factor-kappa B(NF-κB) signaling pathway. A Zucker diabetic fatty(ZDF) rat model was induced by Purina 5008 feed, and inflammation models of adipocytes and human umbilical vein endothelial cells(HUVECs) were induced by glucose combined with oxidized low-density lipoprotein(ox-LDL). ZDF rats were randomly allocated into model, low-dose Qingxue Xiaozhi Formula, high-dose Qingxue Xiaozhi Formula, and metformin groups, with Zucker lean(ZL) rats serving as the normal group. After 7 weeks of drug intervention, the body weight, Lee's index, fasting blood glucose, oral glucose tolerance, serum lipid levels, and liver enzyme levels were measured. Hepatic pathological changes were observed via hematoxylin-eosin(HE) and oil red O staining. miRNAs sequencing was used to quantify the expression of miRNAs in exosomes derived from the visceral adipose tissue. Immunofluorescence staining was employed to detect aortic vascular cell adhesion molecule-1(VCAM-1) expression. The cell counting kit-8(CCK-8) method was adopted to examine cell viability. qRT-PCR, enzyme-linked immunosorbent assay(ELISA), and Western blot were employed to measure the mRNA and protein levels of miR-27b-3p, PPARα, NF-κB p65, VCAM-1, and interleukin-6(IL-6). The results showed that compared with the model group, Qingxue Xiaozhi Formula reduced Lee's index, fasting blood glucose, serum lipid levels, and liver enzyme levels in ZDF rats(P<0.05), improved oral glucose tolerance and the expression profile of miRNAs in exosomes derived from the visceral adipose tissue, and alleviated hepatic lipid deposition and aortic endothelial inflammation. In cell experiments, Qingxue Xiaozhi Formula increased the viability of HUVECs and adipocytes, downregulated the mRNA and protein levels of miR-27b-3p, NF-κB p65, VCAM-1, and IL-6 in endothelial cells, and upregulated the mRNA and protein levels of PPARα(P<0.05). These effects could be mimicked by treatment with an miR-27b-3p antagonist or exosome synthesis/release inhibitors. The results indicate that Qingxue Xiaozhi Formula can alleviate endothelial inflammation and vascular complications in glucose and lipid metabolism disorders by regulating visceral adipose tissue exosome miR-27b-3p, activating PPARα, and inhibiting the NF-κB signaling pathway.
Tumor recurrence and progression occur in many patients with renal cell carcinoma, highlighting a clinical demand for safe and effective treatment options, particularly by employing plant-derived bioactive compounds. Limonene, an aromatic monoterpene commonly found in citrus peels, has been shown to possess anticancer effects in malignancies such as prostate and bladder cancers. This study aimed to explore the potential anticancer effects of limonene on ACHN papillary renal cancer cells (pRCC). The viability of ACHN, human embryonic kidney (HEK293), and human dermal fibroblast (HDF) cells treated with limonene was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cellular migration, colony formation, 3D spheroid development, morphological alterations, and caspase 3/7 activity were evaluated in vitro. Reactive oxygen species (ROS) levels, apoptosis, and cell cycle alterations were analyzed by flow cytometry. The mRNA levels of BAX, BCL2, E-cadherin, N-cadherin, Caspase-3, Caspase-9, NF-κB, Akt1, MMP-2, MMP-9, mTOR, TNF-α, COX-2, and IL-6 were quantified using quantitative polymerase chain reaction. NF-κB protein levels and IκB phosphorylation were assessed by Western blot. Nuclear NF-κB levels were measured by ELISA. The viability of ACHN cells was reduced by limonene in a dose- and time-dependent manner, with comparatively limited effects on HEK293 and HDF cells. Limonene significantly reduced migration, colony formation, and spheroid growth in ACHN cells. Furthermore, limonene elevated ROS levels and increased apoptosis in ACHN cells without affecting cell cycle progression. After limonene exposure, mRNA expression of Akt1, mTOR, NF-κB, MMP-2, N-cadherin, BCL2, TNF-α, COX-2, and IL-6 decreased, whereas BAX and E-cadherin expression increased. Caspase-3 and Caspase-9 mRNA levels were not significantly altered, whereas caspase 3/7 activity increased. Limonene also reduced NF-κB protein levels, IκB phosphorylation, and NF-κB nuclear translocation. These preliminary in vitro findings support further investigation of limonene as a therapeutic candidate in additional pRCC models.
The androgen receptors (Ar) are fundamental mediators of androgen signaling, critical for male fertility. Environmental endocrine disruptors (EEDs) can impair male fertility; however, it remains unclear whether EED-induced fertility impairments are linked to DNA methylation alterations in the ar promoter within germ cells, or if a germline-to-soma transfer of these epigenetic profiles affects ar transcription in the testis leading to fertility impairment. To address this, we exposed medaka (Oryzias latipes) to bisphenol A (BPA, 100 µg/l, an EED) and determined fertility defects, testis histology, DNA methylation alterations in the CpG island of arα promoter, and mRNA levels of arα mRNA in the testicular germ cells and soma. Reduced fertility was observed in F0 and F2 generations, which was accompanied by alterations in the testicular tubular structure and germ cell landscape. In F0 males, the testicular germ cells maintained increased levels of DNA methylation on the CpG island of the arα promoter at the ground state of epigenetic reprogramming of germ cells (15 days post-fertilization, dpf) and as such in sperm. In F2 males, testis showed elevated expression of DNA methyltransferase enzyme genes. F2 germ cells maintained significantly higher levels of DNA methylation. The elevated DNA methylation profile was maintained in testicular somatic cells and correlated with significantly decreased arα mRNA levels. The present results demonstrate that ancestral BPA exposure induces transgenerational male subfertility by altering the methylation profile of arα promoter and gene expression, highlighting the profound risk EEDs pose to reproductive health in vertebrates and population stability in wildlife.
Cardiac hypertrophy is a pivotal pathological process leading to heart failure, driven by chronic stress, pressure overload, and neurohormonal stimulation, such as Angiotensin II (Ang II). Although METTL3-mediated m6A modification has been implicated in cardiovascular diseases, the precise role and underlying mechanism of the KLF5 in Ang II-induced myocardial hypertrophy remain poorly understood. H9C2 and AC16 cardiomyocytes were treated with Ang II to establish an in vitro hypertrophy model. The expression of METTL3, KLF5, and hypertrophic markers (ANP, BNP, and β-MHC) was quantified using RT-qPCR and Western blot. Functional validation was performed via lentiviral-mediated knockdown and overexpression. The direct interaction and m6A modification of KLF5 mRNA were validated using RIP-qPCR and MeRIP-qPCR assays. Cardiomyocyte surface area and α-actinin distribution were assessed by immunofluorescence. mRNA stability was determined by Actinomycin D assays. Ang II stimulation significantly upregulated the expression of METTL3 and KLF5 in a dose-dependent manner. Silencing of either METTL3 or KLF5 effectively attenuated Ang II-induced cardiomyocyte enlargement and suppressed the fetal gene program. Mechanistically, METTL3 was found to promote KLF5 expression by increasing its m6A modification levels and enhancing its mRNA stability. Furthermore, rescue experiments demonstrated that the inhibitory effect of METTL3 knockdown on cardiomyocyte hypertrophy was substantially reversed by KLF5 overexpression, confirming that KLF5 is a functional downstream effector of METTL3. METTL3 facilitates Ang II-induced cardiac hypertrophy by modulating KLF5 expression through an m6A-dependent mechanism. Targeting the METTL3/KLF5 axis may offer a novel therapeutic strategy for the treatment of pathological myocardial hypertrophy.
This study aimed to investigate the therapeutic effect of Bushen Yisui Shengxue Formula on aplastic anemia(AA) and to explore the mechanism by which this prescription alleviates bone marrow failure in AA mice through modulation of the Hippo/Yes-associated protein(Yap) pathway. BALB/c mice were randomly assigned to control, model, and Bushen Yisui Shengxue Formula(traditional Chinese medicine) groups. The AA model was established by X-ray irradiation combined with DBA/2 mouse lymphocyte infusion. After successful modeling, the traditional Chinese medicine group received Bushen Yisui Shengxue Formula via gavage for 14 consecutive days, while the model and control groups received normal saline. Following the intervention, general conditions such as mental status, activity, and body weight were observed in each group. Blood samples were collected from the orbital vein for peripheral blood cell counts. Bone marrow pathology was examined by hematoxylin-eosin(HE) staining, while Wright-Giemsa staining was used to assess the number, classification, and morphology of bone marrow cells. Bone marrow nucleated cells were counted under a microscope, and apoptosis levels were measured by flow cytometry. The mRNA expression of Hippo/Yap pathway: Yap, transcriptional enhanced associate domain(TEAD), and telomerase reverse transcriptase(TERT) was detected using quantitative polymerase chain reaction(qPCR), and their protein expression levels were evaluated by Western blot. The results showed that, compared with the control group, mice in the model group exhibited weight loss, weakness, and lethargy; reduced peripheral blood cell counts; decreased bone marrow hematopoietic cells with increased adipocyte infiltration; elevated apoptosis; lower bone marrow nucleated cell counts; and downregulated mRNA and protein expression of TERT, Yap, and TEAD. In contrast, Bushen Yisui Shengxue Formula treatment significantly restored body weight, mental state, and physical activity in the AA mice. It also increased peripheral blood WBC, RBC, and PLT counts; enhanced bone marrow hematopoiesis while reducing fat infiltration; decreased apoptosis; raised bone marrow nucleated cell counts; and upregulated both mRNA and protein levels of TERT, Yap, and TEAD compared with the model group. These findings suggest that Bushen Yisui Shengxue Formula can ameliorate bone marrow failure in AA by modulating the Hippo/Yap pathway, which is implicated in AA pathogenesis, indicating its potential as a promising therapeutic agent for AA treatment.
This study aims to explore the role of harmane in ameliorating pulmonary fibrosis in mice by inhibiting the transformation of lung fibroblasts into myofibroblasts. Transforming growth factor-β_1(TGF-β_1, 5 ng·mL~(-1)) was used to treated MRC-5 cells for the modeling of pulmonary fibrosis. The inhibitory rates of different concentrations of harmane on model cells were measured by the CCK-8 method, and the median inhibitory concentration(IC_(50)) was determined. Harmane at IC_(50) and the adjacent concentrations was used for intervention. The cell myofilament immunofluorescence method, scratch experiment, and flow cytometry were adopted to evaluate the inhibitory effects of harmane on the proliferation, differentiation, and migration of model cells. Then, qRT-PCR and immunofluorescence methods were used to analyze the effects of harmonine on the expression of α-smooth muscle actin(α-SMA), type Ⅰ collagen(collagen Ⅰ), and fibronectin at mRNA and protein levels. Fifty SPF-grade male C57BL/6 mice were randomly assigned into a normal control group and a modeling group. A mouse model of pulmonary fibrosis was constructed by intranasal dropping of 5.0 μg·kg~(-1) bleomycin. The modeled mice were randomly allocated into model, nintedanib(30 mg·kg~(-1)) and harmane(30, 45 mg·kg~(-1)) groups, with 10 mice in each group. The mice in harmane and nintedanib groups were administrated with aqueous solution by gavage. After 14 days of continuous administration, the respiratory function of each mouse was measured with a non-invasive whole-body plethysmography system. The pathomorphological characteristics of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining. The hydroxyproline(HYP) level in the lung tissue was measured by a HYP assay kit. Enzyme-linked immunosorbent assay was employed to determine the content of TGF-β_1, interleukin(IL)-6, and IL-1β in the lung tissue. Western blot was employed to quantify the expression levels of α-SMA and collagen Ⅰ in the lung tissue. The results showed that harmane significantly inhibited the proliferation of MRC-5 cells stimulated with TGF-β_1, with an IC_(50) value of 29.01 μg·mL~(-1). Harmane inhibited the phenotypic differentiation and decreased the scratch migration rate of lung fibroblasts, while significantly promoting the occurrence of apoptosis. In addition, harmane significantly down-regulated the mRNA levels of α-SMA, collagen Ⅰ, and fibronectin. The animal experiment showed that compared with the control group, the model group exhibited significant reductions in body weight and lung function, deposition of collagen fibers, an increase in fibrosis area, elevations in levels of HYP, TGF-β_1, IL-6, and IL-1β, up-regulation in protein levels of α-SMA and collagen Ⅰ, and obvious pulmonary fibrosis. Compared with the model group, the administration groups showed increases in body weight and lung function, reductions in collagen fiber deposition and fibrosis area, declines in levels of HYP, TGF-β_1, IL-6, and IL-1β, and alleviated pulmonary fibrosis. Harmane slows down pulmonary fibrosis in both in vivo and in vitro experiments by inhibiting the transformation of lung fibroblasts and reducing the inflammatory response.