Small Interfering RNA Targeting Research Articles

Kidney Arteriolar Responses to Liver-Targeted Small Interference RNA Targeting Angiotensinogen in Diabetic Rats: Comparison With Other Renin-Angiotensin System Blockers.

Kidney Arteriolar Responses to Liver-Targeted Small Interference RNA Targeting Angiotensinogen in Diabetic Rats: Comparison With Other Renin-Angiotensin System Blockers.

ALOX15 Aggravates Metabolic Dysfunction-Associated Steatotic Liver Disease in Mice with Type 2 Diabetes via Activating the PPARγ/CD36 Axis.

Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent hepatic disorder worldwide. Arachidonic acid 15-lipoxygenase (ALOX15), an enzyme catalyzing the peroxidation of polyunsaturated fatty acids, plays a crucial role in various diseases. Here, we sought to investigate the involvement of ALOX15 in MASLD. Results: In this study, we observed upregulation of ALOX15 in the liver of high-fat diet (HFD)- and streptozotocin (STZ)-induced mice. Metabolomic analysis revealed elevated levels of ALOX15 metabolites, 12(S)-hydroperoxyeicosatetraenoic acid and 15(S)-hydroperoxyeicosatetraenoic acid. Transcriptomic analysis showed that the increased fatty acid uptake regulated by the PPARγ/CD36 pathway predominated in lipid accumulation. To elucidate the mechanism underlying ALOX15-induced lipid accumulation, HepG2 cells were transfected with a lentivirus expressing ALOX15 or small interfering RNA targeting ALOX15 and exposed to palmitic acid (PA). Both ALOX15 overexpression and PA exposure led to increased intracellular free fatty acid and triglyceride, resulting in lipotoxicity. ALOX15 overexpression aggravated the effect of PA, while the knockdown of ALOX15 attenuated PA-induced lipotoxicity. Moreover, the treatment with PPARγ antagonist GW9662 or CD36 inhibitor sulfosuccinimidyl oleate sodium effectively reduced lipid accumulation and lipotoxicity resulting from ALOX15 overexpression and PA exposure, indicating the involvement of the PPARγ/CD36 pathway in ALOX15-mediated lipid accumulation. Furthermore, liraglutide, a widely used glucagon-like peptide 1 receptor (GLP-1R) agonist (GLP-1RA), improved hepatic lipid accumulation in HFD/STZ-induced mice by suppressing the ALOX15/PPARγ/CD36 pathway. Innovation and Conclusion: Our study underscores the potential of ALOX15 as an emerging therapeutic target for MASLD. In addition, the GLP-1RA may confer hepatoprotection by regulating ALOX15, enhancing our comprehension of the mechanisms underpinning their protection on MASLD. Antioxid. Redox Signal. 00, 000-000.

MRI-Based Multifunctional Nanoliposomes for Enhanced HCC Therapy and Diagnosis.

The morbidity and mortality rates of hepatocellular carcinoma (HCC) are high and continue to increase. The antitumor effects of single therapies are limited because of tumor heterogeneity and drug resistance, and the lack of real-time monitoring of tumor progression during the treatment process leads to poor therapeutic outcomes. Therefore, novel nanodelivery platforms combining tumor therapy and diagnosis have garnered extensive attention. In this study, we developed a multifunctional nanodelivery vector, LPSD-DOX/siRNA, which was loaded with oleic acid-modified superparamagnetic iron oxide nanoparticles (OA-SPION) and the antitumor drug doxorubicin (DOX), further modified by DOTAP to carry small interfering RNA targeting phosphatidylinositol proteoglycan-3 (Glypican-3, GPC3) (siRNA-GPC3). These components were utilized for the combined treatment of HCC and tumor monitoring with magnetic resonance imaging. LPSD-DOX/siRNA exhibited high drug loading, high gene transfection efficiency, and low toxicity. Pharmacokinetic and in vivo distribution experiments showed that LPSD-DOX/siRNA significantly prolonged the circulation time of DOX and enhanced drug accumulation at the tumor site. Magnetic resonance imaging demonstrated that LPSD-DOX/siRNA can serve as a T2 imaging contrast agent to enhance the imaging contrast between the tumor site and other tissues and facilitate the imaging monitoring of tumor tissues. Antitumor experiments revealed that the effects of DOX were promoted by inhibiting the expression of GPC3 protein in HepG2 cell-transplanted tumors, with increased tumor apoptosis. In conclusion, LPSD-DOX/siRNA serves as a promising strategy for combination therapy and monitoring of HCC, with significant potential in antitumor therapy.

Rescuing ACE2-Deficiency-Mediated Nucleus Pulposus Senescence and Intervertebral Disc Degeneration by a Nanotopology-Enhanced RNAi System.

Nucleus pulposus cell (NPC) senescence contributes to intervertebral disc degeneration (IVDD). However, the underlying molecular mechanisms are not fully understood. In this study, it is demonstrated that angiotensin-converting enzyme 2 (ACE2) counteracted the aging of NPCs and IVDD at the cellular and physiological levels. The expression of ACE2 correlates negatively with the degree of NPC senescence and IVDD. Using both loss- and gain-of-function mouse models, it is revealed that ACE2 deficiency increased the senescence of NPCs and exacerbated injury- or instability-induced IVDD, whereas ACE2 overexpression counteracted these detrimental effects. Mechanistically, integrated analysis of single-cell and bulk transcriptomics shows that ACE2 deficiency results in the activation of TGFβ2/Smads signaling pathway and the transcription of Serpine1, ultimately triggering NPC senescence and IVDD. A nanomedical delivery system (virus-like nanovectors, VNs) composed of nanovectors with a virus-like surface topology and small interfering RNA targeting Serpine1 (VN-siSer) is developed. With nanotopology-enhanced transfection efficiency, RNA-interfering treatment by VN-siSer effectively alleviated NPC senescence and IVDD at both the cellular and animal levels. Overall, the data reveal the underlying mechanisms of ACE2 in NPC senescence and IVDD pathogenesis and propose a distinct paradigm of precise nanomedical senescence-blockade RNAi for IVDD treatment.

Evaluating the specific STAT3 inhibitor YHO-1701 in ovarian cancer cell lines and patient-derived cell models: efficacy, mechanisms, and therapeutic potential.

Signal transducer and activator of transcription 3 (STAT3) plays key roles in regulating cancer cell proliferation, survival, and metastasis. We aimed to determine the effects of YHO-1701, an oral STAT3 inhibitor, in ovarian cancer (OC). We evaluated the impact of YHO-1701 on cell growth in patient-derived cells (PDCs) and OC cell lines using standard cell proliferation assays. Spheroid models derived from PDCs were assessed using three-dimensional (3D) cell viability assays. Antitumor activity was performed in SKOV3 xenograft mice treated orally administrated YHO-1701 with 20 mg/kg. Changes in STAT3 signaling were analyzed by western blotting. The molecular mechanisms of STAT3 inhibition were investigated by sequencing RNA and analyzing pathways in the SKOV3 using a small interfering RNA targeting STAT3 (STAT3 siRNA) and YHO-1701. YHO-1701 inhibited the growth of OC cell lines by preventing STAT3 dimerization and decreasing the expression of its downstream signaling molecule, survivin. The growth of PDCs and spheroids obtained from patients with primary and recurrent OCs was significantly inhibited. Antitumor effect was observed in the SKOV3 xenograft mice with YHO-1701. YHO-1701 induced apoptosis in OC cells. Additionally, p53 and/or MAPK signaling pathways were upregulated in SKOV3 cells incubated with YHO-1701 and in those with STAT3 siRNA. Our results showed that YHO-1701 suppressed cell growth in PDCs of OC, accompanied by survivin inhibition, and a decrease in the number of peritoneal metastasis in the mice by YHO-1701, compared with those treated with control. Therefore, YHO-1701 could be a promising candidate agent for treating OC.

Interaction Between YTH Domain-Containing Family Protein 2 and SET Domain-Containing Lysine Methyltransferase 7 Suppresses Autophagy in Osteoarthritis Chondrocytes, Exacerbating Cartilage Damage.

Osteoarthritis (OA) is characterized by progressive cartilage degeneration mediated by various molecular pathways, including inflammatory and autophagic processes. SET domain-containing lysine methyltransferase 7 (SETD7), a methyltransferase, has been implicated in OA pathology. This study investigates the expression pattern of SETD7 in OA and its role in promoting interleukin-1 beta (IL-1β)-induced chondrocyte injury through modulation of autophagy and inflammation. The expression of SETD7 in cartilage tissues from OA patients and healthy controls was quantified using quantitative reverse transcription PCR and Western blot analysis. Small interfering RNA targeting SETD7 (si-SETD7) was transfected into human articular chondrocytes (HACs) treated with IL-1β to examine its impact on cellular viability, apoptosis, inflammatory responses, and autophagy. Functional assays including Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, and commercial kits were employed to assess biochemical changes. Interaction between YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) and SETD7 was explored using RNA immunoprecipitation and co-immunoprecipitation assays. SETD7 was overexpressed in OA cartilage compared with controls and increased further upon IL-1β treatment. Knockdown of SETD7 in IL-1β-treated HACs improved cellular viability, decreased apoptosis, and reversed the adverse effects on lactate dehydrogenase release and inflammatory markers (tumor necrosis factor-alpha and interleukin-6) while enhancing antioxidant enzymes (catalase, malondialdehyde, and superoxide dismutase). Additionally, autophagy was restored, as evidenced by changes in the levels of autophagy related 5, Beclin1, and sequestosome 1. Interfering with autophagy using chloroquine negated the protective effects of SETD7 knockdown. Furthermore, YTHDF2 was found to stabilize SETD7 mRNA, influencing its expression and enhancing IL-1β-induced chondrocyte injury. SETD7 plays a critical role in the pathogenesis of OA by modulating chondrocyte survival, apoptosis, inflammation, and autophagy. The interaction between YTHDF2 and SETD7 exacerbates chondrocyte injury under inflammatory conditions, highlighting potential therapeutic targets for OA treatment. The YTHDF2/SETD7 axis offers a novel insight into the molecular mechanisms governing cartilage degeneration in OA.

Augmented epigenetic repression of hepatitis B virus covalently closed circular DNA by interferon-α and small-interfering RNA synergy.

Since there are currently no approved drugs targeting and silencing covalently closed circular DNA (cccDNA), achieving a "functional cure" remains difficult. This study aims to comprehensively compare the effects of IFNα, small-interfering RNA targeting hepatitis B virus (HBV), and their combination on the activity, accessibility, and epigenetic modifications of cccDNA minichromosomes in cell models. A more durable and stable inhibition of HBV RNAs and antigens expression by IFNα and HBx-targeting siRNA (siHBx) synergy was observed, associated with augmented epigenetic repression of the cccDNA minichromosome. Besides, in an extracellular humanized IFNAR mouse model harboring recombinant cccDNA with an intact response to human IFNα, the synergistic effect of clinically used pegylated IFNα2 and in-house-developed GalNac-siHBx was further clarified.

SDC1 Influences Keloid Fibroblasts Migration and Invasion via Targeting Wnt/β-Catenin Signaling Pathway Mediated EMT.

Keloid is the result of abnormal wound healing, puzzled by the invasive growth and high recurrence rate attributed to its complex pathogenic mechanism. Syndecan1 (SDC1) contributes to regulating cell migration and invasion by activating epithelial-mesenchymal transition (EMT) in tumor and fibrotic disease. Herein, using western blot analysis, the authors assessed the role of SDC1 on EMT in keloid and its underlying mechanism. The authors found keloid fibroblasts exhibited a higher proportion of mesenchymal phenotypes, and SDC1 was significantly upregulated in keloid fibroblasts. Then, the authors transfected small interfering RNA targeting SDC1 in keloid fibroblasts and tested the abilities of cell migration and invasion, as well as the expression of EMT-related markers, including N-cadherin, vimentin, and E-cadherin. The results showed that the knockdown of SDC1 markedly suppressed the migration and invasion abilities of keloid fibroblasts and reduced the phenotypes of EMT by inhibiting Wnt/β-catenin signaling pathway. The authors' findings suggest that SDC1 may influences keloid fibroblasts migration and invasion through targeting Wnt/β-catenin signaling pathway mediated EMT, which supports its potential value as a therapeutic target for the treatment of keloid.

SLC1A3 knockdown in inhibiting the proliferation, apoptosis resistance, and migration of ovarian cancer cells.

Ovarian cancer accounts for 3% of all malignancies in women and kills about 140,000 women worldwide each year, representing the fifth leading cause of cancer-related death in women. At diagnosis, 70% of patients with ovarian cancer are already at stage III or IV disease, with a 5-year survival rate of less than 45%. Studies have found that solute carrier family 1 member 3 (SLC1A3) is highly expressed in various cancers and is associated with the poor prognosis of these cancers. However, the role of SLC1A3 in ovarian cancer remains unknown. The purpose of this study was to investigate the role of the SLC1A3 gene in the proliferation, apoptosis, migration, and outcomes of ovarian cancer. The expression level of SLC1A3 was measured via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Knockdown experiments were performed with small interfering RNA targeting SLC1A3 in ovarian cancer cells. After the knockdown of SLC1A3, proliferation was evaluated with Cell Counting Kit 8 (CCK8) assay, apoptosis was measured by flow cytometry, and migration was evaluated via wound-healing assay. Kaplan-Meier method was used to analyze the effect of SLC1A3 expression on the prognosis of patients with ovarian cancer. High expression of SLC1A3 was associated with poor prognosis in ovarian cancer patients, and the expression of SLC1A3 in ovarian cancer cells was higher than that in ovarian epithelial cells. In vitro experiments demonstrated that knockdown of SLC1A3 restrained the proliferation activity of ovarian cancer cells, enhanced cell apoptosis, and inhibited cell migration. High expression of SLC1A3 is linked to poor prognosis in ovarian cancer patients. SLC1A3 activity impedes apoptosis while enhancing the proliferation and migration of ovarian cancer cells, suggesting its potential as a therapeutic target for drug development.

HMGB1 Modulates Macrophage Metabolism and Polarization in Ulcerative Colitis by Inhibiting Cpt1a Expression.

Macrophage polarization is involved in the development of ulcerative colitis (UC). This study investigated the mechanism by which high mobility group box-1 protein (HMGB1) regulates macrophage polarization through metabolic reprogramming, thereby contributing to the pathogenesis of UC. Dextran sulfate sodium (DSS) was used to induce colitis in mice. RAW264.7 cells were polarized to M1 or M2 macrophages in vitro by stimulating with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) or Interleukin-4 (IL-4), respectively. Macrophage infiltration and distribution within colon tissue were assessed by immunohistochemistry and flow cytometry. Glycolysis, fatty acid oxidation (FAO), and inflammatory factors were evaluated using relevant reagent kits. Chromatin Immunoprecipitation (ChIP) and luciferase reporter experiments were performed to study the regulation of Carnitine palmitoyltransferase 1A (Cpt1a) promoter transcriptional activity by HMGB1. The mouse UC model showed upregulated HMGB1 and increased macrophage infiltration. Overexpression of HMGB1 promoted M1 macrophage polarization, increased glycolysis, and reduced FAO, whereas knockdown of HMGB1 promoted M2 macrophage polarization, reduced glycolysis, and increased FAO. HMGB1 negatively regulated Cpt1a expression by inhibiting transcription of the Cpt1a promoter. Knockdown of Cpt1a reversed the effects of small interfering RNA targeting HMGB1 (si-HMGB1) on macrophage metabolism and polarization. Administration of adeno-associated virus (AAV)-shHMGB1 in vivo caused a reduction in UC symptoms and inflammation. HMGB1 modulates macrophage metabolism in UC by inhibiting Cpt1a expression, leading to increased M1 polarization. This provides a theoretical basis for the clinical application of HMGB1 inhibitors in the treatment of UC.

Zerlasiran—A Small-Interfering RNA Targeting Lipoprotein(a)

Elevated lipoprotein(a) increases the risk of atherosclerotic cardiovascular disease (ASCVD) and aortic stenosis. To evaluate the effects of zerlasiran, a small-interfering RNA targeting hepatic synthesis of apolipoprotein(a), on lipoprotein(a) serum concentration. A multicenter trial in patients with stable ASCVD with serum lipoprotein(a) concentrations greater than or equal to 125 nmol/L at 26 sites in Europe and South Africa between January 3, 2023, and April 27, 2023, with last follow-up on July 1, 2024. Participants randomized to receive a subcutaneous dose of placebo every 16 weeks for 3 doses (n = 23) or every 24 weeks for 2 doses (n = 24) or zerlasiran 450 mg every 24 weeks for 2 doses (n = 45), 300 mg every 16 weeks for 3 doses (n = 42), or 300 mg every 24 weeks for 2 doses (n = 44). The primary outcome was the time-averaged percent change in lipoprotein(a) concentration from baseline to 36 weeks, with follow-up to 60 weeks. Among 178 patients, mean (SD) age was 63.7 (9.4) years, 46 (25.8%) were female, with a median (IQR) baseline lipoprotein(a) concentration of 213 (177-282) nmol/L; 172 patients completed the trial. Compared with the pooled placebo group, the least-squares mean time-averaged percent change in lipoprotein(a) concentration from baseline to week 36 was -85.6% (95% CI, -90.9% to -80.3%), -82.8% (95% CI, -88.2% to -77.4%), and -81.3% (95% CI, -86.7% to -76.0%) for the 450 mg every 24 weeks, 300 mg every 16 weeks, and 300 mg every 24 weeks groups, respectively. Median (IQR) percent change in lipoprotein(a) concentration at week 36 was -94.5% (-97.3% to -84.2%) for the 450 mg every 24 weeks group, -96.4% (-97.7% to -92.3%) for the 300 mg every 16 weeks group, and -90.0% (-93.7% to -81.3%) for the 300 mg every 24 weeks group. The most common treatment-related adverse effects were injection site reactions, with mild pain occurring in 2.3% to 7.1% of participants in the first day following drug administration. There were 20 serious adverse events in 17 patients, none considered related to the study drug. Zerlasiran was well-tolerated and reduced time-averaged lipoprotein(a) concentration by more than 80% during 36 weeks of treatment in patients with ASCVD. ClinicalTrials.gov Identifier: NCT05537571.

Advances in Development of Drug Treatment for Hemophilia with Inhibitors.

Patients with hemophilia A and B who have inhibitors face limited treatment options, because replacement therapy with clotting factor VIII or IX concentrates is ineffective, particularly for patients with high-titer inhibitors. Current mainstay therapies include immune tolerance induction (through frequent injections of clotting factor VIII or IX concentrates) to eradicate inhibitors and bypassing agents (such as recombinant activated clotting factor VII and activated prothrombin complex concentrates) for the prevention and treatment of bleeding episodes. The use of these agents typically requires intravenous injections and sometimes hospitalization, which can be burdensome for patients. More recently, emicizumab, a bispecific antibody that mimics the function of activated clotting factor VIII, has demonstrated favorable efficacy for prophylaxis in patients with hemophilia A and inhibitors, representing a promising new therapeutic strategy. Ongoing research aims to discover and develop easy-to-use nonfactor agents for managing hemophilia with inhibitors. This review summarizes the current understanding of the pathophysiology of inhibitor development in hemophilia, outlines existing treatment options, and discusses advancements in novel therapeutic biologics, including a recombinant activated clotting factor VII variant (marzeptacog alfa), a new bispecific antibody (Mim8), antitissue factor pathway inhibitor antibodies (concizumab and marstacimab), and small interfering RNA targeting antithrombin (fitusiran). All of these agents are administered subcutaneously, with some offering the convenience of less frequent dosing (e.g., weekly or monthly). These potential drug candidates may provide significant benefits for the prophylaxis or treatment of bleeding disorders in patients with hemophilia and inhibitors.

ALKBH5 Regulates Corneal Neovascularization by Mediating FOXM1 M6A Demethylation.

This study aims to explore the regulatory role and potential mechanisms of ALKBH5-mediated N6-methyladenosine (m6A) demethylation modification in corneal neovascularization (CNV). A mouse CNV model was established through corneal alkali burns. Total m6A levels were measured using an m6A RNA methylation quantification kit. The mRNA expression of candidate m6A-related enzymes was quantified by quantitative RT-PCR. Small interfering RNA targeting ALKBH5 was injected subconjunctivally into alkali-burned mice. The CNV area, corneal epithelial thickness, and pathological changes were evaluated. Protein expression was detected by western blot and immunofluorescence. Human umbilical vein endothelial cells (HUVECs) were treated with IL-6. Plasmid transfection knocked down ALKBH5 or overexpressed FOXM1 in IL-6-induced HUVECs. The assays of CCK8, wound healing, and tube formation evaluated the cell proliferation, migration, and tube formation abilities, respectively. The dual-luciferase assay examined the binding between ALKBH5 and FOXM1. Methylated RNA immunoprecipitation-qPCR detected the m6A levels of FOXM1. Significant CNV was observed on the seventh day. Total m6A levels were reduced, and ALKBH5 expression was increased in CNV corneas and IL-6-induced HUVECs. ALKBH5 knockdown alleviated corneal neovascularization and inflammation and countered IL-6-induced promotion of cell proliferation, migration, and tube formation in HUVECs. ALKBH5 depletion increased m6A levels and decreased VEGFA and CD31 expression both in vivo and in vitro. This knockdown in HUVECs elevated m6A levels on FOXM1 mRNA while reducing its mRNA and protein expression. Notably, FOXM1 overexpression can reverse ALKBH5 depletion effects. ALKBH5 modulates FOXM1 m6A demethylation, influencing CNV progression and highlighting its potential as a therapeutic target.

Knockdown of nuclear protein 1 delays pathological pro-gression of osteoarthritis through inhibiting chondrocyte ferroptosis.

To investigate the effect of nuclear protein 1 (Nupr1) on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes. Chondrocytes of mouse knee were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and real-time RT-PCR. Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of long-chain acyl-coenzyme A synthetase 4 (ACSL4), tumor protein 53 (P53), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into 4 groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-Short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA targeting Nupr1 (shNupr1) group, DMM+AAV5-shRNA group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of Nupr1, matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), cyclooxygenase 2 (COX2), and glutathione peroxidase 4 (GPX4) were detected by real-time RT-PCR. In vitro experiments showed that knocking down Nupr1 alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered the lipid peroxidation, downregulated ACLS4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all P<0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, in vivo animal experiments demonstrated that knocking down Nupr1 delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all P<0.01). Knockdown of Nupr1 can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.

Empagliflozin alleviates obesity-related cardiac dysfunction via the activation of SIRT3-mediated autophagosome formation

BackgroundEmpagliflozin (EMPA) has demonstrated efficacy in providing cardiovascular benefits in metabolic diseases. However, the direct effect of EMPA on autophagy in obesity-related cardiac dysfunction remains unclear. Therefore, this study aimed to determine changes in cardiac autophagy during diet-induced obesity and clarify the exact mechanism by which EMPA regulates autophagic pathways.MethodsMale C57BL/6J mice were fed a 12-week high-fat diet (HFD) followed by 8 weeks of EMPA treatment. Body composition analysis and echocardiography were performed to evaluate metabolic alterations and cardiac function. Histological and immunofluorescence staining was used to evaluate potential enhancements in myocardial structure and biological function. Additionally, H9c2 cells were transfected with small interfering RNA targeting sirtuin 3 (SIRT3) and further treated with palmitic acid (PA) with or without EMPA. Autophagy-related targets were analyzed by western blotting and RT‒qPCR.ResultsEMPA administration effectively ameliorated metabolic disorders and cardiac diastolic dysfunction in HFD-fed mice. EMPA prevented obesity-induced myocardial hypertrophy, fibrosis, and inflammation through the activation of SIRT3-mediated autophagosome formation. The upregulation of SIRT3 triggered by EMPA promoted the initiation of autophagy by activating AMP-activated protein kinase (AMPK) and Beclin1. Furthermore, activated SIRT3 contributed to the elongation of autophagosomes through autophagy-related 4B cysteine peptidase (ATG4B) and autophagy-related 5 (ATG5).ConclusionsEMPA promotes SIRT3-mediated autophagosome formation to alleviate damage to the cardiac structure and function of obese mice. Activated SIRT3 initiates autophagy through AMPK/Beclin1 and further stimulates elongation of the autophagosome membrane via ATG4B/ATG5. These results provide a new explanation for the cardioprotective benefits of EMPA in obesity.

Role of caspase-11 non-canonical inflammasomes in retinal ischemia/reperfusion injury

BackgroundRetinal ischemia/reperfusion (IR) injury is a common pathological process in many ophthalmic diseases. Interleukin-1β (IL-1β) is an important inflammatory factor involved in the pathology of retinal IR injury, but the mechanism by which IL-1β is regulated in such injury remains unclear. Caspase-11 non-canonical inflammasomes can regulate the synthesis and secretion of IL-1β, but its role in retinal IR injury has not been elucidated. This study aimed to evaluate the role of caspase-11 non-canonical inflammasomes in retinal IR injury.MethodsRetinal IR injury was induced in C57BL/6J mice by increasing the intraocular pressure to 110 mmHg for 60 min. The post-injury changes in retinal morphology and function and in IL-1β expression were compared between caspase-11 gene knockout (caspase-11−/−) mice and wild-type (WT) mice. Morphological and functional changes were evaluated using hematoxylin–eosin staining and retinal whole mount staining and using electroretinography (ERG), respectively. IL-1β expression in the retina was measured using enzyme-linked immunosorbent assay (ELISA). The levels of caspase-11-related protein were measured using western blot analysis. The location of caspase-11 in the retina was determined via immunofluorescence staining. Mouse type I astrocytes C8-D1A cells were used to validate the effects of caspase-11 simulation via hypoxia in vitro. Small-interfering RNA targeting caspase-11 was constructed. Cell viability was evaluated using the MTT assay. IL-1β expression in supernatant and cell lysate was measured using ELISA. The levels of caspase-11-related protein were measured using western blot analysis.ResultsRetinal ganglion cell death and retinal edema were more ameliorated, and the ERG b-wave amplitude was better after retinal IR injury in caspase-11−/− mice than in WT mice. Further, caspase-11−/− mice showed lower protein expressions of IL-1β, cleaved caspase-1, and gasdermin D (GSDMD) in the retina after retinal IR injury. Caspase-11 protein was expressed in retinal glial cells, and caspase-11 knockdown played a protective role against hypoxia in C8-D1A cells. The expression levels of IL-1β, cleaved caspase-1, and GSDMD were inhibited after hypoxia in the si-caspase-11 constructed cells.ConclusionsRetinal IR injury activates caspase-11 non-canonical inflammasomes in glial cells of the retina. This results in increased protein levels of GSDMD and IL-1β and leads to damage in the inner layer of the retina.

Hypertension Cure Research progress: The use of Zilebesiran in Hypertension

Chronic hypertension is a lifelong condition, and the patients need lifelong treatment with oral anti-hypertensive medications to say healthy and to prevent the development of serious complications. Demands for changing this situation of lifelong dependence on daily medications have been increasing. The aim of this paper is to highlight the recent breakthrough in hypertension research. Expert opinion: Zilebesiran, a small interfering RNA targeting angiotensinogen has the main advantage of long duration of action which allows meeting many hypertensions patients’ demand of avoiding the inconvenience of talking oral hypertensive medication on daily basis. It is worth mentioning that a more optimal blockade of renin-angiotensin-aldosterone system is not the only advantageous therapeutic approach for the treatment of hypertension. Impairment of endothelium-dependent vasodilation has been increasingly recognized as an important contributor for the development of essential hypertension’s-arginine is an amino acid that is considered the biological precursor of nitric oxide which is an important contributor to microvascular vasodilation and to the reduction atherosclerosis. Oral L-arginine has been emerging as a new effective approach of reducing blood through improving endothelial function in hypertensive patients.

Abstract P104: Kidney arteriolar responses to liver-targeted small interference RNA (siRNA) targeting angiotensinogen in diabetic rats: comparison with other renin-angiotensin system blockers

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin’s fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan + captopril group only (P&lt;0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 (14%) diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

SiTGF-β1 and pirfenidone contained Ionizable-Liposomal nanodrug for enhanced treatment of Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a challenging interstitial lung disease characterized by heterogeneous manifestations and significant mortality. Due to the relative complexity of IPF pathogenesis, currently approved clinical drugs mainly for anti-inflammatory or anti-fibrotic present limited disease containment and insufficient damage repair, falling far short of previous expectations. To address this limitation, we developed an ionizable liposome nanodrug (named as TPNPs) co-loaded with small interfering RNA targeting TGF-β1 (siTGF-β1) and pirfenidone (PFD) for enhanced IPF therapy. The siTGF-β1 (blocking TGF-β signaling pathway) and PFD were carefully selected and integrated in consideration of their crucial roles in endothelial mesenchymal transition, alveolar epithelial cell apoptosis, and fibroblast differentiation and migration, which are closely related to IPF development. As a result, TPNPs exhibited synergistic enhancement effects against the damage of alveolar epithelial cells and fibroblasts in vitro. Notably, repeated administration of TPNPs mitigated the injury to alveolar epithelium, inhibited the excessive accumulation of extracellular matrix (ECM), and alleviated the destruction of lung, thereby synergistically ameliorating the lung function in a bleomycin (BLM)-induced murine model. This work provides a promising therapeutic strategy that combines nucleic acid with small molecule for enhanced treatment of IPF.

Role of hypoxia-inducible-factor-1α (HIF-1α) in ferroptosis of adipose tissue during ketosis

Postpartum cows experience lipolysis in adipose tissue due to negative energy balance (NEB), and accumulation of free fatty acids (FFA) leads to metabolic stress in adipose tissue. Ferroptosis is a type of cell death triggered by excessive buildup of iron-dependent lipid peroxides, which is involved in the occurrence and development of various metabolic diseases in nonruminants. However, it is still unclear whether ferroptosis occurs in the adipose tissue of ketotic cows and the regulatory mechanisms behind ferroptosis. Despite multiple studies demonstrating the significant involvement of hypoxia-inducible-factor-1α (HIF-1α) in regulating cellular dysfunction, its specific function in adipose tissue of ketotic dairy cows remains uncertain, particularly its regulation of oxidative stress and ferroptosis. The study aimed to explore the impact of HIF-1α on oxidative stress and ferroptosis in bovine subcutaneous adipose tissue and isolated adipocytes. The adipose tissue of clinical ketosis cows (n = 15) with a serum BHB concentration of 3.13 mM (interquartile range = 0.14) and healthy cows (n = 15) with a serum BHB concentration of and 0.58 mM (interquartile range = 0.13) was collected. The results showed that the concentrations of lipid peroxidation malondialdehyde (MDA), reactive oxygen species (ROS), Fe2+ and total iron were increased in adipose tissue of cows with ketosis, while the contents of glutathione (GSH) were reduced. Furthermore, the protein levels of HIF-1α, heme oxygenase 1 (HMOX1), catalase (CAT), superoxide dismutase 1 (SOD1), acyl-CoA synthetase 4 (ACSL4), and nuclear factor erythroid-derived 2-like 2 (NFE2L2) exhibited higher abundance in adipose tissue obtained from cows with ketosis, whereas the protein abundance of solute carrier family 7 member 11 (SLC7A11), glutamate cysteine ligase catalytic subunit (GCLC), kelch-like ECH-associated protein 1 (KEAP1), glutamate cysteine ligase regulatory subunit (GCLM) and glutathione peroxidase 4 (GPX4) were lower. To simulate the ferroptosis state of adipose tissue in ketotic cows, primary bovine adipocytes were isolated from the adipose tissue of healthy cows and cultured with erastin to construct ferroptosis model. Adipocytes were cultured with either an adenovirus overexpressing HIF-1α or small interfering RNA targeting HIF-for 48 h, followed by exposure to erastin (1 μM) for 24 h. Treatment with erastin led to higher protein abundance of CAT, SOD1, NFE2L2 and HMOX1, while it inhibited the protein expression levels of GCLC, SLC7A11, GCLM, GPX4 and KEAP1. Furthermore, erastin treatment elevated the levels of ROS, MDA, Fe2+, total iron and reduced the content of GSH. The overexpression of HIF-1α reversed the erastin-induced decreases in the protein abundance of GPX4 and SLC7A11, as well as the levels of MDA, ROS, Fe2+ and total iron, while significantly increasing protein abundance and content of CAT, SOD1, NFE2L2, HMOX1, GCLC, GCLM, GPX4, SLC7A11 and GSH. Conversely, the silencing of HIF-1α further exacerbated the erastin-induced levels of MDA, ROS, Fe2+ and total iron, while inhibiting the upregulation of SOD1, CAT, NFE2L2 and HMOX1. Collectively, these findings suggest that activation of HIF-1α may function as an adaptive mechanism to mitigate ferroptosis and alleviate oxidative stress in adipose tissue.