Dichloroacetate Prevents Sepsis Associated Encephalopathy by Inhibiting Microglia Pyroptosis through PDK4/NLRP3.

Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, is often used to treat lactic acidosis and malignant tumors. Increasing studies have shown that DCA has neuroprotective effects. Here, we explored the role and mechanism of DCA in Sepsis associated encephalopathy (SAE). Single-cell analysis was used to determine the important role of PDK4 in SAE and identify the cell type. GO and GSEA analysis were used to determine the correlation between DCA and pyroptosis. Through LPS + ATP stimulation, a microglia pyroptosis model was established to observe the expression level of intracellular pyroptosis-related proteins under DCA intervention, and further detect the changes in intracellular ROS and JC-1. Additionally, a co-culture environment of microglia and neuron was simply constructed to evaluate the effect of DCA on activated microglia-mediated neuronal apoptosis. Finally, Novel object recognition test and the Morris water maze were used to explore the effect of DCA on cognitive function in mice from different groups after intervention. Based on the above experiments, this study concludes that DCA can improve the ratio of peripheral and central M1 macrophages, inhibit NLRP3-mediated pyroptosis through ROS and mitochondrial membrane potential (MMP). DCA can reduce neuron death caused by SAE and improve cognitive function in LPS mice. In SAE, DCA may be a potential candidate drug for the treatment of microglia-mediated neuroinflammation.

The comparison of meat yield, quality, and flavor between small-tailed Han sheep and two crossbred sheep and the verification of related candidate genes.

Introduction: In Northeast China, Dorper and Australian White rams are commonly crossbred with small-tailed Han (STH) ewes to improve the offspring's meat yield and quality. However, the differences in traits and the flavor between the crossbred sheep and STH sheep remain unclear. In addition, the candidate genes potentially influencing the meat quality in the three sheep breeds require further verification. Methods: A total of 18 2-month-old healthy rams were raised over a period of 5 months, which included 6 STH, 6 Dorper and small-tailed Han crossbred (Do × STH), and 6 Australian white and small-tailed Han crossbred (Au × STH) offspring. The differences in slaughter, meat quality traits, fatty acid and amino acid composition in the muscular longissimus dorsi (MLD), and volatile compounds in the semitendinosus muscle were compared between the sheep breeds. The candidate genes related to intramuscular fat (IMF) content and fatty acids were validated. Results: The results of this study revealed that the crossbred sheep had higher body weight, carcass weight, bone weight, net meat weight, and IMF content than the STH sheep (p < 0.05). The Do × STH offspring had a higher pH value (24 h), moisture content, and cooking percentage; they also had redder and brighter meat color. The content of myristate, palmitic, and margaric acids in the crossbred sheep was higher than that in the STH sheep (p < 0.05). The Do × STH offspring had the highest saturated fatty acid content (p < 0.05). The Au × STH offspring had the highest protein content (p < 0.05). The arachidonic acid and amino acid (Asp, Ala, Ile, Leu, Lys, Thr, and essential amino acid) contents were higher in the STH sheep than in the crossbred sheep (p < 0.05). The odor activity value (OAV) analysis showed that most of the aldehydes in the Au × STH offspring had higher values. The PDK4 gene expression was positively associated with the IMF content and was negatively correlated with the linoleic acid content in the Do × STH sheep (p < 0.05). The TMEM273 gene expression was positively associated with linoleic and arachidonic acid contents and was negatively correlated with oleic and palmitic acid contents in the Do × STH sheep (p < 0.05). Discussion: The results showed the differences between the crossbred sheep and STH sheep and provided the candidate genes related to meat quality in sheep.

Post-translational modifications in prion diseases.

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.

Unveiling the PDK4-centered rituximab-resistant mechanism in DLBCL: the potential of the "Smart" exosome nanoparticle therapy.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor, with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance, primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4's resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative, effective treatment modalities for DLBCL. METHODS: We utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing, aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence, immunohistochemistry, and Western blotting were employed to determine PDK4's localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore, we engineered advanced exosome nanoparticle complexes, aCD20@ExoCTX/siPDK4, through cellular, genetic, and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM), and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles' effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally, their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model. RESULTS: We found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site, suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies, simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment, showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model. CONCLUSIONS: This study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway, representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.

12-O-deacetyl-phomoxanthone A inhibits ovarian tumor growth and metastasis by downregulating PDK4.

AIMS: The xanthone dimer 12-O-deacetyl-phomoxanthone A (12-ODPXA) was extracted from the secondary metabolites of the endophytic fungus Diaporthe goulteri. The 12-ODPXA compound exhibited anticancer properties in murine lymphoma; however, the anti-ovarian cancer (OC) mechanism has not yet been explored. Therefore, the present study evaluated whether 12-ODPXA reduces OC cell proliferation, metastasis, and invasion by downregulating pyruvate dehydrogenase kinase (PDK)4 expression. METHODS: Cell counting kit-8, colony formation, flow cytometry, wound healing, and transwell assays were performed to examine the effects of 12-ODPXA on OC cell proliferation, apoptosis, migration, and invasion. Transcriptome analysis was used to predict the changes in gene expression. Protein expression was determined using western blotting. Glucose, lactate, and adenosine triphosphate (ATP) test kits were used to measure glucose consumption and lactate and ATP production, respectively. Zebrafish xenograft models were constructed to elucidate the anti-OC effects of 12-ODPXA. RESULTS: The 12-ODPXA compound inhibited OC cell proliferation, migration, invasion, and glycolysis while inducing cell apoptosis via downregulation of PDK4. In vivo experiments showed that 12-ODPXA suppressed tumor growth and migration in zebrafish. CONCLUSION: Our data demonstrate that 12-ODPXA inhibits ovarian tumor growth and metastasis by downregulating PDK4, revealing the underlying mechanisms of action of 12-ODPXA in OC.

Abrogating PDK4 activates autophagy-dependent ferroptosis in breast cancer via ASK1/JNK pathway.

BACKGROUND: Targeting ferroptosis mediated by autophagy presents a novel therapeutic approach to breast cancer, a mortal neoplasm on the global scale. Pyruvate dehydrogenase kinase isozyme 4 (PDK4) has been denoted as a determinant of breast cancer metabolism. The target of this study was to untangle the functional mechanism of PDK4 in ferroptosis dependent on autophagy in breast cancer. METHODS: RT-qPCR and western blotting examined PDK4 mRNA and protein levels in breast cancer cells. Immunofluorescence staining appraised light chain 3 (LC3) expression. Fe (2 +) assay estimated total iron level. Relevant assay kits and C11-BODIPY (591/581) staining evaluated lipid peroxidation level. DCFH-DA staining assayed intracellular reactive oxygen species (ROS) content. Western blotting analyzed the protein levels of autophagy, ferroptosis and apoptosis-signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) pathway-associated proteins. RESULTS: PDK4 was highly expressed in breast cancer cells. Knockdown of PDK4 induced the autophagy of breast cancer cells and 3-methyladenine (3-MA), an autophagy inhibitor, countervailed the promoting role of PDK4 interference in ferroptosis in breast cancer cells. Furthermore, PDK4 knockdown activated ASK1/JNK pathway and ASK1 inhibitor (GS-4997) partially abrogated the impacts of PDK4 absence on the autophagy and ferroptosis in breast cancer cells. CONCLUSION: To sum up, deficiency of PDK4 activated ASK1/JNK pathway to stimulate autophagy-dependent ferroptosis in breast cancer.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Ginsenoside Rh2 shifts tumor metabolism from aerobic glycolysis to oxidative phosphorylation through regulating the HIF1-α/PDK4 axis in non-small cell lung cancer.

BACKGROUND: Ginsenoside Rh2 (G-Rh2), a steroidal compound extracted from roots of ginseng, has been extensively studied in tumor therapy. However, its specific regulatory mechanism in non-small cell lung cancer (NSCLC) is not well understood. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in various malignant tumors. We investigated the impact of G-Rh2 on the malignant progression of NSCLC and how it regulated PDK4 to influence tumor aerobic glycolysis and mitochondrial function. METHOD: We examined the inhibitory effect of G-Rh2 on NSCLC through I proliferation assay, migration assay and flow cytometry in vitro. Subsequently, we verified the ability of G-Rh2 to inhibit tumor growth and metastasis by constructing subcutaneous tumor and metastasis models in nude mice. Proteomics analysis was conducted to analyze the action pathways of G-Rh2. Additionally, we assessed glycolysis and mitochondrial function using seahorse, PET-CT, Western blot, and RT-qPCR. RESULT: Treatment with G-Rh2 significantly inhibited tumor proliferation and migration ability both in vitro and in vivo. Furthermore, G-Rh2 inhibited the tumor's aerobic glycolytic capacity, including glucose uptake and lactate production, through the HIF1-α/PDK4 pathway. Overexpression of PDK4 demonstrated that G-Rh2 targeted the inhibition of PDK4 expression, thereby restoring mitochondrial function, promoting reactive oxygen species (ROS) accumulation, and inducing apoptosis. When combined with sodium dichloroacetate, a PDK inhibitor, it complemented the inhibitory capacity of PDKs, acting synergistically as a detoxifier. CONCLUSION: G-Rh2 could target and down-regulate the expression of HIF-1α, resulting in decreased expression of glycolytic enzymes and inhibition of aerobic glycolysis in tumors. Additionally, by directly targeting mitochondrial PDK, it elevated mitochondrial oxidative phosphorylation and enhanced ROS accumulation, thereby promoting tumor cells to undergo normal apoptotic processes.

circFTO from M2 macrophage-derived small extracellular vesicles (sEV) enhances NSCLC malignancy by regulation miR-148a-3pPDK4 axis.

BACKGROUND: Accumulation studies found that tumor-associated macrophages (TAMs) are a predominant cell in tumor microenvironment (TME), which function essentially during tumor progression. By releasing bioactive molecules, including circRNA, small extracellular vesicles (sEV) modulate immune cell functions in the TME, thereby affecting non-small cell lung cancer (NSCLC) progression. Nevertheless, biology functions and molecular mechanisms of M2 macrophage-derived sEV circRNAs in NSCLC are unclear. METHODS: Cellular experiments were conducted to verify the M2 macrophage-derived sEV (M2-EV) roles in NSCLC. Differential circRNA expression in M0 and M2-EV was validated by RNA sequencing. circFTO expression in NSCLC patients and cells was investigated via real-time PCR and FISH. The biological mechanism of circFTO in NSCLC was validated by experiments. Our team isolated sEV from M2 macrophages (M2Ms) and found that M2-EV treatment promoted NSCLC CP, migration, and glycolysis. RESULTS: High-throughput sequencing found that circFTO was highly enriched in M2-EV. FISH and RT-qPCR confirmed that circFTO expression incremented in NSCLC tissues and cell lines. Clinical studies confirmed that high circFTO expression correlated negatively with NSCLC patient survival. Luciferase reporter analysis confirmed that miR-148a-3p and PDK4 were downstream targets of circFTO. circFTO knockdown inhibited NSCLC cell growth and metastasis in in vivo experiments. Downregulating miR-148a-3p or overexpressing PDK4 restored the malignancy of NSCLC, including proliferation, migration, and aerobic glycolysis after circFTO silencing. CONCLUSION: The study found that circFTO from M2-EV promoted NSCLC cell progression and glycolysis through miR-148a-3p/PDK4 axis. circFTO is a promising prognostic and diagnostic NSCLC biomarker and has the potential to be a candidate NSCLC therapy target.

PDK4 inhibits osteoarthritis progression by activating the PPAR pathway.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease caused by the deterioration of cartilage. However, the underlying mechanisms of OA pathogenesis remain elusive. METHODS: Hub genes were screened by bioinformatics analysis based on the GSE114007 and GSE169077 datasets. The Sprague-Dawley (SD) rat model of OA was constructed by intra-articular injection of a mixture of papain and L-cysteine. Hematoxylin-eosin (HE) staining was used to detect pathological changes in OA rat models. Inflammatory cytokine levels in serum were measured employing the enzyme-linked immunosorbent assay (ELISA). The reverse transcription quantitative PCR (RT-qPCR) was implemented to assess the hub gene expressions in OA rat models. The roles of PDK4 and the mechanism regulating the PPAR pathway were evaluated through western blot, cell counting kit-8 (CCK-8), ELISA, and flow cytometry assays in C28/I2 chondrocytes induced by IL-1ß. RESULTS: Six hub genes were identified, of which COL1A1, POSTN, FAP, and CDH11 expressions were elevated, while PDK4 and ANGPTL4 were reduced in OA. Overexpression of PDK4 inhibited apoptosis, inflammatory cytokine levels (TNF-α, IL-8, and IL-6), and extracellular matrix (ECM) degradation protein expressions (MMP-3, MMP-13, and ADAMTS-4) in IL-1ß-induced chondrocytes. Further investigation revealed that PDK4 promoted the expression of PPAR signaling pathway-related proteins: PPARA, PPARD, and ACSL1. Additionally, GW9662, an inhibitor of the PPAR pathway, significantly counteracted the inhibitory effect of PDK4 overexpression on IL-1ß-induced chondrocytes. CONCLUSION: PDK4 inhibits OA development by activating the PPAR pathway, which provides new insights into the OA management.

Inhibition of Pyruvate Dehydrogenase Kinase 4 Protects Cardiomyocytes from lipopolysaccharide-Induced Mitochondrial Damage by Reducing Lactate Accumulation.

Mitochondrial dysfunction is considered one of the major pathogenic mechanisms of sepsis-induced cardiomyopathy (SIC). Pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of mitochondrial metabolism, is essential for maintaining mitochondrial function. However, its specific role in SIC remains unclear. To investigate this, we established an in vitro model of septic cardiomyopathy using lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Our study revealed a significant increase in PDK4 expression in LPS-treated H9C2 cardiomyocytes. Inhibiting PDK4 with dichloroacetic acid (DCA) improved cell survival, reduced intracellular lipid accumulation and calcium overload, and restored mitochondrial structure and respiratory capacity while decreasing lactate accumulation. Similarly, Oxamate, a lactate dehydrogenase inhibitor, exhibited similar effects to DCA in LPS-treated H9C2 cardiomyocytes. To further validate whether PDK4 causes cardiomyocyte and mitochondrial damage in SIC by promoting lactate production, we upregulated PDK4 expression using PDK4-overexpressing lentivirus in H9C2 cardiomyocytes. This resulted in elevated lactate levels, impaired mitochondrial structure, and reduced mitochondrial respiratory capacity. However, inhibiting lactate production reversed the mitochondrial dysfunction caused by PDK4 upregulation. In conclusion, our study highlights the pathogenic role of PDK4 in LPS-induced cardiomyocyte and mitochondrial damage by promoting lactate production. Therefore, targeting PDK4 and its downstream product lactate may serve as promising therapeutic approaches for treating SIC.

Deficiency of TOP1MT enhances glycolysis through the stimulation of PDK4 expression in gastric cancer.

BACKGROUND: Abnormal glucose metabolism is one of the determinants of maintaining malignant characteristics of cancer. Targeting cancer metabolism is regarded as a new strategy for cancer treatment. Our previous studies have found that TOP1MT is a crucial gene that inhibits glycolysis and cell metastasis of gastric cancer (GC) cells, but the mechanism of its regulation of glycolysis remains unclear. METHODS: Transcriptome sequencing data, clinic-pathologic features of GC from a variety of public databases, and WGCNA were used to identify novel targets of TOP1MT. Immunohistochemical results of 250 patients with GC were used to analyze the relative expression relationship between TOP1MT and PDK4. The function of TOP1MT was investigated by migration assays and sea-horse analysis in vitro. RESULTS: We discovered a mitochondrial topoisomerase I, TOP1MT, which correlated with a higher risk of metastasis. Functional experiments revealed that TOP1MT deficiency promotes cell migration and glycolysis through increasing PDK4 expression. Additionally, the stimulating effect of TOP1MT on glycolysis may be effectively reversed by PDK4 inhibitor M77976. CONCLUSIONS: In brief, our work demonstrated the critical function of TOP1MT in the regulation of glycolysis by PDK4 in gastric cancer. Inhibiting glycolysis and limiting tumor metastasis in GC may be accomplished by suppressing PDK4.

Inhibition of Pyruvate Dehydrogenase Kinase 4 Attenuates Myocardial and Mitochondrial Injury in Sepsis-Induced Cardiomyopathy.

BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is a cardiac dysfunction caused by sepsis, with mitochondrial dysfunction being a critical contributor. Pyruvate dehydrogenase kinase 4 (PDK4) is a kinase of pyruvate dehydrogenase with multifaceted actions in mitochondrial metabolism. However, its role in SIC remains unknown. METHODS: Serum PDK4 levels were measured and analyzed in 27 children with SIC, 30 children with sepsis, and 29 healthy children. In addition, for mice exhibiting SIC, the effects of PDK4 knockdown or inhibition on the function and structure of the myocardium and mitochondria were assessed. RESULTS: The findings from the analysis of children with SIC revealed that PDK4 was significantly elevated and correlated with disease severity and organ injury. Nonsurvivors displayed higher serum PDK4 levels than survivors. Furthermore, mice with SIC benefited from PDK4 knockdown or inhibition, showing improved myocardial contractile function, reduced myocardial injury, and decreased mitochondrial structural injury and dysfunction. In addition, inhibition of PDK4 decreased the inhibitory phosphorylation of PDHE1α (pyruvate dehydrogenase complex E1 subunit α) and improved abnormal pyruvate metabolism and mitochondrial dysfunction. CONCLUSIONS: PDK4 is a potential biomarker for the diagnosis and prognosis of SIC. In experimental SIC, PDK4 promoted mitochondrial dysfunction with increased phosphorylation of PDHE1α and abnormal pyruvate metabolism.

MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

BACKGROUND: To investigate the regulatory role of microRNA (miR)-148a-3p in mouse corpus cavernous pericyte (MCPs)-derived extracellular vesicles (EVs) in the treatment of diabetes-induced erectile dysfunction (ED). METHODS: Mouse corpus cavernous tissue was used for MCP primary culture and EV isolation. Small-RNA sequencing analysis was performed to assess the type and content of miRs in MCPs-EVs. Four groups of mice were used: control nondiabetic mice and streptozotocin-induced diabetic mice receiving two intracavernous injections (days - 3 and 0) of phosphate buffered saline, MCPs-EVs transfected with reagent control, or MCPs-EVs transfected with a miR-148a-3p inhibitor. miR-148a-3p function in MCPs-EVs was evaluated by tube-formation assay, migration assay, TUNEL assay, intracavernous pressure, immunofluorescence staining, and Western blotting. RESULTS: We extracted EVs from MCPs, and small-RNA sequencing analysis showed miR-148a-3p enrichment in MCPs-EVs. Exogenous MCPs-EV administration effectively promoted mouse cavernous endothelial cell (MCECs) tube formation, migration, and proliferation, and reduced MCECs apoptosis under high-glucose conditions. These effects were significantly attenuated in miR-148a-3p-depleted MCPs-EVs, which were extracted after inhibiting miR-148a-3p expression in MCPs. Repetitive intracavernous injections of MCPs-EVs improved erectile function by inducing cavernous neurovascular regeneration in diabetic mice. Using online bioinformatics databases and luciferase report assays, we predicted that pyruvate dehydrogenase kinase-4 (PDK4) is a potential target gene of miR-148a-3p. CONCLUSIONS: Our findings provide new and reliable evidence that miR-148a-3p in MCPs-EVs significantly enhances cavernous neurovascular regeneration by inhibiting PDK4 expression in diabetic mice.

Ruangan Lidan decoction inhibits the growth and metastasis of liver cancer by downregulating miR-9-5p and upregulating PDK4.

A growing number of studies have suggested that traditional Chinese medicine (TCM) plays an essential role in the development and occurrence of liver cancer. However, the function of Ruangan Lidan decoction (RLD) in liver cancer are not yet adequately identified and manifested, which attracted our attention. The key genes related to liver cancer and RLD and the upstream miRNAs of PDK4 were obtained based on bioinformatics analysis, followed by verification of the targeting relationship between miR-9-5p and PDK4. Next, Huh7 cells were treated with RLD to detect cell proliferation, colony formation, migration, invasion, and apoptosis by multiple assays with gain- and loss-of-function experiments. Moreover, subcutaneous transplanted tumor model and lung metastasis model of liver cancer in nude mice were established to further verify the functional role of RLD in liver cancer growth and metastasis via miR-9-5p/PDK4 axis. Bioinformatics analysis found that PDK4 and miR-9-5p were related to liver cancer, and PDK4 may be a downstream regulator of RLD. miR-9-5p could target and inhibit PDK4. In vitro cell experiments demonstrated that RLD suppressed liver cancer cell proliferation, invasion and migration, and promoted apoptosis by inhibiting miR-9-5p expression and promoting PDK4 expression. In vivo animal experiments further confirmed that RLD inhibited liver cancer growth and metastasis via upregulation of miR-9-5p-dependent PDK4. RLD downregulated miR-9-5p and upregulated PDK4 to inhibit the proliferation, migration, invasion, and induce apoptosis, thereby suppressing the growth and metastasis of liver cancer, highlighting a potential novel target for treatment of liver cancer.

PDK4 facilitates fibroblast functions and diabetic wound healing through regulation of HIF-1α protein stability and gene expression.

Fibroblast activation disorder is one of the main pathogenic characteristics of diabetic wounds. Orchestrated fibroblast functions and myofibroblast differentiation are crucial for wound contracture and extracellular matrix (ECM) formation. Pyruvate dehydrogenase kinase 4 (PDK4), a key enzyme regulating energy metabolism, has been implicated in modulating fibroblast function, but its specific role in diabetic wounds remains poorly understood. In this study, we investigated the impact of PDK4 on diabetic wounds and its underlying mechanisms. To assess the effect of PDK4 on human dermal fibroblasts (HDFs), we conducted CCK-8, EdU proliferation assay, wound healing assay, transwell assay, flow cytometry, and western blot analyses. Metabolic shifts were analyzed using the Seahorse XF analyzer, while changes in metabolite expression were measured through LC-MS. Local recombinant PDK4 administration was implemented to evaluate its influence on wound healing in diabetic mice. Finally, we found that sufficient PDK4 expression is essential for a normal wound-healing process, while PDK4 is low expressed in diabetic wound tissues and fibroblasts. PDK4 promotes proliferation, migration, and myofibroblast differentiation of HDFs and accelerates wound healing in diabetic mice. Mechanistically, PDK4-induced metabolic reprogramming increases the level of succinate that inhibits PHD2 enzyme activity, thus leading to the stability of the HIF-1α protein, during which process the elevated HIF-1α mRNA by PDK4 is also indispensable. In conclusion, PDK4 promotes fibroblast functions through regulation of HIF-1α protein stability and gene expression. Local recombinant PDK4 administration accelerates wound healing in diabetic mice.

PPAR/PDK4 pathway is involved in the anticancer effects of cGMP in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with a high mortality rate. Current treatments for PDACs often have side effects, and drug resistance in cancer stem cells (CSCs) would be also a problem. Cyclic guanosine monophosphate (cGMP) suppresses the mitochondrial function of PDACs and inhibits their CSC properties. Metabolic regulation plays a crucial role in the maintenance of CSC phenotype, and we hypothesized that cGMP induction suppresses cancer stem cell properties in the cancer cell through energy-related signaling pathways. We demonstrated that induction of cGMP upregulated the PPARα/PDK4 pathway and suppressed CSC properties in PDAC, and patients with pancreatic cancer with high PDK4 gene expression had a better prognosis than those with low gene expression. Therefore, these mechanisms may provide new therapeutic targets for the eradication of pancreatic CSCs.

Identification of PDK4 as Hub Gene for Diabetic Nephropathy Using Co-Expression Network Analysis.

INTRODUCTION: Diabetic nephropathy (DN) is related to type 1 and type 2 diabetes. They are the leading cause of end-stage renal disease, but the underling specific pathogenesis of DN is not yet clear. Our study was conducted to explore how DN changed the transcriptome profiles in the kidney. METHODS: The gene expression profile of microdissected glomeruli of 41 type 2 DN patients and 20 healthy controls were included. The sample dataset GSE96804 was obtained from the GEO database. Differentially expressed genes (DEGs) were analyzed in R with the limma package and the important modules were found by weighted gene co-expression network analysis (WGCNA) clustering. The modules were then analyzed based on Gene Ontology (GO) gene set enrichment analysis, and the hub genes were found out. We next validated the hub gene, PDK4, in a cell model of DN. We also constructed the PDK4-related PPI network to investigate the correlation between PDK4 expression and other genes. RESULTS: Heatmap and volcano map were drawn to illustrate the mRNA expression profile of 1,204 DEGs in both samples of DN patients and the control group. Using WGCNA, we selected the blue module in which genes showed the strongest correlation with the phenotype and the smallest p value. We also identified PDK4 as a hub gene. PDK4 expression was upregulated in human diabetic kidney tissue. Moreover, PDK4 was speculated to play a role in glomerular basement membrane development and kidney development according to the enrichment of functions and signaling pathways. Furthermore, PDK4 and two key genes GSTA2 and G6PC protein expression were verified highly expressed in the cell model of DN. CONCLUSION: During the pathogenesis of DN, many genes may change expression in a coordinated manner. The discovery of PDK4 as key gene using WGCNA is of great significance for the development of new treatment strategies to block the development of DN.

Acetate controls endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-ß signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-ß signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-ß-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-ß-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-ß receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-ß signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.