As yet, there was no effective pharmacological therapy approved for non-alcoholic fatty liver disease (NAFLD). Here, we aimed to evaluate the therapeutic potential of puerarin against NAFLD and explored the underlying mechanisms. C57BL/6J mice were fed with a high-fat high-sucrose (HFHS) diet with or without puerarin coadministration intragastrically. The levels of hepatocellular injury, steatosis, fibrosis, and mitochondrial and metabolism alteration were detected. First, puerarin ameliorated histopathologic abnormalities due to HFHS. We observed a marked increase in hepatic lipid content, inflammation, and fibrosis level, which were attenuated by puerarin. Possible mechanisms were related to puerarin-mediated activation of PI3K/AKT pathway and further improvement in fatty acid metabolism. Puerarin restored the NAD+ content and beneficially affected the hepatic mitochondrial function, which attenuated HFHS-induced steatosis and metabolic disturbances. Finally, hepatic PARP-1 was activated due to excessive fat intake. Puerarin attenuated the PARP-1 expression in HFHS-fed mice, and PJ34, the PARP inhibitor, could mimic these protections of puerarin. However, pharmacological inhibition of PI3K disabled the protection of puerarin or PJ34 toward NAD+ refilling and mitochondrial homeostasis. In conclusion, our findings indicated that puerarin could be a promising and practical therapeutic strategy in NAFLD through modulating PARP-1/PI3K/AKT signaling pathway and further facilitating mitochondrial function.
Abstract Iron is essential for growth and proliferation of mammalian cells. The maintenance of cellular iron homeostasis is regulated by iron regulatory proteins (IRPs) through binding to the cognate iron-responsive elements in target mRNAs and thereby regulating the expression of target genes. Irp1 or Irp2 -null mutation is known to reduce the cellular iron level by decreasing transferrin receptor 1 and increasing ferritin. Here, we report that Irp1 or Irp2 -null mutation also causes downregulation of frataxin and IscU, two of the core components in the iron-sulfur cluster biogenesis machinery. Interestingly, while the activities of some of iron-sulfur cluster-containing enzymes including mitochondrial aconitase and cytosolic xanthine oxidase were not affected by the mutations, the activities of respiratory chain complexes were drastically diminished resulting in mitochondrial dysfunction. Overexpression of human ISCU and frataxin in Irp1 or Irp2 -null cells was able to rescue the defects in iron-sulfur cluster biogenesis and mitochondrial quality. Our results strongly suggest that iron regulatory proteins regulate the part of iron sulfur cluster biogenesis tailored specifically for mitochondrial electron transport chain complexes.
Trans-differentiation of quiescent hepatic stellate cells (HSC) into myofibroblast cells is considered the linchpin of liver fibrosis. A myriad of signaling pathways contribute to HSC activation and consequently liver fibrosis. Epidermal growth factor (EGF) family of cytokines signal through the cognate receptor EGFR to promote HSC activation. In the present study we investigated the transcription regulation of epiregulin (EREG), an EGFR ligand, during HSC activation. We report that EREG expression was significantly up-regulated in activated HSCs compared to quiescent HSCs isolated from mice. In addition, there was an elevation of EREG expression in HSCs undergoing activation in vitro . Of interest, deficiency of myocardin-related transcription factor A (MRTF-A), a well-documented regulator of HSC trans-differentiation, attenuated up-regulation of EREG expression both in vivo and in vitro . Further analysis revealed that MRTF-A interacted with serum response factor (SRF) to bind directly to the EREG promoter and activate EREG transcription. EREG treatment promoted HSC activation in vitro , which was blocked by MRTF-A depletion or inhibition. Mechanistically, EREG stimulated nuclear trans-location of MRTF-A in HSCs. Together, our data portray an EREG-MRTF-A feedforward loop that contributes to HSC activation and suggest that targeting the EREG-MRTF-A axis may yield therapeutic solutions against liver fibrosis.
Afatinib is mainly used to treat advanced non-small cell lung cancer, but its therapeutic effect on hepatocellular carcinoma is still unclear.Over 800 drugs were screened by CCK8 technology and afatinib was found to have a significant inhibitory effect on liver cancer cells. The expression of PDL1 in tumor cells treated with drugs were detected by qRT-PCR and Weston Blot experiments. The effects of afatinib on the growth, migration and invasion of HCC cells were evaluated using wound healing, Transwell, and cell cloning assays. The in vivo effects of afatinib in combination with anti-PD1 were evaluated in C57/BL6J mice with subcutaneous tumorigenesis. Bioinformatics analysis was performed to explore the specific mechanism of afatinib's inhibition of ERBB2 in improving the expression level of PD-L1, which was subsequently verified through experiments.Afatinib was found to have a significant inhibitory effect on liver cancer cells, as confirmed by in vitro experiments, which demonstrated that it could significantly suppress the growth, invasion and migration of HCC cells. qRT PCR and Weston Blot experiments also showed that Afatinib can enhance the expression of PD-L1 in tumor cells. In addition, in vitro experiments confirmed that afatinib can significantly enhance the immunotherapeutic effect of hepatocellular carcinoma. Afatinib's ability to increase PD-L1 expression is mediated by STAT3 activation following its action on HCC cells.Afatinib enhances PD-L1 expression in tumor cells through the STAT3/PD-L1 pathway. The combination of afatinib and anti-PD1 treatment significantly increases the immunotherapeutic effect of HCC.
Nonalcoholic fatty liver disease (NAFLD) is more sensitive to ischemia and reperfusion injury (IRI), while there are no effective methods to alleviate IRI. Necroptosis, also known as "programmed necrosis," incorporates features of necrosis and apoptosis. However, the role of necroptosis in IRI of the fatty liver remains largely unexplored. In the present study, we aimed to assess whether necroptosis was activated in the fatty liver and whether such activation accelerated IRI in the fatty liver. In this study, we found that the liver IRI was enhanced in HFD-fed mice with more release of TNFα. TNFα and supernatant of macrophages could induce necroptosis of hepatocytes in vitro. Necroptosis was activated in NAFLD, leading to more severe IRI, and such necroptosis could be inhibited by TN3-19.12, the neutralizing monoclonal antibody against TNFα. Pretreatment with Nec-1 and GSK'872, two inhibitors of necroptosis, significantly reduced the liver IRI and ROS production in HFD-fed mice. Moreover, the inhibition of necroptosis could decrease ROS production of hepatocytes in vitro. Inflammatory response was activated during IRI, and necroptosis inhibitors could suppress signaling pathways of inflammation and the soakage of inflammation cells. In conclusion, TNFα-induced necroptosis played an important role during IRI in the fatty liver. Our findings demonstrated that necroptosis might be a potential target to reduce the fatty liver-associated IRI.
Background This study aimed to explore the regulatory effect of anserine on HUVEC cell injury and thrombosis in deep venous thrombosis (DVT) rats, and to elucidate the underlying molecular mechanisms. Methods Non-targeted metabolomics data analyses were conducted using an ultra-performance liquid chromatography system Vanquish UHPLC and mass spectrometer to detect plasma metabolism profiles. The transcriptome sequencing and gene intervention experiments were performed to verify the regulatory effect. Further in vivo and in vitro experiments were performed. Enzyme-linked immunosorbent assay was used to detect the levels of P-selectin, E-selectin, and vWF, hematoxylin-eosin (HE) staining was performed to observe thrombotic and inflammatory cell infiltration, flow cytometry and TUNEL assays were performed to detect apoptosis, and qPCR and WB assays were conducted to determine the gene and protein expression. Results Anserine alleviated HUVECs injury, reduced adhesion molecule expression, and inflammation. It decreased P-selectin, E-selectin, vWF, THBD, TFPI levels, and apoptosis while promoting NOS3, ET-1, and NO release in HUVECs. In DVT rats, anserine reduced P-selectin, E-selectin, vWF, thrombosis, cell infiltration, apoptosis, and promoted NO release. Transcriptome sequencing and gene intervention confirmed anserine’s regulation of the PI3K-Akt pathway and coagulation via MYB. CARNMT1, a regulatory enzyme for anserine metabolism, increased anserine content, inhibiting coagulation, thrombosis, cell infiltration, and promoting NO release in rats. Conclusion This study confirmed anserine could alleviate DVT by improving the inflammatory response, inhibiting blood agglutination, and promoting vasodilation, providing new potential therapeutic targets, important scientific evidence for the development of DVT management, and new clues for an in-depth understanding of its molecular mechanisms.
Abstract Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, with complex etiology and mechanism, and a high mortality rate. Tumor‐associated macrophages (TAMs) are an important part of the HCC tumor microenvironment. Studies in recent years have shown that TAMs are involved in multiple stages of HCC and are related to treatment and prognosis in HCC. The specific mechanisms between TAMs and HCC are gradually being revealed. This paper reviews recent advances in the mechanisms associated with TAMs in HCC, concentrating on an overview of effects of TAMs on drug resistance in HCC and the signaling pathways linked with HCC, providing clues for the treatment and prognosis determination of HCC.
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