Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with disordered lipid and iron metabolism. Our previous study has substantiated the pivotal role of Caveolin-1 (Cav-1) in protecting hepatocytes and mediating iron metabolism in the liver. This study aimed to explore the specific mechanisms underlying the regulation of iron metabolism by Cav-1 in NAFLD. METHODS: Hepatocyte-specific Cav-1 overexpression mice and knockout mice were used in this study. Cav-1-knockdown of RAW264.7 cells and mouse primary hepatocytes were performed to verify the changes in vitro. Moreover, a high-fat diet and palmitic acid plus oleic acid treatment were utilized to construct a NAFLD model in vivo and in vitro, respectively, while a high-iron diet was used to construct an in vivo iron overload model. Besides, iron concentration, the expression of Cav-1 and iron metabolism-related proteins in liver tissue or serum were detected using iron assay kit, Prussian blue staining, Western blotting, immunofluorescence staining, immunohistochemical staining and ELISA. The related indicators of lipid metabolism and oxidative stress were evaluated by the corresponding reagent kit and staining. RESULTS: Significant disorder of lipid and iron metabolism occurred in NAFLD. The expression of Cav-1 was decreased in NAFLD hepatocytes (P < 0.05), accompanied by iron metabolism disorder. Cav-1 enhanced the iron storage capacity of hepatocytes by activating the ferritin light chain/ferritin heavy chain pathway in NAFLD, subsequently alleviating the oxidative stress induced by excess ferrous ions in the liver. Further, CD68+CD163+ macrophages expressing Cav-1 were found to accelerate iron accumulation in the liver, which was contrary to the effect of Cav-1 in hepatocytes. Positive correlations were also observed between the serum Cav-1 concentration and the serum iron-related protein levels in NAFLD patients and healthy volunteers (P < 0.05). CONCLUSIONS: These findings confirm that Cav-1 is an essential target protein that regulates iron and lipid metabolic homeostasis. It is a pivotal molecule for predicting and protecting against the development of NAFLD.

Ginsenosides Rg1 regulate lipid metabolism and temperature adaptation in Caenorhabditis elegans.

Background: Obesity is a risk factor for aging and many diseases, and the disorder of lipid metabolism makes it prominent. This study aims to investigate the effect of ginsenoside Rg1 on aging, lipid metabolism and stress resistance. Methods: Rg1 was administered to Caenorhabditis elegans (C. elegans) cultured in NGM or GNGM. The lifespan, locomotory activity, lipid accumulation, cold and heat stress resistance and related mRNA expression of the worms were examined. Gene knockout mutants were used to clarify the effect on lipid metabolism of Rg1. GFP-binding mutants were used to observe the changes in protein expression. Results: We reported that Rg1 reduced lipid accumulation and improved stress resistance in C. elegans. Rg1 significantly reduced the expression of fatty acid synthesis-related genes and lipid metabolism-related genes in C. elegans. However, Rg1 did not affect the fat storage in fat-5/fat-6 double mutant or nhr-49 mutant. Combined with network pharmacology, we clarified the possible pathways and targets of Rg1 in lipid metabolism. In addition, Rg1-treated C. elegans showed a higher expression of anti-oxidative genes and heat shock proteins, which might contribute to stress resistance. Conclusion: Rg1 reduced fat accumulation by regulating lipid metabolism via nhr-49 and enhanced stress resistance by its antioxidant effect in C. elegans.

Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/ß-Catenin and Akt/NF-κB signaling pathways.

BACKGROUND: Surgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PURPOSE: This study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. METHODS: The potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. RESULTS: Network pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/ß-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. CONCLUSION: Our study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/ß-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Collagen VI antibody reduces atherosclerosis by activating monocyte/macrophage polarization in ApoE-/- mice.

Atherosclerosis (AS) has been regarded as an autoimmune disease. However, studies on immunotherapy against AS are limited. We previously found that IgG in AS patients serum binding to alpha 5 and 6 chain of collagen VI (COL6A5 or COL6A6) was significantly higher than that in healthy subjects, here we tried to identify whether they are AS-protective, and tried to develop human antibodies against them. ApoE-/- mice were immunized with COL6A5 or COL6A6 and COL6A6 was found a protective antigen against atherosclerosis. A phage display human single-chain antibody (scFv) library was constructed and COL6A6-specific scFv was obtained, and cloned into a modified pcDNA3 vector to express full-length human antibodies. ApoE-/- mice were fed a high-fat diet (HFD) for 20 weeks and administered three weekly injections of CVI monoclonal antibody (mAb) or isotype control antibody, CVI mAb was found to be able to reduce plaque area by 45 % via aorta oil red O staining. Flowcytometry method predicted that CVI mAb induced monocyte/macrophage polarization from M1 to M2. Furthermore, CVI mAb induced decreases of pro-inflammatory cytokines of MCP-1and IL-1ß, and increases of IL-4 and IL-10 levels in animal serum by using theLuminexassay. Overall, we found a novel atherosclero-related antigen - Collagen VI, and its protective fragment - Collagen VI alpha 6 chain (COL6A6) and proved that humanized antibody against COL6A6 therapy regresses atherosclerosis and induces monocyte/macrophage polarization from M1 to M2 in ApoE-/- mice animal model.

Modified Shenlingbaizhu Decoction represses the pluripotency of colorectal cancer stem cells by inhibiting TGF-ß mediated EMT program.

BACKGROUND: The Modified Shenlingbaizhu Decoction (MSD) utilizes various phytomedicines has been applied to treat colorectal cancer (CRC). Colorectal cancer stem cells (CSCs) have proven to be tightly associated with CRC progression and metastasis. The mechanism of MSD's inhibitory effect on CSCs has not been determined. PURPOSE: To figure out how MSD inhibits the pluripotency of CSCs and impedes the EMT program. METHODS: The ingredients of MSD extracts were characterized by high-performance liquid chromatography (HPLC). BALB/c-nu mice were transplanted into EGFP labeled SW480 CRC cells and the tumor weight and volume were recorded before and after various doses of MSD treatment. The concentration of TGF-ß1 was quantified with an Enzyme-linked immunosorbent assay. To delineate the logical relationship between EMT and CSCs regulated by MSD, TGF-ß/Smad inhibitor and activator were adopted in tumor-bearing mice and diverse CRC cell lines. Cancer stem cell markers were analyzed by flow cytometry. In vitro analysis of cell motility and viability were done using CCK-8, wound healing, and invasion assay. Immunohistochemistry (IHC) and western blotting (WB) were used for detecting protein expression. The collected results were statistically analyzed with GraphPad Prism 8.0. RESULTS: MSD treatment significantly reduced the size of colorectal cancer tumors and lowered the serum content of TGF-ß1 in mice. Importantly, MSD markedly reduced the expression of pluripotent factors and depressed CD133+ stem cells in the tumor tissues. The TGF-ß/Smad inhibitor neutralized the EMT signaling and lowered the pluripotency by dephosphorylation of SMAD2/3. Similarly, MSD attenuated the pluripotency by limiting TGF-ß/Smad signaling-induced EMT in vivo. MSD inhibited colorectal cancer cell proliferation, migration, and invasion. CONCLUSIONS: MSD inhibits the growth of colorectal cancer. It dampens the pluripotency of CSCs by repressing the TGF-ß-induced EMT program.

Poria cocos polysaccharides rescue pyroptosis-driven gut vascular barrier disruption in order to alleviates non-alcoholic steatohepatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Poria cocos polysaccharides (PCP) are abundant in Poria cocos (Schw.) Wolf (Poria). This is a common traditional Chinese medicine used to treat gastrointestinal and liver diseases. Poria cocos dispel dampness and enhance gastrointestinal functions, strongly affecting the treatment of non-alcoholic fatty liver disease. Still, the mechanism is not yet clear. AIM OF THE STUDY: The latest research found that protecting the integrity of the intestinal barrier can slow down the progression of non-alcoholic fatty liver disease (NAFLD). Hence, our research ought to explore the protective mechanism of PCP on the intestinal barrier under a high-fat diet and to clarify the relationship between intestinal barrier damage and steatohepatitis. MATERIALS AND METHODS: H&E staining was done to evaluate pathological damage, whereas Nile red and oil red O staining was conducted to evaluate hepatic fat infiltration. Immunofluorescence staining and immunohistochemical staining were used to detect protein expression and locations. Bone marrow-derived macrophages were isolated for in vitro experiments. ONOO- and ROS fluorescent probes and MDA, SOD, and GSH kits assessed the levels of nitrogen and oxidative stress. LPS levels were detected with a Limulus Amebocyte Lysate assay. The Western blot analysis and reverse transcription-quantitative PCR detected the expression of related proteins and genes. The Elisa kit detected the level of the inflammatory factors in the cell supernatant. For the vivo NAFLD experiments, in briefly, mice were randomly chosen to receive either a High-fat diet or control diet for 12 weeks. Drug treatments started after 4 weeks of feeding. Zebrafish larvae were raised separately in fish water or 7 mM thioacetamide as the control or model group for approximately 72 h. In the therapy groups, different concentrations of PCP were added to the culture environment at the same time. RESULTS: In zebrafish, we determined the safe concentration of PCP and found that PCP could effectively reduce the pathological damage in the liver and intestines induced by the NAFLD model. In mice, PCP could slow down weight gain, hyperlipidemia, and liver steatosis caused by a high-fat diet. More importantly, PCP could reduce the destruction of the gut-vascular barrier and the translocation of endotoxins caused by a high-fat diet. Further, we found that PCP could inhibit intestinal pyroptosis by regulating PARP-1. Pyroptosis inhibitors, such as MCC950, could effectively protect the intestinal and liver damage induced by a high-fat diet. We also found that pyroptosis mainly occurred in intestinal macrophages. PCP could effectively improve the survival rate of bone marrow-derived macrophages in a high-fat environment and inhibit pyroptosis. CONCLUSIONS: These results indicated that PCP inhibited the pyroptosis of small intestinal macrophages to protect the intestinal barrier integrity under a high-fat diet. This resulted in decreased endotoxin translocation and progression of steatohepatitis.

Isoliquiritigenin alleviates liver fibrosis through caveolin-1-mediated hepatic stellate cells ferroptosis in zebrafish and mice.

BACKGROUND: Liver fibrosis is a major disease that threatens people's health around the world. However, there is a lack of effective treatment to completely reverse liver fibrosis. Liver transplantation is currently the only curative option for patients with advanced cirrhosis. Ferroptosis is a newly discovered type of cell death and plays an important role in the process of liver fibrosis, but the specific mechanism needs to be clarified. HYPOTHESIS/PURPOSE: To explore the regulatory mechanism of isoliquiritigenin (ISL) in the process of liver fibrosis and the relationship between Cav-1 and ferroptosis. METHODS: In this research, zebrafish, HSC-T6 cells, and mice were used as the research object. Different ROS probes to visually detect the content and distribution of ROS in live zebrafish and cells. Lentivirus and siRNA-mediated transfection techniques were used for the construction of Cav-1 overexpression and knockdown cell lines to verify the important role of Cav-1 in vitro. RESULTS: Generally, we first elucidated that ISL relieved liver fibrosis by inducing hepatic stellate cells (HSCs) ferroptosis through repressing GPX4 expression and increasing the expression of TFR and DMT1, thus producing a large number of ROS, we also found that Cav-1 exerted its anti-hepatic fibrosis effect by promoting HSCs ferroptosis. CONCLUSION: Our results have shown that Cav-1-mediated HSCs ferroptosis is necessary for ISL to play an anti-fibrotic effect in vitro and in vivo.

Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway.

BACKGROUND: Glioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear. PURPOSE: This study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells. METHODS: CCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting. RESULTS: Here we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. CONCLUSION: We first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT+ GBM patients.

Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

Acetaminophen (APAP) is the leading cause of acute liver failure (ALF), which is characterized by GSH depletion, oxidative stress and mitochondrial dysfunction. However, the specific mechanism of APAP-induced ALF remains to be clarified. In this study, we demonstrated that indoleamine 2,3-dioxygenase 1 (IDO1) aggravated APAP-induced ALF associated with excess lipid peroxidation, which was reversed by lipid peroxidation inhibitor (ferrostatin-1). Meanwhile, IDO1 deficiency effectively decreased the accumulation of reactive nitrogen species. Additionally, IDO1 deficiency prevented against APAP-induced liver injury through suppressing the activation of macrophages, thereby reduced their iron uptake and export, eventually reduced iron accumulation in hepatocytes through transferrin and transferrin receptor axis. In summary, our study confirmed that APAP-induced IDO1 aggravated ALF by triggering excess oxidative and nitrative stress and iron accumulation in liver. These results offer new insights for the clinical treatment of ALF or iron-dysregulated liver diseases in the future.

Caveolin-1 dictates ferroptosis in the execution of acute immune-mediated hepatic damage by attenuating nitrogen stress.

Ferroptosis is a new regulated cells death manner defined as results of iron-dependent accumulation of lipid peroxidation. However, the specific mechanisms of regulating ferroptosis remain unclear. In our present study, we demonstrated that Caveolin-1 (Cav-1) played a central role in protecting hepatocytes against ferroptosis in autoimmunity-mediated hepatitis (AIH). The down-regulated Cav-1 in liver tissues, accompanied by ferroptotic events and RNS production, were contributed to the outcome of ConA-induced hepatic damage, which were rescued by ferrostatin-1 (an inhibitor of ferroptosis) in vivo and in vitro. Additionally, Cav-1 deficiency aggravated ConA-induced hepatocellular death and ferroptosis associated with excessive nitrogen stress response. Short hairpin RNA of Cav-1 in hepatocytes promoted ferroptosis and nitrative stress in response to erastin in vitro, which was ameliorated by Cav-1 over-expression. Meanwhile, administration of the iNOS inhibitor (1400W) or ONOO- scavenger (Fe-TMPyP), diminished reactive nitrogen species (RNS), remarkably reduced hepatocytes ferroptosis and attenuated ConA-induced liver damage. Furthermore, immune inhibition by gadolinium chloride (GdCl3), a well-known Kupffer cell depletor, elevated hepatic Cav-1 but inhibited ferroptosis and nitrative stress under ConA exposure. In conclusion, these data revealed a novel molecular mechanism of ferroptosis with the Cav-1 regulation was essential for pathogenesis of ConA-induced hepatitis. Downstream of Cav-1, RNS-mediated ferroptosis was a pivotal step that drives the execution of acute immune-mediated hepatic damage.