SQSTM1/p62 Knockout by Using the CRISPR/Cas9 System Inhibits Migration and Invasion of Hepatocellular Carcinoma.

Migration and invasion play crucial roles in the progression of hepatocellular carcinoma (HCC), but the underlying mechanisms are not clear. Analysis of clinical samples indicates that SQSTM1/p62 is highly expressed in HCC and seriously affects the prognosis of patients. Subsequently, we showed that SQSTM1/p62 knockout using the CRISPR/Cas9 system led to impaired migration and invasion of HCC, upregulated Keap1, and promoted the inhibitory effect of Keap1 on Nrf2. Then, the inactivation of Nrf2 inhibited the expression of matrix metalloproteinases (MMPs), thus attenuating the migration and invasion of HCC. We also found that SQSTM1/p62 knockout significantly inhibited migration and invasion in a lung metastasis model of nude mice with HCC. Furthermore, we found that cisplatin not only significantly inhibited the expression of SQSTM1/p62 but also slowed down the migration and invasion of HCC, while the inflammatory microenvironment accelerated the migration and invasion of HCC. These results suggest for the first time that SQSTM1/p62 knockout inhibits the migration and invasion of HCC through the Keap1/Nrf2/MMP2 signaling pathway. SQSTM1/p62 may be developed into a key drug target to regulate the migration and invasion of HCC cells.

Empagliflozin-Enhanced Antioxidant Defense Attenuates Lipotoxicity and Protects Hepatocytes by Promoting FoxO3a- and Nrf2-Mediated Nuclear Translocation via the CAMKK2/AMPK Pathway.

Lipotoxicity is an important factor in the development and progression of nonalcoholic steatohepatitis. Excessive accumulation of saturated fatty acids can increase the substrates of the mitochondrial electron transport chain in hepatocytes and cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired triphosphate (ATP) production, and fracture and fragmentation of mitochondria, which ultimately leads to hepatocellular inflammatory injuries, apoptosis, and necrosis. In this study, we systematically investigated the effects and molecular mechanisms of empagliflozin on lipotoxicity in palmitic acid-treated LO2 cell lines. We found that empagliflozin protected hepatocytes and inhibited palmitic acid-induced lipotoxicity by reducing oxidative stress, improving mitochondrial functions, and attenuating apoptosis and inflammation responses. The mechanistic study indicated that empagliflozin significantly activated adenosine 5'-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through Calcium/Calmodulin dependent protein kinase kinase beta (CAMKK2) instead of liver kinase B1 (LKB1) or TGF-beta activated kinase (TAK1). The activation of empagliflozin on AMPKα not only promoted FoxO3a phosphorylation and thus forkhead box O 3a (FoxO3a) nuclear translocation, but also promoted Nrf2 nuclear translocation. Furthermore, empagliflozin significantly upregulated the expressions of antioxidant enzymes superoxide dismutase (SOD) and HO-1. In addition, empagliflozin did not attenuate lipid accumulation at all. These results indicated that empagliflozin mitigated lipotoxicity in saturated fatty acid-induced hepatocytes, likely by promoting antioxidant defense instead of attenuating lipid accumulation through enhanced FoxO3a and Nrf2 nuclear translocation dependent on the CAMKK2/AMPKα pathway. The CAMKK2/AMPKα pathway might serve as a promising target in treatment of lipotoxicity in nonalcoholic steatohepatitis.

Dapagliflozin attenuates steatosis in livers of high-fat diet-induced mice and oleic acid-treated L02 cells via regulating AMPK/mTOR pathway.

Dapagliflozin (DAPA), a kind of sodium-glucose cotransporter 2(SGLT2) inhibitor is used to treat diabetes mellitus by inhibiting urine glucose reuptake. Recent clinical outcomes indicate that SGLT2 inhibitors may exert pharmacological activities against non-alcoholic fatty liver diseases. Nonetheless, the underlying molecular mechanisms are still poorly elucidated. In this study, we investigated the potential anti-fatty liver effects of DAPA in vivo and in vitro and assayed their underlying mechanisms. Male NIH (National Institutes of Health) mice were fed with a high-fat diet (HFD) and then treated with DAPA by gavage for 4 weeks. In the following experiments, L02 cells were treated with oleic acid (OA) and different concentrations of DAPA to assess lipid metabolism. Our results revealed that DAPA administration could remarkably suppress excessive fat accumulation in the liver tissues of HFD-fed mice and OA-treated L02 cells. Importantly, DAPA could downregulate the expression levels of proteins related to lipid synthesis and upregulate the expression levels of genes associated with fatty acid oxidation in vitro and in vivo. We also found that DAPA intervention could activate adenosine monophosphate-activated protein kinase (AMPK) phosphorylation but inhibit mammalian target of rapamycin (mTOR) phosphorylation in vitro and in vivo. AMPK activation might be mediated by increasing liver kinase B1 activity and decreasing ATP level. Furthermore, these ameliorative effects were completely eliminated by an AMPK inhibitor, compound C. This study suggested that DAPA might remarkably ameliorate hepatic steatosis mediated through the AMPK/mTOR pathway and thus could be a potential drug candidate for the treatment of fatty liver diseases.

Canagliflozin attenuates lipotoxicity in cardiomyocytes and protects diabetic mouse hearts by inhibiting the mTOR/HIF-1α pathway.

Lipotoxicity plays an important role in the development of diabetic heart failure (HF). Canagliflozin (CAN), a marketed sodium-glucose co-transporter 2 inhibitor, has significantly beneficial effects on HF. In this study, we evaluated the protective effects and mechanism of CAN in the hearts of C57BL/6J mice induced by high-fat diet/streptozotocin (HFD/STZ) for 12 weeks in vivo and in HL-1 cells (a type of mouse cardiomyocyte line) induced by palmitic acid (PA) in vitro. The results showed that CAN significantly ameliorated heart functions and inflammatory responses in the hearts of the HFD/STZ-induced diabetic mice. CAN significantly attenuated the inflammatory injury induced by PA in the HL-1 cells. Furthermore, CAN seemed to bind to the mammalian target of rapamycin (mTOR) and then inhibit mTOR phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression. These results indicated that CAN might attenuate lipotoxicity in cardiomyocytes by inhibiting the mTOR/HIF-1α pathway and then show protective effects on diabetic hearts.

Intragastric and atomized administration of canagliflozin inhibit inflammatory cytokine storm in lipopolysaccharide-treated sepsis in mice: A potential COVID-19 treatment.

To date, drugs to attenuate cytokine storm in severe cases of Corona Virus Disease 2019 (COVID-19) are not available. In this study, we investigated the effects of intragastric and atomized administration of canagliflozin (CAN) on cytokine storm in lung tissues of lipopolysaccharides (LPS)-induced mice. Results showed that intragastric administration of CAN significantly and widely inhibited the production of inflammatory cytokines in lung tissues of LPS-induced sepsis mice. Simultaneously, intragastric administration of CAN significantly improved inflammatory pathological changes of lung tissues. Atomized administration of CAN also exhibited similar effects in LPS-induced sepsis mice. Furthermore, CAN significantly inhibited hypoxia inducible factor 1α (HIF-1α) and phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) protein levels in LPS-treated lung tissues. These results indicated that CAN might attenuate cytokine storm and reduce the inflammatory symptoms in critical cases in COVID-19. Its action mechanism might involve the regulation of HIF-1α and glycolysis in vivo. However, further studies about clinical application and mechanism analysis should be validated in the future.

Proteomic and microRNA-omic profiles and potential mechanisms of dysfunction in pancreatic islet cells primed by inflammation.

Diabetes is an inflammatory disease that induces pancreatic islet dysfunction. However, to the best of our knowledge, the potential underlying molecular mechanisms of this inflammatory process remains unknown. The present study investigated microRNA (miRNA/miR) and protein expression profiles through proteomics and miRNA-omics. Lipopolysaccharide-induced macrophage cell medium (LRM) was used to stimulate inflammation in mouse Beta-TC-6 islet cells. Protein analysis revealed that 87 proteins were upregulated and 42 proteins were downregulated in LRM-treated Beta-TC-6 cells compared with control cells. Additionally, miRNA analysis revealed that 11 miRNAs were upregulated, while 28 miRNAs were downregulated in LRM-treated Beta-TC-6 cells compared with control cells. Islet cells exposed to inflammation exhibited markedly downregulated protein levels of transcription factor MafA, pancreatic and duodenal homeobox 1, paired box 6, homeobox protein Nkx-2.2, synaptosomal-associated protein 25, glucagon and insulin-2, while the expression of miR-146a-5p and miR-21a-5p were upregulated. It was also determined that upregulated miR-146a-5p and miR-21a-5p levels may be mediated by NF-κB activation. The downregulation of islet functional factor mRNA was partially reversed by treating islet cells with an inhibitor of miR-21a-5p. However, treatment with an miR-146a-5p inhibitor did not exert the same effect. Overall, the present study determined the molecular profiles of islet cell inflammation based on proteomics and miRNA-omics, and indicated that the proteins and miRNAs with altered expressions may form a large network that serves a role in islet dysfunction. Particularly, miR-21a-5p upregulation in response to inflammation may contribute to islet cell dysfunction. However, how these miRNAs regulated the expression of certain mRNAs and proteins in islet cell inflammation requires further investigation.

Canagliflozin inhibits p-gp function and early autophagy and improves the sensitivity to the antitumor effect of doxorubicin.

Cancer easily induces resistance to most chemotherapy drugs. In this study, we investigated the combination cytotoxic and antitumor effects of canagliflozin (CAN) and doxorubicin (DOX) in vitro and in vivo. CAN significantly increased the cytotoxicity of DOX in HepG2, HepG2-ADR (adriamycin or doxorubicin-resistant) and MCF7 cells. CAN significantly promoted the intracellular uptake of DOX in HepG2 cells. CAN also reduced the P-glycoprotein (P-gp) level in HepG2 cells. The function of P-gp required ATP, but CAN significantly reduced the intracellular ATP level. CAN might inhibit the function of p-gp, increase the intracellular DOX concentration and contribute to an enhanced cytotoxic activity. Autophagy plays a protective role in chemotherapy-induced cell survival. However, CAN significantly inhibited DOX-induced autophagy in HepG2 cells, and the mechanism appeared to be mediated by promoting ULK1 ser 757 phosphorylation. The downregulation of P-gp may be associated with protein degradation but is independent of the autophagy pathway. Furthermore, in HepG2-xenograft BALB/c nude mice, CAN significantly increased the antitumor effect of DOX. This study is the first to report that a classical antidiabetic drug, CAN improved the sensitivity to the antitumor effect of DOX, and the potential molecular mechanisms of CAN may involve the inhibition of P-gp function and the autophagy pathway.

Effects of Mogrosides on High-Fat-Diet-Induced Obesity and Nonalcoholic Fatty Liver Disease in Mice.

Obesity and nonalcoholic fatty liver disease (NAFLD) are highly prevalent and cause numerous metabolic diseases. However, drugs for the prevention and treatment of obesity and NAFLD remain unavailable. In this study, we investigated the effects of mogrosides (luo han guo, LH) in Siraitia grosvenorii saponins on high-fat-diet-induced obesity and NAFLD in mice. We found that compared with the negative control, LH reduced body and liver weight. LH also decreased fat accumulation and increased AMP-activated protein kinase (AMPK) phosphorylation (pAMPK) levels in mouse livers. We also found that high-purity mogroside V upregulated pAMPK expression in HepG2 cells. In addition, high-purity mogroside V inhibited reactive oxygen species production and upregulated sequestosome-1 (SQSTM1, p62) expression in THP-1 cells. These results suggest that LH may affect obesity and NAFLD by enhancing fat metabolism and antioxidative defenses. Mogroside V may be a main component of LH. However, the exact molecular mechanisms and active components responsible for the inhibitory effects of LH on obesity and NAFLD require further investigation.

Hepatoprotective Effects of Antrodia cinnamomea: The Modulation of Oxidative Stress Signaling in a Mouse Model of Alcohol-Induced Acute Liver Injury.

In the present study, the components of A. cinnamomea (AC) mycelia were systematically analyzed. Subsequently, its hepatoprotective effects and the underlying mechanisms were explored using a mouse model of acute alcohol-induced liver injury. AC contained 25 types of fatty acid, 16 types of amino acid, 3 types of nucleotide, and 8 types of mineral. The hepatoprotective effects were observed after 2 weeks of AC treatment at doses of 75 mg/kg, 225 mg/kg, and 675 mg/kg in the mouse model. These effects were indicated by the changes in the levels of aspartate aminotransferase, alanine aminotransferase, several oxidation-related factors, and inflammatory cytokines in serum and/or liver samples. AC reduced the incidence rate of necrosis, inflammatory infiltration, fatty droplets formation, and cell apoptosis in liver detecting via histological and TUNEL assay. In addition, AC reduced the expression of cleaved caspase-3, -8, and -9 and the levels of phosphor-protein kinase B (Akt) and phosphor-nuclear factor-κB (NF-κB) in the liver samples. Collectively, AC-mediated hepatoprotective effects in a mouse model of acute alcohol-induced liver injury are the result of reduction in oxidative stress. This may be associated with Akt/NF-κB signaling. These results provide valuable evidence to support the use of A. cinnamomea as a functional food and/or medicine.