Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death.

ETHNOPHARMACOLOGICAL RELEVANCE: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases. AIM OF THE STUDY: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms. MATERIALS AND METHODS: A CCl4-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFß1 were used as in vitro models. RESULTS: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFß1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl4-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3ß is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3ß levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFß1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3ß/Nrf2 cascade and eliminated its protective effects against TGFß1. CONCLUSIONS: Our findings reveal that WEE improves mouse LF triggered by CCl4 and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3ß/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

Ethanol Extract of Rosa rugosa Ameliorates Acetaminophen-Induced Liver Injury via Upregulating Sirt1 and Subsequent Potentiation of LKB1/AMPK/Nrf2 Cascade in Hepatocytes.

Acetaminophen (APAP)-induced liver injury is a common hepatic disease resulting from drug abuse. Few targeted treatments are available clinically nowadays. The flower bud of Rosa rugosa has a wide range of biological activities. However, it is unclear whether it alleviates liver injury caused by APAP. Here, we prepared an ethanol extract of Rosa rugosa (ERS) and analyzed its chemical profile. Furthermore, we revealed that ERS significantly ameliorated APAP-induced apoptosis and ferroptosis in AML-12 hepatocytes and dampened APAP-mediated cytotoxicity. In AML-12 cells, ERS elevated Sirt1 expression, boosted the LKB1/AMPK/Nrf2 axis, and thereby crippled APAP-induced intracellular oxidative stress. Both EX527 and NAM, which are chemically unrelated inhibitors of Sirt1, blocked ERS-induced activation of LKB1/AMPK/Nrf2 signaling. The protection of ERS against APAP-triggered toxicity in AML-12 cells was subsequently abolished. As expression of LKB1 was knocked down, ERS still upregulated Sirt1 but failed to activate AMPK/Nrf2 cascade or suppress cytotoxicity provoked by APAP. Results of in vivo experiments showed that ERS attenuated APAP-caused hepatocyte apoptosis and ferroptosis and improved liver injury and inflammation. Consistently, ERS boosted Sirt1 expression, increased phosphorylations of LKB1 and AMPK, and promoted Nrf2 nuclear translocation in the livers of APAP-intoxicated mice. Hepatic transcriptions of HO-1 and GCLC, which are downstream antioxidant genes of Nrf2, were also significantly increased in response to ERS. Our results collectively indicated that ERS effectively attenuates APAP-induced liver injury by activating LKB1/AMPK/Nrf2 cascade. Upregulated expression of Sirt1 plays a crucial role in ERS-mediated activation of LKB1.

Ergothioneine suppresses hepatic stellate cell activation via promoting Foxa3-dependent potentiation of the Hint1/Smad7 cascade and improves CCl4-induced liver fibrosis in mice.

Ergothioneine (EGT) is a bioactive compound derived from certain edible mushrooms. The activation of hepatic stellate cells (HSCs) is critically involved in the etiology of liver fibrosis (LF). Here, we report that in LX-2 HSCs, EGT upregulates the expression of Hint1 and Smad7 and suppresses their activation provoked by TGFß1. The EGT-triggered inhibition of HSC activation is abolished by knocking down the expression of Hint1. Overexpression of Hint1 increases Smad7 and represses TGFß1-provoked activation of LX-2 HSCs. In silico predictions unveiled that in the promoter region of the human Hint1 gene, there are two conserved cis-acting elements that have the potential to interact with the transcription factor Foxa3 termed hFBS1 and hFBS2, respectively. The knockdown of Foxa3 obviously declined Hint1 expression at both mRNA and protein levels. Transfection of Foxa3 or EGT treatment increased the activity of the luciferase reporter driven by the Hint1 promoter in an hFBS2-dependent manner. The knockdown of Foxa3 eliminated EGT-mediated upregulation of Hint1 promoter activity. Additionally, EGT triggered the nuclear translocation of Foxa3 without obviously affecting its expression level. Molecular docking analysis showed that EGT has the potential to directly interact with the Foxa3 protein. Moreover, Foxa3 played a critical role in EGT-mediated hepatoprotection. EGT modulated the Foxa3/Hint1/Smad7 signaling in mouse primary HSCs and inhibited their activation. The gavage of EGT considerably relieved CCl4-induced LF in mice. Our data provide new insights into the anti-LF activity of EGT. Mechanistically, EGT triggers the nuclear translocation of Foxa3 in HSCs, which promotes Hint1 transcription and subsequently elevates Smad7.

Theaflavine inhibits hepatic stellate cell activation by modulating the PKA/LKB1/AMPK/GSK3ß cascade and subsequently enhancing Nrf2 signaling.

Activation of hepatic stellate cells (HSCs) constitutes a crucial etiological factor leading to liver fibrosis. Theaflavine (TF) is a characteristic bioactive compound in fermented tea. Here, we found that TF attenuated the activation of LX-2 HSCs induced by transforming growth factor-ß1 (TGF-ß1). TF potentiated nuclear factor erythroid 2-related Factor 2 (Nrf2) signaling. Knockdown of Nrf2 abrogated TF-mediated resistance to TGF-ß1. Liver kinase B1 (LKB1), AMP-activated kinase (AMPK), and glycogen synthase kinase-3ß (GSK3ß) are upstream regulators of Nrf2. TF modulated the LKB1/AMPK/GSK3ß axis. Inhibition of AMPK or knockdown of LKB1 crippled TF-mediated potentiation of Nrf2. Protein kinase A (PKA) catalyzes LKB1 phosphorylation. In LX-2 cells, TF increased the LKB1/PKA interaction without affecting their contents. Inhibition of PKA abolished TF-mediated potentiation of LKB1/Nrf2 and abrogated the inhibitory effects of TF on their activation. TF also enhanced direct binding between purified catalytic subunit α of PKA (PKA-Cα) and LKB1 proteins in vitro. Molecular docking indicated that TF showed binding activity with both LKB1 and PKA-Cα proteins. In mouse primary HSCs, TF elevated LKB1/PKA-Cα binding, boosted LKB1 phosphorylation, potentiated Nrf2 and suppressed their spontaneous activation. PKA inhibition or LKB1 knockdown eliminated TF-mediated induction of Nrf2 and suppression of HSC activation. Furthermore, TF considerably alleviated CCl4-induced mouse liver fibrosis. In mouse livers, TF increased the LKB1/PKA-Cα interaction, upregulated LKB1 phosphorylation and modulated its downstream AMPK/GSK3ß/Nrf2 cascade. Our findings collectively indicated that TF suppresses HSC activation. Mechanistically, TF elevated the LKB1/PKA interaction in HSCs, which increased LKB1 phosphorylation and subsequently modulated the downstream AMPK/GSK3ß/Nrf2 axis.

New Aclyphloroglucinols and geranyl-α-pyrones from Hypericum hengshanense.

Hypericum hengshanense is a previously uninvestigated endemic plant species of China. Three new aclyphloroglucinols, hengshanols A-C (1-3), and two new geranyl-α-pyrones, hengshanpyol D and E (4 and 5), together with three known compounds were isolated from the aerial parts of H. hengshanense. The structure of these compounds were elucidated by NMR, MS, optical rotation, and ECD data. All compounds were isolated from H. hengshanense for the first time. Among them, compounds 2-4 may have anti-laryngeal cancer activity. Compounds isolated were tested for glucose uptake in L6 cells, and compound 4 showed the most potent glucose uptake with 1.62-fold enhancement.

Limonin relieves TGF-ß-induced hepatocyte EMT and hepatic stellate cell activation in vitro and CCl4-induced liver fibrosis in mice via upregulating Smad7 and subsequent suppression of TGF-ß/Smad cascade.

Liver fibrosis is a pathological process as a result of intrahepatic deposition of excessive ECM. EMT of hepatocytes and activation of HSCs both play important roles in the etiology of liver fibrosis. Here, we found that limonin repressed TGF-ß-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. Limonin suppressed TGF-ß-provoked Smad2/3 C-terminal phosphorylation and subsequent nuclear translocation. However, limonin exerted few effects on Smad2/3 phosphorylation atlinker region. Mechanistically, limonin increased Smad7 in both AML-12 and LX-2 cells. Knockdown of Smad7 abrogated inhibitory effects of limonin on TGF-ß-induced changes in both two cells. Further studies revealed that limonin upregulated Smad7 and declined C-terminal phosphorylation and nuclear translocation of Smad2/3 to alleviate mouse CCl4-induced liver fibrosis. Our findings indicated that limonin inhibits TGF-ß-induced EMT of hepatocytes and activation of HSCs in vitro and CCl4-induced liver fibrosis in mice. Upregulated Smad7 which suppresses Smad2/3-dependent gene transcription is implicated in the hepatoprotective activity of limonin.

Piperine inhibits AML-12 hepatocyte EMT and LX-2 HSC activation and alleviates mouse liver fibrosis provoked by CCl4: roles in the activation of the Nrf2 cascade and subsequent suppression of the TGF-ß1/Smad axis.

Piperine (PIP) is an alkaloid derived from peppercorns. Herein, we assessed its effects on hepatocyte EMT and HSC activation in vitro and CCl4-elicited liver fibrosis in mice. Further experiments were performed to unveil the molecular mechanisms underlying the hepatoprotective activity of PIP. We found that PIP inhibited TGF-ß1-provoked AML-12 hepatocyte EMT and LX-2 HSC activation. Mechanistically, in AML-12 and LX-2 cells, PIP evoked Nrf2 nuclear translocation and increased transcriptions of Nrf2-responsive antioxidative genes. These events decreased TGF-ß1-induced production of ROS. Moreover, PIP increased the expression of Smad7, suppressed phosphorylation and nuclear translocation of Smad2/3, and decreased the transcriptions of Smad2/3-downstream genes. Knockdown of Nrf2 abrogated the protective activity of PIP against TGF-ß1. Modulatory effects of PIP on the TGF-ß1/Smad cascade were also crippled, which suggested that activation of Nrf2 played critical roles in the regulatory effects of PIP on TGF-ß1/Smad signaling. Experiments in vivo unveiled that PIP ameliorated mouse liver fibrosis provoked by CCl4. PIP modulated the intrahepatic contents of the markers of EMT and HSC activation. In mouse livers, PIP activated Nrf2 signaling and reduced Smad2/3-dependent gene transcriptions. Our findings collectively suggested PIP as a new chemical entity with the capacity of alleviating liver fibrosis. The activation of the Nrf2 cascade and subsequent suppression of the TGF-ß1/Smad axis are implicated in the hepatoprotective activity of PIP.

A characterized saponin extract of Panax japonicus suppresses hepatocyte EMT and HSC activation in vitro and CCl4-provoked liver fibrosis in mice: Roles of its modulatory effects on the Akt/GSK3ß/Nrf2 cascade.

BACKGROUND AND PURPOSE: Liver fibrosis constitutes a pathologic condition resulting in a series of advanced liver diseases. Oleanane-type saponins are distinctive active constituents in the medicinal plant Panax japonicus C. A. Mey (P. japonicus). Herein, we assessed protective effects of a characterized saponin extract of rhizomes of P. japonicus (SEPJ) on hepatocyte EMT and HSC activation in vitro and liver fibrosis in mice. We also investigated molecular mechanisms underlying the hepatoprotective activity of SEPJ. METHODS: EMT of AML-12 hepatocytes was evaluated by observing morphology of cells and quantifying EMT marker proteins. Activation of LX-2 HSCs was assessed via scratch assay, transwell assay, and EdU-incorporation assay, and by quantifying activation marker proteins. Liver fibrosis in mice was evaluated by HE, SR, and Masson staining, and by measuring related serum indicators. Immunoblotting and RT-PCR were performed to study mechanisms underlying the action of SEPJ. RESULTS: SEPJ inhibited TGF-ß-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. SEPJ elevated Akt phosphorylation at Ser473 and GSK3ß phosphorylation at Ser9 in these cells, giving rise to a descent of the catalytic activity of GSK3ß. These events increased levels of both total and nuclear Nrf2 protein and upregulated expressions of Nrf2-responsive antioxidative genes. In addition, enhanced phosphorylation of Akt and GSK3ß acted upstream of SEPJ-mediated activation of Nrf2. Knockdown of Nrf2 or inhibition of Akt diminished the protective activity of SEPJ against TGF-ß in both AML-12 and LX-2 cells. Our further in vivo experiments revealed that SEPJ imposed a considerable alleviation on CCl4-provoked mouse liver fibrosis. Moreover, hepatic Akt/GSK3ß/Nrf2 cascade were potentiated by SEPJ. Taken together, our results unveiled that SEPJ exerted protective effects against fibrogenic cytokine TGF-ß in vitro and ameliorated liver fibrosis in mice. Mechanistically, SEPJ regulated the Akt/GSK3ß/Nrf2 signaling which subsequently enhanced intracellular antioxidative capacity. CONCLUSIONS: SEPJ inhibits hepatocyte EMT and HSC activation in vitro and alleviates liver fibrosis in mice. Modulation of the Akt/GSK3ß/Nrf2 cascade attributes to its hepatoprotective effects. Our findings support a possible application of SEPJ in the control of liver fibrosis.

Methylgerambullin derived from plant Glyccsmis pentaphylla (Retz) correa. Mediates anti-hepatocellular carcinoma cancer effect by activating mitochondrial and endoplasmic reticulum stress signaling and inhibiting AKT and STAT3 pathways.

Hepatocellular carcinoma (HCC) is one of the most common fatal malignant tumors. Glycosmis pentaphylla is used by traditional medical practitioners worldwide to treat various diseases. We isolated and identified a chemical component with potential anti-hepatocellular carcinoma (HCC) effects. Methylgerambullin is a sulfur containing amine and has significant antihepatoma activity in vitro and in vivo. Methylgerambullin was significantly cytotoxic to HCC cells and induces apoptosis in HCC cells. In addition, methylgerambullin is able to inhibit the growth of transplanted tumors in nude mice without significant toxicity. Regarding the anti-cancer mechanism of methylgerambullin, treatment with methylgerambullin increased the expression of caspase-3, caspase-9 and Bax in vitro and in vivo and reduce the expression of B-cell lymphoma-2 (Bcl-2). Simultaneously, methylgerambullin can also affect ERS-related proteins, inhibit Protein Kinase B (Akt) activity, cause dephosphorylation of downstream Bad, and inhibit the expression of the Signal Transducer and Activator of Transcription 3 (STAT3) protein to inhibit HCC cells proliferation. Overall, these results suggest that methylgerambullin can inhibit HCC cells proliferation by inducing mitochondrial apoptosis, activating ERS signaling pathways and inhibiting the Akt and STAT3 pathways.

Anti-esophageal Cancer Effect of Corilagin Extracted from Phmllanthi Fructus via the Mitochondrial and Endoplasmic Reticulum Stress Pathways.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Corilagin (ß-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose) is a tannin isolated from the traditional ethnopharmacological plant Phmllanthi Fructus, which is widely used in not only traditional Chinese medicine but also tropical and subtropical medicine to ameliorate various diseases. AIM OF THE STUDY: This study was designed to isolate the potential anti-esophageal cancer (EC) component corilagin from Phmllanthi Fructus and explain its anti-EC mechanism. MATERIALS AND METHODS: Corilagin was isolated from Phmllanthi Fructus by extraction and chromatographic procedures, and its anti-esophageal cancer effect was evaluated by in vitro and in vivo experiments. In vitro experiments included MTT analysis, flow cytometry, and the Transwell assay and were used to observe corilagin-mediated inhibition of EC cell growth. Western blotting was used to analyze the apoptotic pathway of EC cells. In vivo experiments used tumor-bearing nude mice to evaluate the antitumor effect of corilagin, and its potential mechanism was explored by Western blotting. RESULTS: Corilagin showed significant anti-EC activity in vitro and in vivo. Corilagin was significantly cytotoxic to EC cells and induced apoptosis in EC cells. Corilagin induced G0/G1 phase arrest by altering key G0/G1 cell cycle regulatory markers and significantly reducing the migration of EC cells and the number of cells in a time- and dose-dependent manner. Additionally, corilagin inhibited the growth of transplanted tumors in nude mice without significant toxicity. Regarding the anticancer mechanism of corilagin, the results showed that corilagin inhibited esophageal cancer progression by activating mitochondrial and endoplasmic reticulum stress signaling pathways. CONCLUSIONS: Corilagin shows significant anti-EC activity in vitro and in vivo. The mechanism of the anti-EC activity of corilagin may be due to activating mitochondrial and endoplasmic reticulum stress signaling pathways.

γ-Oryzanol alleviates acetaminophen-induced liver injury: roles of modulating AMPK/GSK3ß/Nrf2 and NF-κB signaling pathways.

Acetaminophen (APAP) overdose is a major cause of drug-induced liver injury worldwide. Our current study was performed to assess the potential protective effects of γ-oryzanol (ORY) on APAP-induced liver injury in mice and explore the underlying molecular mechanisms. We unveiled that ORY alleviated the APAP-induced death of HL-7702 hepatocytes in vitro and liver injury in mice. Moreover, ORY promoted the nuclear translocation of Nrf2, increased the expressions of Nrf2-downstream antioxidative enzymes, including HO-1, NQO1, GCLC, and GCLM, and thereby restrained APAP-induced oxidative stress in hepatocytes. Moreover, ORY modulated the AMPK/GSK3ß axis that acts upstream of Nrf2 in hepatocytes. Compound C, an inhibitor of AMPK, prevented the ORY-mediated activation of Nrf2 and protection against APAP toxicity in HL-7702 hepatocytes. Additionally, in the liver of mice receiving APAP, ORY suppressed the nuclear translocation of the NF-κB p65 subunit, downregulated the expressions of iNOS and COX-2, and reduced the levels of pro-inflammatory factors including TNF-α, IL-1ß, IL-6, and NO. Taken together, our findings revealed that ORY is capable of ameliorating APAP-induced liver injury. The modulation of AMPK/GSK3ß/Nrf2 and NF-κB signaling pathways is implicated in the hepatoprotective activity of ORY.

Chlorogenic acid methyl ester exerts strong anti-inflammatory effects via inhibiting the COX-2/NLRP3/NF-κB pathway.

The objective of this study was to investigate the anti-inflammatory effect of chlorogenic acid methyl ester (CME) and the molecular mechanism involved, through using non-infectious inflammation and infectious inflammation animal models as well as lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cell models. Our results demonstrated that CME markedly inhibited ear swelling, paw swelling and granuloma swelling, and decreased intraperitoneal capillary permeability in non-infectious inflammation. Moreover, CME significantly alleviated the pathological damage of the lung tissue, reduced the levels of PGE2 and IL-1ß in the serum and the protein expression levels of related-inflammatory factors in the lung tissue of LPS-induced mice with acute lung injury (ALI). In addition, CME affected the RAW264.7 cell cycle and inhibited the protein expressions of COX-2 and NLRP3 and prevented the phosphorylation of NF-κB p65 in RAW264.7 cells treated with LPS. These observations not only validated the anti-inflammatory effects of CME, but also revealed the underlying molecular basis, which involves the down-regulation of the expression of inflammatory factors and blockade of the COX-2/NLRP3/NF-κB signaling pathway.

Dauricine upregulates the chemosensitivity of hepatocellular carcinoma cells: Role of repressing glycolysis via miR-199a:HK2/PKM2 modulation.

Dauricine (Dau) is a natural alkaloid exhibiting anti-proliferative activity against several different types of malignant cell. However, effects of Dau on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. In this study, we found that Dau elevated the sensitivities of HCC cells to chemotherapeutic reagents, including cisplatin, sorafenib, and isoliensinine. Moreover, Dau promoted apoptosis of HCC cells triggered by these chemotherapeutic reagents. Consistently, in a xenograft mouse model, Dau sensitized HCC cells to sorafenib. In HCC cells, Dau dose-dependently inhibited glucose glycolysis and increased oxidative phosphorylation. Mechanistically, Dau downregulated the expression of hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2). HK2 and PKM2 can be directly targeted by miR-199a. Dau dose-dependently increased miR-199a expression in HCC cells. Transfection of anti-miR-199a abrogated Dau-mediated suppression of HK2 and PKM2. Dau-induced metabolic shift was thereby severely crippled by anti-miR-199a. In addition, the incremental activity of Dau on sorafenib sensitivity of HCC cells was diminished in response to the transfection of anti-miR-199a. Taken together, our findings provided novel insights into the impact of Dau on HCC cells and supported considering Dau as an adjuvant reagent in the clinical treatment of HCC.

Antitumor immunostimulatory activity of polysaccharides from Panax japonicus C. A. Mey: Roles of their effects on CD4+ T cells and tumor associated macrophages.

In this study, chemical properties of polysaccharides from rhizomes of Panax japonicus C. A. Mey (PSPJ) were investigated and the antitumor immunostimulatory activity of PSPJ was assessed in mice bearing H22 hepatoma cells. Chemical properties of PSPJ were determined by GC, FT-IR, 1H NMR and 13C NMR analysis. Furthermore, we showed that PSPJ repressed H22 tumor growth in vivo with undetectable toxic effects on tumor-bearing mice. PSPJ upregulated host thymus/spleen indexes and ConA/LPS-induced splenocyte proliferation. Cytotoxic activities of natural killer and CD8+ T cells against H22 hepatoma cells were also elevated. Tumor transplantation led to substantial apoptosis of CD4+ T cells and dysregulation of the cytokine profile secreted by CD4+ T cells. These abnormalities were alleviated by PSPJ in a dose-dependent manner. In tumor-associated macrophages (TAMs), PSPJ reduced the production of immunosuppressive factors such as TGF-ß, IL-10 and PEG2. In addition, M2-like polarization of TAMs was also considerably declined in response to PSPJ. Our findings clearly demonstrated the antitumor immunostimulatory activity of PSPJ and supported considering PSPJ as an adjuvant reagent in clinical treatment of malignant diseases.

Activity of Isoliensinine in Improving the Symptoms of Type 2 Diabetic Mice via Activation of AMP-Activated Kinase and Regulation of PPARγ.

This study was designed to explore the effects and mechanism of isoliensinine (isolie) from embryos of Nelumbo nucifera on type 2 diabetes and dyslipidemia in vivo and in vitro. The in vitro study showed that isolie increased the GLUT4 translocation by 2.5-fold in L6 cells. Furthermore, after 4 weeks of treatment, the in vivo biochemical study indexes revealed that isolie had a positive effect on decreasing serum insulin level (42.2 ± 5.10 vs 55.7 ± 6.33 mU/L, P < 0.05) and reducing fast blood glucose (9.4 ± 1.5 vs 18.7 ± 2.3 mmol/L, P < 0.001) and body weight (37.8 ± 2.9 vs 46.9 ± 5.4 g, P < 0.05) compared with the KK-Ay model mice. Isolie treatment led to significant increases in GLUT4 proteins (∼2.7-fold in skeletal muscle and ∼2.4-fold in WAT) and phosphorylated AMP-activated protein kinase (∼1.4-fold in skeletal muscle, ∼3.1-fold in WAT, and ∼2.3-fold in liver). However, isolie caused a significant decrease in lipogenesis protein expressions of PPARγ and SREBP-1c, and decreased the activity of ACC by increasing the phospho-ACC level. Our findings showed that isolie has the potential to alleviate type 2 diabetes associated with hyperlipidemia in KK-Ay mice. Regulation of GLUT4, SREBP-1c, PPARγ, AMPK phosphorylation, and ACC phosphorylation is implicated in the antidiabetic effects of isolie.

Astragalin Reduces Hexokinase 2 through Increasing miR-125b to Inhibit the Proliferation of Hepatocellular Carcinoma Cells in Vitro and in Vivo.

Astragalin (ASG) can be found in a variety of food components. ASG exhibits cytotoxic effects on several different types of malignant cells. However, its effects on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. Here, we revealed that ASG remarkably suppressed the proliferation of HCC cells. In HCC cells, ASG inhibited glucose glycolysis and promoted oxidative phosphorylation, resulting in a surge of reactive oxygen species (ROS). Mechanistically, ASG suppressed the expression of hexokinase 2 (HK2). This event was indispensible for ASG-mediated metabolic reprogramming, ROS accumulation, and subsequent growth arrest. Our further investigations unveiled that ASG repressed HK2 expression via increasing miR-125b. In vivo experiments showed that gavage of ASG decreased the proliferation of Huh-7 HCC xenografts in nude mice and inhibited the growth of transplanted H22 HCC cells in Kunming mice. Declined HCC tumor growth in vivo was associated with boosted miR-125b and reduced expression of HK2 in tumor tissues. Collectively, our results demonstrated that ASG is able to suppress the proliferation of HCC cells both in vitro and in vivo. Inhibition of HK2 through upregulating miR-125b and subsequent metabolic reprogramming is implicated in the antiproliferative effects of ASG on HCC cells.

Isoliensinine induces dephosphorylation of NF-kB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction.

Our previous study discovered that isoliensinine (isolie) triggers hepatocellular carcinoma (HCC) cell apoptosis via inducing p65 dephosphorylation at Ser536 and inhibition of NF-κB. Here, we showed that isolie promoted p65/PP2A interaction in vitro and in vivo. Repression of PP2A activity or knockdown of the expression of PP2A-C (the catalytic subunit of PP2A) abrogated isolie-provoked p65 dephosphorylation. I2PP2A is an endogenous PP2A inhibitor. Isolie directly impaired PP2A/I2PP2A interaction. Knockdown of I2PP2A boosted p65/PP2A association and p65 dephosphorylation. Overexpression of I2PP2A restrained isolie-induced p65 dephosphorylation. Untransformed hepatocytes were insensitive to isolie-induced NF-κB inhibition and cell apoptosis. In these cells, basal levels of I2PP2A and p65 phosphorylation at Ser536 were lower than in HCC cells. These findings collectively indicated that isolie suppresses NF-κB in HCC cells through impairing PP2A/I2PP2A interaction and stimulating PP2A-dependent p65 dephosphorylation at Ser536.

Amplexicaule A exerts anti-tumor effects by inducing apoptosis in human breast cancer.

Chemotherapy is the main treatment for patients with breast cancer metastases, but natural alternatives have been receiving attention for their potential as novel anti-tumor reagents. Amplexicaule A (APA) is a flavonoid glucoside isolated from rhizomes of Polygonum amplexicaule D. Don var. sinense Forb (PADF). We found that APA has anti-tumor effects in a breast cancer xenograft mouse model and induces apoptosis in breast cancer cell lines. APA increased levels of cleaved caspase-3,-8,-9 and PARP, which resulted from suppression of MCL-1 and BCL-2 expression in the cells. APA also inactivated the Akt/mTOR pathway in breast cancer cells. Thus, APA exerts a strong anti-tumor effect on breast cancer cells, most likely through induction of apoptosis. Our study is the first to identify this novel anti-tumor compound and provides a new strategy for isolation and separation of single compounds from herbs.

Protective effects of protostemonine on LPS/GalN-induced acute liver failure: Roles of increased hepatic expression of heme oxygenase-1.

Here, we explored protective effects of protostemonine (PSN), on mouse acute liver failure induced by lipopolysaccharide/d-galactosamine (LPS/GalN). PSN dose-dependently declined LPS/GalN-induced lethality of mice as well as increase of ALT/AST activities in their serum. Hepatoprotective effects of PSN were also supported by liver histopathological examinations. After LPS/GalN treatment, severe oxidative stresses in the liver could be detected by boosted MDA and ROS as well as decreased GSH. Moreover, hepatic expression of pro-inflammatory cytokines, including TNF-α, IL-1ß and IL-6, were sharply elevated. These symptoms were dose-dependently ameliorated by PSN. Mechanistically, PSN promoted the transcription and translation of heme oxygenase-1 (HO-1) in hepatocytes and liver Kupffer cells. Nrf2 is a master transcription factor contributing to the expression of HO-1. PSN elevated Nrf2 nuclear accumulation and enhanced Nrf2/HO-1 promoter interaction. Suppressing enzyme activity of HO-1 by co-treating mice with HO-1 inhibitor ZnPP abolished protective effects of PSN. ZnPP also abrogated alleviative impacts of PSN on LPS/GalN-mediated hepatic oxidative stresses and inflammatory responses. Finally, we showed that PSN exhibited undetectable toxic effects on vital organs of mice. Our findings suggested that PSN is able to attenuate LPS/GalN-induced acute liver failure and upregulating HO-1 expression is implicated in its hepatoprotective activity.

Isoliensinine, a Bioactive Alkaloid Derived from Embryos of Nelumbo nucifera, Induces Hepatocellular Carcinoma Cell Apoptosis through Suppression of NF-κB Signaling.

Isoliensinine (isolie) is an alkaloid produced by the edible plant Nelumbo nucifera. Here, we unveiled that isolie was able to provoke HepG2, Huh-7, and H22 hepatocellular carcinoma (HCC) cell apoptosis. Isolie decreased NF-κB activity and constitutive phosphorylation of NF-κB p65 subunit at Ser536 in HCC cells. Overexpression of p65 Ser536 phosphorylation mimics abrogated isolie-mediated HCC cell apoptosis. Furthermore, intraperitoneal injection of isolie inhibited the growth of Huh-7 xenografts in nude mice. Additionally, isolie given by both intraperitoneal injection and gavage diminished the proliferation of transplanted H22 cells in Kunming mice. Reduced tumor growth in vivo was associated with inhibited p65 phosphorylation at Ser536 and declined NF-κB activity in tumor tissues. Finally, we revealed that isolie was bioavailable in the blood of mice and exhibited no detectable toxic effects on tumor-bearing mice. Our data provided strong evidence for the anti-HCC effect of isolie.