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.

Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance.

Insulin resistance, a hallmark of type 2 diabetes, is a defect of insulin in stimulating insulin receptor signalling, which has become one of the most serious public health threats. Upon stimulation by insulin, insulin receptor recruits and phosphorylates insulin receptor substrate proteins, leading to activation of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. Activated Akt phosphorylates downstream kinases and transcription factors, thus mediating most of the metabolic actions of insulin. Beta-arrestins mediate biological functions of G-protein-coupled receptors by linking activated receptors with distinct sets of accessory and effecter proteins, thereby determining the specificity, efficiency and capacity of signals. Here we show that in diabetic mouse models, beta-arrestin-2 is severely downregulated. Knockdown of beta-arrestin-2 exacerbates insulin resistance, whereas administration of beta-arrestin-2 restores insulin sensitivity in mice. Further investigation reveals that insulin stimulates the formation of a new beta-arrestin-2 signal complex, in which beta-arrestin-2 scaffolds Akt and Src to insulin receptor. Loss or dysfunction of beta-arrestin-2 results in deficiency of this signal complex and disturbance of insulin signalling in vivo, thereby contributing to the development of insulin resistance and progression of type 2 diabetes. Our findings provide new insight into the molecular pathogenesis of insulin resistance, and implicate new preventive and therapeutic strategies against insulin resistance and type 2 diabetes.

Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling.

Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis.

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.

Water extract of earthworms mitigates kidney injury triggered by oxidative stress via activating intrarenal Sirt1/Nrf2 cascade and ameliorating mitochondrial damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemia-reperfusion (IR) injury can result in acute renal failure. Oxidative stress is a major factor in IR-induced cell death in the kidneys. According to traditional Chinese medicine, earthworms (Pheretima aspergillum) can be used to treat various kidney diseases. AIM OF THE STUDY: The present study was designed to understand the protective effects of the water extract of earthworms (WEE) against oxidative stress on the kidneys and the crucial molecular events associated with its nephroprotective activity. MATERIALS AND METHODS: Cytotoxicity caused by H2O2 in HEK293, HK2, and primary mouse renal tubular epithelial cells (TECs) was used to investigate the effect of WEE on oxidative stress-induced renal injury in vitro. IR-induced kidney injury was established using rats as an in vivo model. The WEE-mediated protection of the kidneys against oxidative stress was compared with that of glutathione, a common antioxidant used as a positive control. RESULTS: In HEK293 cells, HK2 cells, and primary mouse TECs, WEE relieved H2O2-induced mitochondrial damage, apoptosis, and ferroptosis. In kidney cells, WEE increased the expression of Sirt1, boosted LKB1 and AMPK phosphorylation, and upregulated nuclear Nrf2. Suppression of Sirt1 and LKB1 knock down abrogated WEE-induced protection against H2O2. WEE ameliorated IR-induced kidney injury and intrarenal inflammation in rats. In rat kidneys, WEE mitigated mitochondrial damage and suppressed IR-induced apoptosis and ferroptosis. Mechanistically, WEE increased Sirt1 expression, enhanced the phosphorylation of LKB1 and AMPK, and increased intranuclear Nrf2 levels in IR kidneys. IR treatment resulted in considerable increase in renal MDA levels and a prominent decrease in antioxidative enzyme activity. These lesions were significantly alleviated by WEE. CONCLUSIONS: WEE mitigated H2O2-induced cytotoxicity in kidney cells in vitro and improved IR-induced kidney damage in rats. Mechanistically, WEE potentiated the Sirt1/Nrf2 axis and relieved mitochondrial damage in the kidney cells. These events inhibited the apoptosis and ferroptosis induced by oxidative stress. Our findings support the potential application of WEE for the clinical treatment of kidney diseases caused by intrarenal oxidative stress.

Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4+ T cells.

Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearing mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4+ T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8+ T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4+ T cells but not in CD8+ T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4+ T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4+ T cells is implicated in its immunomodulatory activity.

Phaseoloideside E, a novel natural triterpenoid saponin identified from Entada phaseoloides, induces apoptosis in Ec-109 esophageal cancer cells through reactive oxygen species generation.

Phaseoloideside E (PE), a new oleanane-type triterpene saponin, was isolated from the seed kernels of Entada phaseoloides (Linn.) Merr. PE had strong cytotoxic activity against an array of malignant cells. Typical morphological and biochemical features of apoptosis were observed in PE-treated Ec-109 cells. PE induced a dose-dependent increase in the sub-G1 fraction of the cell cycle and DNA fragmentation. Decreases in the mitochondrial membrane potential, SOD activity, and GSH content were also observed. Further investigations revealed that PE reduced the ratio of Bcl-2 to Bax and increased the activities of caspase-3 and -9, but this was prevented by Z-VAD-fmk. PE also induced a decrease of the sub-G1 fraction. Furthermore, PE-induced apoptosis was mediated by up-regulating cellular ROS, which was suppressed by cotreating the cells with N-acetylcysteine (NAC). NAC also attenuated the ratio of sub-G1, the generation of DNA fragmentation and the expression of Bcl-2, Bax, caspase-3, and caspase-9. Interestingly, PE did not up-regulate ROS or induce cell death in untransformed cells. These data showed that PE induces cell death through up-regulation of cellular ROS production. Our investigation provides the scientific basis for the traditional application of this herb and suggests the possibility that PE may be used for a treatment of esophageal carcinoma. [Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.12193FP].

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.

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.

Antimicrobial constituents from the tubers of Bletilla ochracea.

A new 2-(2-methylpropyl)butanedioic acid derivative, bletillin A ( 1), and a new bibenzyl, bletillin B ( 2), together with seventeen known compounds ( 3- 19), were isolated from the tubers of Bletilla ochracea Schltr. Their structures were established by detailed spectroscopic analyses. Phenanthrenes ( 12- 14) exhibited antibacterial activities against gram-positive bacteria Staphylococcus aureus, S. epidermidis, and Bacillus subtilis, with MIC values of 12.5-25 µg/mL.

[Preliminary study on mechanisms of total saponins from Entada phaseoloides against diabetes].

OBJECTIVE: To study the effect of total saponins from Entada phaseoloides (TSEP) on islet morphology and skeletal muscle PI3K pathway-related protein expression of type 2 diabetic rats. METHOD: Type 2 diabetic rats were induced by high-fat diet and low-dose streptozotocin and then randomly divided into 5 groups, i.e. the normal control, the model group, the positive control drug (200 mg x kg(-1) metformin), the low-dose TSEP (25 mg x kg(-1)) group and the high-dose TSEP (50 mg x kg(-1)). Three weeks later, the islet morphology of rat pancreas were observed by HE staining, and protein expressions of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), protein tyrosine phosphatase-1B (PTP-1 B) and glucose transporter 4 (GLUT4) in rat skeletal muscle were detected by Western blot. RESULT: Compared with the modal group, TSEP administration groups showed relatively normal structures, clear pancreatic cells and intact capsula structures in pancreatic tissue pathological sections, with the number of pancreatic islets close to the normal control group. Meanwhile, above TSEP administration groups showed increased IRS-1, PI3K and GLUT4 protein expressions in their skeletal muscle tissues and decreased PTP-1B protein expression compared with the model group. CONCLUSION: TSEP has an effect on protecting pancreatic tissues of type 2 diabetic rats and intervening in abnormal expression of proteins in skeletal muscle tissues.

[Phaseoloideside E induces human hepatoma HepG2 cells apoptosis].

OBJECTIVE: To study in vitro anti-tumor activity of phaseoloideside E (PE) with human hepatoma HepG2 cells as the objective. METHOD: MTT assay was adopted to detect the cytotoxic effect of PE of different concentrations on HepG2 cells after being processed for 48 h. Changes in morphology of PE-processed cells were observed under an optical microscope and fluorescence microscope. DNA agrose gel electrophoresis was used to detect the DNA ladder, an important characteristic of cell apoptosis. The expression levels of Bax and Bcl-2 were determined by western blot assay. RESULT: PE dramatically repressed the viability of HepG2 cells. Typical morphological changes of apoptosis had been detected by both direct microscopic observation and Hoechst 33,258 staining. Typical DNA Ladder was also observed by agarose gel electrophoresis in the administration group, but it did not exist in the control group. Western blot showed that the expression of Bax was up-regulated and Bcl-2 was down-regulated. CONCLUSION: Above data demonstrates that PE can induce apoptosis in human hepatoma HepG2 cells, and indicate that PE-induced expression level changes of Bax and Bcl2 may be related to the apoptosis-induction effect.

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.

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.

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.

Signal transducer and activator of transcription 3 (STAT3) regulates adipocyte differentiation via peroxisome-proliferator-activated receptor gamma (PPARgamma).

BACKGROUND INFORMATION: STAT3 (signal transducer and activator of transcription 3) is an important transcription factor involved in many biological events, including apoptosis, tumorigenesis, angiogenesis and epithelial-to-mesenchymal transition. However, no direct evidence for a role of STAT3 in 3T3-L1 adipocyte differentiation has been reported. RESULTS: In the present study, we found that rapid activation of STAT3, lasting for more than 48 h, was elicited upon induction of adipogenesis. Both the STAT3-selective inhibitor stattic and the JAK2 (Janus kinase 2)/STAT3-selective inhibitors AG490 and Gö6976 inhibited STAT3 activation, leading to the suppression of adipocyte differentiation. Adipocyte differentiation was also suppressed by STAT3 siRNA (small interfering RNA) or dominant-negative STAT3. Interestingly, the PPARgamma (peroxisome-proliferator-activated receptor gamma) agonist TAZ (troglitazone) abolished the STAT3-inhibitor- and RNAi (RNA interference)-mediated suppression of adipogenesis. However, TAZ treatment had no effect on the stattic- and AG490-mediated down-regulation of STAT3 activation, suggesting that STAT3 regulates adipocyte differentiation through signalling that occurs upstream of PPARgamma. CONCLUSION: These data indicate that STAT3 functions as a critical factor for adipogenesis via a mechanism involving the PPARgamma activation pathway.

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.