The Regulatory Mechanism of Smilax China L. Saponins against Nonalcoholic Fatty Liver Is Revealed by Metabolomics and Transcriptomics.

This study was to investigate the effects of Smilax China L. saponins (SCS) on non-alcoholic fatty liver disease (NAFLD). Rats were fed a high-fat diet (HFD) for 8 weeks to induce NAFLD, followed by SCS treatment for 8 weeks. The effect of SCS on liver injury was observed by H&E staining and the regulative mechanism of SCS on lipid formation was exposed by detecting Oil red O, insulin resistance (IR), and fatty acids synthesis (FAS). Furthermore, transcriptomics and metabolomics were performed to analyze the potential targets. The experimental results indicated that SCS exerted a positive curative effect in alleviating HFD-induced overweight, hepatic injury, steatosis, and lipid formation and accumulation in rats, and the preliminary mechanism studies showed that SCS could alleviate IR, inhibit FAS expression, and reduce Acetyl-CoA levels. Besides, the integrative analysis of transcriptomics and metabolomics exposed the targets of SCS to regulate lipid production likely being the sphingolipid metabolism and glycerophospholipid metabolism pathways. This study demonstrates that SCS significantly ameliorates lipid metabolic disturbance in rats with NAFLD by relieving insulin resistance, inhibiting the FAS enzymes, and regulating the sphingolipid and glycerophospholipid metabolism pathways.

[Retracted] Inhibition of RKIP aggravates thioacetamide­induced acute liver failure in mice.

[This retracts the article DOI: 10.3892/etm.2018.6542.].

Integrative Analysis of Transcriptome and Metabolome to Illuminate the Protective Effects of Didymin against Acute Hepatic Injury.

Based on the multiomics analysis, this study is aimed at investigating the underlying mechanism of didymin against acute liver injury (ALI). The mice were administrated with didymin for 2 weeks, followed by injection with lipopolysaccharide (LPS) plus D-galactosamine (D-Gal) to induce ALI. The pathological examination revealed that didymin significantly ameliorated LPS/D-Gal-induced hepatic damage. Also, it markedly reduced proinflammatory cytokines release by inhibiting the TLR4/NF-κB pathway activation, alleviating inflammatory injury. A transcriptome analysis proved 2680 differently expressed genes (DEGs) between the model and didymin groups and suggested that the PI3K/Akt and metabolic pathways might be the most relevant targets. Meanwhile, the metabolome analysis revealed 67 differently expressed metabolites (DEMs) between the didymin and model groups that were mainly clustered into the glycerophospholipid metabolism, which was consistent with the transcriptome study. Importantly, a comprehensive analysis of both the omics indicated a strong correlation between the DEGs and DEMs, and an in-depth study demonstrated that didymin alleviated metabolic disorder and hepatocyte injury likely by inhibiting the glycerophospholipid metabolism pathway through the regulation of PLA2G4B, LPCAT3, and CEPT1 expression. In conclusion, this study demonstrates that didymin can ameliorate LPS/D-Gal-induced ALI by inhibiting the glycerophospholipid metabolism and PI3K/Akt and TLR4/NF-κB pathways.

Didymin Ameliorates Liver Fibrosis by Alleviating Endoplasmic Reticulum Stress and Glycerophospholipid Metabolism: Based on Transcriptomics and Metabolomics.

Introduction: Origanum vulgare L. is a traditional Chinese herb, having a strong hepatoprotective effect. In our previous experiments, we have isolated an ingredient from this herb and identified it as didymin. This study aimed to investigate the effects and underlying mechanisms of didymin on liver injury and fibrosis, elucidating whether it was the pharmacodynamic material basis of Origanum vulgare L. Methods: Mice were injected with CCl4 for 10 weeks to induce liver fibrosis, followed by didymin treatment for 6 weeks. Then, biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of didymin in alleviating fibrosis. Next, the possible mechanisms of didymin were predicted by transcriptomics and then verified by the multiple relevant examinations. Results: The pharmacodynamic experiments indicated that didymin significantly attenuated CCl4-induced hepatic injury and fibrogenesis, as evidenced by the ameliorative pathological tissue, low transaminase activity, and decreased collagen accumulation. Interestingly, the transcriptome analysis predicted that the potential targets were likely to be endoplasmic reticulum stress (ERS), inflammation, apoptosis, and metabolic pathways. And the predictions were then verified by the following examinations: (1) didymin significantly inhibited ERS by regulating the ATF6, IRE1α, and PERK pathways; (2) didymin markedly alleviated hepatocyte apoptosis by restoring the expression of Bcl-2 and caspase families, as well as the mitochondrial dysfunction; (3) didymin significantly decreased the production of the pro-inflammatory cytokines (IL-1ß and IL-6); (4) didymin inhibited the glycerophospholipid metabolism pathway by decreasing the synthesis of phosphatidylethanolamines and phosphatidylcholines. Conclusion: Our findings demonstrate that didymin can ameliorate liver fibrosis, which is mainly attributed to the inhibition of ERS, inflammation, and glycerophospholipid metabolism.

Tormentic Acid Ameliorates Hepatic Fibrosis in vivo by Inhibiting Glycerophospholipids Metabolism and PI3K/Akt/mTOR and NF-κB Pathways: Based on Transcriptomics and Metabolomics.

This study aimed to investigate the effects and underlying mechanisms of tormentic acid (TA) on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. The rats were intragastrically administered with 50% CCl4 for 9 weeks to induce hepatic fibrosis, followed by various agents for 6 weeks. Transcriptomic analysis was carried out to predict the potential targets, and then multiple examinations were performed to verify the prediction. The results showed that TA significantly alleviated liver injury and fibrosis, as evidenced by the ameliorative pathological tissue, low transaminase activity, and decreased collagen accumulation. Besides, TA markedly reduced hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. The transcriptomic analysis revealed 2,173 differentially expressed genes (DEGs) between the TA and model groups, which could be enriched in the metabolic pathways and the PI3K/Akt and NF-κB signaling pathways. The metabolomics analysis showed that TA could regulate the glycerophospholipid metabolism pathway by regulating the synthesis of phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines. Moreover, the integrative analysis of the transcriptomics and metabolomics data indicated that TA inhibited the glycerophospholipid metabolism pathway by inhibiting the expression of LPCAT4, PTDSS2, PLA2G2A and CEPT1. In addition, the relevant signaling pathways analysis confirmed that TA inhibited HSCs activation by blocking the PI3K/Akt/mTOR pathway and ameliorated inflammatory injury by inhibiting the NF-κB pathway. In conclusion, TA significantly alleviates liver fibrosis in vivo by inhibiting the glycerophospholipid metabolism pathway and the PI3K/Akt/mTOR and NF-κB signaling pathways.

Comprehensive analysis of transcriptomics and metabolomics to illustrate the underlying mechanism of helenalin against hepatic fibrosis.

This study aimed to investigate the protective mechanisms of helenalin on hepatic fibrosis. In brief, rats were intragastrically administrated with 50% CCl4 for 9 weeks to induce liver fibrosis, followed by treatment with various agents for 6 weeks. The effects of helenalin on hepatic injury were assessed by pathological examinations. The potential targets were predicted by the "Drug-Disease" bioinformatic analysis and then verified by multiple experiments. Moreover, the underlying mechanism was investigated by transcriptomics and metabolomics as a whole. The results showed that helenalin significantly alleviated hepatocyte necrosis and hepatic injury, as proved by the pathological examinations. Also, helenalin markedly attenuated hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. Besides, helenalin could significantly reduce collagen accumulation, as evidenced by the decreased contents of collagen, hyaluronic acid and laminin. Moreover, helenalin significantly down-regulated the phosphorylation of PI3K, Akt, FAK, mTOR and P70S6K, and PTEN protein expression, suggesting that helenalin inhibited the PI3K/Akt signaling cascade. Meanwhile, helenalin inhibited the NF-κB signaling pathway by reducing the phosphorylation of IκBα, NF-κB p65 and IKKα/ß, alleviating inflammation response. Interestingly, the analysis of transcriptomics and metabolomics indicated that helenalin inhibited the glycerophospholipid metabolism pathway by down-regulating the target genes (CHKA, ETNPPL, LYPLA1, PCYT2, PLD4 and PNPLA6), ultimately ameliorating hepatocyte damage. In conclusion, helenalin ameliorates hepatic fibrosis by regulating the PI3K/Akt and NF-κB signaling pathways and the glycerophospholipid metabolism pathway.

[Tormentic acid inhibits proliferation and promotes apoptosis of LX-2 human hepatic stellate cells via blocking NF-κB signaling pathway].

Objective To investigate the regulating function of tormentic acid (TA) on the NF-κB signaling pathway and analyze its effect on proliferation and apoptosis of LX-2 human hepatic stellate cells and its mechanism. Methods The cultured LX-2 cells were randomized into normal control group, platelet-derived growth factor BB (PDGF-BB) stimulated group (20 ng/mL), PDTC group (20 ng/mL of PDGF-BB combined with 10 mmol/L of PDTC), and TA treated groups (20 ng/mL of PDGF-BB combined with 35, 25, 15 µmol/L of TA). The effects of TA on cell proliferation were detected by MTT assay. The activities of ASL, ALT, and TBIL were detected by using commercially available kits. Cell apoptosis was determined by flow cytometry. The mRNA of NF-κB p65 was detected by real-time quantitative PCR; and the protein expressions of NF-κB p65, phosphorylated NF-κB p65(p-NF-κB p65), IκBα, α-SMA, TGF-ß, Bcl2, and Bax were detected by Western blot. Results Compared with PDGF-BB, TA significantly inhibited the activation and proliferation of LX-2 cells, and decreased the protein expressions of α-SMA and TGF-ß. TA treatment reduced the levels of ASL, ALT, and TBIL in the cells and the cellular damage. TA significantly induced LX-2 cells apoptosis by down-regulating the expression of the anti-apoptotic protein Bcl2 and up-regulating the expression of the pro-apoptotic protein Bax. In addition, TA markedly inhibited the expressions of NF-κB p65 mRNA and protein, and the phosphorylation of NF-κB p65 and IκBα proteins. Conclusion TA inhibits proliferation and promotes apoptosis of LX-2 cells by blocking the NF-κB signaling pathway.

Prediction and verification of target of helenalin against hepatic stellate cell activation based on miR-200a-mediated PI3K/Akt and NF-κB pathways.

Hepatic stellate cell (HSC) activation is a crucial event in the progress of liver fibrosis. In this study, the target of helenalin was firstly predicted by bioinformatics analysis, and then the prediction was verified by various experiments. HSC-T6 cells were activated by interleukin-1 beta (IL-1ß) and then treated with helenalin. Moreover, HSC-T6 cells were transfected with miR-200a mimic or inhibitor, and the effect of helenalin on the miR-200a-mediated PI3K/Akt and NF-κB signaling pathways was investigated. The bioinformatics analysis indicated that miR-200a might regulate the PI3K/Akt pathway, NF-κB activation, Bcl-2 family and Caspases, ultimately affecting cell survival and apoptosis. Interestingly, the molecular docking demonstrated that the target of helenalin might be miR-200a-mediated the PI3K/Akt and NF-κB pathways. Moreover, the experiments showed that helenalin administration led to the inactivation of HSC-T6 cells, as evidenced by the inhibition of cell proliferation, α-SMA expression and collagen production. The mechanism studies showed that helenalin reduced collagen accumulation by restoring the balance of MMPs/TIMPs. Moreover, helenalin markedly suppressed HSC activation by inhibiting the PI3K/Akt pathway and alleviated inflammatory response by blocking the NF-κB signal transduction. Further study indicated that helenalin up-regulated miR-200a expression, thus leading to the inhibition of the PI3K/Akt and NF-κB signaling pathways. In conclusion, helenalin inhibits HSC activation via inhibiting the miR-200a-mediated PI3K/Akt and NF-κB pathways, and it may be developed as a potential medicine for the treatment of liver fibrosis.

Tormentic acid inhibits hepatic stellate cells activation via blocking PI3K/Akt/mTOR and NF-κB signalling pathways.

The present study was to investigate the inhibitory effect and underlying mechanism of Tormentic acid (TA) on hepatic stellate cells (HSCs). HSC-T6 cells were stimulated with Platelet-derived growth factor-BB (PDGF-BB) and TA, and then cell proliferation, apoptosis, inflammatory factor, and collagen-related indicators were detected. In order to elucidate the potential mechanism, the PI3K/Akt/mTOR and NF-κB signalling pathways were also detected. The results showed that TA treatment markedly inhibited PDGF-BB-stimulated HSC-T6 cell activation, as evidenced by the inhibition of cell proliferation, migration and colony formation, as well as the decreased expression of TGF-ß and α-SMA. TA treatment caused a significant increase in the activity of lactate dehydrogenase and significantly promoted cell apoptosis. TA treatment significantly reduced aspartate aminotransferase, alanine aminotransferase and total bilirubin activity. Importantly, TA inhibited the expression of collagen type I and III, alleviating the excessive deposition of extracellular matrix (ECM). Further experiments showed that TA administration significantly inhibited the phosphorylation of PI3K, Akt, FAK and mTOR and the protein expression of P70S6K, indicating the inhibition of the PI3K/Akt/mTOR pathway. Moreover, treatment with TA markedly decreased the phosphorylation of IκBα, NF-κB p65 and IKKα/ß, thereby blocking the NF-κB signal transduction. In summary, this study demonstrates that TA significantly inhibits HSC activation and promotes cell apoptosis via the inhibition of the PI3K/Akt/mTOR and NF-κB signalling pathways. SIGNIFICANCE OF THE STUDY: Tormentic acid (TA) could inhibit HSC activation and alleviate collagen-based ECM deposition, suggesting that TA exerted anti-hepatic fibrosis. Further mechanism research revealed that the inhibition of TA on HSC activation might be through blocking the PI3K/Akt/mTOR and NF-κB signalling pathways. These findings provided a new cue to understand the protective effect of TA against liver fibrosis, which may provide a potential nature medicine for the treatment of liver fibrosis.

Didymin ameliorates dexamethasone-induced non-alcoholic fatty liver disease by inhibiting TLR4/NF-κB and PI3K/Akt pathways in C57BL/6J mice.

The present study aimed to investigate the protective effects and mechanisms of Didymin from Mentha spicata on non-alcoholic fatty liver disease (NAFLD) induced by dexamethasone and high-fat diet (DEX/HFD) in C57BL/6J mice. Briefly, mice were acclimated for 5 days and then subjected to DEX/HFD from days 5 to 28; meanwhile, the animals were treated with Didymin or Silibinin from days 12 to 28. Key indicators of NAFLD were then detected, including the pathological changes of liver tissues, serum biochemical indicators, inflammation, oxidative stress, apoptosis and lipid metabolism. Besides, the expressions of pivotal genes and proteins of the TLR4/NF-κB and PI3K/Akt pathways were examined to further elucidate the mechanisms of Didymin. The results demonstrated that Didymin significantly extenuated hepatocyte damage and lipid disorder. Moreover, Didymin markedly decreased hepatocyte apoptosis by regulating the expressions of B-cell lymphoma-2 (Bcl-2) family and the expressions of the caspase family. Further study elucidated that Didymin decreased the expressions of toll-like receptor 4 (TLR4), as well as the phosphorylation of inhibitor of nuclear factor kappa-B (IκB) and nuclear factor kappa-B p65 (NF-κB p65), suggesting the inhibition of Didymin on the TLR4/NF-κB pathway. Similarly, the PI3K/Akt pathway was also inhibited by Didymin, as evidenced by the decrease in the phosphorylation levels of PI3K and Akt. In summary, this study indicates that Didymin mitigates NAFLD by alleviating lipidosis and suppressing the TLR4/NF-κB and PI3K/Akt pathways, which may be a potential natural medicine for the treatment of NAFLD.

Helenalin from Centipeda minima ameliorates acute hepatic injury by protecting mitochondria function, activating Nrf2 pathway and inhibiting NF-κB activation.

Acute liver injury is a life-threatening syndrome that often caused by hepatocyte damage and is characterized by inflammatory and oxidative responses. Helenalin isolated from Centipeda minima (HCM) has been found to have anti-inflammatory and anti-oxidative effects. Here, this study aimed to investigate the effects and underlying mechanisms of HCM on Lipopolysaccharide/D-Galactosamine (LPS/D-GalN)-induced acute liver injury. Mice were intragastrically administered with various dose of HCM for 10 days; 2 h after the final treatment, the mice were injected with 50 µg/kg LPS and 800 mg/kg D-GalN. The histopathological changes, hepatocyte apoptosis, serum cytokines, oxidative stress and inflammatory cytokines were assessed. The results showed that HCM significantly ameliorated the hepatic injury, as evidenced by the attenuation of histopathological changes and the decrease in serum aminotransferase and total bilirubin activities. HCM markedly decreased hepatocyte apoptosis by modulating the mitochondria-dependent pathway, including the increase in the Bcl-2/Bax ratio, the inhibition of caspase-3, -8 and -9, and the inhibition of cytochrome C release. Moreover, HCM strongly alleviated oxidative stress, lipid peroxidation and reactive oxygen species (ROS) generation by activating the Nrf2 signaling pathway. In addition, HCM significantly attenuated inflammatory cytokines including TNF-α, IL6 and IL-1ß as well as NO production by inhibiting TLR4 signaling transduction and NF-κB activation. In conclusion, HCM protects hepatocytes from damage induced by LPS/D-GalN, which may contribute to its ability to alleviate hepatocyte apoptosis by protecting the mitochondrial function, inhibit oxidative stress by activating the Nrf2 pathway, and attenuate inflammation by inhibiting NF-κB activation. This study demonstrates that HCM may be developed as a potential agent for the treatment of acute liver failure.

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway.

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.