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.

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.

Role of RKIP in human hepatic stellate cell proliferation, invasion, and metastasis.

The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, apoptosis, invasion, and metastasis of human hepatic stellate cell line (LX-2). A recombinant plasmid (pcDNA3.1-RKIP) or RKIP-targeting small interfering RNA (siRNA) vector (siRNA-RKIP) was transfected into LX-2 cells to interfere with the RKIP expression. The results demonstrated that increased RKIP expression significantly reduced cell viability, clonogenic growth, and invasion. Further, it promoted cell apoptosis and induced cell cycle arrest in the G1 phase. Overexpression of RKIP led to inactivation of LX-2 cells, as evidenced by the decrease in the expression levels of collagen I and α-smooth muscle actin (α-SMA). In addition, increased RKIP expression significantly reduced the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), the transcriptional activity of nuclear factor-κB (NF-κB), and the levels of matrix metalloproteinases-1 and -2. In conclusion, these findings clearly demonstrate that RKIP inhibits LX-2 cell growth, metastasis, and activation, primarily by downregulating the ERK/MAPK and NF-κB signaling pathways.

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.

Methyl Helicterate Inhibits Hepatic Stellate Cell Activation Through Modulation of Apoptosis and Autophagy.

BACKGROUND/AIMS: Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix (ECM). Therefore inhibiting HSC activation is considered as an effective strategy to inhibit the process of liver fibrosis. This study aimed to investigate the underlying mechanism of methyl helicterate (MH) isolated from Helicteres angustifolia on the activation of HSCs. METHODS: HSC-T6 cells were treated with various concentration of MH and autophagy was inhibited by 3-Methyl adenine (3-MA) or RNA interference. Cell viability was observed by MTT assay and cell colony assay. Cell cycle and apoptosis were analyzed using flow cytometry. Autophagic vacuoles were observed by transmission electron microscopy and monodansyl cadaverine (MDC) staining. Moreover, autophagy-related genes and proteins were detected using real-time PCR and Western blot assays, respectively. RESULTS: MH significantly inhibited HSC activation, as evidenced by the inhibition of cell viability, colony formation and the expression of α-SMA and collagen I. MH caused cell cycle arrest in G2/M phase. Moreover, MH significantly induced apoptosis through regulating the mitochondria-dependent pathway and the activity of caspases. MH treatment significantly increased lysosomes and autophagosomes, and enhanced the formation of autophagic vacuoles and autophagic flux. Interestingly, inhibiting autophagy by 3-MA or RNA interference abolished the ability of MH in inhibiting HSC activation. On the other hand, induction of autophagy promoted MH-induced HSC apoptosis. Further study showed that MH-induced HSC apoptosis and autophagy was mediated by the JNK and PI3K/Akt/mTOR pathways. CONCLUSION: Our results demonstrate that MH-induced HSC apoptosis and autophagy may be one of the important mechanisms for its anti-fibrosis effect.

Trolline Ameliorates Liver Fibrosis by Inhibiting the NF-κB Pathway, Promoting HSC Apoptosis and Suppressing Autophagy.

BACKGROUND/AIMS: Previous studies have shown that trolline possesses various forms of pharmacological activity, including antibacterial and antiviral potency. The present paper addressed the putative hepatoprotective effects of trolline. METHODS: Rats received 2 ml/kg CCl4 (mixed 1: 1 in peanut oil) intragastrically twice a week for 8 weeks to induce hepatic fibrosis. The animals were then treated with trolline for additional 4 weeks. Liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminase activity and collagen-related indicator level were determined by commercially available kits. NF-κB pathway activation was also examined. Moreover, the effects of trolline on hepatic stellate cell (HSC-T6) apoptosis, mitochondrial membrane potential (MMP), and autophagy were assessed. RESULTS: Trolline significantly alleviated CCl4-induced liver injury and notably reduced the accumulation of collagen in liver tissues. Trolline treatment also markedly decreased inflammatory cytokines levels by inhibiting the NF-κB pathway. Trolline strongly inhibited HSC-T6 activation and notably induced cell apoptosis by modulating the Bax/Bcl-2 ratio, caspase activity, and MMP. Moreover, trolline significantly inhibited HSC-T6 autophagy, as evidenced by the decrease in the formation of autophagic vacuoles and the number of autophagosomes, by regulating the expression levles of LC3, Beclin-1, P62, Atg 5 and 7. CONCLUSION: Our study demonstrates that trolline ameliorates liver fibrosis, possibly by inhibiting the NF-κB pathway, promoting HSCs apoptosis and suppressing autophagy.

Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein.

BACKGROUND: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. METHODS: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP) and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. RESULTS: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. CONCLUSION: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.

Raf Kinase Inhibitory Protein Down-Expression Exacerbates Hepatic Fibrosis In Vivo and In Vitro.

BACKGROUND/AIMS: Raf kinase inhibitory protein (RKIP) is closely associated with numerous tumors and participates in their development through regulating the growth, apoptosis, invasion and metastasis of tumor cells. However, the role of RKIP in chronic liver injury and particularly in liver fibrosis is still unclear. METHODS: In the present study, hepatic fibrosis was induced by porcine serum (PS) in rats and primary hepatic stellate cells (HSCs) were isolated from rat livers. Moreover, locostatin was used to interfere with RKIP expression. RESULTS: RKIP expression was significantly inhibited by locostatin in both liver tissues of rats and primary HSCs. Down-regulating RKIP expression resulted in serious liver injury, extensive accumulation of collagen, and significant increase in the levels of ALT, AST and TNF-α during liver fibrosis in rats. Moreover, down-regulating RKIP significantly promoted HSCs proliferation and colony formation in vitro. Reduced RKIP significantly increased the production of collagen and the level of α-SMA as well as the expression of MMP-1 and MMP-2 in both liver tissues and primary HSCs. Furthermore, down-regulating RKIP promoted the activation of the ERK and TLR4 signaling pathways. CONCLUSION: Our findings clearly indicate an inverse correlation between RKIP level and the degree of the liver injury and fibrosis. The decrease in RKIP expression may exacerbate chronic liver injury and liver fibrosis.

[Protective effect of asiatic acid from Potentilla chinensis on alcohol hepatic injury in rats].

To study the protective effect and the mechanism of asiatic acid (AA) from Potentilla chinensis on alcohol hepatic injury in rats. Male Wistar rats were randomly divided into six groups: the normal control group, the AA control group (8 mg · kg(-1) AA), the model group (5.0-9.0 g · kg(-1) alcohol) and high, medium and low-dose AA-treated groups (alcohol + 8, 4, 2 mg · kg(-1) AA). Each group was orally administered with the corresponding drugs once a day for 24 weeks. Approximately 1. 5 hours after the final administration, all rats were killed, and their blood samples and hepatic tissues were collected. The AST and ALT in rat serum and the contents of MPO, TNF-α, IL-1ß, SOD, GSH-Px, GSH-Rd and MDA in hepatic tissues were detected. The expressions of NF-κB, TLR4, CD14, MyD88, TRIF and protein expression in hepatic tissues were measured by western blot. The pathological changes in liver tissues were observed by histological examination. The results showed that compared with the model group, the AA-treated groups showed significant decreases in serum ALT, AST and MDA and increases in the activities of SOD, GSH-Px, GSH-Rd and MPO. Moreover, AA markedly inhibited the expressions of TNF-α, IL-1ß, TLR4, CD14, MyD88 and NF-κB. The histological examination showed alleviated hepatic issue ijury to varying degrees. In short, asiatic acid (AA) from P. chinensis could protect alcohol-induced hepatic injury in rats. Its mechanism may be related to the inhibition of NF-κB inactivation and the reduction of inflammatory response.

Protective effect of genistein on lipopolysaccharide/D-galactosamine- induced hepatic failure in mice.

This study examined the effect of genistein from Hydrocotyle sibthorpioides on lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute hepatic failure. Compared to the model control, genistein treatment significantly protected against LPS/D-GalN-induced liver injury, as evidenced by the decrease in serum alanine and aspartate aminotransferases activities and the attenuation of histopathological changes. Furthermore, genistein alleviated the pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO)/inducible nitric oxide synthase (iNOS) by inhibiting nuclear factor-κB (NF- κB) activity. Genistein attenuated the elevated level of caspases-3, while augmented the expression of Bcl-2. In addition, LPS/D-GalN induced significant increase of heme oxygenase (HO), carbon monoxide and bilirubin levels and these alterations were augmented by genistein treatment. In conclusion, the protective effect of genistein on LPS/D-GalN-induced liver damage was mainly due to its ability to block NF-κB signaling pathway for anti-inflammation response, attenuate hepatocellular apoptosis and increase HO level. These findings suggest that genistein can be considered as a potential agent for preventing acute hepatic failure.