Ginkgetin exhibits antifibrotic effects by inducing hepatic stellate cell apoptosis via STAT1 activation.

Liver fibrosis affects approximately 800 million patients worldwide, with over 2 million deaths each year. Nevertheless, there are no approved medications for treating liver fibrosis. In this study, we investigated the impacts of ginkgetin on liver fibrosis and the underlying mechanisms. The impacts of ginkgetin on liver fibrosis were assessed in mouse models induced by thioacetamide or bile duct ligation. Experiments on human LX-2 cells and primary mouse hepatic stellate cells (HSCs) were performed to explore the underlying mechanisms, which were also validated in the mouse models. Ginkgetin significantly decreased hepatic extracellular matrix deposition and HSC activation in the fibrotic models induced by thioacetamide (TAA) and bile duct ligation (BDL). Beneficial effects also existed in inhibiting hepatic inflammation and improving liver function. In vitro experiments showed that ginkgetin markedly inhibited HSC viability and induced HSC apoptosis dose-dependently. Mechanistic studies revealed that the antifibrotic effects of ginkgetin depend on STAT1 activation, as the effects were abolished in vitro after STAT1 silencing and in vivo after inhibiting STAT1 activation by fludarabine. Moreover, we observed a meaningful cross-talk between HSCs and hepatocytes, in which IL-6, released by ginkgetin-induced apoptotic HSCs, enhanced hepatocyte proliferation by activating STAT3 signaling. Ginkgetin exhibits antifibrotic effects by inducing HSC apoptosis via STAT1 activation and enhances hepatocyte proliferation secondary to HSC apoptosis via the IL-6/STAT3 pathway.

Hydroalcoholic extract of Scrophularia striata has a significant therapeutic effect on thioacetamide-induced liver cirrhosis in rats.

OBJECTIVES: Liver cirrhosis is one of the most important causes of death from liver diseases. Nowadays, the use of herbal medicines has increased due to its availability, less side effects and cheapness for the treatment of liver diseases. The present study was conducted to examine therapeutic effects of hydroalcoholic extract of Scrophularia striata (S. striata) on thioacetamide-induced liver cirrhosis in rats through evaluate its effects on oxidative stress markers and the expression of metalloproteinase 1 (TIMP 1), toll-like receptor-4 (TLR-4), and Mitofusin (MFN2) genes. METHODS: 24 male rats were selected by simple random sampling. Rats were randomly assigned to four groups: group I: healthy rats, group II: thioacetamide (TAA) injected rats, group III: TAA injected rats+100 mg/kg bw of S. striata and group IV: TAA injected rats+200 mg/kg bw of S. striata. Liver cirrhosis was induced in rats by a 300 mg/kg bw TAA administration twice with an interval of 24 h. After 8 weeks of treatment by S. striata at doses of 100 and 200 mg/kg bw, biochemical factors and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) were measured using spectrophotometric methods. Also, gene expression of TIMP 1, TLR-4, and MFN2 were analyzed using real-time PCR. ANOVA and Bonferroni post hoc test analysis were applied to evaluate the data. RESULTS: The results showed the S. striata extract significantly improve the serum ALT, AST and ALP levels, TIMP 1, TLR-4, and MFN2 genes and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) in the liver tissues when compared to control group (p<0.05). Also, it was found that the beneficial effects of the S. striata were dose-dependent. CONCLUSIONS: Based on the results obtained S. striata by reducing the expression of TIMP 1, TLR-4, and MFN2 genes and improving oxidative stress might be used as adjuvant treatment for liver cirrhosis.

Effects of Arum dioscoridis Extract on Hepatic Toxicity Caused by Thioacetamide in Rats.

BACKGROUND: The aim of this study was to investigate the prophylactic and therapeutic effects of Arum dioscoridis (tirsik) plant extract against thioacetamide-induced experimental liver toxicity. METHODS: In this study, 35 male Wistar-Albino rats, of 12-14 weeks old, weighing between 200 and 270 g, were used. Rats were divided into 5 groups of 7 each. The first group was determined as the control group, the second group as the hepatotoxicity group, the third group as the prophylaxis group, the fourth group as the intraperitoneal treatment group, and the fifth group as the oral treatment group. Hepatotoxicity was achieved with a single intraperitoneal dose of 350 mg/kg of thioacetamide (TAA). On the seventh day, the rats were sacrificed under general anesthesia. Their blood was taken and liver enzymes were studied. Malondialdehyde (MDA), glutathyon peroxi dase (GPx), catalase (CAT), superoxit dismutase (SOD) enzymes were studied from liver tissues. In addition, liver tissues were evaluated histopathologically. RESULTS: With Arum dioscoridis treatment and prophylaxis, improvements in all parameters and increases in tissue antioxidant levels were detected. CONCLUSION: It was determined that Arum dioscoridis plant extract has prophylactic and therapeutic effects on liver toxicity. In cases of acute liver injury and hepatotoxicity, we suggest the potential application of Arum dioscoridis for effective and inexpensive treatment.

Flavonoid constituents and protective efficacy of Citrus reticulate (Blanco) leaves ethanolic extract on thioacetamide-induced liver injury rats.

Context: Oxidative stress leads to deleterious processes in the liver that resulted in liver diseases.Objective: To evaluate antioxidant activity and hepatoprotective potential of ethanolic leaves extract of Citrus reticulate against hepatic dysfunction induced by thioacetamide (TAA).Materials and Methods: Flavonoid constituents were isolated from the ethanol extract by chromatographic techniques and identified by the spectroscopic analyses. Antioxidant activity was determined using DPPH assay. Hepatotoxicity was induced in rats via intraperitoneal injection of TAA and the ethanol extract was orally administrated at a dose of 100 mg/kg/day for four weeks. Serum biomarkers, hepatic antioxidant enzymes, tumour necrosis factor-alpha (TNF-α), hepatic hydroxyproline levels, and histopathology were examined.Results: Ten known flavonoids were identified, among of them, 6,3`-dimethoxyluteolin and 8,3`-dimethoxyluteolin possessed the highest antioxidant activity. The substantially elevated serum enzymatic levels of ALT, ALP, and bilirubin were found to be restored towards normalisation significantly by the plant extract. Furthermore, the markers including MDA, GSH, SOD, NO, and protein carbonyl which were close to oxidative damage, were restored. Meanwhile, the extract treatment decreased TNF-α level and also was able to reverse the induced fibrosis by significantly reducing the hydroxyproline content. Moreover, histopathological studies further substantiate the protective effect of the extract.Conclusion: C. reticulate leaves extract is a rich source of phytochemicals with in vitro and in vivo protective effects.

[Icariin inhibits thioacetamide-induced osteoclast differentiation through RANKL-p38/ERK-NFAT pathway].

This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.

Molecular Changes Following Induction of Hepatocellular Carcinoma by Diethylnitrosamine and Thioacetamide, and Subsequent Treatment with Dioscorea membranacea Extract.

Hepatocellular carcinoma (HCC) is a primary liver cancer commonly found in adults. Previously, we showed the anticancer effects of Thai herbal plant extract, Dioscorea membranacea Pierre (DM), in HCC-bearing rats. In the present study, we further examined the proposed mechanism of DM, including apoptosis and antioxidant activity. Moreover, we used RNA sequencing (RNA-seq) to analyze molecular pathways in the rat model in which HCC was induced by diethylnitrosamine (DEN) and thioacetamide (TAA). The HCC-bearing rats were then treated with 40 mg/kg of DM for 8 weeks, after which experimental and control rats were sacrificed and liver tissues were collected. The RNA-seq data of DEN/TAA-treated rats exhibited upregulation of 16 hallmark pathways, including epithelial mesenchymal transition, inflammatory responses, and angiogenesis (p<0.01). DM extract expanded the Bax protein-positive pericentral zone in the tumor areas and decreased hepatic malondialdehyde levels, implying a decrease in lipid peroxidation in liver. However, DM treatment did not ameliorate the molecular pathways induced in DEN/TAA-treated livers. Our findings indicate that DM extract has antioxidant activity and exerts its pro-apoptotic effect on rat HCCs in vivo at the (post-)translational level.

Phytochemical constituents and protective efficacy of Schefflera arboricola L. leaves extract against thioacetamide-induced hepatic encephalopathy in rats.

CONTEXT: Hepatic encephalopathy (HE) is a severe neuropsychiatric syndrome resulting from liver failure. OBJECTIVE: To evaluate the protective effect of Schefflera arboricola L. leaves methanol extract against thioacetamide (TAA) induced HE in rats. MATERIALS AND METHODS: GC/MS, LC-ESI-MS, and the total phenolic and flavonoid contents were determined. The methanol extract was orally administrated (100 and 200 mg/kg body weight) for 21 days. TAA (200 mg/kg body weight) was given intraperitoneally on day 19 and continued for three days. The evaluation was done by measuring alanine aminotransferase (ALT), alkaline phosphatase (ALP), ammonia, reduced glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), toll-like receptor 4 (TLR4), interleukin-1 beta (IL-1ß), interlukin-6 (IL-6), cyclooxygenase 2 (COX2), B cell lymphoma 2 (BCL2), alpha-smooth muscle actin (α-SMA), and the cluster of differentiation 163 (CD163). The histological features of the liver and brain were conducted. RESULTS: Forty-five compounds were identified from the n-hexane fraction, while twenty-nine phenolic compounds were determined from the methanol extract. Pre-treatment with the plant extract returned most of the measurements under investigation to nearly normal. CONCLUSION: Due to its richness with bioactive compounds, Schefflera arboricola L. leaves methanolic extract succeeded to exert anti-fibrotic, anti-inflammatory, and antioxidants properties in TAA-induced HE in rats with more efficacy to its high protective dose.

Flavonoids from Barnebydendron riedelii leaf extract mitigate thioacetamide-induced hepatic encephalopathy in rats: The interplay of NF-κB/IL-6 and Nrf2/HO-1 signaling pathways.

Phytochemical investigation of the butanol fraction (BUF) derived from the 70% aqueous methanolic leaf extract of Barnebydendron riedelii led to the isolation of three flavonoid glycosides; kaempferol-3-O-α-l-rhamnopyranosyl-(1 â†’ 6)-ß-d-glucopyranoside, quercetin-3-O-α-l-rhamnopyranosyl-(1 â†’ 6)-ß-d-galactopyranoside and quercetin-3-O-α-l-rhamnopyranosyl-(1 â†’ 6)-ß-d-glucopyranoside. Docking studies were fulfilled to validate the possible bio-properties of BUF toward nuclear factorkappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). The protective role of BUF against behavioral, biochemical, molecular, histopathological and immunohistochemical alterations in thioacetamide (TAA)-induced hepatic encephalopathy in rats was investigated. The toxicological studies indicated that BUF was safe up to 2000 mg/kg bw. Prior to TAA intoxication, rats were orally treated with either BUF at multiple doses (70, 140 and 280 mg/kg bw) or lactulose (8 mL/kg bw) for 14 consecutive days. On the 13th and the 14th day, TAA (200 mg/kg bw/day) was intraperitoneally injected. The BUF significantly improved motor impairment, ameliorated cognitive deficits and attenuated TAA-induced hepatotoxicity. Moreover, BUF controlled the inflammatory processes by suppressing the hepatic inflammatory cytokine; interleukin-6 (IL-6) as well as its pro-inflammatory mediator; NF-κB supporting the molecular docking assessment. The brain neurotransmitters; dopamine, serotonin and noradrenaline, as well as ammonia levels were improved in BUF-treated TAA-intoxicated animals in a dose-dependent manner. Furthermore, BUF administration to TAA-intoxicated rats modulated the Nrf2 and heme oxygenase 1 (HO-1) genes expression in liver and brain tissues. The histological evaluation showed that pretreatment of TAA-intoxicated rats with BUF ameliorated the degenerative effects of TAA on liver and brain tissues as well as reduced the activation of cellular apoptotic marker; caspase-3 and glial fibrillary acidic protein (GFAP+) astrocytes. In conclusion, the observed hepato-neuroprotective effect of BUF is attributed to its flavonoidal content through its modulatory effects on of NF-κB/IL-6 and Nrf2/HO-1 signaling pathways.

Protective effects of kaerophyllin against liver fibrogenesis in rats.

BACKGROUND: We previously demonstrated that kaerophyllin, a lignan, isolated from a widely used traditional Chinese herb, Bupleurum scorzonerifolium, leading to the inhibition of hepatic stellate cells (HSCs) activation in vitro. This current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms. MATERIALS AND METHODS: Liver fibrosis in Sprague-Dawley rats was induced by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Animals were divided into five groups: vehicle control, TAA control, TAA + low dose kaerophyllin, TAA + high dose kaerophyllin and TAA + curcumin groups. Kaerophyllin (10 or 30 mg/kg) or curcumin (150 mg/mL) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against tumour necrosis factor α (TNF-α) in vitro. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs. RESULTS: Kaerophyllin significantly protected liver from injury by reducing serum aspartate transaminase and alanine transaminase levels and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, interleukin-1ß (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of intracellular adhesion molecule-1 (ICAM-1), MCP-1 and IL-1ß genes, which were reversed by small interfering RNA knockdown of PPAR-γ gene. CONCLUSIONS: Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.

A standardized aqueous extract of Anoectochilus formosanus ameliorated thioacetamide-induced liver fibrosis in mice: the role of Kupffer cells.

Anoectochilus formosanus is used in traditional folk medicine as an hepatoprotective agent. The purpose of this study was to investigate the effects of a standardized aqueous extract of A. formosanus (SAEAF) on thioacetamide (TAA)-induced liver fibrosis. An in vitro study showed that the inhibitive effect of kinsenoside, a major component of SAEAF, on tumor necrosis factor alpha (TNF-alpha) secretion from Kupffer cells might be derived at least partly from downregulation of LPS-receptor Toll-like receptor 4 (TLR4) signaling. Hepatic fibrosis was produced by TAA (200 mg/kg, i.p.) 3 times per week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SAEAF (1.0, 0.2 g/kg) via gastrogavage throughout the experimental period. The mice that received the SAEAF treatment had significantly reduced plasma alanine aminotransferase activity, relative liver weights, and hepatic hydroxyproline contents. A histological examination also confirmed that SAEAF reduced the degree of fibrosis caused by TAA treatment. RT-PCR analysis showed that SAEAF treatment reduced mRNA expression of collagen (alpha1)(I), lipopolysaccharide-binding protein, CD14, TLR4, and TNF receptor 1. An immunohistochemical examination also indicated that SAEAF reduced the number of CD68-positive cells (macrophages). In conclusion, oral administration of SAEAF significantly reduced TAA-induced hepatic fibrosis in mice, probably through inhibition of hepatic Kupffer cell activation.

The urease-catalyzed hydrolysis of thiourea and thioacetamide.

Jack bean urease catalyzes the hydrolysis of thiourea with a second-order rate constant (kcat/Km) of 1.6 (+/- 0.2) x 10(-3) M-1 S-1 at pH7, 25 degrees C. This value is lower than that for urea by a factor of 3 x 10(8). The corresponding substitution of S for O in acetamide reduces the kcat/Km value by only a factor of 33. This greater reactivity of the oxo compounds than of the corresponding thiono compounds, and the tighter binding of urea (Ks = 2.9 mM) than of either the guanidinium ion (Ki = 30 mM) or thiourea (Ki = 70 mM), suggests that the substrate chalcogen (S or O) is more likely to be stabilized in the transition state by coordination to the enzyme via a neutral hydrogen-bond donor (i.e., Brønsted acid catalysis) than by coordination via one of the active-site nickel ions (i.e., Lewis acid catalysis).