Comparison of in vitro Toxicities of 8-Prenylnaringenin, Tartrazine and 17ß-Estradiol, Representatives of Natural and Synthetic Estrogens, in Rat and Human Hepatoma Cell Lines.

BACKGROUND: Phytoestrogens have been praised for their beneficial health effects, whereas synthetic xenoestrogens have been connected to ailments. AIMS: To ascertain whether the toxicities of natural and synthetic estrogens differ, we examined the potent phytoestrogen 8-prenylnaringenin (8-PN), the common synthetic xenoestrogen tartrazine, and the physiological estrogen 17ß-estradiol (E2). METHODS: These three compounds were tested for cytotoxicity, cell proliferation and genotoxicity in human HepG2 and rat H4IIE hepatoma cells. RESULTS: All three estrogens elicited cytotoxicity at high concentrations in both cell lines. They also inhibited cell proliferation, with E2 being the most effective. They all tended to increase micronuclei formation. CONCLUSION: Natural estrogens were no less toxic than a synthetic one.

An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Background: Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. Materials and Methods: After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Results: Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. Conclusion: LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Isorhamnetin exerts anti-tumor activity in DEN + CCl4-induced HCC mice.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer and the main cause of cancer death globally. The use of medicinal herbs as chemotherapeutic agents in cancer treatment is receiving attention as they possess no or minimum side effects. Isorhamnetin (IRN), a flavonoid, has been under attention for its anti-inflammatory and anti-proliferative properties in a number of cancers, including colorectal, skin, and lung cancers. However, the in vivo mechanism of isorhamnetin to suppress liver cancer has yet to be explored. METHODS AND RESULT: HCC was induced by N-diethylnitrosamine (DEN) and carbon tetrachloride (CCL4) in Swiss albino mice. Isorhamnetin (100 mg/kg body weight) was given to examine its anti-tumor properties in HCC mice model. Histological analysis and liver function assays were performed to assess changes in liver anatomy. Probable molecular pathways were explored using immunoblot, qPCR, ELISA, and immunohistochemistry techniques. Isorhamnetin inhibited various pro-inflammatory cytokines to suppress cancer-inducing inflammation. Additionally, it regulated Akt and MAPKs to suppress Nrf2 signaling. Isorhamnetin activated PPAR-γ and autophagy while suppressing cell cycle progression in DEN + CCl4-administered mice. Additionally, isorhamnetin regulated various signaling pathways to suppress cell proliferation, metabolism, and epithelial-mesenchymal transition in HCC. CONCLUSION: Regulating diverse cellular signaling pathways makes isorhamnetin a better anti-cancer chemotherapeutic candidate in HCC. Importantly, the anti-TNF-α properties of isorhamnetin could prove it a valuable therapeutic agent in sorafenib-resistant HCC patients. Additionally, anti-TGF-ß properties of isorhamnetin could be utilized to reduce the EMT-inducing side effects of doxorubicin.

Chemopreventive and Therapeutic Efficacy of Enhalus acoroides against Diethylnitrosamine Induced Hepatocellular Carcinoma in Wistar Albino Rats.

Hepatocellular carcinoma is the second most cause of death among the various cancers worldwide. Recent research searching an alternative therapy for cancer treatment without or less side effects. Many studies indicated the beneficial effects of Enhalus acoroides. There has been no scientific validation on antioxidant and chemopreventive potential of ethanolic extract E. acoroides against hepatoma. To assess the hepatoprotective activity of E. acoroides (EEEA) against DEN-induced hepatoma using Wistar albino rats. Animals were distributed into five groups, each containing six rats. To Group I - control rats - normal saline given. Groups II, III, IV and V rats were injection of DEN at a dose of 100 mg/kg body weight i.p. to induce liver cancer. At the commencement of 6th week, Group III rats supplemented with EEEA at a dose of 200 mg/kg body weight/day upto 16 weeks. Group IV rats supplemented with EEEA for 1 week before the administration of DEN and continued till the sixteenth week. Group V supplementation of silymarin at a dose of 100 mg/kg body weight at the beginning of 6th week after the injection of DEN and continued upto 16 weeks and considered as positive control rats. The efficiency of E. acoroides for its antioxidant hepatoprotective and activity evaluated in rats against DEN-induced liver damage. The hepatoprotective ability of EEEA at a dose of 200 mg/kg was examined against DEN at a dose of 100 mg/kg/b.w. induced hepatotoxicity and analysed by evaluating serum liver and kidney marker levels, lipid profile (TG, HDL, LDL and total cholesterol) and serum tumour markers (DNA, RNA, AFP and CEA). Supplementation of EEEA to DEN treated rats was determined by evaluating various antioxidant biomarkers (SOD, CAT, GPx, GSH, Vit E and Vit C). Histopathological studies and morphometric gross analysis were also support the consequences of this study. A significant improvement of antioxidant defence and declined MDA levels within the serum of EEEA treated animals compared to the DEN-induced hepatoma. The supplementation of EEEA declined the serum liver, kidney and serum tumour marker levels and lipid profile as comparatively to Group I rats. The histopathological changes were changed on supplementation of EEEA demonstrating its protecting effects on hepatocytes as comparatively to Group I rats. Our significances recognized that crude extract (ethanol) of E. acoroides revealed a potential impact against DEN-induced hepatoma and assists as a superior choice for chemopreventive treatments.

Tropaeolum majus L. and low dose gamma radiation suppress liver carcinoma development via EGFR-HER2 signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers around the world and remain asymptomatic in early stage. An alcoholic extract prepared from leaves of Tropaeolum majus L. (Tropaeolaceae) was assessed for its potential activity against diethylnitrosamine-induced liver carcinoma in vivo. Oral administration of the extract significantly decreased the inflammatory marker translation NF-kB and supressed HCC progression in combination with 0.5 Gy gamma radiation via EGF-HER-2 pathway. Histopathological and immunohistopathological features also showed the recovery of a hepatic architecture. Immunohistochemical study showed the T. majus and LDR enhancement effect on proapoptotic markers (caspase-3 and Bax) and inhibition of anti-apoptotic factor (BCl2). HPLC-DAD-MSn analysis of the extract revealed the annotation of twelve compounds. T. majus could mediate a defensive influence against diethylnitrosamine-induced hepatocarcinogenesis and serve as a respectable option in amelioration of the hepatocellular carcinoma development in combination with low dose of gamma radiation.

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.

Chemoprotective Effect of Decalactone on Hepatic Cancer via Diminishing the Inflammatory Response and Oxidative Stress.

Hepatocellular Carcinoma (HCC) is the 5th most common type of cancer in all types of cancers, globally. It is well known that the frequency of inflammatory reaction and oxidative stress increases during the HCC. The goal of this study was to see if decalactone could prevent rats against HCC caused by diethylnitrosamine (DEN). Single intraperitoneal administration of DEN (200 mg/kg) used as inducer and weekly intraperitoneal injection of phenobarbital (8 mg/kg) was used as promotor for induction the HCC in rats. Serum alpha fetoprotein (AFP) was used for the confirmation of HCC. Different doses of decalactone (5, 10 and 15 mg/kg) were orally administered to the rats. The body weight was determined at regular time. The hepatic, non-hepatic, antioxidant markers and inflammatory mediators were scrutinized. All groups of animals were scarified and macroscopically examination of the liver tissue was performed and the weight of organ (hepatic tissue) were estimated. Decalactone increased body weight while also suppressing hepatic nodules and tissue weight. Decalactone treatment reduced AFP, total bilirubin, and direct bilirubin levels while increasing albumin and total protein levels in a dose-dependent manner. Decalactone reduced lipid peroxidation (LPO) and increased catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels significantly (p < 0.001) (SOD). Decalactone lowered the levels of significantly (p < 0.001) inflammatory cytokines and inflammatory markers in the liver. Based on the findings, we may conclude that decalactone inhibited HCC in DEN-induced HCC animals via reducing oxidative stress and inflammatory mediators.

Risk Assessment of Hepatocellular Carcinoma with Aflatoxin B1 Exposure in Edible Oils.

Contamination of edible oils with aflatoxins (AFs) is a universal issue due to the detrimental effects of aflatoxins on human health and the fact that edible oils are a major source of fungal growth, particularly storage fungi (Aspergillus sp.). The objective of this study was to assess aflatoxin B1 (AFB1) in edible oil used in fried food in order to determine the risk of cancer from AFB1 exposure through cooked food using the FAO/WHO's and EFSA's margin of exposure (MOE) quantitative liver cancer risk approaches. Using Mycosep 226 columns and HPLC-FLD, 100 samples of cooking oils (soybean, canola, and sunflower oil) from different food points were analyzed for contamination with aflatoxins. Of all the samples tested, 89% were positive for total aflatoxins and AFB1, with 65% indicating AF concentrations beyond permitted levels. Canola oil was found to contain higher levels of AFB1 and AFs than soybean and sunflower oil. Almost 71 percent of canola oil samples (range of 54.4-281.1 µg/kg) were contaminated with AF levels higher than the proposed limits of the European Union (20 µg/kg). The consumption of canola oil samples used in fried foods had MOE values that were significantly lower as compared to sunflower and soybean oils, indicating that risk reduction is feasible. Additionally, compared to soybean and sunflower oil, canola oil exhibited a greater threat of liver cancer cases linked to AFB1 exposure (17.13 per 100,000 males over 35 and 10.93 per 100,000 females over 35). Using a quantitative liver cancer approach, health risk valuation demonstrated that males and females over the age of 35 are at significant risk of developing liver cancer. The health risk assessment exposed that the males and female over the age of 35 are at considerable risk of liver cancer by using a quantitative liver cancer approach. The innovation of this study lies in the fact that no such study is reported related to liver cancer risk evaluation accompanied with AFB1 exposure from consumed edible oil. As a result, a national strategy must be developed to solve this problem so that edible oil products are subjected to severe regulatory examination.

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine.

Several fractions of Calotropis gigantea extracts have been proposed to have potential anticancer activity in many cancer models. The present study evaluated the anticancer activity of C. gigantea stem bark extracts in liver cancer HepG2 cells and diethylnitrosamine (DEN)-induced primary liver cancer in rats. The carcinogenesis model induced by DEN administration has been widely used to study pathophysiological features and responses in rats that are comparable to those seen in cancer patients. The dichloromethane (CGDCM), ethyl acetate, and water fractions obtained from partitioning crude ethanolic extract were quantitatively analyzed for several groups of secondary metabolites and calactin contents. A combination of C. gigantea stem bark extracts with doxorubicin (DOX) was assessed in this study to demonstrate the enhanced cytotoxic effect to cancer compared to the single administration. The combination of DOX and CGDCM, which had the most potential cytotoxic effect in HepG2 cells when compared to the other three fractions, significantly increased cytotoxicity through the apoptotic effect with increased caspase-3 expression. This combination treatment also reduced ATP levels, implying a correlation between ATP and apoptosis induction. In a rat model of DEN-induced liver cancer, treatment with DOX, C. gigantea at low (CGDCM-L) and high (CGDCM-H) doses, and DOX + CGDCM-H for 4 weeks decreased the progression of liver cancer by lowering the liver weight/body weight ratio and the occurrence of liver hyperplastic nodules, fibrosis, and proliferative cells. The therapeutic applications lowered TNF-α, IL-6, TGF-ß, and α-SMA inflammatory cytokines in a similar way, implying that CGDCM had a curative effect against the inflammation-induced liver carcinogenesis produced by DEN exposure. Furthermore, CGDCM and DOX therapy decreased ATP and fatty acid synthesis in rat liver cancer, which was correlated with apoptosis inhibition. CGDCM reduced cleaved caspase-3 expression in liver cancer rats when used alone or in combination with DOX, implying that apoptosis-inducing hepatic carcinogenesis was suppressed. Our results also verified the low toxicity of CGDCM injection on the internal organs of rats. Thus, this research clearly demonstrated a promising, novel anticancer approach that could be applied in future clinical studies of CGDCM and combination therapy.

Protective effects of Persea americana fruit and seed extracts against chemically induced liver cancer in rats by enhancing their antioxidant, anti-inflammatory, and apoptotic activities.

This study aims to explore the chemopreventive mechanisms of hydroethanolic extracts from avocado (Persea Americana) in diethylnitrosamine (DEN)/2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. Chemical induction of hepatocarcinogenesis was induced by intraperitoneal injection of DEN at 150 mg/kg body weight (b.w.) twice a week for a fortnight, followed by oral administration of 2AAF at 20 mg/kg b.w. four times a week for 3 weeks. Rats administered DEN/2AAF were orally treated with hydroethanolic extracts of avocado fruits and seeds at a dose of 50 mg/kg b.w. every other day for 20 weeks. Moreover, rats administered DEN/2AAF and treated with avocado extracts revealed a marked decrease in liver enzyme activities, total bilirubin levels, and elevated liver tumor markers, but revealed an increase in total protein and albumin levels. The hepatocytes with hyperchromatic and bile duct cystadenoma observed in the liver of rats administered DEN/2AAF were reduced due to treatment with avocado extracts. Furthermore, the treatments prevented the elevation of lipid peroxidation levels and ameliorated the lowered glutathione peroxidase, glutathione-S-transferase, superoxide dismutase activities, and glutathione content in the liver tissues. Also, antigen Ki-67, cyclooxygenase-2, and nuclear factor kappa-B expression levels were decreased, but of the suppressor proteins p53 and BAX levels were increased in the liver of rats administered DEN/2AAF and treated with avocado extracts. In conclusion, the current results demonstrated that avocado extracts could abate hepatocarcinogenesis in rats administered DEN/2AAF through activation of antioxidant, anti-inflammatory, and apoptotic properties.

Fuzheng Xiaozheng prescription relieves rat hepatocellular carcinoma through improving anti-inflammation capacity and regulating lipid related metabolisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng Xiaozheng prescription (FZXZP) is a traditional Chinese medicine (TCM) that was derived from Sanjiasan, a famous decoction documented in the book of Wenyilun in Ming dynasty. Based on our years' clinic application, FZXZP demonstrated satisfactory therapeutic effects in cirrhosis and hepatocellular carcinoma (HCC) treatments. However, the underlying mechanisms are still largely unknown. AIM OF STUDY: In this study, we aim to systematically evaluate the intervention effects of FZXZP on rat HCC and deeply elucidate the underlying regulative mechanisms on rat HCC. MATERIALS AND METHODS: The HCC rats were induced by using diethylnitrosamine (DEN) and two doses of FZXZP were adopted to treat the HCC rats. Liver phenotype, blood chemistry and liver histopathology were used to evaluate the intervention effects. High performance liquid chromatography (HPLC) was conducted to analyze the components of FZXZP. Finally, miRNA-Seq and mRNA-Seq were performed to investigate the regulative mechanisms of FZXZP on rat HCC and qRT-PCR was carried out to verify the accuracies of the two RNA-Seqs. RESULTS: Results of liver phenotypes, blood chemistry and liver histopathology demonstrated that FZXZP significantly alleviated the liver damage, inhibited the progresses of HCC. Nine potential components were identified from FZXZP, and anti-cancer prediction suggested that almost all of them were reported to show an anti-cancer effect. Mechanistically, FZXZP was found to promote the lipid related metabolisms, improve the anti-inflammation ability by activating PPAR signaling pathway, arachidonic acid metabolism, bile secretion, etc. CONCLUSION: our results suggested that FZXZP significantly alleviated the rat HCC, mechanistically by improving the anti-inflammation ability and promoting the lipid related metabolisms.

Protective role of naringin loaded solid nanoparticles against aflatoxin B1 induced hepatocellular carcinoma.

This study examines the hepatoprotective activity of naringin loaded solid nanoparticles (NRG-SLNs) and compared with free naringin (FNRG) against aflatoxin B1 (AFB1) induced hepatocellular carcinoma. The liver's self-healing ability was studied using a self-recovery group that received no therapy. Following AFB1 therapy, rats were given NRG-SLNs produced using the ion-gelation technique. Histology, serum injury indicators, oxidative stress biomarkers, a pro-inflammatory response biomarker, and tumor indicators were used to evaluate the liver tumor and its responsiveness to therapy. At a dosage of 6.18 mg/kg BW, NRG-SLNs (128 ± 4 nm) provided substantially greater hepatoprotection than free NRG. The actions of NRG-SLNs were equivalent to those of silymarin (SILY), which was given at a dosage of 20 mg/kg BW. The lack of regeneration potential of liver tissue after the damage was verified by the self-recovery group. NRG's efficiency in treating hepatic cancer was increased by using SLN's approach. The increased impact is most likely due to: a) enhanced oral bioavailability, b) the regulated and sustained action of enclosed NRG, and c) a decrease in discomfort and toxicity if any after orally administered. NRG-SLNs may be considered as a therapeutic option for hepatic ailments as effectiveness post-induction of liver carcinoma, is demonstrated presently.

Vitamin D3 supplementation alleviates chemically-induced cirrhosis-associated hepatocarcinogenesis.

Vitamin D3 (VD3) deficiency has been associated with increased risk for cirrhosis and hepatocellular carcinoma, a highly incident malignant neoplasia worldwide. On the other hand, VD3 supplementation has shown some beneficial effects in clinical studies and rodent models of chronic liver disease. However, preventive effects of dietary VD3 supplementation in cirrhosis-associated hepatocarcinogenesis is still unknow. To investigate this purpose, male Wistar rats submitted to a combined diethylnitrosamine- and thioacetamide-induced model were concomitantly supplemented with VD3 (5,000 and 10,000 IU/kg diet) for 25 weeks. Liver samples were collected for histological, biochemical and molecular analysis. Serum samples were used to measure 25-hydroxyvitamin D [25(OH)D] and alanine aminotransferase levels. Both VD3 interventions decreased hepatic collagen deposition and pro-inflammatory p65 protein levels, while increased hepatic antioxidant catalase and glutathione peroxidase activities and serum 25(OH)D, without a clear dose-response effect. Nonetheless, only the highest concentration of VD3 increased hepatic protein levels of VD receptor, while decreased the number of large preneoplastic glutathione-S-transferase- (>0.5 mm²) and keratin 8/18-positive lesions, as well the multiplicity of hepatocellular adenomas. Moreover, this intervention increased hepatic antioxidant Nrf2 protein levels and glutathione-S-transferase activity. In summary, dietary VD3 supplementation - in special the highest intervention - showed antifibrotic and antineoplastic properties in chemically-induced cirrhosis-associated hepatocarcinogenesis. The positive modulation of Nrf2 antioxidant axis may be mechanistically involved with these beneficial effects, and may guide future clinical studies.

Anti-hepatocellular carcinoma efficacy of Fuzheng Xiaozheng prescription and its interventional mechanism studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng Xiaozheng prescription (FZXZP), a traditional Chinese medicine, which was derived from the famous decoction, Sanjiasan, in the book of "Wenyilun" in Ming dynasty. Due to its function of invigorating the circulation of blood in Chinese medicine, it was usually used for treating the liver cirrhosis, hepatocellular carcinoma (HCC), etc. Clinical application found that FZXZP exhibited satisfactory therapeutic effects in HCC treatments. However, we still know little about the underlying mechanisms. AIM OF STUDY: In this study, we aim to gain a deeper insight into the inhibiting effects of FZXZP on HCC rats and preliminarily elucidate the underlying intervention effects. MATERIALS AND METHODS: Two doses of FZXZP were adopted to evaluate the therapeutic effects on rat HCC, and then the intervention effects were evaluated from different aspects. High performance liquid chromatography (HPLC) was used for the active compounds prediction in FZXZP. Finally, the mRNA-Seq was conducted to reveal the intervention mechanisms and the mechanisms were further validated by quantitative Real-time PCR (qRT-PCR) and lipid contents analyses. RESULTS: The results showed that FZXZP significantly alleviated the serum biochemical indicators and improved the pathological characteristics of HCC rats. Mechanistically, FZXZP could regulate some lipid related metabolisms, including arachidonic acid, linoleic acid and retinol, as well as improving the steroid hormone biosynthesis, to improve the inflammatory statuses and restoring ability of HCC livers, and these were further confirmed by our following analyses on serum lipid contents and cytokine expressions. In addition, FZXZP could also negatively regulate four extracellular growth factors which could result in the blocking of two cancer-related signaling pathways, Ras/MAPK and Ras/PI3K-Akt. CONCLUSION: Our results suggested that FZXZP demonstrated significant inhibiting effects on rat HCC progresses, and these may be realized by improving the inflammatory statuses and blocking the Ras/MAPK and Ras/PI3K-Akt signaling pathways.

Ethanol supernatant extracts of Gynura procumbens could treat nanodiethylnitrosamine-induced mouse liver cancer by interfering with inflammatory factors for the tumor microenvironment.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura procumbens (Lour.) Merr, (Family Asteraceae), which serves as both medicine and food in traditional ethnic medicine, has the effects of diminishing inflammation, relieving cough, reducing blood glucose and lipids levels, mitigating hepatotoxicity, and can be used for liver cancer prevention and treatment. AIM OF THE STUDY: To explore how the ethanol extract of Gynura procumbens stems (EEGS) can effectively intervene in the tumor microenvironment, it is necessary to study the mechanism of EEGS on the chemical toxicant nanodiethylnitrosamine (nanoDEN) that induces liver cancer. MATERIALS AND METHODS: EEGS contains large quantities of caffeoylquinic acid (CAC) and non-caffeoylquinic acid (n-CAC), which can be separated by high-performance liquid chromatography. The liver cancer model that was induced by the chemical toxin, nanoDEN, was used to clarify the effective mechanism for tumor intervention of the EEGS and its active ingredients. RESULTS: (1) after interventions with the four drugs on liver cancer, the tumor nodules were obviously reduced and inflammation levels improved. (2) The immunohistochemical staining results showed that both the EEGS and its active ingredients could significantly reverse the abnormal changes in inflammation, proliferation, aging and hypoxia-related proteins in mouse liver tissues that were caused by nanoDEN. (3) Real-time PCR results showed that compared with the nanoDEN group, the expression levels of inflammatory, fatty, and fibrosis-related factors in each group after drug intervention were decreased. (4) The transmission electron microscopy measurements showed that the EEGS significantly reversed the nanostructure changes in hepatocytes that were induced by nanoDEN. CONCLUSION: The EEGS component of Gynura procumbens is effective in preventing and treating liver cancer by interfering with the inflammatory microenvironment during oncogenesis induced by nanoDEN.

Mulberry fruit polysaccharides alleviate diethylnitrosamine/phenobarbital-induced hepatocarcinogenesis in vivo: the roles of cell apoptosis and inflammation.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and chemoprevention represents a feasible treatment to reduce the mortality of this carcinoma. Mulberry fruit polysaccharides (MFP) possess immunoregulatory and anti-inflammatory effects, which have been reported to alleviate liver damage evoked by CCl4 or alcohol in previous reports. However, its chemopreventive effect against liver carcinogenesis is insufficient. The present study was aimed to investigate the possible role of MFP as a pro-apoptosis, and anti-inflammatory agent to possess its chemoprevention property. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB) for 14 weeks. The DEN/PB-administered rats were co-treated with different doses of MFP (50 or 100 mg/kg body weight) by oral gavage for 14 weeks. Basic hepatic function indexes (AST, ALT, ALP, GGT, total bilirubin, and albumin), and hepatic tumor biomarkers (AFP, CEA, and CA19.9), together with histological assessment were performed. Besides, the hepatic apoptosis markers (Bcl-2, Bax, caspase3, and caspase9), inflammation markers (IL-1ß, TNF-α, and NF-κB), and mutT homologue gene 1 (MTH1) were examined. Oral gavage of MFP inhibited the elevations of hepatic function indexes and hepatic tumor biomarkers and alleviated pathological changes in hepatic tissue. In addition, the hepatic apoptosis markers, inflammation markers, and the mRNA level of MTH1 were abnormal in DEN/PB group, which were reversed by MFP treatment. In conclusion, MFP is an effective agent that provides chemoprevention against DEN/PB-evoked hepatocarcinogenesis via inhibition of inflammation and induction of apoptosis.

Safflower yellow reduces DEN-induced hepatocellular carcinoma by enhancing liver immune infiltration through promotion of collagen degradation and modulation of gut microbiota.

Safflower yellow (SY) is the main active ingredient isolated from the traditional Chinese medicine Carthamus tinctorius, which is a valuable natural edible pigment that is widely used to treat cerebrovascular and cardiovascular diseases. However, the effect of SY on hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that SY decreased the degree of injury and inhibited the release of inflammatory factors in the liver of a diethylnitrosamine (DEN)-induced HCC mouse model. Flow cytometry and immunoblotting showed that SY increased the infiltration of CD8+ T cells and Gr-1+ macrophages to improve the immune microenvironment by affecting the expression of collagen fibers. Further cellular experiments showed that SY degraded the collagens in the liver cells through the TGF-ß/Smad signalling pathway. SY also regulated the gut microbiota which may contribute to the immune microenvironment. In conclusion, SY exhibited a potent effect on the development of HCC by enhancing liver immune infiltration by promoting collagen degradation and modulating the gut microbiota. This study provides novel insights into the mechanism of SY as a candidate for the treatment of HCC in the future.

Pharmacokinetic herb-drug interactions between Aidi injection and doxorubicin in rats with diethylnitrosamine-induced hepatocellular carcinoma.

BACKGROUND: Aidi Injection (ADI), a Chinese herbal preparation with anti-cancer activity, is used for the treatment of hepatocellular carcinoma (HCC). Several clinical studies have shown that co-administration of ADI with doxorubicin (DOX) is associated with reduced toxicity of chemotherapy, enhanced clinical efficacy and improved quality of life for patients. However, limited information is available about the herb-drug interactions between ADI and DOX. The study aimed to investigate the pharmacokinetic mechanism of herb-drug interactions between ADI and DOX in a rat model of HCC. METHODS: Experimental HCC was induced in rats by oral administration of diethylnitrosamine. The HCC rats were pretreated with ADI (10 mL/kg, intraperitoneal injection) for 14 consecutive days prior to administration of DOX (7 mg/kg, intravenous injection) to investigate pharmacokinetic interactions. Plasma concentrations of DOX and its major metabolite, doxorubicinol (DOXol), were determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: Preadministration of ADI significantly altered the pharmacokinetics of DOX in HCC rats, leading to increased plasma concentrations of both DOX and DOXol. The area under the plasma drug concentration-time curve (AUCs) of DOX and DOXol in rats pretreated with ADI were 3.79-fold and 2.92-fold higher, respectively, than those in control rats that did not receive ADI. CONCLUSIONS: Increased levels of DOX and DOXol were found in the plasma of HCC rats pretreated with ADI.

Rhubarb granule promotes diethylnitrosamine-induced liver tumorigenesis by activating the oxidative branch of pentose phosphate pathway via G6PD in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb is a natural herbal medicine widely used clinically with numerous pharmacological activities including anti-cancer. Specifically, several studies reported that free anthraquinones from Rhubarb suppressed the proliferation of hepatoma cells. Nonetheless, recent studies revealed that Rhubarb caused hepatotoxicity in vivo, confirming its "two-way" effect on the liver. Therefore, the efficacy and safety of Rhubarb in the in vivo treatment of liver cancer should be further elucidated. AIM OF THE STUDY: This study investigated the presence of hepatoprotection or hepatotoxicity of Rhubarb in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. MATERIAL AND METHODS: A total of 112 male Sprague-Dawley rats weighing 190-250 g were enrolled. The rats were induced hepatocarcinogenesis using diethylnitrosamine (0.002 g/rat) until 17 weeks. Starting at week 11, Rhubarb granules (4 g/kg and 8 g/kg) were intragastrically administered daily for 7 weeks. All rats were euthanized at week 20 and the livers were analyzed via non-targeted metabolomics analysis. We established hepatic glucose 6 phosphate (6PG) levels and glucose 6 phosphate dehydrogenase (G6PD) activities to assess the pentose phosphate pathway (PPP). And the liver injuries of rats were analyzed via histological changes, hepatic function, as well as hepatic protein levels of alpha-fetoprotein (AFP), pyruvate kinase isozyme type M2 (PKM2), and proliferating cell nuclear antigen (PCNA). Furthermore, polydatin (0.1 g/kg/d) as a specific inhibitor of G6PD was used to treat rats. Notably, their histological changes, hepatic function, hepatic 6PG levels, hepatic G6PD activities, PCNA levels, and PKM2 levels were recorded. RESULTS: Non-targeted metabolomics revealed that Rhubarb regulated the PPP in the liver of Rhubarb-DEN-treated rats. Besides, Rhubarb activated the oxidative branch of the PPP by activating G6PD (a rate-limiting enzyme in the oxidative PPP) in the liver of Rhubarb-DEN-treated rats. Meanwhile, Rhubarb promoted DEN-induced hepatocarcinogenesis. Moreover, polydatin attenuated the promoting effect of Rhubarb on DEN-induced hepatocarcinogenesis. CONCLUSIONS: Rhubarb promoted DEN-induced hepatocarcinogenesis by activating the PPP, indicating that the efficacy and safety of Rhubarb in the treatment of liver cancer deserve to be deliberated.