Comparison of growth features and cancer stem cell prevalence in intrahepatic and extrahepatic cholangiocarcinoma cell lines.

Aim of the study: Intra- and extrahepatic cholangiocarcinoma (I-CCA and E-CCA respectively) exhibit different growth features that contribute to different clinical outcomes. Cancer stem cells (CSCs) influence tumor growth and thereby may be responsible for these differences. The aim of this study was to document and compare the growth features of human I-CCA and E-CCA cell lines and determine whether any differences observed could be explained by differences in the prevalence and/or stem cell surface marker (SCSM) expression profiles of CSCs within the tumor cell lines. Material and methods: Six CCA cells lines, three I-CCA and three E-CCA, were studied. Tumor cell growth features including cell proliferation, colony/spheroid formation, migration and invasion were documented. CSC prevalence and SCSM expression profiles were examined by flow cytometry. Results: I-CCA cells had significantly increased proliferative activity, shorter doubling times and were more invasive than E-CCA cells, while colony/spheroid formation and migration were similar in the two cell populations. There were no significant differences in CSC prevalence rates or SCSM expression profiles. Conclusions: These findings suggest that I-CCA cells proliferate at a more rapid rate and are more invasive than E-CCA cells but the differences cannot be explained by differences in the prevalence or SCSM expression profiles of CSCs within the tumor cell population.

The Ciji-Hua'ai-Baosheng II Formula Attenuates Chemotherapy-Induced Anorexia in Mice With H22 Hepatocellular Carcinoma.

Background: Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medicine formula, which specifically targets different aspects of chemotherapy-induced adverse effects in patients with cancer. In our clinical application, CHB-II-F significantly alleviated chemotherapy-induced anorexia (loss of appetite) and improved the quality of life for patients with tumor during and after chemotherapy. However, the mechanism of CHB-II-F in alleviation of chemotherapy-induced anorexia remains to be further investigated. Aim of Study: To explore the therapeutic effect and mechanism of CHB-II-F on chemotherapy-induced anorexia in the mice model of H22 hepatoma. Materials and Methods: A total of 72 Kunming mice of SPF grade were inoculated subcutaneously with H22 hepatoma cells into the right anterior armpit of the mice. After 1 week of seeding, mice were injected intraperitoneally with a high dose of 5-fluorouracil (200 mg/kg 5-FU) to establish the model of chemotherapy. The mice were randomly divided into six groups: untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji capsule (YZXJC) group, and three groups of 5-FU plus different concentrations of CHB-II-F. All the mice in each group were treated for 14 days. The body weight, food intake, tumor volume, and tumor weight of mice were measured, and pathological examinations of tumor tissue, stomach, and duodenum were carried out. Expressions of serum Leptin, Neuropeptide Y (NPY), epidermal cell growth factor (EGF), Motilin (MTL), Orexin A (OXA), Gastrin (GAS), Ghrelin, Prostaglandin E2 (PGE2), and jejunum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined. The protein and mRNA levels of proopiomelanocortin (POMC), Orexin receptor 1 (OX1R), neuropeptide Y (NPY), cocaine and amphetamine regulated transcript peptide (CART), Agouti gene-related protein (AgRP), Leptin receptor (Ob-R), and Ghrelin receptor (GHSR) were examined in hypothalamus, and the protein levels of substance P (SP) and 5-hydroxytryptamine (5-HT) in duodenum were measured. Results: The combination of CHB-II-F and 5-FU could enhance the inhibitory effect of 5-FU on tumor. The tumor inhibition rates of 5-FU group, YZXJC group, CHB-II-F(H) group, CHB-II-F(M) group, and CHB-II-F(L) group were 58.88, 28.08, 54.96, 37.69, and 28.61%, respectively. Compared with untreated group and 5-FU group, CHB-II-F significantly increased the body weight and food intake of tumor-bearing mice; increased the content of NPY, Orexin A, Ghrelin, GAS, MTL, EGF, and PGE2 in serum and the activity of SOD in jejunum; and decreased the content of Leptin in serum and the content of MDA in jejunum. Compared with untreated group and 5-FU group, CHB-II-F also enhanced the expression of OX1R, GHSR, NPY, and AgRP protein and gene and decreased the expression of Ob-R, POMC, and CART protein and gene in hypothalamus of mice, and the gene expression was consistent with the protein expression. In addition, CHB-II-F decreased the expression of 5-HT and SP protein in duodenum. Conclusion: In the murine model of H22 hepatocellular carcinoma (HCC) receiving chemotherapy, CHB-II-F enhances the inhibitory effect of 5-FU on tumor, significantly improves the pathological injury of gastrointestinal tract caused by chemotherapy, and regulates the secretion of gastrointestinal hormones. It may alleviate chemotherapy-induced anorexia by affecting appetite regulatory factors in the feeding area of hypothalamus central nervous system and peripheral appetite regulatory factors.

Alterations in chemokine receptor CCR5 activity influence tumor cell biology in human cholangiocarcinoma cell lines.

INTRODUCTION AND OBJECTIVES: Intrahepatic (I-CCA) and extrahepatic (E-CCA) cholangiocarcinoma (CCA) have different growth patterns and risks for tumor metastasis. Inhibition and/or activation of the chemokine receptor CCR subclasses have been reported to alter tumor cell biology in non-CCA cancers. In this study we documented CCR expression profiles in representative human I-CCA and E-CCA cell lines and the in vitro effects of CCR antagonists and agonists on tumor cell biology. MATERIALS AND METHODS: CCR expression profiles were documented by real-time reverse transcription polymerase chain reaction; cell proliferation by WST-1; spheroid formation by sphere dimensions in anchorage-free medium; cell migration by wound healing and invasion by Transwell invasion chambers. RESULTS: All 10 CCR motifs (CCR1-10) were expressed in the I-CCA, HuCCT1 cell line and six (CCR4, 5, 6, 8, 9 and 10) in the E-CCA, KMBC cell line. In HuCCT1 cells, CCR5 expression was most abundant whereas in KMBC cells, CCR6 followed by CCR5 were most abundant. The CCR5 antagonist Maraviroc significantly inhibited cell proliferation, migration and invasion in HuCCT1 cells, and spheroid formation and invasion in KMBC cells. The CCR5 agonist RANTES had no effect on HuCCT1 cells but increased cell proliferation, migration and invasion of KMBC cells. CONCLUSION: These results suggest that CCR expression profiles differ in I-CCA and E-CCA. They also indicate that CCR5 antagonists and agonists have cell-specific effects but in general, CCR5 inactivation inhibits CCA tumor cell aggressiveness. Additional research is required to determine whether CCR5 inactivation is of value in the treatment of CCA in humans.

Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that tends to be resistant to chemotherapy. Multidrug resistance proteins (MRPs) contribute to the chemoresistance of these tumors. The objectives of the study were to document MRP expression profiles in two representative human intrahepatic and extrahepatic CCA cells lines (HuCCT1 and KMBC, respectively) and gemcitabine-induced cytotoxicity prior to and following MRP knockdown. METHODS: Multidrug resistance protein mRNA and protein expression were documented by real-time reverse transcription-polymerase chain reaction and western blots, respectively. MRP knockdown was achieved with lentivirus small hairpin RNA constructs. RESULTS: Prior to gemcitabine exposure, MRP1, MRP2, MRP4, MRP5, and MRP6 mRNA were expressed in HuCCT1 cells and MRP1, MRP3, MRP4, and MRP5 in KMBC cells. Following gemcitabine exposure, MRP5 and MRP6 expressions were significantly upregulated in HuCCT1 cells and MRP5 in KMBC cells. In HuCCT1 cells, although MRP5 knockdown had no effect, MRP6 knockdown significantly increased gemcitabine-induced cytotoxicity. In KMBC cells, MRP5 knockdown significantly increased gemcitabine cytotoxicity. CONCLUSIONS: Inhibition of MRP6 expression in intra-hepatic and MRP5 in extra-hepatic should be explored as potential treatments for CCA in humans.

Long-term application of hydroxychloroquine could not prevent the infection of COVID-19.

INTRODUCTION: Current pandemic of the coronavirus induced disease 2019 (COVID-19) presents an urgent issue to the world due to lack of vaccine and medication. Hydroxychloroquine (HCQ) has generated a lot of controversies whether it is effective in prevention and treatment of COVID-19. Current report presents a 63-year-old woman who has taken HCQ for many years but still infected by COVID-19. CASE PRESENTATION: A patient with rheumatoid arthritis came to the clinic with fever and sore throat. The patient has been treated with 200 mg HCQ per day since 2016. Laboratory tests showed that the patient had lymphopenia, increased levels of high-sensitive C-reactive protein (hs-CRP) and serum Interleukin-6 (IL-6). Chest radiography showed that the patient had pneumonia. Throat swab test confirmed COVID-19 positive. On admission, she was treated with nebulized interferon alfa-2b, oral Lopinavir/Ritonavir, and ceftriaxone sodium for the COVID-19 in addition to HCQ. The patient stayed in hospital for 18 days, recovered from oxygen intake, and eventually discharged from hospital. Follow up investigation showed the patient developed antibody against COVID-19. CONCLUSIONS: Long-term application of HCQ could not prevent COVID-19 infection, but whether HCQ exerts benefit to alleviation of clinical symptoms and duration of hospital stays remains to be further investigated.

Role of Traditional Chinese Medicine in the Management of Viral Pneumonia.

Viral pneumonia is one kind of acute respiratory tract infection caused by the virus. There have been many outbreaks of viral pneumonia with high contagiousness and mortality both in China and abroad, such as the great influenza in 1918, the severe acute respiratory syndrome (SARS) coronavirus in 2003, the Influenza A (H1N1) virus in 2009, and the Middle East Respiratory Syndrome coronavirus (MERS-CoV) in 2012 and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. These outbreaks and/or pandemic have significant impact on human life, social behaviors, and economic development. Moreover, no specific drug has been developed for these viruses. Traditional Chinese medicine (TCM) plays an important role in the treatment of viral pneumonia during these outbreaks especially in SARS and SARS-CoV-2 because studies suggest that TCM formulations may target several aspects of the disease and may have lesser side effects than manufactured pharmaceuticals. In recent years, a lot of clinicians and researchers have made a series of in-depth explorations and investigations on the treatment of viral pneumonia with TCM, which have understood TCM therapeutic mechanisms more specifically and clearly. But critical analysis of this research in addition to further studies are needed to assess the potential of TCM in the treatment of viral pneumonia.

Chaiyu-Dixian Formula Exerts Protective Effects on Ovarian Follicular Abnormal Development in Chronic Unpredictable Mild Stress (CUMS) Rat Model.

BACKGROUND: Chronic stress has been known to impair the female reproductive function, but the mechanism remains to be further investigated. Chaiyu-Dixian Formula (CYDXF) has been reported to regulate human endocrine disorders clinically. However, whether this formula can affect chronic stress-induced ovarian follicular development is not clear. AIM OF THE STUDY: To examine effects of CYDXF on follicular development and explore possible mech anisms in a chronic unpredictable mild stress (CUMS) model. MATERIALS AND METHODS: Adult female rats were randomly divided into 5 groups control group, CUMS group (saline treatment), CUMS+Estradiol (E2) (0.1 mg/kg) group, CUMS+CYDXF (2.73 g/kg) group, and CUMS+CYDXF (5.46 g/kg) group. Body weights and behavioral tests were documented. Serum hormone levels were determined by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the protein levels in the PI3K/Akt pathway and brain-derived neurotrophic factor (BDNF). The follicles were analyzed and classified according to their morphological characterization. RESULTS: CYDXF relieved depression-like behaviors and ameliorated the abnormality in rat estrous cycle within the rat model of CUMS. Moreover, CYDXF could regulate endocrine disorders, increase the proportion of antral follicles as well as decrease the proportion of follicular atresia, which suggested that CYDXF could alleviate abnormal follicular development and improve overall ovarian function. Furthermore, CYDXF also activated the BDNF-mediated PI3K/Akt signaling pathway. CONCLUSIONS: CYDXF (at dose of both 2.73 and 5.46 g/kg) attenuated chronic stress-induced abnormal ovarian follicular development by relieving depression-like behaviors and improving ovarian function through partly the regulation of the BDNF-mediated PI3K/Akt pathway.

Attenuation of Experimental Autoimmune Hepatitis in Mice with Bone Mesenchymal Stem Cell-Derived Exosomes Carrying MicroRNA-223-3p.

MicroRNA-223-3p (miR-223-3p) is one of the potential microRNAs that have been shown to alleviate inflammatory responses in pre-clinical investigations and is highly encased in exosomes derived from bone mesenchymal stem cells (MSC-exosomes). MSC-exosomes are able to function as carriers to deliver microRNAs into cells. Autoimmune hepatitis is one of the challenging liver diseases with no effective treatment other than steroid hormones. Here, we examined whether MSC-exosomes can transfer miR-223-3p to treat autoimmune hepatitis in an experimental model. We found that MSC-exosomes were successfully incorporated with miR-223-3p and delivered miR-223-3p into macrophages. Moreover, there was no toxic effect of exosomes on the macrophages. Furthermore, treatments of either exosomes or exosomes with miR-223-3p successfully attenuated inflammatory responses in the liver of autoimmune hepatitis and inflammatory cytokine release in both the liver and macrophages. The mechanism may be related to the regulation of miR-223-3p level and STAT3 expression in the liver and macrophages. These results suggest that MSC-exosomes can be used to deliver miR-223-3p for the treatment of autoimmune hepatitis.

Rosenroot (Rhodiola): Potential Applications in Aging-related Diseases.

Aging is a progressive accumulation of changes in the body, which increases the susceptibility to diseases such as Alzheimer's disease, Parkinson's disease, cerebrovascular disease, diabetes, and cardiovascular disease. Recently, Chinese medicinal herbs have been investigated for their therapeutic efficacy in the treatment of some aging-related diseases. Rhodiola, known as 'Hongjingtian' in Chinese, has been reported to have anti-aging activity. Here, we provide a comprehensive review about its origin, chemical constituents, and effects on aging-related diseases.

Safety Evaluation of a New Traditional Chinese Medical Formula, Ciji-Hua'ai-Baosheng II Formula, in Adult Rodent Models.

BACKGROUND: Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a new traditional Chinese medical formula that has been shown to reduce toxicity and side effects of chemotherapy and increase the probability of cancer patient survival. Whether CHB-II-F is safe as an adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined. PURPOSE: To evaluate the acute and subchronic toxic effects of CHB-II-F in rodent models. METHODS: In acute toxicity test, 24 Kunming mice were divided into 2 groups: untreated control and CHB-II-F 1.05 g/mL (31.44 g/kg) treated group. Treatment was administered to the treated group 3 times a day for 14 days. The overall health, adverse reactions, and mortality rate were documented. In subchronic toxicity test, 96 Sprague-Dawley rats were divided into 4 groups: untreated control, high dose CHB-II-F (H) (26.20 g/kg), medium dose CHB-II-F (M) (13. 10 g/kg), and low dose CHB-II-F (L) (6.55 g/kg) [equal to 24.375 g (dried medicinal herb)/kg] treated groups. Treated groups were given the treatments once a day for 4 weeks. The overall health and mortality rate were recorded every day. Body weight and food consumption were measured once a week. Hematologic and biochemical parameters, organ weights, and histopathologic markers were analyzed after 4 weeks. An additional 2 weeks were given as the treatment recovery period before end-point euthanization, and biochemical analyses were performed. RESULTS: The maximum tolerated dose (MTD) of CHB-II-F on mice was found to be 94.31 g/kg [equal to 351 g (dried medicinal herb)/kg], which is 108 times the human adult dose. In the acute toxicity test, administration of CHB-II-F 31.44 g/kg showed no adverse effect and did not cause mortality. In the subchronic toxicity test, after 4 weeks of treatment, compared to the controls, total cholesterol (TCHO) level, cardiac and splenic indexes, body weights of female rats, and mean corpuscular hemoglobin concentration (MCHC) in the CHB-II-F (H) group were significantly increased; triglyceride (TG) in the CHB-II-F (M) group and liver and splenic indexes in the CHB-II-F (L) group were increased. After the two-week recovery period, biofluid analyses, food consumption, and histopathologic examinations showed no abnormalities. CONCLUSION: Administration of CHB-II-F had no obvious adverse effect on the overall health of rodent models. A daily maximum dose of less than 94.31 g/kg or 6.55 g/kg CHB-II-F for 4 continuous weeks was considered safe.

The Antioxidant Activity of Recombinant Rat Hepatic Fatty Acid Binding Protein T94A Variant.

PURPOSE: Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies. METHODS: Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate. RESULTS: Although the FABP1 T94A (20 µM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 µM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 µM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only. CONCLUSIONS: The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.

The effects of Ciji-Hua'ai-Baosheng on immune function of mice with H22 hepatocellular carcinoma receiving chemotherapy.

BACKGROUND: Ciji-Hua'ai-Baosheng Decoction (CHBD) is a traditional Chinese formula that may attenuate the toxicity and side-effects of chemotherapy. The formula may also prolong the life of cancer patients. Whether CHBD should be employed as adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined as does the mechanism whereby CHBD exerts its beneficial effects. AIM OF THE STUDY: To document the potential effects of CHBD on tumor growth and immune function in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty Kunming mice were injected subcutaneously with H22 hepatoma cells in the right anterior armpit. After seven days, the mice with formed tumors were injected with Cytoxan (CTX) (200 mg/kg) to establish the chemotherapy model. These mice were randomly divided into 5 groups: model (untreated controls), control (CTX,33.33 mg/kg), and high CHBD (H) (117 g/kg), moderate CHBD (M) (58.5 g/kg) and low CHBD (L) (29.25 g/kg) treated groups. Tumor weights and inhibitory ratio (decrease in tumor dimensions), histology of tumor, colon, spleen and liver, and biochemical tests of liver and kidney function were documented after 10 days. Serum and tumor IL-2, IFN-γ, IL-6, and TNF-α levels were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot respectively. The potential bioactive compounds in CHBD were characterized by UHPLC-MS. RESULTS: Although tumor weights were decreased in CTX alone and CHBD (H) and CHBD (M) groups (-66%, -41% and -25% respectively), tumor cell density was reduced to the greatest extent in the CHBD (H) group. CHBD had no evident effects on liver and kidney function. CTX-induced colon inflammation and decrease in spleen lymphocytes were attenuated with CHBD treatment. CHBD increased serum IL-2, IFN-γ and TNF-α, but decreased IL-6 levels in serum and tumor tissue. UHPLC-MS analysis of CHBD revealed the presence of 11 bioactive compounds. CONCLUSIONS: In this murine model of HCC receiving chemotherapy, CHBD inhibited tumor growth, improved immune function and pro-inflammatory cytokine responses while attenuating CTX-associated side effects.

High fiber dietary and sodium butyrate attenuate experimental autoimmune hepatitis through regulation of immune regulatory cells and intestinal barrier.

Autoimmune hepatitis (AIH) is chronic autoimmune liver disease accompanied with the imbalance of Treg/Th17 and increased intestinal permeability. We investigated the effects of a high fiber diet and sodium butyrate on the Treg/Th17 and intestinal barrier function in an experimental autoimmune hepatitis. Intraperitoneal injection of hepatic antigen (S100) was used to induce experimental autoimmune hepatitis mice model and mice were divided into normal control, S100 model control, S100 plus high fiber diet and S100 plus sodium butyrate. Serum aminotransferases and liver histology were examined. Short chain fatty acids in feces were determined by HPLC. The ratio of CD4 + C25 + Foxp3+ Treg and CD4 + IL-17 + Th17 were evaluated by flow cytometry. Tight junction proteins Zonula ocluden, Occludin and Claudin-1 were used to assess intestinal barrier function, so does Escherichia coli protein in the liver. Mice fed with either high fiber diet or sodium butyrate showed significantly lower levers of serum aminotransferases and minor liver injury compared to that of model control. Moreover, the ratio of Treg/Th17 was significantly higher in high fiber diet and sodium butyrate fed mice than that in model control. Furthermore, high fiber diet and sodium butyrate significantly increased intestinal tight junction proteins and decreased Escherichia Coli protein in the liver. In conclusion, high fiber diet and sodium butyrate can attenuate development of autoimmune hepatitis through regulation of immune regulatory cells and intestinal barrier function.

Inhibition of BMP4 and Alpha Smooth Muscle Actin Expression in LX-2 Hepatic Stellate Cells by BMP4-siRNA Lipid Based Nanoparticle.

PURPOSE: To develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to hepatic stellate cells (HSC). VA was selected to increase the uptake by HSC based on their function in the storage of VA. METHODS: DOTAP/DOPE liposomes were prepared by film hydration method and their surfaces were decorated with VA. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 assay. Inhibition of BMP4 and α-SMA was determined by PCR and ELISA. RESULTS: VA-coated lipoplexes exhibited an average particle sizes less than 200 nm and zeta potential around +25 mV both determined using ZetaPALS. Inclusion of VA to liposomal surfaces significantly enhanced their cellular uptake without affecting cytotoxicity. VA-coupled liposomes carrying BMP4-siRNA resulted in a significant reduction in BMP4 and α-SMA at both mRNA and protein levels.  Conclusion: VA-coated liposomes were successfully designed to deliver BMP4-siRNA to specifically target HSC. The novel delivery system discussed herein may serve as a potential therapeutic strategy for the treatment of liver fibrosis in the future. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The Protective Effects of Ciji-Hua'ai-Baosheng II Formula on Chemotherapy-Treated H22 Hepatocellular Carcinoma Mouse Model by Promoting Tumor Apoptosis.

Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medical formula that has been shown in clinical practice to relieve side effects of chemotherapy and improve quality of life for cancer patients. In order to understand the mechanism of its protective effects on chemotherapy, mice with transplanted H22 hepatocellular carcinoma were employed in this study. Ninety-two mice were injected subcutaneously with H22 HCC cell suspension into the right anterior armpit. After mice were treated with 5-fluorine pyrimidine (5-FU), they were divided into six groups as untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji Capsule group and three groups of 5-FU plus different concentrations of CHB-II-F. Twenty mice were euthanized after 7 days of treatment in untreated and medium concentration of CHB-II-F groups and all other mice were euthanized after 14 days of treatment. Herbal components/metabolites were analyzed by UPLC-MS. Tumors were evaluated by weight and volume, morphology of light and electron microscope, and cell cycle. Apoptosis were examined by apoptotic proteins expression by western blot. Four major components/metabolites were identified from serum of mice treated with CHB-II-F and they are ß-Sitosterol, Salvianolic acid, isobavachalcone, and bakuchiol. Treatment of CHB-II-F significantly increased body weights of mice and decreased tumor volume compared to untreated group. Moreover, CHB-II-F treatment increased tumor cells in G0-G1 transition instead of in S phase. Furthermore, CHB-II-F treatment increased the expression of pro-apoptotic proteins and decreased the expression anti-apoptotic protein. Therefore, CHB-II-F could improve mice general condition and reduce tumor cell malignancy. Moreover, CHB-II-F regulates apoptosis of tumor cells, which could contribute its protective effect on chemotherapy.

BMSCs-derived miR-223-containing exosomes contribute to liver protection in experimental autoimmune hepatitis.

Autoimmune hepatitis is a chronic inflammatory disease in the liver with potential to the development of liver fibrosis. Recent evidences suggest that bone marrow derived mesenchymal stem cells (BMSCs) may exert its therapeutic activity through exosomes. Moreover, miR-223 is highly expressed in BMSCs and plays an important role in autoimmune diseases. Therefore, in this study, hepatoprotective role of BMSCs and miR-223 was investigated in both mice and hepatocytes. Liver antigen S100 was used to establish autoimmune hepatitis model in mice while LPS and ATP were used to establish cell injury model in hepatocyte. Before the experiments, BMSCs were infected with pre-miR-223 and transfected with miR-223 inhibitor respectively. Exosomes from bone marrow stem cells were isolated by ultracentrifugation. Liver injury was evaluated by serum levels of ALT and AST as well as liver histology. Inflammation and cell death were examined by inflammatory cytokines and lactase dehydrogenase respectively. Both BMSCs-exo and BMSCs-exomiR-223(+) significantly reversed either S100 or LPS/ATP induced injury in mice and hepatocytes. Meanwhile, the expressions of cytokines, NLRP3 and caspase-1 were also downregulated by BMSCs-exo and BMSCs-exomiR-223(+) at both protein and mRNA levels in mice and hepatocytes. Moreover, BMSCs-exomiR-223(-) reverses the effects of BMSCs-exo and BMSCs-exomiR-223(+) in mouse AIH and in hepatocytes. In conclusion, bone marrow stem cell derived exosomes can protect liver injury in an experimental model of autoimmune hepatitis and the mechanism could be related to exosomal miR-223 regulation of NLRP3 and caspase-1.

Glycochenodeoxycholic Acid Does Not Increase Transforming Growth Factor-Beta Expression in Bile Duct Epithelial Cells or Collagen Synthesis in Myofibroblasts.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a chronic, progressive hepatobiliary disorder characterized by extensive fibrosis and stricturing of the intra- and/or extra-hepatic bile ducts: Previous studies have documented low phosphatylcholine (PC) concentrations in PSC bile. The aim of this study was to determine whether low PC levels in bile facilitate toxic bile acid induced injury of biliary tract epithelial cells resulting in enhanced transforming growth factor-beta (TGF-ß) expression and increased collagen synthesis by myofibroblasts. METHODS: TGF-ß mRNA expression was documented in bile duct epithelial cells exposed to varying concentrations of the toxic bile acid; glycochenodeoxycholic acid (GCDCA) ± PC. RESULTS: In these experiments, as well as in co-culture experiments where bile duct epithelial cells were cultured with peripheral blood mononuclear cells and myofibroblasts, TGF-ß mRNA expression remained unaltered in the presence or absence of PC. Moreover, collagen type Iα1 mRNA expression by myofibroblasts also remained unaltered. CONCLUSION: The results of this study do not support the hypothesis that PC deficiency contributes to toxic bile acid-induced bile duct injury and/or myofibroblast activation.

Long-Term, Low-Dose Exposure to Microcystin-LR Does not Cause or Increase the Severity of Liver Disease in Rodents.

BACKGROUND: Acute exposure to high concentrations of microcystin-LR (MC-LR) can cause significant hepatocyte injury. AIM: To document the effects of long-term, low-dose MC-LR exposure on hepatic inflammation and fibrosis in mice with healthy and diseased livers. MATERIAL AND METHODS: Male CD1 mice (N = 20/group) were exposed to 1.0 µg/L of MC-LR in drinking water; 1.0 µg/L MC-LR plus 300 mg/L of the hepatotoxin thioacetamide (MC-LR/TAA); or 300 mg/L TAA alone for 28 weeks. Liver biochemistry and histology were documented at the end of the study period. In addition, hepatic stellate cells (HSCs), were exposed in vitro to MC-LR (0.1-10,000 µg/L) and monitored for changes in cell metabolism, proliferation and activation. RESULTS: Liver biochemistry and histology were essentially normal in MC-LR alone exposed mice. MC-LR/TAA and TAA alone exposed mice had significant hepatic inflammation and fibrosis but the extent of the changes were similar in the two groups. In vitro, MC-LR had no effect on HSC metabolism, proliferation or activation. CONCLUSION: Long-term, low-dose exposure to MC-LR is unlikely to lead to chronic liver disease in the setting of a normal liver or exacerbate existing liver disease in the setting of ongoing hepatitis.

Sodium butyrate ameliorates S100/FCA-induced autoimmune hepatitis through regulation of intestinal tight junction and toll-like receptor 4 signaling pathway.

BACKGROUND AND AIM: Recent investigation revealed that dysbiosis in the gut flora and disruption of permeability of intestinal barrier are possible causes for the development of autoimmune hepatitis. Supplementation of sodium butyrate has been suggested to protect liver injury from disrupted permeability of small intestine. In current study, we employed S100/Freund's complete adjuvant induced autoimmune hepatitis to investigate therapeutic efficacy of sodium butyrate and its mechanism in the liver and upper small intestine. METHODS: C57BL/6 mice were employed and divided into three groups - control group (n=8), autoimmune hepatitis group (n=12) and autoimmune hepatitis with treatment of sodium butyrate group (n=12). Histological staining and western blot analyses were employed to evaluate liver and upper small intestine morphology and gene expression respectively. RESULTS: The findings revealed that S100/Freund's complete adjuvant caused liver injury and disruption of upper small intestine villi. Sodium butyrate attenuated the injuries and prevented migration of Escherichia coli into the liver. Moreover, the effect of sodium butyrate on protection of injuries of the liver and upper small intestine could be due to inhibition of toll-like receptor 4 signaling pathway, as well as its down-regulation of inflammatory cytokines - interleukin-6 and tumor necrosis factor-a. CONCLUSIONS: Sodium butyrate can prevent liver injury by maintaining the integrity of small intestine and inhibiting inflammatory response in S100/Freund's complete adjuvant induced autoimmune hepatitis.

Active immunization against transforming growth factor beta1 prevents hepatic fibrosis in a rat model of liver disease.

Transforming growth factor beta1 (TGF-ß1) plays an important role in hepatic fibrogenesis. In this study, we documented the effects of active immunization against TGF-ß1 on hepatic fibrosis in an animal model of chronic liver disease. BALB/c mice were immunized against 3 different peptides of TGF-ß1 ligated into hepatitis B virus core protein (HBVc). Titers of TGF-ß1 antibodies were documented by enzyme linked immunoassays and antibody activity by cell membrane receptor binding and proliferation assays. The most immunogenic recombinant HBVc + TGF-ß1 peptide (HBVc + C) then served as a vaccine in Sprague-Dawley rats with dimethylnitrosamine-induced chronic liver disease. Hepatic fibrosis was documented by serum hyaluronic acid levels, liver histology, and reverse transcriptase polymerase chain reaction for hepatic collagen I (α1) and smooth muscle alpha actin mRNA expression. Relative to control rats vaccinated with HBVc alone, recombinant HBVc + C vaccinated animals had significantly lower serum hyaluronic acid levels, less histologic evidence of hepatic fibrosis, and reduced expression of collagen I (α1) and smooth muscle alpha actin mRNA in the liver. The results of this proof-of-concept study suggest that active immunization against TGF-ß1 is a worthwhile strategy to pursue in efforts to prevent hepatic fibrosis associated with chronic liver disease.