Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However, it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here, we synthesized a novel schiff base zinc coordination compound (SBZCC) and investigated its effects on the growth, proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression, mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover, SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis, accompanied with increased Bax/Bcl-2 and FlasL/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways, suggesting that SBZCC is a promising agent for the development as anticancer drugs.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Coordination Complexes ; chemical synthesis ; pharmacology ; Fas Ligand Protein ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; pathology ; Osteoblasts ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Schiff Bases ; chemistry ; Signal Transduction ; Zinc ; chemistry ; bcl-2-Associated X Protein ; genetics ; metabolism ; fas Receptor ; genetics ; metabolism

The aim of the present study was to investigate the regulatory effects of histone methylation modifications on the expression of miR-200c, as well as invasion and migration of gastric carcinoma cells. Gastric carcinoma cell line, MGC-803, were treated by 2.5 μmol/L histone methyltransferase inhibitor, DZNep. The expression of miR-200c was detected by real-time quantitative PCR (qRT-PCR). The epithelial-mesenchymal transition (EMT) indicators (ZEB1/2 and E/N-cadherin), EZH2, EED, SUZ12 and H3K27me3 expressions were detected by Western blot. Cell migration and invasion abilities were detected by Transwell and scratch tests. The result showed that, compared with DMSO (control) group, DZNep significantly increased the expression of miR-200c to about 2.1 times, inhibited ZEB1, ZEB2, and N-cadherin expressions, and activated E-cadherin expression; Also, DZNep decreased the protein expressions of EZH2, EED, SUZ12 and H3K27me3; Moreover, DZNep could inhibit MGC-803 cell invasive and migrative abilities, as well as MMP9 expression. These results suggest DZNep raises miR-200c expression to delay the invasion and migration of gastric carcinoma cells, and the underlying mechanisms involve the regulations of EMT-related proteins and polycomb repressive complex 2.
Adenosine ; analogs & derivatives ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Homeodomain Proteins ; metabolism ; Humans ; MicroRNAs ; metabolism ; Protein Methyltransferases ; antagonists & inhibitors ; Repressor Proteins ; metabolism ; Transcription Factors ; metabolism ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo investigate the effects of bisdemethoxycurcumin (BDMC) on non-small cell lung cancer (NSCLC) cell line, A549, and the highly metastatic lung cancer 95D cells.

METHODSCCK-8 assay was used to assess the effect of BDMC on cytotoxicity. Flow cytometry was used to evaluate apoptosis. Western blot analysis, electron microscopy, and quantification of GFP-LC3 punctuates were used to test the effect of BDMC on autophagy and apoptosis of lung cancer cells.

RESULTSBDMC inhibited the viability of NSCLC cells, but had no cytotoxic effects on lung small airway epithelial cells (SAECs). The apoptotic cell death induced by BDMC was accompanied with the induction of autophagy in NSCLC cells. Blockage of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) repressed the growth inhibitory effects and induction of apoptosis by BDMC. In addition, BDMC treatment significantly decreased smoothened (SMO) and the transcription factor glioma-associated oncogene 1 (Gli1) expression. Furthermore, depletion of Gli1 by siRNA and cyclopamine (a specific SMO inhibitor) induced autophagy.

CONCLUSIONAberrant activation of Hedgehog (Hh) signaling has been implicated in several human cancers, including lung cancers. The present findings provide direct evidence that BDMC-induced autophagy plays a pro-death role in NSCLC, in part, by inhibiting Hedgehog signaling.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cell Line, Tumor ; Curcumin ; analogs & derivatives ; chemistry ; pharmacology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Hedgehog Proteins ; genetics ; metabolism ; Humans ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Signal Transduction ; drug effects ; Zinc Finger Protein GLI1

Hypoxia-inducible factor 1alpha (HIF-1alpha), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1alpha is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1alpha. The poly-ubiquitination of HIF-1alpha was resumed by restoration of free ubiquitin, which suggests that the HIF-1alpha stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1alpha with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1alpha.
Cell Hypoxia/physiology ; Cell Line, Tumor ; HCT116 Cells ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis/*metabolism ; Leupeptins/pharmacology ; Nickel/chemistry ; Proteasome Endopeptidase Complex/*metabolism ; Proteasome Inhibitors/*pharmacology ; Ubiquitin/*metabolism ; Ubiquitination/*physiology ; Up-Regulation ; Zinc/chemistry

OBJECTIVETo investigate the effects of zinc deficiency on the relevant immune function in rats with LPS-induced sepsis.

METHODSSixty rats were divided into low zinc group (LZ), normal zinc pair-fed group (NP), and normal zinc control group (NC) according to the random number table, with 20 rats in each group. The rats in group LZ were fed with low zinc diet, and the rats in group NP were fed with normal zinc diet, with the same intake as that of group LZ by manual control, and the rats in group NC were fed with normal zinc diet freely. After being fed for 7 d, the rats all fasted and were further divide into the below subgroups named LZ-LPS, LZ-normal saline (NS), NP-LPS, NP-NS, NC-LPS, and NC-NS according to the random number table, with 10 rats in each subgroup. Rats in the LPS subgroups were intraperitoneally injected with 1 mg/mL LPS solution with the dosage of 5 mg/kg, rats in the corresponding NS subgroups were intraperitoneally injected with equivalent NS. The rats were sacrificed at post injection hour 6 to collect blood, spleen, and thymus. The serum level of zinc was detected by inductively coupled plasma mass spectrometry, and the serum alkaline phosphatase (ALP) activity was detected by automatic blood biochemical analyzer. The body weight and weight of spleen and thymus of rats were weighed, and the indices of spleen and thymus were calculated. Six routine blood indices were examined by automatic blood cell analyzer. The serum levels of interferon gamma (IFN-γ), TNF-α, IL-4, and IL-10 were determined with ELISA, and the ratio of IFN-γ to IL-4 was calculated. Data were processed with one-way analysis of variance and SNK test.

RESULTS(1) Serum levels of zinc and ALP activity in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (2) Body weight, spleen and thymus weight, indices of spleen and thymus in the LPS subgroups were similar with those in the corresponding NS subgroups (with P values above 0.05). The 4 former indices, except for body weight, in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The 4 former indices, except for body weight, in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). (3) Levels of leucocyte count in subgroups LZ-LPS and NP-LPS were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). Level of leucocyte count in subgroup NC-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Level of leucocyte count in subgroup NC-LPS was significantly lower than that in subgroup LZ-LPS (P<0.05). Levels of neutrophilic granulocyte count (NGC) and NG in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-LPS were significantly lower than those in subgroup LZ-LPS (with P values below 0.05). Level of NG in subgroup NC-NS was significantly lower than that in subgroup LZ-NS (P<0.05). Levels of lymphocyte count and lymphocyte in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (1.8 ± 0.4) × 10⁹/L, (1.0 ± 0.3)× 10⁹/L, (2.6 ± 0.7) × 10⁹/L, (1.4 ± 0.4) × 10⁹/L, (3.3 ± 0.6) × 10⁹/L, (1.5 ± 0.5) × 10⁹/L, and 0.39 ± 0.10, 0.11 ± 0.03, 0.47 ± 0.12, 0.14 ± 0.04, 0.50 ± 0.09, 0.24 ± 0.07. The two former indices in the LPS subgroups were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroup NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05). Level of lymphocyte count in subgroup NP-NS was significantly higher than that in subgroup LZ-NS (P<0.05). Levels of platelet count (PC) in subgroups NP-LPS and NC-LPS were significantly lower than those in the corresponding NS subgroups (with P values below 0.05). Levels of PC in subgroups NP-NS and NC-NS were significantly higher than those in subgroup LZ-NS (with P values below 0.05). Level of PC in subgroup NC-LPS was significantly higher than that in subgroup LZ-LPS (P<0.05). (4) Serum levels of TNF-α, IL-4, and IL-10 in each subgroup showed no significant differences (with P values above 0.05). Serum levels of IFN-γ and ratios of IFN-γ to IL-4 in subgroups LZ-NS, LZ-LPS, NP-NS, NP-LPS, NC-NS, and NC-LPS were respectively (75 ± 21), (233 ± 40), (80 ± 14), (345 ± 74), (66 ± 7), (821 ± 189) pg/mL, and 3.1 ± 1.0, 6.6 ± 1.7, 3.9 ± 1.7, 20.2 ± 8.3, 3.4 ± 1.5, 45.7 ± 7.6. The two former indices in the LPS subgroups were significantly higher than those in the corresponding NS subgroups (with P values below 0.05). The two former indices in subgroups NP-NS and NC-NS were similar with those in subgroup LZ-NS (with P values above 0.05). The two former indices in subgroups NP-LPS and NC-LPS were significantly higher than those in subgroup LZ-LPS (with P values below 0.05).

CONCLUSIONSZinc deficiency can induce the atrophy of spleen and thymus, and reduction of peripheral blood lymphocyte. In sepsis, zinc deficiency can further decrease the production of IFN-γ, thus making the cytokines of Th1/Th2 shift to Th2 and the immune imbalance worse.

Animals ; Cytokines ; Interferon-gamma ; Interleukin-10 ; Interleukin-4 ; Lipopolysaccharides ; pharmacology ; Rats ; Sepsis ; chemically induced ; Tumor Necrosis Factor-alpha ; metabolism ; secretion ; Zinc ; deficiency

OBJECTIVETo investigate the effect of tetrandrine (TET) on zinc finger protein 139 (ZNF139) and multidrug resistance (MDR) of human gastric carcinoma cell lines and possible mechanisms.

METHODSCultured SGC7901 and SGC7901/ADR were treated with TET (0.5, 1.0, 1.5, 2.0, and 2.5 microg/mL), then inhibition rates were measured by MTT assay in vitro. The expressions of ZNF139, MRP-1, MDR1, and GST-pi were detected by RT-PCR. The correlation between ZNF139 and each multidrug resistance factor was analyzed using Spearman correlation analysis, and the coefficient correlation was calculated.

RESULTSThe inhibition rate of TET (< or = 2.0 microg/mL) for SGC7901 and SGC7901/ADR was less than 10% with MTT assay. Expressions of ZNF139, MRP-1, MDR1, and GST-pi mRNA were higher in SGC7901/ADR than in SGC7901 (all P < 0.05). The expressions of ZNF139, MRP-1, MDR1, and GST--pi were down-regulated in SGC7901/ADR cells efficiently (all P < 0.01). Positive correlation existed between ZNF139 and MRP-1, ZNF139 and MDR1 before treated by TET in SGC7901/ADR, and this relationship also existed in SGC7901/ADR cells after treated by TET (all P < 0.05).

CONCLUSIONTET could achieve MDR reversion in gastric cancer cells by down-regulating the expression of ZNF139, MRP-1, and MDR1.

ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Benzylisoquinolines ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Multiple ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Humans ; Kruppel-Like Transcription Factors ; metabolism ; Multidrug Resistance-Associated Proteins ; metabolism ; Stomach Neoplasms ; metabolism ; Zinc Fingers ; genetics

OBJECTIVETo investigate the antiproliferative and anti-metastasis effect of Xihuang Pill (, XP) on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects.

METHODSHighly metastatic human colorectal cancer cell line LoVo was treated with low-, medium-, and highdose XP-containing serum (XP-L, XP-M, XP-H) groups for 48 h, cells intervened with no drug rat serum and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor] as negative and positive controls (NC and PC) groups. Cell proliferation assay was made using cell counting kit-8 (CCK8). The 8 μm pore-size transwell chamber and 4', 6-diamidino-2-phenylindole (DAPI) staining were applied to examine the ability of invasion and migration of the cells. The protein expression of ERK1/2, zinc fifi nger E-box-binding homeobox 1 (ZEB1), Scrib and lethal giant larvae homolog 2 (Lgl2) was detected by Western blotting while the relative mRNA quantity of E-cadherin, N-cadherin, Occludin and junctional adhesion molecule-1 (JAM1) was measured by realtime fluorescent quantitative polymerase chain reaction (RT-qPCR).

RESULTSXP induced a dose-dependent suppression on the proliferation of LoVo cells (P <0.05 or P<0.01), with the inhibition rates varied from 27.30% to 31.08%. Transwell assay showed that when preprocessed with PD98059 and XP-containing serum, the number of cells that passed the filter decreased significantly compared with that of NC group (P <0.05 or P<0.01). Moreover, XP inhibited the protein expression of ERK1/2 and ZEB1 (P <0.05); and up-regulated the protein expression of Scrib and Lgl2 (P <0.05). The mRNA levels of E-cadherin, Occludin and JAM1 of the XP intervened groups and PC group markedly ascended (P <0.05) while that of N-cadherin showed a descending tendency (P>0.05).

CONCLUSIONXP intervention suppressed the ability of proliferation, invasion and migration of the LoVo cells. Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.

Animals ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Polarity ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Homeodomain Proteins ; metabolism ; Humans ; Intercellular Junctions ; drug effects ; metabolism ; Membrane Proteins ; metabolism ; Neoplasm Invasiveness ; Phenotype ; RNA, Messenger ; genetics ; metabolism ; Rats, Wistar ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism ; Zinc Finger E-box-Binding Homeobox 1

OBJECTIVETo determine the role of zinc finger protein 1 (ZEB 1) in liver fibrosis and in regards to expression of the tumor growth factor-beta (TGFb) signaling factor using a rat model system.

METHODSSprague-Dawley rats were randomly divided into a normal (control) group, liver fibrosis (model) group and a liver fibrosis + therapy (ZEB1 intervention) group. The model group and the ZEB1 intervention group were given intraperitoneal injections of dimethylnitrosamine (DMN) for the first 3 days of each week over a 7-week period; starting at week 5, the ZEB 1 intervention group was started on a routine of every other day tail vein injections of recombinant ZEB1. During this 7-week period, the control group was given intraperitoneal injections of 0.9% NaC1 alone on the DMN schedule. Liver tissues were collected for pathological examination (with hematoxylin-eosin and Masson staining) and for detection of TGFb1 and ZEB 1 expression (by RT-PCR and western blotting). Measurement data were compared between groups using the single-factor analysis of variance test, followed by the least significant difference LSD test. Count data were analyzed by Fisher's exact test.

RESULTSThe model group's liver tissues showed degeneration and necrosis, as well as obvious fibrous septa accompanied by pseudo lobules. The ZEB 1 intervention group's liver tissues showed a significantly higher degree of fibrosis (x²=21.63, P=0), with more coarse fiber cords. The expression of ZEB1 and TGFb1 was significantly higher in the model group than in the control group (both P less than 0.05). However, the ZEB 1 intervention group showed the highest levels of ZEB 1 and TGFb1 expression (vs. model group, P less than 0.05).

CONCLUSIONZEB 1 may promote the development of liver fibrosis in rats through a mechanism involving the TGFb/Smad signaling pathway.

Animals ; Homeodomain Proteins ; pharmacology ; Liver ; drug effects ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; pharmacology ; Transforming Growth Factor beta1 ; metabolism ; Zinc Fingers

OBJECTIVETo investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism.

METHODSTo establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.

RESULTSThe fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group.

CONCLUSIONSThe overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.

Alanine Transaminase ; analysis ; Animals ; Aspartate Aminotransferases ; analysis ; Dimethyl Sulfoxide ; pharmacology ; Disease Models, Animal ; Fluoride Poisoning ; drug therapy ; metabolism ; Fluorosis, Dental ; diagnosis ; Hedgehog Proteins ; antagonists & inhibitors ; metabolism ; Hepatocytes ; metabolism ; Kruppel-Like Transcription Factors ; metabolism ; Liver ; metabolism ; Liver Diseases ; drug therapy ; metabolism ; RNA, Messenger ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Veratrum Alkaloids ; pharmacology ; Zinc Finger Protein GLI1