Sodium Ferulate Inhibits Rat Cardiomyocyte Hypertrophy Induced by Angiotensin II Through Enhancement of Endothelial Nitric Oxide Synthase/Nitric Oxide/Cyclic Guanosine Monophosphate Signaling Pathway.

ABSTRACT: Sodium ferulate (SF) is the sodium salt of ferulic acid, which is one of the effective components of Angelica sinensis and Lignsticum chuanxiong , and plays an important role in protecting the cardiovascular system. In this study, myocardial hypertrophy was induced by angiotensin II 0.1 µmol/L in neonatal Sprague-Dawley rat ventricular myocytes. Nine groups were designed, that is, normal, normal administration, model, L-arginine (L-arg 1000 µmol/L), SF (50, 100, 200 µmol/L) group, and N G -nitro-L-arg-methyl ester 1500 µmol/L combined with SF 200 µmol/L or L-arg 1000 µmol/L group, respectively. Cardiomyocyte hypertrophy was confirmed by observing histological changes and measurements of cell diameter, protein content and atrial natriuretic factor, and ß-myosin heavy chain levels of the cells. Notably, SF could inhibit significantly myocardial hypertrophy of neonatal rat cardiomyocytes in a concentration-dependent manner without producing cytotoxicity, and the levels of nitric oxide, NO synthase (NOS), endothelial NOS, and cyclic guanosine monophosphate were increased, but the level of cyclic adenosine monophosphate was decreased in cardiomyocytes. Simultaneously, levels of protein kinase C beta, Raf-1, and extracellular regulated protein kinase 1/2 (ERK1/2) were downregulated, whereas levels of mitogen-activated protein kinase phosphatase-1 were significantly upregulated. All the beneficial effects of SF were blunted by N G -nitro-L-arg-methyl ester. Overall, these findings reveal that SF can inhibit angiotensin II-induced myocardial hypertrophy of neonatal rat cardiomyocytes, which is closely related to activation of endothelial NOS/NO/cyclic guanosine monophosphate, and inhibition of protein kinase C and mitogen-activated protein kinase signaling pathways.

GC-MS Profile of Hua-Feng-Dan and RNA-Seq Analysis of Induced Adaptive Responses in the Liver.

Background: Hua-Feng-Dan is a patent Chinese medicine for stroke recovery and various diseases. This study used GC-MS to profile its ingredients and RNA-Seq to analyze the induced adaptive response in the liver. Methods: Hua-Feng-Dan was subjected to steam distillation and solvent extraction, followed by GC-MS analysis. Mice were orally administered Hua-Feng-Dan and its "Guide drug" Yaomu for 7 days. Liver pathology was examined, and total RNA isolated for RNA-Seq, followed by bioinformatic analysis and quantitative real-time PCR (qPCR). Results: Forty-four volatile and fifty liposoluble components in Hua-Feng-Dan were profiled and analyzed by the NIST library and their concentrations quantified. The major components (>1%) in volatile (5) and liposoluble (10) were highlighted. Hua-Feng-Dan and Yaomu at hepatoprotective doses did not produce liver toxicity as evidenced by histopathology and serum enzyme activities. GO Enrichment revealed that Hua-Feng-Dan affected lipid homeostasis, protein folding, and cell adhesion. KEGG showed activated cholesterol metabolism, bile secretion, and PPAR signaling pathways. Differentially expressed genes (DEGs) were identified by DESeq2 with p < 0.05 compared to controls. Hua-Feng-Dan produced more DEGs than Yaomu. qPCR on selected genes largely verified RNA-Seq results. Ingenuity Pathways Analysis of the upstream regulator revealed activation of MAPK and adaptive responses by Hua-Feng-Dan, and Yaomu was less effective. Hua-Feng-Dan-induced DEGs were highly correlated with the Gene Expression Omnibus database of chemical-induced adaptive transcriptome changes in the liver. Conclusion: GC-MS primarily profiled volatile and liposoluble components in Hua-Feng-Dan. Hua-Feng-Dan at the hepatoprotective dose did not produce liver pathological changes but induced metabolic and signaling pathway activations. The effects of Hua-Feng-Dan on liver transcriptome changes point toward induced adaptive responses to program the liver to produce hepatoprotective effects.

Zuotai (ß-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice.

Background: Zuotai (mainly ß-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases.  We have shown that 70W protected against CCl 4 hepatotoxicity.  CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation.   Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects.  Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate.

Mercury sulfide-containing Hua-Feng-Dan and 70W (Rannasangpei) protect against LPS plus MPTP-induced neurotoxicity and disturbance of gut microbiota in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Mercury sulfides (HgS) are frequently included in Ayurveda, Tibetan and Chinese medicines to assist the presumed therapeutic effects, but the ethnopharmacology remains elusive. The present study examined the protective effects of α-HgS-containing Hua-Feng-Dan and ß-HgS-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) against Parkinson's disease mice induced by lipopolysaccharide (LPS) plus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). METHOD: A single injection of LPS (5 mg/kg ip) was given to adult male C57BL/6 mice, and 150 days later, the low dose of MPTP (15 mg/kg, ip, for 4 days) was given to produce the "two-hit" Parkinson's disease model. Together with MPTP treatment, mice were fed with clinically-relevant doses of Hua-Feng-Dan (0.6 g/kg) and 70W (0.2 g/kg) for 35 days. Rotarod test was performed to examine muscle coordination capability. At the end of the experiment, brain was transcardially perfused with paraformaldehyde, the substantia nigra was sectioned for microglia (Iba1 staining) and dopaminergic neuron (THir staining) determination. Colon bacterial DNA was extracted and subjected to qPCR analysis with 16S rRNA probes. RESULTS: The low-grade, chronic neuroinflammation produced by LPS aggravated MPTP neurotoxicity, as evidenced by decreased motor activity, intensified microglia activation and loss of dopaminergic neurons. Both Hua-Feng-Dan and 70W increased rotarod activity and ameliorated the pathological lesions in the brain. In gut microbiomes examined, LPS plus MPTP increased Verrucomicrobiaceae, Methanobacteriaceae, Pronicromonosporaceae, and Clostridaceae species were attenuated by Hua-Feng-Dan and 70W. CONCLUSIONS: α-HgS-containing Hua-Feng-Dan and ß-HgS-containing 70W at clinical doses protected against chronic LPS plus MPTP-induced toxicity to the brain and gut, suggesting HgS-containing traditional medicines could target gut microbiota as a mechanism of their therapeutic effects.

Age-associated changes of cytochrome P450 and related phase-2 gene/proteins in livers of rats.

Cytochrome P450s (CYPs) are phase-I metabolic enzymes playing important roles in drug metabolism, dietary chemicals and endogenous molecules. Age is a key factor influencing P450s expression. Thus, age-related changes of CYP 1-4 families and bile acid homeostasis-related CYPs, the corresponding nuclear receptors and a few phase-II genes were examined. Livers from male Sprague-Dawley rats at fetus (-2 d), neonates (1, 7, and 14 d), weanling (21 d), puberty (28 and 35 d), adulthood (60 and 180 d), and aging (540 and 800 d) were collected and subjected to qPCR analysis. Liver proteins from 14, 28, 60, 180, 540 and 800 days of age were also extracted for selected protein analysis by western blot. In general, there were three patterns of their expression: Some of the drug-metabolizing enzymes and related nuclear receptors were low in fetal and neonatal stage, increased with liver maturation and decreased quickly at aging (AhR, Cyp1a1, Cyp2b1, Cyp2b2, Cyp3a1, Cyp3a2, Ugt1a2); the majority of P450s (Cyp1a2, Cyp2c6, Cyp2c11, Cyp2d2, Cyp2e1, CAR, PXR, FXR, Cyp7a1, Cyp7b1. Cyp8b1, Cyp27a1, Ugt1a1, Sult1a1, Sult1a2) maintained relatively high levels throughout the adulthood, and decreased at 800 days of age; and some had an early peak between 7 and 14 days (CAR, PXR, PPARα, Cyp4a1, Ugt1a2). The protein expression of CYP1A2, CYP2B1, CYP2E1, CYP3A1, CYP4A1, and CYP7A1 corresponded the trend of mRNA changes. In summary, this study characterized three expression patterns of 16 CYPs, five nuclear receptors, and four phase-II genes during development and aging in rat liver, adding to our understanding of age-related CYP expression changes and age-related disorders.

HgS and Zuotai differ from HgCl2 and methyl mercury in intestinal Hg absorption, transporter expression and gut microbiome in mice.

Mercury (Hg) is generally considered as a toxic metal; yet the biological outcomes of Hg-containing compounds are highly dependent upon their chemical forms. We hypothesize that mercury sulfide (HgS) is different from HgCl2 and methylmercury (MeHg) in producing intestinal Hg absorption and disruption of gut microbiome. To test this hypothesis, mice were given orally with HgS (α-HgS, 30 mg/kg), Zuotai (ß-HgS, 30 mg/kg), HgCl2 (33.6 mg/kg, equivalent Hg as HgS), or MeHg (3.1 mg/kg, 1/10 Hg as HgS) for 7 days. Accumulation of Hg in the duodenum and ileum after HgCl2 (30-40 fold) and MeHg (10-15 fold) was higher than HgS and Zuotai (~2-fold). HgCl2 and MeHg decreased intestinal intake peptide transporter-1 and Ost-ß, and increased ileal bile acid binding protein and equilibrative nucleoside transporter-1. The efflux transporters ATP-binding cassette sub-family C member-4 (Abcc4), Abcg2, Abcg5/8, and Abcb1b were increased by HgCl2 and to a lesser extent by MeHg, while HgS and Zuotai had minimal effects. Bacterial DNA was extracted and subjected to 16S rDNA sequencing. Operational taxonomic unit (OTU) results showed that among the 10 phyla, HgS increased Firmicutes, Proteobacteria, while HgCl2 increased Bacteroidetes, Cyanobacteria and decreased Firmicutes; among the 79 families, HgS increased Rikenellaceae, Lactobacillaceae, Helicobacteraceae, and decreased Prevotellaceae, while HgCl2 increased Odoribacteraceae, Porphyromonadaceae, and decreased Lactobacillaceae; among the 232 genus/species, HgS and Zuotai affected gut microbiome quite differently from HgCl2 and MeHg. qPCR analysis with 16S rRNA confirmed sequencing results. Thus, chemical forms of mercury are a major determinant for intestinal Hg accumulation, alterations in transporters and disruption of microbiome.

Ginsenoside Re inhibits PDGF-BB-induced VSMC proliferation via the eNOS/NO/cGMP pathway.

Ginsenoside Re (GS-Re), which is a major monomeric member of the ginseng trialcohol saponin family, is one of the main active components of ginseng and plays an important role in protecting the cardiovascular system. Here, we report a novel function by which GS-Re regulates the eNOS/NO/cGMP pathway, which affects the platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of vascular smooth muscle cells (VSMCs). GS-Re inhibited PDGF-BB-induced VSMC proliferation in a concentration-dependent manner without cytotoxicity, and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) antagonized the antiproliferative effect of GS-Re. The flow cytometry analysis suggested that GS-Re regulates VSMC proliferation by influencing the cell cycle transition from G0/G1 to S phase and decreasing the expression of G0/G1-specific regulatory proteins, including proliferating cell nuclear antigen (PCNA), cyclin D1, and CDK4, in PDGF-BB-treated VSMCs, consequently upregulating the protein expression of p21. After GS-Re treatment, the levels of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) and the phos-eNOS Ser1177/eNOS protein ratio were obviously increased. In addition, treatment with L-NAME blocked the eNOS/NO/cGMP signaling pathway, and the protein levels of PCNA, cyclin D1, and CDK4 were markedly increased in GS-Re-treated VSMCs, while p21 expression was decreased in PDGF-BB-induced VSMCs. Overall, these findings reveal that GS-Re can inhibit the proliferation of VSMCs through G0/G1 cell cycle arrest, which is closely related to eNOS/NO/cGMP pathway activation. The present results provide basic pharmacological evidence of the potential prevention and treatment of cardiovascular diseases by GS-Re.

Sodium ferulate inhibits myocardial hypertrophy induced by abdominal coarctation in rats: Involvement of cardiac PKC and MAPK signaling pathways.

Sodium ferulate (SF) is the sodium salt of ferulic acid which is an active ingredient of Radix Angelica Sinensis and Ligusticum chuanxiong hort. Here, we investigated SF inhibition in a rat model of myocardial hypertrophy induced by coarctation of the abdominal aorta. Following coarctation, rats were given SF (20, 40, and 80 mg/kg/day) for 25 consecutive days. We characterized myocardial hypertrophy using myocardial hypertrophic parameters, histopathology, and gene expression of atrial natriuretic factor (ANF) -a gene related to myocardial hypertrophy. We detected the levels of angiotensin II (Ang II) and endothelin-1 (ET-1), protein kinase C beta (PKC-ß), Raf-1, extracellular regulated protein kinase 1/2 (ERK1/2), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in myocardium. Notably, coarctation of the abdominal aorta increases myocardial hypertrophic parameters, cardiac myocyte diameter, the concentration of Ang II and ET-1 in myocardium, and gene expression of ANF. SF significantly ameliorates myocardial hypertrophy caused by coarctation of the abdominal aorta; reduces concentrations of Ang II and ET-1; suppresses the overexpression of ANF, PKC-ß, Raf-1, and ERK1/2; and increases the expression of MKP-1. These results indicate that SF alleviates myocardial hypertrophy induced by coarctation of the abdominal aorta, and these protective effects could be related to the inhibition of PKC and mitogen-activated protein kinase (MAPK) signaling pathways.

Inhibitory effects of total ginsenoside on bleomycin-induced pulmonary fibrosis in mice.

Pulmonary fibrosis is the final outcome of a variety of diffuse pulmonary interstitial diseases, and it has an unclear pathogenesis. There is no effective drug treatment, so the clinical prognosis is poor. As an effective component of ginseng, total ginsenoside (TG) inhibits acute lung injury. This study determined whether TG has protective effects on pulmonary fibrosis and investigated its protective mechanisms. A pulmonary fibrosis model in BALB/c mice was established by injecting the bleomycin chemotherapeutic agent into the trachea. TG (40, 80, and 160 mg/kg/d) was given continuously for 28 days from the second day after development of the model. Pulmonary fibrosis was determined by measuring the lung coefficient, haematoxylin and eosin, and Masson's trichrome staining of lung samples, and detection of alpha smooth muscle actin expression in lung tissues. To investigate the mechanisms of anti-pulmonary fibrosis by TG, we detected the genes and proteins of the transforming growth factor-ß1 (TGF-ß1)/Smad signalling pathway and matrix metalloproteinase (MMP) system. Treatment with TG (40, 80, and 160 mg/kg/d) ameliorated pulmonary fibrosis induced by bleomycin in mice, downregulated the expression of TGF-ß1, Smad2, Smad3, MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1, and upregulated the protein expression of Smad7. These results suggest that the protective effects of TG on pulmonary fibrosis induced by bleomycin are related to regulation of the TGF-ß1/Smad signalling pathway and MMP system.

Oleanolic acid reprograms the liver to protect against hepatotoxicants, but is hepatotoxic at high doses.

Oleanolic acid (OA) is a triterpenoid that exists widely in fruits, vegetables and medicinal herbs. OA is included in some dietary supplements and is used as a complementary and alternative medicine (CAM) in China, India, Asia, the USA and European countries. OA is effective in protecting against various hepatotoxicants, and one of the protective mechanisms is reprogramming the liver to activate the nuclear factor erythroid 2-related factor 2 (Nrf2). OA derivatives, such as CDDO-Im and CDDO-Me, are even more potent Nrf2 activators. OA has recently been shown to also activate the Takeda G-protein-coupled receptor (TGR5). However, whereas a low dose of OA is hepatoprotective, higher doses and long-term use of OA can produce liver injury, characterized by cholestasis. This paradoxical hepatotoxic effect occurs not only for OA, but also for other OA-type triterpenoids. Dose and length of time of OA exposure differentiate the ability of OA to produce hepatoprotection vs hepatotoxicity. Hepatotoxicity produced by herbs is increasingly recognized and is of global concern. Given the appealing nature of OA in dietary supplements and its use as an alternative medicine around the world, as well as the development of OA derivatives (CDDO-Im and CDDO-Me) as therapeutics, it is important to understand not only that they program the liver to protect against hepatotoxic chemicals, but also how they produce hepatotoxicity.

Ginsenoside Re inhibits vascular neointimal hyperplasia in balloon-injured carotid arteries through activating the eNOS/NO/cGMP pathway in rats.

Ginsenoside Re (GS-Re) is one of the main ingredients of ginseng, a widely known Chinese traditional medicine, and has a variety of beneficial effects, including vasorelaxation, antioxidative, anti-inflammatory, and anticancer properties. The aims of the present study were to observe the effect of GS-Re on balloon injury-induced neointimal hyperplasia in the arteries and to investigate the mechanisms underlying this effect. A rat vascular neointimal hyperplasia model was generated by rubbing the endothelium of the common carotid artery (CCA) with a balloon, and GS-Re (12.5, 25 or 50 mg/kg/d) were subsequently continuously administered to the rats by gavage for 14 days. After GS-Re treatment, the vessel lumen of injured vessels showed significant increases in the GS-Re 25.0 and 50.0 mg/kg/d (intermediate- and high-dose) groups according to H.E. staining. Additionally, a reduced percentage of proliferating cell nuclear antigen (PCNA)-positive cells and an increased number of SM α-actin-positive cells were detected, and the levels of NO, cyclic guanosine monophosphate (cGMP), and eNOS mRNA as well as the phos-eNOSser1177/eNOS protein ratio were obviously upregulated in the intermediate- and high-dose groups. Moreover, the promotive effects of GS-Re on NO and eNOS expression were blocked by L-NAME treatment to different degrees. These results suggested that GS-Re can suppress balloon injury-induced vascular neointimal hyperplasia by inhibiting VSMC proliferation, which is closely related to the activation of the eNOS/NO/cGMP pathway.

Ontogeny and aging of Nrf2 pathway genes in livers of rats.

The Nrf2/Keap1 antioxidant system plays important roles in protecting against oxidative stress and toxic stimuli, which may vary in infants, elderly, and females. AIM: The constitutive expression of the Nrf2 genes during development and aging in both sexes would help our understanding of the Nrf2/Keap1 pathway in toxicological studies. MAIN METHODS: Sprague Dawley rat livers were collected at 11 age points from prenatal (-2 d), neonatal (1, 7, 14 and 21 d), at puberty (28 and 35 d), at adulthood (60 and 180 d), to aging (540 and 800 d) from both sexes. Total RNA and proteins were extracted for real-time RT-PCR and Western-blot analysis. KEY FINDINGS: The abundant mRNA expression was in the order of Nrf2, Gclm, Nqo1, Gclc, Ho-1, and Keap1. The expression of these genes except Gclc was high in fetal livers, decreased at birth, reached the first peak at 7 days of age, and gradually decreased to adult levels till 180 days of age. All these genes remained high at 540 days of age, but declined at 800 days of age, with more increases with Nqo1 and Ho-1. Females had lower fetal, neonatal, and aged levels than males. Protein expressions of Nrf2, Nqo1, Ho-1, GCLC and GCLM agree with mRNA analysis. SIGNIFICANCE: This study characterized the age- and sex-related changes of Nrf2-related gene/proteins in livers of rats, and higher expressions in newborns and aged rats could cope with increased oxidative stress in infants and elderly.

Xiaochaihutang Inhibits the Activation of Hepatic Stellate Cell Line T6 Through the Nrf2 Pathway.

Xiaochaihutang (XCHT) is one of classic prescriptions in Treatise on Febrile Diseases in China which was reported to have the effect of anti-hepatic fibrosis in vivo. Activation of hepatic stellate cells (HSCs) is now well established as a central driver of fibrosis in liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important element for anti-oxidative damage which is one of the key factors responsible for occurrence. This study was to investigate the effect of XCHT compound serum on HSCs activation and focus on the Nrf2 pathway. Rats in treatment groups were given the appropriate doses of XCHT granules (5 g/kg) and Silybin (50 mg/kg) for 6 days, and the serum were obtained. The compound serum was used to intervene HSCs. The results found that XCHT compound serum significantly inhibited the proliferation of HSCT6 cells. The number of α-SMA positive stained cells in HSCT6 cells and the content of Collagen type I (collagen-I) in supernatant were significantly decreased indicating suppression of activated HSCs. Compared with the control group, the nuclear transcription of Nrf2 and the expressions of Nqo1, GCLC, and GCLM were significantly increased in XCHT group. However, the effects of XCHT were inhibited in Nrf2-siRNA transfected HSCT6 cells. These studies demonstrated that XCHT could inhibit HSCT6 cell proliferation and activation. The mechanism might be related to up-regulation of the Nrf2 pathway against oxidative stress.

[Effect of Zuotai and HgS on gene expression of drug-metabolizing enzymes in livers of mice].

Zuotai and cinnabar(96%HgS) are contained in many traditional medicines. To examine their potential effects on drug metabolism genes, mice were orally given Zuotai or HgS at doses of 10, 30, 100, 300 mg•kg⁻¹ for 7 days. HgCl2(33.6 mg•kg⁻¹) was gavaged for control. Twenty-four hour later after the last administration, livers were collected, and expressions of genes related to metabolic enzymes and transporters were examined. Zuotai and HgS had no effects on major phase-1, phase-2 and transporter genes; HgCl2 increased the expressions of CYP2B10, CYP4A10, OATP1A4, UGT1A1, UGT2A3, SULT1A1, SULT2A1, MRP1, MRP3 and MRP4; expression of OATP1A1 was decreased by HgCl2, but not by Zuotai and HgS. Therefore, Zuotai and HgS have different adverse effects on drug-metabolizing genes from HgCl2.

Oeanolic acid protects against the hepatotoxicity of D-galactosame plus endotoxin in mice.

Oleanolic acid (OA) is a triterpenoid contained in many herbal medicines. The aim of this study was to investigate the protective effect of OA against D-galactosame plus lipopolysaccharides (D-GalN/LPS)-induced acute liver injury and the underling mechanisms. Mice were randomly divided into normal control with vehicles only (corn oil), D-GalN/LPS only (700mg/10µg/kg, ip), OA-po (200µmol/kg in corn oil, po) plus D-GalN/LPS, and OA-sc (50µmol/kg in 2% tween 80, sc) plus D-GalN/LPS groups. OA pretreatment was conducted twice daily for 4 consecutive days. Hepatotoxicity was evaluated by histopathology, serum enzyme activity, hepatic lipid peroxidation and GSH levels. To reveal the possible mechanisms of the protection, mRNA and protein expressions of toxicity-relevant genes and proteins were examined by real-time RT-PCR and western-blot analysis. Both OA-po and OA-sc at therapeutic doses successfully protected liver injury induced by D-GalN/LPS, as evidenced by reduced serum enzyme activities, prevented liver hemorrhage, massive necrosis, and reduced degenerative lesions. OA increased hepatic GSH contents and decreased lipid peroxidation (MDA) levels. Furthermore, OA significantly inhibited the mRNA expression of the tumor necrosis factor-α (TNF-α) and ER responsive gene Gadd45. D-GalN/LPS-induced activation of p-JNK and NF-κB p65, and protein overexpression of caspase-3, caspase-8, and COX2 were significantly suppressed by OA. These results clearly demonstrated OA-po is effective as OA-sc in protecting against D-GalN/LPS-induced liver injury, and the protection mechanisms are related to reduction of oxidative damage, suppression of TNFα-triggered signaling through the NF-kB and JNK pathways, thus reducing apoptosis and hepatocellular death.

Ontogeny, aging, and gender-related changes in hepatic multidrug resistant protein genes in rats.

Multidrug resistance proteins (Mrps) are efflux transporters playing important roles in endogenous substances and xenobiotics transport out of the liver. Children, elderly, gender and physio-pathological conditions could influence their expression and result in changes in drug disposition. AIM: This study was aimed to examine the development-, aging-, and sex-dependent changes in Mrp1-4 and ATP-binding cassette sub-family G member 2 (Abcg2) gene expressions in livers of rats. MAIN METHODS: The livers from male and female SD rats at development (-2, 1,7,14,21,28,35, and 60d) and aging (28, 60, 180 and 540d) were collected and total RNA was isolated, purified and subjected to real-time RT-PCR analysis. KEY FINDINGS: Results showed that expression of Mrp1 was low, while Abcg2 and Mrp2 were the high in the liver. Mrp1 expression decreased with maturity but remained constant to 540d, while Mrp3 and 4 increased with liver development, reached the peak with maturity at 35-60days of age, and slightly reduced with aging. Mrp2 and Abcg2 were high at 7days of age and maintained at relative high levels till maturity, while Abcg2 was reduced during aging. Females had higher Mrp3 and Abcg2 mRNA expression than male rats, while male rats had higher Mrp2 and Mrp4 mRNA expression. SIGNIFICANCE: The expression of hepatic Mrp1-4 and Abcg2 mRNA during development, aging in male and female rats was characterized, which could be fundamental to our understanding of age- and sex-associated variations in drug disposition in children, elderly, and women.

Age-associated differences in transporter gene expression in kidneys of male rats.

Kidney transporters are involved in the secretion and reabsorption of endogenous and exogenous molecules. Numerous factors may influence their expression and affect drug disposition, efficacy and toxicity. The present study aimed to examine the development­ and age­associated variations in primary renal transporters in rats, including 6 uptake transporters: Organic anion transporter (OAT) 1 and 3, organic cation transporter (OCT) 1, 2 and 3 and organic anion­transporting polypeptide (OATP) 4C1, and 6 efflux transporters: Multidrug resistance protein 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance­associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2­K. Kidneys from male Sprague Dawley rats during development (­2, 1, 7, 14 and 21 days), maturation (28, 35 and 60 days) and aging (180, 540 and 850 days) were collected and total RNA was extracted, purified and subjected to reverse transcription­quantitative polymerase chain reaction analysis. Total proteins were extracted for western blot analysis. OAT1 and 3, OCT1, BCRP, MRP2 and 4 and MATE2­K expression levels were low in fetal kidneys, increased gradually following birth and markedly increased on maturation and adulthood. High levels were maintained until 850 days. OCT2, OATP4C1, Mdr1b and MATE1 expression levels were low in fetal kidneys, increased gradually following birth, and increased markedly on weaning, maturation and adulthood; however, levels were decreased on aging. OCT3 mRNA expression levels were low in fetal and newborn kidneys, and had two peaks at 35 and 850 days. The selected OAT1 and 3 and MDR1 protein expression levels revealed a similar expression pattern. Thus, kidney transporter expression is affected by ontogeny and aging, which may impact drug and toxicant disposition in children and the elderly.

Xiaochaihutang attenuates liver fibrosis in rats through activation of Nrf2 pathway / 中国药理学与毒理学杂志

OBJECTIVE Oxidative sress is one of the key factor responsible for occurrence and development of hepatic fibrosis, a common consequence of chronic liver injury of multiple etiology. Nuclear factor erythroid 2-related factor 2 (Nrf2) serves as a major regulator of a celular defense system against oxidative stress. Xiaochaihutang (XCHT), a compound of seven botanical extracts used for liver diseases traditionally in East Asia. However, few studies have investigated its anti-hepatic fibrosis effects and pathophysiological mechanism of action. The present study was designed to confirm the anti-hepatic fibrosis effects and explore its potential mechanism of action by investigating the intervention of Nrf2 pathway. METHODS Liver fibrosis was induced by repeated injection of Carbon tetrachloride (CCl4) over a period of 9 weeks. Starting from the 6th week, the animals in treatment groups were given the appropriate dose of XCHT granules and silybin. Biochemical parameters, histological changes of the liver and alpha-smooth muscle actin (α-SMA) were determined. The expressions of Nrf2, Keap1, Nqo1, HO-1, Gclc and Gclm were assessed by RT-PCR and Western blot. RESULTS CCl4 caused a significant fibrosis damage in the rat liver and the liver functions and fibrosis degree were significantly improved by XCHT (5 g·kg- 1 and 10 g·kg- 1). XCHT (5 g·kg- 1 and 10 g·kg- 1) treatment significantly decreased the number of cells labeled with α-SMA antibodies. Moreover, XCHT (5 g·kg-1 and 10 g·kg-1) significantly increase Nqo1, HO-1, Gclc and Gclm expressions in the liver. CONCLUSION These studies establish XCHT is a potentially useful therapeutic agent for treatment of hepatic fibrosis and it might be via regulation of Nrf2 pathway in rats against oxidative stress, making further efforts to inhibiting the activated HSCs. Activation or up-regulation of Nrf2 pathway may be an alternative treatment strategy for liver fibrosis.

Age- and sex-related differences of organic anion-transporting polypeptide gene expression in livers of rats.

Organic anion-transporting polypeptides (Oatps) play important roles in transporting endogenous substances and xenobiotics into the liver and are implicated in drug-drug interactions. Many factors could influence their expression and result in alterations in drug disposition, efficacy and toxicity. This study was aimed to examine the development-, aging-, and sex-dependent Oatps expression in livers of rats. The livers from SD rats during development (-2, 1, 7, 14, 21, 28, 35, and 60 d) and aging (60, 180, 540 and/or 800 d) were collected and total RNAs were extracted, purified, and subjected to real-time PCR analysis. Total proteins were extracted for western-blot analysis. Results showed that Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 were all hardly detectable in fetal rat livers, low at birth, rapidly increased after weaning (21 d), and reached the peak at 60 d. The Oatps remained stable during the age between 60-180 d, and decreased at elderly (540 and/or 800 d). After birth, Oatp1a1, Oatp1a4, and Oatp1b2 were all highly expressed in liver, in contrast, Oatp1a5 expression was low. Oatp expressions are male-predominant in rat livers. In the livers of aged rats, the Oatp expression decreased and shared a consistent ontogeny pattern at the mRNA and protein level. In conclusion, this study showed that in rat liver, Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 gene expressions are influenced by age and gender, which could provide a basis of individual variation in drug transport, metabolism and toxicity in children, elderly and women.

Effect of icariin on UDP-glucuronosyltransferases in mouse liver.

Icariin is a flavonol glycoside isolated from Epimedium genus and has been used in the treatment of sexual dysfunction and osteoporosis. Our laboratory has shown that icariin is beneficial in brain disorders and cardiovascular diseases. Since icariin is widely used with other herbs and drugs, to understand its potential herb-drug interactions is of importance. Recently, icariin was shown to inhibit UDP-glucuronosyltransferases, particularly the Ugt1 family enzymes in vitro, but little is known about such effects in vivo. This study investigated the effects of icariin on the expression of UDP-glucuronosyltransferases and cytochrome P450 enzymes in the livers of mice. Adult mice were treated with icariin at doses of 0, 40, 80, 160, and 320 mg/kg, p. o., for 7 days. Phenobarbital (120 mg/kg, p.o.) and rifampin (360 mg/kg, p. o.) were given twice daily for 3 days as positive controls. The livers were removed to determine UDP-glucuronosyltransferase activity and total RNA isolation. The UDP-glucuronosyltransferase activities towards 2-aminophenol were basically unaltered by the treatments. The expression of Cyp2b10 was increased 35-fold by phenobarbital, and Cyp3a11 was increased 4.5-fold by rifampin. Icariin did not affect Cyp2b10 and Cyp3a11 expression, but unexpectedly increased Cyp4a14 expression. Both phenobarbital and rifampin increased Ugt1a1, Ugt1a6, Ugt1a9, and icariin but did not show any suppressive effects on the Ugt1 family genes. Icariin at the highest dose (320 mg/kg) slightly increased Ugt2b1, Ugt2b5, and Ugt2b36. These findings indicate that icariin did not suppress UDP-glucuronosyltransferase expression, instead, it increased the mRNA of Cyp4a14 and slightly increased Ugt2b isoforms in mouse livers.