Weirong Li

Geniposidic acid protected against ANIT-induced hepatotoxity and acute intrahepatic cholestasis, due to Fxr-mediated regulation of Bsep and Mrp2.

ETHNOPHARMACOLOGICAL RELEVANCE Geniposidic acid (GPA) is the main constituent of Gardenia jasminoides Ellis (Rubiaceae), which has long been used to treat inflammation, jaundice and hepatic disorders. The cholagogic effect of Gardenia jasminoides Ellis (Rubiaceae) and GPA have been widely reported, but the underlying occurrence mechanism remains unclear. AIM OF THE STUDY This investigation was designed to evaluate the hepatoprotection effect and potential mechanisms of GPA derived from Gardenia jasminoides Ellis (Rubiaceae) on fighting against α-naphthylisothiocyanate (ANIT) caused liver injury with acute intrahepatic cholestasis. MATERIALS AND METHODS Sprague-Dawley (SD) rats were intragastrically (i.g.) administered with the GPA (100, 50 and 25mg/kg B.W. every 24h) for seven consecutive days, and then they were treated with ANIT (i.g. 65mg/kg once in the 5th day) which induced liver injury with acute intrahepatic cholestasis. Serum and bile biochemical analysis, bile flow rate and liver histopathology were measured to evaluate the protective effect of GPA fight against ANIT treatment. The protein and mRNA expression levels of farnesoid X receptor (Fxr), bile-salt export pump (Bsep), multidrug resistance associated protein2 (Mrp2), were evaluated to study the effect of liver protection about GPA against ANIT induced hepatotoxicity and underlying mechanisms. RESULTS Some abnormalities were observed on ANIT treated rats including weight loss, reduced food intake and hair turned yellow. Obtained results demonstrated that at dose 100 and 50mg/kg B.W. (P<0.01) and 25mg/kg B.W. (P<0.05) of GPA pretreated dramatically prevented ANIT induced decreased in bile flow rate. Compared with ANIT treated group, the results of bile biochemical parameters about total bile acid (TBA) was increased by GPA at groups with any dose (P<0.01), glutathione (GSH) was increased significantly at high dose (P<0.01) and medium dose (P<0.05), total bilirubin (TB) was increased at high and medium dose (P<0.05), direct bilirubin (DB) was only increased at high dose (P<0.01). Serum levels of glutamic-Oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-glutamyltranspeptidase (γ-GT), TB, DB and TBA in comparison with ANIT treated group (P<0.01) were reduced by GPA (between 100 and 50mg/kg B.W.) pretreatment. Histopathology of the liver tissue showed that pathological damages and hepatic portal area filled with bile were relieved after GPA pretreatment compared with ANIT treated group. The protein and mRNA expression of Fxr, Bsep and Mrp2 were decreased in ANIT treated group. On the contrary, the protein and mRNA of Fxr, Bsep and Mrp2 were up regulated significantly pretreatment by GPA at dose of high and medium groups. On protein level of Bsep and Mrp2 the result shown no statistical difference in GPA (25mg/kg B.W.), but it was not same shown in mRNA level. CONCLUSION The results of this investigation have demonstrated that the GPA exerts a dose dependent hepatoprotection effect on ANIT induced liver damage with acute intrahepatic cholestasis in rats, which may due to Fxr mediated regulation of bile transporters like Bsep and Mrp2.

Toxicity of Evodiae fructus on rat liver mitochondria: the role of oxidative stress and mitochondrial permeability transition.

Evodiae fructus (EF) has been used in China for thousands of years as an analgesic, antiemetic, anti-inflammatory and antidiarrheal drug. EF is a toxic drug and causes hepatotoxicity in humans. Although recent chronic toxicity studies performed on aqueous extract of EF has revealed that it can produce obvious cumulative hepatotoxicity, the mechanism behind this toxicity is still uncertain. In the present study, we investigated the influence of EF on oxidative stress, mitochondrial permeability transition, adenosine triphosphate (ATP), and cytochrome C release of hepatic mitochondria. Rats were divided into four groups and fed distilled water, 6, 12, 24 g/kg of aqueous extract of EF daily for 15 days. Evodiamine, rutaecarpine and evodine were quantified in the aqueous extract by high performance liquid chromatography with ultraviolet detection (HPLC/UV). The results showed that aqueous extract of EF could significantly (p < 0.05) decrease MnSOD levels to 56.50%, 46.77% and 19.67% of control group, GSH level was decreased to 74.24%, 53.97% and 47.91% of control group and MDA level was increased to 131.55%, 134.34% and 150.81% of control group in the 6, 12 and 24 g/kg groups, respectively; extract also induced mitochondria swelling, vacuolation, MPT pore opening and a significant decrease (p < 0.05) in mitochondrial potential, while ATP levels were significant decreased (p < 0.05) to 65.24%, 38.08% and 34.59% of control group in the 6, 12 and 24 g/kg groups, respectively, resulting in ATP depletion and CytC release, finally trigger cell death signaling, which are the partial hepatotoxicity mechanisms of EF.

Alteration of glutamate/GABA balance during acute alcohol intoxication in rats: Effect of Xingnaojing injection

ETHNOPHARMACOLOGICAL RELEVANCE Xingnaojing Injection (XNJI) is a modern Chinese formula came from famous Chinese medicine An Gong Niu Huang Pill. XNJI has been used for treatment of cerebral diseases and stroke in China, and is approved by the State Food and Drug Administration of China for the treatment of acute alcohol intoxication (AAI). XNJI belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanisms of the XNJI effect on AAI. AIM OF THE STUDY To investigate the effects of XNJI on glutamate, gamma-aminobutyric acid (GABA) and related receptor in lateral hypothalamic area (LHA) of AAI rat. MATERIAL AND METHODS Adult male Sprague-Dawley rats were implanted with microdialysis probes in LHA. Rats were randomly divided into control, model, 1.36mg/kg XNJI, 0.68mg/kg XNJI and 0.34mg/kg XNJI groups. During microdialysis, baseline samples were collected from 1h to 2.5h; thereafter, the rats were given an intraperitoneal injection of 52% ethanol, 5.2g/kg, or saline for control group. Twenty minutes later, three doses of XNJI was given by unilateral injection respectively, while saline for control and model groups, and samples were collected for the next 4h. The extracellular glutamate and GABA levels were measured in the LHA by a high performance liquid chromatography coupled with fluorescence detector (HPLC-FLU). The expression levels of related receptors N-methyl-d-aspartate receptor (NR) subunit NR2A, NR2B and GABAA were analyzed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS Ethanol (5.2g/kg) significantly decreased the extracellular levels of glutamate and increased extracellular GABA in LHA. On the other hand ethanol significantly decreased NR2A and NR2B mRNAs expression, and increase GABAA mRNA expression. XNJI could increase the extracellular level of glutamate and decrease that of GABA; moreover, induced an increase in NR2A and NR2B mRNA expression, and a decrease in GABAA mRNA expression in LHA. CONCLUSIONS The current changes in glutamate, GABA and mRNA expressions of related receptors in LHA after injection of XNJI suggest that changes in these neurotransmitters and receptors as a potential mechanism of action for AAI.

Study on Pharmacokinetics of Three Preparations from Levistolide A by LC–MS-MS

A rapid sensitive analytical method was established and validated to investigate levistolide A in rat plasma by liquid chromatography-tandem mass spectrometry operated in the positive ion mode. Levistilide A (LA) and internal standard (IS) andrographolide (AD), mixed with the plasma sample, were separated on a reversed phase Spursil™ C18 5 µm column. The precursor/product transitions (m/z) were 398.5/381.3 for LA and (m/z) 368.0/351.1 for AD. The calibration curve was linear over the range from 5 to 1,250 ng/mL for oral administration and 10-4,000 for intravenous administration with a correlation coefficient (r) ≥0.9993. The lower limit of quantification was 5 ng/mL for LA in plasma. The inter- and intra-day accuracy and precision were less than ±15% of the relative standard deviation. In this study, the developed method is successfully applied to the comparative pharmacokinetic study of LA in rats after oral administration of LA alone, Rhizoma Chuanxiong, and Danggui-Shaoyao-San along with the bioavailability study of LA in rats. Our study shows that low bioavailability (7.5%) is observed after oral administration of LA. Traditional formula compatibility of Danggui-Shaoyao-San could significantly enhance LA bioavailability compared with LA alone and Rhizoma Chuanxiong.

Toxicity study of oxalicumone A, derived from a marine-derived fungus Penicillium oxalicum, in cultured renal epithelial cells

Oxalicumone A (POA), a novel dihydrothiophene-condensed chromone, was isolated from the marine-derived fungus Penicillium oxalicum. Previous reports demonstrated that POA exhibits strong activity against human carcinoma cells, thus it has been suggested as a bioactive anticancer agent. To research the toxic effect of POA on cultured normal epithelial human kidney-2 (HK-2) cells and evaluate its clinical safety, cell survival was evaluated by the Cell Counting Kit-8 assay and apoptosis was evaluated by Hoechst 33258 staining, flow cytometry, caspase-3 activity assay and western blotting. 2′,7′-Dichlorofluorescin diacetate and JC-1 dye staining was used to evaluate reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP), respectively. The results indicated that POA inhibited HK-2 cell growth and promoted apoptosis, by increasing levels of Fas cell surface cell receptor and the B-cell lymphoma 2 associated protein X apoptosis regulator (Bax)/B-cell lymphoma 2 apoptosis regulator (Bcl-2) ratio. POA treatment also induced release of ROS and loss of MMP in HK-2 cells. Compared with untreated control, a significant decrease was also demonstrated in superoxide dismutase activity and glutathione content with POA treatment, accompanied by enhanced release of N-acetyl-β-D-glucosaminidase, increased leakage of lactate dehydrogenase, increased malondialdehyde formation and increased release of nitric oxide. In conclusion, the present in vitro study revealed that POA exhibits antiproliferation activity on HK-2 cells, through stimulation of apoptosis and oxidative stress injury, which may be relevant to its clinical application. The present study may, therefore, offer valuable new information regarding the use of POA as a candidate novel antitumor drug for clinical use.

Do Chinese Researchers Conduct Ethical Research and Use Ethics Committee Review in Clinical Trials of Anti-Dementia Drugs? An Analysis of Biomedical Publications Originating from China

BACKGROUND Medical research using human participants must conform to the basic ethical principles found in the Declaration of Helsinki (DoH) of the World Medical Association. OBJECTIVE The purpose of this review was to assess whether journals in China have improved in regard to the fulfillment of ethical disclosure procedures for clinical trials of anti-dementia drugs. METHODS Four medical databases were searched for articles reporting clinical trials of oral anti-dementia drugs published in China in 2003, 2009, and 2014. The frequencies of reporting of informed consent from participants (ICP), approval of a regional ethical committee (REC), reference to DoH, and study registration were estimated respectively. Statistical analyses were conducted with SPSS v21 software. RESULTS Among those randomized controlled trials published in 2003, 2009, and 2014, disclosure of REC approval was present for 2.67%, 1.15%, and 6.84%; statements of ICP were included in 9.33%, 7.76%, and 17.34%; reference to DoH was found for 4.00%, 1.44%, and 7.45%; and study registration reporting was included in 2.67%, 2.59%, and 9.28%, respectively. Improvements to reporting rates between 2009 and 2014 were seen, with more than twice as many trials reporting REC approval, ICP, reference to DoH, and study registration compared with 2009. CONCLUSION Compared with 2003 and 2009, reporting rates for REC approval, ICP, reference to DoH, and study registration for clinical trials of anti-dementia drugs were enhanced in 2014 in the major medical journals of China. However, biomedical publications without definite statements of ethical considerations remain common, and this continues to be seen in Chinese journals. It is imperative that measures are taken to reinforce the ethical protection in clinical trials in China.

Antinociceptive Activity of Aqueous and Alcohol Extract of Evodia Rutaecarpa

Water, methanol and ethanol extracts of Evodia rutaecarpa were tested for antinociceptive activity, which were correlated with the contents of evodiamine, rutaecarpine and evodine. Determination of contents was achieved by chromatographic techniques. Extracts were evaluated for antinociceptive activities using hot-plate test; acetic acid-induced writhing test and formalin test. All three extracts of Evodia rutaecarpa showed antinociceptive activities but the ethanol extract exhibited better effect. The better antinociceptive activity appeared to be related to higher contents of evodiamine, rutaecarpine and evodine in ethanol extract of Evodia rutaecarpa.

Anticancer activity of polysaccharide from Glehnia littoralis on human lung cancer cell line A549

The purpose of this study was to investigate the anticancer activity of polysaccharide (PGL) from Glehnia littoralis on human lung cancer cell line A549. Based on MTT assay, the results suggested that PGL could significantly reduce A549 cells proliferation in a time- and dose-dependent manner. In addition, PGL displayed an inhibitory activity for the A549 cells migration in Transwell migration assay. The results from both flow cytometry analysis and Hochst 3342 staining of apoptotic cells indicated that PGL could promote apoptosis, and induce cycle arrest of A549 cells. Moreover, immunofluorescence assay elucidated PGL could also down-regulate expression of proliferating cell nuclear antigen (PCNA). Overall, these results showed that PGL exerts a strong anticancer action through inhibiting the A549 cells migration, proliferation and inducing cell apoptosis. It could be a new source of natural anticancer agent against lung cancer with potential value in supplements and medicine.

Hepatocellular toxicity of oxalicumone A via oxidative stress injury and mitochondrial dysfunction in healthy human liver cells

The marine-derived oxalicumone A (POA) has been demonstrated as a potent anti-tumor bioactive agent for a variety of human carcinoma, but to the best of our knowledge, remains to be evaluated in healthy liver cells. As many drugs distribute preferentially in the liver, the present study aimed to investigate the effects of POA on apoptosis, oxidative stress and mitochondrial function in L-02 healthy liver cells. A Cell-Counting kit-8 assay demonstrated that POA inhibits the proliferation of L-02 cells in a dose- and time-dependent manner. Furthermore, POA induced apoptosis by increasing the percentage of cells in early apoptosis and the sub-G1 cell cycle, along with causing S-phase arrest in L-02 cells. Additionally, POA activated caspase 3, increased the protein expression levels of Fas ligand and B-cell lymphoma X-associated protein, and decreased the expression of the anti-apoptotic protein B-cell lymphoma 2. POA additionally reduced the content of GSH and the activity of superoxide dismutase, elevated malondialdehyde and nitric oxide levels, increased reactive oxygen species production and the levels of alanine aminotransferase and aspartate aminotransferase, which suggested that POA induced lipid peroxidation injury in L-02 cells and that oxidative stress serves an important role. Furthermore, POA caused alternations of mitochondrial function, including an abrupt depletion of adenosine triphosphate synthesis, mitochondrial permeability transition pore opening and depletion of mitochondrial membrane potential in L-02 cells. These data suggested that POA exerts cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial function and oxidative stress by POA may therefore be critical in POA-induced toxicity in L-02 cells.

Pharmacokinetic and Pharmacodynamic Properties of Rosmarinic Acid in Rat Cholestatic Liver Injury

The objective of this study was to evaluate the hepatoprotective and metabolic effects of rosmarinic acid (RA) in rats. RA [100 mg/kg body weight (BW)] was intragastrically (i.g.) administered to Sprague-Dawley (SD) rats once a day for seven consecutive days. The rats were then i.g. administered α-naphthylisothiocyanate (ANIT) (80 mg/kg once on the 5th day) to induce acute intrahepatic cholestasis after the last administration of RA. Blood samples were collected at different time points (0.083 h, 0.17 h, 0.33 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 3 h, 4 h, 6 h, 8 h, 12 h, 20 h) after administration, and the levels of RA were estimated by HPLC. Plasma and bile biochemical analysis, bile flow rate, and liver histopathology were measured to evaluate the hepatoprotective effect of RA. The PK-PD curves showed obviously clockwise (AST and ALT) or anticlockwise (TBA, TBIL). Pretreatment with RA at different doses significantly restrained ANIT-induced pathological changes in bile rate, TBA, TBIL, ALT, AST (p < 0.05 or p < 0.01). The relationship between RA concentration and its hepatoprotective effects on acute cholestasis responses was assessed by PK-PD modeling.