[Therapeutic effect of a natural squamosamide derivative FLZ on Parkinson's disease model mice induced by LPS plus MPTP].

The aim of this study is to investigate the protective effect of N-[2-(4-hydroxyphenyl)ethyl]-2-(2, 5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl)acrylamide (FLZ), a novel synthetic squamosamide cyclic derivative, against Parkinson's disease (PD) model mice induced by the inflammatory bacterial endotoxin, lipopolysaccharides (LPS) and the neurotoxin 1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). C57/BL mice were ip injected LPS (5 mg x kg(-1)) once. One week following the LPS injection, mice received a subcutaneous injection of MPTP (25 mg x kg(-1)) once daily for 2 days. Eight weeks later, FLZ (25, 50 and 75 mg x kg(-1)) was orally administered to mice once daily for 60 days. The motor ability of the mice was evaluated by rod climbing test and footprint test. The dopamine (DA) levels in mouse striatum were determined by high performance liquid chromatography system. The tyrosine hydroxylase (TH)-positive cells were showed by immunohistochemical analysis. FLZ treatment significantly improved motor dysfunction of mice challenged by LPS plus MPTP. The increase of TH-positive cell numbers and elevation of DA levels may be contributed to the beneficial effects of FLZ on motor behavior. This study showed FLZ has significant therapeutic effect on LPS plus MPTP induced chronic PD model, which indicates its potential as a new candidate drug to treat PD.

Scutellarin protects against lipopolysaccharide-induced acute lung injury via inhibition of NF-kappaB activation in mice.

This paper investigates the effect of natural scutellarin on acute lung injury (ALI) induced by Escherichia coli endotoxin lipopolysaccharide (LPS) in mice and its mechanism of action. Mouse ALI was induced by the injection of LPS (15 mg/kg) via the tail vein, and mice were intraperitoneally injected with 50 and 25 mg/kg of scutellarin before the LPS injection. The lung index, serum NO2(-)/NO3(-), and tumor necrosis factor-alpha (TNF-alpha) levels were determined using kits. The lung lesions were examined by light microscope. The mRNA levels of TNF-alpha, inducible nitric oxide synthase (iNOS), and FasL in pulmonary tissues were detected by RT-PCR. c-Fos, c-Jun, IkappaB, and iNOS proteins were detected by the western blotting method. Pretreatment with 25 and 50 mg/kg of scutellarin significantly reduced lung injury induced by LPS, which expressed in the decrease in lung morphological lesions, serum NO2(-)/NO3(-), TNF-alpha levels, lactate dehydrogenase release, and total protein in the lavage fluid of bronchoalveolar of the lung. The mRNA level of TNF-alpha, iNOS, the protein content of c-Fos, iNOS, and the activation of NF-kappaB in pulmonary tissues were all inhibited, while the lung glutathione level increased. In conclusion, scutellarin has protective action against LPS-induced lung damage in mice, and its underlying mechanism might be the inhibition of IkappaB alpha degradation and the expression of TNF-alpha mRNA.

[Protective action of ulinastatin against lipopolysaccharides-induced acute lung injury in mice and the relation of it to iNOS and c-Jun expressions].

AIM: To study the protective action of ulinastatin against lipopolysaccharide (LPS)-induced acute lung injury in mice and the mechanism of its action. METHODS: Mice were intraperitoneally injected with ulinastatin (50 and 100 ku x kg(-1)) or saline at a period of 12 h, separately, 30 min after the last injection of ulinastatin, except normal control, all mice of other groups were injected a dose of LPS 15 mg x kg(-1) via tail vein. The levels of TNFalpha in serum and lung were measured by ELISA. The expression of TNFalpha mRNA and iNOS mRNA in lung was assayed by RT-PCR. The expression of c-Fos and c-Jun protein in lung was measured by Western blotting method. And the NO2- / NO3- level in serum and MDA in lung were measured with kits. RESULTS: The levels of NO2- / NO3- and TNFalpha in serum, MDA and TNFa in lung all increased after iv injection of LPS. The expressions of TNFa mRNA, iNOS mRNA, c-Fos and c-Jun in lung of LPS-injected mice were enhanced. Pretreatment with ulinastatin 100 ku x kg(-1) decreased the levels of NO2- / NO3- in serum and lung, reduced the index of lung, and inhibited the expressions of iNOS mRNA and c-Jun in lung induced by LPS in mice, while ulinastatin showed no effect on TNFa level in serum and lung. CONCLUSION: Ulinastatin protected mice from acute lung injury induced by lipopolysaccharides via inhibiting the activation of c-Jun and iNOS mRNA expression.

Toxicity of novel anti-hepatitis drug bicyclol: a preclinical study.

AIM: To study the toxicity of bicyclol to animals. METHODS: Acute toxicity test was performed in Kunming strain mice that were orally given bicyclol at the doses of 3 and 5 g/kg body weight, respectively. Wistar rats were orally administered bicyclol at a dose of 5 g/kg body weight. Death and clinical symptoms of animals were recorded within 7 d. Sub-acute toxicity test was carried out in rats that were treated with various doses of bicyclol (150, 300, 600 mg/kg) once daily for 14 d. Animal behaviors, blood biochemical markers, blood and urine pictures were examined. Chronic toxicity test was conducted in 80 Wistar rats of both sexes. The animals were orally administered with various doses of bicyclol (150, 300, 600 mg/kg, 100-400 folds corresponding to the proposed therapeutic dose (1.5 mg/(kg.d)) of bicyclol for patients) once daily for 6 mo except for Sunday. The control group was given the same volume of 0.2% sodium carboxyl methylcellulose (Na-CMC). Twenty-one beagle dogs received bicyclol (25, 75, 225 mg/kg, 16.6, 50, 150 folds corresponding to the proposed therapeutic dose of bicyclol for patients) once a day for 6 mo except for Sunday. The body weight, food intake, urine and feces, blood picture, blood biochemical markers, and pathological examination of main organs were determined. Mutagenicity and teratogenicity were determined. Mutagenicity assay included Ames's test, chromosome aberration test in CHL cells and micronucleus test in mice. For the teratogenicity assay, pregnant Wistar rats weighing 200-250 g were treated with 0.2, 1.0 g/kg bicyclol once daily from the 7th d of gestation for 10 d. RESULTS: The oral LD(50) of bicyclol was over 5 g/kg in mice and rats. No noticeable alterations in subacute and chronic toxicity of rats and dogs were demonstrated. No mutagenicity and teratogenicity of bicyclol were found. CONCLUSION: Bicyclol has no detectable chronic toxicity as well as mutagenicity and teratogenicity in animals.

Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel(7402).

AIM: To investigate the resistance mechanism of 5-fluorouracil (5-FU) in Bel(7402)/5-FU cells which was established in our lab by in vitro continuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel(7402) to 5-FU. METHODS: The expression of multidrug resistance-associated protein (MRP) and thymidylate synthase (TS) in Bel(7402) cells was detected by immonocytochemistry. The fluorescein (FLU) accumulation, an index of MRP functional activity, was determined by flow cytometry. The distribution of FLU was observed by confocal laser scanning microscope. The spectrofluorometry was used to show the intracelluar content of glutathione (GSH). Cell growth inhibition was determined by MTT assay. The activity of glutathione S-transferases (GSTs) was determined by spectrophotometry. RESULTS: A higher expression of MRP in the Bel(7402)/5-FU cells was observed by using monoclonal mouse anti-MRP antibody, MRPr-1, in comparison with Bel(7402) cells. Bel(7402)/5-FU cells also showed a significant decrease of FLU accumulation. FLU mainly accumulated in the nucleus with a high nuclear/cytoplasmic ratio in Bel(7402) cells, whereas there was no difference of FLU accumulation between the nucleus and cytoplasm in Bel(7402)/5-FU cells. The intracellular GSH content in Bel(7402)/5-FU cells was almost 3 folds higher than that in Bel(7402) cells. Addition of D, L-buthione-S, R-sulfoximine (BSO) dose-dependently reduced the GSH content in Bel(7402)/5-FU cells, however, only a weak enhancement on the cytotoxicity of 5-FU and doxorubicin (Dox) to Bel(7402)/5-FU cells was observed. Bel(7402)/5-FU cells also exhibited 29.1 % higher total GSTs activity than Bel(7402) cells. Immunocytochemical staining by using anti-TS monoclonal antibody TS 106 showed that the level of TS in Bel(7402)/5-FU cells elevated markedly as compared with Bel(7402) cells. CONCLUSION: The continuous exposure of Bel(7402) cells to 5-FU led to overexpression of TS and MRP, as well as increased intracellular GSH content and total GST activity.

[Synthesis and antioxiactivity of squamosamide cyclic analogs].

OBJECTIVE: To design and synthesize a series of squamosamide cyclic analogues and to test their antioxidation activity. METHODS: Eleven 3-substituted indole-2-one derivatives were designed and synthesized through 9 steps with p-hydroxyphenylacetic acid as the starting material and their structures were confirmed by nuclear magnetic resonance and mass spectrometry. RESULTS: Eleven compounds showed antioxidation activity and the activities of compounds 9 and 13 matches the positive control FLZ-52. CONCLUSION: Cyclic reconstruction with FLZ-52 as the lead compound have some antioxidation activity.

Antioxidant phenolic glycosides from the roots of Illicium dunnianum.

Eight new phenolic glycosides, dunnianosides A-H (1-8), and nine known phenolic glycosides (9-17), were isolated from the roots of Illicium dunnianum. The structures of these new compounds were elucidated by spectroscopic methods including 1D and 2D NMR, HRESIMS, and chemical methods. Compounds 1-5, 7, and 9 exhibited potent antioxidant activities against Fe(2+)-cystine-induced rat liver microsomal lipid peroxidation, with IC(50) values ranging from 3.8 ± 0.6 to 23.0 ± 2.2 µM.

Long-term deprivation of gonadal hormone accelerates brain aging in mice.

OBJECTIVE: The purpose of this study was to assess the effect of long-term deprivation of gonadal hormone on brain aging in mice to develop a model of gonadectomy-accelerated brain aging. METHODS: Male and female mice at 2 months old were orchiectomized (ORX) or ovarectomized (OVX) bilaterally or sham operated, and then they were fed for 10 months. The spatial learning and memory ability was tested using Morris Water Maze. The biomarkers of brain neuropathology were examined by Western blotting and immunohistochemistry. RESULTS: Ovarectomy mildly impaired spatial learning and memory of mice, while the impairment in ORX-mice was not significant. The amount of Nissl bodies decreased in the hippocampus and cortex of gonadectomied mice. The expression of beta-amyloid (Aß), beta-site APP cleaving enzyme 1 and phosphorylated-Tau increased in gonadectomied mice. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) decreased in the brain of OVX-mice, but neurotrophin-3 (NT-3) showed no change. We detected no decrease of NGF, BDNF or NT-3 in ORX-mice. TrkA expression decreased and p75(NTR) increased in the brain of gonadectomied mice. In all the above tests, there were no significant differences between young (2 months old) and sham operated (12 months old) mice. Alternations in the brain aging parameters were more obvious in OVX-mice than in ORX-mice. CONCLUSION: Long-term gonadal hormone deprivation by young-age gonadectomy accelerated mouse brain aging, which could serve as a valuable mouse model to study brain aging and aging-related pathological changes.

Establishment of a HPLC method for preclinical pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rats.

An HPLC method was established and validated for the determination of compound FLZ, a synthetic novel anti-Parkinson's disease candidate drug, in rat plasma. FLZ and the internal standard bicyclol were extracted from plasma by solid-phase extraction method and analyzed on a Restek C18 column (4.6 x 250 mm, 5 microm) with a mobile phase consisting of methanol and water (60:40, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 320 nm. The calibration curve was linear within the concentration range from 25 to 500 ng/mL (r2 > 0.999), the limit of quantitation was 25 ng/mL and the average recovery was 92.0% with the RSD less than 5.9%. The relative standard deviation for intra- and inter-day precision was less than 3.8 and 6.9%, respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and applied to study the preclinical pharmacokinetics of FLZ in rat plasma, and it was the first time that the pharmacokinetics of FLZ had been investigated.

Mechanism of protective action of bicyclol against CCl-induced liver injury in mice.

Bicyclol is a novel synthetic drug for the treatment of chronic viral hepatitis in China. This paper reports the protective action of bicyclol against experimental liver injury in mice and its mechanism of action. Oral administration of bicyclol markedly reduced the elevated serum transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and the hepatic morphologic changes induced by CCl(4) in mice. Mechanistic studies demonstrated that bicyclol significantly inhibited CCl(4)-induced lipid peroxidation of liver microsomes and (14)CCl(4) covalent binding to microsomal lipids and proteins in vitro, and decreased the level of the trichloromethyl free radical (*CCl(3)) generated from CCl(4) metabolism by NADPH-reduced liver microsomes. On the other hand, bicyclol neither directly inhibited the activity of ALT or AST in vitro nor affected hepatic ALT protein content in mice. These results suggest that bicyclol has remarkable hepatoprotective effects and its mechanism of action may be related to a decrease in free radical-induced damage to hepatocytes.