OBJECTIVETo observe the effect of total flavones of Epimedium leptorrhizum (YYH-C) on osteoporosis in ovariectomized rats.

METHODOvariectomized female rats were randomly divided into the model group, YYH-C lower, middle and high dose (0.7, 1.4, 2.8 g x kg(-1)) groups, the positive drug Bujiale (0.15 mg x kg(-1)) group, and the sham group. The rats were orally ad-ministrated with drugs for three months. Parathyroid hormone (PTH), procollagen I N-terminal peptide (PINP), alkaline phosphatase (ALP), calcium (Ca) and phosphrous (P) in serum were detected. Femur bones and vertebrae bones of left side were collected to determined bone metrological indexes, including bone mineral density (BMD), bone Ca, and bone ash weight/dry weight percentage. Femur bones of right side were collected to for a morphological observation of bone.

RESULTCompared with the sham group, the model group showed significantly higher PTH and ALP content but obviously lower PINP and Ca content. The three YYH-C 3 groups could resist the decrease of PINP. Specifically, low and middle dose groups could remarkably inhibit the increase of PTH, and the high dose group could increase the Ca content in serum, but without significant effect on the rise in ALP. There was no significant difference in P content in serum in each group. BMD, ash weight/dry weight percentage, Ca and P content of the model group were significantly lower than those in the sham group. The high dose YYH-C group could significantly increase BMD. All of the three YYH-C groups could notably increase ash weight/dry weight percentage and Ca, P content in femur bones and vertebrae bones. YYH-C could significantly increase average thickness, area, area percentage of bony trabeculae, cortical bone area percentage of femoral shaft and the number of osteoblasts on the surface of bony trabeculae, and decrease the number of osteoclasts.

CONCLUSIONYYH-C can effectively control the bone mass loss of rats with ovariectomy-induced osteoporosis, prevent the changes in bone microstructure, and inhibit bone absorption, so as to resist high turn-over osteoporosis after ovariectomy. [Key words] total flavones of Epimedium leptorrhizum; ovariectomized rat; osteoporosis

Alkaline Phosphatase ; metabolism ; Animals ; Bone Density ; drug effects ; Calcium ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Epimedium ; chemistry ; Female ; Flavones ; administration & dosage ; Humans ; Osteoporosis, Postmenopausal ; drug therapy ; metabolism ; physiopathology ; Ovariectomy ; Parathyroid Hormone ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To explore the association of Crohn's disease (CD) with T cell immunoglobulin and mucin domain 3 (Tim-3) gene polymorphisms in patients of Zhejiang Han population in China.Methods A total of 308 CD patients and 573 age-and sex-matched healthy controls were enrolled in our study.Two single nucleotide polymorphisms (SNPs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique (iMLDR).Analyses of linkage disequilibrium and haplotype were also performed by Haploview 4.2 software in all study subjects.Results In general,the allele and genotype frequencies of Tim-3 (rs1036199 and rs10515746) were not statistically different between CD patients and the controls (all P ＞0.05).According to the Montreal Classification,CD patients were divided into different subgroups.The variant allele (C) and genotype (AC + CC) of rs1036199 were more frequent in CD patients with penetrating diseases than in the controls (10.4％ vs 1.7％,P =0.002;20.8％ vs 3.5％,P =0.023).Similar conclusions were also drawn for the variant allele (A) and genotype (CA + AA) of rs10515746 in patients with penetrating diseases when compared with the controls (10.4％ vs 2.2％,P =0.000;20.8％ vs 4.2％,P =0.033,respectively).The two SNPs of Tim-3 were in strong linkage disequilibrium (D'=1.0,r2 =0.928).The haplotype (AC) formed by their wild-type alleles (A) and (C) was decreased in patients with penetrating CD compared with the controls (89.6％ vs 98.3％,P =0.000).However,the haplotype (CA) formed by their variant alleles was more frequent in patients with penetrating CD than in the controls (10.4％ vs 1.6％,P =0.000).Conclusions Tim-3 (rs1036199 and rs10515746) variations might be correlated with the enhanced risk of penetrating diseases in CD patients.Furthermore,the haplotype (AC) and (CA) formed by the two SNPs might be a protective and a risky factor for penetrating CD respectively.

OBJECTIVETo compare the sensitivity of Brown Norway rats (BN) with Guinea pigs (GP) as allergen assessment animal models.

METHODBN rats and GP were randomly assigned to 1 control group, 2 Bovine serum albumin group (BSA), respectively. Animals in BSA groups of BN rats and GPs were sensitized by intraperitoneal injection of 0.6% BSA 1 ml on day 1, 3, 5, respectively, and irritated by intravenous injection of 2.4% BSA 1 ml on day 7 and day 14 after the last sensitization, while the same volume of normal saline was given to control group on each time point mentioned above. The allergic reactions were scored within 1 h after each irritation treatment, and the sera of both BN rats and GPs were collected to detect IgE concentration by using ELISA. The sera were also applied for passive cutaneous anaphylaxis test (PCA test) in SD rats.

RESULTNo obvious allergic reactions were observed in BSA group of GPs after each irritation treat, however, the score of allergic response in BSA group of BN rats was evidently higher than that in control group after first irritation. PCA test by using sera from BSA group of BN rats after both irritations showed the strong positive result characterized as large amount of subcutaneous effusions of Evans blue in SD rats, however, the sera from BSA group of GP were negative in PCA test. Serum IgE concentration did not increase after each irritation in BSA group of both BN rats and GP.

CONCLUSIONBN rats were more sensitive than GPs on initiative systemic anaphylaxis test and passive cutaneous anaphylaxis test. Meanwhile, BN rats has an advantage in experimental treatment compared with Guinea pigs.

Allergens ; administration & dosage ; toxicity ; Anaphylaxis ; chemically induced ; Animals ; Guinea Pigs ; Hypersensitivity ; etiology ; Male ; Models, Animal ; Rats ; Rats, Inbred BN ; Rats, Sprague-Dawley

The industry of germ-free animals has been a hot spot in research along with the rapid development of studies on the relationship between microbiota and host diseases. Because it is pathogen?free, and the high degree of simi?larity in anatomy, physiology, pathogenesis to humans, germ?free pig is considered a clinical relevant model to be widely used in life science research. Based on the current state of research of germ?free pig cultivation at home and abroad and the experimental studies carried out in our laboratory as well, this article gives a simple discussion on germ?free technique of domestic pigs.

OBJECTIVETo investigate the mercury cumulation following single dose or long-term use of Cinnabar to rats.

METHODThe Cinnabar which was used in the study contains 98% insoluble mercuric sulfide (HgS) and 21.5 mg x kg(-1) soluble mercuric compounds. Two separate experiments were performed: (1) Tweenty-eight fasting SD rats were orally given a single dose of Cinnabar at the dose of 0.8 g x kg(-1) and the other four rats were given ultra-filtrated water served as control group. Blood, livers, kidneys and brains of four rats were taken out at 0.5, 1, 2, 4, 8, 16, 36 h respectively after treatment. Mercury quantity of each organ or blood sample was measured. (2) Forty SD rats were randomly divided into four groups: control group and Cinnabar 0.1, 0.4, 0.8 g x kg(-1) groups, each group containing 5 females and 5 males. The rats were intra-gastrically treated with Cinnabar once a day for successively 90 days, while the control group was given ultra-filtrated water. Mercury contents in blood, livers, kidneys and brain of each rat were measured at 16 h of fasting after last dosing.

RESULTMercury contents of blood, liver, kidney and brain increased slightly after single dosing of Cinnabar at dose of 0.8 g x kg(-1), with the order from high to low liver > blood > brain > kidney. Whereas 90-day oral treatment of Cinnabar led to significant cumulation of mercury in organs but not in blood. Kidney' s cumulation of mercury was much higher than any other tested organs and blood. Brain's mercury cumulation was also very high. The contents of mercury in kidney and brain of 0.8 g x kg(-1) group (total intake of soluble mercury within 90 days was 1 548 microg x kg(-1)) were respectively 71.2 and 27.4 times higher than control group. Even though in the lowest dose 0.1 g x kg(-1) group (total intake of soluble mercury 194 microg? kg(-1)), the mercury cumulation folds in kidney and brain were 16.77 and 20.43 respectively. However, liver got lower mercury cumulation than kidney and brain, which led to only 2 folds mercury cumulation at dose of 0.8 g x kg(-1). Our previous study showed that 90-day administration of Cinnabar at the dose > or = 0.1 g x kg(-1) (total intake of soluble mercury 194 microg x kg(-1)) could cause pathological changes in kidney and liver, indicating both were the toxicity targets for Cinnabar. Those manifested that liver could be more sensitive than kidney to mercury. Though brain got 20 times mercury cumulation after 90 day treatment, the animals showed no abnormal signs in general behavior and brain histomorphology,which indicated that rat brain was not sensitive to mercury.

CONCLUSIONSoluble mercury in Cinnabar can be absorbed causing high cumulated in some organs, such as kidney and brain after long-term use of Cinnabar. Liver had also mercury cumulation, but was much lower than kidney. Total intake of soluble mercury for > or = 194 microg x kg(-1) within 90 days could cause toxicosis by mercury cumulation.

Administration, Oral ; Animals ; Brain ; metabolism ; Female ; Kidney ; metabolism ; Liver ; metabolism ; Male ; Mercury ; pharmacokinetics ; Mercury Compounds ; administration & dosage ; pharmacokinetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Distribution

OBJECTIVEBy using RAW 264.7 macrophage cell line, we studied the dose-effect relationship of endotoxin induced RAW 264.7 cells to release TNF-alpha, and then detected the content of endotoxin in 8 kinds of injections, so that we can investigate the feasibility and the interference factors of the novel test.

METHODBy using endotoxin of different concentrations to induce RAW 264. 7 cells to release TNF-a, we drew the curve of dose-effect relationship between endotoxin and generated TNF-alpha. Then we detected the content of TNF-alpha in yuxingcao, shuanghuanglian, qingkailing, gegensu, xiangdan, qianrongmei and jiangxianmei injections and shuanghuanglian powder injection, and calculated their content of endotoxin.

RESULTThe endotoxin could induce the cells to release TNF-alpha in a good dose-dependent manner, even at a very low concentration. In the range of maximum available dilution multiple, the content of endotoxin in the rest 7 kinds of injections was less than 1.0 EU x mL(-1) except qingkailing injection of two batch.

CONCLUSIONCytokine revulsion has the advantage of wide detection range, high sensitivity, simple operation, and the detected endotoxin is of bioactivity. This method provides another technical mean for pyrogen test of injections.

Animals ; Biological Assay ; methods ; Cell Line ; Drug Contamination ; Drugs, Chinese Herbal ; analysis ; Endotoxins ; adverse effects ; analysis ; Macrophages ; drug effects ; immunology ; Mice ; Tumor Necrosis Factor-alpha ; analysis ; immunology

OBJECTIVETo detect content of bacterial endotoxin in Yuxingcao and Qingkailing injections by specific and nonspecific tachypleus amebocyte lysate technique for in order to investigate the feasibility of specific tachypleus amebocyte lysate technique for detecting bacterial endotoxin in traditional Chinese drug injections.

METHODDifferent batches of Yuxingcao and Qingkailing injections were detected by specific and nonspecific tachypleus amebocyte lysate kits.

RESULTYuxingcao injection could be detected by specific and nonspecific tachypleus amebocyte lysate technique, Whereas Qingkailing injection could be detected only by specific tachypleus amebocyte lysate.

CONCLUSIONUsing specific tachypleus amebocyte lysate as a substitute for nonspecific tachypleus amebocyte lysate is an effective method for detecting content of bacterial endotoxin in Qingkailing injection.

Animals ; Drug Contamination ; Drugs, Chinese Herbal ; analysis ; Endotoxins ; analysis ; Horseshoe Crabs ; Limulus Test ; methods

OBJECTIVETo investigate the effect of cytochrome P450 isozymes on aristolochic acid induced cytotoxicity on renal proximal tubular epithelial cell (cell line HK-2).

METHODHuman renal tubular cells (cell line HK-2), were treated with aristolochic acid (AA) alone or in combination with cytochrome P450 isozymes inhibitors, including alpha-naphthoflavone (CYP450 1A1 and 1 A2 inhibitors), ketoconazole (CYP450 3A4 inhibitor), sodium diethyldithiocarbamate (CYP450 2A6 and 2E1 inhibitors), quinidine (CYP450 2D inhibitor), alpha-lipoic acid (NADPH: P450 reductase inhibitor), sulfaphenazole (CYP450 2C inhibitor) in the presence or absence of liver microsome(S9). The inhibition of cell proliferation rate was studied by MTT assay and the lactate dehydrogenase release rate was determined with continuous monitoring method.

RESULTAA inhibits cell proliferation and promotes the release of LDH over the range of 12.5-100 mg x L(-1), in a dose-dependent manner. Addition of S9 into the culture system reduced AA cytotoxicity, with the cell proliferation inhibition reducing and the release of LDH decreasing (AA + S9 group vs the same concentration of AA alone group, P < 0.05). In the absence of S9, ketoconazole or alpha-naphthoflavone has no obvious effect on AA cytotoxicity, however,under the conditions of adding S9, ketoconazole or alpha-naphthoflavone enhances AA cytotoxicity. Other inhibitors of CYP450 isozymes has no distinct effect on AA cytotoxicity.

CONCLUSIONThe microsomal enzyme of Liver can reduce the AA cytotoxicity, and CYP450 3A, CYP450 1A may be the major cytochrome P450 isozymes which impact AA cytotoxicity.

Animals ; Aristolochic Acids ; toxicity ; Cell Line ; Cell Proliferation ; drug effects ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; Kidney Tubules ; drug effects ; enzymology ; immunology ; Male ; Rats ; Rats, Wistar

OBJECTIVETo modify the empirical method of precision-cut liver slice technique, and study the hepatotoxicity of monocrotaline by this technique.

METHODLiver slices were prepared by the domestic shaking slicer. The technique of precision-cut liver slice was established by detecting MTT reduction used as the slice viability under different culture medium, thickness of slices, pH and culture temperature. After monocrotaline and liver slices co-culture for 6, 24 h, the slice viability, enzyme activity of GPT, GOT, LDH, GGT and protein concentration were detected by MTT reduction, enzyme kinetics method and BCA protein assay method, respectively.

RESULTWhen the thickness of slices was 200 microm and pH of medium was 6.8, culture temperature was 37 degrees C, BPM culture medium, the viability of slices could maintain on a steady level. LDH leakage was significantly increased and protein content was obviously decreased after monocrotaline co-culture for 24 h with final concentration 0.02, 0.1 and 0.5 g x L(-1). No statistically significant difference between control group and monocrotaline 3 dose groups was observed in the slice viability and the content of GPT, GOT, LDH, GGT and protein after monocrotaline co-culture for 6 h.

CONCLUSIONThe slice viability could retain 24 h in modified BPM medium surroundings; monocrotaline displayed liver toxicity in some degree after co-culture for 24 hours in 0.02, 0.1 and 0.5 g x L(-1) concentration.

Animals ; Hydrogen-Ion Concentration ; L-Lactate Dehydrogenase ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Monocrotaline ; toxicity ; Rats ; Rats, Sprague-Dawley ; Temperature ; Toxicity Tests

OBJECTIVETo establish a simple and feasible method of anaphylactoid test on awaked small animals for screening and assessing anaphylactoid reaction of traditional Chinese medicine (TCM) injection with different concentration of tween 80.

METHODTest substances containing 0.4% Evans blue were intravenously injected into mice at volume of 20 mL x kg(-1) or guinea pigs at a volume of 30 mL x kg(-1). The behaviors were observed and the vascular permeability of ears evaluated by the extent of ear blue staining and absorbance of Evans blue extraction of ears were tested at 30 min after injection.

RESULTTween 80 solution, Yuxingcao injection with tween 80, and Shuanghuanglian powder injection obviously increased vascular permeability of ears characterized as ear blue staining and increased absorbance of the Evans blue extract from ears extracted by acetone saline both in mice and in guinea pigs in a concentration-dependent (in the case of tween 80) or a dose-dependent (Shuanghuanglian) manner.

CONCLUSIONEar vascular permeability test in mice and guinea pigs can be used as animal models to screen and test anaphylactoid reaction induced by injections.

Anaphylaxis ; chemically induced ; Animals ; Capillary Permeability ; drug effects ; Guinea Pigs ; Male ; Medicine, Chinese Traditional ; adverse effects ; Mice ; Mice, Inbred ICR ; Models, Animal