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 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

OBJECTIVETo investigate the characteristics, sensitizin and the mechanism of pseudo allergic reaction induced by Yuxingcao injection.

METHODBeagle dogs were randomly assigned to control group, 0.5% tween 80 group, Yuxingcao injection without tween 80 group, Yuxingcao injection included 0.5% tween 80 group. The animals in control group were intravenously injected with saline. The other group were intravenously injected with the corresponding test substances. Observe pseudo anaphylaxis of Beagle dogs within 30 min after administration. Blood pressure and respiration rate of Beagle dogs were measured before and after injection drugs 10 min and 30 min respectively. The pseudo allergic reactions were scored at same time points, and the sera of animals were collected to determine the HIS, CH50 and C5b-9 concentration using ELISA.

RESULTThe scores of allergic reaction in 0.5% tween 80 group and Yuxingcao injection included 0.5% tween 80 group was evidently higher than that in control group in 2-5 min after administration. Animals of above two groups showed the symptoms of red swelling on ear part, pruritus, throwing the head, nausea, lapping the tongue, dysphoria and bradykinesia. Some of them had behaved with repose, urination, defecation, cyanosis, the frequency of breathes accelerating and blood pressure decreasing. The rate of pseudo allergic reactions was 100%. Serum CH50 concentration of 0.5% tween 80 group decreased 10 min after injection, while C5b-9 concentration increased. No obvious differences were observed 30 min after injection. There was no significant difference in HIS concentration between control group and treatment groups.

CONCLUSIONThe pseudo allergic reactions appeared after intravenous 0.5% tween 80 and Yuxingcao injection when mixed with tween 80. Furthermore, Yuxingcao injection without tween 80 did not induce pseudo allergic reactions. It was suggested that the pseudo allergic reactions of Yu Xing Cao Injection was related to the cosolvent tween 80. The pseudo allergic reactions of tween 80 may relate to the activation of complement.

Animals ; Disease Models, Animal ; Dogs ; Drug Hypersensitivity ; etiology ; immunology ; Drug Interactions ; Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; Humans ; Male ; Polysorbates ; administration & dosage ; adverse effects ; Random Allocation

OBJECTIVETo search new effective compounds, the different hemostatic effects of Flos Sophorae, Flos Sophorae Carbonisatus and principal constituent were observed.

METHODUsing the bleeding time (BT) and the recalcification time (RT) as the specificity indicators for the hematischesis function, the hemostatic effects of the following were observed. Flos Sophorae, Flos Sophorae Carbonisatus, characteristic value extraction thing A and B (SCE A and B) and the principal constituent after orally administered in normal rats in order to analyze the new hemostatic compounds.

RESULTFlos Sophorae, and Flos Sophorae Carbonisatus can obviously reduce BT and RT in rats, in which the effect of Carbonisatus is stronger than the crude. Otherwhile, SCE A and SCE B can also obviously reduce BT and RT in rats, in which the effects of SCE B surpassed those of SCE A. Furthermore, two characteristic compounds extracted from SCE B (kaikasaponin I called compound 1 and isorhamnetin-3-O-rutinoside named called 2) and other nominated principal constituents (rutin, tannin), can obviously shorten BT and RT in rats, among which compound 2 is most superior.

CONCLUSIONThe Flos Sophorae, Flos Sophorae Carbonisatus and their character compounds can shorten! the BT and RT in rats. The compound 2 from SCE B has the most superior effect. Study showed that compound 2 should be the new hemostatic compounds after Flos Sophorae carbonized. The results also indicated that the increase of hemostatic effect after Flos Sophorae carbonized should be related with the coordination of various kinds of ingredient.

Animals ; Bleeding Time ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Flowers ; chemistry ; Hemostatics ; administration & dosage ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sophora ; chemistry

OBJECTIVETo investigate the preclinical evaluation method of pseudoanaphylactoid reactions for Chinese herbal injections.

METHODBeagle dogs were divided into control group (C), 0.5% tween 80 group (T), Yuxincao injection containing 0.5% tween 80 (YT), distilled solution from Yuxincao (Y). Various groups of Beagle dogs were given 3 mL x kg(-1) of the test articles intravenously. The anaphylactoid reactions were observed immediately, while blood pressure, respiratory frequencies and heart rates were tested at 10 min and 30 min after administration.

RESULTA variety of symptoms that range from cutaneous and mucosa signs to bronchospasma and cardiovascular collapse, including angioedema at lip, conjunctiva, ear and circumoral skin, somnolence, lethargy, breathless or dyspnea, severe hypotension etc were observed in T and TY groups from immediately post-injection to at least 30 min after administration. These reactions occurred at both first injection or repeated injections at 24 weeks intervals, manifesting that it was pseudoanaphylactoid reaction mediated by non-immune mechanisms.

CONCLUSIONBeagle dogs could be used as an animal model for preclinical evaluation of pseudoanaphylactoid reactions of Chinese herbal injection with sensitivity, reproducibility, and high clinic consistency.

Anaphylaxis ; chemically induced ; etiology ; immunology ; Animals ; Dogs ; Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; Injections, Intravenous ; Male ; Models, Animal ; Random Allocation