NMR Assignments of Six Asymmetrical N-Nitrosamine Isomers Determined in an Active Pharmaceutical Ingredient by DFT Calculations.

N-nitrosamines, which are well-known pro-mutagens, are found in drugs, pickled food and tobacco. Therefore, controlling their concentrations is very important. When an HPLC, GC or NMR analysis is conducted to investigate certain asymmetrical N-nitrosamines, two sets of signals attributed to the asymmetric N-nitrosamine isomers are usually observed. However, few reports on the NMR assignment of asymmetrical N-nitrosamine isomers have been published. In this study, we investigated the NMR assignments of the Z/E isomers of six asymmetrical N-nitrosamines by means of density functional theory (DFT) calculations. The configuration of the major isomer of asymmetrical N-nitrosamine 3 was the Z-configuration. The configuration of the major isomers of asymmetrical N-nitrosamines 4-7 was the E-configuration. Then, we determined the Z/E ratios of these asymmetrical N-nitrosamines by means of variable temperature (VT) and room temperature (RT) 1H-NMR experiments. The ratios of the Z/E isomer 3 quickly increased beyond 100% in the VT 1H NMR experiments. The ratios of Z/E isomers 4-7 were increased in the range of 10-60% in the VT 1H NMR experiments. The results of this study indicate that identifying the isomers of asymmetrical N-nitrosamine is necessary to control the quality of N-nitrosamines for active pharmaceutical ingredients (APIs).

[Mechanism of traditional Chinese medicine balancing Yin-Yang by targeting ERα/ERß and its application in treatment of menopausal syndrome].

The coordination and unification of Yin and Yang are the basis of normal human life activities. Along with the age growth and aging of the body, women will suffer from menopausal syndrome during menopause. In addition to the significant changes in the genital system, there are also pathological manifestations in estrogen target points including bone, nerve and cardiovascular systems, due to the imbalance of Yin and Yang. Besides the insufficiency of estrogen, the main cause of menopausal syndrome is the changes in the response of target organs to estrogen. In other words, the biological effects mediated by estrogen receptor(ER) alpha and beta subtypes in target cells are often different or even opposite; the changes of expression level and ratio of ERα and ERß are also important causes for the abnormal estrogenic effects in target organs and the imbalance of Yin and Yang of the body. Therefore, on one hand, the therapeutic mechanism of drugs is ER-mediated estrogenic effect. On the other hand, the drugs have a regulatory effect on ER subtype expression in target cells and Yin-Yang state in target organs and even organisms, so as to cause further changes in the response of target cells to estrogen or estrogenic components, and exert its therapeutic effects. This paper reviews the pharmacological mechanism of gynecological traditional Chinese medicine in harmonizing Yin and Yang in estrogen-positive target cells and the clinical efficacy in the following aspects, including estrogen and its mechanism, the estrogenic effect of ER in traditional Chinese medicine and the mechanism of ER subtype in balancing Yin and Yang and mediating and regulating the main target tissues in menopausal syndrome treatment.

[Molecular mechanism of apoptosis of breast cancer SKBR-3 cells induced by cryptanshinone via G protein coupled estrogen receptor( GPER) mediated pathway].

The study aimed to illuminate the role of G protein coupled estrogen receptor( GPER) and its mediated PI3 K/AKT signaling pathway in cryptotanshinone( CPT) induced apoptosis of breast cancer SKBR-3 cells,which is GPER positive and ER negative.The apoptosis rate of SKBR-3 cells was tested by Annexin V-FITC/PI staining and apoptosis effector caspase-3 was determined by Western blot. The key proteins in PI3 K/AKT signaling pathway mediated by GPER were detected by Western blot and immunofluorescence technique. Meanwhile,the agonist G1 and antagonist G15 of GPER and antagonist LY294002 of PI3 K were employed in the test to further clarify the effect of GPER and PI3 K/AKT pathway. The results indicated that the apoptosis rate was increased from 4. 7% to46. 1% and 69. 0% after treatment with 0,5,10 µmol·L~(-1) CPT for 48 h( P<0. 01). The expression of PI3 K,AKT and p-AKT were inhibited( P<0. 05 or P<0. 01),while caspase-3 level increased obviously after treatment with CPT( P<0. 01). Importantly,inhibitory effect of PI3 K/AKT signaling pathway by CPT was further enhanced by G1 and attenuated by G15. LY294002 also induced a further inhibition of expression of AKT and p-AKT. The mean fluorescence intensity of AKT and p-AKT could be decreased by CPT. Furthermore,CPT could downregulate GPER expression in SKBR-3 cells( P<0. 01),which could be inhibited by G1 and enhanced by G15.In conclusion,CPT could induce the apoptosis of ER negative and GPER positive breast cancer SKBR-3 cells and the molecular mechanism is related to its regulatory effect of GPER and its mediated PI3 K/AKT signaling pathway.

Fructus Ligustri Lucidi modulates estrogen receptor expression with no uterotrophic effect in ovariectomized rats.

BACKGROUND: Accumulating evidence suggests that Fructus Ligustri Lucidi (FLL) plays a beneficial role in preventing the development of osteoporosis. However, the effects of FLL on estrogen receptor (ER) α and ERß expressions remain unknown. Therefore, in the current study we attempted to probe into the effects of FLL on ERα and ERß expressions in femurs, tibias and uteri of ovariectomized (OVX) rats. METHODS: The OVX rats were orally administrated with FLL water extract (3.5 g/kg/day) for 12 weeks. The uteri, femurs, tibias and serum were harvested from rats. The serum levels of estrogen (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by ELISA. The expressions of ERα and ERß in the femurs and tibias as well as uteri were analysed by western blot and immunohistochemical staining. RESULTS: FLL treatment did not increase uterus relative weight in OVX rats. Further, FLL treatment increased ERα expression in the femurs and tibias, and enhanced ERß expression in the uteri of OVX rats. However, the resulted expression of ERα was stronger than that of ERß in OVX rats in response to FLL treatment. Meanwhile, administration with FLL to OVX rats increased FSH and LH but did not increase E2 level in the serum. CONCLUSION: FLL treatment shows tissue selection on ERα and ERß expressions in the femurs and tibias as well as uteri of OVX rats without uterotrophic effect, which may offer the scientific evidence of the efficiency and safety of its clinical application.

Curcumin treatment suppresses CCR7 expression and the differentiation and migration of human circulating fibrocytes.

BACKGROUND/AIM: Recent studies have demonstrated that circulating fibrocytes contribute to the formation and development of fibrosis. Curcumin, a polyphenolic compound isolated from turmeric, has been shown to have anti-fibrotic effects in various organs. We and others have demonstrated that curcumin beneficially affects the development of fibrosis. However the effect of curcumin on circulating fibrocytes has not been reported. METHODS: Human circulating fibrocytes were isolated from leukocyte concentrates of healthy human donors and identified based on the expression of CD34, CD45, collagen I (COLI), and chemokine receptor CCR7 (CCR7) via flow cytometry. Cell Counting Kit-8 was used to evaluate cell viability. The effect of curcumin on the differentiation and migration of human circulating fibrocytes was evaluated by immunofluorescence staining, flow cytometry and a transwell migration assay. Transforming growth factor (TGF)-ß1 secretion was examined by ELISA. RESULTS: Curcumin treatment (72 h; 20 µM) significantly decreased the expression of COL I, α-SMA and CCR7, as well as TGF-ßl secretion, in human circulating fibrocytes. The inhibitory effect of curcumin on the differentiation and migration of human circulating fibrocytes is likely via regulating the CCR7/CCL21 signaling pathway, in particular by reducing CCR7 expression. These observed effects may be beneficial in resolving fibrosis by suppressing TGF-ß1 secretion. CONCLUSION: Our results suggest that curcumin has the potential to suppress the differentiation and migration of circulating fibrocytes, which would provide new explanation for curcumin's application in the development of fibrosis in various organs.

[Effect of carnosol against proliferative activity of breast cancer cells and its estrogen receptor subtype's mediation and regulation mechanisms].

Carnosol has been proved to have anti-breast cancer effect in previous research. But its ER subtype's specific regulation and mediation mechanisms remain unclear. The aim of this study is to observe the effect of carnosol on cell proliferation and its estrogen receptor α and ß's specific regulation and mediation mechanisms with ER positive breast cancer T47D cell. With estrogen receptor α and ß antagonists MPP and PHTPP as tools, the MTT cell proliferation assay was performed to observe the effect of carnosol on T47D cell proliferation. The changes in the T47D cell proliferation cycle were detected by flow cytometry. The effect of carnosol on ERα and ERß expressions of T47D cells was measured by Western blot. The findings showed that 1 x 10(-5)-1 x 10(-7) mol x L(-1) carnosol could significantly inhibit the T47D cell proliferation, which could be enhanced by MPP or weakened by PHTPP. Meanwhile, 1 x 10(-5) mol x L(-1) or 1 x 10(-6) mol x L(-1) carnosol could significantly increase ERα and ERß expressions of T47D cells, and remarkably increase ERα/ERß ratio. The results showed that carnosol showed the inhibitory effect on the proliferation of ER positive breast cancer cells through target cell ER, especially ERß pathway. In the meantime, carnosol could regulate expressions and proportions of target cell ER subtype ERα and ERß.

The antioxidant effect of carnosol in bovine aortic endothelial cells is mainly mediated via estrogen receptor α pathway.

Antioxidant action is critical for maintaining the normal cardiovascular function and vascular endothelial cell is an important target of estrogen action through estrogen receptor (ER) pathway. This study is carried out to explore the antioxidant effect of carnosol in bovine aortic endothelial cells (BAECs) via ER pathway. The ER subtype specific estrogenic effect of carnosol was further demonstrated by luciferase reporter gene assay in human embryonic kidney (HEK) 293 cells. Carnosol was extracted from Chinese medicine Rosmarinus officinalis. ER positive BAECs were employed in cell proliferation assay and cell apoptosis tests. Oxidative stress by intracellular reactive oxygen species (ROS) were measured via 2'7'-dichlorofluorescein (DCF) production. ERα and ERß specific antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-yl]phenol (PHTPP) were employed as tools in the experiment. ER negative HEK 293 cells were employed in luciferase reporter gene assay. The results indicate that carnosol can effectively attenuate H(2)O(2) induced slowing down of cell growth and increasing of cell apoptosis. At the meantime, carnosol pretreating can also effectively reduce the H(2)O(2) induced intracellular ROS elevation in BAECs. ERα and ERß antagonist, especially ERα antagonist, can effectively decrease the above antioxidant effects of carnosol. The reporter gene analysis further demonstrates that the action of carnosol on inducing ERE dependent luciferase expression is realized via ER pathway. The conclusion is that carnosol can exert antioxidant effects towards oxidative stress induced by H(2)O(2) in BAECs. And such effects are realized via ER, especially ERα pathway. The results contribute to explain the mechanism of cardiovascular protective function of carnosol in postmenopausal women.

[Effects of curcumin on bleomycin-induced damages in pulmonary functions in rats].

OBJECTIVE: To determine the effects of curcumin on bleomycin (BLM)-induced pulmonary fibrosis in rats. METHOD: One hundred and forty-four male Sprague-Dawley rats were randomized into 6 groups (24 rats in each group, model group, sham group, prednisone group (0.56 mg x kg(-1) x d(-1)), curcumin with low dose 5 mg group, curcumin with middle dose group 10 mg and curcumin with high dose group 20 mg per 100 g of body weight). Rats in all groups except in sham group were injected with BLM intratracheally. Curcumin with different doses were given by gavage one time everyday for 7, 14 and 28 days. Prednisone were given to rats in prednisone group, po, serving as the positive treatment group. On the 7th, 14th, 28th day, the lung functions (inspiratory resistance, maximal volutary ventilation, forced vital capacity, Fev 0.2/FVC, peak expiratory flow) were determinated in experimental rats, respectively, and the concentrations of hydroxyproline in lung homogenates of each rat were assayed. RESULT: Administration of curcumin in different doses improved lung functions of BLM-induced fibrotic rats in the all experimental days; and it decreased the concentration of hydroxyproline in lung homogenates compared with those levels in model control group; and it also lessened the hyperplasia of BLM-induced pulmonary fibrosis in rats. CONCLUSION: Administration of curcumin can suppress BLM induced pulmonary fibrosis indicated by improved respiratory function, as well as companied with low content of hydroxyproline in lung tissue of rats.

[Experimental study on effect and mechanism of soybean protease inhibitor on endotoxin-induced acute lung injury].

OBJECTIVE: To study the protective effect of soybean protease inhibitor on LPS-induced lung injury in rats. METHOD: Fifty male SD rats were randomly divided in five groups, 10 rats in each group as sham-operation group, model control group, positive medicine group, and high, moderate SBTI groups. Except the sham-group, other groups were induced by intratracheal instillation of LPS with a dose of 6 mg x kg(-1). All rats were given drug throughout intraperitoneal injection except the model controlled group, the positive medicine group was given PMSF with a dose of 50 mg x kg(-1), the high dose group of SBTI was given SBTI with a dose of 100 mg x kg(-1), a dose of the moderate group is 50 mg x kg(-1). We examined all rats in seven days. Index exam: cell quantity, activity of neutrophilic granulocyte released elastic protease proteins in BALF, histopathological examination and so on. RESULT: Soybean protease inhibitor can level down the level of total protein, cell quantity, PMN percent, activity of neutrophilic granulocyte in BALF. SBTI level down the content of NF-kappa B in nucleoprotein, while increase the content of I kappa B alpha in plasmoprotein. CONCLUSION: SBTI is useful in protecting experimental pulmonary injury induced by LPS in rats.

A novel triterpenoid saponin from Polygala tenuifolia Willd.

A new triterpenoid saponin, tenuifoside A, was isolated together with three known triterpenoid saponins 2, 3, and 4 from the roots of Polygala tenuifolia Willd. With the help of chemical and spectral analyses (IR, MS, 1D-NMR, and 2D-NMR), the structure of the new saponin was elucidated as 3-O-beta-d-glucopyranosyl presenegenin 28-O-beta-d-xylopyranosyl-(1 --> 3)-beta-d-xylopyranosyl-(1 --> 4)-[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-p-methoxycinnamoyl]-[alpha-l-rhamnopyranosyl-(1 --> 3)]-beta-d-fucopyranosyl ester (1). Three known triterpenoid saponins (2-4) were identified on the basis of spectroscopic data.

A new triterpenoid saponin from Albizia julibrissin Durazz.

A new triterpenoid, saponin hehuanoside A, was isolated together with the known triterpenoid saponins 2, 3, and 4 from the stem bark of Albizia julibrissin. With the help of chemical and spectral analyzes (IR, MS, 1D-NMR, and 2D-NMR), the structure of the new triterpenoid saponin was elucidated as 21-O-[(6S)-2-trans-2,6-dimethyl-6-O-beta-d-quinovopyranosyl-2,7-octadienoyl]-3-O-beta-d-xylopyranosyl-(1 --> 2)-beta-d-fucopyranosyl-(1 --> 6)-beta-d-2-deoxy-2-acetamidoglucopyrasyl acacic acid 28-O-alpha-l-arabinofuranosyl-(1 --> 4)-[-beta-d-glucopyranosyl-(1 --> 3)]-alpha-l-rhamnopyranosyl-(1 --> 2)-beta-d-glucopyranosyl ester (1). Three known triterpenoid saponins 2-4 were identified on the basis of spectroscopic data.

Two new furostanol saponins from Tribulus terrestris L.

Two new furostanol saponins, tribufurosides B (1) and C (2), were isolated from the fruits of Tribulus terrestris L. With the help of chemical and spectral analyses (IR, MS, 1D NMR and 2D NMR), the structures of two new furostanol saponins were established as 26-O-beta-d-glucopyranosyl-(25S)-5alpha-furost-20(22)-en-2alpha,3beta,26-triol-3-O-beta-d-galactopyranosyl(1 --> 2)-beta-d-glucopyranosyl(1 --> 2)-beta-d-galactopyranoside (1) and (25S)-5alpha-furost-20(22)-en-12-one-3beta, 26-diol-26-O-beta-d-glucopyranoside (2).

A novel steroidal glycoside, ophiofurospiside A from Ophiopogon japonicus (Thunb.) Ker-Gawl.

A new furospirostanol saponin, ophiofurospiside A (1), was isolated together with the known steroidal glycosides 2, 3, and 4 from the tubers of Ophiopogon japonicus (Thunb.) Ker-Gawl. Using chemical and spectral analyses (IR, MS, 1D NMR, and 2D NMR), the structure of 1 was established as 26-O-beta-d-glucopyranosyl-(22S, 25R)-furospirost-5-ene-3beta, 17alpha, 26-triol-3-O-[alpha-l-rhamnopyranosyl-(1 --> 2)]-[beta-d-xylopyranosyl-(1 --> 4)]-glucopyranoside (1). Three known steroidal saponins 2-4 were identified on the basis of spectroscopic data.

[Research on phytoestrogenic effects and its mechanism of psoralen].

OBJECTIVE: To observe and evaluate the phytoestrogenic effects and its mechanism of psoralen in estrogen receptor (ER) alpha and beta positive T47D and ishikawa cells. METHOD: The proliferation rate of T47D influenced by 1 x 10(-5) mol x L(-1) to 1 x 10(-9) mol x L(-1) psoralen and that of Ishikawa influenced by 1 x 10(-6) mol x L(-1) and 1 x 10(-7) mol x L(-1) psoralen were analyzed by MTT assay. PR mRNA expression in T47D was quantified by RT-PCR assay. Estrogen receptor antagonist ICI 182, 780 was employed as a tool. ER-alpha and ER-beta expression of T47D was measured by flow cytometry. RESULT: The proliferation rates of T47D cells treated with 1 x 10(-5) mol x L(-1) to 1 x 10(-7) mol x L(-1) psoralen and ishikawa cells treated with 1 x 10(-6) mol x L(-1) to 1 x 10(-7) mol x L(-1) psoralen were increased significantly. The RT-PCR result showed that 1 x 10(-7) mol x L(-1) and 1 x 10(-6) mol x L(-1) psoralen could increase PR expression in T47D cells. The above effects could be blocked by ICI 182,780. Psoralen could also induce the augment of ER-alpha and ER-beta expression in T47D cells significantly. CONCLUSION: Psoralen has phytoestrogenic effects. The effects are attained through ER pathway.

Two new triterpenoid saponins from Pulsatilla cernua (Thunb.) Bercht. et Opiz.

Two new triterpenoid saponins, named cernuaside C and D, have been isolated from Pulsatilla cernua (Thunb.) Bercht. et Opiz. The structures of the two new triterpenoid saponins were elucidated as 3-O-beta-D-xylopyranosyl(1 --> 2)-[alpha-L-rhamnopyranosyl(1 --> 3)]-alpha-L-arabinopyranosyl oleanolic acid 28-alpha-L-rhamnopyranosyl(1 --> 4)-beta-D-glucopyranosyl(1 --> 6)-beta-D-glucopyranoside. (1) and 3-O-alpha-L-arabinopyranosyl (1 --> 3)-alpha-L-rhamnopyranosyl(1 --> 2)-alpha-L-arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranoside (2) by 1D, 2D-NMR techniques, ESI-MS analysis as well as chemical methods.

[Treatment of Alzheimer disease: an evidence-based review].

OBJECTIVE: The purpose of this research was mainly two-fold: first, to get an understanding of current researches conducted on Alzheimer disease in China; second, to systematically evaluate and compare Alzheimer's treatment delivered by traditional Chinese medicine (TCM) and Western medicine. METHODS: Two steps were employed in this research. They were data collection and cleaning, followed by systemic review and qualitative analysis. The data were selected from the following two databases: CNKI (http://www.cnki.net) and Wanfang Data (http://www.wanfangdata.com.cn). Inclusion criteria were: (1) Chinese literature; (2) Published between year 1994 and year 2004; (3) Using TCM as treatment and Western medicine as control; (4) Similar research purposes and methodology; (5) Subjects were diagnosed as Alzheimer disease. Descriptive analysis, homogeneity test, meta analysis, sensitivity analysis and subgroup analysis were performed in the second step. RESULTS: Supposing all qualified studies were of high quality, we got the following conclusion: the advantage of TCM was losing because of the newly-developed acetylcholinesterase inhibitors came in market. Moreover, the studies conducted after year 2002 were more homogeneous in comparison with those conducted in early years. Those studies using mini-mental status examination (MMSE) as outcome measurement were also more homogeneous than non-MMSE measurement groups. Combined odds ratio in comparative studies was 1.5 fold higher than that in experimental studies. Regarding to different outcome measurement, those studies using TCM assessment profile were 2.58(4.79/1.86) fold higher than those using MMSE as outcome measurement. CONCLUSIONS: After systemic literature search, we found that only 40 out of 2,403 studies met our inclusion criteria. Moreover, those qualified studies were of low quality. Therefore, the external validity of this study would be compromised. The solution to this would be to improve study quality by strengthening study design and carefully select more homogeneous subjects in terms of syndrome differentiation, by so doing, the results of meta analysis will be more convincing and easily recognized by international society.

[Evaluation on phytoestrogen effects of ten kinds of Chinese medicine including flos carthami].

OBJECTIVE: To explore the phytoestrogenic effects of ten kinds of Chinese medicine including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus ligustri lucidi, fructus lycii, radix clycyrrhizae, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae. METHOD: 240 female Kunming mice weighting 9 - 12 g were randomly divided into two main groups A and B. A group was divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine groups. B group was also divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine antagonistic groups. Mice in ten antagonistic groups were administered both Chinese medicine and diethylstilbestrol everyday. After administered(op) for 4 days, blood was collected and serum was separated. The effect of the pharmacological serum on proliferation rate of MCF-7 (ER+) was analyzed by MTT-assay. RESULT: In A group, proliferation rates of MCF-7 cells treated with serum from eight Chinese medicine groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus lycii, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae were coued markedly increase respectively. While serum from fructus ligustri lucidi group could markedly decrease the proliferation rate of MCF-7 cells. In B group, the increased proliferation rate of MCF-7 cells caused by diethylstilbestrol was significantly reduced in seven Chinese medicine antagonistic groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, radix clycyrrhizae, herba epimedii, fructus psoraleae and semen cuscutae. While the increased proliferation rate could be markedly enhanced in herba cistanches group. CONCLUSION: Six kinds of Chinese medicine such as flos carthami, radix cyathulae, radix salviae miltiorrhizae, herba epimedii, fructus psoraleae and semen cuscutae show both estrogenic effects (when administered indepently) and antiestrogenic effects (when administered together with diethylstilbestrol). Such bidirectional effects depends on the internal estrogen level.

[Apoptosis of type II alveolar epithelial cell induced by bleomycin in lung fibrotic rat].

OBJECTIVE: To investigate the effects of bleomycin (BLM) on the apoptosis of type II alveolar epithelial cell (AT II) in lung fibrotic rats and its possible mechanisms. METHODS: Totally 32 male Sprague-Dawley rats were randomly divided into sham group (n = 8) and BLM group (n = 24). Rats in sham group or BLM group were intratracheally instillated with saline or 5 mg/kg of bleomycin, respectively. One, three, and seven days after the instillation of bleomycin, 8 rats in BLM group were taken for AT II isolation and purification. Rats in sham group were used to isolate and purify AT II on 7 days after the instillation of saline. The cell cycle and apoptosis, intracellular free calcium concentration, and mitochondrial membrane potential (MMP) in AT II were determined by flow cytometry. Immunohistochemistry was performed to observe the expressions of Bax, Bcl-2, and Fas. Caspase-3, Caspase-8, and Caspase-9 activities were measured by Caspase activity detection kit. RESULTS: The ratio of S phase AT II in BLM group was significantly lower than in sham group (P < 0.05). AT II apoptosis rates on day 1 and 3 were significantly higher in BLM group than in sham group (P < 0.01). Intracellular free calcium concentrations in BLM group were significantly higher than in sham group (P < 0.05). However, MMP was significantly lower than sham group (P < 0.05). The positive rates of Bax, Fas and Caspase-3, Caspase-8, and Caspase-9 activities of BLM group were significantly higher than those of sham group (P < 0.05, P < 0.01). The positive rates of Bcl-2 on day 1 and 3 were significantly lower than those of sham group (P < 0.05). CONCLUSION: Early AT II apoptosis may be induced by bleomycin, which may be explained by the increase of intracellular free calcium concentration, depression of MMP, increased expressions of Fas and Bax, and increase of Caspase-3, Caspase-8, and Caspase-9 activities.

[Changes of matrix metalloproteinases activities in pulmonary fibrosis rats].

OBJECTIVE: To observe the changes of matrix metalloproteinases (MMPs) activities in pulmonary fibrosis rats. METHODS: Eighty male SD rats were randomly divided into sham group (n = 40) and bleomycin group (BLM, n = 40), in which SD rats were injected with a single intratracheal dose of sham saline or bleomycin respectively. On day 1, 3, 7, 14, and 28 following bleomycin or saline instillation, rats were randomly killed, and serum from abdominal aorta, alveolar fluid from the bronchoalveolar lavage, and the lung homogenate were collected and then stored at -80 degrees C. MMPs activity was determined by zymography. RESULTS: Compared with sham group, the levels of MMP-9 in all samples were augmented. MMP-9 activities in the serum were highest on day 3 than those on day 1 and day 7, and in lung tissue homogenate were highest on day 7; however, no significant differences were found between BLM group and sham group on day 14 and day 28; and that of bronchoalveolar lavage fluid (BALF) was highest on day 7 than those on day 1 and day 14, while no significant difference existed between BLM group and sham group on day 28. Serum MMP-2 level did not change from day 1 to day 28, while the level of BALF MMP-2 began to increase after day 14, even on day 28. Lung tissue homogenate MMP-2 level began to increase early on day 3 and continued to day 28. CONCLUSION: The sources and effects of MMP-2 and MMP-9 differ in BLM-induced rat pulmonary fibrosis.

[Experimental study of estrogenic activities of five kinds of Chinese herbal medicines].

OBJECTIVE: To evaluate the estrogenic activity of several kinds of Chinese herbal medicines. METHOD: Use zoopery and reporter gene technique to study the estrogenic activity of five Chinese herbal medicines. Zoopery: weanling female Kunming mice weight 9-12 g were administrated botanical extracts of Selaginella tamariscina, Pinus Massoniana, Corallodiscus flabellate, Dryopteris sublaeta and Leonurus heterophyllus, the positive control group with Nilestriol tablets and control group with water, respectiely. On the eighth day, the animals were sacrificed and the uteri were separated solely and weighed. Reporter gene technique: Induce the expression of reporter gene controlled by ERE and measure the activity of luciferase on cell's clear supernatant. RESULT: The botanical extracts of S. tamariscina can increase weights of mice (P < 0.01); In the expression of reporter gene controlled by ERE, Either ERalpha or ERbeta's has estrogenic activity (P < 0.01). Follow in the zoopery we find the water part and the n-butanol part of S. tamariscina are the two active parts. CONCLUSION: S. tamariscina and it's water part and n-butanol part have estrogenic activities, effect on ERbeta is greater than ERalpha.