[Study on time-toxicity relationship and mechanism of Gardeniae Fructus extract on hepatoxicity in rats based on proteomics].

To study the time-toxicity relationship and mechanism of Gardeniae Fructus extract on the hepatoxicity in rats. Rats were randomly divided into C group(0 day), D5 group(5 days), D12 group(12 days), D19 group(19 days), and D26 group(7 days recovery after 19 days of administration). The rats in normal group received normal saline through intragastric administration, and the rats in other groups received 10 g·kg~(-1 )Gardeniae Fructus extract through intragastric administration. After the final administration, the livers were collected. Hematoxylin-eosin staining was used to observe the histopathological changes in the liver tissue. Total liver proteins were extracted for proteomic analysis, detected by the Nano-ESI liquid-mass spectrometry system and identified by Protein Disco-very software. SIEVE software was used for relative quantitative and qualitative analysis of proteins. The protein-protein interaction network was constructed based on STRING. Cytoscape software was used for cluster analysis of differential proteins. Kyoto encyclopedia of genes and genomes(KEGG) database was used to perform enrichment signal pathway analysis. Pearson correlation analysis was performed for the screened differential protein expression and liver pathology degree score. The results showed that the severity of liver injury in D5, D12 and D19 groups was significantly higher than that in group C. The degree of liver damage in D5 group was slightly higher than that in D12 and D19 groups, with no significant difference between group D26 and group C. Totally 147 key differential proteins have been screened out by proteomics and mainly formed 6 clusters, involving in drug metabolism pathways, retinol metabolism pathways, proteasomes, amino acid biosynthesis pathways, and glycolysis/gluconeogenesis pathways. The results of Pearson correlation analysis indicated that differential protein expressions had a certain temporal relationship with the change of liver pathological degree. The above results indicated that the severity of liver damage caused by Gardeniae Fructus extract did not increase with time and would recover after drug with drawal. The above pathways may be related to the mechanism of liver injury induced by Gardeniae Fructus extract.

[Proteomics research of intervention effect of "Qi enriching" herbs on "Qi deficiency" rats based on technology of NanoLC-LTQ-Orbitrap].

Proteomics method, based on NanoLC-LTQ-Orbitrap technology, was applied to explore the biological basis of intervention effect of "Qi enriching" herbs on "Qi deficiency" rats. The "Qi deficiency" rat model was established with caloric restriction combined with excessive swimming. Muscle proteins of vastus lateralis from the blank group, the model group and the ginseng group were detected by NanoLC-LTQ-Orbitrap system. The data were imported into Protein Discovery software to identify the proteins and all the raw datum were analyzed by SIEVE software. Compared with model group, 26 significant difference proteins were found in ginseng group, which the variation trend was consistent with the blank group. Through the biological function analysis, the found proteins could be classified into proteins involved in energy metabolism, proteins involved in glucose metabolism, electrolyte balance and material transfer related proteins, inflammation related protein and cytoskeleton protein. The above target proteins and their regulation pathways may be the biological basis which ginseng played a role of tonifying "Qi" of "Qi deficiency" symptom.

[Study on discrimination mode of cold and hot properties of traditional Chinese medicines based on biological effects].

OBJECTIVE: To observe the effect of cold or hot properties of traditional Chinese medicines (TCM) on biological effect indexes, and analyze the contribution of variables on cold or hot properties, in order to preliminarily establish the discrimination mode for the biological effects of cold or hot properties. METHOD: Rats were randomly divided into the blank control group, cold TCM groups (Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex, Gardeniae Fructus, Sophorae Flavescentis Radix and Gentianae Radix) and hot TCM groups (Aconiti Lateralis Preparata Radix, Zingiberis Rhizoma, Alpiniae Officinarum Rhizoma, Zanthoxyli Pericarpium, Cinnamomi Cortex and Evodiae Fructus), and orally administered with 10 mL x kg(-1) of corresponding TCM water decoctions for 30 d, twice a day. Altogether 53 biological effect indexes correlated to cold or hot properties of traditional Chinese medicines were founded by searching literatures. The data warehouse were established by using data-mining software Clementine12.0. Data of the blank control group, cold TCM groups (Coptidis Rhizoma, Phellodendri Cortex, Gardeniae Fructus, Sophorae Flavescentis Radix, Gentianae Radix) and hot TCM groups (Aconiti Lateralis Preparata Radix, Zingiberis Rhizoma, Alpiniae Officinarum Rhizoma, Zanthoxyli Pericarpium, Cinnamomi Cortex) were selected into a training set. C5.0 algorithm and C&R classification and regression algorithm were adopted to define the importance of variable, create the decision trees, and test hot or cold properties of Evodiae Fructus and Scutellariae Radix. RESULT: According to C&R classification and regression algorithm, SDH activity of livers was the most important hot or cold property, with the significance closed to 30%. It was followed by triglyceride, liver Na' -K' -ATPase enzyme, muscle glycogen and platelet distribution width, with the accuracy up to 97.39% in models. C5.0 algorithm showed that liver SDH activity was the most important hot or cold property, with the significance closed to 40%. It was followed by triglyceride, GOT, muscle glycogen and liver Na(+)-K(+)-ATPase enzyme, with the accuracy up to 98.26% in models. The possibilities that Evodiae Fructus is in hot property and Scutellariae Radix is in cold property were 100. 00% and 77.78% by using both C&R classification and regression algorithm and C5.0 algorithm. CONCLUSION: The SDH activity of liver is the most important biological effect index to distinguish cold and hot properties of TCMs. The discrimination pathway or mode of cold and hot properties is closely related to energy metabolism.

Interaction of baicalin with berberine for glucose uptake in 3T3-L1 adipocytes and HepG2 hepatocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalin and berberine are important coexisting constituents of the combination of Radix Scutellariae and Rhizoma Coptidis, known as scutellaria-coptis herb couple (SC), which has heat clearing and detoxifying effects. The aims of the present study were to investigate the effects of the combination of baicalin+berberine on glucose uptake in 3T3-L1 adipocytes or HepG2 cells. MATERIALS AND METHODS: Insulin-resistant adipocytes and hepatocytes models were established. Glucose consumption was assayed to evaluate the effects of berberine, baicalin, and berberine+baicalin on glucose uptake, and the interaction of baicalin with berberine for glucose uptake was evaluated in 3T3-L1 adipocytes or HepG2 cells. Moreover, the effects of baicalin on the dose-effect relationship of berberine for glucose uptake was also evaluated in 3T3-L1 adipocytes. RESULTS: The results of the present study demonstrated that berberine increased glucose consumption in 3T3-L1 adipocytes and HepG2 hepatocytes in a dose-dependent manner. In contrast, statistical analyses indicated that baicalin (in doses up to 100µmol/L) produced no obvious effect. The effect of berberine+baicalin on glucose uptake was better than that of berberine or baicalin alone, which indicated that berberine and baicalin had the trend of synergetic effect on glucose uptake. Furthermore, these results showed that the synergistic effect occurred in a specific dose range, while the antagonistic effect was present in another dose range in the presence of 10µmol/L baicalin. Interestingly, the entire dose-response curves of berberine shifted down in the presence of 100µmol/L baicalin, and baicalin antagonised the effect of berberine on glucose uptake in 3T3-L1 adipocytes. CONCLUSIONS: The results of the present study showed that berberine dose-dependently increased glucose consumption in 3T3-L1 adipocytes and HepG2 hepatocytes. Furthermore, interaction of baicalin with berberine was additive at low doses of baicalin and antagonistic at higher baicalin doses. Thus, it is possible that baicalin is a partial agonist. These results provided a basis for the study of the TCM compatibility mechanism and a new insight into the application for Gegen Qinlian Decoction (GGQLD) or SC in the clinic.

Anti-diabetic activities of Gegen Qinlian Decoction in high-fat diet combined with streptozotocin-induced diabetic rats and in 3T3-L1 adipocytes.

Gegen Qinlian Decoction (GGQLD) is one of the well-known traditional Chinese medicines. Recently, it was reported that GGQLD had good clinical effects on type 2 diabetes mellitus. However, few studies have confirmed in detail the anti-diabetic activities of GGQLD in vivo and in vitro. In the present study, we investigated the anti-diabetic effects of GGQLD in high-fat diet combined with streptozotocin-induced diabetic rats and in 3T3-L1 adipocytes. The present results suggested GGQLD (4.95, 11.55 and 18.15 g/kg) decreased significantly fasting blood glucose, glycosylated serum protein, and glycosylated hemoglobin of diabetic rats (p<0.05), and GGQLD (4.95 and 18.15 g/kg) decreased significantly fasting serum insulin levels of diabetic rats (p<0.05); in 3T3-L1 adipocytes, Gegen Qinlian Decoction-containing serum (GGQLD-CS) (4%, 8% and 16%) enhanced glucose consumption, triglyceride (TG) content, adiponectin protein concentration and the mRNA expression of adiponectin. Adiponectin contributes to the regulation of lipid and glucose metabolism, and can play a critical role in the development of diabetes mellitus; the mechanisms of action of GGQLD might be related to augmentation of adiponectin protein concentration and up-regulation of the mRNA expression of adiponectin. However, the multi-target mechanisms of action of GGQLD need to be clarified further. The present study further validated the beneficial effects of GGQLD as an anti-diabetic agent. These findings provide a new insight into the anti-diabetic application for GGQLD in clinic and display the potential of GGQLD as a new drug candidate for the treatment of diabetes mellitus.

[Effect of euodiae fructus on hepatic energy metabolism-related mechanisms of reserpine-induced asthenia cold syndrome rats].

OBJECTIVE: To discuss the effect of Euodiae Fructus on hepatic energy metabolism-related mechanisms of mitochondria of hepatic tissues of asthenia cold syndrome rats. METHOD: Rats were subcutaneously injected with Reserpine to establish the model. After the oral administration with Euodiae Fructus for 12 d, the oxygen electrode method was adopted to determine the respiration efficiency. The expressions of Cox4, Atp5b, Ucp2,Pgc-1alpha, Nrf1, Tfam mRNA were assayed by using RT-PCR method. RESULT: Euodiae Fructus 4.2 g x kg(-1) could obviously increase ST3 and RCR of asthenia cold syndrome rats, and expressions of Cox4, Ucp2 Nrf1 mRNA. It could also increase expressions of Atp5b and Pgc-1alpha mRNA, but with no statistical significance. No obvious change was observed in Tfam mRNA expression. Euodiae Fructus 4.2 g x kg(-1) could significantly increase ST3 and RCR of asthenia cold syndrome rats and Pgc-1alpha mRNA and Nrf1 mRNA expressions, and significantly decrease P/O, with no obvious impact on Cox4, AtpSb, Ucp2, Tfam mRNA expressions. CONCLUSION: Euodiae Fructus can promote mitochondrial respiratory function and oxidative phosphorylation efficiency by improving Pgc-1alpha mRNA and Nrf1 mRNA expressions and regulating Cox4 and Atp5b mRNA in mitochondrial respiratory chain. It can also strengthen mitochondrial uncoupling respiration and add heat production by activating Ucp2 mRNA expression in liver.

[Recombinant human tumor necrosis factor-alpha promotes human adipose-derived stromal cells transforming into osteoblast in vitro].

OBJECTIVE: To investigate the effect of recombinant human tumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced human adipose-derived stromal cells (hASCs) in vitro. METHODS: hASCs at passage 4 were divided into four groups according to culturing conditions: basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 µg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + ß-glycerophosphate) and OM with 10 µg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . RESULTS: rhTNF-α promoted ALP activities of induced hASCs on day 14 (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells. CONCLUSION: The findings indicate that 10 µg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.

[Effect of cold property Chinese medicine radix scutellariae on energy metabolism of rats].

OBJECTIVE: To observe the effect on energy metabolism of rats with cold property Chinese medicine Radix Scutellariae. METHODS: The body weight gain, temperature, hydroposia content were determined before administration and every five days after administration. The activities of Na4(+)-K(+)-ATPase, Ca(2+)-ATPase and SDH, LPL, HP, the contents of NEAF, T3, T4, TSH were measured after having been administrated with water extracts of Radix Scutullaxiae at the dose of 6.0, 3.0 g/kg for 43 days. RESULTS: The body weight gains were raised and the hydroposia contents have been decreased. The activities of SDH were increased significantly while Na(+)-K(+)-ATPase, Ca(2+)-ATPase of liver had little change. The content of NEAF, the activity of LPL, HP were decreased significantly, and the contents of T3, T4, TSH and the body weight, temperature had no significant change. CONCLUSION: Radix Scutellariae can inhibit the energy metabolism of rat. The mechanism may not be related to thyroxine pathway.

[Effects of rhTNF-alpha on the secretion of angiogenesis-related growth factors of human adipose tissue-derived stromal cells before and after osteogenic differentiation].

OBJECTIVE: To investigate the proliferation and the secretion of vascular endothelial growth factor(VEGF), fibroblast growth factor-2(FGF-2) and insulin-like growth factor-1(IGF-1) of human adipose tissue-derived stromal cells(hADSCs) before and after osteogenic differentiation under the stimuli of recombinant human tumor necrosis factor alpha (rhTNF-alpha). METHODS: hADSCs were obtained from human lipoaspirates. All the cells used were at passage four. The proliferation of hADSCs was measured with MTT assays 48, 72, 96 hours after being treated with 0, 1, 5, 10, 50 or 100 microg/L rhTNF-alpha respectively. The secretion of VEGF, FGF-2 and IGF-1 of the undifferentiated hADSCs under stimuli of rhTNF-alpha with the above 5 concentration grades was observed and the secretion of these 3 growth factors of hADSCs at different stages of osteogenic differentiation under stimuli of 10 microg/L rhTNF-alpha was also observed. All the supernatants were harvested for measuring after 24 hours' incubation with rhTNF-alpha. The secretion of VEGF, FGF-2 and IGF-1 was measured with ELISA, and the values were normalized to the cell number of the corresponding wells. RESULTS: The effect of rhTNF-alpha on the proliferation of hADSCs varied with the concentration and time. Compared with the control(0 microg/L), 10 microg/L rhTNF-alpha showed no suppression or acceleration on proliferation of hADSCs at hour 48, but significantly promoted the proliferation at hour 96 (0.903+/-0.042 vs 0.810+/-0.011, P<0.01), 100 microg/L rhTNF-alpha seemed to suppress the proliferation at hour 48 (0.317+/-0.024 vs 0.458+/-0.046, P<0.01), but appeared to promote it (0.956+/-0.030 vs 0.810+/-0.011, P<0.01) at hour 96. rhTNF-alpha(1, 5, 10, 50 and 100 microg/L) significantly increased VEGF, FGF-2 and IGF-1 production of hADSCs versus the control (0 microg/L) (P<0.01). After osteogenic differention, the secretion of the three growth factors of hADSCs (without rhTNF-alpha treated) was elevated with the days increasing. Under the stimulus of 10 microg/L rhTNF-alpha, the hADSCs after 1 day of osteogenic differentiation significantly increased the secretion of VEGF (P<0.01) compared with the group without rhTNF-alpha treated; after 3 and 7 days of osteogenic differentiation, the hADSCs significantly increased the secretion of VEGF (P<0.01), FGF-2 (P<0.05)and IGF-1 (P<0.05). However, after 14 days of osteogenic differentiation, 10 microg/L rhTNF-alpha appeared to suppress the production of VEGF (P<0.01), FGF-2 (P<0.05) and IGF-1 (P<0.05) of the differentiated hADSCs. CONCLUSION: Within certain concentration range, rhTNF-alpha can promote the proliferation of hADSCs and the production of VEGF, FGF-2 and IGF-1. The effect of 10 microg/L rhTNF-alpha on the production of VEGF, FGF-2 and IGF-1 of the differentiated hADSCs varied at different stages of osteogenic differentiation.

[Silver-ion release and particle distribution of denture base resin containing nanometer-sized silver-supported antimicrobial agent].

OBJECTIVE: To investigate the distribution of antimicrobial agent STR-1 of nanometer level which was incorporated with ball-grinding method in the polymethylmethacrylate (PMMA) denture base, and to study the release mode of silver ions from the base. METHODS: The distribution of the antimicrobial agent in the PMMA denture base containing STR-1 at concentrations of 0 g/L, 5 g/L, and 10 g/L was examined with scanning electronic microscopy. Then, PMMA resin bases containing STR-1 at the three concentrations were respectively immersed in artificial saliva at 37 degrees C for 54 days. The release of silver ions from the resin bases was surveyed with inductively coupled plasma-mass spectroscopy (ICP-MS) every 24 hours. RESULTS: The antimicrobial agent incorporated by ball-grinding method was even-distributed with individual particles of nanometer level in the PMMA resin base. The release of silver ions from the PMMA resin with antimicrobial agent was extremely slow during the test, a very small fraction of the silver ions released. At the beginning of the test, the release speed was extremely slow, the speed increased rapidly in the middle of the test, and at the end of the test, the speed returned to slow and steady. The cumulative release curve of silver ions was of "S" type. CONCLUSIONS: STR-1 can be even-distributed in the denture base, and the silver ions release from the base with extremely slow speed. It also indicates that biological safety and long-term antimicrobial efficacy of denture base containing silver-supported antimicrobial agents of nanometer level are possibly obtained based on their slow release of silver ions.

[Biocompatibility test of polymethylmethacrylate denture base resin containing silver-supported antimicrobial agent STR-1 at nanometer level].

OBJECTIVE: To evaluate the biocompatibility of polymethylmethacrylate(PMMA) denture base resin containing silver-supported antimicrobial agent STR-1 of nanometer level in vitro. METHODS: According to the national standards for biological evaluation of dental materials, the cytotoxicity of denture base resin containing STR-1 at concentrations of 5 g/L and 10 g/L was examined by molecular filtrating method, and the hemolysis of STR-1, denture base resin containing STR-1 at concentrations of 5 g/L and 10 g/L was also surveyed. RESULTS: The control denture base resin without containing STR-1 and the denture base resins containing STR-1 at concentrations of 5 g/L and 10 g/L were not cytotoxic to L929 cells. Two hours and 24 hours after cell culturing, the filter membranes of the control and experimental groups were stained evenly with blue color. The staining intensity was not decreased and the fading areas were 0 mm2 during the culturing. The cytotoxicity grades were 0. The hemolysis rates of the antimicrobial agent STR-1 and the denture base resins containing STR-1 at concentrations of 5 g/L and 10 g/L were 1.7%, 3.5% and 3.7% respectively. They were less than the national guild standard 5% which represent no hemolysis. CONCLUSION: The PMMA denture base resins containing silver-supported antimicrobial agents STR-1 of nanometer level at concentrations of 5 g/L and 10 g/L exhibit good biocompatibility.