Extraction and identification of collagen-derived peptides with hematopoietic activity from Colla Corii Asini.

ETHNOPHARMACOLOGICAL RELEVANCE: Colla Corii Asini is a widely used traditional Chinese medicine to treat anemia with a long history due to its stimulating effect in hematopoiesis, but the components contributing to this effect are still unknown. In this study, we aimed to establish a methodology to isolate the bioactive components and provide pharmacological basis for its usage in treating anemia. METHODS: 5-FU and γ-ray radiation induced anemic mice models were generated by treating with 5-FU at 150mg/kg body weight and γ-rays by a 4MV linear accelerator by total body irradiation using female ICR mice respectively. Oral administration of fraction A was performed by gastric lavage at 1g/kg and 2g/kg body weight for 12 days and 25 days and peripheral blood sample was collected from ocular sinus red blood cell (RBC) and white blood cell (WBC) counts every 3 days and 5 days for 5-FU and radiation induced models, respectively. Next, fraction A was separated to A1 and A2 using cation exchange chromatography (IEC) based on ionic strength. Fraction A1 was further separated using reverse phase chromatography (RPC) based on the hydrophobicity first with 0-10% linear gradient, then 20%, 30%, 50% constant gradient of 60% acetonitrile in neutral Na2HPO4 buffer. Peak fractions were pooled, evaporatively dried, and dissolved in ultrapure water. Finally, fraction A11 was analyzed combining tandem mass spectrometry and proteomic tools and two peptides (peptide 11 and 16) were identified. The hematopoietic effects of multiple fractions and the two peptides were measured using colony-forming units-erythroid (CFU-E), an indication of late erythroid progenitor cells and colony-forming units granulocyte-monocyte (CFU-GM), an indication of granulocyte and monocyte progenitor cells respectively on hematopoietic progenitor cells prepared from bone marrow (Till and Mcculloch 1961). RESULTS: Fraction A at 1g/kg and 2g/kg could increase RBC and WBC counts in 5-FU and radiation induced anemic mice models. Fraction A1 at 0.1mg/ml and 0.5mg/ml, exhibited stronger hematopoietic activity than fraction A2, both of which were subfractions from fraction A using IEX, by elevated CFU-E and CFU-GM of mouse bone marrow cells. Furthermore, fraction A11 at 0.1mg/ml showed stronger CFU-E and CFU-GM than fractions A12 to A14 from RPC separation. Finally, peptide 11 and peptide 16 were identified from tandem mass spectrometry and peptide 11 increased CFU-E and CFU-GM in a dose dependent manner. CONCLUSIONS: We combined multiple approaches including chromatography, mass spectrometry, cell-based assays, as well as animal studies to identify and demonstrate that the hematopoietic effect of Colla Corii Asini is at least in part from the peptidic components identified using our methodology. This is the first time to isolate peptidic components from Colla Corii Asini, and to provide molecular basis for its usage in treating anemia, which may particularly have the potential to benefit cancer patients suffering from myelosuppression due to radiotherapy or chemotherapy.

Assay development of inducible human renal phosphate transporter Npt2A (SLC34A1) in Flp-In-Trex-HEK293 cells.

Hyperphosphatemia is associated with severe decline of renal function in chronic kidney disease and elevates cardiovascular mortality. Type II sodium dependent phosphate transporter 2A (Npt2A) plays a major role in renal phosphate reabsorption and could be explored as a target for anti-hyperphosphatemia therapy. Human Npt2A transporter activity was examined upon transfection into CHO, MDCK, HEK293, Flp-In-CHO and Flp-In-HEK293 cells. Only kidney-derived cells expressed functional Npt2A. HEK293 and Flp-In-HEK293 cell lines stably transfected with hNpt2A could be selected, but these cells were inactive in phosphate transport. This suggests that high-level, constitutive Npt2A expression has deleterious effects on the cell. By using the conditional promoter in the Flp-In-Trex vector, functional expression of Npt2A was achieved by doxycycline induction in HEK293 cells. The EGFP tagged and non-tagged, inducible stable hNpt2A-HEK293 cell lines afforded development of a robust phosphate uptake assay mediated by hNpt2A, which can be used to screen hNpt2A inhibitors and inducers of hNpt2A expression. Using this assay, the small molecule LC-1 was identified as a potent inhibitor of hNpt2A, suggesting that it is feasible to develop potent specific hNpt2A inhibitors to control phosphate overloading for hyperphosphatemia therapy.

Therapeutic effect of puerarin on non-alcoholic rat fatty liver by improving leptin signal transduction through JAK2/STAT3 pathways.

In order to investigate the mechanism of the therapeutic effect of puerarin on non-alcoholic fatty liver disease, a non-alcoholic fatty disease male rat model was induced by a high fat diet, all rats were randomly divided into a blank group, model group, simavastatin group and puerarin group. After 4 weeks of drug treatment, the liver was slided to investigate pathological morphology. Elisa was used to measure the total cholesterol (TC), triglyeride (TG) in liver, and leptin content in serum. RT-PCR and Western blotting were employed to detect liver leptin mRNA receptor expression and P-JAK2, P-STAT3 expression levels in the liver respectively. The results showed that puerarin significantly decreased the TG, TC content in liver of the non-alcoholic fatty disease rats, ameliorated steatosis in liver, lowered liver inflammatory reaction, decreased leptin level in serum, and enhanced the expression of leptin receptor mRNA and P-JAK2/P-STAT3 level. All the results demonstrated that puerarin can exhibit therapeutic effect on non-alcoholic fatty liver disease by improving leptin signal transduction through JAK2/STAT3 pathways.

Effect of constituents from Fructus Aurantii Immaturus and Radix Paeoniae Alba on gastrointestinal movement.

Fructus Aurantii Immaturus and Radix Paeoniae Alba Powder (FPP) is a popular Chinese herbal prescription. The combination of Fructus Aurantii Immaturus and Radix Paeoniae Alba has been used to treat gastrointestinal disorders for hundreds of years. To our interest, this combination shows a bilateral effect on gastrointestinal peristalsis. Our present study was focused on the bilateral role of this combination on the gastrointestinal tract. The effective constituents and mechanisms were explored. Six monomer constituents from Radix Paeoniae Alba and Fructus Aurantii Immaturus were screened by intestinal transit assay. The bilateral roles of three effective constituents were authenticated by gastric emptying assay, and the combination of three constituents showed a bilateral effect. Then, the mediating receptors and the role of NO and NF- kappaB p65 were examined to determine the mechanism involved. The overall results suggest that the major effective constituents of this combination are synephrine, hesperidin and paeoniflorin. Synephrine inhibits the gastrointestinal movement, while hesperidin stimulates it. Paeoniflorin shows different effects on intestinal and gastric activity. The effect of synephrine relies on the alpha-adrenergic receptor, and the effect of hesperidin is mediated via the H1 histamine receptor. The regulation of hesperidin and synephrine on NF- kappaB p65 translocation and NO production through the alpha-receptor and the H1 receptor, respectively, is involved in the bilateral effect of the Fructus Aurantii Immaturus-Radix Paeoniae Alba combination.

Resveratrol inhibits hypoxia-induced metastasis potential enhancement by restricting hypoxia-induced factor-1 alpha expression in colon carcinoma cells.

Resveratrol has been shown recently to exhibit antimetastatic effect on various human solid tumors. However, the possible molecular mechanism for its antimetastatic action needs to be elucidated. In this study, we investigated the effect of resveratrol on metastasis potential of colon carcinoma cells under normoxia and hypoxia in vitro. These results showed that, resveratrol can restrict the migration, adhesion, invasion and MMP-9 and MMP-2 secretion in Lovo cells cultured under normoxia and hypoxia. Hypoxia and iron chelator 2,2'-dipyridyl treatment can stimulate the invasion and migration enhancement of Lovo cells, while resveratrol exhibited substantial resistance on the metastasis potential stimulation by inhibiting the mRNA expression of VEGF and MMP-9 in colon carcinoma cells under normoxia and hypoxia, reducing HIF-1 alpha protein expression under hypoxia. Also, iron chelator 2,2'-dipyridyl treatment showed approximately the same effect on metastasis potential as Lovo cells cultured under hypoxia. These data demonstrated that, the antimetastatic effect of resveratrol under hypoxia were associated with the restriction of HIF-1 alpha protein expression and stabilization, which could be a promising drug target for resveratrol in the development of an effective chemopreventive and anticancer therapy in human tumors.

Hematopoietic effect of fractions from the enzyme-digested colla corii asini on mice with 5-fluorouracil induced anemia.

Effects of fractions A and B from enzyme-digested traditional Chinese medicine colla corii asini on mice with 5-fluorouracil-induced anemia were investigated. The purpose of this study was to further understand the hematopoietic activities and mechanisms of colla corii asini. The fractions A and B were administered to anemic mice for 12 days. After confirming the anti-anemic effect of fractions A and B, we examined the effects of fractions A and B on immature granulocyte and erythroid cell activity and plasma cytokine level. Fraction A administration at 2 g/kg and 1 g/kg and fraction B administration at 1.6 g/kg and 0.8 g/kg activated granulocyte and erythrocyte progenitor cells in bone marrow and erythrocyte progenitors in spleen, led to the recovery of white blood cell and red blood cell counts, and increased the percentage of peripheral reticulocytes in red cells. The GM-CSF and EPO production determined by examining GM-CSF mRNA and EPO mRNA in the kidney and liver of the anemic mice were also enhanced. This treatment significantly increased serum GM-CSF and EPO level and lowered serum transforming growth factor (TGF-beta) level. These results suggested that fractions A and B promoted hematopoiesis by activating immature granulocyte and erythroid cells, partly by stimulating GM-CSF and EPO secretion and suppressing TGF-beta release. Identification of a specific peptide or protein is still required for the development of a novel medicine for anemia caused by malignancy or chemotherapy.