AR-3

To study the effect of an acidic region-3 (AR-3), capable of improving the secretion of heavy chain of coagulation factor VIII (fVIII), on the secretion of protein spicing ligated full-length fVIII.

A pair of vectors was used to deliver intein fused heavy and light chain genes of a full-length fVIII gene with an additional AR-3 incorporated on the end of heavy chain into cultured 293 cells. The intracellular protein splicing was observed by Western blot. And the secretion of spliced fVIII and activity in culture supernatant were quantified by enzyme-linked immunosorbent assay (ELISA) and Coatest assay respectively.

A noticeable spliced fVIII protein band was observed from the gene co-transfected cells. The culture supernatant displayed a spliced fVIII of (112 ± 18) ng/ml with an activity of (0.76 ± 0.13) U/ml greater than that of cells co-transfected with AR-3-free heavy chain and light chain genes [(64 ± 11) ng/ml and (0.37 ± 0.05) U/ml]. And a spliced fVIII of (27 ± 7) ng/ml with an activity of (0.16 ± 0.05) U/ml was detected in the culture supernatant from combined cells separately transfected with AR-3-fused heavy chain gene and light gene.

AR-3 can enhance the fVIII gene transfer by improving the secretion of intein spliced full-length fVIII.

The extract from the root of Angelica acutiloba Kitagawa (AR), which is used as herbal medicine in Japan, has been reported to be clinically effective for postmenstrual blood loss and erythropoietin (EPO)-resistant anemia in chronic renal failure, although the pharmacological mechanisms underlying its clinical efficacy are unknown. We prepared an animal model of anemia by bolus injection of 5-fluorouracil (5FU) at 150 mg/kg to mice (8- to 12-week-old female C57BL/6J), and then administered orally the water-soluble fraction of AR to the anemic mice for 10 days. After confirming the anti-anemic effect of the water-soluble fraction of AR (AR-3) containing polysaccharides, we examined the effects of AR-3 on immature erythroid cell activity, EPO production, and plasma cytokine levels. AR-3 administration at 50 mg/kg activated erythroid progenitor cells in bone marrow on day 10, increased the percentage of peripheral reticulocytes in red blood cells on day 15, and led to the recovery of red blood cell count to a value that was almost equal to the basal level on day 20. Although EPO production, which was determined by examining EPO mRNA expression in kidney and liver, remained unaltered by AR-3 administration, this treatment significantly lowered plasma interferon-gamma level, which may suppress the activity of erythroid progenitor cells. These results suggest that the polysaccharides in AR promote hematopoiesis by activating immature erythroid cells, in part, by suppressing cytokine secretion. Since the hematopoietic effect was achieved by high-dose AR-3, identification of specific polysaccharides is still required for the development of a novel medicine for anemia caused by a malignancy or chemotherapy.