Fo Shou San (FSS; 佛手散) is an ancient paired-herb decoction that comprises of Angelica Sinensis Radix (ASR; 當歸) and Chuanxiong Rhizoma (CR; 川芎) in a ratio of 3:2, which has been used in China for treating patients who suffer from blood deficiency and blood stasis for about one thousand years. Being one of the most popular “paired-herbs” in Chinese medicines, the scientific evidences available today are very limited to support the biological functions of this decoction. Here, the effects of FSS were measured in different cell culture systems, and the underlying molecular action mechanisms were revealed.
The first critical requirement for the study of FSS is to set up its quality standard, which can be used to control the consistency for its biological functions. Here, ferulic acid, senkyunolide I, senkyunolide H and Z-ligustilide were selected as chemical markers to standardize the chemical property of FSS preparation. In our studies, the standardized FSS should contain no less than 3.9 mg ferulic acid, 3.7 mg senkyunolide I, 0.7 mg senkyunolide H and 1,0 mg Z-ligustilide per one g dried weight of FSS.
“Nourishing Blood (补血)” and “Promoting Blood Circulation (活血)”, the two major biological functions of FSS, were determined in my study. Firstly, erythropoietic precursor K562 cells and liver Hep3B cells were used to reveal the “Nourishing Blood” function of FSS. The results showed that FSS could inhibit the proliferation of K562 cells and, subsequently, induce the production of hemoglobin, a differentiation marker for the erythropoiesis in K562 cells. Additionally, erythropoietin (EPO) is a critical protein for erythropoietic precursor to survive. In cultured Hep3B cells, the mRNA level of EPO could be induced in a dose-dependent manner by FSS through inducing the transcriptional activation of hypoxia responsive element (HRE) on EPO gene. To account for the regulation of HRE, FSS could increase the protein expression of hypoxia-inducible factor-1 alpha (HIF-1α) via blocking the degradation pathway of HIF-1α protein. According to our study, the up-regulation of EPO expression, which was induced by the application of FSS, was mediated by HIF signaling pathway. Taken together, the current results supported that the “Nourishing Blood” function of FSS was achieved through the enhancement of erythropoietic activity. In addition, the up-regulation of EPO expression by FSS was mediated by HIF signaling pathway. Secondly, “Promoting Blood Circulation” function of FSS was evaluated in the blood platelets and human umbilical vein endothelial (HUVEC) cells. The results showed that FSS could inhibit the ADP-mediated platelet aggregation in a dose-dependent manner. In HUVEC cells, FSS induced the production of nitric oxide (NO) and the phosphorylation of endothelial nitric oxide synthase (eNOS) protein, which therefore, could trigger the vasodilatation and reduce the platelet aggregation. To account for the phosphorylation of eNOS protein, FSS activated the phosphorylation of Akt and increased the concentration of intracellular calcium: both of them were the eNOS regulators, which suggested that Akt and intracellular calcium were involved in FSS-induced NO production. In addition, FSS protected the reactive oxygen species (ROS)-induced endothelial dysfunction in rat aortic ring. Taken together, our results supported “Promoting Blood Circulation” function of FSS via facilitating the blood flow and preventing the blood blockage, which revealed the action mechanisms of FSS in the circulatory systems.
In order to find the active ingredients in FSS, pharmacokinetic study was employed. Ferulic acid, which is reported to have bio-activities, is a major effective compound in both ASR and CR. Therefore, the pharmacokinetic studies of ferulic acid in FSS could illustrate its mechanisms of action in vivo. After oral administration of FSS, a quick absorption of ferulic acid was observed in rat plasma, as compared to that of single administration of ferulic acid. The pharmacokinetic parameters indicated that the bioavailability and AUC (area under the curve) values of ferulic acid in FSS were higher during the oral administration.
Our study, in agreement with the traditional and current clinical usage, strongly supported the preparation and biological efficacies of FSS: (i) the chemical assessment of traditional preparation of FSS was proved to be reasonable; (ii) the effects and mechanism of “Nourishing Blood” function of FSS were elucidated, and this could further validate the ancient functions of FSS; and (iii) the vasodilation function of FSS and its underlying action mechanisms were determined, which provided evidences to support the “Promoting Blood Circulation” function of FSS. In summary, we have developed a systematic approach in addressing the chemical and biological assessment of FSS: these methodologies could be applied in other TCM formulae.
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