Blood-regulating formulae based on TCM theory has been observed to exert beneficial effects such as regulating blood circulation, removing blood stasis, cooling blood, and analgesic. It is believed that blood-regulating formulae have the effects of treating trauma, inflammation, gynecological diseases, cardiovascular diseases, and cancer. We prove that the JH-CX-DG formula (Ligusticum chuanxiong-Angelica sinensis) is a key formula containing JH in blood-regulating TCM formulae. Furthermore, JH, CX, and DG belong to blood-regulating formulae based on TCM. Our results by using the Arrowsmith knowledge discovery tool revealed that the JH-CX-DG formula can target the AKT, TLR4, caspase-3, PI3K, mTOR, p38 MAPK, VEGF, iNOS, Nrf2, BDNF, NF-κB, Bcl-2, Bax 13 drug targets. We also predicted that the JH-CX-DG formula can regulate the expression of 13 miRNAs inclduing hsa-miR-124-3p and hsa-miR-29a-3p. Zhao et al. [8] demonstrated that curcumin (a compound from JH) can cause apoptosis in SKOV3 cells by upregulating miR-124 by employing a cell Counting Kit-8 (CCK-8) and a colony formation assay. Zhang et al. [9] found that curcumin inhibited TGFβ1-induced cell proliferation and collagen synthesis in NIH-3T3 cells via upregulating miR-29a by a conventional molecular biology experiment. Curcumin has been observed to possess an anti-proliferation effect on leukemic cells by up-regulation of miR-15a and miR-16. It can also inhibit cell proliferation of laryngeal cancer cells by preventing Bcl-2 and PI3K/Akt signaling pathway via upregulation of miRNA-15a [10, 11]. Moreover, Liu et al. [12] found that curcumin has a protective effect on Alzheimer's disease via upregulated miR-15b-5p by RT-qPCR. These studies indicate that JH can directly regulate the expression of various miRNA Our results were shown to be consistent with the discussed studies.
Our PPI and the location of targets analysis via pathways enrichment predicted that the JH-CX-DG formula have potential pharmacological effects including anti-inflammatory, improving microcirculation, protecting brain nerves, anti-tumor and anti-tumor angiogenesis via the regulation of the PI3K/Akt, MAPK, Toll-like receptor, T cell receptor, EGFR, VEGFR, Apoptosis, HIF-1 pathways. Liu et al. [13] indicated JH could lead to cell apoptosis in ovarian cancer cell lines SK-OV-3 by inhibiting the AKT/mTOR pathway by using an MTT assay. Ashrafizadeh et al. [13] found that JH possess various effects including antioxidant, antibacterial, antineoplastic and anti-inflammatory via targeting the Nrf2 signaling pathway. There are reports that show Chuanxiong (Ligusticum chuanxiong) and Danggui (Angelica sinensis) also can exert anti-tumor effects. Shen J et al. [14] used RT-qPCR and western blot analysis to show that CX significantly decreased the Akt expression and activity, increased the activity of caspase-3, and was also shown to have anti-tumor effects in several types of cancer. DG can induce gastric cancer cell death through inhibiting mTOR protein and can significantly prolong the survival rate of cancer patients [15]. DG also affected a variety of apoptotic factors, Bcl-2 Associated X Protein (Bax), Bcl-2 and caspase-3 expression and promoted apoptosis in human breast cancer cells, therefore it has proved to be a promising therapeutic agent for breast cancer treatment [16]. Additionally, the JH-CX-DG formula may have an anti-tumor angiogenesis effect. An ELISA assay revealed that JH suppressed VEGF secretion from tumor cells and inhibited cancer angiogenesis [17]. Shen et al. [18] found that DG could prevent cancer cell proliferation and cancer angiogenesis by inhibiting the PI3K/AKT/mTOR signaling pathway and downregulating the expression of HIF-1α and VEGF. These studies indicate that the JH-CX-DG formula can exert anti-tumor effects. Our results demonstrate a strong agreement with the aforementioned studies.
We inferred that the JH-CX-DG formula could also display anti-inflammatory, protecting brain nerves effects, and improving microcirculation effects. Zhu et al. [19] used immunohistochemistry, ELISA, and Western blot, to demonstrate that JH have the potential to alleviate acute inflammatory injury in experimental traumatic brain injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway. Data demonstrated that DG can upregulate TLR4 protein, TLR4, and miRNA expression, suggesting this could represent an effective therapy for anti-inflammatory and anti-nociceptive afflictions [20]. Experiments have demonstrated that CX dimisnished the expression of inflammatory cytokines and adhesion molecules on Vascular Endothelial Cells by Inhibiting p38 MAPK and NF-κB Signaling Pathways. These studies confirm our hypotheses regarding the anti-inflammatory effect of the components of JH-CX-DG formula. Wu et al. [21] demonstrated that JH can exert beneficial effects on ischemic cerebral injury by upregulating the caspase3 and Bcl-2 expression, downregulating the Bax expression, and promoting PI3K/AKT pathway activation. Hurley et al. [22] showed that JH also resulted in a dose-dependent increase in hippocampal BDNF and protected the brain nerves. Cheng et al. [23] found that DG pretreatment protected patients from cerebral infarct and improved neurological damage by downregulating the caspase3 and upregulated p-p38 MAPK, hypoxia-inducible factor (HIF-1), vascular endothelial growth factor-A (VEGF-A). Chen et al. [24] found that a Danggui Sini decoction could ebe effectively used to protect microvascular endothelial cells of islet microvasculature via PI3K/Akt/iNOS pathway. CX can also significantly enhance BCL-2, VEGF and reduced caspase-3 and BAX and protects against cerebral ischemia by anti-oxidation, apoptosis inhibition, and angiogenesis [25]. Our results are in agreement with the above studies, demonstrating the pharmacological effects of protecting brain nerves and improving of the JH-CX-DG formula.