Mechanism of Shuangyang Houbitong granules against acute erythroleukemia through PI3K/AKT and JAK/STAT signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) posits that the onset of leukemia is often attributed to internal deficiencies and imbalances between yin and yang. Shuangyang Houbitong granules (SY) have been used in folk medicine to treat leukemia by balancing the yin-yang. However, the molecular mechanisms in which SY exerts anti-leukemia effects remain largely unexplored.

AIM OF THE STUDY: To investigate the molecular mechanisms and bioactive ingredients of SY for the treatment of acute erythroleukemia (AEL).

MATERIALS AND METHODS: A mouse model of erythroleukemia was established using Friend murine leukemia virus (F-MuLV). The therapeutic effect of SY in erythroleukemic mice was assessed through various methods, including examining spleen morphology, measuring biochemical parameters, evaluating erythrocyte differentiation, analyzing splenic immune cell markers, conducting histopathological staining, and recording survival rates. Erythroleukemia cell viability and apoptosis were measured using MTT assays, flow cytometry, and JC-1 staining. Furthermore, network pharmacology and RNA sequencing (RNA-seq) were employed to identify differentially expressed genes and explore regulatory pathways. Western blotting was utilized to detect relevant protein expression, and the components of SY were analyzed using UHPLC-Q-TOF/MS.

RESULTS: The vivo experiments revealed that SY significantly repaired F-MuLV-induced splenic injuries in erythroleukemic mice. Flow cytometry analysis indicated that SY promoted differentiation towards mature erythrocytes, markedly reducing the ratio of CD71/Ter119 in the spleens of the treated mice. In addition, SY enhanced the proportion of immune cells (CD3, CD4, CD8a, B220, and CD11b), effectively activating the immune system and prolonging the survival of the mice. In vitro, SY significantly induced apoptosis in human erythroleukemia (HEL) cells in a time- and dose-dependent manner, inhibiting HEL cell proliferation. The results of network pharmacology and RNA sequencing suggested that SY may inhibit HEL cell proliferation by affecting the PI3K/AKT, JAK/STAT signaling pathways. Consistent with the above findings, SY increased the expression of apoptosis-related proteins (Bad, cleaved caspase-3/9, and cleaved PARP) and key proteins of the JAK/STAT signaling pathway (p-JAK2, p-STAT3), while down-regulating Bcl-xl and proteins related to the PI3K/AKT signaling pathway (p-PI3K and p-AKT) in the HEL cells. Using UHPLC-Q-TOF/MS analysis, a total of 144 compounds were identified in the component of SY that entered the serum. We evaluated the anti-erythroleukemia activity of eight selected compounds (luteolin, genistin, isofraxidin, glabridin, isovitexin, bergenin, diosmetin, and xanthohumol) from the initial 144 compounds. Notably, xanthohumol (XAN) exhibited the most significant inhibition of HEL cell proliferation, inducing apoptosis and activating apoptosis-related proteins in HEL cells.

CONCLUSION: SY increased the survival rate and prolonged life span in erythroleukemia mice without inducing hepatorenal toxicity. SY inhibits HEL cell proliferation and induces apoptosis through PI3K/AKT and JAK/STAT signaling pathways. A total of 144 compounds were identified in the component of SY that entered the serum, XAN showed a significant inhibition of HEL cell proliferation.