Integration of Network Pharmacology and Experimental Validation to Explore Jixueteng - Yinyanghuo Herb Pair Alleviate Cisplatin-Induced Myelosuppression

Abstract

Jixueteng, the vine of the bush Spatholobus suberectus Dunn., is widely used to treat irregular menstruation and arthralgia. Yinyanghuo, the aboveground part of the plant Epimedium brevicornum Maxim., has the function of warming the kidney to invigorate yang. This research aimed to investigate the effects and mechanisms of the Jixueteng and Yinyanghuo herbal pair (JYHP) on cisplatin-induced myelosuppression in a mice model. Firstly, ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) screened 15 effective compounds of JYHP decoction. Network pharmacology enriched 10 genes which may play a role by inhibiting the apoptosis of bone marrow (BM) cells. Then, a myelosuppression C57BL/6 mice model was induced by intraperitoneal (i.p.) injection of cis-Diaminodichloroplatinum (cisplatin, CDDP) and followed by the intragastric (i.g.) administration of JYHP decoction. The efficacy was evaluated by blood cell count, reticulocyte count, and histopathological analysis of bone marrow and spleen. Through the vivo experiments, we found the timing of JYHP administration affected the effect of drug administration, JYHP had a better therapeutical effect rather than a preventive effect. JYHP obviously recovered the hematopoietic function of bone marrow from the peripheral blood cell test and pathological staining. Flow cytometry data showed JYHP decreased the apoptosis rate of BM cells and the western blotting showed JYHP downregulated the cleaved Caspase-3/Caspase-3 ratios through RAS/MEK/ERK pathway. In conclusion, JYHP alleviated CDDP-induced myelosuppression by inhibiting the apoptosis of BM cells through RAS/MEK/ERK pathway and the optimal timing of JYHP administration was after CDDP administration.

Keywords: cisplatin; epimedium sagittatum; myelosuppression; suberect Spatholobus stem; traditional Chinese medicine.

Figure 1.

Flow chart of JYHP alleviating myelosuppression: (A) flow chart of network pharmacology analysis and experiment validation and (B) flow chart of vivo experiment.

Figure 2.

Effect of JYHP on hematopoiesis: (A) body weight in each group, (B) RET count, (C) RBC count, (D) HGB count, (E) PLT count, (F) WBC count, (G) LYM count, (H) NEUT count. *P < .05, **P < .01, ***P < .001, ****P < .0001. Data was shown as mean ± SEM; n = 6.

Figure 3.

Basic condition of C57BL/6 mice and pathological staining: (A) body weight in each group, (B-I) peripheral blood examination in each group, (J) HE staining of bone marrow, and (K) HE staining of the spleen, arrow refers to megakaryocytes. *P < .05, **P < .01, ***P < .001, ****P < .0001. Data was shown as mean ± SEM; n = 6.

Figure 4.

Determination of JYHP components: (A) UPLC-Q-TOF/MS chromatogram of JYHP decotion and (B) identifiable components of JYHP.

Figure 5.

Network pharmacology analysis reasoning possible mechanism of JYHP alleviating myelosuppression. (A) The Venny diagram shows potential targets for JYHP in the treatment of myelosuppression. (B) PPI network of potential targets for JYHP in alleviating myelosuppression. The circle represents the target protein. The darker the color, the larger the diameter of the circle, representing a greater degree value. The lines represent the interaction relationship between proteins. (C) Hub genes for JYHP treating myelosuppression. (D) GO-BP, GO-CC, GO-MF term analysis of hub genes in Metascape. (E) KEGG pathway enrichment in Metascape.

Figure 6.

mRNA expression levels of target genes. Quantification of mRNA expression levels of AKT1 (A), TP53 (B), TNF (C), IL6 (D), RELA (E), EGFR (F), CASP3 (G), MAPK1 (H), MYC (I), VEGFA (J). Data were presented as the mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001; n = 3.

Figure 7.

JYHP alleviated CDDP induced apoptosis of BM cells. (A) Apoptosis of BM cells was examined by flow cytometry analysis with Annexin V APC and 7-AAD double staining. (B) The apoptosis rate calculated as early apoptosis (Q3) plus late apoptosis (Q2) and shown in the histogram. (C/D) Apoptosis protein expression measured by Western blotting. (E/F) Cell cycle of each group was examined by flow cytometry analysis. Data were presented as the mean ± SEM. *P < .05, **P < .01, n = 3.

Figure 8.

Affect of JYHP administration on protein expression of RAS/MEK/ERK pathway. (A) Expressions of β-Actin, ERK, p-ERK, MEK, p-MEK measured by Western blotting. Quantification of protein expression levels of RAS (B), p-MEK/MEK (C), p-ERK/ERK (D) Data were presented as the mean ± SEM. *P < .05, **P < .01, n = 3.