Bioinformatics-based Prediction of the mechanism and 1 targets for BushenjieduDecoction in preventing relapse 2 of acute leukemia

bioinformatics analysis, and selected some genes for experimental verification. 35 These studies demonstrated the effect of BSJD on MSC. We hope that this research 36 method could provide a new way of systematically studying the effects of 37 traditional Chinese medicine on diseases .


14
Leukemia is a lethal myeloproliferative disorder, its' relapse following 15 chemotherapy is the major concern in clinical practice. For a long time, we found 16 that traditional Chinese medicines such as Bushenjiedudecoction (BSJD) have 17 significant effects on delaying relapse. However, the underlying mechanisms are 18 not clear, which limits the clinical application of BSJD decoction. 19

20
Therefore, we tried to make some explorations in this study. We isolated 21 mesenchymal stem cells (MSC) after treated them with BSJD for proteomic 22 analysis. And then 109 targets were screened out through analysis of the shared 23 proteins of that affected by BSJD and those related to leukemia. Subsequently, the 24 data were analyzed by GO functions, KEGG pathways, PPI network and 25 topological analysis, and then some nodes were selected for animal experiment. 26

27
As a result, we demonstrated the effective targets of BSJD on MSC through 28 bioinformatics analysis and explored the potential mechanism of BSJD from its and drug resistance, which eventually become an important cause of clinical 60 recurrence (9). Treatment to hijacked or transformed niches may provide no 61 vel therapies to delay the leukemia relapse(10). 62 As an important part of Chinese health system, traditional Chinese medicine 63 (TCM) has been used extensively. In disease free interval, we use a variety of 64 TCMs as adjuvant treatments to delay the recurrence. In these Chinese patent 65 medicines we found that Bushenjiedudecoction (BSJD) has a more obvious efficacy, 66 but the specific mechanism is still unclear. Although the research of TCM is facing 67 challenges, the developments of new technologies provide new methods to study 68 the complex mechanism of it. Especially, with the emerging of high-throughput 69 sequencing technology, it makes it possible to obtain large-scale protein data and 70 make comparative analysis of proteomic in a single experiment (11, 12).As a next-generation of high-throughput technology through relative and absolute 72 quantitative proteomics analysis with high sensitivity and repeatability, iTRAQ has 73 been widely used in cancer research (13,14). Meanwhile, various bioinformatics 74 databases based on the proteomics also provide powerful support for functional 75 annotation of proteins, classifying protein sequences into families, and comparative   100 Leukemia as the key word was searched in two data bases: NCBI (https:// 101 www.ncbi.nlm.nih.gov/gene/)and GENE CARD(https://www.genecards.org/).We 102 removed duplicates and non-mouse genes from the search results and found 103 2045 common targets in two databases. Subsequently, these differential protei 104 ns were compared with 728 differential proteins after BSJD treatment.    All data were represented in mean ± SD. Statistical analysis was performed using 155 GraphPad Prism 8 software with student's t test (P < 0.05 was considered with 156 statistical significance). Histogram was made using the Graphpad software.

159
In order to obtain the BSJD target genes, we performed high-throughput 160 proteomics analysis by iTRAQ. First, the 6-week old mice were divided 161 randomly into two groups (n=6). BSJD and Control groups were treated with 162 decoction of BSJD (0.2ml every day ) and normal saline (0.2ml every day) 163 respectively. After 28 days treatment, mice were killed and the BM cells were 164 collected. Then MSC were cultured in selective medium for mouse MSC. (Fig.2)

165
A comparative proteomic analysis was carried out between the two groups after 166 three generations. We finally got 728 differential proteins, 167 including303down-regulations and 425up-regulations. processes (BP). (Fig.4, table 1)In the higher ranking results, the words related to 193 the "binding" appeared more frequently.     an in vitro colony formation assay, we found that the colony-forming ability 266 of BSJD treated MSC was significantly reduced (Fig.8c).  then we screened out the targets of BSJD from these differential proteins through 327 bioinformatics analysis. Finally, the potential targets were preliminarily verified by qRT-PCR. These results demonstrated the effects of BSJD on MSC in leukemia. 329 We will systematically validate other potential targets in the core sub-network in 330 our following studies. We hope to systematically, comprehensively and objectively 331 explain the regulatory effect of BSJD on MSC after chemotherapy in leukemia, by 332 verifying the interaction between potential targets. 333 6. Conclusion 334 We screened the potential targets of BSJD on MSC through proteomics and 335 bioinformatics analysis, and selected some genes for experimental verification. 336 These studies demonstrated the effect of BSJD on MSC. The authors declare no conflicts of interest.