Abstract

Objective

   The objective of this study was to assess the pharmacological activity
   and therapeutic mechanism of Dahuang Mudan Decotion (DHMDD) for
   colorectal cancer using ultra-performance liquid chromatography tandem
   mass spectrometry (UPLC-MS), network pharmacology and in vitro
   experiments.

Methods

   The chemical components of DHMDD were identified by UPLC-MS. Network
   pharmacological analysis was utilized to screen the active ingredients
   and targets associated with DHMDD for colorectal cancer. Based on the
   results of network pharmacology, the potential mechanism of DHMDD on
   colorectal cancer predicted was experimentally studied and verified in
   vitro.

Results

   DHMDD primarily exerts its effects on colorectal cancer through 52
   active ingredients. AKT1, ESR1, HSP90AA1, JUN, PIK3CA, PIK3CB, PIK3R1,
   SRC, STAT3, TP53 were the top 10 targets. The top 10 ingredient nodes
   were Quercetin, Physcione, Pontigenin, Crysophanol, Linolenic acid,
   Piceatannol, Adenosine, Emodin, Sambunigrin, and Prunasin. The main
   compounds and the target proteins exhibited strong binding ability in
   molecular docking studies. The results of cell experiments demonstrated
   that DHMDD can inhibit the proliferation, invasion and migration of CRC
   cells through the PI3K/Akt pathway.

Conclusion

   Through network pharmacology analysis and cell experiments, this study
   suggests that DHMDD can exert its therapeutic effects on colorectal
   cancer through a combination of multiple components and targets.

   Keywords: Dahuang Mudan Decotion, Colorectal cancer, Network
   pharmacology, Molecular docking, Traditional Chinese medicine

1. Introduction

   Colorectal cancer (CRC) is a common malignancy of the digestive tract
   that poses a significant health risk. The 2020 Cancer Statistics report
   published by the International Agency for Research on Cancer (IARC)
   revealed that CRC is the third most frequently diagnosed cancer and the
   second leading cause of cancer-related mortality worldwide. In 2023,
   approximately 153,020 individuals were diagnosed with colorectal
   cancer. China has higher incidence and mortality rates of CRC compared
   to the global average [[35]1,[36]2]. In recent years, dietary and
   lifestyle changes have occurred alongside socio-economic development
   and improvements in living standards. There is a distinct trend of
   colorectal cancer patients getting younger, as the number of
   individuals under 50 years old continues to rise annually [[37]2]. CRC
   is characterized by a subtle onset, challenging early detection, and a
   bleak prognosis, resulting in many patients missing the opportunity for
   optimal surgical intervention. By the time typical symptoms manifest,
   the tumor is already in the intermediate or advanced stages, with a
   five-year survival rate of less than 50 % [[38]3]. Therapeutic
   strategies for CRC encompass surgery, chemotherapy, radiation therapy,
   and immunotherapy. However, the clinical efficacy and prognosis of
   these approaches are unsatisfactory, often accompanied by severe side
   effects and high toxicity [[39]4]. Traditional Chinese Medicine (TCM)
   is extensively utilized as an adjunct therapy for CRC in China.
   Additionally, TCM has been found to impact cancer prognosis. Clinical
   studies have demonstrated the significant role of TCM in ameliorating
   patients' clinical symptoms, boosting immunity, reversing chemotherapy
   resistance, and preventing tumor recurrence and metastasis [[40]5].
   Therefore, there is promising potential in exploring therapeutic drugs
   for CRC derived from TCM that exhibit notable efficacy with minimal
   side effects.

   Dahuang Mudan Decotion (DHMDD) is a renowned traditional Chinese herbal
   prescription that was originally documented in ShanghanLun, a
   well-known text on TCM authored by Zhang Zhongjing [[41]6]. It is
   composed of five TCMs, including Rheum potaninii Losinskaja (Dahuang in
   Chinese), Moutan Cortex (Moudanpi in Chinese), Prunus persica L.
   Batsch. (Taoren in Chinese), Semen Benincasae（Dongguazi in Chinese）and
   Natrii Sulfas (Mangxiao in Chinese). DHMDD had been found to have an
   anti-cancer effects for CRC related intestinal diseases [[42][7],
   [43][8], [44][9]]. DHMDD mitigates ulcerative colitis in mice by
   modulating ILC3 function to repair intestinal barriers [[45]10], and by
   adjusting gut microbiota to restore Th17/Treg balance [[46]11]. The
   main pharmacological ingredients include emodin, rhein, aloe emodin,
   chrysophanol, emodin methyl ether, paeoniflorin, paeonol, amygdalin,
   prunasin etc. Emodin reduces tumor burden in colorectal cancer by
   decreasing M2-like macrophages [[47]12]. Rhein enhances the sensitivity
   of human colorectal cancer cells to EGFR inhibitors by blocking the
   STAT3 pathway [[48]13]. Aloe emodin inhibits EGF-induced neoplastic
   cell transformation by targeting the ERK/MSK1 and AKT/GSK3β signaling
   pathways [[49]14]. Chrysophanol suppresses proliferation, invasion, and
   tumorigenesis of colon cancer SW480 cells in nude mice via the
   AMPK-dependent signaling pathway [[50]15]. Emodin methyl ether
   effectively suppresses the release of inflammatory factors during
   intestinal epithelial cell injury, mitigating LPS-induced intestinal
   epithelial cell damage by boosting autophagy proteins like Beclin-1 and
   LC3 while inhibiting the expression of gap junction protein Cx43
   [[51]16]. Paeoniflorin combats colorectal cancer cell stemness through
   the miR-3194–5p/catenin beta-interacting protein 1 axis [[52]17].
   However, there is a limited number of scientific studies of DHMDD on
   the mechanisms of CRC treatment. There is also a lack of systematic and
   comprehensive analysis and identification of DHMDD chemical components.
   Therefore, the aim of this study was to characterize the chemical
   components of DHMDD and explore the role of DHMDD in colorectal cancer
   and its molecular mechanism. It provides theoretical basis for clinical
   application of DHMDD and elucidation of its pharmacological substance
   basis.

   Network pharmacology has emerged as a valuable approach for
   comprehending the intricate interactions between molecules and
   proteins, offering significant potential in elucidating the
   pathogenesis and progression of diseases, TCM syndromes, and the
   mechanisms underlying TCM's therapeutic effects. The integration of
   network pharmacology and in vitro experiments represents an effective
   strategy for elucidating the anti-cancer effects of traditional Chinese
   medicine. These findings provide novel insights into the mechanism of
   action of DHMDD in CRC treatment and offer a scientific foundation for
   its clinical application.

2. Materials and methods

2.1. Materials and reagents

   Formic acid was obtained from Tianjin Kermel Chemical Reagent Co., Ltd.
   in Tianjin, China. Methanol and Acetonitrile (HPLC grade) were supplied
   by Fisher Scientific Corporation in Pittsburgh, PA, USA.
   Chromatographic pure Leucineenkephalin was purchased from Sigma-Aldrich
   in St Louis, MO, USA. Ultra-pure water was produced using a Milli-Q
   Ultra-pure water system from Millipore in Billerica, USA. Other
   reagents and chemicals were of analytical grade. The crude drugs,
   including Dahuang, Mangxiao, Taoren, Mudanpi, and Dongguazi, were
   obtained from Harbin Tongrentang Drug Store in Harbin, China. This
   study was conducted at the Faculty of Pharmacy, Heilongjiang University
   of Chinese Medicine.

   The CRC cell line HCT116 was purchased from the Cell Bank of the
   Chinese Academy of Sciences (Shanghai, China). Cells were cultured in
   MCCOY’S 5A medium (Solarbio, China) supplemented with Fetal bovine
   serum (CELL-BOX, China). Penicillin-streptomycin was obtained from.
   Beyotime Biotechnology (Shanghai, China). CCK-8 Kit from Seven biotech
   (Beijing, China)was used to mesure the cell Cytoactive. The 24-well
   Transwell insert system (Corning, NY, USA, aperture 8 mm) was used to
   measure the ability of cells to invade. Matrigel was purchased from
   Corning (NY, USA). Total RNA Extraction Kit from Seven biotech
   (Beijing, China)was used to extract total RNA from cells. Two Step RT
   and qPCR Kit from Seven biotech (Beijing, China) was used to detect
   mRNA level expression in cells.Primary antibody β-Actin, AKT1, p-AKT
   were purchased from Beyotime Biotechnology (Shanghai, China) and Bcl-2,
   BAX were purchased from Affinity Biosciences (Jiangsu, China).
   HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from
   Beyotime Biotechnology (Shanghai, China).

2.2. Preparation of Dahuang Mudan decoction

   The DHMDD was prepared according to the method and procedure described
   in Shang-Han-Lun and was optimized: Dahuang (55.2 g), Mudanpi (13.8 g),
   Taoren (15 g), and Dongguazi (30 g) were soaked in double-distilled
   water for 30min at a ratio of 1:10 (m/v), decocted for 45 min, then
   added Mangxiao (48 g), decocted for 2min. The resulting filtrates were
   mixed, condensed by heating evaporation, and dried using a vacuum
   freeze drier.

2.3. Component identification analysis of DHMDD decoction

   A 500 mg DHMDD freeze-dried powder was weighed and then sufficiently
   dissolved in 20 ml 75 % methanol, the solution was sonicated for 30 min
   and centrifuged at 13000 rpm for 15 min at 4 °C, and then filtered
   through a 0.22um filter and analyzed by UPLC. Chromatographic
   separation of the metabolites was performed on the Acquity UPLC system
   (Waters Corporation, Milford, Massachusetts) by an Acquity UPLC BEH C18
   column (2.1 × 100 mm, 1.7 μm, Waters) at 35 °C column temperature.

   Chromatographic conditions: Column temperature was 35 °C, injection
   volume was 4 μL, flow rate was 0.3 mL/min, Mobile phase: Mobile phase A
   consisted of water and 0.1 % formic acid; mobile phase B consisted of
   methanol with 0.1 % formic acid with the gradient elution mode
   (0–1.5 min, 99-95 % A; 1.5–12 min, 95–85 % A; 12–20min, 85-64%A;
   20–22min, 64-50%A; 22–25min, 50-1%A, 25–27min, 1%A; 27–28 min, 1–99%A;
   28–30min, 99%A).

   Mass spectrometry conditions: Capillary voltage was 3.0 kV in
   ESI + mode and 2.8 kV in ESI- mode, sampling cone voltage was 30 V,
   desolvation temperature was 350 °C, desolvation gas flow was 600L/h.
   The Q-TOF Premier acquisition rate was fixed to 0.4 s/scan, with 0.1s
   inter scan delay. The mass range was set at m/z 50–1500 using extended
   dynamic range. Leucine-enkephalin was used for mass accuracy correction
   during the whole experiment.

2.4. Network pharmacology

2.4.1. Establishment of component target database for DHMDD

   The constituents identified from DHMDD were entered into the PubChem
   database [[53]18]([54]https://www.ncbi.nlm.nih.gov/pubmed/) to obtain
   the SMILES and SDF files. Subsequently, these files were input into the
   Swiss ADME database ([55]http://www.swissadme. ch/) [[56]19] for
   further analysis. The active constituents were then screened using
   Lipinski's rule of five [[57]20], the selection criteria were as
   follows: (1) Molecular weight <500, (2) Rotatable bonds ≤10, (3) H-bond
   acceptors ≤10, (4) H-bond donors ≤5, (5) Log Po/w ≤ 5. If a compound
   violates more than two of the five rules, it should be further
   investigated by conducting a literature search to identify the main
   active components that have been previously reported. Then, the
   component targets were found by inputting them into the Swiss Target
   Prediction Database ([58]http://www.swisstargetprediction.ch/). targets
   <0.5 probability were removed. In order to gather more information
   about the targets, the names of the ingredients were entered into the
   Traditional Chinese Medicine Systems Pharmacy Database and Analysis
   Platform (TCMSP) [[59]21].

2.4.2. Establishment of therapeutic target database for CRC

   The information on therapeutic target was searched by using “colorectal
   cancer” as the keyword. The therapeutic targets database can be
   obtained from OMIM [[60]22], GeneCards [[61]23], TCMSP, and TTD
   [[62]24] databases.

2.4.3. Enrichment analysis and construction of the components-targets-pathway
network

   We employed the cluster profile R package to analyze the potential role
   of active components in DHMDD in gene function and signaling pathways,
   specifically focusing on the Gene Ontology (GO) and Kyoto Encyclopedia
   of Genes and Genomes (KEGG) pathways. GO terms and KEGG pathways were
   deemed statistically significant if they had a p-value <0.05. We
   constructed a components-targets network for DHMDD in the treatment of
   CRC using the network-visualization software Cytoscape v3.8.0.

2.4.4. Molecular docking study of target proteins and active components of
DHMDD

   Suitable target protein structures were selected from the PDB database
   [[63]25], with the species defined as “Homo sapiens,” fine resolution
   less than 2.5, and small molecular compounds attached as reference
   standards. Obtain the SDF file of the 3D structure of the active
   ingredients of DHMDD from the PubChem database. AutoDock software
   [[64]26] was utilized to dehydrate the target protein, adjust the size
   and position of the docking box, and optimize the conformation of the
   ligand and receptor. The results were outputted in the “pdbqt” format.
   PyMol 2.4.0 [[65]27]was employed to visualize the results of molecular
   docking, and the docking affinity, hydrogen bonding, and bond length
   reflected the stability of intermolecular interactions.

2.5. Experimental validation in vitro

2.5.1. CCK8 assay

   Colorectal cancer HCT116 cells in the logarithmic growth phase were
   harvested, and the cell density was adjusted to 5 × 10^3 cells/mL.
   Subsequently, 100 μL of cell suspension was seeded into each well of a
   96-well plate. DHMDD was dissolved in fresh culture medium and
   configured with different concentrations. Following a 24-h incubation
   at 37 °C with 5 % CO2, the culture medium was aspirated, and 100 μL of
   fresh culture medium containing various concentrations of DHMDD (0, 1,
   2, 4, 8, 16, 32, 64 mg/ml) was added to each well. Incubate the plate
   in a 37 °C incubator with 5 % CO2 for 24 h. Add 10 μL of CCK-8 reagent
   to each well, mix thoroughly, and incubate for 60 min in the incubator.
   Measure the absorbance at a wavelength of 490 nm using a microplate
   reader and calculate the results. The inhibitory effect of DHMDD on the
   proliferation of colorectal cancer cells was evaluated, and a line
   graph was plotted.

2.5.2. Colony formation assay

   The experiment was divided into a control group and an experimental
   group. The experimental group was treated with DHMDD for 24 h.
   Monolayer-cultured cells in the logarithmic growth stage were
   dissociated into single cells using 0.25 % trypsin, counted, and then
   suspended in culture medium containing 10 % fetal bovine serum.
   700 cells were seeded per well in a six-well plate and cultivated for
   14 days until colonies were visible to the naked eye. The number of
   cell clones formed in the 6-well plate was quantified using ImageJ
   software and the difference between the two groups was calculated.

2.5.3. Transwell assay

   Cell invasion ability was assessed using the transwell assay. The
   Matrigel was taken out in advance and placed it in a 4 °C refrigerator
   to thaw for later use. The bottom of the upper side of the Transwell
   chamber was coated with Matrigel. Placed in the incubator for 3 h and
   taken out for use when a white layer appeared. Colorectal cancer
   HCT116 cells were divided into control group and DHMDD group and
   treated for 24 h. Then the HCT116 cells were adjusted to
   2.0 × 10^5 cells/ml in the serum-free medium and culture medium
   containing 10 % FBS was added to the lower chamber and incubated for
   48 h in a cell incubator. The Transwell chamber was taken out, and the
   culture medium was aspirated. It was washed twice with PBS, and then
   4 % paraformaldehyde was added to fix it for 10–15 min. After that, it
   was stained with 0.1 % crystal violet for 5–10 min. The number of cells
   passing through the membrane were counted by an inverted microscope.
   ImageJ software was used for image analysis.

2.5.4. Wound healing assay

   The cell migration ability was assessed using a wound-healing assay.
   Briefly, HCT116 cells were seeded in 6-well plates
   (1 × 10^6 cells/well). When reaching 100 % confluence, a scratch was
   generated using a 10-μl pipette tube. The debris was removed by washing
   with phosphate buffered saline (PBS), then incubated with serum-free
   medium and medium containing DHDDT for 24 h. Images were captured using
   a light microscope at 0 h and 24 h. ImageJ software was used for image
   analysis, then the migration rate was assessed.

2.5.5. Quantitative real-time polymerase chain reaction (qRT-PCR)

   Based on the findings of network pharmacology, we chose AKT, PI3K,
   PI3KCA, and PI3KR1 to investigate the mechanism by which DHMDD promotes
   cell proliferation in colorectal cancer (CRC). Total RNA was isolated
   from the cultured cells using the SevenFast Total RNA Extraction Kit.
   Subsequently, reverse transcription was performed, and qRT-PCR was
   carried out using the SevenFast Two Step RT and qPCR Kit. β-actin as
   internal reference. Fluorescence signal was confirmed by melting curve
   analysis, and mRNA levels of target genes were calculated using the 2
   ^–ΔΔCT method. The primer sequences utilized for qRT-PCR are provided
   in [66]Table 1.

Table 1.

   Real-Time polymerase chain reaction primers.
     Gene           Sequence (5′-3′)
   AKT1-F    5′-AGAAGCAGGAGGAGGAGGAG-3′
   AKT1-R    5′-CCCAGCAGCTTCAGGTACTC-3′
   PI3K–F    5′-AAGCAGTGCCTGTAGGAGGA-3′
   PI3K–R    5′-TGTCGATGAGCTTTGGTGAG-3′
   PIK3CA-F  5′-CATGCATTGTTTTGCACCCC-3′
   PIK3CA-R  5′-ATGGAAGACGGGAGATTCACAT-3′
   PIK3R1-F  5′-TCTACCCAGTGTCCAAATACCAG-3′
   PIK3R1-R  5′-TAAATGCTTCGATAGCCGTTC-3′
   β-actin-F 5′-CAGATGTGGATCAGCAAGCAGGA-3′
   β-actin-R 5′-CGCAACTAAGTCATAGTCCGCCTA-3′
   [67]Open in a new tab

2.5.6. Western blotting

   Colorectal cancer HCT116 cells in the logarithmic growth phase were
   harvested and seeded in 6 cm culture dishes at a density of
   3 × 10^5 cells per dish. After 24 h, the cells were divided into two
   groups: the Control group and the DHMDD group. Cells from each group
   were collected 24 h later. The cells were washed twice with 1 × PBS,
   and total proteins were extracted using RIPA buffer. The protein
   concentrations were determined using a BCA protein assay kit. The
   protein samples were transferred onto PVDF membranes. Subsequently, the
   membranes were incubated overnight at 4 °C with primary antibodies
   targeting β-Actin, AKT1, p-AKT, Bcl-2, and BAX, and then incubated with
   HRP-conjugated secondary antibodies. The grayscale values were
   quantified using Image Lab software.

2.5.7. Statistical analysis

   Data are presented as the mean ± SD from ≥3 independent experiments.
   Data were analyzed using one-way ANOVA among multiple groups. The
   experimental results were subjected to statistical analysis using
   GraphPad Prism 8.0 software. Differences in experimental data among
   groups were compared using single-factor analysis of variance (ANOVA)
   followed by Dunnett's test. A significance level of α = 0.05 was used
   as the threshold for statistical significance.

3. Results

3.1. Identification of chemical compounds in DHMDD

   The chemical constituents of DHMDD were analyzed using a rapid analysis
   technique combined with UPLC-QTOF-MS/MS ([68]Fig. 1A and B). Both
   positive and negative ionization modes were employed, and the analysis
   involved comparing the retention behavior, MS/MS data, and mass
   spectrometry characteristics of known reference compounds. A total of
   81 ingredients in DHMDD were identified by matching them against a
   chemical library, as presented in [69]Supplementary Table S1. Out of
   these, 35 compounds were identified as Dahuang, 3 as Dongguazi, 34 as
   Mudanpi, 7 as Tairen, and 2 as a combination of Dahuang and Mudanpi.

Fig. 1.

   [70]Fig. 1
   [71]Open in a new tab

   Representative base peak intensity (BPI) chromatograms of DHMDD in (A)
   positive and (B) negative modes by UPLC-ESI-Q-TOF/MS. The numbering of
   identified compounds (1−81) is the same as in [72]Supplementary Table
   S1.

3.2. Targets and network analysis of DHMDD against CRC

   A total of 65 active compounds underwent ADME screening ([73]Fig. 2).
   The SMILES of the 65 active compounds were used to predict their
   potential targets by querying SwissTargetPrediction and TCMSP
   databases. After removing duplicates, a total of 562 genes related to
   the compounds were collected. Additionally, a database search retrieved
   a total of 1900 genes related to CRC ([74]Supplementary Table S2). The
   analysis revealed 224 overlapping genes when comparing the 562
   compound-related genes with the 1900 disease-related genes ([75]Fig.
   2A; [76]Supplementary Table S2). A compound-target network of DHMDD
   against CRC was constructed using Cytoscape 3.8.0, incorporating the 65
   active compounds and 224 overlapping genes ([77]Fig. 2B). The network
   demonstrated the differential contributions of these active compounds
   and genes to DHMDD against CRC. The targets were uploaded to the String
   database, restricting the species to “Homo sapiens”, and a confidence
   level of 0.900 was used to construct a PPI network graph ([78]Fig. 3A).
   The protein interactions were imported into Cytoscape 3.8.0 software,
   and topological analysis was conducted using the NetworkAnalyzer tool
   ([79]Fig. 3B). The top 10 targets based on the degree parameter were
   AKT1, ESR1, HSP90AA1, JUN, PIK3CA, PIK3CB, PIK3R1, SRC, STAT3, and TP53
   ([80]Fig. 3C). The top 10 ingredient nodes with the highest degree,
   betweenness centrality, and closeness centrality in the topological
   analysis were Quercetin, Physcione, Pontigenin, Crysophanol, Linolenic
   acid, Piceatannol, Adenosine, Emodin, Sambunigrin, and Prunasin,
   indicating their strong associations with multiple targets. GO and KEGG
   enrichment analyses were conducted on the aforementioned 224 targets to
   investigate their involvement in the treatment of CRC. [81]Fig. 4A/B/C
   displays the top 20 terms in the GO biological process (BP), cellular
   component (CC), and molecular function (MF) ranked by p-value. The
   analysis revealed that these potential targets were primarily
   associated with the membrane raft, membrane microdomain, and membrane
   region, and they were involved in regulating molecular functions such
   as protein serine/threonine kinase activity, protein tyrosine kinase
   activity, phosphatase binding, oxidoreductase activity under oxidative
   stress, protein kinase B signaling, peptidyl-tyrosine phosphorylation,
   inflammatory response, and protein kinase B signaling. Furthermore, the
   results of the KEGG analysis showed significant enrichment of 224
   overlapping genes between DHMDD and CRC in 20 signaling pathways
   (p < 0.05). These pathways include the PI3K-Akt signaling pathway,
   Lipid and atherosclerosis, Proteoglycans in cancer, Chemical
   carcinogenesis-receptor activation, Fluid shear stress and
   atherosclerosis, Endocrine resistance, AGE-RAGE signaling pathway in
   diabetic complications, HIF-1 signaling pathway, and EGFR tyrosine
   kinase inhibitor resistance ([82]Fig. 4D). Results were visualized with
   a heatmap ([83]Fig. 5A), revealing that DHMDD's core target and main
   active ingredients exhibit good binding ability, suggesting a
   synergistic effect on the core target through multi-component action.
   High scores in DHMDD (Physcione, Crysophanol, Quercetin, and Emodin)
   and their targets were visualized using PyMol 2.4.0 ([84]Fig. 5B).

Fig. 2.

   [85]Fig. 2
   [86]Open in a new tab

   (A)the Venn Diagram between constituents-related targets and
   CRC-related targets; (B)The network of 53 constituents and overlapped
   CRC-related targets.

Fig. 3.

   [87]Fig. 3
   [88]Open in a new tab

   (A) Protein-Protein Interaction Networks of potential targets; (B)
   topological analysis of potential targets; The color from light to dark
   indicates the Degree value from small to large; (C) the Interaction
   Networks of ten core targets.

Fig. 4.

   [89]Fig. 4
   [90]Open in a new tab

   Enrichment analysis of potential targets from main active ingredients
   of DDMDD: (A) Gene ontology terms for biological process; (B) Gene
   ontology terms for molecular function; (C) Gene ontology terms for
   cellular component; (D) KEGG pathways.

Fig. 5.

   [91]Fig. 5
   [92]Open in a new tab

   (A) Molecular docking heat map; (B)Schematic diagram of molecular
   docking.

3.3. Experimental validation in vitro

3.3.1. DHMDD effect on colorectal cancer cells proliferation

   The concentration-dependent toxic effect of DHMDD on HCT116 cells was
   observed to gradually intensify by CCK-8 assay, as indicated by the
   increasing inhibition rate. Based on the curve in [93]Fig. 6A, the IC50
   of DHMDD on HCT116 cells was estimated to be approximately 17.8 mg/ml.
   These findings confirm the toxic effect of DHMDD on HCT116 cells, and
   the concentration corresponding to half inhibition rate was selected
   for subsequent experiments. The same effect was also detected in a
   colony formation assay ([94]Fig. 6B). The DHMDD group exhibited a
   significant decrease in the number of cell clones compared to the
   control group, and this difference was statistically significant
   (p＜0.01).

Fig. 6.

   [95]Fig. 6
   [96]Open in a new tab

   (A) The graph of DHMDD inhibiting HCT116 growth. (B) Effect of DHMDD on
   CRC cell clonal formation (**p＜0.01). (C) Effect of DHMDD on CRC cell
   invasion (**p＜0.01).(D) Effect of DHMDD on CRC cell migration
   (**p＜0.01).(E) Effect of DHMDD on PI3K, AKT1, PIK3CA and PIK3R1 mRNA
   expression.(F) Effect of DHMDD on AKT1, p-AKT1, Bcl 2 and Bax protein
   expression (*p < 0.05, **p < 0.01).

3.3.2. DHMDD effect on colorectal cancer cells invasion and migration

   In view of cellular invasion and migration were the important
   characteristics of tumor metastasis, HCT116 cells were observed for
   assessing the capability of invasion and migration by using
   Matrigel-coated transwell plates and wound healing assay. The results
   demonstrated a significant reduction in the invasion and migration
   abilities of Colorectal Cancer Cells following treatment with DHMDD
   ([97]Fig. 6C and D).

3.3.3. DHMDD induced apoptosis via PI3K/AKT signaling pathway

   The mRNA expression levels of key targets, including AKT1, PI3K,
   PIK3R1, and PIK3CA, were assessed using qRT-PCR to investigate the
   effect of DHMDD. The DHMDD group exhibited significantly higher mRNA
   expression levels of AKT1, PI3K, PIK3R1, and PIK3CA compared to the
   control group (p＜0.05), as shown in [98]Fig. 6E. The results of western
   blotting analysis showed the AKT1 protein expression in the DHMDD group
   was not significantly different from that in the control group, while
   the expression of p-AKT1 was significantly decreased (p＜0.05). The
   DHMDD group exhibited higher Bax protein expression compared to the
   control group (p＜0.05), and there was no statistically significant
   difference in Bcl-2 protein expression between the two groups. However,
   the ratio of Bax/Bcl-2 proteins was higher in the DHMDD group than in
   the control group (p＜0.05) ([99]Fig. 6F).

4. Discussion

   Colorectal cancer is a highly prevalent and lethal malignancy
   worldwide. The primary treatment modalities for this disease include
   surgery, radiotherapy, chemotherapy, and targeted therapy. However,
   these approaches have limitations such as extensive surgical trauma,
   significant side effects of radiotherapy and chemotherapy, and limited
   efficacy of targeted therapy based on the molecular profile of the
   tumor. Therefore, the current research focus is to discover novel
   treatment strategies that can enhance the quality of life and survival
   rates in patients with colorectal cancer [[100]4]. DHMDD is a
   traditional Chinese medicine formulation comprising Rheum potaninii
   Losinskaja, Moutan Cortex, Prunus persica L. Batsch, Semen Benincasae,
   and Natrii Sulfas. It is known for its efficacy in treating intestinal
   diseases. However, the precise mechanism of action of DHMDD in the
   treatment of colorectal cancer remains incompletely elucidated.

   In this study, UPLC-Q/TOF-MS technology was employed to analyze and
   identify 81 compounds present in DHMDD. To establish the correlation
   between these compounds and the clinical efficacy, network pharmacology
   methods were utilized to screen and identify 224 targets. The network
   pharmacology study found the connection between the active ingredients
   of traditional Chinese medicine, potential targets and diseases,
   predicted the key targets of traditional Chinese medicine in the
   treatment of related diseases, and combined with molecular docking,
   could preliminarily verify the stability of the combination of
   effective active ingredients and key targets. Among the 52 active
   ingredients found in DHMDD, this study revealed that quercetin,
   physcione, pontigenin, crysophanol, linolenic acid, piceatannol,
   adenosine, emodin, sambunigrin, and prunasin are the main active
   ingredients derived from DDMDD for the treatment of colorectal cancer,
   as determined from a network pharmacology perspective.

   The PPI network screening results show that AKT1, ESR1, HSP90AA1, JUN,
   PIK3CA, PIK3CB, PIK3R1, SRC, STAT3 and TP53 are the top ten targets.
   AKT1 is a serine/threonine protein kinase that plays an important role
   in regulating cell growth, proliferation, and survival [[101]28]. ESR1
   is the most important regulatory gene in the occurrence, development
   and metastasis of CRC, and is a potential mechanism for inducing CRC
   [[102]29]. HSP90AA1 is closely linked to tumor cell cycle, apoptosis,
   invasion, and differentiation, making it a key protein in CRC treatment
   [[103]30]. JUN, a member of the AP-1 family of transcription factors,
   plays a crucial role in cell proliferation, apoptosis, and
   transcriptional regulation. Abnormal expression of JUN in colorectal
   cancer is linked to tumor invasion and metastasis [[104]31]. The PI3K
   family, a class of phosphatidylinositol kinases, plays a vital role in
   cell proliferation, survival, and metabolic regulation. PIK3CA, PIK3CB,
   and PIK3R1, important members of the PI3K family, are frequently
   mutated and abnormally activated in colorectal cancer [[105]32]. SRC, a
   non-receptor tyrosine kinase, is involved in cell signaling and
   transcriptional regulation. Abnormal activation of SRC is associated
   with tumor growth, invasion, and metastasis [[106]33,[107]34]. STAT3, a
   transcription factor, plays a critical role in cell proliferation,
   survival, and immune regulation [[108][35], [109][36], [110][37],
   [111][38]]. TP53, a tumor suppressor gene, is essential for apoptosis,
   DNA repair, and cell cycle regulation. Mutations and loss of function
   in TP53 are common in various cancers [[112]39,[113]40]. These targets
   have significant implications in the onset and progression of
   colorectal cancer. The therapeutic efficacy of DHMDD may be closely
   related to its regulatory effects on these targets.

   KEGG pathway enrichment analysis showed that DHMDD treatment of CRC may
   involve PI3K/Akt signaling pathway, AGE-RAGE signaling pathway, HIF-1
   signaling pathway and other pathways in cancer. Among them, the highly
   enriched PI3K/Akt signaling pathway is one of the most important
   signaling pathways in various cancers, and its abnormal activation can
   promote the development of tumors. The PI3K/Akt signaling pathway is
   frequently mutated and abnormally activated in various tumor cells
   [[114][41], [115][42], [116][43]]. The activation of PI3K/AKT signaling
   pathway is involved in the regulation of biological processes related
   to cell proliferation, differentiation, motility, survival and various
   metabolic processes [[117]44,[118]45]. Molecular docking showed that
   DHMDD had the lowest binding energy and the most stable conformation
   with PI3KCA and AKT1. Therefore, based on GO function, KEGG pathway
   enrichment analysis and molecular docking results, the core target of
   the PI3K/Aktsignalingpathway was selected for cell verification in this
   study.

   We conducted in vitro experimental studies to investigate the potential
   mechanism of DHMDD in colorectal cancer, based on the results of
   network pharmacology. Cell experiments demonstrate that DHMDD can
   enhance apoptosis in colorectal cancer cells by modulating the PI3K/Akt
   pathway. This suggests that DHMDD has the potential to suppress the
   growth and metastasis of colorectal cancer by modulating the PI3K/Akt
   signaling pathway. These findings offer valuable insights into the
   potential mechanism of DHMDD as a therapeutic approach for colorectal
   cancer. This also validates the reliability of network pharmacological
   predictions and molecular docking results.

   In summary, this study demonstrates that DHMDD has the potential to
   treat colorectal cancer through a multi-component and multi-target
   approach, as evidenced by network pharmacology analysis and cell
   experiments. The findings of this study provide additional support for
   considering DHMDD as a potential therapeutic drug for colorectal
   cancer. Nevertheless, this study has certain limitations. First of all,
   this study primarily relies on in vitro experiments and lacks
   validation in clinical trials and animal models. Therefore, additional
   studies are required to verify the effectiveness and safety of DHMDD in
   real clinical settings. Secondly, this study solely examined the
   pharmacological activity and a portion of the therapeutic mechanism of
   DHMDD, necessitating further comprehensive investigation into its
   overall mechanism of action in colorectal cancer.

   Overall, our in-depth exploration of the pharmacological activity and
   therapeutic mechanism of DHMDD revealed its potential to inhibit the
   growth and metastasis of colorectal cancer through the regulation of
   the PI3K/Akt signaling pathway. These findings offer novel insights for
   the clinical application of DHMDD and are expected to stimulate further
   research and development in the field of colorectal cancer treatment.
   Future research can explore the synergistic effects of combining DHMDD
   with other treatments to enhance therapeutic outcomes. Additionally,
   research on drug metabolism, drug interactions, as well as the
   toxicological and side effects of DHMDD, is imperative. By employing a
   comprehensive approach that integrates multidisciplinary research
   methods, we can achieve a more comprehensive understanding of the
   mechanism underlying DHMDD's efficacy in colorectal cancer treatment,
   thereby offering more effective therapeutic strategies for clinical
   practice.

5. Conclusion

   This study comprehensively evaluated the pharmacological activity and
   therapeutic mechanism of DHMDD in treating colorectal cancer using
   network pharmacology and in vitro experiments. The research findings
   demonstrate that DHMDD inhibits the proliferation, invasion and
   migration of colorectal cancer cells and promotes cell apoptosis by
   regulating the PI3K/Akt signaling pathway. This finding holds
   significant importance in enhancing our understanding of the
   anti-colorectal cancer mechanism of DHMDD. DHMDD, a traditional Chinese
   medicine, shows potential as an adjuvant treatment for colorectal
   cancer, offering significant efficacy and minimal side effects.
   However, despite the important findings obtained in this study, there
   are still certain limitations. Future research should focus on further
   verifying the therapeutic efficacy and safety of DHMDD, utilizing
   clinical experiments and animal models. Furthermore, additional studies
   are needed to fully comprehend the therapeutic effects of DHMDD in
   colorectal cancer by investigating other mechanisms. In conclusion,
   this study establishes a scientific foundation for considering DHMDD as
   a potential drug for colorectal cancer treatment. The findings of this
   study offer significant support for considering DHMDD as a potential
   drug for colorectal cancer treatment and provide valuable guidance for
   further research and development of DHMDD's clinical applications. This
   will help enhance the treatment outcomes and quality of life for
   individuals diagnosed with colorectal cancer, while also presenting
   novel concepts and strategies for personalized treatment within the
   field of colorectal cancer.

Data availability statement

   Data will be made available on request.

Funding

   This work was supported by the Natural Science Foundation of Shanxi
   Province (No: 202303021222376, No：202303021212366), the Shanxi
   Provincial Health Commission Research Project (No:2023141), the Natural
   Science Foundation of Heilongjiang Province (No: LH2019H049).

CRediT authorship contribution statement

   Xinghua Li: Writing – review & editing, Writing – original draft,
   Conceptualization. Xinyue Liu: Writing – review & editing, Validation.
   Fan Yang: Validation. Tianwei Meng: Data curation. Xiang Li:
   Validation. Yan Yan: Validation. Keyuan Xiao: Validation, Supervision,
   Funding acquisition.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Footnotes

   ^Appendix A

   Supplementary data to this article can be found online at
   [119]https://doi.org/10.1016/j.heliyon.2024.e32136.

Appendix A. Supplementary data

   The following are the Supplementary data to this article:
   Multimedia component 1
   [120]mmc1.docx^ (46.7KB, docx)
   Multimedia component 2
   [121]mmc2.xlsx^ (5.4MB, xlsx)
   Multimedia component 3
   [122]mmc3.docx^ (758.2KB, docx)

References