Abstract

Background

   JianPiTongLuo Recipe (JPTL Recipe) is a traditional Chinese medicine
   formula commonly used in the clinical treatment of colorectal cancer.
   Clinical studies have found that it can significantly improve the
   prognosis of patients with colorectal cancer. However, its mechanisms
   of action are not well understood, which has limited its further
   clinical application.

Methods

   We investigated the potential mechanisms of action of the JianPiTongLuo
   (JPTL) Recipe on colorectal cancer (CRC) using a multi-step approach.
   Initially, network pharmacology and bioinformatics analyses were
   conducted using databases such as TCMSP, HERB, BATMAN-TCM, and STRING
   to identify active components of JPTL Recipe and predict their
   therapeutic targets. Interaction networks and functional enrichment
   analyses were constructed to hypothesize relevant biological processes
   and pathways. In vitro studies involved treating human CRC cell lines
   HCT116, LoVo and SW480 with varying concentrations of JPTL Recipe
   extract, measuring cell viability with the CCK-8 assay, assessing
   apoptosis via flow cytometry, and analyzing signaling pathways through
   Western blotting. To corroborate these findings, in vivo experiments
   were performed on BALB/c nude mice implanted with HCT116 cells, divided
   into control, JPTL Recipe-treated, 5-fluorouracil (5-FU)-treated, and
   JPTL Recipe combined with 5-FU groups, with tumor growth and
   histological changes monitored. Mechanistic studies focused on the
   PI3K/AKT signaling pathway, examining the phosphorylation status of key
   pathway proteins using immunofluorescence and Western blot analyses to
   elucidate JPTL Recipe 's interaction with pathway activity.

Results

   We demonstrated that JPTL Recipe effectively inhibits colorectal cancer
   cell proliferation, anti-apoptotic ability, and exerts synergistic
   therapeutic effects with fluorouracil. Further analysis revealed that
   JPTL Recipe affects the activity of colorectal cancer cells by
   inhibiting the phosphorylation of the PI3K/AKT signaling pathway.

Conclusion

   In summary, we have discovered and confirmed that the traditional
   Chinese medicine compound JPTL Recipe can serve as a novel adjuvant
   therapy for colorectal cancer, offering a new treatment approach for
   the integration of traditional Chinese and Western medicine in the
   treatment of colorectal cancer.

   Keywords: Network pharmacology, PI3K/AKT signaling pathway, Cell
   proliferation, Apoptosis, Colorectal cancer

1. Introduction

   Colorectal cancer (CRC) is one of the highly prevalent malignant tumors
   of the digestive system worldwide [[43]1] According to global health
   statistics, colorectal cancer ranks third in global cancer incidence
   and second in cancer mortality [[44]2]. Multiple factors, including
   lifestyle factors (such as obesity, dietary habits, alcohol
   consumption) and genetic factors, are known to be associated with its
   increased incidence [[45]3,[46]4]. Despite significant progress in
   diagnosis and treatment in recent years, the survival rate and quality
   of life for CRC patients still needs improvement due to high disease
   recurrence rates and treatment side effects [[47]5].

   Current modern medical treatments for colorectal cancer typically
   include: surgical procedures, chemotherapy, radiation therapy, and
   targeted therapy [[48][6], [49][7], [50][8], [51][9]]. Although
   effective to some degree, these methods also pose risks of side effects
   and recurrence. In recent years, there has been increasing interest in
   exploring the potential value of traditional Chinese medicine in the
   treatment of colorectal cancer. Traditional Chinese medicine emphasizes
   a holistic approach and individualized treatment, and its compound
   formulations often contain multiple components that can act on multiple
   targets, thus improving treatment efficacy and reducing side effects
   [[52][10], [53][11], [54][12]].

   A recent study explored the impact of a Chinese medicine formula
   (JianPiTongLuo Recipe) on colorectal cancer (CRC) [[55]13]. In our
   study, we have discovered through network pharmacology and in vitro/in
   vivo experiments that JPTL Recipe can significantly inhibit the
   proliferation and growth of colorectal tumors. In terms of molecular
   mechanisms, we also found that JPTL Recipe exerts its effects through
   the PI3K/AKT signaling pathway, providing experimental evidence for
   elucidating the mechanism of action of this traditional Chinese
   medicine Recipe.

2. Materials and methods

2.1. Cell line

   The human colon cancer cell lines HCT116 (Grade III), LoVo (Grade
   II-III), and SW480(Grade II) were obtained from the American Type
   Culture Collection (ATCC, Manassas, USA). HCT116 and LoVo cells were
   grown in McCoy's 5a modified medium with 10 % FBS, which was prepared
   by ATCC (#30-2007). The cells were cultured at 37 °C in a 5 % CO[2]
   environment.

2.2. Traditional Chinese medicine formulation

   The JPTL Recipe was obtained from Jiangyin Tianjiang Pharmaceutical
   Co., Ltd. (Jiangyin, China) and authenticated by the Chinese Medicine
   Pharmacy of the Shanghai Traditional Chinese Medicine Hospital. The
   JPTL Recipe consists of 30 g Astragalus propinquus, 15 g Sophora
   flavescens, 15 g Codonopsis pilosula, 15 g Atractylodes macrocephala,
   15 g Poria cocos, 15 g Coix lacryma-jobi, 30 g Hedyotis diffusa and 6 g
   Glycyrrhiza uralensis. After alcohol extraction, the filtrate was
   concentrated under reduced pressure and dried. The residual liquid was
   extracted with ethyl acetate, and both layers were further concentrated
   under reduced pressure to dryness.

2.3. CCK-8 assay

   HCT116 cells in the logarithmic growth phase were seeded in a 96-well
   plate at a concentration of 8000 cells per well. After cell adhesion,
   the serum-free medium was replaced and cells were starved overnight.
   Different concentrations of JPTLR alcohol extract, specifically
   12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 300 μg/mL,
   400 μg/mL, and 500 μg/mL, were added to the cells and a control group
   was established. After 48 h, a CCK-8 solution was added at a
   concentration of 10 %, and the cells were cultured at 37 °C for 1 h.
   The absorbance of HCT116 cells was measured at 450 nm using an enzyme
   immunoassay analyzer to calculate cell viability.

2.4. Western Blot analysis

   Antibodies against PI3K (AF6241), pan-AKT1/2/3 (AF6261), p-PI3K
   (AF3241), p-pan-AKT1/2/3 (AF0016) from Affinity, and Bax (#14796), Bcl2
   (#3498), Cleaved Caspase-3 (#9664), and β-Actin (#4970) from CST were
   used. Western blot experiments were conducted according to the
   previously described method. Primary antibodies were diluted to 1:1000,
   and the secondary antibody (ab6721; Abcam) was diluted to 1:10000. RIPA
   lysis buffer (Beyotime) containing 1 % protease inhibitor and 1 %
   phosphatase inhibitor (Beyotime) was used. Protein concentration was
   quantified using the Enhanced BCA Protein Assay Kit (Beyotime)
   following the manufacturer's instructions. Equal amounts of protein
   (25 μg) were then separated by 10 % SDS-PAGE (Solarbio) and transferred
   onto PVDF membranes (Millipore, MA, USA). After blocking with 5 % skim
   milk at room temperature for 1.5 h, the membranes were incubated with
   primary antibodies overnight at 4 °C. Next, the membranes were washed
   three times with 1 × TBST (Solarbio) and incubated with secondary
   antibodies at room temperature for 2 h. After three washes with
   1 × TBST, all protein bands were visualized using Immobilon Western
   Chemiluminescent HRP Substrate (Millipore). In brief, the reaction
   liquid was evenly added to the membrane (protein surface), and the
   membrane (protein surface) was gently placed in a colorimeter. The film
   was scanned or photographed, and band intensity was evaluated using
   Image Lab 3.0 software (Bio-Rad Laboratories).

2.5. Immunofluorescence (IF)

   Primary antibodies against pan-AKT1/2/3 (AF6261) and p-PI3K (AF3241)
   were purchased from Affinity. Secondary antibodies, including goat
   anti-rabbit IgG H&L (CoraLite594; SA00013-4) and goat anti-rabbit IgG
   H&L (CoraLite488; SA00013-2), were purchased from Proteintech. The DNA
   dye DAPI (4,6-diamidino-2-phenylindole) was obtained from Molecular
   Probes. Immunostaining of cells was performed according to the
   previously described method, and cell images were captured using Leica
   SP5 confocal fluorescence microscope.

2.6. Ethynyl deoxyuridine staining

   Cell proliferation was measured using the EdU detection kit (Beyotime).
   Cells were seeded in a 6-well plate (2 × 104 cells/well) and cultured
   with DMEM (10 % FBS). After 24 h of drug treatment, EdU (50 μM) was
   added. Following the protocol, cells were incubated at 37 °C for 2 h,
   fixed with 4 % paraformaldehyde for 30 min, permeabilized with 0.5 %
   Triton X-100 at room temperature for 10 min, and washed with PBS. Click
   reaction solution (500 μL/well) was added and incubated at room
   temperature in the dark for 30 min. After removing the Click reaction
   solution, cells were washed three times with washing buffer and stained
   with the DNA dye DAPI (Molecular Probes) for 30 min to visualize cell
   nuclei. Cell images were captured using a Leica SP5 confocal
   fluorescence microscope.

2.7. Flow cytometry (FC) apoptosis

   Early and late-stage cell apoptosis were determined using the Annexin
   V-FITC Cell Apoptosis Detection Kit (Beyotime) according to the
   manufacturer's instructions. HCT116 and LOVO cells were treated with
   different concentrations (0, 100, 200, and 250 μg/mL) of JPTL24 for
   24 h, collected, and resuspended in 300 μl 1× binding buffer
   (Beyotime). Annexin V-FITC (5 μl) and propidium iodide (PI) (10 μl)
   were added to each sample and incubated in the dark for 15 min.
   Apoptotic cells were then detected using flow cytometry (FACSCanto II;
   BD Biosciences) and analyzed using FlowJo 7.6 software (FlowJo LLC) to
   determine the percentage of cells in each subpopulation.

2.8. Animal experiments

   In the in vivo experiments, male BALB/c nude mice
   (specific-pathogen-free, SPF, 4 weeks old, female, weight ∼ 18 ± 2 g)
   were subcutaneously injected with 100 μl HCT116 cell suspension
   (1 × 10^6 cells/mouse). The nude mice were randomly divided into four
   groups. The JPTL group and 5-Fu group were treated with JPTL recipe and
   5-Fu intervention, respectively. The JPTL+5-Fu group was treated with
   half-dose JPTL recipe and 5-Fu intervention, while the NC group
   received an equal volume of normal saline. The intervention lasted for
   a total of 30 days. H&E staining and immunohistochemistry were
   performed. The experimental protocol was approved by the Animal Care
   and Use Committee of Shanghai Traditional Chinese Medicine Hospital
   (2023072, July 21, 2023).

2.9. Statistical analysis

   All experiments were performed in triplicate. The mean ± SEM of three
   or more independent experiments was reported. A value of P ≤ 0.05 was
   considered statistically significant.

3. Results

3.1. Mass spectrometry combined with network pharmacology reveals the targets
of JPTL recipe

   JPTL Recipe is an empirical formula that has the effect of invigorating
   the spleen and promoting circulation, resolving lumps and tumors, and
   anti-cancer. Based on the key pathogenesis of “spleen deficiency as the
   root cause" in colorectal cancer (CRC), the research team summarized
   the full recipe of JPTL, which has the combined effects of invigorating
   the spleen and promoting circulation, resolving lumps and tumors, and
   anti-cancer. It embodies the treatment principles of invigorating the
   spleen to protect the body's vitality and promoting circulation to
   dispel the toxic pathogen in the treatment of CRC ([56]Fig. 1A). Due to
   the uncertainty of the targets and mechanisms of multi-component
   Chinese medicine formulations, in order to study the efficacy and
   potential targets of JPTL Recipe in treating CRC in-depth, the
   potential effective monomers in JPTL Recipe were first analyzed by mass
   spectrometry and combined with TCMSP, HERB, BATMAN-TCM and other TCM
   databases and relevant literature reports. A total of 361 effective
   ingredients were found in the 8 ingredients of JPTL Recipe ([57]Fig.
   1B). Then, we used the PubChem database to verify the structures of
   related active ingredient compounds, and screened a total of 1232
   potential targets of JPTL Recipe through the TCMSP database. Based on
   the network topology analysis of the computer and using the Cytoscape
   3.8.0 software to visualize the data, the core targets were selected
   ([58]Fig. 1C).

Fig. 1.

   [59]Fig. 1
   [60]Open in a new tab

   Component analysis of the JPTL Recipe A. Composition of the JPTL
   Recipe; B. Mass spectrometry analysis combined with database
   exploration to identify the components of the JPTL Recipe; C.
   "Component-Target" diagram of the JPTL Recipe.

3.2. Analysis of the function and targets of JPTL recipe in the treatment of
colorectal cancer (CRC)

   In order to clarify the possible mechanisms of JPTL Recipe in treating
   CRC, we searched for CRC-related targets through the disease database
   using the keyword “Colorectal cancer". Among them, a total of 10746
   CRC-related targets were obtained from the GeneCards disease database,
   and 1490 CRC-related disease targets were obtained with “Relevance
   Score>10″ as the standard; 166 CRC-related targets were obtained from
   the DisGeNET database; 1109 CRC-related targets were obtained from the
   MalaCards database, and 1074 CRC-related disease targets were obtained
   by screening with “Score>25″ as the standard; a total of 489
   CRC-related targets were obtained from the OMIM database; and 110
   CRC-related targets were obtained from the TTD database. After
   excluding the duplicated targets, a total of 2187 targets related to
   CRC were obtained ([61]Fig. 2A). After taking the intersection with the
   potential targets of active ingredients in JPTL Recipe, 279 potential
   targets of JPTL Recipe in the treatment of CRC were obtained ([62]Fig.
   2B). The protein-protein interaction (PPI) analysis was performed on
   the 279 potential targets of JPTL Recipe using the STRING database, and
   the core targets were screened based on the topology network analysis
   using the MCC algorithm of the cytoHubba plugin of the Cytoscape 3.8.0
   software. The darker the color, the higher the assigned value of the
   target, and the more critical the regulatory role it may play. Based on
   the algorithm, we found that VEGFA, AKT1, TNF, IL6, PTGS2 and other
   protein nodes have higher parameter values, and may play a key role in
   JPTL Recipe's treatment of CRC ([63]Fig. 2C). Subsequently, we
   performed GO and KEGG biological function analysis on the core targets
   and found that the mechanism of JPTL Recipe in treating CRC may be
   related to the inhibition of tumor cell proliferation and promotion of
   apoptosis ([64]Fig. 2D).

Fig. 2.

   [65]Fig. 2
   [66]Open in a new tab

   Network pharmacology analysis discovered the potential anti-tumor
   mechanism of the JPTL Recipe A. Venn diagram of colorectal cancer
   disease targets; B. Venn diagram of effective targets of the JPTL
   Recipe in the treatment of colorectal cancer; C. MCC algorithm of the
   cytoHubba plugin of the Cytoscape 3.8.0 software to screen for core
   targets; D. GO and KEGG functional enrichment analysis of targets of
   the JPTL Recipe in the treatment of colorectal cancer.

3.3. Functional enrichment analysis and prognostic analysis of the core
targets of JPTL recipe in the treatment of colorectal cancer

   To further explore the biological functions of JPTL Recipe in treating
   CRC, we used R 3.5.0 software and the ClusterProfiler and ggplot2 R
   packages to perform GO and KEGG biological function enrichment analysis
   on the 279 potential targets of JPTL Recipe against CRC. Biological
   functions with P < 0.05 were selected as the screening criteria, and
   the possible biological functions were analyzed and visually displayed.
   The biological process (BP) analysis of gene ontology (GO) enrichment
   showed that JPTL Recipe may exert its anti-CRC effect by inhibiting
   tumor cell proliferation, kinase activity, and tumor apoptosis. The
   molecular function (MF) analysis showed that JPTL Recipe is also
   involved in molecular biological processes such as phosphorylation
   regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis showed that PI3K-AKT and apoptosis signaling
   pathways play a critical role in JPTL Recipe's treatment of CRC
   ([67]Supplementary Figure 1).

3.4. JPTL recipe suppresses the activity and proliferation of colorectal
cancer cells

   To verify our predicted results of the systematic network pharmacology,
   we first prepared purified JPTL Recipe powder by vacuum freeze-drying
   ([68]Fig. 3A). In vitro experiments showed that JPTL Recipe has a good
   anti-tumor activity against colorectal cancer HCT116 and LoVo cells,
   with IC50 values of 129.51 μg/mL and 112.27 μg/mL, respectively
   ([69]Fig. 3B). Subsequently, the CCK8 proliferation experiments showed
   that JPTL Recipe can effectively inhibit the proliferation of CRC cells
   ([70]Fig. 3C). The clone formation experiments also confirmed the
   inhibitory effect of JPTL Recipe on CRC cell proliferation ([71]Fig.
   3D). Finally, by using the EDU staining technique, we confirmed the
   regulatory effect of JPTL Recipe on CRC cell proliferation ([72]Fig.
   3E). The above in vitro experiments preliminarily confirmed that JPTL
   Recipe may have a good inhibitory effect on CRC cell proliferation.

Fig. 3.

   [73]Fig. 3
   [74]Open in a new tab

   JPTL Recipe inhibits the activity and proliferation of colorectal
   cancer cells A. Preparation of purified freeze-dried powder of the JPTL
   Recipe; B. IC50 value of the JPTL Recipe in inhibiting the growth of
   colorectal cancer cells; C. CCK8 experiment to detect the effect of the
   JPTL Recipe on the activity of colorectal cancer cells; D. Clone
   formation experiment to detect the effect of the JPTL Recipe on the
   proliferation of colorectal cancer cells; E. Edu immunofluorescence
   staining to detect the effect of the JPTL Recipe on the proliferation
   ability of colorectal cancer cells.

3.5. JPTL recipe promotes apoptosis in colorectal cancer cells through the
PI3K/AKT signaling pathway

   Subsequently, we conducted apoptosis flow cytometry on HCT116 and LoVo
   cells intervened with different JPTL Recipe. The results likewise
   indicated that JPTL Recipe effectively promoted the apoptosis levels of
   HCT116 and LoVo cells ([75]Fig. 4A). Due to previous network
   pharmacology analyses suggesting that the PI3K/AKT signaling pathway
   may play a crucial role in the treatment of CRC with JPTL Recipe, we
   performed Western blot experiments to detect the expression of the
   PI3K/AKT signaling pathway and apoptosis-related proteins after JPTL
   Recipe intervention. The results confirmed that JPTL Recipe effectively
   inhibited the phosphorylation of key molecules in the PI3K/AKT
   signaling pathway and promoted tumor cell apoptosis ([76]Fig. 4B).
   Immunofluorescence experiments similarly confirmed our hypothesis,
   showing a significant decrease in PI3K phosphorylation levels within
   HCT116 and LoVo cells following JPTL Recipe intervention ([77]Fig. 4C).
   Therefore, we speculate that JPTL Recipe may exert its therapeutic
   effects on colorectal cancer by inhibiting the phosphorylation of the
   PI3K/AKT signaling pathway.

Fig. 4.

   [78]Fig. 4
   [79]Open in a new tab

   JPTL Recipe promotes apoptosis of colorectal cancer cells through the
   PI3K/AKT signaling pathway A. Flow cytometry experiments to detect the
   effect of the JPTL Recipe on the apoptosis of colorectal cancer cells;
   B. WB experiment to detect the effect of the JPTL Recipe on the
   PI3K/AKT signaling pathway and apoptosis-related proteins; C.
   Immunofluorescence experiment to detect the effect of the JPTL Recipe
   on p-PI3K (Tyr524).

3.6. Rescue experiments confirmed that JPTL recipe exerts therapeutic effects
by regulating the PI3K/AKT signaling pathway

   To further confirm our conclusion, we conducted rescue experiments
   using the PI3K/AKT signaling pathway activator insulin-like growth
   factor 1 (IGF-1) following JPTL Recipe treatment. The results indicated
   that IGF-1 effectively impeded the regulatory effect of JPTL Recipe on
   the PI3K/AKT signaling pathway ([80]Fig. 5A). Subsequent CCK8 cell
   proliferation, colony formation experiments, and EDU proliferation
   experiments revealed that with the hindrance of JPTL Recipe's
   regulatory effect on the PI3K/AKT signaling pathway, its inhibitory
   effect on the proliferation of HCT116 and LoVo cells was significantly
   reduced ([81]Fig. 5B–D). Apoptosis flow cytometry experiments also
   demonstrated that the promoting effect of JPTL Recipe on apoptosis in
   HCT116 and LoVo cells was markedly blocked by the activation of the
   PI3K/AKT signaling pathway ([82]Fig. 5E). In conclusion, we found that
   JPTL Recipe can function by inhibiting the phosphorylation of the
   PI3K/AKT signaling pathway, thereby exerting its anti-proliferative and
   pro-apoptotic effects on colorectal cancer cells.

Fig. 5.

   [83]Fig. 5
   [84]Open in a new tab

   In vivo experiments confirm that the JPTL Recipe treats colorectal
   cancer through the PI3K/AKT signaling pathway A. Effect of the JPTL
   Recipe on the PI3K/AKT signaling pathway and apoptosis-related proteins
   after intervention of the PI3K/AKT signaling pathway activator IGF-1;
   B. Effect of IGF-1 intervention on the inhibitory effect of the JPTL
   Recipe on tumor cell activity; C-D. Effect of IGF-1 intervention on the
   inhibitory effect of the JPTL Recipe on tumor cell proliferation; E.
   Effect of IGF-1 intervention on the pro-apoptotic effect of the JPTL
   Recipe on tumor cells.

3.7. In vivo experiments on the treatment of colorectal cancer by JPTL recipe
combined with 5-fu

   To clarify whether JPTL Recipe can also exert its therapeutic effect in
   vivo, we constructed a subcutaneous mouse model of colorectal cancer
   and administered JPTL Recipe and chemotherapy drug 5-fluorouracil
   (5-Fu) alone or in combination ([85]Fig. 6A). Through continuous
   monitoring of the tumor body size and weighing the tumor after
   dissection, the experimental results showed that JPTL Recipe combined
   with 5-Fu can exert a better anti-tumor effect ([86]Fig. 6B–D). Western
   Blot experiments targeting PI3K/AKT signaling pathway also showed that
   JPTL Recipe combined with 5-Fu can effectively inhibit the
   phosphorylation of key molecules in PI3K/AKT signaling pathway and
   promote tumor cell apoptosis ([87]Fig. 6E). At the same time, we also
   checked the tumor and liver and kidney tissues of each group of mice by
   H&E staining, and confirmed that JPTL Recipe not only inhibits tumor
   growth but also cause no obvious liver and kidney damage to mice at the
   corresponding dose ([88]Fig. 6F).

Fig. 6.

   [89]Fig. 6
   [90]Open in a new tab

   In vivo experiments confirm that the JPTL Recipe combined with 5-Fu
   inhibits the growth of colorectal cancer subcutaneous tumors A.
   Schematic diagram of nude mouse colorectal cancer subcutaneous tumors;
   B. Monitoring of body weight of nude mice in each group; C. Monitoring
   of the size and weight of subcutaneous tumors in each group of nude
   mice; D. The condition of subcutaneous tumors in each group of nude
   mice after treatment; E. Effect of the JPTL Recipe, combined with 5-FU
   or alone, on the PI3K/AKT signaling pathway and apoptosis-related
   proteins in each group of nude mice; F. Representative images of HE
   staining of tumors, liver and kidney after treatment with the JPTL
   Recipe combined with 5-FU or alone in each group of nude mice.

3.8. In vivo experiments confirmed that JPTL recipe promotes apoptosis of
colorectal cancer cells through the PI3K/AKT signaling pathway

   Finally, to confirm again the effect of JPTL Recipe on tumor tissue
   apoptosis and the PI3K/AKT signaling pathway in vivo, we performed
   Tunel staining on the tumor tissues of HCT116 and LoVo cells after
   different interventions. The results showed that JPTL Recipe combined
   with 5-Fu can significantly increase the degree of apoptosis in HCT116
   and LoVo cells ([91]Fig. 7A). Immunofluorescence staining against
   p-PI3K (Tyr524) and p-AKT (Ser473) in the tumor tissues of each group
   of mice also confirmed the inhibitory effect of JPTL Recipe on the
   PI3K/AKT signaling pathway in vivo ([92]Fig. 7B). Based on the results
   of the above in vivo and in vitro experiments, we confirmed that JPTL
   Recipe may exert its anti-colorectal cancer effect by inhibiting the
   phosphorylation of key molecules in the PI3K/AKT signaling pathway and
   promoting the apoptosis of tumor cells ([93]Fig. 8).

Fig. 7.

   [94]Fig. 7
   [95]Open in a new tab

   In vivo experiments confirm that the JPTL Recipe treats colorectal
   cancer through the PI3K/AKT signaling pathway A. Tunel experiment to
   detect the effect of the JPTL Recipe, combined with 5-FU or alone, on
   the apoptosis of subcutaneous tumors in each group of nude mice; B.
   Immunofluorescence experiment to detect the effect of the JPTL Recipe,
   combined with 5-FU or alone, on the expression of p-PI3K (Tyr524) in
   subcutaneous tumors in each group of nude mice; C. Immunofluorescence
   experiment to detect the effect of the JPTL Recipe, combined with 5-FU
   or alone, on the expression of p-AKT (Ser473) in subcutaneous tumors in
   each group of nude mice.

Fig. 8.

   [96]Fig. 8
   [97]Open in a new tab

   Schematic diagram of the mechanism of the JPTL (JPTL) Recipe in
   regulating anti-apoptosis and cell proliferation in colorectal cancer
   through the PI3K/AKT signaling pathway.

4. Discussion

   Colorectal cancer (CRC) is considered one of the most common malignant
   tumors in the digestive system and ranks among the deadliest cancers
   worldwide [[98]14]. Despite the existence of effective diagnostic and
   treatment methods for CRC, it still poses a serious threat to millions
   of people globally. The phosphoinositide 3-kinase (PI3K)/protein kinase
   B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, also
   known as the PAM signaling pathway, is involved in various
   physiological processes and plays a crucial role in the development of
   many diseases, especially cancer [[99]15]. It regulates cell survival,
   metastasis, and metabolism, and also plays a role in angiogenesis and
   recruitment of inflammatory factors [[100]16]. PI3K phosphorylates PIP2
   to generate PIP3, which then recruits downstream signaling proteins,
   including serine/threonine kinase Akt [[101]17]. Activated Akt
   phosphorylates many substrates, with mTOR being one of the most common
   downstream effectors of Akt. The PI3K/Akt signaling pathway regulates
   processes such as tumor cell growth, proliferation, survival, and
   angiogenesis, and plays an important role in the occurrence and
   development of colorectal cancer [[102]18,[103]19]. Additionally,
   excessive activation of the PAM signaling pathway is one of the
   important mechanisms of resistance to immune therapy, chemotherapy, and
   targeted therapy [[104]20]. Mechanisms by which the PI3K/AKT signaling
   pathway inhibits apoptosis in cells include the activation of
   anti-apoptotic proteins by Akt [[105]21] Akt can phosphorylate and
   activate the protein Bad, which is a pro-apoptotic protein that can
   bind to the anti-apoptotic proteins Bcl-2 or Bcl-XL and prevent their
   protective effects on mitochondria. When Akt is activated, it
   phosphorylates Bad, preventing its binding to Bcl-2 or Bcl-XL
   [[106]22,[107]23].

   5-Fluorouracil (5-FU) mediates its anticancer effects in colorectal
   cancer cells through diverse mechanisms. Initially, its active
   metabolites form a stable complex with thymidylate synthase (TS),
   inhibiting TS activity, thereby blocking the production of
   deoxythymidine monophosphate (dTMP) and consequently hindering DNA
   synthesis [[108]24]. Furthermore, 5-FU metabolites incorporate into RNA
   as 5-fluorouridine triphosphate (FUTP) and into DNA as
   5-fluoro-2′-deoxyuridine-5′-triphosphate (FdUTP), thereby disrupting
   RNA function and DNA stability [[109]25]. These actions result in DNA
   damage and RNA dysfunction, activating DNA damage response pathways,
   such as p53, and other pro-apoptotic signaling pathways, including the
   Fas pathway [[110]26]. Additionally, 5-FU modulates the PI3K/AKT and
   MAPK/ERK signaling pathways, thereby further promoting apoptosis
   [[111]27,[112]28]. These interconnected mechanisms collectively inhibit
   cancer cell growth and induce apoptosis. Accumulating evidence suggests
   that CRC develops resistance to 5-FU through the PI3K/AKT signaling
   pathway, which leads to recurrent metastasis of CRC.

   This study employed an innovative approach using network pharmacology
   in combination with comprehensive in vivo and in vitro experiments,
   providing evidence for the ability of JPTL Recipe to inhibit the growth
   and metastasis of CRC cells. Despite these promising findings, the
   specific role and potential mechanisms of JPTL Recipe in the treatment
   of colorectal cancer remain to be fully elucidated. In this study, we
   demonstrated that JPTL Recipe effectively inhibits colorectal cancer
   cell proliferation, anti-apoptotic ability, and exerts synergistic
   therapeutic effects with fluorouracil. Further analysis revealed that
   JPTL Recipe affects the activity of colorectal cancer cells by
   inhibiting the phosphorylation of the PI3K/AKT signaling pathway.
   Moreover, JPTL could affect on this pathway and reduce the resistance
   of CRC to 5-FU and thus promote the inhibitory effect of 5-FU on CRC.
   In summary, we have discovered and confirmed that the traditional
   Chinese medicine compound JPTL Recipe can serve as a novel adjuvant
   therapy for colorectal cancer, offering a new treatment approach for
   the integration of traditional Chinese and Western medicine in the
   treatment of colorectal cancer.

5. Conclusion

   In conclusion, we have found that JPTL Recipe can suppress the activity
   and proliferation of colorectal cancer cells, promote tumor cell
   apoptosis, and exert better synergistic therapeutic effects when
   combined with 5-FU in the treatment of colorectal cancer. Mechanism
   studies have shown that JPTL Recipe inhibits the phosphorylation of key
   molecules in the PI3K/AKT signaling pathway to hinder the progression
   of colorectal cancer. Our research data provide a new combined Chinese
   and Western medicine treatment plan for clinical colorectal cancer
   treatment, effectively expanding the application of TCM in the clinical
   treatment of colorectal cancer.

Ethics approval and consent to participate

   The animals experimental was approved by the Animal Care and Use
   Committee of Shanghai Hospital of Traditional Chinese Medicine
   (2023072, July 21, 2023).

Funding

   This work was supported by Project of Clinical Three-Year Action Plan
   of Shanghai Shen-Kang Hospital Development Centre (Grant No.
   SHDC2020CR2047B to Jianlin Ren), Project of the National Natural
   Science Foundation of China (Grant Nos. 82174452, 81873279 to Jianlin
   Ren), Project of Shanghai Science and Technology Innovation Action Plan
   Biomedical Science and Technology Support Project (Grant No.22S21901000
   to Jianlin Ren), Project of Shanghai Municipal Hospital of Traditional
   Chinese Medicine (Grant No. WLJH2021ZY-MZY026 to Jianlin Ren) and
   Project of Shanghai Municipal Hospital of Traditional Chinese Medicine
   (Grant No. WL-YSXM-2022001K to Chenyue Yuan).

Data availability statement

   All data generated or analyzed during this study are included in this
   published article and supplementary information files. All data
   associated with this study has not been deposited into a publicly
   available repository.

CRediT authorship contribution statement

   Jinyan Chu: Writing – original draft, Visualization, Data curation.
   Chenyue Yuan: Writing – review & editing, Writing – original draft,
   Visualization, Resources, Investigation, Funding acquisition, Formal
   analysis, Conceptualization. Lin Zhou: Visualization, Software,
   Methodology. Yong Zhao: Formal analysis, Data curation,
   Conceptualization. Xingli Wu: Project administration. Yuting Yan:
   Formal analysis. Yi Liu: Supervision. Xiangjun Liu: Visualization,
   Supervision, Project administration, Funding acquisition, Data
   curation. Lin Jing: Software, Methodology, Data curation. Tiangeng
   Dong: Visualization, Funding acquisition, Formal analysis, Data
   curation. Jianlin Ren: Writing – review & editing, Resources,
   Methodology, 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
   [113]https://doi.org/10.1016/j.heliyon.2024.e35490.

Contributor Information

   Lin Jing, Email: jinglin1004@163.com.

   Tiangeng Dong, Email: dong.tiangeng@zs-hospital.sh.cn.

   Jianlin Ren, Email: renjianlin666@126.com.

Appendix A. Supplementary data

   The following are the supplementary data to this article:

figs1.

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figs2.

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figs3.

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figs4.

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figs5.

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figs6.

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References