Hepatocellular Carcinoma (HCC) ranks as the third leading cause of cancer-related mortality, with a 5-year survival rate of $\le$5% in advanced stages, posing a severe threat to human life and health [1]. The HCC, one of the most critical primary liver cancers, is a major contributor to cancer-related deaths, exhibiting a male-to-female incidence ratio ranging from 2:1 to 8:1 [2, 3]. The complex etiology of HCC remains unclear and its occurrence is closely associated with factors such as viral infections, fatty liver disease, diabetes, alcoholic liver disease, non-alcoholic fatty liver disease and consumption of aflatoxin-contaminated food [4]. Despite advancements in the diagnosis and treatment of HCC in recent years, the insidious onset, difficulty in early detection and the diagnosis of over 70% of patients at an advanced stage pose significant challenges [5].

The expression of Toll-Like Receptor 4 (TLR4) has been linked to the progression, invasion, metastasis and adverse prognosis of HCC [6, 7]. The TLR4 is expressed on the surface of tumor cells in many different tissues and is associated with the establishment and maintenance of tumor immune microenvironment. The activation of TLR4 signaling pathway in tumor tissue can cause the increase of immunosuppressive factors, promote cancer cell proliferation and immune escape and lead to the growth and metastasis of cancer cells [8, 9]. The TLR4 can activate downstream signals through myeloid differentiation primary response protein 88 (MyD88), triggering the activation of important proteins such as nuclear factor-kappa B (NF-$\kappa$B). It further promotes the synthesis and release of downstream cytokines such as tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interleukin (IL)-2, IL-8 and participates in the occurrence and development of liver cancer and the production of inflammation [10]. Thus, the TLR4/MyD88/NF-$\kappa$B cascade reaction is considered to be associated with the induction of liver inflammation and the progression of HCC.

Chemotherapy remains one of the most effective treatment options for advanced-stage HCC and 5-Fluorouracil (5-FU) is a commonly used chemotherapy drug in clinical practice [11]. The 5-FU intervenes in the biological behavior of HCC cells through multiple pharmacological mechanisms. Firstly, it is converted into active metabolites that disrupt the synthesis of purines and pyrimidines, causing abnormal DNA and RNA synthesis, thereby hindering the normal proliferation of cancer cells. Secondly, the binding of active metabolites to DNA induces DNA strand damage, triggering apoptosis in cancer cells [12]. Additionally, the 5-FU metabolic product, 5-Fluoro-2^′-Deoxyuridine Monophosphate (FdUMP), inhibits thymidylate synthase, affecting DNA synthesis and repair [13]. However, despite the significant inhibitory effects of 5-FU in HCC treatment, its usage is associated with a range of potential toxic side effects. The 5-FU can lead to bone marrow suppression, immune suppression, gastrointestinal toxicity, immune system suppression, skin reactions and neurotoxicity [9, 14]. Moreover, with prolonged treatment courses, issues such as decreased drug sensitivity and drug resistance leading to poor efficacy may arise [15].

Numerous studies have indicated that the combination of traditional Chinese medicine (TCM) and chemotherapy can synergistically enhance therapeutic effects, alleviate chemotherapy side effects, improve the quality of life for patients and prolong survival time [16, 17]. The TCM can suppress the occurrence of HCC by inhibiting the expression of MyD88, TRAF6, NF-$\kappa$B and TNF-$\alpha$ [18, 19]. The TCM can also modulate immune responses, inhibit the proliferation and metastasis of tumor cells [20] and rapidly identify and eliminate tumor cells, reshape the tumor microenvironment and inhibit tumor growth. The TCM achieves this by activating the functions of immune cells [21, 22]. The Periplaneta americana has a long history of medicinal use and exhibits pharmacological activities such as anti-tumor and immune modulation [23]. The CII-3, an active component extracted from Periplaneta americana, has been identified as possessing anti-tumor efficacy and research suggests that its anti-tumor effects may be related to its immunomodulatory function [24]. A total of 571 peptide sequences were discovered and 329 precursor proteins were identified in CII-3 [25]. The CII-3 can enhance the immune organ indices in tumor-bearing mice, increase the number of peripheral blood immune cells, regulate the body’s immune function and inhibit tumor growth [26].

Given this background, this study explored the synergistic effects and immunomodulatory actions of the Periplaneta americana extract CII-3 in combination with 5-fluorouracil in the treatment of HCC. This study aimed to investigate the changes in the TLR4/MyD88/NF-$\kappa$B signaling pathway within the tumor and elucidate its underlying mechanisms.

In the classification of liver cancer based on clinical symptoms in traditional Chinese medicine, it is categorized into patterns such as qi stagnation and blood stasis, liver depression and spleen deficiency, liver-gallbladder damp-heat, liver-kidney yin deficiency, Zheng deficiency blood stasis, spleen-kidney yang deficiency and damp-heat toxin accumulation [28]. The Periplaneta americana, as documented in the “Shennong Bencao Jing”, is described as having effects such as “controlling blood stasis, solidifying lumps, addressing cold and heat, dispersing accumulations, relieving throat obstruction and treating internal cold and infertility” [29]. Acting on the liver meridian, the Periplaneta americana is believed to possess functions corresponding to breaking down blood stasis, promoting blood circulation and dispelling stagnation, aligning with the patterns seen in traditional Chinese medicine for liver cancer [26, 30]. Modern medical research indicates significant potential of the Periplaneta americana in the treatment of various cancers, particularly in studies related to liver and lung cancers [23].

The CII-3 may exert a comprehensive inhibitory effect on liver cancer through multiple pathways, including immunomodulation and anti-inflammatory effects. Firstly, inflammation in liver cancer has been identified as a crucial factor, playing diverse roles in cancer initiation and progression [31]. Inflammatory processes involve the release of pro-inflammatory cytokines such as TNF-$\alpha$, IL-6 and IL-1$\beta$, which are closely associated with tumor growth, invasion and angiogenesis [32]. The IL-8, an important inflammatory mediator, is linked to tumor development and progression [33]. A significant reduction in IL-8 levels in the 5-FU combined with CII-3 group was observed. Thus, CII-3 may enhance the efficacy of chemotherapy by inhibiting IL-8 production, regulating inflammatory responses and influencing the tumor microenvironment.

Secondly, under the context of 5-FU treatment for liver cancer, the impact on the host immune system becomes a critical consideration. Current experimental results indicated that the combination of 5-FU and CII-3 has a pronounced regulatory effect on the murine immune system, particularly on immune cells and factors. Elevated levels of IgA, IgM and IgG in the 5-FU combined with CII-3 group suggest a potential role of CII-3 in modulating humoral immunity, enhancing the body’s defense against infections. Moreover, significant changes were observed in the quantity and activity of immune cells after 5-FU treatment. The NK cells, crucial for nonspecific immune activity, play a vital role in overall immune function [34]. The activation of immune cells is also part of the inflammation in liver cancer [35]. Current results demonstrate that CII-3 can enhance the cytotoxic activity of NK cells, potentially compensating for the inhibitory effect of 5-FU on NK cell activity. This discovery provides a new mechanistic explanation for the immunomodulatory effects of 5-FU combined with CII-3 treatment. The T lymphocytes, especially CD³⁺, CD⁴⁺ and CD⁸⁺ cells, may decrease after 5-FU treatment, leading to an imbalance in T lymphocyte subgroups. This aligns with previous research results, indicating the direct impact of chemotherapy drugs on T lymphocytes.

Research suggested that the activation of the TLR4 signaling pathway in liver cancer cells can increase the production of immune inhibitory factors, promoting tumor cell proliferation and immune escape, leading to the growth and metastasis of liver cancer cells [36, 37]. The MyD88-dependent signaling pathway downstream of TLR4 can activate NF-$\kappa$B, inducing the migration of tumor cells, a phenomenon verified in various cancers [38]. Inhibiting NF-$\kappa$B can suppress the growth and migration of cancer cells while enhancing their sensitivity to chemotherapy drugs [39]. The TRAF6, as a key factor in the NF-$\kappa$B pathway, also serves as a regulatory factor for adaptive and innate immunity [40, 41]. The TNF-$\alpha$, regulated by the TLR4/NF-$\kappa$B pathway, can, under certain conditions, alter the tumor microenvironment, promoting tumor growth, proliferation, invasion, metastasis and tumor angiogenesis [42]. Overexpression of TNF-$\alpha$ may lead to poor prognosis in liver cancer patients [43]. Research results indicated that the mice in the 5-FU combined with CII-3 group exhibited significantly smaller tumor mass and volume than those in the 5-FU group alone, suggesting a tumor-inhibitory effect of the combination. The mRNA and protein levels of TLR4, NF-$\kappa$B, TRAF6, MYD88 and TNF-$\alpha$ in the 5-FU combined with CII-3 group were significantly downregulated, indicating that CII-3 may enhance the efficacy of chemotherapy by inhibiting the TLR4/MyD88/NF-$\kappa$B signaling pathway, thereby exerting an inhibitory effect on liver cancer.

This study revealed that 5-FU combined with CII-3 can modulate immune factors and immune cells, improving the immune function of tumor-bearing mice and alleviating the immune suppression state. The 5-FU combined with CII-3 can effectively inhibit the growth of liver cancer and the mechanism may involve the inhibition of the TLR4/MyD88/NF-$\kappa$B signaling pathway to exert antitumor effects. Further research will delve into the mechanisms of CII-3 in inhibiting tumor growth and modulating immunity, providing experimental and theoretical foundations for the development and application of CII-3 derived from the Periplaneta americana. However, it is essential to note some limitations in our study. The experiments primarily focused on murine models and the biological characteristics of mice differ from those of humans. Therefore, caution should be exercised in interpreting the results when applying them to human subjects. Additionally, although we observed the regulatory effect of CII-3 on IL-8, further in-depth research is needed to elucidate the specific molecular mechanisms and signaling pathways involved.

In summary, this research outcomes unveil the multifaceted actions of CII-3 in the treatment of liver cancer, encompassing immunomodulation, anti-inflammatory effects and tumor inhibition. This provides a theoretical foundation for the clinical application of CII-3, but further investigations are warranted to comprehensively understand its mechanisms of action and its potential prospects in clinical therapy.

The combination of Periplaneta americana extract CII-3 with 5-Fluorouracil (5-FU) demonstrates synergistic anti-tumor and immunomodulatory effects in H22 liver cancer-bearing mice. The CII-3, a complex mixture comprising 25 identified compounds, enhances the inhibitory efficacy on tumor growth, as evidenced by reduced tumor mass and volume. Additionally, CII-3 exerts anti-inflammatory effects by significantly lowering IL-8 levels. These findings provide insights into the potential of CII-3 as an adjunctive therapeutic agent for liver cancer, complementing the effects of conventional chemotherapy. However, caution should be exercised in generalizing these results to human subjects due to species-specific variations. Further research is necessary to unravel the intricate molecular mechanisms underlying CII-3’s actions and to explore its clinical applications.

Hepatocellular Carcinoma (HCC) presents a significant threat to human health and life. While chemotherapy stands as a cornerstone treatment for advanced-stage HCC, its efficacy is often hindered by adverse reactions such as immunosuppression. This study aims to identify therapeutic approaches that can enhance chemotherapy effectiveness while mitigating adverse effects. Our research highlights the synergistic anti-tumor and immunomodulatory effects of combined CII-3 and 5-FU treatment, potentially mediated through the regulation of the TLR4/NF-$\kappa$B signaling pathway. These findings suggest a promising adjunctive role for CII-3 in liver cancer treatment, offering a novel therapeutic strategy.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

RG and GW designed the research study. RY, GW, MG, and XL performed the experiments and curated the data. MG and XL provided guidance on experimental procedures. YW, RG and RY conducted the formal analysis and data visualization. RG and GW developed the methodology. MG acquired the funding. RG, RY, and YW drafted the original manuscript. MG, XL, and YW searched references. MG and XL reviewed and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All the experiments were performed based on the approved animal protocols and guidelines established by the Medicine Ethics Review Committee for Animal Experiments of Dali University under the approval (number: 2023P2260).

This work was supported by the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (202101BA070001-119), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (202101BA070001-121), the Open project of Yunnan Provincial Key Laboratory of Insect Biomedicine Research and Development (AT2024002) and the Team Project of Yunnan Revitalization Talent Support Program (No.202305AS350001).

The authors declare no conflict of interest.