The Immunoregulatory Effect of Aconite Treatment on H22 Tumor-Bearing Mice via Modulating Adaptive Immunity and Natural Killer-Related Immunity

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and, in its advanced stages, has a 5-year survival rate of only 3% to 5%. Despite novel mechanisms and treatments being uncovered over the past few years, effective strategies for HCC are currently limited. Previous studies have proven that aconite can suppress tumor growth and progression and prevent the recurrence and metastasis of multiple cancers, but the underlying molecular mechanisms are largely unknown. In this study, different doses of aconite were applied to mice bearing subcutaneous HCC tumors. It was found that aconite had a therapeutic effect on H22 tumor-bearing mice in a dose-dependent manner by reducing tumor volumes and prolonging survival times, which could be attributed to the immunoregulatory effect of aconite. Furthermore, results showed that high-dose administration of aconite could enhance adaptive immunity and natural killer (NK) cell-mediated immunity by regulating the secretion of interferon-γ, upregulating T cells and NK cells, and modulating the expression of the NK cytotoxicity biomarker CD107a and the inhibitory receptor TIGIT. This study revealed a novel mechanism through which aconite exerts antitumor effects, not merely through apoptosis induction pathways, providing more sound evidence that aconite has the potential to be developed into an effective anti-HCC agent.


Introduction
Primary liver cancer is one of the most fatal malignancies, causing more than 2,814,000 deaths in China annually [1,2]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for 75% to 85% of all cases [2]. Although novel mechanisms and treatments have increasingly been uncovered over the past decade, efective strategies for HCC are currently limited, leading to a 5-year survival rate of only 3% to 5% in advanced HCC [3,4]. Currently, the 5-year survival rates of HCC patients who receive hepatectomy are still below 60%, with a high recurrence rate of 60% to 80% [3]. Te dismal cure rates of HCC can be attributed not only to the aggressiveness of HCC but also to the poor overall status of patients, especially impaired tumor immunity. Currently, the immunological status of patients with HCC is increasingly regarded as the most critical factor that afects cancer progression. In the immune microenvironment of HCC patients, cells including CD4 + /CD8 + T cells, dendritic cells (DCs), and natural killer (NK) cells can promote immune response and exert antitumor efects, which contributes to efective immunological surveillance and mediates residual tumor cell eradication after radical surgery [5]. Nevertheless, most HCC patients present with impaired immunological status, which can result in failed tumor eradication and, subsequently, HCC progression.
Several immunotherapy schemes have been promoted, including tumor vaccines, immune modulators, adoptive immune cell transfers, and immune checkpoint inhibitors. High rates of drug resistance and severe immune-related adverse events remain the major obstacles to the development of such immune-targeting therapies. Te efcacy of immunotherapy in liver cancer is relatively low compared with other cancers due to the peculiar immunotolerance characteristics of the liver [6]. Serving as antitoxic, cytotoxic, and ftness-enhancing agents, traditional Chinese medicines (TCMs) have long been proven to be efective in alleviating clinical symptoms, improving quality of life and immune function, and prolonging the survival of cancer patients. Te liver-specifc immunotolerance that leads to tumor escape is hard to reverse with single-target treatments [6]. However, the multitarget characteristics of TCM compounds may provide new ideas and methods for HCC immunotherapy.
In TCM theory, the immunodefcient condition of patients with advanced HCC can be recognized as "Yangdefciency and Yin-excessiveness," which can be characterized by symptoms including drowsiness, fatigue, edema, chilliness in the abdomen and limbs, and diarrhea. One of the principles of TCM treatment is "Yang-warming." As a typical herb used in Yang-warming methods, Aconitum carmichaeli Debx. (aconite) has been shown to have clinical efcacy in treating advanced HCC [7]. Studies have shown that aconite can suppress tumor growth and progression, prevent recurrence and metastasis of multiple cancers, and alleviate pain and other symptoms, including diarrhea and chilliness, in patients undergoing chemotherapy [8,9]. Tese antitumor efects of aconite can be attributed to inducing apoptosis, inhibiting infammation, and suppressing epithelial-to-mesenchymal transformation [10,11]. Nevertheless, few studies on the immunoregulation efect of aconite in HCC have been reported.
Tis study was designed to evaluate the efect of aconite on the immune status of mice with subcutaneous HCC tumors. Our results showed that aconite had a therapeutic efect on H22 tumor-bearing mice that could be attributed to its immunoregulatory efects, involving adaptive and NK cell-mediated immunity. Tis study revealed a novel mechanism through which aconite exerts an antitumor efect, not merely through apoptosis induction pathways, providing evidence that aconite has the potential to be developed into an efective anti-HCC agent.

Materials and Methods
All animal studies were conducted in accordance with protocols from the Animal Care and Use Committee of the Naval Medical University. Also, all possible steps were taken to avoid animal sufering at each stage of the experiment.

Preparation of Aconite Decoction.
Aconite herbs were purchased from the Pharmacy of TCM, Changhai Hospital, afliated with the Naval Medical University, Shanghai, China. Te original solution of aconite decoction was prepared as follows: (1) 100 g of crude aconite was immersed and boiled in 1000 mL of diluted water for 2 h and then fltered; (2) this was repeated again; and (3) the two decoctions were mixed and concentrated to 100 mL. Aliquots of the original solution (1 g/mL) were stored at 4°C for further experiments.

HCC Xenografts and Grouping of Mice.
In total, 50 4week-old male BALB/c mice were purchased from SLAC Co., Ltd. (Shanghai, China). Te mice were housed in a specifc pathogen-free animal laboratory of Changhai Hospital, with a 12 h light/dark cycle lighting condition, 15×/h air ventilation, and 21 ± 1°C controlled temperature. During the experimental period, all mice had free access to standard fodder and distilled water, which were provided by SLAC Co., Ltd. and the animal laboratory. H22 cells (kindly provided by the TCM Laboratory of Changhai Hospital, Shanghai) were cultured for three passages and confrmed to be uncontaminated by a mycoplasma test. Ten, 1 mL of H22 cells (1 × 10 7 cells/mL) were intraperitoneally injected into BALB/c mice. After 5 days, the ascitic fuid was drawn from the abdominal cavity and intraperitoneally reinjected into another healthy BALB/c mice. When the mice exhibited abdominal bulging, 5 mL of ascites (faxen-colored) were collected and diluted with saline solution to a concentration of 1 × 10 7 cells/mL suspension.
Except for mice in the blank group, which were inoculated with saline solution, all other mice were subcutaneously inoculated in the left inguinal region with the aforementioned H22 cell suspension (0.2 mL/mice). Tis was followed by a 3-week treatment, starting the day after H22 inoculation, with saline solution or aconite in diferent concentrations (0.2 mL/20 g body weight per day, intragastric administration (i.g.)). Te mice were then divided into fve groups (10 mice/group): blank (no tumor grafted; saline solution i.g.), subcutaneous tumor-bearing model mice (saline solution i.g.), low dose (LD; aconite at 0.125 g/ mL i.g.), medium dose (MD; aconite at 0.25 g/mL i.g.), and high dose (HD; aconite at 0.5 g/mL i.g.). During the administration period, the general condition and body weight dynamics of the mice were recorded daily. Mice were sacrifced after 3 consecutive weeks of aconite administration, and the survival times of mice in each group were documented. After all the mice were sacrifced, the tumors in each group were excised and weighed. Te body mass changes of mice in each group were calculated as weight at sacrifce/ natural death with tumor)−(weight of mice before modeling).

Calculating Tumor Volumes and Inhibition Rates.
Tumor volumes were measured every 2 days for 3 weeks by recording the longest diameter (d) and maximum vertical diameter (vd) of the tumors. Te following formula was used to calculate the tumor size: volume � 1/2 × d × vd 2 . Tree weeks after inoculation, the mice were sacrifced by cervical dislocation, and the tumors were removed, cleaned with distilled water, dried with absorbent paper, and weighed. Te average tumor weights in the model (M) and experimental (E) groups were calculated; tumor inhibition rates (IRs) in each group were calculated as IR (%) � (M−E)/M × 100%.
BALB/c mice were grouped, inoculated with H22 suspension, and administered as aforementioned. Te death dates of mice in each group were recorded. Mice in the blank group were sacrifced the day after any tumor-bearing mice died naturally.

Hematoxylin and Eosin
Staining. Tumor tissues were fxed in 10% formalin for 24 h and embedded in parafn. Te tissues were then cut into 3 μm thick slices, incubated at 65°C for 1 h, and subjected to hematoxylin (0.5%, 5 min) and eosin (1%, 1 min) staining at room temperature.

Flow Cytometry.
Flow cytometry was used to detect the number of NK cells and the expression of the NK cell receptors (NKp46, NKG2D, and TIGIT) in peripheral blood and spleen samples, the expression of CD107a in the spleen, and the number of B and T lymphocytes in peripheral blood from each group. Briefy, freshly isolated splenocytes and peripheral blood samples were prepared. Red blood cell lysate was added to cell suspensions for lysis, and samples were centrifuged at 400 × g for 5 min, and then washed twice with PBS. Te samples were incubated with CD49b-APC antibodies in the dark at 4°C for 30 min and then washed with PBS bufer once. Cells were resuspended in 250 μL PBS for detection on the fow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA).

Enzyme-Linked Immunosorbent Assay (ELISA).
ELISA was used to detect the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-c in serum. Briefy, the wells of ELISA plates were coated with samples at an appropriate concentration and then incubated in the dark at 37°C for 90 min. Next, the solution was diluted with double-distilled water, gently shaken for 30 s, and then repeated cleaned three times. Ten, 100 μL of biotinylated secondary antibody working solution and incubated with the samples in the dark at 37°C for 1 h. Ten, 100 μL of streptomycin HRP working solution was added and incubated in the dark at 37°C for 30 min. Next, 100 μL of chromogen was added and incubated at 37°C for 30 min. Finally, the termination solution was added when there was an obvious color gradient. Te absorbance at 450 nm was detected with a Bio-Rad model 680 microplate reader (Bio-Rad, Hercules, CA, USA).

Statistical
Analysis. Statistical analyses were performed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). Te data are expressed as mean ± standard deviation. Signifcant diferences between groups were analyzed by one-way analysis of variance, or the Student's t-test. A twoway analysis of variance was applied to analyze signifcant diferences between two groups afected by time. P < 0.05 was considered to represent a signifcant diference.

Qualitative Test and Phytochemical Screening of Aconite
Extract. UHPLC-HRMS results showed the chemical composition of aconite extract. Several compounds, including isotalatizidine, songorine, and hokbusine B, were identifed from aconite extract. Te compounds and corresponding retention times are shown in Table 1.

Te Efect of Aconite on Tumor Growth and Survival Time.
During aconite administration, no toxic reactions or abnormal behaviors were found in any group. Te subcutaneous tumors decreased body mass in each group, which can be attributed to tumor progression, and aconite alleviated this weight loss. Compared with the M group, mice in the MD and HD groups (P < 0.05 for MD and P < 0.01 for HD) gained signifcant weight (Table 2).
After 3 weeks of treatment, the mice were euthanized, and tumor tissues were resected and weighed. As shown in Figure 2(a), tumor sizes were decreased in the aconite groups in a dose-dependent manner (P > 0.05 for LD vs. blank; P < 0.05 for MD vs. blank; and P < 0.01 for HD vs. blank). Te IR in each group was 13.44% (LD), 28.63% (MD), and 42.17% (HD). Figure 2

Aconite Strengthened Adaptive Tumor Immunity.
First, the spleen and thymus indices of each group were calculated. Te spleen index of each H22-bearing group was signifcantly higher than the blank group, while the thymus index Te concentrations of safomin A, benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, aconitine, and hypaconitine were detected by the UHPLC-HRMS method and were 4.1 mg/g, 5.63 mg/g, 6.45 mg/g, 1.22 mg/g, and 1.35 mg/g, respectively, in the extracts. Te chromatographic profle of the extract is shown in Figure 1.    was lower. Compared with M, the spleen index of aconitetreated mice presented a dose-dependent decrease, while the thymus index steadily increased (Figure 3(a) and 3(b)). Aconite also afected levels of serum cytokines that lead to an immunefavored environment. As shown in Figure 3(c)-3(e), compared with the M group, aconite increased serum levels of TNF-α, IL-1β, and IFN-c in a signifcantly dose-dependent manner. Te efect of aconite on total B cells and T cells in peripheral blood was also tested. As shown in Figure 3(f), peripheral B cell numbers in tumor-bearing mice were signifcantly decreased compared with the blank group, indicating that aconite did not have signifcant efects on the total number of B cells. Te numbers of peripheral Tcells in tumor-bearing mice were higher than those in the nontumor group, indicating that high-dose aconite treatment signifcantly upregulated peripheral T cells.

Aconite Enhanced the Quantity and Activity of NK Cells.
As shown in Figure 4(a), the proportion of NK cells in the spleen of tumor-bearing mice was lower than that in the blank group, while high-dose aconite treatment signifcantly increased the NK cell percentage. Te splenic receptor CD107a is a potent biomarker of NK cell cytotoxicity. In this study, only high-dose aconite increased the expression of CD107a in the spleen (Figure 4(b)). We also investigated whether aconite afected the expression of several receptors on NK cells to perform its immune-enhancing efect. In the spleen, NKp46 (CD335) and NKG2D levels were signifcantly reduced in tumor-bearing mice, while aconite had no efect on either ( Figure 5). TIGIT expression was upregulated in tumor-bearing mice, and aconite (all doses) signifcantly increased its expression.

Evidence-Based Complementary and Alternative Medicine
In peripheral blood, NKp46, NKG2D, and TIGIT expression were signifcantly upregulated in tumor-bearing mice, while high doses of aconite treatment downregulated the expression of all three receptors ( Figure 6).

Discussion
Several studies have shown that aconite and its main components can suppress the progression of HCC through multiple pathways [7][8][9]11]. Taipeinine A, which is derived from aconite, has been found to induce apoptosis in the HCC cell line HepG2 [9]. Aconitum polysaccharide can exert antitumor efects by inducing apoptosis of H22 cells, which is mediated by pituitary tumor transforming gene 1 (PTTG1)-related suppression of P13K/Akt signaling and activation of the downstream MAPK signaling pathway [12].
Aconitine has been proven to inhibit the proliferation of HCC by activating the production of reactive oxygen species, which leads to an increased release of cytochrome c from mitochondria and the activation of apoptosis [7]. Nevertheless, few studies on the immunoregulatory efect of aconite in HCC have been reported. In this study, aconite was found to inhibit H22 tumor growth in vivo and to alleviate the body weight reduction efects of subcutaneous tumors. Meanwhile, indices of adaptive tumor immunity status were improved in tumor-bearing mice, with the thymus index and IFN-c signifcantly upregulated. Strangely, TNF-α and IL-1β are extensively regarded as immunosuppressive cytokines and both were unexpectedly upregulated following aconite treatment, which may be attributed to M2-polarized macrophages. Tis unexpected phenomenon requires further experimental validation.   Evidence-Based Complementary and Alternative Medicine 7 NK cells are a signifcant component of the nonspecifc immune response that exert antitumor efects by both direct cytolytic activity and the secretion of immunostimulatory cytokines [13][14][15][16]. Studies have confrmed that the occurrence of many tumors is related to the loss of the recognition function of NK cells, which is also one of the main mechanisms of tumor immune evasion [17][18][19][20]. Te release of lytic granules containing granzymes and perforin mediates the cytolytic activity of NK cells via binding death receptors on tumor cells, and this can be demonstrated by the expression of CD107a on the surface of NK cells. Te expression of CD107a can also be evaluated as a marker of the activation of efector NK cells [21][22][23]. Innovatively, we found that aconite treatment enhanced NK-mediated immune responses by increasing both the quantity and activity of NK cells. In this study, aconite was proven to be efective in increasing the NK cell percentage in the spleen. Moreover, the expression of CD107a in the spleen could be upregulated under high-dose aconite treatment, which indicated the direct oncolytic activity of NK cells. Activated NK cells can also release TNF-α [24]; however, the role of TNF-α signaling in HCC progression remains controversial. For one thing, TNF-α can inhibit tumors via activating programmed death cascades in targeted cells, with devastating efects on the function of lysosomal enzymes and/or lysosomal membrane integrity. TNF-α signaling can also be used to induce B7-H1 and PD-L1 expression on CD8 + T cells via several pathways, thereby causing immune resistance [25][26][27]. We found that three diferent doses of aconite all increased TNF-α levels in the serum of tumor-bearing mice. Te activation and proliferation of NK cells mediated by aconite treatment may be responsible for the release of TNF-α.
Te functions of NK cells depend on the regulation of activated receptors and inhibitory receptors expressed on their surfaces. Te former are represented by the NCR family (NKp30, NKp44, NKp46, and NKp80), NKG2D, NKG2C, CD226 (DNAM-1), 2B4, and CD16, while the latter mainly includes TIGIT, KIRs, CD96, and CD94/NKG2A [28,29]. NKp46 is involved in the killing and activation of NK cells against pathogens, tumor cells, virus-infected cells, and their own cells [30]. NKG2D is currently recognized as an activated receptor that plays a key role in antitumor immune responses and is also a key target for tumor immune escape [31]. TIGITcan restrict the proliferation of T cells, inhibit the activation of Tcells and NK cells, and block multiple steps of the cancer immune cycle [32]. In this study, we found that inhibitory receptors on NK cells such as TIGIT were signifcantly downregulated in peripheral blood following high-dose aconite treatment, thus promoting the activation of NK cells. Te increased secretion of IFN-c may also be related to this change. To our surprise, TIGIT was upregulated in the spleen following aconite treatment. Tis may be related to the increased expression of CD155, the shared ligand of TIGIT and CD226 [33,34]. Te activated receptor CD226 plays a dominant role, thus increasing the quantity and activity of NK cells. Nevertheless, this hypothesis requires further experimental validation.
Aconite and its components have been found to conduct bidirectional regulation of immune status in diseases, including chronic glomerulonephritis, rheumatic arthritis, osteoarthritis, and allergic rhinitis [35][36][37]. Tis efect can be explained by both immune stimulation and antiinfammation efects. On the one hand, aconite upregulates CD4 + /CD8 + T cells and stimulates related cytokines, including IL-4, IL-10, and IFN-c in patients with septicopyemia [35]. As for cancerous diseases, aconite can also stimulate tumor immunity in patients with breast cancer undergoing chemotherapy, which is mediated by the upregulation of CD4 + /CD8 + T cells and NK cells [38]. On the other hand, aconite can inhibit the release of infammatory cytokines, including IL-6, IL-8, and IL-10, in several autoimmune diseases [39,40]. Our study found that aconite had an immunomodulatory efect on H22 tumor-bearing mice. Combined with the previous research of our group and relevant literature, we speculate that aconite may enhance adaptive immunity and NK cell-mediated immunity by regulating the secretion of immunostimulatory cytokines, upregulating T cells and NK cells, and altering the expression of CD107a and NK receptors. Te deep mechanism may be related to the p38 MAPK signal transduction pathway, which could activate the E2F1 transcription factor and upregulate the expression of ligand CD155 (Figure 7). Te specifc mechanism and main functional component still need further study.

Conclusions
Te results of our study showed that aconite had a therapeutic efect on HCC subcutaneous tumor-bearing mice, which could be attributed to the immunoregulatory efect of  Figure 7: Te possible mechanism through which aconite treatment modulates adaptive immunity and NK cell-mediated immunity. 8 Evidence-Based Complementary and Alternative Medicine aconite, including adaptive immunity and NK-mediated immunity. Tis study revealed a novel mechanism through which aconite exerts an antitumor efect, not only through apoptosis induction pathways. Considering the high incidence and poor prognosis of HCC patients, our fndings could be of valuable clinical signifcance in the treatment of HCC. However, the bidirectional regulation of adaptive immunity mediated by aconite as well as the deep mechanism through which aconite activates NK cells still require further investigation. Tese studies may provide more sound evidence before the introduction of this treatment into routine clinical practice.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure
Huan Wang, Xiuzhong Qi, and Wentao Jia are the co-frst authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.