Effects of Jianpi Huayu Decoction on Th1/Th2 Immune Balance in Mice With Liver Cancer-Related Fatigue via the IL- 27/STAT1 Signaling Pathway

Abstract

Objective: The Chinese medicine Jianpi-Huayu decoction (健脾化瘀方, JPHY) can alleviate cancer-related fatigue in patients with liver cancer. However, its mechanism remains unclear. In this study, we used BALB/c mice with liver cancer model to investigate whether JPHY alleviates cancer-related fatigue by regulating Th1/Th2 immune balance; and the possible association with the IL-27/STAT1 signaling pathway. Methods: We established a mouse model of liver cancer fatigue. Mice were gavaged with physiological saline, low, medium, or high concentrations of JPHY respectively; and intraperitoneal injection of fludarabine (STAT1 pathway inhibitor) with JPHY for 21 days. We recorded the general condition of the mice, and assessed fatigue using scoring criteria and Exhausted Swimming Test. We calculated the spleen and thymus indices, performed H&E staining and immunohistochemical analysis on liver tumor tissues to observe the tumor proliferation marker ki67. We quantified the secretion levels of IFN-γ and IL-2 produced by Th1 cells in serum and splenic lymphocytes, as well as the secretion of IL-4, IL-10 by Th2 cells, and IL-27 in the signaling pathway through ELISA analysis. We evaluated the expression levels of p-STAT1 and STAT1 in spleen tissues using Western blot analysis. Results: JPHY exhibits a therapeutic effect on hepatocellular carcinoma-induced splenomegaly in murine models by upregulating the pro-inflammatory cytokines IFN-γ and IL-2 and downregulating the anti-inflammatory cytokines IL-4 and IL-10. Moreover, JPHY suppresses ki67 expression, reduces tumor-related inflammation infiltration, and ameliorates cancer-associated fatigue. Additionally, the expression of phosphorylated protein p-STAT1 is down-regulated. Conclusion: JPHY may improve the Th1/Th2 immune balance through its anti-inflammatory effects and promotion of IL-27-induced STAT1 phosphorylation, thereby alleviating fatigue in mice with liver cancer.

Introduction

Cancer-related fatigue (CRF) is the most prevalent clinical symptom among cancer patients. ¹ Studies have indicated that over 75% of cancer patients experience CRF, ² with 25% to 30% of them enduring CRF for up to 10 years after cancer diagnosis.^3,4 CRF can be induced by the tumor itself or exacerbated by anti-tumor therapies, including radiotherapy, chemotherapy, and endocrine therapy.^5,6

However, the exact pathogenesis of CRF remains unclear, and currently the focus of research on the biological basis of CRF lies in the inflammation theory. Research has demonstrated that serum inflammatory cytokines, such as IL-2, IL-6, TNF-α, and IFN-γ, are linked to the emergence and development of CRF. ⁷ In animal experiments, it has been found that giving anti-inflammatory drugs to mice models to reduce endocrine IL1RA or IL-10 can improve fatigue, weight loss, and hypersomnia. ⁸ The secretion of these inflammatory cytokines is linked to the body’s immune cells, with Th1 and Th2 cells playing a crucial role. The imbalance between Th1/Th2 in cancer patients has implications for the development, progression, and metastasis of tumors themselves. ⁹ Research further suggests that the disparity between Th1 and Th2 cells could be linked to the STAT1 signaling pathway. ¹⁰ IL-27 can induce STAT1 and promote the secretion of IFN-γ by T cells. ¹¹ Studies have revealed that the combination of traditional Chinese medicine spleen-tonifying herbs and FOLFOX chemotherapy regimens can effectively reduce cancer-related fatigue in postoperative colon cancer patients, while preserving the balance of Th1/Th2 immune responses. ¹² However, the mechanism of this has yet to be explored.

Jianpi-Huayu decoction (JPHY) is a traditional Chinese medicinal formula composed of Baizhu (Rhizoma Atractylodis Macrocephalae), Ezhu (Curcuma zedoaria Roscoe), Fuling (Poria cocos), Foshou (fingered citron), Kushen (Radix Sophorae Flavescentis), and Baihuasheshecao (Hedyotis diffusa Willd.). Studies have shown that Rhizoma Atractylodis Macrocephalae can increase the expression level of IL-2 on the surface of T cells, thereby affecting the structure of T cell subpopulations and inhibiting tumor growth. ¹³ Hedyotis diffusa Willd. demonstrates a significant capacity to elevate serum levels of IFN-γ, TNF-α, and IL-2 in mice, exerts inhibitory effects on tumor microvasculature, and augments the anti-tumor immune response in mice. ¹⁴ Our preliminary research found that the network pharmacology analysis of the active ingredients of JPHY identified the potential target pathway JAK-STAT associated with immune inflammation in treating primary liver cancer. ¹⁵ After 48 hours of treatment, JPHY exhibits significant inhibitory effects on HepG-2 cells, showing pronounced inhibition of cell migration and induction of apoptosis in HepG-2 cells. ¹⁶ Experimental evidence shows that JPHY effectively inhibits tumor growth. ¹⁷ In clinical practice, the survival rates of primary liver cancer patients treated with JPHY at 1, 3, and 5 years were 73.58%, 38.66%, and 22.57% respectively; Additionally, JPHY treatment can ameliorate symptoms such as fatigue, intractable vomiting, pain, insomnia, and decreased appetite in postoperative liver cancer patients, thereby enhancing their quality of life. ¹⁸ However, the potential role and mechanism of JPHY in managing liver cancer fatigue still remain unclear.

In this study, a mouse model of hepatocellular carcinoma with cancer-related fatigue (CRF) was established by injecting fluorescent-labeled murine hepatocellular carcinoma cells (H22-LUC) in situ. The therapeutic effect of JPHY on this mouse model was observed to observe the impact of JPHY on the regulation of Th1/Th2 balance in the IL-27/STAT1 pathway on cancer fatigue in mice.

Materials and Methods

Reagents

Baizhu (No. 20211202), Foshou (No. 20210802), and Kushen (No. 20210601) were purchased from Guangdong Huiqun Traditional Chinese Medicine Pieces Co., LTD. Ezhu (No. B21102001-01) was purchased from Hebei Chufeng Traditional Chinese Medicine Decoction Pieces Co., LTD. Fuling (No. 211104101) was purchased from Kangmei Pharmaceutical Co., LTD. The Hedyotis diffusa Willd. (HDW) was provided by Guangzhou University of Traditional Chinese Medicine. The fluorescence-labeled murine hepatoma cell line H22-luc (RRID: CVCL_C8Y4) was donated by the Key Laboratory of Traditional Chinese Medicine, Southern Medical University. D-luciferin (sodium salt) was purchased from Glpbio (Guangzhou, China) (No. GC43497-100). Servicebio rabbit antigen ki67 (No.GB111141), Servicebio HRP-conjugated goat anti-rabbit IgG (No.GB23303). ELISA Test IFN-γ (No. JL10967-96T), IL-2 (No. JL20256-96T), IL-4 (No. JL20266-96T), IL-10 (No. JL20242-96T), and IL-27 (No. JL13159-96T) kits were purchased from Jianglai Biological (Shanghai, China). P-STAT1 (No. AF3300), STAT1 (No. AF6300), and GADPH (No. AF7021) antibodies were purchased from Jiangsu Affinity Biology Research Center Co., LTD. (Jiangsu, China). Goat Anti-Rabbit IgG (H + L) HRP provided by the Public Laboratory of Guangdong Provincial People’s Hospital.

Animal Procedures and Treatment

The experimental protocols involving animals were approved by the Animal Experiment Ethics Inspection Committee of Guangzhou University of Chinese Medicine. Thirty-six male SPF BALB/C mice, 6 weeks old, weighing 18 ± 1 g, were procured from the Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine [License No. SCXK (Guangdong) 2018-0034]. Prior to commencement of the study, all mice underwent a 1-week acclimatization period under controlled conditions of temperature (22℃-26℃), relative humidity (40%-60%), and a 12-hour light-dark cycle, with ad libitum access to water. The animal facility maintained a tranquil environment.

Preparation of JPHY

The 6 herbs in JPHY (a single dose containing 12 g of Ezhu, 12 g of Baizhu, 12 g of Foshou, 20 g of Fuling, 20 g of HDW, and 20 g of Kushen) were prepared in the appropriate proportions and crushed using a Chinese medicine grinder prior to decoction. ¹⁵

Extraction of volatile oil

JPHY contains a higher concentration of volatile oil components in Baizhu, Ezhu, and Foshou, making it suitable for extracting volatile oil. A flask of round-bottom composition, filled with the 3 herbs in their respective proportions, is employed to extract the volatile oil, and 8 times the volume of ultra-pure water is then added. The traditional Chinese medicine is then soaked in the flask for 6 hours. Once the volatile oil extraction device is set up, the round-bottom flask is heated using an electric heating sleeve. At 220w, the Chinese medicine water decoction is heated until it reaches boiling point. The voltage is then adjusted to 50w, and the Chinese medicine is boiled for approximately 8 hours, or until the volatile oil in the extractor stops increasing. Heating is then stopped. After boiling, 1 ml of volatile oil is collected from the extractor using an EP tube, which is then sealed with a sealing film and stored in a light-free refrigerator at 4°C. Sterile gauze is employed to filter the liquid, which is then transferred to a beaker for future use. The remaining residue is then poured into a round-bottomed flask of 10 000 ml for the following step.

Preparation of water decoction

Add the remaining 3 herbal components of JPHY into a 10 000 ml round-bottom flask. About 8 times the volume of ultra-pure water is added in advance (including the water decoction saved during the extraction of volatile oil, which is added to the water solution before frying) to soak the Chinese medicine for 3 hours. An electric heating jacket is used to heat the round-bottom flask until it reaches a gentle boil, and timing is commenced. It is left to simmer for 1.5 hours. After the decoction, sterile gauze is used to filter it and it is collected in a large beaker. Subsequently, 5 times the amount of ultra-pure water is added and the Chinese medicine continues to simmer. This step is repeated 2 times. After 3 rounds of decoction, the decoction of Chinese medicine is thoroughly mixed and set aside.

The rotary evaporator was employed to concentrate the decoction of Chinese medicine collected, with the temperature of the water bath kept below −20℃ and the concentration set to 2.5 g/ml. Subsequently, the drug was divided into 50 ml centrifuge tubes and stored in a refrigerator at −20℃ for future use.

Preparation of Cells

H22-luc luciferase-labeled mouse hepatocellular carcinoma cell lines were cultured in a CO₂ incubator at 37℃ with PMSI-1640 base culture medium supplemented with 10% fetal bovine serum and 1% streptomycin. Routinely, the cell culture medium and passages were altered. ¹⁹

Establishing a Mouse Model of Cancer-Related Fatigue Induced by Hepatic Carcinoma In Situ

By adhering to the techniques outlined in the pertinent literature, we constructed a mouse model of hepatic carcinoma in situ. ²⁰ Briefly, the culture medium in the bottle was extracted using a pipette, and the H22-luc cells were gently dispersed to form a cell suspension. The cell density was then adjusted to 2 × 10⁷ cells/ml using a cell counting plate.

After randomly dividing the mice into 6 groups, the following method was employed after a week of adaptive feeding: the control group (CON), the model group (MOD), the low-dose of JPHY group (JPHL), the medium-dose of JPHY group (JPHM), the high-dose of JPHY group (JPHH), and the medium-dose of JPHY combined with fludarabine group (JPHF), all using a random number table method (n = 6). After conventional anesthesia, 30 male BALB/c mice, excluding the control group, were anesthetized and disinfected with iodine, followed by a midline incision along the lower abdomen from the xiphoid process, revealing the left lobe of the liver. Using a sterile micropipette, 20 μl of H₂₂-luc cell suspension was extracted and slowly injected into the liver. After pressing with a sterile cotton swab for 30 s, the abdomen was closed. Upon awakening from anesthesia, the mice were brought back to their cages.

Dosing Method

The conversion ratio of 0.0026 between humans (70 kg) and mice (0.020 kg) in terms of body surface area, as detailed in the fourth edition of Pharmacological Experimental Methodology, was applied to calculate the equivalent dose for mice (12.48 g/kg) (96 g divided by 0.0026/0.020 kg = 12.48 g/kg). This dose was then used to determine the administration dose of JPHY, resulting in doses of 6.24, 12.48, and 24.96 g/kg, respectively. ¹⁵ Before administering, the drug-to-extract ratio was used to determine the correct amount of extract, and a suspension was made with normal saline at a volume of 20 ml/kg. The suspension was thoroughly mixed before each administration. Grouping the drug, intragastric administration was conducted for 21 days, with normal saline administered to the CON and MOD groups. The JPHL, JPHM, and JPHH groups each received low, medium, and high concentrations of JPHY. The JPHF group received intraperitoneal injections of a specific STAT1 inhibitor and medium concentrations of JPHY. Fludarabine was dissolved in sterile phosphate solution (PBS) starting from the seventh day of tumor growth, at a dosage of 80 mg/kg, and continued for 21 days.

Observation Index

2.7.1 General observations were made on the 0th day of administration to assess the mental state, eating habits, hair color, stool condition, and weight of the mice. The liver cancer fatigue mice were scored based on the grading standard (Table 1) for their basic status index. ²¹

Table 1.

Classification standard of basic state indicators in mice with liver cancer-related fatigue.

items	Standard of classification
	1	2	3	4
Food intake	Normal	Decrease	Halve	Barely eating
Water intake	Normal	Decrease	Halve	Barely drinking
Spirit	Normal	Sleepy	Drowsy	Malaise
State of body	Normal	Gather together	Arch back up	Vertical hair shiver
Breath	Normal	A little wheeze	Tachypnea	Hypopnea
Luster of hair	Normal	Dull	Fluffy and dull	Yellow and rare
Grip strength	Normal	Slight decline	Severe decline	Inability to grasp

2.7.2 The body weight of the mice was recorded as a percentage of their initial body weight.

2.7.3 In vivo optical molecular imaging technology ²² was used to observe the tumor formation in a mouse model of orthotopic liver transplantation, 7 days after the transplantation surgery.

2.7.4 A behavioral test was conducted during the swimming experiment to assess weight and exhaustion.

After 1 week of adaptive feeding, behavioral tests were performed on day 7, 12, 17, and 21 after tumor inoculation. A lead wire, equivalent to approximately 7% of the mouse’s body weight, was tied to its tail for the weight exhaustion swimming experiment. ²³ Subsequently, the mouse was placed in a round beaker with a diameter of 10 cm and a depth of 10 cm, filled with warm water at 25℃ ± 1℃. The time elapsed from the mouse’s initiation of swimming until exhaustion (defined as the head submerging in the water for 5 seconds without resurfacing) was documented.

Extracting Animal Organ Tissues

After administering medication for 12 hours, mice were anesthetized with 0.3% pentobarbital sodium, and blood samples were taken from their eyeballs. The serum was allowed to coagulate at 37℃ for 15 minutes in a water bath, followed by centrifugation at 5000 rpm and 4℃ for 15 minutes. The separated serum was then stored at −80℃. Subsequently, the mice were euthanized through cervical vertebrae dislocation. Under aseptic conditions, the liver, thymus, and spleen were rapidly excised. Rapid excision of the liver, thymus, and spleen occurred under aseptic conditions, and the thymus and spleen were weighed after the excess water had been absorbed with filter paper.

Spleen index = spleen mass (mg)/body mass (g);

Thymus index = thymus mass (mg)/body mass (g).

Hematoxylin-Eosin Staining (H&E)

The liver was immersed in 4% formaldehyde solution for fixation over a period of 72 hours. The liver cancer and adjacent tissues were excised, trimmed to appropriate dimensions, and subjected to dehydration processing in an automated tissue processor. Following dehydration, the tissues were embedded in paraffin, cooled at 4℃ for 12 hours post-embedding, and then tissue sectioning proceeded. The tissue sections were deparaffinized using xylene, rehydrated in varying concentrations of ethanol, and stained with hematoxylin for 2 minutes. Differentiation was conducted in 1% hydrochloric acid ethanol for 20 minutes, counterstaining with eosin proceeded for 10 minutes, and the sections were washed with tap water. After sealing with a neutral resin sealer, the sections were viewed under a microscope to capture images. ²⁴

Immunohistochemistry of Tumor Tissue (IHC)

Deparaffinization of tissue paraffin sections was completed, followed by a 3 minutes wash in PBS (PH7.4) for slides. Subsequently, the slides were placed in a 3% hydrogen peroxide solution, and left to incubate in the dark for 25 minutes at room temperature. Finally, a decolorization shaker was used on a rocker with PBS (PH7.4) for a 3-minute wash, followed by a 30-minute incubation at room temperature. Last, a 3% BSA solution was evenly added to the histology ring, covering the tissue. The slides were washed in a decolorization shaker on a rocker with PBS (PH7.4) for 3 and 5 minutes each, then the blocking solution was gently shaken off. The primary ki67 antibody, diluted in PBS at 1:500, was then added to the slides, which were laid flat in a humidified box and incubated overnight at 4°C. Finally, HRP-labeled goat anti-rabbit IgG, diluted in PBS at 1:200, was added to the ring to cover the tissue and left to incubate at room temperature for 50 minutes. After 3 washings in a PBS (PH7.4) decolorization shaker on a rocker for 5 minutes each, the slides were slightly dried. Subsequently, a freshly prepared DAB chromogenic solution was added to the ring, and the chromogenic time was monitored under a microscope. When a brownish-yellow staining was observed, the slides were rinsed with tap water to cease the staining. Hematoxylin was used to counterstain for approximately 3 minutes; then, tap water was used to wash, followed by a few seconds of differentiation in the differentiation solution. Afterward, tap water was used to rinse, and blue with bluing reagent, followed by a rinse with running water. The slides were dehydrated, making them transparent, air-dried, sealed, and placed under a bright field microscope to interpret results. ²⁵

Preparation of Lymphocyte Suspension

Isolation of mouse spleens occurred in aseptic conditions, with the fat and connective tissue surrounding the spleen being removed. Subsequently, the tissue was washed with RPMI-1640 medium for a period of 2 to 3 cycles. The spleen was ground and run through a 200-mesh sieve. Centrifugation of the collected cell suspension at 1000 r/min for 5 minutes was followed by the addition of 3 times the volume of red cell lysate, which was then gently blown, cooled on ice for 10 minutes, and spun at 4℃. The cells were then washed twice with PBS. After this, the medium of RPMIS-1640 (containing 10% fetal bovine serum and 1% double antibody) was used to re-suspend them, and Trypan blue staining was applied. The cell count, which was found to be more than 95%, was then adjusted to 2.5 × 10⁶/ml. Planting the cells in 6-well plates, they were then incubated in a 5% CO₂ environment at 37℃. ²⁶

ELISA Analysis

ELISA was used to measure the concentrations of IFN-γ, IL-2, IL-4, IL-10, and IL-27 in serum and spleen lymphocytes of mice. About 200 μl of the sample was diluted and added to the reaction wells of a 96-well ELISA plate. This plate was then incubated at 37℃ for 90 minutes. Following this, the plate was washed and enzyme-labeled antibodies were added freshly. The plate was then incubated again at 37℃ for 1 hour, followed by a further wash. At 37℃, 0.1 ml of TMB substrate solution was blended and the plate was left to incubate for 30 minutes. The absorbance was measured at a wavelength of 450 nm using an enzyme-linked instrument. ²⁷

Western-Blot Analysis

The levels of pSTAT1 and STAT1 in the spleen were determined using Western blot analysis. ²⁸ Two hours after the final dose, the eyeballs were removed and blood was collected. Subsequently, the mice were euthanized, and their spleens were excised and weighed. The spleens were then cut into small pieces using tissue scissors and homogenized in a glass homogenizer. Lysate was added at a ratio of 100 to 200 μl per 20 mg of tissue, and thorough homogenization was achieved. The homogenate was centrifuged at 4℃ and 12 000 r/min for 5 minutes, and the supernatant was collected and stored for further use. The protein concentration was determined using the BCA protein concentration assay kit, and the denatured samples were stored at −20℃. The required separation and concentration gels were prepared according to the instructions provided in the SDS-PAGE gel preparation kit. Gel electrophoresis was performed after loading the protein samples, and the resulting film was transferred using the wet rotation method. The target protein fragments were excised and placed in an antibody incubator box, which was then sealed and incubated overnight with p-STAT1 antibody or STAT1 antibody (diluted 1:1000). This was followed by incubation with Goat Anti-Rabbit IgG (H + L) HRP (diluted 1:3000), TBST washing, and exposure in the gel imaging system. ImageJ software was then employed to analyze the pSTAT1 and STAT1 protein expression in cells, with GAPDH serving as the internal reference for quantitative protein expression.

Data Analysis

Using SPSS 19.0 software, a statistical analysis was conducted, with the measurement results presented as mean ± standard deviation. T-test was used to compare within-group differences in measurement data, and ANOVA was used to compare differences among groups. Chi-square test was used to compare counting data among groups, and a P-value of less than .05 was deemed statistically significant.

Results

As shown in Figure 1A and B, we have successfully established a mouse liver cancer model, and the liver cancer presents irregular shapes in the liver. As shown in Figure 1C, the body weight of mice in all groups increased. During the perioperative period, the body weight of mice in the liver cancer mice decreased. We found that compared to the other groups, mice in the MOD group were more likely to develop hepatic ascites in the early stage. The diet and activity levels of mice in the JPHL, JPHM, and JPHH groups improved after treatment, resulting in an increase in body weight. However, as the experiment progressed, more liver ascites appeared in the later stage of the modeling mice, leading to an increase in body weight in all groups. Mice in the JPHF group were in poor condition and gained weight slowly. As shown in Figure 1D, compared to the CON group, the fatigue score of liver cancer mice increased. After 21 days of treatment, the fatigue scores of the JPHM and JPHH groups were significantly lower than the MOD group (P < .05). As depicted in Figure 1E and F, the swimming time with weight exhaustion was initially balanced among all groups before modeling. However, after modeling, the MOD group exhibited a significant decrease in exhaustive swimming time compared to the CON group (P < .001). Additionally, the medium group showed an increase in exhaustive swimming time compared to the MOD group on days 7, 12, 17, and 20 (P < .01). On the 12th and 17th days, the high group also demonstrated an increase in exhaustive swimming time compared to the MOD group (P < .05). On the seventh day, the swimming times of each group were similar. Considering that performing laparotomy surgery on mice causes certain damage, and the effects of Chinese medicine require a certain amount of accumulation time, the fatigue levels of the mice in the early stages were similar. However, the middle-dose group seemed to show a significant decrease in fatigue as the administration time increased, while the low-dose and high-dose groups did not exhibit this trend. Therefore, the appropriate concentration of JPHY may be the key to reducing fatigue.

Figure 1.

(A) Establishment of a mouse liver in situ cancer model. (B) The use of optical molecular imaging technology for the observation of small animals in vivo. (C) Mouse weight change chart (weight percentage) (n = 6). (D) Graph of Daily Fatigue Scores for Mice (n = 6); *: compared to the MOD group, P < .05. (E) Schematic diagram of the exhaustion swimming test for mice with weight loading. (F) Graph of changes in swimming duration of mice under varying gravitational loads (n = 6); compared to the MOD group, *P < .05, **P < .01, ***P < .001.

As shown in Figure 2A and B, CON group has intact liver lobular structure with clear boundaries. Hepatocytes are complete and arranged radially around the central vein, with large and round nuclei centrally located. Chromatin is sparse and lightly stained, and nucleoli are clearly visible. MOD group shows multiple cancer nodules with tumor cells exhibiting invasive growth and accompanied by a large number of inflammatory cells infiltrating, disrupting the normal liver lobular structure. The central vein and liver lobular structure in the adjacent tissue are incomplete. JPHL, JPHM, JPHH groups have fewer cancer nodules compared to the MOD group, with minimal inflammatory cell infiltration. Normal central veins and liver lobular structures are visible in the adjacent tissue. As shown in Figure 2C and D, The tumor proliferation index ki67 is significantly increased in the MOD group, while ki67 positivity rates in the JPHM and JPHH groups are lower than in the MOD group (P < .001). These findings indicate that JPHY treatment is beneficial in suppressing HCC tumors.

Figure 2.

Liver cancer tissue (A) stained with HE in mice and cancer adjacent tissue (B); Immunohistochemical staining for ki67 in liver cancer tissue of mice (C) and ki67 positivity rate (D), (n = 3), compared to the MOD group, *P < .05, **P < .01, ***P < .001.

As shown in Figure 3, the results of the spleen index showed that compared with the CON group, the spleen index of the MOD group was significantly increased (P < .001). However, the spleen index of the JPHM and JPHH groups was significantly decreased compared to the MOD group (P < .001 for JPHM group, P < .01 for JPHH group). JPHY treatment effectively reduced the spleen index and improved splenomegaly in mice with hepatocellular carcinoma. The results of the thymus index showed that compared with the CON group, the thymus index of the MOD group was significantly decreased (P < .01). Furthermore, the thymus index of the JPHL and JPHM groups was also significantly decreased compared to the MOD group (P < .05). JPHY treatment resulted in a reduction in the thymus index in mice with hepatocellular carcinoma.

Figure 3.

(A) The spleen of mice. (B) The thymus of mice. (C) Spleen index of mice (n = 6), compared to the MOD group, *P < .05, **P < .01, ***P < .001. (D) Thymus index of mice (n = 6), compared to the MOD group, *P < .05, **P < .01, ***P < .00.

As shown in Figure 4, compared with the MOD group, the serum IL-2 of JPHH group was increased (P < .001), while serum IL-4 and IL-10 of JPHM and JPHH was decreased (P < .001). There were no significant differences in serum IFN-γ levels among all groups (P > .05). Also, the lymph IFN-γ of JPHH group was increased (P < .01); the lymph IL-2 of JPHL, JPHM, and JPHH groups was increased (P < .001); while lymph IL-4 and IL-10 of JPHM and JPHH was decreased (P < .001). Serum IL-27 levels in the JPHM group were increased (P < .001) and lymph IL-27 levels in the JPHH group were increased (P < .01). In short, using JPHY can enhance the secretion of IFN-γ and IL-2 levels in Th1 cells, while inhibiting the levels of IL-4 and IL-10 secretion in Th2 cells.

Figure 4.

The result of ELISA analysis. (A) Serum IFN-γ level, n = 3. (B) Serum IL-2 level, n = 3; compared with MOD group, ***P < .001. (C) Serum IL-4 level, n = 3; compared with MOD group, ***P < .001; (D) Serum IL-10 level, n = 3; compared with MOD group, ***P < .001, **P < .01. (E) Serum IL-27 level, n = 3, compared with MOD group, ***P < .001. (F) Lymph IL-2 level, n = 3; compared with MOD group, **P < .01. (G) Lymph IL-2 level, n = 3, compared with MOD group, n = 3, ***P < .001. (H) Lymph IL-4 level, n = 3, compared with MOD group, n = 3, ***P < .001. (I) Lymph IL-10 level, n = 3, compared with MOD group, ***P < .001. (J) Lymph IL-27 level, n = 3, compared with MOD group, **P < .01, ***P < .001.

The results shown in Figure 5 indicate that there was no significant difference in STAT1 levels between the different groups (P > .05). However, the level of p-STAT1 decreased in the MOD group compared to the CON group (P < .001). On the other hand, the level of p-STAT1 increased in the JPHL, JPHM and JPHH groups compared to the MOD group (P < .05). Furthermore, the level of p-STAT1 protein in the JPHF group decreased compared to the MOD group (P < .05).

Figure 5.

The result of Western-blot analysis. (A) Expression of p-STAT1, STAT1, and the internal reference GADPH protein. (B and C) Western blot strip gray scale histogram of STAT1 and p-STAT1 protein, n = 3, compared with model group, *P < .05, **P < .01. (D) Phosphorylation level of STAT1 (p-STAT1/STAT1), n = 3, compared with model group, *P < .05.

Discussion

Combined with the clinical symptoms and signs, cancer-related fatigue (CRF) can be classified as a “deficiency and exhaustion” category in traditional Chinese medicine. It also exhibits symptoms similar to “depression syndrome,” “Lily disease,” and “Zang dryness.”^29,30 The majority of Chronic Renal Failure (CRF) syndromes are characterized by deficiency patterns such as Kidney Deficiency, Spleen Deficiency, and Qi and Blood Deficiency, whereas there are fewer syndromes based on empirical patterns, such as Qi Stagnation, Blood Stasis, and Phlegm Coagulation. ³¹ For the treatment of CRF, although there are related studies in Western medicine, there is currently no specific drug. Traditional Chinese medicine has shown positive effects in enhancing the efficiency and reducing the toxicity of tumor radiotherapy and chemotherapy, preventing tumor recurrence and metastasis, improving quality of life, and extending overall survival. ³²

This study found that the food and water intake increased in mice with liver cancer treated with JPHY, the duration of weight-bearing forced swimming increased, and fatigue symptoms improved. Enhancing Qi in the spleen and stimulating blood circulation to break the stasis of blood, JPHY is employed in clinical practice to prevent the recurrence of postoperative liver cancer. Previous studies have shown that clinical administration of JPHY can improve symptoms such as fatigue, severe vomiting, pain, sleep disorders, and loss of appetite in patients after liver cancer surgery, ultimately enhancing their quality of life. ¹⁸ In basic studies, it has been observed that JPHY can reduce the expression level of TNF-α in the tumor microenvironment, thus reversing the epithelial-mesenchymal transition (EMT) and desiccation of liver cancer cells. Moreover, JPHY can also inhibit the expression of IL-10 and TGF-β in tumor cells.^33,34 These findings suggest that JPHY can alleviate fatigue symptoms caused by liver cancer treatment and modulate the concentration of related cytokines to inhibit tumor recurrence and immune escape.

The exact mechanism of CRF has not yet been clarified. The role of inflammatory cytokines in the pathogenesis of cancer-related fatigue has been widely studied, as persistent and severe fatigue is a common occurrence in various diseases, implying a shared inflammation mechanism underlying the fatigue symptoms. The STAT family members have been implicated in the onset, progression, metastasis, and treatment resistance of human cancers, and the occurrence and development of CRF are affected by multiple factors. STAT1 has been identified as a tumor suppressor, ³⁵ and is of particular importance in physiological processes such as inhibiting cell growth, promoting cell apoptosis, and controlling the immune system. Studies have shown that STAT1 can cause CD4+ T cells to differentiate into Th1, and its signaling pathway blocks GATA-3 transcription, thus suppressing Th2 differentiation and controlling Th1/Th2 balance.^36,37 IL-27, a member of the IL-12 family, has a significant part in the immune response and can directly trigger apoptosis in tumor cells by engaging with various immune cells in the body, such as CD4+ T cells, CD8+ T cells, regulatory T cells, natural killer cells, and dendritic cells. Moreover, IL-27 stimulates immune cells to release particular cytokines, thus controlling both cellular and humoral immunity and indirectly restraining tumor growth. Studies both domestically and internationally have indicated a correlation between the aberrant activation of the JAK-STAT signaling pathway and the biological behavior of liver cancer. In comparison to adjacent tissues, human liver cancer tissues display significantly higher expression levels of STAT1 and STAT3. A positive correlation between the clinical stage and pathological grade of liver cancer and the expression level of STAT3 protein is observed, and a greater amount of phosphorylated STAT3 (p-STAT3) is linked to a poorer prognosis for patients. ³⁸

JPHY’s efficacy in this study was demonstrated to reduce splenomegaly in mice with hepatocellular carcinoma, to some degree enhance the harm inflicted on paracancer hepatocytes, stimulate the secretion of IFN-γ and IL-2 by spleen cells, impede the release of IL-4 and IL-10, thus altering the Th1/Th2 balance toward Th1, and to partially ameliorate the fatigue caused by cancer in these mice. The comparison of STAT1 phosphorylation levels in spleen tissues further confirms that JPHY can up-regulate p-STAT1 levels and increase STAT1 pathway phosphorylation. However, the addition of the STAT1 inhibitor Fludarabine prevented JPHY from reversing the down-regulation of p-STAT1 levels, symptoms of fatigue in mice did not improve, and there was an imbalance in Th1/Th2 equilibrium. We found that IL-27 secretion from JPHH group mice lymphocytes increased, TNF-γ secretion from Th1 cells increased, and the fatigue state of the mice was reduced. IL-27 activates STAT1 by recruiting JAK1 or JAK2 via the pg130 subunit on the receptor. The self-phosphorylation of JAK leads to further phosphorylation and dimerization of STAT1 molecules, resulting in the dimerized pSTAT1 which then translocates into the nucleus, binds to particular target gene sequences, and serves as a transcription factor. ³⁹ It has been confirmed that the therapeutic effect of JPHY is related to STAT1 pathway phosphorylation. And the secretion level of IL-27 is consistent with the phosphorylation level of STAT1. Therefore, this study suggests that the mechanism by which JPHY affects liver cancer fatigue is through the IL-27-mediated STAT1 pathway to improve the Th1/Th2 imbalance.

This study has many shortcomings. Firstly, we did not use a positive drug group for treating CRF. During the treatment process, we were unable to observe the advantages and disadvantages of JPHY compared to positive drugs, or whether JPHY has fewer side effects than positive drugs. Secondly, we did not set an IL-27 negative control group. Using an IL-27 inhibitor would better illustrate the role of JPHY in the IL-27/STAT1 pathway. Thirdly, due to experimental conditions, we could not dynamically observe the secretion of Th1 and Th2 cytokines in the spleen and serum of animals. Regular blood sampling may better illustrate the impact of JPHY on the TH1/TH2 balance. Fourthly, this study did not directly detect Th1 and Th2 levels, nor did it validate the pathway from the transcription level. Using flow cytometry to detect TH1/TH2 can more directly illustrate their balance, and q-PCR experiments validate the pathway at the transcription level.

In future studies, we must take into account these deficiencies. Our research revealed that IL-27 primarily catalyzes STAT1 phosphorylation and amplifies the expression of PD-L1 gene and surface protein on tumor cells in vitro, as well as the liberation of sPD-L1. ⁴⁰ PD-1 is a co-inhibitory molecule that plays a suppressive role in regulating peripheral T cell activation. PD-L1 and PD-L2 may have different functions in regulating type 1 and type 2 responses. Th1 cells and microbial products can enhance PD-L1 expression on many different macrophage subsets, while Th2 cells only induce the upregulation of PD-L2 on inflammatory macrophages. ⁴¹ Whether JPHY influences the Th1/Th2 balance through the IL-27/STAT1 pathway via a certain type of exosome delivery, and the involvement of PD-1, will be the focus of our future research. In addition, the treatment of traditional Chinese medicine is based on syndrome differentiation and treatment. Clinical medication prescriptions need to consider multiple factors in order to implement appropriate measures and achieve the best therapeutic effects.