The Effects of Tibetan Medicine Renqing Changjue Extracts on Cyclophosphamide-Induced Immunosuppression in a Mouse Model

Abstract

Objectives

Renqing Changjue (RQCJ) is a form of Tibetan medicine recognized for its immunomodulatory effects. However, the underlying mechanisms through which it exerts these effects remain to be elucidated.

Methods

We randomly divided 180 male Kunming mice into five groups: control, model (CTX), and high-, medium-, low-dose RQCJ (RQCJH, RQCJM, and RQCJL respectively). The mice received appropriate medications for 15 days. We measured body weight, thymus, spleen index, immune-related cytokines, T cells, and antibodies to evaluate the immunomodulatory effects. Finally, the impact of RQCJ on immunocompromised mice was examined using spleen transcriptome sequencing analysis.

Results

The RQCJ can significantly enhance immune function by regulating specific and non-specific immunity. Transcriptome analysis revealed significant changes in the cytosolic DNA-sensing pathway, TNF signaling pathway, ECM-receptor interactions, and Chagas disease within the RQCJH group. The Jag1 and Tgtp1 genes showed significant upregulation and demonstrated noteworthy correlations with the proportions of T-cell subsets and levels of cytokine expression. Additionally, these genes, along with Rtp4, Irf7, Dhx58, Il1b, Ifi44, and Ifi35 may be the important gene targets for the action of RQCJ.

Conclusion

This study provides new insights into the mechanisms by which RQCJ modulates immune responses, offering theoretical foundations for tumor treatment and clinical application.

Introduction

On a global scale, the burden of malignant tumors is increasing daily, with both incidence and mortality rates rising each year. The challenges of prevention and control are compounded by an aging population, which will further escalate the cancer burden, placing immense pressure on public health and the economy.^1,2 The occurrence, development, and outcomes of diseases are contingent upon fluctuations in beneficial and detrimental factors, which are closely intertwined with the body’s immune function and self-defense mechanisms. In recent years, progressive advancements in immunomodulatory drugs have partially alleviated the adverse reactions associated with radiotherapy and chemotherapy, thereby enhancing patient compliance, optimizing the efficacy of tumor treatments, and improving patients’ quality of life.

Tibetan medicine primarily consists of natural remedies characterized by mild pharmacological properties, low drug resistance and minimal adverse reactions. It offers distinct advantages in restoring the body’s overall balance, enhancing disease resistance, and exhibiting anti-tumor effects. Renqing Changjue (RQCJ), a prominent Tibetan medicinal formula, comprises over one hundred medicinal herbs and is documented in The Four Medical Tantras, ³ reflecting a long history of medicinal use. This formulation is effective in treating a variety of ailments, including chronic gastritis, ulcers, atrophic gastritis, cervical cancer, and conditions such as syphilis and leprosy. ⁴ Notably, RQCJ has shown therapeutic benefits that surpass those of Western medicine. ⁵ Recent studies indicate that RQCJ possesses anti-inflammatory properties and helps maintain normal cytokine ratios by regulating T cell subpopulations. In a chemotherapy experiment involving RQCJ-assisted CTX in L615 leukemia mice, a significant prolongation of survival time was observed following RQCJ administration. Additionally, there was a notable increase in the CD4⁺/CD8⁺ T cell subset ratio in the spleen, along with elevated levels of IL-2, IL-6, and IFN-γ in spleen homogenates. These findings suggest that RQCJ ameliorates chemotherapy-induced immunosuppression by modulating T cell subset ratios and enhancing the secretion of cytokines IL-2, IL-6, and IFN-γ post-chemotherapy. ⁶

Our research group has demonstrated that RQCJ significantly reduces the elevated expression of IL-6 and TNF-α in RLE-6TN cells induced by LPS. Additionally, RQCJ notably increases the expression of IL-10 and enhances the Bcl-2/Bax protein ratio while decreasing the expression of caspase 3 protein. These finding suggest that RQCJ may exert its effects by reducing the release of inflammatory factors and promoting anti-apoptotic mechanisms. ⁷ Therefore, we propose that RQCJ has immunomodulatory effects.

To date, the mechanisms underlying the effects of RQCJ on immunoregulation have not been systematically evaluated. Notably, there are currently no transcriptomic studies that have identified its key targets. Therefore, in this study, we generated a CTX-induced immunosuppression model in mice and assessed the immune regulatory function of RQCJ by measuring changes in non-specific and specific immune functions. We also performed transcriptome sequencing analysis of the spleen to provide a theoretical basis for clinical applications.

Materials and Methods

Reagents and Experimental Animals

RQCJ was acquired from Ganlu Tibetan Medicine Co., Ltd. (batch number: 200400302). CTX for injection was acquired from Baxter Oncology GmbH (batch number: HA-80-02-952). Male Kunming mice (18–20 g, 4 weeks old, SPF grade) were purchased from Chengdu Dashuo Experimental Animal Co.LTD. (SCXK (chuan) 2020-030) and kept in the animal house of the Medical College of Xizang Minzu University at 24 ± 1°C and 50% ± 10% humidity. After three days of regular feeding, only mice that met the following criteria were included in the study: a body weight ranging from 18 to 20 grams, body length of 7 to 9 cm, and the absence of visible health abnormalities such as alopecia or lethargy. The exclusion criteria comprised mice whose body weight or body length fell outside the specified range, as well as those exhibiting clinical signs of illness. All procedures were conducted following the ‘Guiding Principles in the Care and Use of Animals in China’ from January to November 2022, and were approved by the Laboratory Animal Ethics Committee of Xizang Minzu University (ethics approved number: 20200-7).

Experimental Treatments

RQCJ pills (4 g) were crushed and soaked in 100 mL of double-distilled water for 12 h, then condensed and refluxed for 3 hours. After filtration, we altered the concentration to 30 mg/mL drug solution for refrigeration.

A total of 180 male Kunming mice were randomly divided into three groups. Based on previous research and literature review,^8,9 each of the 60 mice was allocated to one of the following groups: a control group (Control), a cyclophosphamide group (CTX) (50 mg/kg), an RQCJ high-dose group (RQCJH) (0.3 g/kg RQCJ extract), an RQCJ medium-dose group (RQCJM) (0.15 g/kg RQCJ extract), and an RQCJ low-dose group (RQCJL) (0.075 g/kg RQCJ extract), each group consisted of 12 mice. The dosages of RQCJ were calculated based on the equivalent dose ratio of 9:1 for humans to mice, taking body weight into consideration. ¹⁰ The experimental design of each group is shown in Figure 1. In the first batch, the mice weights were recorded at 0, 3, 6, 9, 12 and 15 days after intragastric administration.

Figure 1.

Experimental design scheme drawing. Gavage (A 10 mL/kg distilled water, B 0.3 g/kg RQCJ extract, C 0.15 g/kg RQCJ extract, D 0.075 g/kg RQCJ extract). Intraperitoneal injection (E 50 mg/kg CTX, F 0.2 mL 20% chicken red blood cell suspension (CBRC), G equal volume normal saline to E).

Compound Identification of RQCJ

Reverse-phase analysis was conducted using an Agilent 1260 Infinity II (CA, USA). The flow rate was maintained at 1 mL/min, with mobile phase A consisting of acetonitrile and mobile phase B comprising of 0.1% phosphoric acid. A gradient elution was employed to achieve sample separation as follows: 0-15 min, 10% A; 15-30 min, 10-15% A; 30-70 min, 15-25% A; 70-75 min, 25-40% A; 75-100 min, 40-60% A; 100-120 min, 60-75% A; 120-125 min, 75-10% A. The column temperature was 25°C, and the injection volume was 10 μL for each analysis. The detection wavelength is 254 nm.

The extraction method for the test substance involved weighing 1.0 g of the sample and placing it into a volumetric flask. Subsequently, 25 mL of methanol was added, and the mixture was subjected to ultrasonication at a power of 250 W and a frequency of 33 kHz for 30 minutes, with three replicates performed for each group.

Carbon Particle Clearance Experiment

On the 15^th day of the experiment, a 5-fold dilution Indian ink was administered via injection into the caudal vein at a dosage of10 mL/kg, following a 3 hours period for the first batch of mice. Mice were injected intraperitoneally with pentobarbital sodium (50 mg/kg), we removed 20 μL of blood from the venous plexus at 2 min (t₁) and 12 min (t₂) and quickly added it into 2 mL 0.1% Na₂CO₃ solution and mixed. The optical densities (OD₁ and OD₂) were measured using a Multiskan GO Full-wavelength ELISA (Thermo, USA) at a wavelength of 600 nm. Following the injections of pentobarbital sodium (50 mg/kg), all mice were euthanized via cervical dislocation. The weights of the livers and spleens were recorded, and the carbon clearance index (K) and phagocytosis index (α) were subsequently calculated. ¹¹

$\mathrm{K}=({\text{lgOD}}_1-{\hspace{0.5em}\text{lgOD}}_2)/({\mathrm{t}}_2-{\mathrm{t}}_1)\hspace{0.17em}({\mathrm{t}}_2-{\mathrm{t}}_1)\text{is}\hspace{0.17em}\text{the}\hspace{0.17em}\text{uncorrected}\hspace{0.17em}\text{phagocytosis}\hspace{0.17em}\text{index}.$

$\mathrm{\alpha }={\mathrm{K}}_{1/3}\times \text{body}\hspace{0.17em}\text{weight}/(\text{liver}\hspace{0.17em}\text{weight}+\text{spleen}\hspace{0.17em}\text{weight})$

Immune Organ Index and Complete Blood Counts

On the 15^th day of the experiment, following 3 hours of administration, blood was collected from the venous plexus after anesthesia for routine blood tests using the BC-2800Vet Veterinary Blood Cell Analyzer (Mindray, China) for the second batch of mice. The counts of white blood cells, lymphocytes, and neutrophils were recorded. Subsequently, the mice were euthanized via cervical dislocation, the thymus and spleen were weighed, with their respective the organ indices recorded.

$\text{Thymus}\hspace{0.17em}\text{index}=\text{thymus}\hspace{0.17em}\text{mass}\hspace{0.17em}\left(\text{mg}\right)/\text{body}\hspace{0.17em}\mathrm{m}\text{ass}\hspace{0.17em}\left(\mathrm{g}\right)\times 10$

$\text{Spleen}\hspace{0.17em}\text{index}=\text{spleen}\hspace{0.17em}\text{mass}\hspace{0.17em}\left(\text{mg}\right)/\text{body}\hspace{0.17em}\text{mass}\hspace{0.17em}\left(\mathrm{g}\right)\times 10$

The Changes of T Lymphocyte Subsets in Mouse Spleen

The second batch of mice was euthanized using cervical dislocation, followed by immersion in 75% alcohol for 3 minutes. Subsequently, the mice were placed on a clean bench for spleen preparation and extraction. The spleens were diced and ground, filtered with a 200-micron screen, and grinding liquid was collected. The supernatant was centrifuged at 4°C for 10 min, and a single-cell suspension was obtained. After the red blood cells were lysed, appropriate amounts of culture medium were added to adjust the cell concentration to 1 × 10⁶ cells/mL. We transferred 100 µL cell suspensions into flow cytometry tubes and added 10 µL of anti-mouse CD4⁺T antibodies (FITC anti-mouse CD4, Clone: RM4-5, Concentration: 0.5 mg/mL) and CD8⁺T antibodies (PE anti-mouse CD8a, Clone: 53-6.7, Concentration: 0.2 mg/mL) labeled with different fluorochromes (Dakewe, China). After mixing, the cells were incubated on ice for 20 min away from light. We added 3 mL phosphate-buffered saline, centrifuged at 1000 rpm for 5 min, and discarded the supernatant. Finally we added 400 µL of phosphate-buffered saline and analyzed the samples using the GALLIOS flow cytometer (Beckman, USA). ¹²

Serum Levels of IL-2, IL-6 and IFN-γ in Mouse

Whole blood from the second batch of mice was centrifuged at 3000 rpm for 20 minutes. The upper serum was carefully separated, and the expression levels of IL-2, IL-6, and IFN-γ expression levels were measured according to enzyme-linked immunosorbent assay kit instructions (Bioswamp, China). Samples and HRP-labeled antibodies were added to ELISA plate wells containing corresponding antibodies and incubated at 37°C. After 60 minutes, the liquid in the plate was discarded and the plate was washed five times, and the enzyme substrate and color developing substrate were added to each well and incubated at 37°C in the dark for 15 minutes. The reaction was terminated by adding the termination solution and the OD value of each well was recorded at 450 nm using an enzyme marker.

Serum Hemolysin Levels in Mouse

On the fifteenth day of the experiment, blood samples were collected according to the previously described methodology. ¹³ The serum was divided into two aliquots. One aliquot was diluted 100-fold, and 0.5 mL of this dilution was mixed with a 5% suspension of chicken red blood cells, 10% guinea pig serum, and 0.5 mL of normal saline. The mixture was incubated in a water bath at 37°C for 1 hour, after which the reaction was stopped by placing it in a refrigerator at 0°C. Following centrifugation at 3000 rpm, the optical densities (ODs) of the supernatant were determined at 540 nm using an enzyme label instrument to determine IgM levels. The other part was added to the same amount of 2-mercaptoethanol in a 37°C water bath for 30 minutes to determine IgG levels. The production levels of hemolysin and IgG were calculated.

${\text{HC}}_{\text{IgM}}=\text{sample}\hspace{0.17em}\text{serum}\hspace{0.17em}\text{OD}\hspace{0.17em}\text{value}\times \text{dilution}\hspace{0.17em}\text{ratio}$

${\text{HC}}_{\text{IgG}}=2-\text{mercaptoethanol}\hspace{0.17em}\text{treated}\hspace{0.17em}\text{serum}\hspace{0.17em}\text{OD}\hspace{0.17em}\text{value}\times \text{dilution}\hspace{0.17em}\text{ratio}$

RNA Extraction, Library Construction and Sequencing

Each group of experiments consisted of four repetitions. Total RNA was isolated from the tissue using TRIzol® Reagent according to the manufacturer’s guidelines, and genomic DNA was eliminated through the application of DNase I (TaKara, China). The quality of RNA was determined by 5300 Bioanalyser (Agilent, USA) and quantified with the NanoDrop-2000 (Thermo, USA). Only RNA samples of high quality were employed for the construction of the sequencing library. ¹⁴ The transcriptome library for RNA sequencing was constructed using the Illumina® Stranded mRNA Prep, Ligation method (San Diego, CA). Messenger RNA was extracted through a polyA selection technique employing oligo(dT) beads, followed by initial fragmentation with a fragmentation buffer. Subsequently, double-stranded cDNA was generated using random hexamer primers. The resulting cDNA underwent end-repair, phosphorylation, and adapter addition according to the library construction protocol. cDNA target fragments were size-selected to a range of 300-400 bp using magnetic beads, followed by PCR amplification for 15 cycles. After quantification with Qubit 4.0, the sequencing library was processed on the NovaSeq X Plus platform (PE150) using the NovaSeq Reagent Kit.

Read Mapping, Differential Expression Analysis and Functional Enrichment

The raw paired-end reads were trimmed and quality-controlled using SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Clean reads were aligned to a reference genome in orientation mode using HISAT2 software. ¹⁵ StringTie assembled the mapped reads of each sample through a reference-based approach. ¹⁶

To identify differentially expressed genes (DEGs) between two samples, the expression level of each transcript was calculated using the transcripts per million reads method. RSEM was employed to quantify gene abundances. ¹⁷ Differential expression analysis was conducted using the DESeq2 and EdgeR with a Q value ≤ 0.05. DEGs with |log2FC| >1 and Q value ≤ 0.05(DESeq2 or EdgeR)/Q value ≤ 0.001(DEGseq) were significant DEGs.^18,19 Functional enrichment analysis, including GO and KEGG, was performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected P-value ≤ 0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analyses were conducted using Goatools and KOBAS. ²⁰

Protein Interaction Network Analysis

We used the STRING database (https://string-db.org/) to construct a protein-protein interaction network analysis (PPI) of DEGs. A selection of protein patterns was made based on a combined score, focusing on the top 50. ²¹

Real-Time Quantitative Polymerase Chain Reaction

According to the manufacturer’s instructions, total RNA was extracted using TRIzol Reagent (Invitrogen, USA), with four replicates for each experiment, consistent with the transcriptome sequencing samples. Subsequently, 1 μg of total RNA was reverse-transcribed using a two-step RT kit (Vazmye, China). Polymerase chain reactions (PCRs) were performed using the CFX96 connect instrument (Bio-Rad, USA) with a reaction mixture that consisted of SYBR Green PCR Master Mix (Toyobo, Japan), cDNA template, and primers. Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The reaction condition: 95°C, 10 s; 55°C, 10 s; 72°C, 30 s; cycle 35 times. Quantification was performed using the efficiency-corrected −2^ΔΔCT method with the housekeeping gene GAPDH as the endogenous control.

Correlation Analysis of Target Genes With Phenotypic Immune Indicators

Based on the Pearson correlation coefficient, a correlation analysis was conducted between the significantly regulated target genes associated with immune regulation identified in the transcriptome sequencing following RQCJ intervention and the phenotypic immune indicators, which included T cell subset proportions and cytokine levels from the experiment. The results were subsequently visualized as a correlation heatmap.

Statistical Analysis

All experimental results are presented as the mean ± SEM. Statistical differences among different groups were assessed with one-way analysis of variance (ANOVA) using SPSS software (version 24.0, Chicago, IL, USA). Differences where P < 0.05 were considered statistically significant. Graphing was performed using GraphPad Prism 8.0 software (San Diego, CA, USA).

Results

Quality Control Analysis of RQCJ

The High-Performance Liquid Chromatography (HPLC) method was established to elucidate the chemical profile of the RQCJ extract and to quantify its primary constituents. The chromatograms of the four herbs of RQCJ were analyzed quantitatively using a standard curve at 254 nm (Figure 2), and the calculated concentrations (mg/g) of each compound are summarized in Table 1. We have detected and calculated four principle components of RQCJ: gallic acid (1.833 mg/g), ferulic acid (0.397 mg/g), glycyrrhizic acid ammonium salt (0.275 mg/g), and chrysophanol (0.024 mg/g) respectively. These medicinal components exhibit significant immunomodulatory effects.

Figure 2.

High-performance liquid chromatogram of the RQCJ extract.

Table 1.

Compounds of RQCJ extract.

​	Gallic acid	Ferulic acid	Glycyrrhizic acid ammonium salt	Chrysophanol
Molecular formula	C7H6O5	C10H10O4	C42H65NO16	C15H10O4
Content (mg/g)	1.833 ± 0.058	0.397 ± 0.010	0.275± 0.001	0.024 ± 0.002

Changes in Mice Weight, Clearance Index, and Phagocytosis Index in Immunosuppressed Mice

Compared with the control group (Figure 3A), the weights of mice in other experimental groups were significantly lower following CTX injection. The weight in the CTX group was notably reduced, with only a slight degree of recovery observed. The body weights in the RQCJ group were significantly greater than the CTX group; the RQCJH group was higher than the other two groups, followed by the RQCJL group. The clearance and phagocytosis indices in the CTX group were significantly lower than the control group (P < 0.05). The RQCJH group’s phagocytic index α was substantially higher (P < 0.01), and the RQCJL group’s clearance index K (P < 0.05) and phagocytic index α (P < 0.05) was greater than that of the CTX group (Figure 3B and C).

Figure 3.

The alteration of mice weight, clearance index, and phagocytosis index in different groups of mice.

The Effect of RQCJ on the Thymus Index, Spleen Index, and Complete Blood Counts

The thymus and spleen indices in the CTX group were significantly lower than those in the control group (P < 0.01 and P < 0.05, respectively). In contrast, the thymus and spleen indices in the RQCJH group were notably higher than those in the CTX group (P < 0.01 and P < 0.05, respectively), surpassing the indices observed in the other two groups. For the RQCJM and RQCJL groups, only the spleen index exhibited a significant increase (P < 0.01 and P < 0.05, respectively) (Figure 4A). The counts of white blood cells, lymphocytes, and neutrophils in the CTX group were significantly lower compared to the control group (P < 0.01) (Figure 4B). Conversely, the RQCJ groups showed a higher number of immune-related cells than the CTX group (P < 0.01 and P < 0.05, respectively). White blood cells were 35.12 × 10⁹ cells/L, which was significantly much higher than neutrophils and lymphocytes. These findings suggest that RQCJ effectively promotes recovery from CTX-induced immunosuppression.

Figure 4.

The effect of RQCJ on the thymus index, spleen index, and complete blood counts.

The Effect of RQCJ on the Proportion of CD4⁺T and CD8⁺T Cells in Immunocompromised Mice

The ratio of CD4⁺/CD8⁺T indicates T cell immune function; lower ratios indicate weaker immune function.^22,23 Flow cytometry analysis demonstrated that the percentage of CD4⁺T cells in splenic lymphocytes and the CD4⁺/CD8⁺T cell ratio in the CTX group were significantly lower than those in the control group (P < 0.01). Following RQCJ treatment, both parameters exhibited a significant increase (P < 0.05) (Figure 5). Notably, The percentage of CD4⁺T cells in splenic lymphocytes and the CD4⁺/CD8⁺T cell ratio increased most significantly in the RQCJH group (P < 0.01), followed by the RQCJL group (P < 0.01) and the RQCJM group (P < 0.05). These findings indicate that the RQCJH group plays a crucial role in the rapid recovery of cellular immunity, with the RQCJL group following, while the RQCJM group demonstrated the least effect.

Figure 5.

The effect of RQCJ on the proportion of CD4⁺T and CD8⁺T cells.

Regulation of Cytokines by RQCJ

T cells can be divided into Th1, Th2, and other cell subtypes. Th1 cells mediate cellular immune responses by secreting cytokines such as IFN-γ and IL-2, which activate effector cells. In contrast, Th2 cells assist the differentiation of B cells in into effector cells by secreting IL-4, IL-6, and other cytokines and participate in humoral immune responses. ²⁴

In the CTX group, serum levels of IL-2, IL-6, and IFN-γ were significantly lower than those in the control group (P < 0.01) (Figure 6). Following RQCJ treatment, these cytokines exhibited a significant increase, with the exception of IL-6 in the RQCJM group when compared to the CTX group. The elevated levels of IL-2, IL-6, and IFN-γ promote the proliferation and differentiation of immune response cells, thereby enhancing their immune function.

Figure 6.

The effect of RQCJ on IL-2, IL-6, and IFN-γ in immunosuppressed mice. Compared with the control group: ^##p < 0.01. Compared with the CTX group: ^*p < 0.05, ^**p < 0.01. (n = 12, one-way ANOVA was used for data analysis).

The Effect of RQCJ on Serum Levels of IgM and IgG in Immunocompromised Mice

The serum hemolysin level in mice sensitized with sheep red blood cells serves as an important indicator for evaluating humoral immune function. ²⁵ After lymphocytes encounter sheep red blood cell antigens, they produce serum hemolysin antibodies to lyse red blood cells. The serum hemolysin level reflects lymphocytes’ proliferation and differentiation level and the secretion of specific antibodies following the encounter with sheep red blood cell antigens.^26,27 In the CTX group, the levels of IgG and IgM were significantly lower compared to the control group (IgG P < 0.01; IgM P < 0.05). Compared with the CTX group, the serum IgM levels were significantly higher in the RQCJH and RQCJL groups (P < 0.01); the serum IgG content in the RQCJM group was also significantly higher (P < 0.01), followed by the RQCJL group (P < 0.05) (Figure 7).

Figure 7.

The effect of RQCJ on IgG and IgM in immunosuppressed mice. Compared with the control group: ^#p < 0.05, ^##p < 0.01. Compared with the CTX group: ^*p < 0.05, ^**p < 0.01. (n = 12, one-way ANOVA was used for data analysis).

RNA Assessment and Basic Statistics of Mouse Spleen

The data quality assessment and sequence comparison of the 5 groups of spleen tissue samples Revealed an error rate ranging from 0.0247 to 0.0253. The minimum Q20 and Q30 content for each sample of the sequencing data was 98.03% and 93.95%, greater than 85% and 80%. All data were deemed reliable. The GC content ranged from 49.85% to 51.68%, while the unique comparison rate of all samples was 96.01%. These findings indicate that the transcriptome sequencing data is of high-quality and reliability, making it suitable for subsequent analyses.

Significant DEGs

We used P-adjust < 0.05 and |log2FC|≥ 2 as criteria to identify DEGs (Figure 8A). Compared with the control group, the CTX group identified 4164 DEGs, of which 1402 genes were upregulated and 2762 genes were downregulated. When Comparing the CTX group to the RQCJH, RQCJM, and RQCJL groups, there were 100, 1, and 3 upregulated genes and 65, 4, and 7 downregulated genes, respectively. The Venn diagram illustrates that 106 genes exhibited consistent regulation in the control and RQCJH groups compared with the CTX group; other similar regulatory genes are shown in Figure 8B.

Figure 8.

Differentially expressed gene histogram (A) and Venn diagram (B).

Gene Ontology Analysis of DEGs

GO functional analysis was conducted on the DEGs after RQCJ intervention at various concentrations. The top 20 genes exhibiting each significant expression patterns are shown in Figure 9. The results are divided into three modules: the cellular component involves cell parts, organelles, and membrane parts; molecular function involves structural molecule activity, binding, catalytic activity, and molecular function regulator; biological processes involve cellular processes, biological regulation, response to stimulus, metabolic processes, and immune system processes. The functional enrichment results for the RQCJH vs. CTX group were consistent with the control vs. CTX group. After RQCJ drug intervention, RQCJH, RQCJM, and RQCJL groups were enriched with 19, 1, and 2 genes associated with immune system processes compared with the CTX group (Table 2).

Figure 9.

GO function annotation map of DEGs. The Y-axis represents the Go term, and the X-axis represents the number of genes.

Table 2.

DEGs related to immune system processes after RQCJ treatment.

Gene name	Regulation	Gene description
RQCJH vs. CTX
Fcgr1	down	Fc receptor, IgG, high affinity I [Source: MGI Symbol; Acc: MGI:95498]
Dhx58	down	DEXH (Asp-Glu-X-His) box polypeptide 58 [Source: MGI Symbol; Acc: MGI:1931560]
Ifitm3	down	Interferon-induced transmembrane protein 3 [Source: MGI Symbol; Acc: MGI:1913391]
Irf7	down	Interferon regulatory factor 7 [Source: MGI Symbol; Acc: MGI:1859212]
Ifi211	down	Interferon activated gene 211 [Source: MGI Symbol; Acc: MGI:3041120]
Jag1	up	Jagged 1 [Source: MGI Symbol; Acc: MGI:1095416]
Ifi44	down	Interferon-induced protein 44 [Source: MGI Symbol; Acc: MGI:2443016]
Cmtm7	down	CKLF-like MARVEL transmembrane domain containing 7 [Source: MGI Symbol; Acc: MGI:2447166]
Rtp4	down	Receptor transporter protein 4 [Source: MGI Symbol; Acc: MGI:1915025]
Bst2	down	Bone marrow stromal cell antigen 2 [Source: MGI Symbol; Acc: MGI:1916800]
Trex1	down	Three prime repair exonuclease 1 [Source: MGI Symbol; Acc: MGI:1328317]
Oas1a	down	2′-5′ oligoadenylate synthetase 1A [Source: MGI Symbol; Acc: MGI:2180860]
Bcl3	down	B cell leukemia/lymphoma 3 [Source: MGI Symbol; Acc: MGI:88140]
Hsh2d	down	Hematopoietic SH2 domain containing [Source: MGI Symbol; Acc: MGI:2676364]
Pilrb1	down	Paired immunoglobin-like type 2 receptor beta 1 [Source: MGI Symbol; Acc: MGI:2450532]
Oas1g	down	2′-5′ oligoadenylate synthetase 1G [Source: MGI Symbol; Acc: MGI:97429]
Tgtp1	up	T cell-specific GTPase 1 [Source: MGI Symbol; Acc: MGI:98734]
Cfb	down	Complement factor B [Source: MGI Symbol; Acc: MGI:105975]
Igkv10-94	down	Immunoglobulin kappa variable 10-94 [Source: MGI Symbol; Acc: MGI:3646140]
RQCJM vs. CTX
Pilrb1	down	Paired immunoglobin-like type 2 receptor beta 1 [Source: MGI Symbol; Acc: MGI:2450532]
RQCJL vs. CTX
Tgtp1	up	T cell-specific GTPase 1 [Source: MGI Symbol; Acc: MGI:98734]
Pilrb1	down	Paired immunoglobin-like type 2 receptor beta 1 [Source: MGI Symbol; Acc: MGI:2450532]

Pathway-Significant Enrichment Analysis

To elucidate the DEGs under the treatments of RQCJ and CTX, we conducted a pathway-significant enrichment analysis of the functions and interactions of these genes (Figure 10). For the control vs. CTX group, we identified the top 20 pathways with statistically significant differences in gene expression. These pathways included primary immunodeficiency, hematopoietic cell lineage, Th17 cell differentiation, the intestinal immune network for IgA production, the T cell receptor signaling pathway, Th1 and Th2 cell differentiation, and asthma, all of which are related to immune responses. CTX treatment profoundly altered immune regulation, especially cellular and humoral immunity. After RQCJ treatment, significant changes were observed in the cytosolic DNA-sensing pathway, the TNF signaling pathway, ECM-receptor interaction, and Chagas disease in the RQCJH group. Conversely, in the RQCJM and RQCJL groups, only the herpes simplex virus type 1 infection exhibited significant alterations.

Figure 10.

Top 20 statistics of KEGG pathway enrichment in DEGs. The Y-axis represents the main pathway, and the X-axis represents the enrichment score.

PPI Network Diagram of DEGs

The PPI network diagram of DEGs after RQCJ intervention is shown in Figure 11. In the comparison between the control and CTX groups, critical genes identified include Igf1, Ndufb7, Ndufa5, Il1b, Rsma7 and Psmb2. After RQCJ treatment, Rtp4, Irf7, Dhx58, Il1b, Ifi44 and Ifi35 were essential genes of the RQCJH vs. CTX group; in the RQCJM vs. CTX group, only Gm6563 and Pps26-ps1 were critical genes; for the RQCJL vs. CTX group, Stfa3 and Stfa2 were the only genes. Fcgr1, Dhx58, Ifitm3, Irf7, Ifi44, Rtp4, Bst2, Trex1, Oas1a, Bcl3, Oas1g and cfb were enriched in the screening differential pathways mentioned above in the RQCJH vs. CTX group and may serve as therapeutic targets for RQCJ to antagonize immune suppression in mice.

Figure 11.

Differentially expressed gene PPI network diagram. The blue nodes in the middle represent the target genes; the darker the color and the larger the node is, the greater the degree value.

Validation Analysis of qRT-PCR

The experimental results indicated that the immunomodulatory effect of the RQCJH group was more significant. The significantly different genes in the RQCJH vs. CTX group were selected for qRT-PCR verification, with the primers listed in Table 3. The results showed that Pilrb1, Rtp4, Irf7, Dhx58, Ifi35 and Ifi44 showed a downward trend, and Tgtp1, Il1b and Jag1 showed an upward trend after RQCJ intervention (Figure 12). This observation aligns with the trends observed in the transcriptomic data, suggesting a high level of reliability in the transcriptomic findings.

Table 3.

The information of qRT-PCR primer sequence.

Gene name	Primer sequence (5′→3′)
Jag1	F: TCT​CTG​TCC​CAC​TGG​TTT​CTC​TG
	R: AAT​AGT​CAC​TGG​CAC​GAT​TGT​AGC
Tgtp1	F: CGC​AGG​GAA​GTC​CAC​TTT​CAT​C
	R: TTG​GGT​GTG​GGT​ATG​GAG​TTC​TC
Pilrb1	F: CCA​AGC​ACT​CAA​CAC​CAC​CAT​G
	R: TTA​GCC​AGG​AGC​ATC​GTG​ACC
Rtp4	F: TGG​AGC​CTG​CAT​TTG​GAT​AAG​AAC
	R: GCA​TCT​GGA​ACA​CTG​GAA​CCT​G
Irf7	F: TGA​GCG​AAG​AGA​GCG​AAG​AGG
	R: CGT​ACA​CCT​TAT​GCG​GAT​CAA​CTG
Dhx58	F: GCT​TCC​TCC​CTC​CTG​CTC​TG
	R: GTG​TGC​TCT​GGC​TTG​TGT​TCC
Il1b	F: CAC​CTC​ACA​AGC​AGA​GCA​CAA​G
	R: GCA​TAA​GAA​ACA​GTC​CAG​CCC​ATA​C
Ifi44	F: CCC​TGC​CAT​TTA​TTC​TGT​GTG​ACT​C
	R: CGG​ATG​GTT​TGA​TGT​GAT​TGG​TTT​C
Ifi35	F: TGT​CCA​GCC​AGC​CAG​ATA​ACC
	R: TCT​ACA​TCC​CCA​CCT​CCA​TTC​TTG

Figure 12.

The mRNA expressions of the RQCJH vs. CTX group were significantly different genes determined by qRT-PCR. Compared with the CTX group: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001. Compared with the control group: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. (n = 4, one-way ANOVA was used for data analysis).

Results of the Correlation Between Target Genes and Phenotypic Immune Indicators

The correlation analysis heatmap revealed that (Figure 13), with the exception of Ifi35, the expression regulation of the remaining eight genes significantly influenced phenotypic immune indicators, including T-cell subset proportions and cytokine levels (p < 0.05). Following the RQCJ intervention, the expression of three genes Tgtp1, Jag1, and I11b was significantly upregulated. Notably, high expression of Tgtp1 significantly enhanced the levels of IgG, IL-6, IFN-γ, and IL-2, as well as increased the CD4⁺/CD8⁺ T cell ratio (p < 0.01). Additionally, elevated expression of the I11b gene significantly promoted the levels of IFN-γ and IL-2, along side an increase in the CD4⁺/CD8⁺ T cell ratio (p < 0.05). Furthermore, high expression of the Tgtp1 gene significantly regulated IgM production (p < 0.05). In contrast, after the RQCJ intervention, the genes Pilrb1, Rtp4, Dhx58, Irf7, and Ifi44 were significantly downregulated, while the expression of IL-6, IFN-γ, and IL-2 was upregulated, accompanied by an increase in the CD4⁺/CD8⁺ T cell ratio (p < 0.05). Detailed results are illustrated in the heatmap.

Figure 13.

Heatmap for correlation analysis between target genes and phenotypic immune indicators, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Discussion

CTX is an anti-metabolic and pro-apoptotic chemotherapy agent used in cancer treatment. ¹² However, while exhibiting anti-tumor properties, it also inhibits normal immune cells by affecting DNA synthesis, leading to immune dysfunction. We injected mice with CTX to create a model of immunosuppression and investigated the immunomodulatory effect of the Tibetan drug RQCJ by gavage and the possible mechanism of its immunomodulatory effect through transcriptomes, which provided evidence for the clinical application of RQCJ.

Non-specific immunity is a critical component of the immune system. White blood cells, including neutrophils and lymphocytes, are essential cell types, while phagocytosis by monocyte macrophages is a standard index to measure non-specific immune function. ²⁸ Our findings indicate that RQCJ effectively restores the decline in immune cell levels caused by CTX, and the level of white blood cells in the RQCJ drug group was close to that in the control group. These findings suggest that RQCJ regulates immune function by accelerating the recovery of immune cells.

The mouse carbon clearance test is a classical experimental method that reflects non-specific immunity. The clearance index (K) measured reflects the phagocytic function of the reticuloendothelial system, while the phagocytic index (α) reflects the phagocytic ability of monocyte macrophages.^11,29 We observed that the low-dose group exhibited increased values of K and α in CTX-treated immunocompromised mice, suggesting that RQCJ improves phagocytotic function in macrophages and promotes the non-specific immune function of immunocompromised mice. It is essential to recognize that a comprehensive assessment of non-specific immunity encompasses multiple facets. In future research, we will evaluate key indicators, including neutrophil phagocytic activity and natural killer (NK) cell cytotoxicity, to further investigate the impact of RQCJ on innate immune function.

The differentiation and proliferation of immune cells contribute to an increase in immune organ mass, while a decrease in body weight and immune organ indices indicate a decline in immune function. To enhance the immune function of immunocompromised mice, the thymic index was significantly reversed in the RQCJH group, and the spleen index showed improvement in the low-, medium-, and high-dose groups of RQCJ.

CD4⁺T is also commonly referred to as helper T lymphocytes (Th),play a crucial role as regulators of both specific and non-specific immunity in the body. They assist B cells in producing antibodies and also promote the differentiation and maturation of other T cells, making them an essential class of immunoregulatory cells. In this experiment, the percentage of CD4⁺T cells in splenic lymphocytes and the CD4⁺/CD8⁺T ratio increased most significantly in the RQCJH group (P < 0.01), followed by the RQCJL group (P < 0.01) and the RQCJM group (P < 0.05). IL-2, IL-6, and IFN-γ significantly increased after RQCJ treatment except IL-6 in the RQCJM group compared with the CTX group. The increase in these cytokines stimulates the proliferation and differentiation of immune response cells and improves their immune function. The serum hemolysin level of immunocompromised mice was significantly increased in the RQCJH and RQCJL groups, and the serum IgG content was significantly greater in the RQCJM and RQCJL groups, suggesting that RQCJ promotes humoral immune function in immunocompromised mice. These results suggest that RQCJ enhances the immune function of immunocompromised mice by regulating the balance of Th1 and Th2, increasing the CD4⁺/CD8⁺T ratio, and elevating IgG and IgM. Studies have demonstrated that in the acute lung injury (ALI) model of sepsis, RQCJ exerts protective effects by regulating the renin-angiotensin system and inhibiting inflammatory factors such as IL-1β, IL-6, and TNF-α. These findings are consistent with our research, ⁹ further confirming the potential of RQCJ as an immunomodulatory agent.

The transcriptomic analysis revealed that, following intervention with various concentrations of RQCJ in immunocompromised mice, the RQCJH, RQCJM and RQCJL groups showed significant regulatory changes in 165, 7 and 10 genes, respectively, in comparison to the CTX group. Notably, one of the co-downregulated genes was Pilrb1. The functional enrichment results of the RQCJH vs. CTX group were consistent with the control vs. CTX group. After RQCJ drug intervention, RQCJH, RQCJM, and RQCJL groups were enriched with 19, 1 and 2 genes associated with immune system processes compared with the CTX group.

The gene regulation and enrichment results suggest that the RQCJH group exhibits a more potent immune regulatory effect, which is consistent with the experimental results. Pathway enrichment analysis identified several significant immune-related pathways, including primary immunodeficiency, hematopoietic cell lineage, Th17 cell differentiation, intestinal immune network for IgA production, the T cell receptor signaling pathway, Th1 and Th2 cell differentiation, and asthma involved in immune-related pathways for the Control vs. CTX group. CTX severely compromises immune regulation in mice, especially cellular and humoral immunity. After RQCJ treatment, significant changes were observed in the cytosolic DNA-sensing pathway, the TNF signaling pathway, ECM-receptor interaction, and Chagas disease in the RQCJH group. The TNF signaling pathway and ECM-receptor interaction-related enriched genes were significantly upregulated (TNF: Jag1, Gm49320, Il1b, Bcl3; ECM: Npnt, Tnxb, Fras1, Itga11). The TNF signaling pathway can enhance the expression of Jag1 by activating RBP-J (recombination signal binding protein for immunoglobulin kappa J region). This TNF-induced Jag1 expression subsequently amplifies the activity of the Notch signaling pathway. In turn, the activation of the Notch signaling pathway influences the function of the TNF signaling pathway. A study has shown that ECM-receptor interaction provides structural support for the normal physiological activities of tissue cells and plays a role in immune regulation through its rich protein components and immune active molecules in the steady-state and pathological states. ³⁰

Compared to the CTX group, 19 immune-related genes were significantly regulated in the RQCJH group, with Jag1 and Tgtp1 genes upregulated considerably. Jag1 is highly expressed on the surface of antigen-presenting cells, including dendritic cells, B cells, and macrophages, mediating dendritic cell maturation and differentiation. ³¹ Jag1 binds to its receptor and activates the Notch signaling pathway, which can induce differentiation of peripheral mature T lymphocytes. ³² Tgtp1 participates in protein sorting during antigen processing and presentation. ³³ Furthermore, the upregulation of the Tgtp1 gene may indirectly influence the activity of the TNF signaling pathway by modulating intracellular signal transduction. Compared with the CTX group, immune-related genes were significantly downregulated in the RQCJH group, partially activating the immune system. These findings suggest that RQCJ may dramatically increase and decrease with early intervention, resulting in a significant downregulation compared to the CTX group.

Furthermore, RQCJL demonstrated significant activity in reversing the effects caused by CTX. The results of the GO analysis in this study indicated that Tgtp1 was upregulated while Pilrb1 was downregulated in RQCJL. Additionally, the KEGG enrichment analysis revealed a significant enrichment of the HSV-1 infection pathway in RQCJL. Thus, we hypothesize that RQCJL may be positioned within an immune-metabolism golden window. On one hand, it may regulate key nodes in the HSV-1 infection pathway, such as cGAS-STING, thereby moderately activating the STING-TBK1 pathway and promoting autophagic flow and the interferon response.^34,35 On the other hand, it may interfere with the interactions between the immune evasion proteins encoded by HSV-1 and host factors, thereby restoring suppressed innate immunity. ³⁶ At this concentration, Tgtp1, an interferon-induced GTPase, was moderately upregulated, which aids in enhancing cell autophagy and intracellular pathogen clearance. Conversely, Pilrb1, an activating receptor, was optimally regulated to promote immune cell activation. However, increasing the concentration of RQCJ may yield the opposite effect. Research has shown that at high infection multiplicities (MOI), HSV-1 can inhibit autophagy, and neurons exhibit resistance to interferon-induced cell death at elevated concentrations. ³⁷ Therefore, an increase in drug concentration beyond a certain threshold may trigger the viral immune escape mechanism or induce negative feedback regulation in the host, ultimately resulting in decreased drug efficacy.

Further research is needed to investigate the underlying mechanism. The significant downregulation of the pilrb1 gene in the three dose groups of RQCJ, compared to the CTX group, may also be elucidated by this finding. In the RQCJM group, Rpl3-ps1, along with Cd300ld2, Tgtp1 and Ptges3-ps in the RQCJL group, exhibited significant upregulation relative to the CTX group. These genes, as well as Rtp4, Irf7, Dhx58, Il1b, Ifi44, Ifi35, and other genes may mediate the effects of RQCJ. We used QPCR to verify that the expression of these genes was consistent with the transcriptome sequencing results. The analysis of the correlation between the expression of the aforementioned immune-related regulatory genes and phenotypic immune indicators, such as T-cell subset proportions and cytokine levels, demonstrates that the regulation of these genes significantly influences the expression levels of IgM, IgG, IL-6, IFN-γ, IL-2, and the CD4⁺/CD8⁺ T cell ratio.

Limitations

The results indicate that, in comparison to other common immunomodulatory herbal medicines, RQCJ, as a compound preparation, exhibits an immunomodulatory effect that transcends mere activation or inhibition of a single pathway. Instead, it operates through a multi-component, multi-target, and multi-pathway action mode, which may confer more comprehensive advantages in immunomodulation. This study represents the first systematic evaluation of RQCJ’s comprehensive influence on T cell subsets and cytokine networks, offering a novel perspective for analyzing its complex mechanism of action and highlighting the innovative nature of this research. It is important to note that transitioning from animal experiments to clinical applications necessitates more thorough evaluations, taking into account individual differences among clinical patients. In both this study and existing toxicological research, no significant toxic reactions were observed for RQCJ, providing supportive evidence for its safety. ⁸ However, as a compound preparation, the specific immunomodulatory effects of its components and the ADME processes within the human body remain unclear. In this study, healthy Kunming mice treated with CTX were utilized to reflect the impact of the drug on the fundamental immune state; however, this does not directly indicate its therapeutic potential in disease conditions. Furthermore, notable differences in immune responses exist when compared to other inbred strains of mice and patients requiring immune intervention in clinical settings. Therefore, future research will delve deeper into these issues, optimize the administration plan for RQCJ, further evaluate its therapeutic significance, and provide a more comprehensive and accurate explanation of RQCJ’s mechanism of action. Lastly, the number of experimental animals was determined based on the fundamental requirements of pharmacological studies, without applying statistical calculations. In subsequent experiments, the number of animals should be established in accordance with calculations derived from the experimental results.

Conclusion

RQCJ enhances the immune function of immunocompromised mice by protecting the immune organs of CTX-treated mice and promoting non-specific immunity, humoral immunity, and cellular immunity. Transcriptomic analysis revealed that the underlying mechanism may regulate Jag1, Tgtp1, Rtp4, Irf7, Dhx58, Il1b, Ifi44, Ifi35, and other genes. It provides theoretical foundations for the treatment of tumors and clinical application.