Compound sophora decoction alleviates ulcerative colitis by regulating macrophage polarization through cGAS inhibition: network pharmacology and experimental validation

Network pharmacological analysis

We used the same oral bioavailability (≥30%) and drug-likeness (≥0.18) thresholds that most researchers use to screen the active components of CSD in the Traditional Chinese Medicine System Pharmacology Database (TCMSP) [24]. The corresponding gene targets of these active components were then identified in TCMSP, and the UniProt knowledge database was used to transform these targets into Homo sapiens gene symbols. We sifted through UC-related disease genes from five databases, including GeneCards, DisGeNet, PharmGkb, Therapeutic Target Database (TTD), and DrugBank. The R software and scripts generated the Venn map of disease-drug-related genes. Based on this, Cytoscape software built the CSD regulatory network for UC treatment, and the STRING database constructed a protein-protein interaction (PPI) network with a high confidence score (≥0.9), manually integrating genes associated with macrophage polarization. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R software to investigate the biological functions related to potential targets.

Preparation of lyophilized powder and complete medium for CSD

The six herbs of CSD were obtained from Wuhan Union Hospital (Wuhan, China). All voucher specimens were deposited in the Laboratory of Integrated Tradition Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) with numbers 2022011001, 2022011002, 2022011003, 2022011004, 2022011005 and 2022011006. We prepared CSD as before into a decoction [25]. The CSD decoction was stored at −80°C. The next day, we quickly placed it in a freeze-drying mechanism to produce lyophilized powder, which we sealed and stored at −20°C. One gram of lyophilized powder was dissolved into a 50 mL complete medium, stored at −80°C for a day, thawed, and centrifuged (3500 r, 10 min). The supernatant was then collected and filtered with a 0.22 μm filter to obtain a drug-containing medium with a concentration of 20 mg/mL [26].

Cell

Mouse tibiofibula were rinsed to prepare bone marrow mesenchymal cell suspension, and cells were stimulated with M-CSF (30 ng/mL) for seven days to induce their differentiation into mouse BMDMs. Cell morphology was observed seven days later, and BMDMs were identified using flow cytometry. The murine RAW264.7 macrophage cell line was purchased from WARNER and incubated at 37°C in a 5% CO₂ incubator. Transfection of ISD (2 μg/mL) with Lip3000 was used to establish an inflammation model of BMDMs. Xiang Gui et al. [15] described the preparation method of ISD. Cell viability is measured using Cell Count Kit-8 (CCK-8, Biosharp, Hefei, China).

Animals and experimental protocols

Male C57BL/6J mice (weighing 20–22 g and aged 6–8 weeks) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China); (Quality certification: SCXK (jing) 2019-0010). Mice were raised at the Experimental Animal Center of Huazhong University of Science and Technology (HUST, Wuhan, China). The mice had free access to feed and water under the specific pathogen-free (SPF) condition and were fed adaptively for five days. The mice were divided into three groups (n = 8), normal control group (Control), model group (DSS), and CSD group (14.56 g/kg). Acute colitis was induced by 3% DSS. CSD was gavaged for seven days from the start of the modeling, and the model group with the same amount of drinking water. Mice were sacrificed on the eighth day, and spleen, mesenteric lymph nodes, and intestinal tissues were collected.

Evaluation of animal inflammation

Starting from the day before modeling, we regularly monitored the weight loss, fecal morphology, blood status in stool, and activity of mice every day until the end of the experiment. The disease activity index (DAI) is calculated based on the severity of symptoms [27]. The intestinal tract of mice was measured after sacrifice, and the intestinal tissue near the anus was transected for Hematoxylin and Eosin (H&E) and Alcian Blue/Phosphoric Acid Schiff (AB/PAS) staining, which were used to assess the degree of pathological damage of intestinal tissue and goblet cell destruction, respectively.

Immunofluorescent (IF)

RAW264.7 cells were fixed with 4% paraformaldehyde after treatment and incubated overnight (4°C) with anti-iNOS (1:200) and anti-Arg1 (1:500). The next day, anti-iNOS was incubated with fluorescein isothiocyanate (FITC) fluorescence secondary antibody, while anti-Arg1 was with Cy3. After dewaxing and dehydrating the paraffin sections of intestinal tissue, the antigen was repaired with citrate buffer and sealed with goat serum. The sections were incubated overnight with anti-dsDNA-antibody (1:200). Before adding 4′,6-diamidino-2-phenylindole (DAPI), the secondary antibody was incubated for 1 h. Finally, an anti-fluorescence quencher was added, and we could observe the fluorescent expression of cells using a fluorescence microscope (Olympus, Tokyo, Japan).

Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA)

The protein concentration was determined by Bicinchoninic Acid (BCA) assay after protein extraction from mouse intestinal tissue and RAW264.7, and the dosage was determined based on the concentration. Primary antibodies include anti-cGAS (1:2000), anti-β-actin (1:1000), anti-GAPDH (1:2000), anti-α-Tubulin (1:2000,), anti-iNOS (1:1000), and anti-Arg1 (1:5000). ImageJ Software was used to quantify WB. The expression levels of IL10, CCL17, CXCL10, and TNF-α in intestinal tissues were quantified by ELISA kits in accordance with the manufacturer’s instructions.

Real-time polymerase chain reaction (PCR)

Total RNA was extracted from cells and tissues following the manufacturer’s instruction. It was then reversely transcribed into cDNA with reverse transcription reagent, and cDNA was amplified using an amplification kit. Supplementary Table 1 lists all mRNA primers.

Flow cytometry

BMDMs were stained, and mononuclear cells were isolated from the intestinal tract, spleen, and mesenteric lymph nodes. The spleen and mesenteric lymph nodes of mice were thoroughly cut up in phosphate buffer saline (PBS) solution containing 2% Bovine Serum Albumin (BSA), strained through a 70 μm strainer, and suspended after centrifugation. A digestive fluid containing Collagenase IV (2 mg/mL) and DNase-I (0.25 mg/mL) was used to digest the intestinal tissue. The digestive fluid was placed in a 38°C water bath for 40 min. Every 5 min, the lid was opened and shaken to aid digestion. Following digestion in a 1640 base medium, an intestinal lamina propria cell suspension was prepared using a 70 μm strainer.

Flow cytometry antibodies, including BV510-conjugated anti-FVD antibody, APC-Cy7-conjugated anti-CD45 antibody, PE-conjugated F4/80 antibody, PC-CY5.5-conjugated CD11b antibody, BV421-conjugated CD11C antibody, and APC-conjugated CD206 antibody were incubated at 4°C for 20 min against light. The stained cells were analyzed by flow cytometry after washing.

Intestinal barrier function assays

The abdominal hair of mice was removed after 1% pentobarbital anesthesia. After fasting for 24 h and keeping mice away from light, FITC-Dex (average molecular weight: 4000, 0.6 mg/g) was administered intragastrically. Fluorescence intensity (excitation 490 nm/emission 535 nm) was measured in 4 h. The 80 μL of serum was taken and then diluted in a ratio of 1:1 with PBS solution. The FITC-Dex content in serum was measured using a fluorescent enzyme label according to the manufacturer’s instructions.

Statistical analysis

The data were presented as mean ± standard deviation (Mean ± SD), with n representing the number of independent experiments. The unpaired two-tailed Student t-test was used to analyze data from two groups. Datasets including data from more than two groups were analyzed with One-way ANOVA with Bonferroni post hoc test, and only results with p < 0.05 were considered statistically significant. GraphPad Prism 8 software (version 8) was used for statistical analysis.

Data availability statement

Data will be made available on request.

Screening the components of CSD in the treatment of UC and macrophage polarization-related targets and signaling pathways

A total of 165 active components were screened from six TCMs of CSD in TCMSP, and eight of them were present in multiple medicines, with quercetin being present in four TCMs (Ku shen, Di yu tan, San qi, and Gan cao), indicating that quercetin was one of the main active components of CSD (Supplementary Table 2). Potential targets corresponding to these active components in TCMSP, in combination with 3211 UC-associated targets in five disease gene databases (Figure 1A), resulted in the identification of 174 UC-CSD-associated targets (Figure 1B). The Cytoscape software was used to establish the compound-target relationship network (Figure 1C).

Figure 1. Pharmacology analysis of CSD. (A) Venn diagram of UC-associated genes in five databases; (B) Venn diagram of CSD target genes and differentially expressed genes in UC; (C) The CSD–active compound–target network; (D) Genes associated with macrophage polarization in PPI network; (E) GO analysis of CSD-targeted genes (p < 0.05); (F) KEGG analysis of CSD-targeted genes.

A PPI network was obtained after importing 174 genes into STRING (Figure 1D), and the targets associated with macrophage polarization were manually labeled. The genes associated with M1 macrophage polarization included NOS2 (iNOS), CXCL10, TNF-α, IL-6, CCL5, CXCL8, CXCL2, and CXCL11. Whereas IL10, VEGF-α, IL-1β, and CCL2 were the genes associated with M2 macrophage polarization (Figure 1D). We performed GO and KEGG analysis on the target genes to further investigate the mechanism of CSD in treating UC. The results revealed that these genes were enriched in the following aspects: oxidative stress response, lipopolysaccharide response, IL-17 signaling pathway, TNF-α signaling pathway, and IBD. The first ten items of biological processes (BP), cell components (CC), and molecular functions (MF) that were significantly enriched in GO analysis were displayed (Figure 1E), as were the first 30 items of KEGG analysis (Figure 1F). The therapeutic effect of CSD on UC was reflected in the regulation of inflammation and oxidative stress.

Inducing BMDMs successfully and determining the intervention concentration of CSD in vitro

BMDMs and RAW264.7 cells were used for experiments to study in vitro macrophage polarization. First, identifying the induced BMDMs using the microscope on the seventh day of induction, the cell morphology was fusiform with two open ends (Figure 2A). Flow cytometry revealed that the proportion of CD11b+F4/80+ cells reached 99.99% (Figure 2B), indicating successful induction of BMDMs [28]. Count Kit 8 (CCK8) test was then performed on RAW264.7 cells to test cell viability. The findings revealed that CSD did not significantly reduce the cell viability at 0.25 mg/mL and 0.5 mg/mL for 6 h after intervention (Figure 2C), nor did it affect the cell viability in BMDMs (Figure 2D). Therefore, 0.25 mg/mL was selected as the low-dose CSD (CSDL) group and 0.5 mg/mL as the high-dose (CSDH) group.

Figure 2. Inducing BMDMs successfully and determining the intervention concentration of CSD in vitro. (A) Morphology of BMDMs at the seventh day of induction, 100X magnification; (B) Flow cytometry figure of BMDMs identification; (C) CCK8 assay of RAW264.7 cells interfered by CSD for 6 h; (D) CCK8 determination of CSD intervention in BMDMs for 6 h. The data represent the mean ± SD of three independent experiments.

CSD restoring macrophage balance in vitro

In vitro experiments were performed first to confirm the effect of CSD on macrophage polarization. iNOS is an M1 macrophage marker, while Arg1 is an M2 macrophage marker [29]. After ISD stimulation, the level of iNOS mRNA increased while Arg1 mRNA decreased in RAW264.7 cells. However, the addition of CSD intervention could reverse this effect, particularly at a concentration of 0.5 mg/mL (Figure 3A, 3B). The expression of iNOS and Arg1 in cell fluorescence assay was identical (Figure 3C, 3D). Flow cytometry also revealed that the M2/M1 ratio of BMDMs decreased after ISD induction but increased after CSD addition (Figure 3E, 3F). These findings suggested that CSD could regulate ISD-induced macrophage polarization in vitro.

Figure 3. CSD restores macrophage balance in vitro. (A) iNOS mRNA levels in RAW264.7 cells; (B) Arg1 mRNA levels in RAW264.7 cells; (C) Fluorescence expression of iNOS in RAW264.7 cells, 100X magnification; (D) Fluorescence expression of Arg1 in RAW264.7 cells, 100X magnification; (E) Flow cytometry diagram of M1 and M2 in BMDMs; (F) M2/M1 cell ratio in BMDMs. The data represented as mean ± SD of three independent experiments. Abbreviation: ns: no significance. ^*p < 0.05, ^***p < 0.001 vs. ISD group.

CSD inhibits cGAS in macrophages and regulates cellular inflammation in vitro

We detected the expression of cGAS, a protein associated with macrophage polarization, to further explore the mechanism by which CSD regulates macrophages. Findings of WB and PCR revealed that ISD induced the expression of cGAS protein and mRNA in RAW264.7 macrophages, and the expression decreased after CSD intervention (Figure 4A–4C). IFN-β is an inflammatory factor in the complex downstream pathway of cGAS [30], and its mRNA level is correlated with cGAS expression (Figure 4D). The mRNA levels of CCL17, IL10, TNF-α, and CXCL10 were used to assess ISD-induced macrophage inflammation. The results revealed that ISD decreased the mRNA levels of anti-inflammatory factors CCL17 and IL10 while increasing the inflammatory factors TNF-α and CXCL10, and CSD changed the outcome (Figure 4E–4H). These findings imply that CSD may regulate macrophage polarization and inhibit inflammation by inhibiting cGAS.

Figure 4. CSD inhibits cGAS in macrophages (RAW264.7) and regulates cellular inflammation in vitro. (A) Representative western blot of cGAS; (B) Statistical map of cGAS protein content; (C) cGAS mRNA levels; (D) IFN-β mRNA levels; (E) CCL17 mRNA levels; (F) IL10 mRNA levels; (G) TNF-α mRNA levels; (H) CXCL10 mRNA levels. The data present the mean ± SD of three independent experiments. Abbreviation: ns: no significance. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. ISD group.

CSD alleviated symptoms and intestinal damage in UC mice

A DSS-induced colitis model was established to study the effects of CSD on the symptoms and pathological changes of UC mice, and CSD was given by gavage on this basis. CSD treatment of DSS-induced UC in mice reduced intestinal bleeding, stabilized stool traits, and increased body weight (Figure 5A). It decreased cumulative (DAI) score (Figure 5B). Inflammation can shorten the colon. The colon length increased significantly after CSD treatment compared to the DSS group (Figure 5C, 5D). In DSS-induced mice, HE staining revealed intestinal pathological injury, intestinal structure destruction, and increased inflammatory infiltration (Figure 5E). The observation of changes in goblet cell number was an important morphological basis for intestinal structural changes [31]. AB/PAS staining demonstrated a depletion in the number of intestinal goblet cells in the DSS group compared to the control group. However, this depletion was reduced after CSD intervention (Figure 5F). These findings imply that CSD could alleviate the progression of DSS-induced UC.

Figure 5. CSD alleviated symptoms and intestinal damage in UC mice. (A) Changes in body weight of mice; (B) Images of the colons of mice; (C) Statistical map of colon length in mice; (D) DAI score of mice; and (E) Mouse intestinal HE staining. (F) Mouse intestinal PAS staining. The results are mean ± SD, n = 6–8, ^**p < 0.01, ^***p < 0.001 vs. DSS group.

CSD decreased the inflammatory phenotype and increased the anti-inflammatory phenotype of macrophages in UC mice

The spleen, mesenteric lymph nodes, and intestine are all important immune system components, and the UC lesion site is in the intestine. Therefore, we focused on detecting macrophage polarization in these sites to investigate the effect of CSD on UC mice. Compared to the control group, DSS could induce macrophages to differentiate into inflammatory phenotypes (M1), and M2/M1 decreased, whereas CSD significantly increased this ratio (Figure 6A–6D). We measured the protein and mRNA levels of iNOS and Arg1 in intestinal tissues, indicating that CSD could lower the content of iNOS while increasing Arg1 in intestinal tissues after DSS-induced UC (Figure 6E–6I). These findings suggest that CSD regulates DSS-induced macrophage polarization in UC mice.

Figure 6. CSD-regulated macrophage polarization in vivo. (A) Flow cytometry diagram of M1 and M2 in the spleen, mesenteric lymph nodes, and intestine of mice; (B) M2/M1 ratio of spleen in mice; (C) M2/M1 ratio of mesenteric lymph nodes in mice; (D) M2/M1 ratio of intestine in mice; (E) Representative western blot of iNOS and Arg1 in intestine; (F) Statistical map of iNOS protein content in intestine; (G) iNOS mRNA levels in intestine; (H) Statistical map of Arg1 protein content in the intestine; (I) Arg1 mRNA levels in intestine. The results are mean ± SD, n = 3–6, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. DSS group.

CSD inhibited the expression of cGAS and the content of inflammatory factors in the intestinal tissues of UC mice

As previously stated, ISD could activate cGAS to induce inflammatory tendencies in macrophages. Therefore, we were interested in whether DSS could influence changes in intestinal inflammation levels in vivo by inducing cGAS expression. WB and PCR results revealed that cGAS expression was increased in the DSS group compared to the control group, and the relative content of IFN-β mRNA was increased, which could significantly inhibit their expression after CSD administration (Figure 7A–7D). The levels of inflammation-related factors in the intestine were measured using ELISA and PCR. Compared with the normal group, CCL17, and IL10 expression decreased in the DSS group, and CSD treatment significantly increased their levels (Figure 7E, 7F, 7I, 7J). However, TNF-α and CXCL10 increased significantly after DSS induction, and CSD could significantly reduce this result (Figure 7G, 7H, 7K, 7L). These findings suggest that CSD could regulate DSS-induced macrophage polarization in UC mice by inhibiting cGAS and alleviating intestinal inflammation.

Figure 7. CSD inhibited the expression of cGAS and the content of inflammatory factors in UC mice intestinal tissues. (A) Representative WB of cGAS in the intestine; (B) Statistical map of cGAS protein content in the intestine; (C) cGAS mRNA levels in the intestine; (D) IFN-β mRNA levels in the intestine; (E) ELISA measured the content of CCL17 in the intestine; (F) ELISA measured the content of IL10 in the intestine; (G) ELISA measured the content of TNF-α in the intestine; (H) ELISA measured the content of CXCL10 in the intestine; (I) CCL17 mRNA levels in intestine; (J) IL10 mRNA levels in intestine; (K) TNF- α mRNA levels in intestine; (L) CXCL10 mRNA levels in intestine. The results are mean ± SD, n = 3–6, ^*p < 0.05, ^***p < 0.001 vs. DSS group.

CSD protects the intestinal barrier

The dsDNA could activate cGAS, initiating IFN-β transcription [30]. Studies in DSS-induced UC mice revealed that the content of dsDNA in intestinal tissues increased, possibly due to DNA damage of intestinal epithelial cells (IEC) [32, 33]. We assumed that DSS-induced cGAS activation in UC mice was related to increased dsDNA after intestinal injury. Therefore, immunofluorescence was used to examine dsDNA expression in intestinal tissues. As expected, dsDNA expression was significantly higher in the DSS group than in the normal group and significantly down-regulated after CSD treatment (Figure 8A). This suggests that CSD may reduce DNA damage in intestinal cells, such as intestinal epithelial cells, which are important for protecting the intestinal barrier. Therefore, live imaging of small animals and the text of Fluorescein isothiocyanate-dextran (FITC-dex) in serum were used to detect intestinal barrier function in mice. In the DSS group, intestinal fluorescence intensity was higher than in the normal group, and serum FITC-dex content significantly increased. In contrast, it was down-regulated after CSD treatment (Figure 8B, 8C). These findings imply that CSD protects the intestinal barrier and may play a role in DNA repair in intestinal cells.

Figure 8. CSD protected the intestinal barrier. (A) Fluorescence expression of dsDNA in intestinal tissue, 100X magnification; (B) FITC-dextran distribution in the intestinal tract of mice; (C) Serum FITC-Dex levels. The results are mean ± SD, n = 3, ^**p < 0.01, ^***p < 0.001 vs. DSS group.