Thymoquinone mediated hedgehog pathway activation and gut microbiota modulation in mouse colitis

Abstract

To investigate the treatment of ulcerative colitis by thymoquinone (TQ) through the Hedgehog signaling pathway and the composition of intestinal microbiota. A total of 24 male Balb/C mice were randomly divided into blank group, model group, thymoquinone low-dose group (20 mg/kg) and thymoquinone high-dose group (80 mg/kg), with 6 mice in each group. The concentrations of IL-1β, TNF-α, IL-10 and TGF-β in the supernatant of mouse serum were determined by enzyme-linked immunosorbent assay, and the expressions of Shh, Smo, Gli1, Ptch1, Atresin -1 (ZO-1) and Occludin in the mouse colon were detected by RT-qPCR, Western blot and immunohistochemistry. RNA-seq was used to detect changes in the intestinal microbial community of the four groups at the same time. TQ slowed down body weight, reduced disease activity index, promoted the expression of Shh, Smo, Gli1, Ptch1, Zo-1 and Occuldin in the colon, and increased the phylum levels of Bacteroidota, Campylobacterota, Desulfobacterota, Verrucomicrobiota, Deferribacterota and the abundances of Bacteroidaceae, Muribaculaceae, Akkermansiaceae, Deferrribacteraceae, Staphylococcaceae at the family level, absent from the abundance of Proteobacteria at the phylum level and Enterobacteriaceae at the family level, and inhibited the expression of IL-1β, TNF-α and TGF-β in peripheral blood, and promoted the expression of IL-10 in peripheral blood. Thymoquinone may play a role in the treatment of ulcerative colitis by upregulating Hedgehog and influencing the composition of gut microbiota.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-10892-4.

Introduction

Ulcerative colitis¹ can be seen at any age, most commonly in the 20 s and 40 s, and in recent years there has been a significant increase in the global incidence² and the rate of proctocolectomy has been increasing annually. The lesions are mainly confined to the mucosa and submucosa of the rectum and colon and are susceptible to cancer. Diarrhea, mucopurulent blood stools and abdominal pain are the main symptoms of ulcerative colitis. Conventional treatment with aminosalicylic acid, glucocorticoids, and immunosuppressive drugs is less effective, has more side effects, and is prone to drug resistance, which increases the pain of patients and the burden on society. Therefore, it is very effective to find a safe and effective medication to treat ulcerative colitis. Thymoquinone is a black seed extract³, which is the main active active monomer in black seed herb, it has antioxidan⁴, antibacteria⁵, anti-inflammatory⁶,hypoglycemic,hypolipidemic,antiasthmatic,immunomodulatory, neuroprotective, antimelancholic, hair growth promotion, hypoglutination and emmenagogue, and diuretic.It has been shown that thymoquinone significantly improves the survival and climbing ability of Drosophila melanogaster in the UC model induced by dextran sodium sulfate ,DSS, and improves the morphology of the adult intestinal tract and the damage of the intestinal wall caused by DSS exposure⁷. Thus, TQ is effective in the treatment of ulcerative colitis.The Hedgehog signaling molecule is a focal protein ligand secreted by signaling cells, and there are three homologs of Hedgehog in mammals:SonicHedgehog (Shh)⁸, IndinHedgehog (Ihh)⁹, and DesertHedgehog (DHH), coded Shh, Ihh, and Dhh proteins, respectively. Among them, SonicHedgehog (Shh) is the most widely studied, which mainly consists of the secreted protein Shh ligand, the transmembrane protein receptors Ptch and Smo and the downstream transcription factor Gli¹⁰ protein. Hedgehog signaling pathway transduction plays a critical role in maintaining intestinal homeostasis and is closely associated with intestinal inflammation, tissue repair, and tumor¹¹ development. In the Hedgehog signaling pathway, Shh (Sonic Hedgehog) is a key ligand for initiating signals, and Gli1 is an important transcriptional activator downstream of the pathway. Upon binding of Hh to Ptc in the presence of Hh ligand, the interaction of Hh ligand with Ptc promotes the internalization and degradation of Ptc, thereby relieving the inhibition of Smo by Ptc¹². Phosphorylation of Smo by GRK2 and CK1 induces a conformational change, followed by the entry of Smo into the primary cilium, causing it to accumulate within the primary cilium.Inside the cilium, activated Smo promotes the dissociation of the Sufu-Gli complex¹³, followed by the translocation of Gli-FL into the nucleus.In the nucleus, Gli-FL is modified into a transcriptional activator, Gli-A, which activates Hh target gene transcription. Thymoquinone acts through the Hedgehog signaling pathway, whereas Shh proteins play an important role in intestinal epithelial renewal and act as paracrine signals expressed in the mesenchyme during intestinal development in ulcerative colitis consistent with the role in Zhuo Xie et al.¹⁴. The aim of this study was to investigate the therapeutic effects of thymoquinone upregulating the Hedgehog signaling pathway and affecting the composition of the intestinal flora in a mouse model of DSS-induced ulcerative colitis. As a classical inflammatory signaling pathway, it has been demonstrated that the Hedgehog¹⁵ signaling pathway and the development of ulcerative colitis are closely related. In this study, we aimed to investigate the role of thymoquinone in the treatment of ulcerative colitis and related mechanisms.

Materials

Twenty-four male SPF grade 6–8 week old BALB/c mice weighing 18–20 g. The mice in the experimental group were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shandong, China), animal license number: SCXK (Henan) 2019-0003, and the mice were placed at room temperature (the temperature was about 25°, 12 h light/dark cycle).All experiments involving animals were approved by Bengbu Medical UniversityExperimental animal management and ethics committee(No. 2021336), and were carried out according to the guiding principles of “animal research: in vivo experiment report” (ARRIVE). Recognizing that all experiments were conducted in accordance with relevant guidelines and regulations.

The reagent consumables thymoquinone was purchased from Maclean’s Biotechnology Co., Ltd. (Shanghai), DSS (MW: 36,000–50,000) was provided by mpbio (USA) Co., Ltd., the mouse polyclonal antibody Gli1 and rabbit polyclonal antibody Shh were purchased from Wuhan Mitaka Biotechnology Co., Ltd., the rabbit polyclonal antibody Ptched and Smo were purchased from Shanghai Thermo Fisher Scientific (USA), and the primary antibody diluent was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The ultra-sensitive ECL chemiluminescence kit was ordered from Heyuan Lee Kee, and the goat anti-rabbit IgG-horseradish peroxidase secondary antibody was purchased from abways technology, the ELISA kit was provided by Quanzhou Ruixin Biotechnology Co., Ltd., the Trizol reagent was purchased from Thermo Fisher Scientific (USA), the RIPA lysate was purchased from Shanghai Biyuntian Biotechnology, the PCR reverse transcription kit was purchased from Novoprotein, and the polymerization fluorescence quantitative reagent was purchased from Zhengzhou Xuanyuan Biotechnology Co., Ltd. The electrophoresis and transfer equipment was purchased from BIO-RAD in the United States.

Methods

Experimental design

Twenty-four mice were randomly divided into a normal group, a model group, a thymoquinone low-dose group (20 mg/kg), and a thymoquinone high-dose group (80 mg/kg), with six mice in each group. The model of ulcerative colitis was established by drinking 3% DSS ad libitum for seven consecutive days in all groups except the normal group, and the thymoquinone low-dose and high-dose groups were gavaged with thymoquinone suspension starting on the second day of drinking DSS ad libitum. The normal and model groups were gavaged with pure water. On the seventh day, UC-related symptoms appeared in the model group, the thymoquinone low-dose group and the high-dose group , respectively, and the mice were withdrawn from the feed and bedding, and were fasted from food and water. On the second day, mice were anesthetized by intraperitoneal injection with ketamine (100 mg/kg)¹⁶, in addition, euthanasia with CO2 to reduce pain in mice. Firstly, blood was taken from the eyeballs for double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and secondly, mice were dissected to remove the colon for collection of feces, the upper section was used for protein extraction for western blotting experiments, the RNA was extracted from the middle section of the colonic tissues and subjected to RT-qPCR, and the lower section of the colons were The lower colon was used for pathology related tests such as (H&E) staining and immunohistochemistry, and the feces was used for microbial community analysis.

Disease Activity Index (DAI): The disease activity index (DAI) was observed and recorded each day of the mice, and the disease activity index score was consistent with the Lei Zhu report¹⁷ and the scoring criteria were implemented according to Table 1.

Table 1.

Disease activity index.

计分	Weight loss (%)	Fecal traits	Blood in the stool
0	no	normal	阴性
1	1–5	Semi-loose stools	+
2	6–10	Semi-loose stools	++
3	11–15	Loose stools	+ ++
4	> 15	Loose stools	++++

Macroscopic assessment of the degree of colon injury: the removed colon is rinsed with normal saline, and the edema of the colon and intestinal wall is observed with the naked eye, whether there is an ulcer in the colon and intestinal wall and the size of the ulcer. The colonic mucosal damage index (CMDI) was scored according to Tejal Gandh¹⁸, and the scoring criteria were shown in Table 2.

Table 2.

Colonic mucosal injury index score.

Type of injury	description	score
	There is no visible injury	0
Macroscopic damage	Mild hyperemia, edema	1
	Moderate erosions or ulcers	2
	Severe mucosal defects or necrosis	3
	The crypt is structurally normal	0
Microscopic damage	Slight changes in crypt structure	1
	Moderately altered crypt structure	2
	Severe changes in crypt structure	3

Histopathologic scoring of the colon

The colonic tissues of mice were subjected to Hematoxylin and Eosin staining (H&E) and then scored according to the report of Fumiaki et al. team¹⁹.

Enzyme-linked immunosorbent assay

After anesthetizing the mice with sodium pentobarbital, the eyeball blood was removed and allowed to stand at room temperature for 30 min, and then centrifuged in a 1200×g 4° centrifuge for 15 min, the precipitate was discarded, and the supernatant was measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to measure the concentrations of IL-1β, TNF-α, IL-10 and TGF-β in the supernatant of the serum of mice in each experimental group.

Western blot

The protein in the colon tissue was extracted and its concentration was measured with a BCA kit, the same amount of protein was electrophoresed and transferred to a PVDF membrane, then blocked with 0.5% skim milk, the primary antibody was incubated overnight in a four-degree freezer, the secondary antibody was incubated at room temperature for 1 h the next day, and finally the copy image was exposed and developed.

RT-qPCR

RT-qPCR was used to detect the genes in Hedgehog pathway and the expressions of Zo-1 and Occuldin genes in colon tissues. RNA in colon tissue was extracted with Trizol and its concentration was determined, and cDNA was reverse transcribed into cDNA by a two-step method using the novoprotein transcription kit, and detected by quantitative PCR using SYBR Green PCR Master Mix (Takara) on a 96-well plate.

Lower intestinal tissue hematoxylin–eosin (H&E)

Fragments of the previously described lower intestinal tissue samples were fixed in 4% buffered formalin, deparaffinized, stained, dehydrated, permeabilized, and mounted to make intestinal tissue sections. Observations and image acquisition.

Immunohistochemistry

The tissue in 4% buffered formalin was fixed and made into wax block sections, and then deparaffinized, antigen retrieval, blocked incubation, primary antibody overnight, incubated with secondary antibody, and the results were observed and images were acquired after counterstaining.

Analysis of intestinal microbial communities

The fecal samples of mice in each experimental group were collected for 16SRNA sequencing, and the sample preparation, DNA extraction, PCR product acquisition, mixing and purification were carried out first, and the library and microbial analysis were constructed.

Statistical analysis

SPSS26.0 statistical software for data analysis, experimental data are displayed in the form of mean + standard deviation, when the number of experimental groups is greater than 2 and in line with the normal distribution, variance chi-square, using one-way analysis of variance (one-way ANOVA) with which want to the opposite is used Mann–Whitney U test. The difference was considered statistically significant at P < 0.05.Results.

TQ slows the weight loss of DSS-induced ulcerative colitis

In this study, a model of ulcerative colitis was established, and we treated it with 3% DSS for 7 days to observe changes in body weight growth and stool status in mice. Among them, diarrhea, mucus, pus and blood were the main epigenetic features of the model, and the increase in colonic epithelial damage and the production of inflammatory cytokines in mice were microscopic manifestations. After the third day of modeling, diarrhea occurred in the DSS group, bloody stools appeared on the fifth day, and most of the mice above the seventh day had bloody stools. Compared with the normal group, the body weight of mice in the model group decreased significantly (Fig. 1A) and DAI was progressively increased (Fig. 1). Compared with the model group, the degree of weight loss was slowed down after thymoquinone administration (Fig. 1), and the rise of DAI was slow (Fig. 1).

Fig. 1.

Changes in body weight and DAI in mice. Note: (A) Percentage of body weight of mice in each group relative to initial value; (B) DAI score of mice in each group; Data were expressed in Mean ± SD, n = 6, and the AB group compared with the model group at the same time point, * P < 0.05, ** P < 0.01.

Thymoquinone can alleviate inflammation and damage of colonic mucosa in mice

From the anatomy of the colon of the mice, it can be seen that the model group has severe bleeding, extensive edema and hemorrhage in the intestinal wall (Fig. 2A). HE staining showed that the crypts in the control group were regular, while the mice in the model group had a disordered crypt arrangement, with a certain amount of erythrocyte exudation and inflammatory cells (plasma cells, lymphocytes, eosinophils and neutrophils) in the interstitium, (Fig. 2C). After high-dose and low-dose TQ treatment, the anatomical mucosal edema of the colon was improved compared with the model group, and the ulcer lesions were reduced or disappeared. Following HE staining, compared with the model group, the thymoquinone-treated group exhibited alleviated colonic mucosal injury, reduced inflammation, decreased inflammatory cell infiltration, fewer cases of cryptitis and crypt abscesses, and increased goblet cells. After modeling, the model group displayed a significantly lower CMDI score (compared with the normal group) and a shortened colon length (Fig. 2B) (P < 0.001) and the HI score increased (Fig. 2D) (P < 0.001).

Fig. 2.

Changes in anatomical lateral and gross scoural scores, HE staining (100X) and histological scores of mice colon. Note: (A,B) Anatomical view of the colon and colonic mucosal injury index scores of mice in each group; (C,D) HE staining and colon histopathological score of colon tissue of mice in each group; Data were expressed as Mean ± SD, n = 6, and compared with the model group, * P < 0.05, ** P < 0.01, *** P < 0.001.

Thymoquinone can reduce IL-1β, TNF-α, TGF-β and increase the concentration of IL-10 in the serum of DSS-induced ulcerative colitis mice.

In the current study, we tested the concentration of thymoquinone inflammatory cytokines in a mouse model established for DSS-induced ulcerative colitis (Fig. 3). Compared with the normal group, the serum levels of IL-1β, TNF-α α and TGF-α β β in the model group increased significantly while the concentration of IL-10 decreased (Fig. 3A–D) (P < 0.001). The concentration of TGF-β was lower than that of the low-dose thymoquinone, while the concentration of IL-10 was higher than that of the low-dose of thymoquinone (Fig. 3A–D) (P < 0.001), and was within a certain concentration range, These effects of thymoquinone increase in a concentration-dependent manner. These results indicated that thymoquinone exerted anti-inflammatory effect by inhibiting the activity of pro-inflammatory factors such as serum IL-1β, TNF-α, and TGF-β and increasing the activity of anti-inflammatory factors such as IL-10.

Fig. 3.

Expression of cytokines in serum of mice in each group. Note: (A) TNF-α expression level in the serum of mice in each group; (B) Serum IL-1β expression level in mice in each group; (C) TGF-β expression level in serum of mice in each group; (D) Serum IL-10 expression level in mice in each group; Data were expressed in Mean ± SD, n = 6, and compared with the model group, *P < 0.05, **P < 0.01, *** P < 0.001.

Thymoquinone can enhance the expression of Shh, Gli1, Smo, Ptch11, Zo-1 and Occuldin genes in ulcerative colitis mice.

RT-qPCR was used to detect the expression of Hedgehog pathway genes Shh, Gli1, Smo, Ptch1, and atresin -1 (ZO-1) and occludin (Occludin) (Fig. 4). The expression of Shh, Gli1, Smo, Ptch1, Zo-1 and Occuldin genes in the model group was lower than that in the control group (Fig. 4A–F) (P < 0.001). Compared with the model group, the expression of Shh, Gli1, Smo, Ptch1, Zo-1 and Occuldin genes was increased in the high- and low-dose thymoquinone groups. Within a certain range, the expression of thymoquinone in the high-dose group was higher than that in the low-dose group (Fig. 4A–F) (P < 0.001).

Fig. 4.

Expression of genes related to Hedgehog signaling pathway in colon tissues of mice in each group. Note: (A) Relative expression of Shh gene in colon tissue of mice in each group, (B) Relative expression of Smo gene in colon tissue of mouse in each group; (C) Relative expression of Gli1 gene in colon tissues of mice in each group; (D) Relative expression of Ptch1 gene in colon tissues of mice in each group; (E) Relative expression of Zo-1 gene in colon tissues of mice in each group; (F) Relative expression of Occludin gene in colon tissues of mice in each group; The data were expressed in Mean ± SD, n = 6; Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

Thymoquinone has a therapeutic effect on ulcerative colitis in mice and can restore the intestinal mucosal barrier.

Expression of Shh, Gli1, Smo, Ptch1, Zo-1 and Occuldin in the Hedgehog Signaling Pathway were detected by Western Blot (Fig. 5A). Compared with the normal group, the expression of Hedgehog-related proteins Shh, Gli1, Smo, Ptch1 and intestinal tight junction proteins Zo-1 and Occuldin in the colon was significantly down-regulated (Fig. 5B–G) (P < 0.05). Compared with the model group, the expression of proteins Shh, Gli1, Smo, Ptch1, Zo-1 and Occuldin was increased in the high-dose and low-dose thymoquinone groups, and the expression in the high-dose thymoquinone group was higher than that in the low-dose thymoquinone group (Fig. 5B–G) (P < 0.05).

Fig. 5.

Expression of Hedgehog signaling pathway-related proteins in colon tissues of mice in each group. Note: (A) Western blot bands of four proteins related to Hdgehog signaling pathway (Shh, Smo, Gli1, Ptch1) and intestinal tight junction proteins (Zo-1, Occuldin) in the colon tissues of each group of mice; (B–G) Western blot was used to detect the expressions of Hedgehog signaling pathways Shh, Smo, Gli1, Ptch1 and intestinal tight junction proteins Zo-1 and Occuldin in mice in each group. The data were expressed in Mean ± SD, n = 6; Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

In ulcerative colitis mice, thymoquinone can alleviate colonic injury

From the immunohistochemistry diagram (Fig. 6), it can be seen that Shh, Smo, Gli1 and Ptch1 were strongly positive expression (++++) in the normal group (Fig. 6. Shh, Smo, Gli1 and Ptch1), low or no expression (+/−) in the model group (Fig. 6. Shh, Smo, Gli1 and Ptch1), weak positive expression (+~ ++) in the low-dose thymoquinone group (Fig. 6. Shh, Smo, Gli1 and Ptch1), and positive or strong positive (++ ~ +++) in the high-dose thymoquinone group (Fig. 6. Shh, Smo, Gli1 and Ptch1).

Fig. 6.

Expression of Hedgehog signaling pathway protein in colon tissues of mice in each group.

Thymoquinone ameliorates ulcerative colitis in mice by alleviating intestinal dysbiosis and upregulating the Hedgehog signaling pathway.

Variation of microbial structure of community in subgroups by 16S RNA sequencing(Fig. 7).The feces of mice were collected in the experimental group and the average number of operational taxona (OUT) and OUT in the normal group, model group, and thymoquinone high-dose group in the gut microbiome were detected by 16SRNA sequencing and visualized by Venn plot (Fig. 7A,B), There were 436 unique communities in the normal group, 242 communities in the model group, 227 unique bacterial communities in the thymoquinone high-dose group, and 374 communities in the three groups. Principal coordinate analysis (PCOA) and non-metric multidimensional (NMDS) on community similarities (Fig. 7C,D) are shown, Compared with the normal group, the model group greatly affected the composition of microorganisms, and the intestinal microbial spectrum changed significantly after thymoquinone treatment, and the abundance of Bacteroidota in the model group decreased and the abundance of Proteobacteria increased in the model group compared with the normal group at the gate level, and after the administration of thymoquinone, the Bacteroidota increased in the high-dose group and decreased in the high-dose group. In addition, we also observed that some neutral bacteria in the thymoquinone high-dose group had higher abundances of Campylobacterota, Desulfobacterota, Verrucomicrobiota, and Deferribacterota than in the model group (Fig. 7E,F). At the family level, compared with the normal group, the abundance of Muribaculaceae, Akkermansiaceae, and Rikenellaceae was observed in the model group, and the abundance of Helicobacteraceae and Enterobacteriaceae microflora increased. The abundance of Deferrribacteraceae and Staphylococcaceae increased, while the abundance of Enterobacteriaceae decreased (Fig. 7G,H). Additionlly, the linear judgment analysis (LDA) effect size (LEfSe) showed that the abundance of Proteobacteria phylum and Enterobacteriaceae family in the model group increased, and the changes of intestinal microbiota induced by DSS were significantly inhibited after thymoquinone treatment. In addition, thymoquinone administration increased the abundance of bacteroidota in ulcerative colitis mice, but slightly lower than that in the normal group (Fig. 7I,J).

Fig. 7.

Effect of thymoquinone on the intestinal microbiota of mice with ulcerative colitis. Note: (A,B): Veen plot for visual analysis of the community in each group of mice, (C) principal coordinates (PCoA) in each group of mice; (D) Non-metric multidimensional calibration analysis (NMDS) in mice in each group; (E,F): community structure of microbial communities at the phylum level in mice in each group; (G,H): community structure of microbial communities at the family level in mice in each group; (I,J): Linear discriminant analysis Effect Size (LEfSe) analysis of intestinal microbial communities in each group.

Discussion

Inflammation is a kind of defensive reaction caused by injury factors²⁰, and the central part of the inflammatory process is the vascular reaction. The causes of inflammation usually include biological factors such as viral infection²¹, physical factors such as high temperature, chemical factors such as strong acids, necrotic tissues, foreign objects such as metal fragments, and abnormalities of immune response such as ulcerative colitis, and so on. Inflammation causes redness, swelling, heat, pain and dysfunction in living tissue.

Ulcerative colitis is a chronic inflammatory bowel disease²², and the treatment of ulcerative colitis is aimed at restoring intestinal function, reducing recurrence, and avoiding the development of cancerous lesions. Currently, the main drugs used in the treatment of ulcerative colitis include aminosalicylic acid preparations, glucocorticoids, and immunosuppressants²³, which not only cannot cure ulcerative colitis but also have high side effects. Therefore, finding a safe and effective medication is necessary for the treatment of ulcerative colitis. Traditional Chinese medicine is also a classic prescription for the treatment of ulcerative colitis, and in recent years, the treatment of ulcerative colitis by traditional Chinese medicine has aroused great interest among researchers, and the typical ones include clearing the intestines and removing the dampness tablet²⁴, compounding the bitter ginseng colonic capsule²⁵, curcumin²⁶, madecassic²⁷, and glaucoma hydrochloride²⁸, and so on. The treatments include herbal oral treatments, herbal enema treatments, acupuncture treatments, and moxibustion treatments, etc. These medicines are characterized by safety, effectiveness, and fewer adverse effects, so the research on herbal medicines has become a great hotspot in the current research, and our point of view focuses on the active substance thymoquinone, which is extracted from the Nigella sativa.

Nigella sativa is an annual herb of the genus Nigella in the buttercup family, native to southern Europe. In China, it is cultivated in some cities, such as Yunnan and Xinjiang, where it is known as Hak Jung Chou²⁹ and can be used as a spice and seasoning, and the black seedpod can also be used to treat a variety of ailments³⁰ such as skin disorders, conjunctivitis, jaundice, rheumatism, high blood pressure, paralysis, diabetes, amenorrhea, and bronchitis. Studies have shown that thymoquinone has strong anti-inflammatory, antioxidant, anticancer and antimutagenic effects. Thymoquinone has been shown to treat neurogenic inflammation behind migraine³¹, antioxidant, and inhibit colon cancer³², pancreatic cancer³³, and bladder cancer³⁴, etc. Currently, there is a study in Drosophila that confirms the protective effect of thymoquinone against DSS-induced ulcerative colitis, but the mechanism of the effect is not clear.

After establishing the mouse UC model, colon histopathology scores and CMDI scores showed that the model group was significantly higher than the normal group in terms of naked eye view and histology scores. Thymoquinone treatment improved intestinal mucosa and lowered scores. After HE staining, it was observed that the crypts in the model group were disorganized, with erythrocytes exuding from the interstitium and covered with inflammatory cells (plasma cells, lymphocytes, eosinophils and neutrophils). After treatment with high and low doses of thymoquinone, the mucosal edema on the anatomical surface of the colon of mice was improved and the ulcer foci were reduced or disappeared compared with the model group. Compared with the model group, high and low doses of TQ treatment improved colonic mucosal injury in mice, with alleviation of inflammation, reduction of inflammatory cell infiltration, reduction of cryptitis and crypt abscess, and increase of cupped cells, which is in agreement with Zhang et al. team study³⁵.

Hedgehog signaling molecules are focal protein ligands secreted by signaling cells. The key ligands for Hedgehog signaling are Shh, the key receptor Smo, the inhibitory receptor Ptch1, and the transcription factor Gli1. The proliferation and differentiation of intestinal stem cells can be regulated by Shh to maintain the integrity of intestinal epithelium, and the ligands Smo and Shh, located in cell membranes. The ligand Smo, located on the cell membrane, activates Smo upon binding to Ptch1, thereby initiating downstream signaling pathways. Ptch1 mainly inhibits the activity of Smo under normal conditions. If Shh binds to Ptch1, the inhibition of Smo by Ptch1 is lifted, and the Hedgehog signaling pathway is activated, Gli1 mainly regulates the expression of downstream target genes, and in ulcerative colitis, the expression of Shh will affect the repair ability of the intestinal epithelial cells if it is affected. Our study showed that the expression of Shh, Smo, Gli1, and Ptch1^36,37 genes and proteins was significantly reduced in the model group in the DSS-induced mouse inflammation model, while their expression was significantly up-regulated after the TQ intervention, which is consistent with the previous studies on the therapeutic effects of cymbopogonin hydrochloride on ulcerative colitis³⁸, and with the study of Helal’s team³⁹ consistent with the study of Helal’s team³⁹. In this study, comparing the model group with the treatment group, thymoquinone promoted the recovery of inflammation by promoting the expression of signaling proteins such as Shh, Smo, Gli1, and Ptch1.

Key proteins of the intestinal barrier include Zo-1 and Occludin. Decreased expression of Zo-1 and Occludin genes and proteins causes impairment of the intestinal barrier function and thus increases intestinal permeability, and is closely related to the occurrence and severity of intestinal inflammation. Disruption of this barrier function allows antigens in the intestine to penetrate the mucosa and enter the lamina propria, thereby activating an immune response that triggers or exacerbates intestinal inflammation. In a mouse model of ulcerative colitis induced by DSS, the expression of Zo-1 and Occludin proteins and genes was significantly decreased. Zo-1 and Occludin were significantly up-regulated after TQ intervention, suggesting that TQ pairs can promote the recovery of intestinal mucosa in intestinal inflammation.

IL-10 is a potent anti-inflammatory cellular pleiotropic factor⁴⁰, which is secreted by T cells, B cells, monocytes, and macrophages, and acts to inhibit the expression of inflammatory factors mainly through the activation of macrophages. IL-1β is a key pro-inflammatory factor⁴¹, IL-1β binds IL-1α and IL-18 and orchestrates the immune response through multiple mechanisms downstream of binding, it is involved in the regulation of IL-6 and TNF-α and activates the vascular adhesion molecule ICAM1. TNF-α is a pleiotropic pro-inflammatory cytokine⁴² that transmits signals by binding to the TNF receptor (TNFR) on the cell surface. In contrast to IL-10, TNF-α is pro-inflammatory, whereas IL-10 is anti-inflammatory, and they interact with each other in the regulation of the immune response and inflammatory processes, working together to maintain immune homeostasis. TGF-β is a multifunctional cytokine⁴³ that regulates the proliferation, differentiation, and activation of immune cells such as lymphocytes, macrophages, and dendritic cells. TGF-β1 is the predominantly expressed form by immune cells, which regulates cellular functions through paracrine and autocrine pathways. In addition, TGF-β regulates the expression of adhesion molecules, which in turn affects the chemotaxis of granulocytes and other inflammatory cells. The enzyme-linked adsorbent assay detected an increase in the concentration of inflammatory factors such as IL-1β, TNF-α, and TGF-β and a decrease in the concentration of IL-10 in the model group, and after the intervention with thymoquinone, the concentration of inflammatory factors such as IL-1β, TNF-α, and TGF-β decreased in the model group and the concentration of IL-10 increased, and the therapeutic benefit increased with the increase in the concentration of thymoquinone.

Intestinal flora play a variety of roles in the intestinal tract^44,45, and play a crucial role in human health and development, and their main roles include participating in the digestion of food and nutrient absorption in the intestinal tract, promoting the synthesis of a variety of vitamins, and breaking down short-chain fatty acids in dietary fibers to produce metabolites that have anti-inflammatory effects, through which they maintain intestinal integrity. It also helps the immune system to mature and develop in a balanced manner, regulates the activity of immune cells to prevent them from generating excessive immune responses, and inhibits the growth of pathogenic bacteria through the production of antimicrobial substances and competitive colonization. In addition, it also affects the development of the nervous system, and dysbiosis is strongly associated with the development of inflammatory bowel disease⁴⁶.

Gut flora dysbiosis may also affect heart health by interfering with neurotransmitter production and neuroinflammation⁴⁷. In the current study, the DSS-induced enterocolitis model showed a dramatic change in the composition of the flora. These changes were reversed to some extent by the TQ intervention, such as a decrease in Bacteroidota abundance and an increase in Proteobacteria abundance in the model group observed at the gate level compared to the normal group, and after thymoquinone administration, an increase in Bacteroidota in the thymoquinone high-dose group, and a decrease in Proteobacteria in the high-dose group, In addition, we we also observed that some neutral bacteria Campylobacterota, Desulfobacterota, Verrucomicrobiota, and Deferribacterota abundance in the thymoquinone high-dose group were higher than in the model group. At the family level, a decrease in the abundance of Muribaculaceae, Akkermansiaceae, and Rikenellaceae, and an increase in the abundance of Helicobacteraceae and Enterobacteriaceae flora were observed in the middle group of the model group compared to the normal group, The increase in abundance of Bacteroidaceae, Muribaculaceae, Akkermansiaceae, Deferrribacteraceae, Staphylococcaceae and the decrease in the abundance of Enterobacteriaceae bacteria after TQ administration suggests the effect of TQ on the intestinal bacterial community community structure restoration has a certain role.

The shortcoming of this study is that the molecular mechanism of thymoquinone up-regulation of the Hedgehog signaling pathway and influence on the composition of the intestinal flora for the treatment of ulcerative colitis was performed only at the animal level, without validating it at the cellular level. In the subsequent studies, we purchased some relevant experimental cells for cell culture, drug interventions such as multiple validation of the therapeutic effect of thymoquinone on ulcerative colitis by targeting the inhibitors of Shh, Smo, Gli1, and Ptch1, respectively, in order to be guided by the later development of drugs for the treatment of ulcerative colitis.

Conclusion

In this study, it was found that thymoquinone exerts a therapeutic effect on mice with ulcerative colitis by promoting the activation of the Hedgehog signaling pathway and affecting the composition of the intestinal microbiota.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

KXQ designed the experiments; HXX performed the experiments and wrote the manuscript; ST Organized data; XLX,LW,interpreted the data ;ZHL，MZZ supervised the project; RZ,LDP,WQW revised the manuscript.

Funding

This work was supported by grants from The Longhu Talent Project of Bengbu Medical University (LH250102004) and Science and Technology Project of Bengbu Medical University (2024byzd055).

Data availability

All data included in this study are available upon request by contact with the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

Approval for this study was obtained from the Animal Ethics Committee of Bengbu Medical University (No. 2021336). All experiments were performed in accordance with relevant guidelines and regulations.