Complanatuside A ameliorates 2,4,6‐trinitrobenzene sulfonic acid‐induced colitis in mice by regulating the Th17/Treg balance via the JAK2/STAT3 signaling pathway

Immunity imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of Crohn's disease (CD). Complanatuside A (CA), a flavonol glycoside, exerts anti‐inflammatory activities and our study aimed to identify its effect on TNBS‐induced colitis and the possible mechanisms. We found that CA alleviated the symptoms of colitis in TNBS mice, as demonstrated by prevented weight loss and colon length shortening, as well as decreased disease activity index scores, inflammatory scores, and levels of proinflammatory factors. Flow cytometry analysis showed that CA markedly reduced the percentage of Th17 cells while increasing the percentage of Treg cells in TNBS mice. Under Th17 cell polarizing conditions, CA inhibited the differentiation of Th17 cells while the Treg cell differentiation was elevated under Treg cell polarizing conditions. Furthermore, it was observed that JAK2 interacted with CA through six hydrogen bonds via molecular docking. The phosphorylation of JAK2/STAT3 was reduced by CA, which might be correlated with the protective effect of CA on colitis. In conclusion, CA reduced the imbalance of Th17/Treg cells by inhibiting the JAK2/STAT3 signaling pathway in TNBS‐induced colitis, which may provide novel strategies for CD treatment.

Crohn's disease (CD) is a chronic inflammatory bowel disease with lifelong recurrent features. 1][4] Immune responses play a crucial role in inducing intestinal inflammation in the pathogenesis of CD. 5 Dysregulation of the immune response triggers inflammatory signals and exacerbates intestinal damage. 6Th17 and Treg cells are critical factors in maintaining intestinal immune homeostasis. 7,8Th17 cells drive intestinal inflammation by secreting proinflammatory cytokines such as IL-17 and IL-22. 9,10Treg cells are responsible for suppressing excessive immune responses and mediating intestinal immune stability through IL-10 and TGF-β production. 11,12For instance, Bassolas Molina demonstrated that the retinoid acid-related orphan nuclear receptor gamma (RORγt) antagonist BI119 reduced Th17 cells while upregulating the Treg response in CD patients and abrogated experimental colitis. 13Additionally, the aryl hydrocarbon receptor (AhR) activation facilitated Treg cell differentiation and inhibited Th17 cell differentiation.Dendritic cells activated by AhR increased the proportion of Treg cells while inhibited the proportion of Th17 cells in TNBS-induced colitis mice. 14Therefore, the imbalance of Th17/Treg cells is considered to be a crucial factor in the pathogenesis of CD.Targeting the regulation of Th17/Treg imbalance is a promising strategy for CD treatment.
It has been reported that traditional Chinese medicine (TCM) intervention could remarkably mitigate experimental colitis and restore the intestinal mucosa immune balance. 15,168][19] Complanatuside A (CA), a natural flavonoid compound extracted from the Semen Astragali Complanati, was recently reported to decrease inflammatory cytokine levels and may be a valuable candidate in ameliorating COVID-19-related skin inflammatory damage. 20CA treatment hindered inflammatory gene expression and inflammasome activation by targeting nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in diabetic mice. 21Additionally, CA displays distinct pharmacological activity in other disease models.For example, CA could improve antioxidation and detoxification activities via regulating SKN-1 and activating Heat shock factor 1 (HSF-1) expression in models of Caenorhabditis elegans, which extended its longevity. 22CA may promote fatty acid oxidation by stimulating the transcription of Recombinant Hepatocyte Nuclear Factor 4 Alpha (HNF4α) to inhibit lipid production, which could maintain lipid metabolism homeostasis and alleviate hepatocyte inflammatory damage. 23owever, the specific effects of CA on CD-like colitis have not been clarified.
In the present study, we aimed to explore the protective effect of CA on colitis of TNBS-induced colitis mice to clarify whether CA could antagonize intestinal inflammation by mediating the Th17/Treg balance, hoping to provide a potential therapy strategy for CD.

| Animals
Male C57BL/6J mice (6-8 weeks old) with a body mass of 22-25 g were selected.All mice were fed in a specific pathogen-free (SPF) environment.CA (C 28 H 32 O 16 , MW = 624.551,CAS No. 116183-66-5, purity: ≥98%) was purchased by Shanghai yuan' ye Bio-Technology Co., Ltd.Mice were randomly divided into three groups (n = 8): control group (WT), model group (TNBS) and CA intervention group (TNBS+CA, 15 mg/kg CA).The model and CA intervention groups were received 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma, USA) to construct colitis models. 24After anaesthetization via intraperitoneal injection with 1% pentobarbital sodium (40 mg/kg), place the mouse head in the supine position, slowly enter the colon with a thin catheter and bolus injection of 2.5% TNBS alcohol solution (5% TNBS mixed 1:1 with absolute ethanol solution) 100 μL.After extubation, mice were kept in place for 3 min to prevent fluid leakage.The control group was given the same amount of 50% ethanol according to the identical steps.The TNBS +CA group was administered CA intragastrically (15 mg/kg, once a day, for 7 consecutive days).The TNBS and control groups were given an equal amount of 0.9% sodium chloride solution.The mice were sacrificed on Day 8, and the samples such as intestinal tubes and

| Disease activity index
Disease activity assessment of colitis was as follows: hair standing (1 point), bloody stool (1 point), loose stool (1 point), rectal prolapse (1 point), severe diarrhea and rectal prolapse that cannot be returned are scored 1 point each. 7isease activity index (DAI) scores were assessed on the seventh day for TNBS mice.The DAI scores of Il-10 −/− was examined once a week for four weeks.

| Body weight
The body mass of mice was measured every day to compare the changes in body mass before and after intervention in each group.

| Colon length
The colon length of mice was measured at the time of inspection.

| Inflammatory score
Inflammatory score was measured by hematoxylin and eosin (H&E) staining as previously described 25 : normal (0 points); mild inflammatory cell infiltration (1 point); monocyte infiltration, crypt separation and mild mucosal hyperplasia (2 points); infiltration of large numbers of inflammatory cells, a structural disorder of the intestinal mucosa, loss of goblet cells, and obvious hyperplasia in the intestinal mucosa (3 points); and crypt abscesses and ulcers (4 points).All sections were graded independently by two blinded histopathologists.

| Gene ontology (GO) enrichment analysis
The DAVID database (https:// david.ncifc rf.gov/ ) was used to analyze the intersecting genes for GO pathways.Restricted species to "Homo sapiens".Biological process, molecular function, and cell component were screened by GO analysis, and the top 20 items for visualization were selected.

| Molecular docking technology
The protein structure of key targets was obtained from the PDB database (http:// www.rcsb.org/ pdb).The 3D structure of CA was obtained from the PubChem database.Autodock 4.2.6 software was used to determine the connection between CA and key targets.If the binding energy was less than 0 kJ/mol, CA could better combine with the protein, and the greater the absolute value of the combination, the stronger the combination ability.PyMOL software was used to visualize the results. 28

| Statistical analysis
The data were analyzed using SPSS v26.0 (SPSS Inc., USA) software and are presented as the mean ± standard deviation (SD).Student's t test or the Mann-Whitney test was used to compare data between two groups (parametric or nonparametric).One-way multivariate analysis of variance (ANOVA) was used for comparisons between multiple groups, and Tukey's comparison test was selected for comparisons between two groups.p < .05 was considered statistically significant.

TNBS-induced model mice
We first explored the protective effect of CA on TNBSinduced colitis.Based on the results of the DAI score, colon inflammation score and weight change, we found that there was no difference between the TNBS+CA (15 mg/ kg) and TNBS+CA (20 mg/kg) groups (Supplementary Figure S1A-C).Thus, we selected CA at a concentration of 15 mg/kg for subsequent examination.As shown in Figure 1A-D, compared with the TNBS group, CA (15 mg/kg) administration decreased the DAI score and alleviated weight loss and colon shortening (Figure 1A-D).Importantly, the inflammatory scores of colon tissues based on H&E staining were markedly reduced in the CA group compared to the TNBS group (Figure 1E,F).Furthermore, the results of another CD model (Il-10 −/− mice) was demonstrated that CA treatment decreased DAI scores, alleviated weight loss, and colon shortening (Supplementary Figure S2A-D).The inflammatory scores of H&E staining were reduced in the CA groups compared with the Il-10 −/− group (Supplementary Figure S2E,F).Moreover, after the establishment of the TNBS-induced colitis model, we found that CA-treated group also significantly improved the DAI scores, colon length and colonic inflammation scores of H&E staining compared to the TNBS group (Supplementary Figure S3A-E).

| CA exerted an anti-inflammatory effect in the colonic musocal tissues in TNBS-induced colitis
Abnormal activation of the immune response and excessive production of inflammatory factors lead to immune imbalance in the intestine, accelerating the progression of colitis. 29To evaluate the anti-inflammatory effect of CA on TNBS-induced colitis in mice, we examined the levels of proinflammatory mediators via qRT-PCR and ELISA.As shown in Figure 2, the mRNA and protein levels of the proinflammatory factors (TNF-α, IL-6, IL-17A and IL-22) in the colonic mucosal tissues were increased after TNBS challenge, along with an apparent reduction in the CAtreated TNBS mice (Figure 2A-H).Taken together, CA administration ameliorated inflammatory responses of the colonic mucosa in TNBS-induced colitis.Additionally, the four proinflammatory mediators (TNF-α, IL-6, IL-17A and IL-22) showed the same expressing levels in the Il-10 −/− mice when treated with CA (Supplementary Figure S4A-H).

Treg imbalance in TNBS-induced experimental colitis
Considering that the intestinal immune balance is crucial for the development of CD, 30,31 we hypothesized that the protective effect of CA on TNBS-induced colitis is associated with the T-cell immune response.As shown in Figure 3A,B, the level of crucial transcription factor RORγt for Th17 cells in colonic lamina propria was increased in TNBS group compared with control group, this trend was reversed by CA group.The expression of transcription factor Foxp3 for Treg cells was decreased in TNBS group, while was increased with CA treatment compared to the TNBS group.Flow cytometry analyses confirmed that CA decreased the proportion of Th17 cells and increased the percentage of Treg cells in the colonic lamina propria of TNBS mice compared to the TNBS group (Figure 3C-F).Meanwhile, the expression of transcription factors (RORγt and Foxp3) and flow cytometry results from Il-10 −/− mice model are consistent with the above results (Supplementary Figure S5A-F).As shown in Figure 3G-J, CA treatment decreased the percentage of single positive RORγt and IL-17A CD4 + T cells and increased the proportion of Foxp3 CD4 + T cells compared with TNBS mice.Additionally, the proportion of IL-17A + IL-22 + CD4 + T cells was markedly inhibited in the CA group than in the TNBS group (Figure 3K,L).Analysis of colonic lamina propria from CA-treated Il-10 −/− mice also revealed a decrease in single positive RORγt and IL-17A CD4 + T cells and increase in Foxp3 cells compared to that in Il-10 −/− mice (Supplementary Figure S5G-J).And the ratio of IL-17A + IL-22 + CD4 + T cells were suppressed in the CA-treated Il-10 −/− group (Supplementary Figure S5K,L).However, no significant changes were found in the proportion of Th1 and Th2 cells between the TNBS group and the CA group (Supplementary Figure S6A-D).Additionally, a similar expression pattern was observed for crucial transcription factors, including T-bet and GATA3, via qRT-PCR (Supplementary Figure S6E,F).These results suggested that CA administration might ameliorate intestinal inflammation possibly through decreasing Th17 and promoting Treg cells in TNBS-induced colitis mice.

Treg differentiation under Th17 and Treg cell polarizing conditions
To further verify the beneficial impact of CA on the Th17/ Treg balance in mice with TNBS-induced colitis, we

| CA may regulate TNBS-induced colitis through JAK2-related signaling
We next attempted to comprehend the potential molecular mechanisms by which CA ameliorates CD development.Network pharmacology analysis showed that there were 11 targets as CD-specific CA target genes: TERT, IL2, PTGS2, ACHE, ADORA3, ALOX5, XDH, CA2, CD38, TNF and AKR1B1 (Figure 5A).In addition, GO enrichment analysis showed that these key targets were mainly related to the inflammatory response, JAK-STAT cascade and immune response (Figure 5B).Based on the above predictive results, we focused on the JAK-STAT cascade in the biological process by which CA alleviates CD.The JAK-STAT cascade and signaling components participate in the regulation of immune and inflammatory responses in 32,33 JAK2, an isoform of tyrosine kinases, is associated with the pathogenesis of IBD and homeostasis of Th17/Treg cells. 34,35The results revealed that JAK2 interacted with CA through six hydrogen bonds through molecular docking, indicating that there was an interaction between CA and JAK2 with hydrogen bonds (Figure 5C).

| CA treatment inhibited the JAK2/STAT3 signaling pathway
The JAK2/STAT3 signaling pathway plays a critical role in CD development. 36Active JAK2/STAT3 signaling functions as a key factor in controlling various Tcell activities, particularly Th17/Treg homeostasis. 37As shown in 6, the phosphorylated levels of JAK2 and STAT3 were dramatically increased in the TNBS group but were reduced by CA treatment (Figure 6A-C).
The protein phosphorylation levels of JAK2 and STAT3 were elevated under Th17-inducing conditions, whereas CA (1.0 μM) administration suppressed this change (Figure 6D-F).In addition, the protein phosphorylation of JAK2 and STAT3 were decreased in the Treg polarizing state, and this reduction was more prominent with CA treatment (Figure 6G-I).The results implied that CA effectively inhibited the activation of JAK2/STAT3 signaling.

| DISCUSSION
Our current study found that CA exerted a protective effect on TNBS-induced colitis, and this protective effect might be regulated by restoring the Th17/Treg imbalance via suppressing the activation of the JAK2/STAT3 signaling pathway.
Although recent studies have confirmed CA exerts anti-inflammatory effects, 20,21 the specific role of CA in TNBS-induced colitis has not been reported.Here, we first reported that CA significantly reduced the symptoms of experimental colitis mice both in TNBS and Il-10 −/− models, as demonstrated by elevated body weight and colon length and decreased inflammatory scores.Moreover, CA treatment inhibited the expression of proinflammatory mediators such as TNF-α, IFN-γ, IL-17 and IL-22.Our results indicated that CA could alleviate the intestinal inflammation of TNBS-induced colitis in mice.
Abnormal immune response in the intestinal system participates in the pathological process of CD. 38,39 The main T-cell subsets, such as Th1, Th2, Th17 and Treg cells, derived from CD4 + T cells, play a crucial role in IBD pathogenesis.Th1 cells are thought to cause intestinal inflammation in CD, while Th2 cells are mainly considered to be mediators in ulcerative colitis (UC). 40,41Th17 cells are involved in CD pathogenesis by releasing the signature cytokine IL-17, causing an excessive inflammatory response. 42,43Treg cells participate in the maintenance of intestinal mucosal homeostasis in CD. 14,44 In this study, we found that CA inhibited Th17 infiltration while promoting Treg infiltration in the colonic lamina propria of TNBS-induced experimental colitis model.The analysis of intracellular cytokines staining showed that CA inhibited Previous studies have shown that flavonoids could influence Th17 and Treg cell differentiation in inflammatory diseases.Acacetin alleviated the generation of Th17 cells and maintained the proportion of Treg cells under Th17-skewing conditions in arthritis mice. 45Baicalin was reported to suppress Th17 differentiation and enhance the Treg response to relieve pancreatic injury. 46Our study found that CA promoted Treg cell differentiation and inhibited Th17 cell differentiation under Treg and Th17 polarization conditions.Therefore, CA served as an effective traditional Chinese medicine (TCM) and mediated the immune homeostasis of the TNBS-induced experimental colitis model by regulating the Th17/Treg ratio.Nevertheless, the specific immunoregulatory mechanisms of CA in TNBS-induced colitis have not been fully elucidated.
To further reveal the molecular mechanism by which CA protects against TNBS-induced colitis, the JAK-STAT cascade was obtained via network pharmacology and GO enrichment.The results of molecular docking showed that there were potential binding sites between CA and JAK2.JAK2/STAT3 signaling, associated with CD4 + Tcell differentiation, has been reported to be activated in an IBD model, and blocking JAK2/STAT3 signaling via Chrysanthemum polysaccharides could be a potential target for CD therapy. 47Activated intracellular JAK2 induces phosphorylation of STAT3 to promote and maintain the expression of the transcription factor RORγt, leading to the differentiation of Th17 cells, upregulating the secretion of IL-17A, and aggravating inflammatory responses. 48TAT3 is an inhibitor of the transcription factor Foxp3 of Treg cells, and its overactivation promotes the transformation of Treg cells into Th17 cells. 49In this study, the results showed that CA blocked the protein phosphorylation of JAK2 and STAT3 in TNBS-induced model mice.In addition, the activation of p-JAK2 and p-STAT3 was crippled after CA treatment in Th17-induced conditions.However, the phosphorylation levels of JAK2 and STAT3 were suppressed more significantly under Treg-skewing conditions.Therefore, CA may play a vital role in regulating immune balance partially through JAK2/STAT3 signaling, which might partially explain the mechanism by which CA ameliorates TNBS-induced experimental colitis.
However, our study has certain limitations.On the one hand, the results showed that CA alleviated colitis in TNBS-induced colitis mice by attenuating the Th17/Treg imbalance, but other mechanisms have not been fully elucidated.On the other hand, CA may relieve mucosal inflammation by inhibiting the activation of the JAK2/ STAT3 signaling pathway, but other molecular mechanisms need to be further investigated.
In summary, we first discovered that CA ameliorated colitis in TNBS-induced colitis mice by reducing the Th17/ Treg imbalance, and this beneficial effect was mediated partially through the JAK2/STAT3 signaling pathway.Our research provides new insights into the therapeutic effects of CA in a mouse model of colitis and suggests potential therapeutic effects in CD.

F I G U R E 1
CA treatment improved TNBS-induced colitis in mice.(A) The DAI scores, (B) body weight change, and (C, D) colon length was examined.(E, F) Representative microscopy images of H&E staining in colon tissues and the corresponding inflammatory scores.N = 8 mice per group.The data are presented as the mean ± SD.Shown are the representative images.*p < .05:compared with Control group, # p < .05:compared with TNBS group.analyzed the effect of CA on the differentiation of CD4 + T cells under Th17 and Treg cell-inducing conditions.The cell counting kit-8 (CCK-8) results showed no significant effect could be seen in terms of CD4 + T-cell viability with different CA concentrations (Supplementary FigureS7).As shown in Figure4, CA treatment significantly inhibited the percentage of Th17 cells, along with a reduction in the specific transcription factor RORγt and cytokines (IL-17A and IL-22) expression levels compared to Th17-polarizing conditions (Figure4A-D).In contrast, CA treatment increased the proportion of Treg cells (Figure4E).The mRNA expression of the Treg cell-specific transcription factor Foxp3 and cytokines (TGF-β and IL-10) were upregulated with CA administration compared with Treg polarizing conditions (Figure4F-H).The results of intracellular staining showed that the protein level of RORγt and IL-22 were increased under Th17-skewing conditions, while were decreased in the CA-treated groups (Figure4I,J).In addition, we noticed no significant effect on the differentiation of Th17 and Treg cells between the 1.0 μM CA and 2.0 μM CA groups.CA (1.0 μM) was used for subsequent experiments.Altogether, these results demonstrated that CA restrained Th17 differentiation while enhancing Treg cell differentiation under Th17 and Treg cell skewing conditions.

F I G U R E 2
CA inhibited the inflammatory response in the colonic mucosal tissues of TNBS mice.(A-D) The mRNA levels of colonic mucosal proinflammatory cytokines, including TNF-α, IL-6, IL-17A and IL-22, were measured by real-time PCR.(E-H) The protein levels of colonic mucosal inflammatory mediators (TNF-α, IL-6, IL-17A and IL-22) were examined by ELISA.N = 8 mice per group.The data are presented as the mean ± SD.Shown are the representative images.*p < .05.F I G U R E 3 CA attenuated Th17 and Treg imbalance in colonic lamina propria of TNBS mice.(A, B) The mRNA levels of RORγt and Foxp3 were measured by qRT-PCR.The number of Th17 (C, E) and Treg (D, F) cells in the Control, TNBS, TNBS+CA groups was analyzed by flow cytometry.(G-L) The frequency of RORγt, Foxp3, IL-17A, IL-22 expression among CD4 + T cells were analyzed by flow cytometry.N = 8 mice per group.The data are presented as the mean ± SD.Shown are the representative images.*p < .05.

F I G U R E 4
CA inhibited Th17 differentiation while enhancing Treg cell differentiation.(A) The proportion of Th17 cells was measured after naïve CD4 + T-cell polarization by flow cytometry.(B-D) The expression of RORγt, IL-17A and IL-22 was measured by qRT-PCR.(E) The proportion of Treg cells was determined using flow cytometry.(F-H) The expression of Foxp3, TGF-β and IL-10 was measured by qRT-PCR.(I, J) The proportions of RORγt and IL-22 were measured by flow cytometry.The data are presented as the mean ± SD from three independent experiments.Shown are the representative images.*p < .05.

F I G U R E 5
Analysis of network pharmacology and molecular docking in CA alleviating Crohn's disease.(A) The common targets of CA and CD.(B) GO enrichment analysis.(C) The binding mode images of CA with CD are shown according to the molecular docking analysis.F I G U R E 6 CA intercepted the activation of JAK2/STAT3 signaling pathway.(A) The protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 was detected by western blotting in colonic tissues.(B, C) Quantitative determination of protein greyscale values was used for the ratio of p-JAK2/JAK2 and p-STAT3/STAT3.(D) The protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 was examined by western blotting under Th17-skewing conditions.(E, F) Quantitative determination of protein greyscale values was used for the ratio of p-JAK2/ JAK2 and p-STAT3/STAT3.(G) The protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 was examined by western blotting under Treg-skewing conditions.(H, I) Quantitative determination of protein greyscale values was used for the ratio of p-JAK2/JAK2 and p-STAT3/ STAT3.N = 8 mice per group for in vivo experiments.The data are presented as the mean ± SD.Shown are the representative images.*p < .05.percentage of single RORγt and 17A CD4 + T cells and increased Foxp3+ regulatory T cells in TNBS mice.And CA could also suppress the secretion of Th17 cell-derived IL-17A and IL-22.However, the Th1 and Th2 ratios showed no significant change.Therefore, we hypothesized that CA may exert an antagonistic effect on TNBSinduced colitis by mediating the Th17/Treg balance, and the expression of their representative transcription factors (GATA3, T-bet, RORγt and Foxp3) further validated the above hypothesis.Notably, the above experimental results from IL-10 −/− mice also proved this hypothesis.

| 3 of 14 WANG
et al. intestinal mucosal tissues were retained for detection and analysis.All animal experimental protocols were approved by the Animal Ethics Committee of Bengbu Medical University (Bengbu, China).The application approval number is 2020_089.