Cynarin ameliorates dextran sulfate sodium-induced acute colitis in mice through the STAT3/NF-κB pathway

Abstract Objective Cynarin is a derivative of hydroxycinnamic acid presented in various medicinal plants, such as Cynara scolymus L. and Onopordum illyricum L. To date, the antioxidant and antihypertensive activities of cynarin have been reported. However, whether cynarin has a therapeutic impact on ulcerative colitis (UC) is unclear. Therefore, the aim of this study was to explore the potential effect of cynarin on dextran sulfate sodium (DSS)-induced acute colitis in vivo and on lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced RAW264.7 and J774A.1 cellular inflammation model in vitro. Methods and Results In this study, we investigated that cynarin alleviated clinical symptoms in animal models, including disease activity index (DAI) and histological damage. Furthermore, cynarin can attenuate colon inflammation through decreasing the proportion of neutrophils in peripheral blood, reducing the infiltration of neutrophils, and macrophages in colon tissue, inhibiting the release of pro-inflammatory cytokines and suppressing the expression of STAT3 and p65. In cellular inflammation models, cynarin inhibited the expression of M1 macrophage markers, such as TNF-α, IL-1β, and iNOS. Besides, cynarin suppressed the expression of STAT3 and p65 as well as the phosphorylation of STAT3, p65. Cynarin inhibited the polarization of RAW264.7 and J774A.1 cells toward M1 and alleviated LPS/IFN-γ-induced cellular inflammation. Conclusion Considering these results, we conclude that cynarin mitigates experimental UC partially through inhibiting the STAT3/NF-кB signaling pathways and macrophage polarization toward M1. Accordingly, cynarin might be a potential and effective therapy for UC.


Introduction
Ulcerative colitis (UC), an idiopathic inflammatory bowel disease (IBD), is a chronic inflammatory disease [1].UC mainly involves the rectum and colon, and the lesions are mostly located in the mucosal and submucosa of the colorectum with a continuous distribution [2].The main clinical manifestations of UC are abdominal pain, diarrhea, and hematochezia, which seriously disturb the lives of patients, and it has the risk of canceration [3,4].In recent decades, the incidence of UC has gradually increased globally and has become an important public health problem [5].At present, the clinical treatment of UC is mainly drug therapy, including aminosalicylic acid agents, glucocorticoids, immunosuppressive agents, biological agents, and so on.However, these drugs cannot achieve satisfactory therapeutic effects and often have a high recurrence rate, in addition, their side effects will prolong the treatment cycle [6,7].Therefore, more novel effective therapeutic strategies are urgently required.
Cynarin (1,3-Dicaffeoylquinic acid or 1,5-Dicaffeoylquinic acid), a derivative of hydroxycinnamic acid, is one of the main active principles of Cynara scolymus L., which is an important plant used both as food and medicinal plant worldwide [8,9].According to reports, cynarin has anti-inflammatory activity.It can reduce the swelling in the hind paws of mice and reduce the release of TNF-α, IL-6, IL-1β, and iNOS pro-inflammatory cytokines in gouty arthritis model induced by monosodium urate crystals [10].Moreover, cynarin also has antioxidant and antiviral properties [11,12].Nevertheless, the anti-inflammatory effect and mechanism of cynarin on UC are still largely understood.Accordingly, the aim of this study was to investigate the effect of cynarin on dextran sulfate sodium (DSS)-induced colitis in vivo or lipopolysaccharide (LPS) and interferon-γ (IFN-γ) in vitro.

Cynarin attenuates DSS-induced experimental colitis in vivo
The structure of cynarin is displayed in Figure 1(A).To explore the impact of cynarin on UC, C57BL/6 mice were orally administered 2.5% DSS for 7 d except for the control group.From the third day, the treatment groups were given the corresponding drugs [cynarin 50 mg/kg, cynarin 100 mg/kg, or sulfasalazine (SASP) 100 mg/kg], respectively, by oral administration from 3 to 7 d.Meanwhile, the control group and the DSS group were given normal saline (Figure 1(B)).As described in Figure 1(C-E), mice receiving cynarin or SASP administration showed less weight loss, disease activity index (DAI), and colon shortening compared to the DSS group.Based on these results, we suggested cynarin could mitigate the acute colitis induced by DSS.

Cynarin alleviates histological inflammatory injury in the DSS-induced colitis model
Results of the histological examination showed that cynarin relieved DSS-induced colitis.The mice with DSS treatment resulted in crypt destruction, mucosal ulcer, submucous edema, and infiltration of numerous inflammatory cells such as neutrophils, macrophages, and lymphocytes in colon tissues [13,14].As shown in Figure 2(A), the colonic epithelial cells of mice in the DSS group were severely damaged, crypts disappeared, submucosa edema, with inflammatory cell infiltration.After cynarin or SASP treatment, epithelial integrity and submucosal edema were ameliorated, as well as infiltration of inflammatory cells was reduced.Moreover, the histological damage score of treatment groups were significantly decreased compared to the DSS group (Figure 2(B)).Collectively, these results revealed that cynarin treatment alleviated the histological injury of DSS-induced colitis.

Cynarin decreases neutrophils and macrophages infiltration in colon of DSS-treated mice
To study the effect of immune cells for DSS-induced colitis, we examined ratio of neutrophils in peripheral blood and neutrophils and macrophages in colon tissues with the neutrophil marker Gr-1 and the macrophage marker F4/80 by flow cytometry.Compared with the DSS group, there were considerably fewer neutrophils in peripheral blood (Figure 3(A,B)) and fewer neutrophils, macrophages in the colon tissues (Figure 3(C-E)) of cynarin-treated mice.Results of the flow cytometry were consistent with the previous report [15,16] and suggest that the treatment of cynarin attenuated DSS-induced inflammatory responses in mice.

Cynarin inhibits the pro-inflammatory cytokines expression in colonic tissues
Increased expression of pro-inflammatory cytokines is an important manifestation of colitis induced by DSS [17].We determined the mRNA expression of TNF-α, IL-1β, IFN-γ, and iNOS in colon tissues using RT-qPCR.The outcomes indicated that DSS resulted in a notable increase in the expression of these genes, and treatment with cynarin markedly suppressed these changes (Figure 4(A-D)).

Cynarin attenuates LPS and IFN-γ induced inflammatory responses in vitro
That M1 macrophages are usually produced in the inflammatory state can enhance the inflammatory response [18].In chronic inflammatory diseases, such as UC, suppressing the polarization of macrophages to M1 can relieve inflammation and protect the body [19].Hence, we selected macrophages RAW264.7 and J774A.1 for anti-inflammatory experiments in vitro.First, both cell lines were treated with different concentrations of cynarin (0, 12.5, 25, 50, 100, and 200 μM) for 24 h, then the relative cell viability was tested by cell counting kit 8 (CCK-8) assay.As described in Figure 5(A,E), cynarin had no significant effect on the proliferation of RAW264.7 and J774A.1 cells.The results indicate that cynarin has no obvious cytotoxicity to RAW264.7 and J774A.1 cell lines.Based on these data, 50, 100, and 200 μM were selected in subsequent experiments.Second, RAW264.7 and J774A.1 cells were induced to M1 macrophages through incubation with LPS (200 ng/mL) and IFN-γ (20 ng/mL) for 6 h.Meanwhile, the corresponding concentrations of cynarin were added to cynarin-treated groups.Two hours before sample collection, BAY 11-7082 (NF-κB pathway inhibitor, 5 μM) was added to M1 + BAY 11-7082 group.Then the mRNA expression of M1 markers including TNF-α, IL-1β, and iNOS was tested.As depicted in Figure 5(B-D,F-H), expression of these genes in M1 group were significantly higher than the control group, and treatment of cynarin notably inhibited these changes.As a whole, our results indicated that cynarin inhibited RAW264.7

Cynarin suppresses activation of the STAT3/NF-кB pathway
According to published studies, NF-κB signaling pathway is activated in the colon tissue of IBD patients [20].Besides, the expression of total STAT3 and p-STAT3 is also increased, which is closely related to the severity of IBD [21].To investigate the mechanism of cynarin alleviating UC, we determined the effect of cynarin on the STAT3/NF-кB signaling pathway.Western blotting assay revelated that the expression of total STAT3, total p65, and total IκBα induced by DSS was increased in colon tissues of mice.However, this expression of total STAT3 and p65 was decreased following cynarin treatment (Figure 6(A-D)).In RAW264.7 and J774A.1 cells stimulated by LPS and IFN-γ, the expression of total STAT3, phosphorylated STAT3, phosphorylated p65 was also increased, which was reduced by treatment of cynarin (Figure 6(E,F)).In general, these findings demonstrated cynarin inhibited the activation of STAT3/NF-кB pathway induced by DSS or LPS and IFN-γ.

Discussion
Although factors such as genetics, geographical environment, imbalance of immune system, and intestinal flora are closely related to the pathogenesis of UC, the etiology of UC is poorly understood [22,23].DSS administration can result in weight loss, diarrhea, blood in the stool, colon shortening, and colon inflammation in experimental animals, which are most similar to the clinical symptoms and pathological changes of human UC [24,25].The DSS-induced UC model serves a crucial function in the study of the pathogenesis of UC and the screening of therapeutic drugs.According to research of pathogenesis in UC, a series of chemical and biological medicines were developed.However, these medicines cannot completely cure UC and have various adverse effects, such as toxicity, headache, and osteoporosis [26,27].Hence, the development of novel effective treatment strategies is urgently needed.
In recent years, evidence from multiple research demonstrated that natural products extracted from plants have alleviative property on UC [28,29].As a monomeric compound, cynarin presents in various plants, such as Cynara scolymus L. and Echinacea angustifolia [30,31].Increasing evidence indicated that cynarin exerts huge beneficial effects on various disease models.For instance, cynarin can reduce the release of iNOS in human coronary artery smooth muscle cells induced by a cytokine mixture [32].Moreover, Hakkou Zineb et al. illustrated that cynarin as one of the main active ingredients of Inula viscosa L. could dilate blood vessels and prevent the increase in systolic blood pressure (SBP) induced by L-NAME [33].To the best of our knowledge, the effect of cynarin on UC has not been researched.In this study, the relationship between cynarin and UC was investigated based on in vivo and in vitro models.SASP is a widely used medicine for the treatment of UC in clinical practice, which can suppress the activation of NF-κB pathway and inhibit the expression of pro-inflammatory cytokines [34].Therefore, we selected SASP as the positive control to evaluate the protective effect of cynarin on UC induced by DSS.
In this study, we found that cynarin significantly decreased UC-related parameters, such as weight loss, colon shortening and DAI compared to the DSS group.Besides, cynarin alleviated colon pathological damage.When the body undergoes an inflammatory response, the proportion of neutrophils in peripheral blood increase.Under the stimulation of chemical factors, they enter the inflammatory site through the endothelium in the form of ameba movement, and have a phagocytic function.Neutrophils produce a large amount of reactive oxygen species in the process of functioning, causing intestinal barrier damage and activating redox-related inflammatory signaling pathways [35].Meanwhile, neutrophils also release a series of proteases and pro-inflammatory cytokines, such as IL-8 and TNF-α, which damage the intestinal barrier and induce monocytes and other neutrophils to enter the site of inflammation [36].The monocytes entering the inflammatory site differentiate into macrophages, and the original macrophages proliferate, resulting in massive infiltration of macrophages [37].In chronic inflammatory diseases, pro-inflammatory factors cannot be quickly cleared and will continue to stimulate the body to produce the inflammatory response.In the process of functioning, leukocytes will release substances, such as prostaglandins and arachidonic acid metabolites to the extracellular matrix in the form of degranulation, causing damage to vascular endothelial cells and tissues, and aggravating the damage caused by the original inflammatory factors.In addition, necrotic and disintegrated leukocytes also release large amounts of toxic substances, causing tissue damage [38].Hence, in chronic inflammatory diseases, such as UC, inhibiting the inflammatory response can alleviate the body damage to a certain extent.In our study, we have demonstrated that cynarin decreased the proportion of neutrophils in peripheral blood and the infiltration of neutrophils and macrophages in colon tissue.Furthermore, our experiments proved that cynarin inhibited the expression of pro-inflammatory cytokines TNF-α, IL-1β, IFN-γ, and iNOS in mice model induced by DSS.According to published studies, many therapeutic agents, such as Qing-dai powder can also relieve UC by inhibiting macrophage infiltration and expression of pro-inflammatory cytokines [39].On the basis of published studies, in the colon tissue of mice stimulated with DSS, the activation of STAT3 and NF-κB signaling pathways is elevated, and the expression of total STAT3, p-STAT3, and NF-κB p65 is increased, which are closely related to the process of disease [40,41].In our study, we demonstrated that cynarin decreased the expression of STAT3 and p65 compared to the DSS group and the expression of IκBα was not changed in vivo experiments.Based on these results, we concluded that cynarin relieves UC at least partially through inhibiting the STAT3 and NF-кB signaling pathways.Nonetheless, more rigorous research is required to explore the precise molecular mechanism of cynarin against UC in the future.
Macrophage polarization is an assessment of the activation state of macrophages in a specific environment.M1 macrophages are usually produced in inflammatory states, which are mediated by Toll-like receptors and interferons, and are involved in immunity to bacteria and intracellular pathogens [42].They can produce a large amount of pro-inflammatory cytokines and reactive oxygen species to promote the inflammatory response [43].Macrophage polarization has the crucial impact on the inflammatory process and prognosis, and the disruption of the balance of M1 and M2 macrophages is an important stimulus for many disorders including IBD [44,45].Therefore, we selected macrophages RAW264.7 and J774A.1 cell lines for in vitro experiments.We found that cynarin suppressed the mRNA expression of M1 macrophage markers, such as TNF-α, IL-1β, and iNOS.Consistent with the results in vivo, cynarin decreased the expression of STAT3, p65, p-STAT3, p-p65, and p-IκBα, and inhibited the activation of STAT3 and NF-κB pathways.

Conclusions
In conclusion, this study illustrated that cynarin clearly alleviated the inflammation induced by DSS in mice as well as mediated by LPS and IFN-γ in RAW264.7 and J774A.1 cells.Moreover, cynarin can mitigate UC through decreasing infiltration of neutrophils and macrophages in colon tissue, reducing the expression of pro-inflammatory cytokines, and inhibiting the activation of STAT3 and NF-кB pathway.Consequently, the natural product, cynarin, might be a potential and effective therapy for UC.monoclonal antibody (30-F11), CD11b monoclonal antibody (M1/70), Ly-6G (Gr-1) monoclonal antibody (1A8-Ly6g), and F4/80 monoclonal antibody (BM8) were purchased from Thermo Fisher Scientific (Waltham, MA).Reverse transcription reagent Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) and quantitative real-time PCR reagent Hieff ® qPCR SYBR Green Master Mix (No Rox) were obtained from Yeasen Biotechnology Co., Ltd (Shanghai, China).

Cell culture
RAW264.7 and J774A.1 cell lines were obtained from the cell repository of School of Medicine, xiamen University, xiamen, China.Both of the cells were incubated in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and cultured in a humidified incubator with 5% CO 2 at 37 °C.For cell passaging, both of the cells were blown off the culture dish by repeated blowing using PBS and continued to be cultured in a 1:3 ratio.

Cell viability assay
The relative cell viability was examined by CCK-8 assay.First, RAW264.7 and J774A.1 cells were distributed in 96-well plates respectively at 7 × 10 3 cells per well.After cell adherence, they were treated with different concentrations of cynarin (0, 12.5, 25, 50, 100, and 200 μM) for 24 h.Second, CCK-8 solution was added to each well and incubated at 37 °C for 4 h.Then, the absorbance at 450 nm was measured to evaluate the relative cell viability.

Animals and treatments
6-8 weeks male C57BL/6 mice, weight 21-26 g, were purchased fromxiamen University Laboratory Animal Center [production license number SCxK(Min)2018-0003]. Mice were maintained in the specific-pathogen-free (SPF) room of Experimental Animal Center of xiamen University.Animal experiments were supported by the Ethics Committee of Medical School of xiamen University (approval number xMULAC20170297).The mice were divided into 5 groups with 6 mice in each group, which were the control group, DSS group, DSS + cynarin (50 mg/kg) group, DSS + cynarin (100 mg/kg) group and positive control group of DSS + SASP (100 mg/kg).Mice were free to drink 2.5% DSS solution (DSS dissolved in drinking water) for 7 dexcept for the control group.The mice in control group drank normal drinking water throughout the experiment.From the third day, mice in the treatment groups were given the corresponding cynarin or SASP by oral administration, lasting for 5 d.Meanwhile, mice in the control group and DSS group were given normal saline.

Disease activity index
DAI can reflect the severity of UC in mice.DAI consists of three parameters: loss of body weight, consistency of stool, and presence of blood in stool.DAI = loss of body weight + consistency of stool + presence of blood in stool.
For details regarding evaluation criteria of DAI, refer to Supplementary Material (Table 1S, Supporting Information).

Assessment of histopathological damage
Pathological damage of colon tissue was assessed through hematoxylin and eosin (H&E) staining.Colon tissue was immersed in 4% paraformaldehyde solution for 48 h at room temperature.Then the tissues were dehydrated, embedded in paraffin, sectioned, and stained.The slices were observed under a microscope and 3 different fields of view were randomly selected for each slice, then the histological damage score was assessed according to the criteria of Cooper et al. [13].

Flow cytometry
Peripheral blood removed erythrocytes and cells of colon tissue filtered through a 300-mesh filter were prepared.Then antibodies were added to the cell suspension and incubated at room temperature under the dark condition for 15 min.The processed samples were analyzed by the flow cytometer Fortessa x-20 (BD).

Quantitative real-time polymerase chain reaction analysis
Total RNA of colon tissue as well as RAW264.7 and J774A.1 cells was extracted by TRIzol reagent, and reverse transcribed into cDNA with reverse transcription reagent.After that the mRNA levels of genes were quantitatively measured using Light Cycler 96 System (Roche, Basel, Switzerland) with qPCR reagent.The relative expression of each gene was calculated with the 2 −ΔΔ Ct method.Details of primer sequences are in Supplementary Material (Table 2S, Supporting Information).

Western blot
Total proteins of colon tissue and RAW264.7 and J774A.1 cells were extracted with RIPA lysis buffer.The concentration of proteins was examined using a BCA protein kit (Beyotime, Shanghai, China).The proteins were separated by 8% SDS-PAGE and transferred to a PVDF membrane.The membrane was incubated in 5% skim milk at room temperature for 1 h and then incubated with the primary antibodies at 4 °C overnight.After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.Finally, the results were detected with enhanced chemiluminescence (ECL) kit (YEASEN, Shanghai, China).

Statistical analysis
All results were presented as mean ± standard error of mean (SEM) and analyzed by Graphpad Prism version 8.4 (GraphPad

Figure 1 .
Figure 1.cynarin treatments mitigated dSS-induced colitis.a. the structure of cynarin.B. Schematic diagram to illustrate the experimental design.c. relative weight change and disease activity index of mice in each group (n = 6).d. morphological images of the colons in each group of mice.E. Statistical results of the length of colons (n = 6).data are expressed as mean ± SEm. ***p < 0.001 and **** p < 0.0001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. the dSS group.Sulfasalazine (SaSp) is a positive control group.

Figure 3 .
Figure 3. Effects of cynarin on ratio of neutrophils in blood and neutrophil or macrophage infiltration in colons of dSS-treated mice.(a, c, E) Flow cytometric analysis of neutrophils in peripheral blood, neutrophils in colon tissues, and macrophages in colon tissues.(B, d, F) Statistical results for ratio of neutrophils to leukocytes in peripheral blood, ratio of neutrophils to leukocytes in colon tissues, and ratio of macrophages to leukocytes in colon tissues (n = 4).data are expressed as mean ± SEm. ***p < 0.001 and **** p < 0.0001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. the dSS group.Sulfasalazine (SaSp) is a positive control group.