Effect Of Cryptotanshinone On Murine Acute And Chronic Ulcerative Colitis And Its Possible Mechanism

Ulcerative colitis(UC)is a form of inflammatory bowel disease(IBD).It is characterized by chronically inflammation of immune response on the gut.At present,the pathogenesis of UC is still unclear,which is generally believed to be the result of the interaction of a variety of factors such as genetics,environment,intestinal flora and immune response.The immune system plays an important role in UC.If the balance of proinflammatory and anti-inflammatory mediators are disrupted,it can lead to overactivation of the immune response.Therefore,T helper cells 17(Th17)and Regulatory T cells(Tregs)are considered to play an important role in the pathogenesis of UC.Interleukin 17(IL-17)is a cytokine secreted specifically by Th17 cells,which can introduce monocytes and neutrophils into the inflammatory site,and at the same time cooperate with pro-inflammatory cytokines such as tumor necrosis factor(TNF)to induce inflammatory response.Therefore,inhibiting the differentiation of Th17 cells is a potential treatment for UC.The Janus kinase/Signal transducers and activators of transcription(JAK/STAT)signaling pathways play a key role in the process of cell growth,differentiation and migration.It is also involved in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease.The role of Signal transduction and activator of transcription 3(STAT3)in UC has been extensively reported,and the expression and activity of STAT3 are significantly increased especially in patients with UC.In addition,the phosphorylation of STAT3 is also significantly increased in T cells of intestinal mucosal.The treatment of UC is usually based on the severity of the disease.Currently,the treatment drugs mainly include amino salicylic acid,glucocorticoids and immunoregulatory drugs.Recently,Pfizer announces that the United States Food and Drug Administration(FDA)has approved the application of tofacitinib for the treatment of patients with moderate to severe ulcerative colitis.Tofacitinib is the first JAK inhibitor currently available for patients with moderate to severe ulcerative colitis,and the STAT3 inhibitor has not been reported for the treatment of ulcerative colitis in the clinical up to date.Cryptotanshinone(CTS)is a kind of tanshinone compounds extracted from the traditional Chinese medicine salvia miltiorrhiza,which posses a variety of pharmacological activities such as anti-inflammatory,anti-tumor,antibacterial,antioxidant.However,its effect on ulcerative colitis has not been reported.In the prophase study,we have shown that CTS can specifically bind to STAT3 by using the Surface Plasmon Resonance technology.Therefore,in order to further study the role of CTS in UC,we investigated the effect of CTS on mice with acute and chronic ulcerative colitis induced by dextran sulfate sodium(DSS)and its mechanism.First,we investigated the effects of CTS on the activation of STAT3 and the differentiation of Th17 cells.Cell Counting Kit-8(CCK8)method was used to detect the effect of CTS on the viability of mouse splenocytes;Enzyme linked immunosorbent assay(ELISA)was used to assess the cytokine levels of interleukin 17A(IL-17A),TNF-α,interleukin 2(IL-2),interleukin 6(IL-6),interferon-γ(IFN-γ)and interleukin 10(IL-10)in the supernatants of splenocytes stimulated with Concavalin A(Con A)with or without CTS;the effect of CTS on the expression of phosphorylation of STAT3 protein was detected by Western blotting(WB);the effect of CTS on nuclear translocation of STAT3 was detected by confocal laser scanning microscopy of Leica,and the effect of CTS on the differentiation from naive CD4~+T cells to Th17 cells was detected by flow cytometry.The results showed that CTS(3,10,30μM)had no significant effect on splenocyte cytotoxicity,and dose-dependently inhibited IL-6-induced phosphorylation and nuclear translocation of STAT3.In addition,CTS could inhibit the production of IL-17A and TNF-αin splenocytes stimulated by ConA,but had no significant effects on the production of IL-2,IL-6,IFN-γand IL-10.Finally,the results of flow cytometry showed that CTS could significantly inhibit the differentiation from naive CD4~+T cells into Th17 cells induced by transforming growth factor-β(TGF-β)and IL-6.These results indicated that CTS could inhibit the activation of STAT3 and the differentiation of Th17 cells.We next established a model of acute and chronic ulcerative colitis in C57BL/6 mice induced by DSS to further evaluate the effect of CTS on UC.In the acute model,mice freely drank 2.5%DSS solution for 7 days,followed by drinking water for 3 days,and were treated with 20mg/kg and 60mg/kg of CTS for 10 days.In the chronic model,mice were given 2.5%DSS solution for 7d,followed by sterile distilled water for 14d,which was one cycle,and a total of two cycles were used to replicate the model.Then the mice were given 2.5%DSS solution again for 7d to establish the chronic colitis model.Starting from the day 22,the mice were administered 20mg/kg of CTS for a total of 28 days.Then the body weight,colon length and disease activity index(DAI)were evaluated,and the colon histopathological changes were observed by Hematoxylin-eosin staining(HE)method.Periodic Acid-Schiff staining(PAS)method was used to observe goblet cells in the colon tissues of the mice;Sirius red/fast green collagen staining was detected the expression of the collagen fibers in the colon,and the flow cytometry was tested the proportion of CD4~+IL-17A~+Th17 cells and CD4~+CD25~+Foxp3~+Treg cells in spleens and mesenteric lymph nodes.Immunohistochemical staining was performed to detect the expression of P-STAT3,myeloperoxidase(MPO),the representative tight junction protein-closed protein(Occludin)andα-smooth muscle actin(α-SMA)in the colon tissues.Western blot was used to detect the expression of P-STAT3 in colon tissues.The results showed that DSS induced obvious diarrhea,blood stool,and weight loss in mice.CTS could effectively improve the symptoms of DSS-induced ulcerative colitis,with the increase in the length of colon,reduced infiltration of inflammatory cell and MPO activity in colon.It could also increase the expression of occludin protein and counts of goblet cells in mice with acute colitis.Flow cytometry showed that CTS reduced the proportion of CD4~+IL-17A~+Th17 cells in the spleen and mesenteric lymph nodes of mice with acute colitis,and increased the proportion of CD4~+CD25~+Foxp3~+Treg cells.In addition,CTS could reduce the increased levels of P-STAT3 in colitis tissues.In chronic colitis,CTS increased the colon length,reduced the expression of collagen fibers,α-SMA and P-STAT3 levels in the colon,and reduced the numbers of CD4~+IL-17A~+Th17 cells in the spleen and mesenteric lymph nodes of mice.These data suggested that CTS could improve acute and chronic ulcerative colitis.In summary,CTS could effectively relieve the inflammation of experimental colitis in mice,alleviate the fibrosis of colon tissue,protect and repair mucosal tissue.Its mechanism may be related to the down-regulation of STAT3 signal transduction pathway and the inhibition of Th17 differentiation.