| Background:Ulcerative colitis is a chronic non-specific intestinal inflammatory disease whose lesions are located in the large intestine,involving the mucous membranes of the colorectal and the lower mucosa,with intestinal inflammation and mucosal injury as the main pathological manifestations.In recent years,the incidence of ulcerative colitis has shown a significant upward trend in China,but there is no effective method to cure ulcerative colitis at present,resulting to the disease is prone to relapse,delay,a large number of patients eventually need to undergo surgical treatment,the health of the people have a serious impact,And to the social health care expenditure to bring a great burden.The crux of the problem is that its pathogenesis has not been clear,so it is v ery important to actively explore the pathogenesis of ulcerative colitis for its clinical precision treatment.Although the pathogenesis of ulcerative colitis is not clear,it is believed that its pathogenesis is mainly caused by the abnormal interaction of patients,such as genetic susceptibility,external environmental impact,autoimmune dysfunction,intestinal flora disorder and damage to intestinal mucosal barrier.It is proved that autophagy and SIRT1/mTOR signaling pathway can participate in many physiological and pathological processes,such as inflammation,tumor and metabolism,but it is not clear whether SIRT1 participates in ulcerative colitis and regulates the level of autophagy of intestinal epithelial tissue.The mechanism of the action of the SIRT1/mTOR signaling pathway in ulcerative colitis is still unclear.Because of the lack of comprehensive and in-depth understanding of the etiology and pathogenesis of ulcerative colitis,it is still impossible to cure the disease,so as soon as possible to expound the pathogenesis of ulcerative colitis,explore new therapeutic targets,improve the prognosis of disease has become an urgent problem to be solved in clinic.In recent years,the active ingredient of traditional Chinese medicine,as a supplementary therapy for the treatment of Ulcerative colitis,has been paid more and more attention because of its good therapeutic effect and less toxic side effects.Curcuma is a herbaceous herb of ginger for many years,and its application as medicinal materi al in the medicine of the motherland has been in the past hundred years.The 2019 ECCO noted that curcumin,as a complementary therapy for 5-ASA,helped induce the remission of mild to moderate active UC;But in 2019 AGA,it was noted that due to limited evidence,for patients with mild-moderate ulcerative colitis treated with 5-ASA,curcumin therapy is not recommended.This shows that the current study on the treatment of ulcerative colitis in turmeric is in its infancy,with few related studies,and its c urative effect and mechanism of action are still controversial,so it needs further exploration and study.Objective:In this study,we discussed the possible mechanism of SIRT1/ mTOR signaling pathway in the occurrence and development of ulcerative colitis induced by sodium dextran(Dextran Sulfate Sodium Salt,DSS)in mice in vivo and in vitro experiments.And then studied the therapeutic effect of curcumin on DSS mice and its regulation mechanism by vivo experiments,and the effect of curcumin on macrophages cells was discussed through in vitro experiments.The related mechanism of the internal and external effects of curcumin was discussed in order to lay a theoretical and experimental basis for the clinical treatment of curcumin for ulcerative colitisMethods:(1)BALB/c mice were randomly divided into 5 groups with 10 mice in each group.The control group was the model control group.The mice in this group were housed regularly without any treatment.The DSS group was treated with dextran sulfate.Sodium(3% DSS)was used to make ulcerative colitis mice model;Resveratrol-1 group: DSS method for the production of ulcerative colitis mice model,40mg/kg(0.4mL)resveratrol was intragastric administration;Resveratrol-2 group: DSS method a murine model of ulcerative colitis was made and 60mg/kg(0.4mL)resveratrol was intragastrically administered;Resveratrol-3 group: DSS method was used to manufacture a mice model of ulcerative colitis and 80mg/kg(0.4mL)resveratrol was intragastric administered.From the date of modeling,the mice were observed daily for stool traits and bloody stools,activity,spirit,diet,and body weight,and the mice disease activity index was calculated.The colon tissues of each group were collected,and histopathological sections were made and stained with HE to observe histopathological changes.ELISA was used to detect the expression of TNF-α and IL-6 in colon tissue culture fluid.Immunofluorescence staining was used to detect the expression of Atg12,Beclin-1,LC3,mTOR and SIRT1 in colon tissues of mice.The mRNA and protein expression of Atg12,Beclin-1,LC3,mTOR and SIRT1 in colon tissues of mice were detected by real-time PCR and western blot.(2)BALB/c mice were randomly divided into 5 groups with 10 mice in each group.The control group was the model control group.The mice in this group were housed regularly without any treatment.The DSS group was treated with dextran sulfate.Sodium(3% DSS)was used to make ulcerative colitis mice model;Curcumin-1 group: The mice model of ulcerative colitis was made by DSS method,and 10 mg/kg(0.4 mL)curcumin was intragastrically administered;Curcumin-2 Group: A mice model of ulcerative colitis was made using the DSS method and 25 mg/kg(0.4 mL)of curcumin was intragastrically administered;Curcumin-3 group: DSS method was used to produce a mice model of ulcerative colitis and given 50 mg/kg.(0.4 mL)curcumin was intragastrically administered.The colon tissues of each group were collected,and histopathological sections were made and stained with HE to observe histopathological changes.ELISA was used to detect the expression of TNF-α and IL-6 in colon tissue culture fluid.Immunofluorescence staining was used to detect the expression of Atg12,Beclin-1,LC3,mTOR and SIRT1 in colon tissues of mice.The mRNA and protein expression of Atg12,Beclin-1,LC3,mTOR and SIRT1 in colon tissues of mice were detected by real-time PCR and western blot.(3)After BALB/c mice were fed for 7 days,they were randomly divided into 6 groups with 10 mice in each group.The control group was the model control group.The mice in this group were fed conventionally without any treatment.The DSS group: dextran was used.A mice model of ulcerative colitis was made by sodium sulfate(3% DSS);DSS+Cur group: a mice model of ulcerative colitis was made by DSS method and 50 mg/kg(0.4 mL)of curcumin was intragastrically administered;DSS+ Res group: The mice model of ulcerative colitis was made by DSS method and 80 mg/kg(0.4 mL)resveratrol was intragastrically administered;Eve+Cur group: a mice model of ulcerative colitis was made by DSS method and given 50 mg/kg.(0.4 mL)curcumin was intragastrically administered,and mice were given daily administration of mTOR inhibitor Everolimus(2.5 mg/kg);Eve+Res group: a mice model of ulcerative colitis was made by DSS method and given 80mg/kg(0.4mL)resveratrol was intragastrically administered,and mice were given daily administration of mTOR inhibitor Everolimus(2.5 mg/kg);Each group of mice peritoneal macrophages and colorectal macrophages were isolated and cultured.CCK-8 method was used to detect the proliferation of cells in each group.Flow cytometry was used to detect the apoptosis.The expression of IL-6 and TNF-α in macrophages culture supernatants was detected by ELISA.The mRNA and protein expressions of LC3A/B,Beclin-1,and Atg12 were detected in macrophages.The mRNA and protein expressions of mTOR and SIRT1 in each group were detected by fluorescence quantitative PCR and western blot respectively.The expression of IL-6 and TNF-α in the culture supernatant of macrophages was detected by ELISA.Results:(1)Compared with the control group,the mortality rate of the DSS group was 50% at day 14,and the Sirt1/mTOR signaling pathway was inhibited.Compared with the control group,the mRNA and protein expression levels of mTOR and SIRT1 in the DSS group were significantly lower(P<0.05);In the resveratroltreatment group,the survival rate of DSS-induced colitis mice was 83.3%,which was significantly higher than that of the DSS group.Compared with the mice in the DSS group,mTOR in the colon tissue of the mice in the resveratrol concentration group were upregulated.The expression levels of SIRT1 mRNA and protein were significantly higher(P<0.05).In the treatment group of resveratrol,the body weight of mice in the DSS group decreased from the 4th day,and was significantly reduced compared with the normal mice before the 8th day(P<0.05).In the group treated with resveratrol,the body weight of DSS-induced colitis mice was significantly less than that of the DSS group(P<0.05).The disease activity index(DAI)of DSS group and mice treated with resveratrol were significantly higher(P<0.05);and DSS group.In comparison,the DAI scores of mice treated with resveratrol were significantly lower(P<0.05).The study also found that resveratrol improved DSS-induced enteritis in mice with diarrhea and rectal bleeding(P<0.05).The colon length of DSS mice was significantly shorter than that of control mice(P<0.05).Compared with the DSS group,the colon length of DSS mice treated with resveratrol was significantly longer than that of the DSS group(p<0.05),indicating an improvement in colon length,and almost completely restored to normal length.In the control group,the mucous layer of the colonic wall was intact,and no obvious infiltration of the eye-shaped cells was observed.Compared with the control group,the colonic mucosal glands in the DSS-induced ulcerative colitis model mice were obviously missing,and there were a large number of inflammatory cells.Infiltration to the mucosal layer and submucosa,some samples can be observed infiltration of inflammatory cells into the muscle tissue,and some even reach the mucosal adipose tissue,submucosal edema,glandular and goblet cell number reduced or even disappeared,mucosal epithelium The structure is not complete;in the mice treated with different concentrations of resveratrol,the colonic mucosal injury is improved compared with the DSS group,and the degree of infiltration of the eye-shaped cells can be observed in the field of vision,and the integrity of the colonic epithelial cells can be observed.As a result,the number of glandular and goblet cells was significantly increased compared with the DSS group,and the submucosal edema was alleviated.Compared with control group,the expression of TNF-α and IL-6 in DSS group was significantly increased(P <0.05).On the 7th day,resveratrol inhibited the increase of TNF-α and IL-6 expression in DSS mice(P <0.05).On the 14 th day,the expression of TNF-α and IL-6 after resveratrol treatment was significantly lower than that of DSS group(P<0.05),similar to that on the 7th day.Compared with control group,the mRNA and protein expression of Atg12,Beclin-1 and LC3 were significantly increased in mice of DSS group(P <0.05).Compared with DSS mice,in each resveratrol the mRNA and protein expression of Atg12,Beclin-1 and LC3 in colon tissues of mice were significantly decreased(P <0.05).Compared with the control group,the mRNA and protein expression levels of mTOR and SIRT1 in the DSS group were significantly lower(P<0.05);compared with the mice in the DSS group,mTOR in the colon tissue of the mice in the resveratrol concentration group.The expression levels of SIRT1 mRNA and protein were significantly higher(P<0.05).(2)In the treatment group of curcumin,the survival rates of DSS-induced colitis mice were 66.7% which were significantly higher than those of the DSS group.The body weight of mice in the DSS group decreased from the 4th day,and was significantly reduced compared with the normal mice before the 8th day(P<0.05).In the group treated with curcumin,the body weight of DSS-induced colitis mice was significantly less than that of the DSS group(P<0.05).The disease activity index(DAI)of DSS group and mice treated with curcumin were significantly higher(P<0.05);and DSS group.In comparison,the DAI scores of mice treated with curcumin were significantly lower(P<0.05).The study also found that curcumin improved DSS-induced enteritis in mice with diarrhea and rectal bleeding(P<0.05).The colon length of DSS mice was significantly shorter than that of control mice(P<0.05).Compared with the DSS group,the colon length of DSS mice treated with curcumin was significantly longer than that of the DSS group(p< 0.05),indicating an improvement in colon length.,and almost completely restored to normal length.In the mice treated with different concentrations of curcumin,the colonic mucosal injury is improved compared with the DSS group,and the degree of infiltration of the eye-shaped cells can be observed in the field of vision,and the integrity of the colonic epithelial cells can be observed.As a result,the number of glandular and goblet cells was significantly increased compared with the DSS group,and the submucosal edema was alleviated.Compared with control group,the expression of TNF-α and IL-6 in DSS group was significantly increased(P <0.05).On the 7th day,curcumin inhibited the increase of TNF-α and IL-6 expression in DSS mice(P <0.05).On the 14 th day,the expression of TNF-α and IL-6 after curcumin treatment was significantly lower than that of DSS group(P<0.05),similar to that on the 7th day.Compared with control group,the mRNA and protein expression of Atg12,Beclin-1 and LC3 were significantly increased in mice of DSS group(P <0.05).Compared with DSS mice,each curcumin or white velvet The mRNA and protein expression of Atg12,Beclin-1 and LC3 in colon tissues of mice with alcohol concentration significantly decreased(P <0.05).Compared with the control group,the mRNA and protein expression levels of mTOR and SIRT1 in the DSS group were significantly lower(P<0.05);compared with the mice in the DSS group,mTOR in the colon tissue of the mice in the curcumin concentration group.The expression levels of SIRT1 mRNA and protein were significantly higher(P<0.05).(3)Compared with control group,the proliferation activity of macrophages in DSS group,DSS+Cur group and DSS+Res group was significantly higher(P<0.05);DSS+Cur group and DSS+Res were compared with DSS group.The proliferation activity of the group cells was significantly reduced(P<0.05).Compared with the control group,the apoptotic rate of macrophages in the DSS group,DSS+Cur group,and DSS+Res group was significantly lower(P<0.05);compared with the DSS group,the DSS+Cur group and the DSS+Res group were large The apoptosis rate of phagocytes was significantly higher(P<0.05).Compared with the control group,the expression levels of Atg12,Beclin-1,and LC3 mRNA in DSS group were significantly increased(P<0.05).Compared with DSS group,DSS + Cur group and DSS + Res group were macrophages.The expression of Atg12,Beclin-1 and LC3 mRNA was significantly decreased in the cells(P<0.05).The expression of Atg12,Beclin-1 and LC3 protein in cells of each group was further detected by western blot.The results showed that the expression of Atg12,Beclin-1 and LC3 protein in cells of DSS group compared with control group.The levels were significantly increased(P<0.05).Compared with the DSS group,the expression levels of Atg12,Beclin-1,and LC3 protein in macrophages of DSS + Cur group and DSS + Res group were significantly lower(P<0.05)..Compared with the control group,the expression levels of mTOR and SIRT1 mRNA and protein in macrophages of the DSS group,Cur group,Res group,Eve+Cur group,and Eve+Res group were significantly decreased(P<0.05);compared with the DSS group.The expression levels of mTOR and SIRT1 mRNA and protein in macrophages in the Cur and Res groups were significantly higher(P<0.05),compared with the Cur and Res groups,macrophages in the Eve+Cur and Eve+Res groups.The mRNA and protein expression levels of mTOR and SIRT1 were significantly lower(P<0.05).Compared with the control group,the expression levels of IL-6 and TNF-α in the macrophage culture supernatant of the DSS group were significantly higher(P<0.05);compared with the DSS group,the macrophage culture supernatant of the Cur group and the Res group was higher than the control group.The expression levels of IL-6 and TNF-α were significantly lower(P<0.05).Compared with Cur and Res groups,IL-6 and TNF-α were found in supernatants of macrophages cultured in Eve+Cur and Eve+Res groups.The expression level of α was significantly increased(P<0.05).Conclusion:(1)DSS-induced SIRT1/mTOR signaling was inhibited in mice with ulcerative colitis.Resveratrol effectively reverses the inhibitory effect of DSS on SIRT1/mTOR signaling pathway.Resveratrol can effectively inhibit the occurrence and development of DSS-induced ulcerative colitis and relieve the disease.(2)Curcumin can effectively inhibit the occurrence and development of DSS-induced ulcerative colitis and relieve the disease.The mechanism of curcumin mentioned above may be related to their ability to relieve inflammatory response,reduce autophagy-related gene expression and promote activation of autophagy-related SIRT1/mTOR signalingpathway.(3)Curcumin can inhibit the proliferation of peritoneal fluid and colonic macrophages in mice with ulcerative colitis,promote their apoptosis,and inhibit the development of inflammation and autophagy.These effects are regulated by SIRT1/mTORPathway implementation. |
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