| Part I:Compound Sophorae Decoction improves UC by inhibiting ferroptosis through autophagy pathwayObjective:To study the mechanism of Compound Sophorae Decoction to relieve ulcerative colitis in mice by inhibiting ferroptosis through activating the autophagy pathway.Methods:C57/B6 mice drank 3%DSS ad libitum for 7 consecutive days to establish a mouse UC model.First,all the mice were adaptively fed for 7 days。And the experiment officially started,the mice were randomly divided into five groups,the control group,the model group,the ferroptosis inhibitor group,the ferroptosis agonist group and the compound Sophorae decoction group.Except the control group and the ferroptosis agonist group continued to drink distilled water,the other three groups were given 3%DSS to drink freely every day,and the ferroptosis inhibitor group was given Fer-1(a ferroptosis inhibitor)intraperitoneal injection once a day.For 7 consecutive days,the compound Sophorae decoction group was given compound Kushen decoction by gavage every day,and the ferroptosis agonist group was given intraperitoneal injection of Erastin(a ferroptosis agonist)on the 6th and 7th days,respectively,with two injections,12 hours apart.During the experiment,the body weight,mental state,and defecation of the mice were recorded daily to judge the disease activity.On the 8th day,the mice were sacrificed after blood collection from the eyeballs,and the colon samples of the mice were collected for subsequent detection,such as the pathological scores of the mice in each group;the detection of apoptosis by immunohistochemistry;the detection of inflammatory indicators by ELISA and qRT-PCR;Western blot to detect markers of ferroptosis and autophagy in intestinal tissue and tissue iron to detect the accumulation of iron ions;immunofluorescence to detect tight junction proteins ZO-1 and Occludin.In the second part of the experiment,four groups were reset,including the control group,the model group,the 3-MA(an autophagy inhibitor)and the compound Sophorae decoction group.The control group was given distilled water to drink freely,and the other three groups were given 3%DSS to drink freely to build a UC model.One intraperitoneal injection of 3-MA was administered for 7 consecutive days.On the 8th day,blood was collected from the eyeball and the colon was also collected.Western blot was used to detect autophagy-related proteins and AMPK,ULK1 and mTOR proteins;transmission electron microscopy was to detect the morphology of autophagy-lysosomes;immunohistochemistry was to detect the expression and distribution of mucus secreted protein MUC2.Results:1.Compound Sophorae Decoction can reduce the weight loss of UC mice,reduce the DAI score and improve intestinal pathology.2.CSD can alleviate inflammation and apoptosis in UC mice.3.Results of ferroptosis-related markers PTGS2 and GPX4 can be modulated by CSD,and increase the gene expression and protein expression of GPX4.4.CSD can up-regulate the expression of autophagy-related genes.5.CSD can up-regulate AMPK/ULK1 pathway and down-regulate AMPK/mTOR pathway.6.CSD can increase the expression and distribution of tight junction proteins ZO-1,Occludin and MUC2.The difference was statistically significant(P<0.05).Conclusion:Compound Sophorae Decoction can activate autophagy by regulating the AMPK/ULK1/mTOR pathway,thereby inhibiting ferroptosis to achieve the purpose of treating UC.Part Ⅱ:EphB2-BMSC-Exo regulate T cell differentiation in UC Objective:To study the mechanism of BMSC-Exo overexpressing EphB2 in regulating UC intestinal barrier function and its effect on T cell differentiation.Methods:A male SD rat weighing 80-100 g was taken,the tibia and fibula were removed,the skin and flesh were removed,the epiphysis at both ends were cut,and the marrow cavity was repeatedly washed with a 5 mL syringe to obtain bone marrow for culture.After 48 hours,the medium was changed for the first time,and the adherent growth was BMSCs(Bone marrow mesenchymal stem cells,BMSCs),and then the medium was changed every 3 days.At the P2 generation,the lentiviral vector encoding EphB2 was transfected into BMSCs,and puromycin was used to select BMSCs that stably overexpressed EphB2.The BMSCs after the drug screening continued to be cultured.When cultured to the P4-P5 generation,the cells overexpressing EphB2 were separated from the culture supernatant of the cells by ultracentrifugation after starvation with serum-free medium for 48 hours.External vesicles(Exosome derived from Bone marrow mesenchymal stem cells,EphB2-BMSC-Exo).The inflammation model of Caco-2 was constructed with 3%DSS,and then co-cultured with EphB2-BMSC-Exo.The effects of EphB2-BMSC-Exo on the proliferation and migration of Caco-2 cells were detected by CCK-8 and scratch experiments;EphB2 was detected by ELISA-The effect of BMSC-Exo on inflammatory response;FITC-4000 to detect the effect of EphB2-BMSC-Exo on the permeability of Caco-2 cell monolayer;Western blot to detect the effect of EphB2-BMSC-Exo on RhoA-ROCK signaling pathway.At the same time,the oxidative damage model of Caco-2 was constructed with hydrogen peroxide to explore the regulatory effect of EphB2-BMSC-Exo on oxidative stress.In addition,a male SD rat with a body weight of 60-80 g was taken,and the spleen of the male SD rat with a body weight of 60-80 g was sacrificed.The spleen was cut into pieces,filtered through a 70 μm mesh,and centrifuged to obtain mouse spleen lymphocytes.Then,the rat spleen CD4+T lymphocytes were obtained by magnetic bead sorting,and various cytokines were added to stimulate the differentiation of CD4+T lymphocytes into T cell subsets.At the same time,EphB2-BMSC-Exo was added for co-culture.The T cell subsets were detected by cytometry to explore the effect of EphB2-BMSC-Exo on T cell differentiation.Results:1.The lentivirus overexpressing EphB2 was successfully constructed and transfected into BMSCs to obtain BMSCs stably expressing EphB2,and EphB2-BMSC-Exo was successfully extracted.2.EphB2-BMSC-Exo can protect the cell activity of Caco-2 cells inhibited by DSS,and promote cell proliferation and migration.3.EphB2-BMSC-Exo can reduce the inflammatory response of Caco-2 cells and increase the activity of antioxidant enzymes,while inhibiting the oxidative stress of Caco-2 cells.4.EphB2-BMSC-Exo restores intestinal barrier function by inhibiting the RhoA/ROCK pathway.5.The CD4+T lymphocytes of rat spleen were successfully obtained by magnetic bead sorting and cultured for 72h.EphB2-BMSC-Exo can promote the differentiation of CD4+T lymphocytes into Treg cells and inhibit their differentiation into Thl and Th17 cells.The difference was statistically significant(P<0.05).Conclusion:EphB2-BMSC-Exo enhances intestinal barrier function by inhibiting RhoA/ROCK pathway in vitro,and can regulate immune balance.Experiments show that EphB2-BMSC-Exo may be used as a novel cell-free therapy for UC or other immune diseases. |
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