| Aim:This study adopts the method of DSS copy reproducible colitis model in mice,in the evaluation of ginseng saponin Rg1 at the same time,the efficacy of colitis in mice to observe the mice flora structure and the changes of macrophage mutation status,and from the Angle of Nogo-B/RhoA signalling pathways,explore Rg1 ginsenosides regulate flora balance in macrophages mutations and possible mechanism of action for the treatment of colitis in mice.Methods:1.Animal model replication and expected methods: 60 SPF male BALB / C mice were randomly divided into 6 groups,10 in each group,normal group(Normal),model group(DSS),high dose(200 mg · kg-1)Ginsenoside Rg1 group(DSS +Rg1-H),medium dose(100 mg · kg-1)ginsenoside Rg1 group(DSS + Rg1-M),low dose(50 mg · kg-1)ginsenoside Rg1 group(DSS + Rg1-L)and mesalazine group(5-ASA).A 3%(w / v)DSS(35000-50 000 k D)solution was prepared by ultrapure water.Except for mice in the normal group,the mice in the other groups drank water normally for 1 week,and then replaced with 2% DSS in water for 5 days.The normal group has been drinking ultrapure water.Mouse were given 3% DSS for the first time on day 1.From the 11 th day,the normal group and the model group was given gavage by ultrapure water according to body weight.The ginsenoside Rg1 high,medium and low dose groups followed the weight of 200 mg · kg-1,100 mg · kg-1,and 50 mg · kg-1 to ginseng.The saponin Rg1 solution was orally administered,and the mesalazine group was administered the mesalazine enteric-coated tablet suspension according to the weight of 300 mg · kg-1.Forty SPF male BALB / C rats wererandomly divided into 4 groups of 10 rats each,the normal group model group,the ginsenoside Rg1 group(DSS + Rg1)and the Y27632 group(DSS + Y27632).The model replication method is the same as above.The alternative method is calculated from the first day of DSS in mice.From the 11 th day,each of the normal group and the model group is administered with ultrapure water according to body weight.The ginsenoside Rg1 solution was orally administered,and the Y27632 group was orally administered with Y27632 solution according to a body weight of 10 mg·kg-1,and adjusted daily for a fixed period of time for a total of 10 days.evaluate the efficacy Shishenwan treatment of ulcerative colitis in rats: weighed daily,and observe its food and water,activity,mental state and symptoms.The colon was isolated,its weight and length were measured,its intestinal weight index was calculated,and the colonic mucosal injury index score was visually observed.2.Evaluation of the efficacy of ginsenoside Rg1 in the treatment of DSS-induced recurrent colitis: body mass,food and water consumption,hair condition and activity index were observed daily.After the mice were anesthetized and killed,the colon mass and colon length were measured,and the colon mass index was calculated as the unit of colon mass.Colonic pathological sections were made,colonic pathological conditions were observed under a microscope and pathological injury score.Detection of IL-6,CCL-2,TNF-α,TGF-β1,IL-4,IL-10,IL-13 and IL-33 inflammatory factors in mouse colon by ELISA method.3.Intestinal flora sequencing: Under sterilization conditions,the feces of the ileocele of the mouse were obtained,quickly placed in dry ice and then transferred to-80 ℃ for storage.DNA was extracted and PCR amplified from the stool to obtain the base sequence,which was compared with the gene pool to obtain the flora information.To explore the correlation between intestinal flora and colitis,as well as the regulatory effect of ginsenoside Rg1 on the flora,and detect the expression of TLR2 protein in the colon by WB method.4.Macrophage polarization analysis: M1 type macrophages i NOS,Tim-1,TLR-4,M2 type macrophages CD163,CD206 were measured by flow cytometry to investigate whether ginsenoside Rg1 regulates macrophage polarities.Treatment ofDSS-induced colitis in mice.5.Detection of Nogo-B / RhoA signaling pathway and macrophage-related proteins: WB method was used to analyze the Nogo-B / RhoA signaling pathway Rock 1,RhoA and Nogo-B proteins,macrophage-related proteins Arg-1.MIF-1 and PIM-1 were tested to investigate the regulatory effect of ginsenoside Rg1 on Nogo-B /RhoA signaling pathway.6.Statistical analysis: SPSS 22.0 statistical software was used for data analysis;measurement data were expressed as mean ± standard deviation(sx ±);single factor analysis of variance was used to determine significance,and then multiple comparison Tukey test was performed;P <0.05 was used to indicate the difference It is significant,and the difference is extremely significant with P <0.01.Results:1.Effective dose of ginsenoside Rg1 for colitis: Compared with the normal group,the mice in the model group began to lose weight on the 3rd day,with slow movements,thin stools,and loss of appetite.On the 5th day,the mouse’s anus was obvious Blood in the stool,the mice gradually recovered weight after stopping drinking DSS.Compared with the model group,the weight recovery rate was accelerated after ginsenoside Rg1 high,medium and low dose and mesalazine group administration.When drinking DSS for the second time,the weight loss of the treatment group was slower than that of the model group.Through statistical analysis of data such as body weight,colon quality,and colon length,it was found that high-dose ginsenoside Rg1 was more effective than medium and low effective.It is suggested that ginsenoside Rg1 can effectively treat recurrent colitis in mice,and the best effect is at high doses.2.Evaluation of the efficacy of ginsenoside Rg1 in the treatment of colitis:Compared with the model group,the body weight and colon quality of the model group significantly increased,and the colon length of the treatment group was restored compared with the model group.When comparing the mass of unit colons,it was found that the model group significantly increased compared with other groups(P <0.05 or P <0.01).Compared with the model group,the ginsenoside Rg1 colonicmass index was significantly reduced(P <0.05 or P <0.01),and the Y27632 group had a decreasing trend but no significant difference.Compared with the normal group and the treatment group,the pathological injury score of the model group was significantly increased(P <0.05 or P <0.01).Observation of pathological sections of colon tissue revealed that the colon mucosa of the normal group of mice was neatly arranged without inflammatory cell infiltration.Compared with the normal group,the DSS model group had a large area of ulcers and submucosal layers.The glands were disordered and partially missing,vascular proliferation,and a large number of inflammatory cells infiltrated with edema.In the ginsenoside Rg1 group and Y27632 group,there was a little inflammatory cell infiltration,a small area of ulcers,and the glands were neatly seen to show glandular hyperplasia and repair.When detecting inflammatory cells,it was found that the levels of IL-6,IL-33 and CCL-2 in the model group were significantly different(P <0.05 or P <0.01).Compared with the model group,IL in the ginsenoside Rg1 group and Y27632 group-6,IL-33,CCL-2and TNF-α levels were significantly decreased(P <0.05 or P <0.01).Compared with the model group,the ginsenoside Rg1 group showed a significant increase in TGF-β1,IL-4,IL-10,and IL-13(P <0.05 or P <0.01).The Y27632 group had TGF-β1,IL-4,and IL-13.Significantly increased(P <0.05 or P <0.01).It is suggested that ginsenoside Rg1 pairs may treat colitis by regulating inflammatory cytokines..3.Intestinal microflora study: The intestinal microflora sequencing showed that the diversity of the model group was decreased compared with that of the normal group,and the number of unique bacteria was decreased.The dominant species in the model group were Staphylococcus,Bacteroides,Bacteroides,and Bacillus,and the richness of the model group was also significant(P <0.05 or P <0.01),although there was no significant difference in the abundance of the model group.A marked increase.In the normal group and ginsenoside Rg1,the abundance of bacteria in the Y27632 group tended to be consistent with that in the model group.showed that the intestinal flora in the colitis mice was disturbed,the pathogenic bacteria increased,the beneficial bacteria decreased,and the abundance of the bacteria decreased.Significant increase in TLR2 expression in the model group(P <0.05 or P <0.01),and theassociation analysis with intestinal flora,found that the model group was the most closely related to TLR2 protein,and the normal group was far related to TLR2 protein.suggesting that intestinal flora disorders can lead to high levels of TLR2 expression,ginsenoside rg1 may treat colitis by regulating the intestinal flora and reducing TLR2 expression,whereas Y27632 does not regulate the flora.4.Macrophage polarization detection: Macrophages were detected by flow cytometry.Compared with the model group,the expression of i NOS,Tim-1,TLR-4 of M1 macrophages in the treatment group was reduced(P <0.05 or P <0.01),the expression of CD163 and CD206 of M2 macrophages increased,which was significantly different(P <0.05 or P <0.01).It shows that ginsenoside Rg1 can effectively regulate macrophage polarization.5.Nogo-B / RhoA signaling pathway and macrophage-related protein detection:colon protein detection using WB method.Compared with the model group,the expression of Arg-1 in the treatment group was significantly increased(P <0.05 or P<0.01).The expressions of MIF-1,PIM-1,Rock 1,RhoA and Nogo-B proteins were significantly reduced(P <0.05 or P <0.01),suggesting that ginsenoside Rg1 can inhibit Nogo-B / RhoA signaling pathway proteins.Concussion:The effective treatment of DSS-induced colitis with ginsenoside Rg1 may be achieved by regulating the intestinal flora balance and inhibiting the Nogo-B / RhoA signaling pathway to regulate macrophage polarization. |
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