| Objective:To clarify the mechanism of JianPiBuShen,QingChangHuaShi decoction for treating ulcerative colitis based on reconstructing the intestinal barrier by promoting bone marrow mesenchymal stem cells homing.Methods:In vitro,MSCs were isolated and cultured, the 3rd generation cells were randomly divided into control group and groups with different concentrations of JianPiBuShen QingChangHuaShi decoction(0.390625 ug/ml-50ug/ml), observe the proliferation ability of MSCs with CCK-8, determine the concentrations for subsequent experiments which contain 0,0.39,0.78,1.56 (ug/ml),observe the transwell ability of MSCs migration. Each compound dose combined ERK kinase inhibitor U0126 as a control, detect the phosphorylation of ERK and CREB protein levels with Western Blot method.In addition, we induced MSCs inflammatory model LPS,cells were respectively treated with Astragaloside, Baicalin and their compatibility.Observe the proliferation ability of MSCs with MTT method,the cell differentiation ability were observed with microscope,useing the flow cytometry to observe the cell cycle situation,Observe the apoptosis ability of MSCs with Annexin-Vflow cytometry.Detect cytokine IL-1β,IL-8 and TNF-a levels and relations with ERK pathway by using Elisa, RT-PCR and Western Blot methods. In vivo experiments, MSCs were cultivated with Jianpi Bushen Qingchang Huashi decoction of 0.78ug/ml,and fluorescent pre-labeling were carried out by DAPI in vitro. UC rat model was established by TNBS method. The rats were divided into 5 groups in random:normal group, model group, MSCs group、intervened MSCs group and combined group. The rats of normal and model groups were received intravenous injection with normal saline separately through tail veins, The rats of MSCs group were received intravenous injection with MSCs through tail veins(1×106/lml), The rats of intervened MSCs group and combined group were received intervened MSCs by the decoction in vitro,the combined group also received the decoction in Oral for 10 days.5 rats were killed 5 and 10days after the transplant respectively. Gross morphological and histological evaluation, observation of the MSCs labeled by DAPI in the colon tissue by confocal microscope separately, detection of the expression of Musashi-1(a marker of intestinal stem cells)by immunohistochemical staining were carried out then.Detection the protein expression of Lgr5,Ephrin-B3 with Western blot respectively then, assayed the levels of IL-6、IL-17and TGF-β with Elisa method..Detect the mRNA and protein expression of Muc2 by Real-Time PCR and Western blot respectively then, assayed the E-cadherin,Mathl and KLF4 by western blot.Results:Compared with the control group, JianPiBuShen QingChangHuaShi decoction group can promote the proliferation of MSCs among a certain concentration range,groups with 0.78and1.56ug/ml concentrations compared 0.39ug/ml group was statistically significant (P <0.01);each dose group were able to promote MSCs migration,also can increased the phosphorylation of ERK and CREB protein levels, and that can be inhibited by U0126(P <0.05).From monomers experimental results, the most obvious effect of proliferation appeared when each drug concentration of 100ng/ml,compatibility group is the best,followed by Astragaloside group,48 hours is better than 24 hours; each group of drugs were able to promote epithelial differentiation and promote cell growth, reduce apoptosis,compatibility group is the best. Astragaloside and Baicalin could reduced IL-1β, IL-8 and TNF-a levels in LPS-induced inflammation MSCs model, compatibility group is the best, and compared with the compatibility group, ERK inhibitor group capable of covering the inflammatory effect.In vivo experiments, confocal microscope could detect MSCs labeled by DAPI in the colon tissue after MSCs were injected through the tail veins of the rats. The expression of Musashi-1 in transplant group was more obvious compared with model group,Each group can improve the gross morphological of colon tissue and histopathology.The number goblet cells were increased in each MSCs transplantation group,with prolonged treatment, the role is more obvious.On the fifth day, Compared with the model group, Musashi-1 positive expression of transplantation group were seen in each group,the combination group were significantly different(P<0.01).Each group could decrease the level of IL-6,the combined group could also decrease the level of IL-17 and increase the level of TGF-β,difference occurred compared with MSCs group (P <0.05).Statistically significant difference occurred between the combined and intervened MSCs relative to the MSCs group of mRNA expression of Muc2.On the 10th day,the expression of Musashi-1 in each group was also more obvious(P<0.01),and the combination group and intervened MSCs group are better than MSCs group(P<0.05).Lgr5 and Ephrin-B3 protein levels were increased in the combined group compared with MSCs group (P<0.05);the combined group can improve the IL-6, TGF-β levels compared with MSCs group (P<0.01), IL-17 and TGF-β levels were improved in the intervened MSCs group(P<0.01),and IL-6 level were decreased(P < 0.05).Difference between the combined group relative to the MSCs group of E-cadherin and MATH1 expression(P<0.05).The intervened MSCs group can also Increased E-cadherin and MATH1 HATH1 expression,better than the MSCs group(P<0.05).Conclusion:JianPiBuShen QingChangHuaShi decoction can promote the proliferation and migration of MSCs among a certain concentration range, the mechanism may be related to ERK /CREB signaling pathway. JianPiBuShen QingChangHuaShi Decoction could not only promote the migration of MSCs to the ulcers of the colon and differentiation, but also can reduced the inflammation and repair the intestinal barrier.Transplantation of MSCs combined with the traditional compound Chinese medicine in oral in the same time is better. Astragaloside and Baicalin could promote epithelial differentiation and promote cell growth, reduce apoptosis and inflammatory effects,the compatibility group is the best. |
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