| Ulcerative colitis (UC) is a chronic, idiopathic, relapsing form ofinflammatory bowel disease (IBD), which is associated with a variety ofextraintestinal manifestations (EIMs), involving hepatobiliary manifestationsthat is increasingly recognized as an important cause of case fatality rate inIBD. Therefore, studying the chronic colitis-associated hepatobiliary disordershas critical significances for the comprehensive treatment of IBD.As we all know, there are amounts of bacteria and endotoxin in intestinecavity. Previous studies have revealed that bacterial translocation alsooccurred in IBD, which is caused by increased intestinal permeability as aresult of a disrupted intestinal barrier, and finally resulted in endotoxemia.Gut-derived lipopolysaccharide (LPS), a gramnegative bacterial cell wallcomponent, via the portal vein traverses directly into the liver, in which LPS iscleared. More and more evidences suggested that LPS/Toll receptor4(TLR4)signaling pathway plays a pivotal role in the pathogenesis of various chronicliver diseases.TLRs are a kind of innate immune receptors that identify pathogens.Within this family of TLRs proteins, TLR4plays a critical role in innate andadaptive immunity system. LPS, a gramnegative bacterial cell wall componentthat is enriched within the intestinal lumen and its associated portal circulation,can be combined with TLR4. Kupffer cells (KC), can directly respond toTLR4ligand. TLR4are cell surface receptors that can swtich LPS into thecells. When KC are activated by LPS, inflammation response are induced andthen inflammation cytokines such as tumor necrosis factor (TNF-α),interferon-γ (IFN-γ), interleukin-1β (IL-1β) and interleukin-17A (IL-17A), arereleased, causing liver damage by LPS/TLR4signaling pathway. Mesenchymal stem cells (MSCs) are derived from mesodermmesenchymal with multiple differentiatral potential. With the ability ofrenewing and potential multiplex differentiation, MSCs can enhance thecapability of repairing intestinal epithelium and adjust the immune respone ofintestinal epithelium, and, therefore, may play a potential role in the therapy ofIBD. Above all, MSCs has significantly ameliorated functions in liver injurycaused by a variety of reasons. In view of the mechanism above, we deducedthat MSCs also has alleviated the hepatobiliary disorders related to chroniccolitis. Therefore, our experiments made further researches into the treatmentand mechanism of MSCs derived from umbilical cord (human umbilical cordMSCs, hUC-MSCs) on chronic colitis-associated hepatobiliary disorders.Objective: To intvestigate the protective role and mechanism of MSCs inchronic colitis-associated hepatobiliary disorders.Methods:(1) C57BL/6mice were used for in vivo studies. Mice wererandomly grouped as follows: control group (n=10); DSS+Vehicle group(n=10) and DSS+MSCs group (n=10). Chronic colitis was induced bymultiple-cycle administration of dextran sodium sulfate (DSS) drinking water.Mice received either regular drinking water (control) or5%(w/v) DSSdrinking water on days1-5,8-12,15-19,22-26,29-33, and36-40. In theDSS+MSCs group,1×106/200μL hUC-MSCs were injected into caudal veinof the mice, while mice in the normal control group and DSS+Vehicle groupwere given volumetric PBS for comparison. Mice were sacrificed at day43;(2)Severity of colitis was evaluated by body weight (BW) changes, diseaseactivity index (DAI), colon length, colon histology changes and pathologyscore;(3) The Haematoxylin and eosin staining (H&E dyeing), Masson'strichrome staining (MT dyeing) were used to observe liver pathology change;(4) Mice liver function change was tested by automatic biochemistryinstrument;(5) Levels of LPS were detected in serum by limulus lysate test;(6)Mesenteric lymph nodes (MLN) were homogenated to observe whether E. colibacterial were found in each group;(7) The expressions of TNF-α, IFN-γ,IL-1β, IL-17A, TLR4, TRAF6, and NF-κB proteins in liver were detected by immunohistochemistry, western blot and real-time Q-PCR, respectively.Results:(1) The results of chronic colitis showed that BW hadsignificantly greater loss in the DSS+Vehicle group compared with that in thecontrol group. However, after the treatment of MSCs, mice in the DSS+MSCsgroup had rapid weight recovery; DAI (4.00±0.65vs0.00±0.00, P<0.05)and pathology scores (11.80±0.96vs0.00±0.00, P<0.05) in theDSS+Vehicle group were significantly higher than that of the control group,and colon length was shorter (3.93cm±0.22cm vs6.30cm±0.55cm,P<0.05); the degree of congestion and edema of the colon wall (2.75±0.50vs0.00±0.00, P<0.05), infiltration into the mucosa superficial layers oflymphocytes and neutrophil and multifocal shallow ulcers were much severerin the DSS+Vehicle group. In the DSS+MSCs group, DAI (0.30±0.46vs3.05±0.25, P<0.05) and pathology score (5.0±0.62vs11.8±0.96, P<0.05)significantly decreased, colon length was longer (5.18cm±0.73cm vs3.93cm±0.22cm, P<0.05); the degree of congestion and edema of the colon wall(1.11±0.45vs2.75±0.50, P<0.05) and infiltration into the mucosasuperficial layers of granulocytes were significantly ameliorated;(2) Livertissue pathology change of H&E and MT dyeing shows that compared withthe control group, lymphocytes and neutrophil of DSS+Vehicle group wereobvious, there have not abnormal pathological changes in the fibers ofDSS+Vehicle group, pathology scores were significantly higher (4.70±0.62vs0.00±0.00, P<0.05), but after MSCs intervention, lymphocytes and neutrophilof DSS+MSCs group obviously reduced, pathology score significantlydecreased (2.56±0.40vs4.70±0.62, P<0.05);(3) The measurement of liverfunction shows, compared with the control group, alanine aminotransferase(ALT) and aspartate aminotransferase (AST) levels in the DSS+Vehicle groupelevated obviously (ALT:56.82U/L±10.40U/L vs34.50U/L±8.23U/L,P<0.05; AST:104.25U/L±17.26U/L vs78.00U/L±10.40U/L, P<0.05),albumin (ALB) level lowered obviously (30.33U/L±1.21U/L vs38.60U/L±1.18U/L. P<0.05), and total bilirubin(TBIL) and direct bilirubin (DBIL)levels increased indistinctively (TBIL:3.08μmol/L±0.57μmol/L vs2.49 μmol/L±0.55μmol/L, P>0.05; DBIL:1.47μmol/L±0.27μmol/L vs1.05μmol/L±0.36μmol/L, P>0.05); But after MSCs intervention, compared withDSS+Vehicle group, ALT and AST levels significantly lowered (ALT:46.00U/L±8.70U/L vs56.82U/L±10.40U/L, P<0.05; AST:87.94U/L±13.51U/Lvs104.25U/L±17.26U/L, P<0.05), ALB level significantly increased (35.43U/L±2.43U/L vs30.33U/L±1.21U/L, P<0.05), and TBIL and DBIL levelslowered indistinctively (TBIL:2.99μmol/L±0.70μmol/L vs3.08μmol/L±0.57μmol/L, P>0.05; DBIL:1.24μmol/L±0.18μmol/L vs1.47μmol/L±0.27μmol/L, P>0.05);(4) The level of serum LPS in DSS+Vehiclegroup significantly increased compared with that of the control group (0.19EU/mL±0.03EU/mL vs0.11EU/mL±0.01EU/mL, P<0.05), while the levelof the LPS remarkedly deseased in DSS+MSCs group compared with that ofDSS+Vehicle group (0.15EU/mL±0.01EU/mL vs0.19EU/mL±0.03EU/mL,P<0.05);(5) Bacterial translocation remarkedly increased in DSS+Vehiclegroup (90%) compared with that of the control group (0%), while bacterialtranslocation to MLN decreased in DSS+MSCs group (15%);(6) The resultsof immunohistochemical staining showed that the expressions of the TNF-α,IFN-γ, IL-1β, IL-17A, TLR4, TRAF6and NF-κB proteins in liver tissuessignificantly increased in DSS+Vehicle group compared with those of thecontrol group (TNF-α:0.79±0.07vs0.20±0.02, P<0.05; IFN-γ:0.82±0.08vs0.15±0.01, P<0.05; IL-1β:0.75±0.08vs0.10±0.01, P<0.05; IL-17A:0.66±0.07vs0.12±0.01, P<0.05; TLR4:0.72±0.08vs0.15±0.02, P<0.05;TRAF6:0.61±0.06vs0.10±0.01, P<0.05; NF-κB:0.67±0.08vs0.13±0.02,P<0.05), while they significantly decreased in DSS+MSCs group comparedwith those of DSS+Vehicle group (TNF-α:0.27±0.03vs0.79±0.08, P<0.05;IFN-γ:0.41±0.05vs0.82±0.08, P<0.05; IL-1β:0.35±0.04vs0.75±0.08,P<0.05; IL-17A:0.35±0.04vs0.66±0.07, P<0.05; TLR4:0.38±0.04vs0.72±0.08, P<0.05; TRAF6:0.29±0.03vs0.61±0.06, P<0.05; NF-κB:0.33±0.04vs0.67±0.08, P<0.05);(2) The results of Western blot and real-timeQ-PCR revealed that the proteins and mRNA expressions of TNF-α, IFN-γ,IL-1β, IL-17A, TLR4, TRAF6and NF-κB in liver tissues significantly increased in the DSS+Vehicle group compared with those of the control group(Proteins:1.50±0.13vs1.05±0.05,1.86±0.10vs1.30±0.06,1.79±0.14vs1.11±0.07,1.51±0.07vs1.01±0.05,2.10±0.26vs1.48±0.17,1.77±0.18vs1.13±0.11,1.86±0.10vs1.42±0.09, P<0.05; mRNA:1.94±0.14vs1.00±0.00,2.11±0.23vs1.00±0.00,2.03±0.19vs1.00±0.00,1.91±0.16vs1.00±0.00,2.46±0.23vs1.00±0.00,1.63±0.20vs1.00±0.00,1.84±0.13vs1.00±0.00,P<0.05), however, after the treatment of MSCs, the proteins and mRNAexpressions of TNF-α, IFN-γ, IL-1β, IL-17A, TLR4, TRAF6and NF-κBsignificantly decreased compared with those of DSS+Vehicle group (Proteins:1.24±0.09vs1.50±0.13,1.43±0.15vs1.86±0.10,1.27±0.13vs1.79±0.14,1.28±0.07vs1.51±0.07,1.40±0.13vs2.10±0.26,1.36±0.15vs1.77±0.18,1.59±0.09vs1.86±0.10, P<0.05; mRNA:1.39±0.17vs1.94±0.14,1.72±0.20vs2.11±0.23,1.33±0.17vs2.03±0.19,1.56±0.15vs1.91±0.16,1.61±0.17vs2.46±0.23,1.46±0.11vs1.63±0.20,1.49±0.19vs1.84±0.13, P<0.05).Conclusions: Chronic colitis-associated hepatobiliary disorders arealleviated by MSCs after intestinal inflammations are ameliorated. Its relevantmechanisms may ameliorate the enterogenous endotoxemia, and thendownregulate the inflammatory lesions of the liver by the mediatation of LPSand TLR4. |
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