The Effect Of HUC-MSCs On Proliferation Of The HSCs And Collagen Metabolism In Liver Fibrotic And Cirrhotic Rats

Hepatic fibrosis (HF) is a repair reaction of hepatic disease caused bychronic inflammation, necrosis or other damage. Liver cirrhosis (LC) is thefinal end of hepatic fibrosis. Its main characteristics are the increased oversynthesis and irregular deposition of extracellular matrix (ECM). At present, itis believed that hepatic stellate cells (HSCs) are the original cell of liverextracellular matrix formation which can produce mass of ECM and cytokineswith plenty of collagen typeⅠ, Ⅲ to make liver fibrosis by activation,proliferation, proliferation and migration, expression of varioussignal-transducing protein. Therefore, restrain HSCs activation and relatedcollagen metabolism is the target for the treatment of liver fibrosis andcirrhosis.Currently, Liver transplantation is the only effective method for thetreatment of end-stage liver disease. However, Donor scarcity, immunologicalrejection and other reasons limit the clinical application of livertransplantation. Therefore, it needs to find a new and effective treatmentmethod. In recent years, with stem cell transplantation for the treatment inliver diseases, and Because of it's rich source, low immunogenicity, smalltraumatic, simple operation etc, it has become an important means in thetreatment of liver disease, and make the regeneration treatment of the liverdisease into a landmark stage.Mesenchymal stem cells (MSCs) are a group of stem cells deriving fromMesoderm mesenchymal with multiple differentiatral potential. MSCs can berecovered from a variety of tissues, including bone marrow, umbilical cordtissue, umbilical cord blood, peripheral blood and adipose tissue et al. MSCshave many advantages, such as easy to extract, low immunogenicity andimmunosuppressive effects, which avoid donor scarcity and immunological rejection compared to liver transplantation. Then MSCs is considered to be themost therapeutic potential donor cells. Research has shown that BM-MSCscan inhibit activation and proliferation of HSCs, prompting HSCs apoptosis,reduce the generation of ECM, decrease the content of collagen typeⅠ, Ⅲ,Thus improving liver fibrosis and cirrhosis. However, human umbilicalcord-derived MSCs as excellent source of MSCs transplantation, the researchin the treatment of liver fibrosis and cirrhosis are few. For this, we constructedrat liver fibrosis and cirrhosis model by intravenous injection of hUC-MSCsto observe the therapeutic effect of hUC-MSCs on liver fibrosis and cirrhosis,discuss the function and mechanism of HSCs in this process, and provideexperimental and theoretical basis for clinical treatment of liver fibrosis andcirrhosis.Objective: To research the therapeutic effect of hUC-MSCs on liverfibrosis and cirrhosis, and discuss the function and mechanism of HSCs inthis process.Methods:120Male Wistar rats were randomly divided into followinggroups: control group: CCl₄/saline0wk; model group (liver fibrosis modelgroup: CCl₄/saline4wks,5wks,7wks; liver cirrhosis model group:CCl₄/saline8wks,10wks,14wks); MSCs transplantation group (liverfibrosis MSCs transplantation group: CCl₄/MSCs1wk,2wks,4wks; livercirrhosis MSCs transplantation group: CCl₄/MSCs2wks,4wks,8wks). n=8.The rat model of liver fibrosis and cirrhosis were established by hypodermicinjection of CCl₄mixed with olive oil at the concentration of40%(2ml/kg,twice a week). After the modeling, model group and MSCs transplantationgroup continued hypodermic injection of CCl₄twice weekly, until executed.The MSCs transplantation group of liver fibrosis and cirrhosis accept theintravenous injection of the hUC-MSCs (5×106cells), after the model weresuccessful (4weeks and7weeks respectively), the rats were killed after MSCsinjection at1,2,4weeks and2,4,8weeks.Serological tests were used to detect the ALT and AST, ALB, TBIL andDBIL in rats and to observe the effect of hUC-MSCs transplantation on rat liver function; Haematoxylin and eosin staining (H&E staining) and sirius redstaining were used to detect liver histopathological changes; The expression ofα-SMA in liver tissues were measured by immunohistochemistry. Theexpression of collagen typeⅠ, collagen type Ⅲ, MMP-13and TIMP-1protein and mRNA in liver tissues were observed by immunohistochemistry,Western blot and real-time Q-PCR respectively.Results:(1) H&E and sirius red staining showed that: CCl₄induction ofrat liver fibrosis and cirrhosis model built successful, with the administratraltime prolonged, degeneration and necrosis of the hepatocytes and depositionof the intrahepatic fibrous tissue gradually increased in CCl₄/saline group;compared to CCl₄/saline group, the degeneration and necrosis of hepatocytesdecreased in CCl₄/MSCs group, The inflammatory score and the quantitativeanalysis of fibrous tissue showed that except liver fibrosis MSCstransplantation CCl₄/MSCs1wk group has no significant decrease comparedwith that in model group, liver fibrosis MSCs transplantation CCl₄/MSCs2wks group,4wks group and liver cirrhosis MSCs transplantation group hassignificant differences compared with that in model group.(2) Serological testshow that: compared with that in model group, liver function in liver fibrosisMSCs transplantation CCl₄/MSCs1wk group has no significant improvement.In liver fibrosis MSCs transplantation CCl₄/MSCs2wks group, ALT, ASTwas significantly improved,while ALB, TBIL and DBIL has no significantdifferences between MSCs transplantation group and model group. In livercirrhosis MSCs transplantation CCl₄/MSCs2wks group, compared with thatin model group, ALB has no significant increase. In the rest MSCstransplantation groups, liver function was significantly improved at each timepoint than that in the model group.(3) In normal rat liver, α-SMA wasoccasionally detected in vascular smooth muscle cells, and the expressionlevel was low, revealing little activated HSCs. After CCl₄administration, theα-SMA spread to the portal area, showing more activated HSCs. The positiveoptical density of α-SMA in CCl₄/saline group significantly increasedcompared with that in the control group, P<0.01. after hUC-MSCs transplantation, the expression of α-SMA except liver fibrosis MSCstransplantation CCl₄/MSCs1wk group has no significant decrease comparedwith that in model group, liver fibrosis MSCs transplantation CCl₄/MSCs2wks group,4wks group and liver cirrhosis MSCs transplantation group hassignificant differences compared with that in model group. However, betweenMSCs transplantation group, with the time of transplantation extended, theexpression of this index were gradually increased.(4) Immunohistochemistryshowed that: In normal rat liver, collagen typeⅠ, collagen type Ⅲ, MMP-13,TIMP-1were expressed only on the portal area, with the time of CCl₄extended, collagen typeⅠ, collagen type Ⅲ, MMP-13, TIMP-1graduallyincreased both in Model groups and MSCs transplantation groups. after thesame CCl₄Injection, MMP-13in MSCs transplantation groups weresignificantly increased, while collagen typeⅠ, collagen type Ⅲ and TIMP-1were significantly decreased, compared with that in the Model group.(5)Using Western blot method, we compared the expression of collagen typeⅠ,collagen type Ⅲ, MMP-13and TIMP-1protein expression in each group of ratliver tissue, Result suggested that, after the hUC-MSCs transplantation, theexpression of the collagen typeⅠ, collagen type Ⅲ, MMP-13and TIMP-1protein have significant differences between the model group and the MSCstranspatation group, except the liver fibrosis MSCs transplantation CCl₄/MSCs1wk group, the expression of the MMP-13protein significantly increased,while the expression of the collagen typeⅠ, collagen type Ⅲ, TIMP-1proteinsignificantly decreased.(6) Using the relative quantification of2-△△Ctmethod,we compared the expression of collagen type Ⅰ, Ⅲ, MMP-13and TIMP-1mRNA expression in each group of rat liver tissue, real time Q-PCR suggestedthat, after the hUC-MSCs transplantation, the expression of the collagentypeⅠ, collagen type Ⅲ, MMP-13and TIMP-1mRNA have significantdifferences between the model group and the MSCs transpatation group,except the liver fibrosis MSCs transplantation CCl₄/MSCs1wk group, theexpression of the MMP-13mRNA significantly increased, while theexpression of the collagen type Ⅰ, Ⅲ, TIMP-1mRNA significantly decreased.Conclusion: MSCs transplantation can improve liver function in liverfibrosis and liver cirrhosis rats, reduce the expression of α-SMA in liver tissue,inhibit the proliferation of the activated HSCs, significantly reduce the contentof collagen typeⅠ and collagen type III; MSCs play a role in the treatment ofliver fibrosis and cirrhosis through upregulating the expression of MMP-13,lowering the expression of TIMP-1; MSCs Transplantation needs a stage toplay a role in the body, with the continued presence of pathogenic factors,MSCs transplantation can not reverse liver fibrosis or cirrhosis, only delay theprocess of liver fibrosis or cirrhosis.