| Liver fibrosis is a necessary stage that all chronic liver diseases evolve to cirrhosis.Liver fibrosis is charactered by the abnormal of extracellular matrix(ECM)acculmulation in the liver tissue.Hepatic stellate cells(HSCs)activation is a pivotal event in the initiation and progression of liver fibrosis and activated HSCs is a major contributor to ECM deposition.Many evidences confirmed that,several cytokines and mi RNAs participate in the development and progression of liver fibrosis.Interleukin-22(IL-22)can be produced by some immune cells,by binding to the IL-22 receptors at the membrane,IL-22 plays its role through the activation of Signal Transducer and Activator of Transcription(STAT)signaling pathway in the cells.mi RNAs,small non-coding RNAs modulating m RNA and protein expression,have emerged as key regulatory molecules in chronic liver disease.Previous studies have suggested that mi R-200 a down-expression in the liver fibrosis tissue of human beings and rat and mice models,and it is also able to inhibit the cell proliferation,activation of HSCs by targeting the down-stream molecular.Our previous study has showed that IL-22 was able to inhibit HSCs activation and suppress liver fibrosis of mice.However,whether there is a cross talk between IL-22 and mi R-200 a remains unknown.If the relation really exist,how this cross talk interact? Currently,no answer is available.Therefore,in order to further understand the mechanism of IL-22 and mi R-200 a in the suppress of liver fibrosis,and elucidate the cross talk between IL-22 and mi R-200 a in liver fibrosis,in this study,the HSCs and liver fibrosis model were undertook the intervention of IL-22 and mi R-200 a,and the down-stream molecular will be detect,our results will provide a new clue for the treatment of liver fibrosis.Part one IL-22 inhibits HSCs activation and suppress liver fibrosis through STAT3 pathwayObjective:The cell proliferation,apoptosis and secreted effect of HSCs after IL-22 intervention was observed,and the effect of IL-22 on liver fibrosis model of mice was test,in order to explore the mechanism of IL-22 inhibits HSCs activation and suppress liver fibrosis.Methods:The rat HSCs line(HSC-T6)was culture to the exponential phase,then IL-22 with the concentration of 250 pg/m L,500 pg/m L,750 pg/m L and1000pg/m L was added to the medium respectively for 48 hours.TGF-?1 with the concentration of 0,2 ng/m L,5 ng/m L was added to the medium for 24 hours for activating HSCs.CCK8 method was used to test the cells proliferation.Flow cytometry was used to test the cells apoptosis.ELISA method was used to test the expression of ?-smooth muscle actin(?-SMA)in the culture supernatant.western-blot method was used to test the protein levels of STAT3 and p-STAT3 in HSCs.Rats were intraperitoneally injected with CCl4 for 8 weeks,then IL-22 was intraperitoneally injected into the the liver fibrosis,HE and Massion stains and Ishak scores system were used to evaluate the degree of liver fibrosis.Immunohistochemical methods was used to test the expression of ?-SMA in the liver tissue.Results:Treatment with higher concentration of IL-22 protein showed stronger inhibitory effect on the proliferation of HSCs(P<0.05).In contrast,the apoptosis rate of HSCs did not change significantly in response to incubation with different concentrations of IL-22 protein.Treatment with either 750 or1000 pg/m L IL-22 significantly induced expression of phosphorylated STAT(p-STAT3)but not STAT3.ELISA showed that the expression of ?-SMA in the culture supernatant was decreased along with the increased of IL-22,the expression of ?-SMA was significantly increased at the concentration of 750pg/m L compared with without IL-22 intervention.The cells proliferation of HSCs was increased with the elevated of TGF-?1,and was significantly increased at the concentration of 5ng/m L compared with without TGF-?1intervention(P<0.05).Expression of STAT3 protein did not change significantly with the intervention of TGF-?1,however,Expression of p-STAT3 protein was deceased clearly after intervention of TGF-?1(P<0.05).Using TGF-?1 pre-treatment HSCs for 12 hours,then IL-22 was used to intervene the HSCs,we found that,expression of STAT3 protein did not change significantly,but expression of p-STAT3 protein showed greatly increased compared with that without IL-22 intervention.In the rat liver fibrosis experiment,we observed that IL-22 was able to significantly ameliorate liver fibrosis compared with that without IL-22 intraperitoneally injection(P<0.05).The expression of STAT3 protein in the liver tissue did not change significantly after IL-22 intraperitoneally injection,but expression of p-STAT3 protein showed greatly increased.Conclusions:IL-22 inhibits the proliferation and activation of HSCs in a concentration dependent manner,but has little effect on the apoptosis of HSCs.IL-22 is able to significantly ameliorate liver fibrosis through the activation of p-STAT3 signaling pathway.Part two mi R-200 a inhibits HSCs proliferation and activation by targeting ?-cateninObjective:To clarify the mechanism of mi R-200 a inhibits HSCs proliferation and activation by targeting ?-catenin,expression of mi R-200 a in HSCs and liver tissue of fibrosis was tested,and the effect of over expression of mi R-200 a on HSCs was observed.Dual luciferase reporter system was used to verify the the effect of mi R-200 a that targeting ?-catenin,and this effect was further verify in HSCs.Methods:TGF-?1 with the concentration of 0 and 5 ng/m L was added to the medium for 24 hours for activating HSCs,then the expression of mi R-200 a in HSCs was tested by RT-PCR.mi R-200 a mimics was transfected into HSCs by lentivirus,CCK8 method was used to test the cells proliferation.Flow cytometry was used to test the cells apoptosis.ELISA method was used to test the expression of ?-SMA in the culture supernatant.western-blot method was used to test the protein levels of ?-catenin in HSCs.Results:After HSCs activation by incubation with TGF-?1,expression of mi R-200 a remarkably decreased,while expression of ?-catenin significantly increased in m RNA and protein levels.The RT-PCR showed that,expression of mi R-200 a was increased over 8 folds after transfection of mi R-200 a mimics compared with that without transfection.Overexpression of mi R-200 a by transfection of mi R-200 a mimics inhibited HSCs proliferation.However,there was no effect on the apoptosis of HSCs after mi R-200 a overexpression.The expression of?-SMA in the culture supernatant was after transfection of mi R-200 a mimics.Target prediction for mi R-200 a suggested that it regulates the expression of?-catenin through a potential seed region in 3'UTR.Using the dual luciferase reporter system,we obtained luciferase-3'UTR reporter constructs for the m RNA and transfected them into HEK293 T cells together with mi R-200 a mimics or a non-targeting control mi RNA.Transfection with mi R-200 a significantly reduced firefly luciferase activity for ?-catenin compared to the negative control.3'UTR mutagenesis of sequence complementary to the mi R-200 a seed region attenuated mi RNA effect,suggesting that ?-catenin is a directly regulated by mi R-200 a in HSCs.The expression of ?-catenin protein levels in HSCs was decreased significantly after transfection of mi R-200 a mimics.Conclusions:Expression of mi R-200 a is decreased during the activation of HSCs,and over expression of mi R-200 a in HSCs can inhibit the proliferation and activation HSCs.?-catenin is a direct target of mi R-200 a.mi R-200 a inhibits HSCs by targeting ?-catenin.Part three Cross-talk between IL-22 and mi R-200 a in HSCs and liver fibrosis tissueObjective:To elucidate the cross talk between IL-22 and mi R-200 a in HSCs and liver fibrosis tissue,the down stream moleculars of IL-22 and mi R-200 a were tested after IL-22 and mi R-200 a inhibitors intervene HSCs or injecting into rat liver fibrosis models.Methods:IL-22 was used to intervene HSCs that activated by TGF-?1,the expression of mi R-200 a and ?-catenin in HSCs was tested by RT-PCR and western-blot,respectively.mi R-200 a inhibitors was transfected into HSCs by lentivirus,then the IL-22 was added to the medium,the proliferation of HSCs,and the expression of ?-SMA in the culture supernatant was tested by ELISA,and the expression of STAT3 and p-STAT3 in HSCs was tested by western-blot.Rats liver fibrosis model was established by intraperitoneally injected with CCl4 for8 weeks,then IL-22 was intraperitoneally injected into the rat,and the mi R-200 a inhibitors was injected via rat tail.HE and Massion stains and Ishak scroes system were used to evaluate the degree of liver fibrosis.Immunohistochemical methods was used to test the expression of ?-SMA in the liver tissue.The expression of ?-catenin,STAT3 and p-STAT3 in the liver tissue was tested by western-blot.Results:HSCs was actived by TGF-?1 for 12 hours,then IL-22 was added to the medium,we observed that mi R-200 a was incrased significantly compared with that without IL-22 intervened,while the expression of ?-catenin protein was derecased.Expression of mi R-200 a was decreased in HSCs that transfected with mi R-200 a inhibitors compared that without mi R-200 a inhibitors transfection,and the ?-catenin was increased.We also found that p-STAT3 was decreased while STAT3 has no obviously changed after mi R-200 a inhibitors transfection.After the establishment of mice liver fibrosis model,IL-22 and mi R-200 a inhibitors and injected into the models alone or together.We observed that the effect of suppress of liver fibrosis of IL-22 was partly offset by mi R-200 a inhibitors.The expression of ?-catenin was relatively increased,while p-STAT3 was decreased in the rat models of combination of IL-22 and mi R-200 a inhibitors compared with that with IL-22 injected alone.Conclusions:IL-22 is able to increased the expression of mi R-200 a and reduced the expression of ?-catenin in HSCs,while inhibited the expression of mi R-200 a can block the effect of IL-22 on HSCs and decreased the expression of p-STAT3.The suppress liver fibrosis effect of IL-22 can be blocked by the inhibition of mi R-200 a,and the expression of p-STAT3 was inhibited either. |
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