| Background:On the basis of pathological damage of chronic liver disease,acute on chronic liver failure(ACLF)refers to the occurrence of new necrotic lesions of liver cells,causing serious dysfunction or decompensation of liver function.The"triple strike"composed of immune injury,ischemia hypoxia and endotoxemia is the main driving mechanism in the development of ACLF.Because of its immunosuppressive,anti-inflammatory,and anti-toxic effects,glucocorticoid(GC)has been used in the treatment of liver failure as early as the 1960s.The application of GC in the treatment of liver failure has been controversial at home and abroad,because many clinical studies have found that GC can not improve the overall survival rate of patients with liver failure,and cannot be used to obtain more time for patients with liver failure to wait for liver transplantation,and may increase the risk of infection and sepsis in patients with liver failure.The reason is closely related to the secondary glucocorticoid resistance induced by inflammation.So,it is of great clinical significance to evaluate the degree of ACLF secondary glucocorticoid resistance and reduce or reverse the secondary glucocorticoid resistance.In recent years,many studies on micro RNAs mediated glucocorticoid resistance have been reported.The detection of micro RNAs is very convenient and reproducible,because micro RNAs are stable in mammalian serum,plasma,urine,saliva,amniotic fluid and other body fluids.It has an important guiding value for the individualized glucocorticoid therapy of ACLF,that looking for micro RNAs of mediate secondary glucocorticoid resistance in liver failure to evaluate the GC sensitivity of ACLF patients.Micro RNAs have broad application prospects in the diagnosis and treatment of secondary glucocorticoid resistance in ACLF,which are expected to be biomarkers.Part 1 Predicting micro RNAs that mediate secondary glucocorticoid resistance in liver failureObjection:To explore micro RNAs related to secondary glucocorticoid resistance in liver failure.Methods:1. All research literatures from the origin to June 31,2019 were retrieved separately,which related to micro RNAs-mediated reduction of glucocorticoid sensitivity and micro RNAs expression in liver failure.Two independent reviewers selected the research literature according to the qualification criteria and counted the relevant data.2.The intersection of micro RNAs was obtained from the two data sets of micro RNAs,which change in expression at liver failure and mediate the reduction of glucocorticoid sensitivity.3.According to the purpose of the research,combined with the existing research results and data,we determined several effective evaluation indexes,and selected the optimal micro RNAs by using analytical hierarchy process(AHP).Results:1. A total of 209 abstracts related to micro RNAs-mediated reduction in glucocorticoid sensitivity were retrieved.According to the selection and exclusion criteria of the literature,40 articles were selected.2.A total of 190 abstracts related to changes in micro RNAs expression in liver failure were retrieved.According to the selection and exclusion criteria of literature,40 articles were selected.3.27 micro RNAs were screened out to target 9 related factors in the negative regulation of GC signal transduction pathway,while satisfying the expression changes in liver failure and mediating the reduction of glucocorticoid sensitivity.4.The comprehensive scoring index GI of the above 27 micro RNAs were obtained by AHP.According to the ranking,mi R-124 was selected as the optimal micro RNA.Summary:Through systematic evaluation and AHP,mi R-124 was predicted to mediate secondary glucocorticoid resistance in liver failure by targeting negative regulatory glucocorticoid receptor.Part 2 Significance of changes in glucocorticoid receptor expression in ACLFObjection:To explore the causes of secondary glucocorticoid resistance in ACLF by detecting the expression of GR?and GR? m RNA and protein in ACLF patients.Methods:1. A total of 50 subjects were included in the study,including 13 healthy controls(HC),13 patients with moderate chronic hepatitis B(CHB),and HBV-related chronic acute liver failure(HBV related).Acute on chronic liver failure(HBV-ACLF)in 14 patients and Alcohol related acute liver failure(A-ACLF)patients in 10 patients.2.Peripheral blood of each group was collected,serum and PBMCs were separated,and CD14~+monocytes were sorted.Quantitative reverse transcription–polymerase chain reaction,q RT-PCR)was used to detect the expression of GR?and GR? m RNA in CD14~+monocytes.Flow cytometry was used to detect the relative expression of GR?and GR? protein.In serum,alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBil),direct bilirubin(DB)and C-reactive protein(CRP)were analyzed by biochemical analyzer.3.Five liver tissues of HC patients and ACLF patients were obtained respectively.The expression levels of GR?and GR? m RNA were detected by q RT-PCR.The relative expression of GR?and GR? protein was detected by Western Blot(WB).Results:1. The levels of ALT,AST,TBil,DB and CRP in serum were compared,which were significantly higher in HBV-ACLF group than in CHB group and HC group,which were higher in CHB group than in HC group,which were higher in HBV-ACLF group and A-ACLF group than in HC group(P<0.05or 0.01),which were no significant difference between HBV-ACLF group and A-ACLF group.2. The relative expressions of GR?m RNA and protein in peripheral blood CD14~+monocytes were compared,which were significantly lower in HBV-ACLF group than in CHB group and HC group,which were lower in CHB group than in HC group,which were lower in HBV-ACLF group and A-ACLF group than in HC group(P<0.01),which were no significant difference between HBV-ACLF group and A-ACLF group.3.In peripheral blood CD14~+monocytes,the relative expressions of GR? m RNA and protein of HBV-ACLF group were higher than that of HC group,GR? protein expressions level was higher than that of CHB group,and the relative expressions of GR? m RNA and protein of CHB group were higher than that of HC group(P<0.05 or 0.01).Compared with HC group,the relative expressions of GR? m RNA and protein of HBV-ACLF group and A-ACLF group were significantly higher(P<0.05 or 0.01),but there was no significant difference between HBV-ACLF group and A-ACLF group.4.The relative expressions of GR?m RNA and protein in liver tissue were compared,which were lower in ACLF group than in HC group(P<0.05or 0.01).5.The relative expressions of GR? m RNA and protein in liver tissue were compared,which were higher in ACLF group than in HC group(P<0.05or 0.01).Summary:1.The expressions of GR?in ACLF patients decrease and the expressions of GR? increase,which lead to secondary glucocorticoid resistance in ACLF.2.The expressions of GR?and GR? in HBV-ACLF group and A-ACLF group have no significant different,which suggested that secondary glucocorticoid resistance in ACLF is not related to the etiology of ACLF.Part 3 Significance of mi R-124a expression changes in ACLFObjection:By detecting the relative levels of mi R-124a in plasma,peripheral blood CD14~+monocytes and liver tissues of ACLF patients,the relationship between mi R-124a and clinical indexes and scores of ACLF was analyzed,and the relationship between mi R-124a and the subtypes expression of GR?and GR? was analyzed.Methods:1.Collect and sort out various indicators related to disease progression in the medical records of ACLF patients:ALT,AST,TBil,DB,CRP,prothrombin time activity(PTA),international normalized ratio(INR),serum ammonia(AMON),albumin(ALB),lactate dehydrogenase(LDH),model for end-stage liver disease-Na score,(MELD-Na).2. Interleukin-1? (IL-1? ),interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?)in plasma of HC group,CHB group,HBV-ACLF group and A-ACLF group were detected separately by Enzyme linked immunosorbent assay(ELISA).3.The relative levels of mi R-124a in the plasma of HC group,CHB group,HBV-ACLF group and A-ACLF group were detected separately by q RT-PCR.4.The relative levels of mi R-124a in liver tissue of HC group and ACLF group were detected separately by q RT-PCR.Results:1.The levels of IL-1? ,IL-6 and TNF-?in plasma of HBV-ACLF group were significantly higher than those in the HC group(P<0.01),and the levels of IL-1? ,IL-6 of HBV-ACLF group were higher than those of CHB group(P<0.05 or 0.01).Compared with HC group,the levels of IL-1? ,IL-6 and TNF-?in plasma of HBV-ACLF group and A-ACLF group were significantly higher(P<0.01),but there was no significant difference between HBV-ACLF group and A-ACLF group.2. The relative levels of mi R-124a in plasma were compared,which were significantly higher in HBV-ACLF group than in CHB group and HC group,which were higher in CHB group than in HC group,which were higher in HBV-ACLF group and A-ACLF group than in HC group(P<0.01),which were no significant difference between HBV-ACLF group and A-ACLF group.3. The relative levels of mi R-124a are correlated with IL-1? ,IL-6 and TNF-?in plasma of ACLF patients,which were found by cluster analysis.4.The relative levels of mi R-124a were positively correlated with IL-1? ,IL-6 and TNF-?in plasma of ACLF patients,which were found by multiple regression analysis.5.The relative levels of mi R-124a in peripheral blood CD14~+monocytes were compared,which were significantly higher in HBV-ACLF group than in CHB group and HC group,which were higher in CHB group than in HC group,which were higher in HBV-ACLF group and A-ACLF group than in HC group(P<0.05 or 0.01),which were no significant difference between HBV-ACLF group and A-ACLF group.6.The relative levels of mi R-124a were negatively correlated with the expressions of GR?m RNA and protein,and positively correlated with the expressions of GR? m RNA and protein in in peripheral blood CD14~+monocytes of ACLF patients.7.The relative levels of mi R-124a in liver tissue were compared,which were higher in ACLF group than in HC group(P<0.05 or 0.01).Summary:1.The plasma,CD14~+monocyte and liver tissue mi R-124a levels in ACLF patients were increased.2.The levels of mi R-124a in plasma of ACLF patients were positively correlated with the levels of IL-1? ,IL-6 and TNF-?.3. The levels of mi R-124a in CD14~+monocyte of ACLF patients were negatively correlated with GR?expression and positively correlated with GR? expression.Part 4 Inflammation injury induced secondary glucocorticoid resistance in cells through the mi R-124a-glucocorticoid receptor pathwayObjection:To explore the effect of inflammatory response on the expression of mi R-124a,GR?and GR? in the cell inflammation injury model established by lipopolysaccharide stimulation.Study the regulatory effects of mi R-124a on the expressions of GR?and GR? by cell transfection and mi RNA interference.Methods:1.Human hepatoma cell line Hep G2 cells and human histiocytic lymphoma cell line U937 cells were cultured and stimulated with lipopolysaccharide(LPS)at 0,100,300,and 500ng/m L for 24 hours to establish a model of cellular inflammatory injury.2.The degree of inflammatory damage was evaluated by detecting the levels of IL-1? ,IL-6 and TNF-?in the culture supernatant of each group.3. The expression of GR?and GR? mRNA in Hep G2 cells and U937cells were detected after 24 hours of LPS stimulation by q RT-PCR.4. The levels of mi R-124a in Hep G2 cells and U937 cells were detected after 24 hours of LPS stimulation by q RT-PCR.5.After transfecting mi R-124-3p mimic,mi R-124-3p inhibitor and transfection reagent blank in Hep G2 cells and U937 cells by liposome transient transfection technique for 6 hours,and adding 500 ng/m L LPS to continue cultivate for 24 hours,the levels of mi R-124a and the m RNA expressions of GR?and GR? were detected in each group.Results:1.With LPS stimulation concentration increased,the levels of IL-1? ,IL-6 and TNF-?in the culture supernatant of treatment groups gradually increased.2.With LPS stimulation concentration increased,GR?m RNA expression gradually decreased,and GR? m RNA expression gradually increased in treatment groups.3.With LPS stimulation concentration increased,mi R-124a levels in treatment groups gradually increased.4.In U937 cells and Hep G2 cells,mi R-124a levels increased,the expression of GR?m RNA decreased,and the expression of GR? m RNA increased in the transfected mi R-124-3p mimic group;mi R-124a levels decreased,the expression of GR?m RNA increased,and the expression of GR? m RNA decreased in the transfected mi R-124-3p inhibitor group.Summary:1.LPS induces increased expression levels of inflammatory factors IL-1? ,IL-6,and TNF-?in Hep G2 cells and U937 cells,proving that the cell inflammation injury model has been successfully established.2.The increase of mi R-124a levels in Hep G2 cells and U937 cells were induced by LPS.3.mi R-124a mediates secondary glucocorticoid resistance in cells by negatively regulating GR?expression and positively regulating GR? expression.Part 5 mi R-124a affects alternative splicing of glucocorticoid receptorsObjection:To explore the effect of mi R-124a on the expression of GR?and GR? by detecting the expression of serine/arginine-rich splicing factor(SRSF)in patients with ACLF and studing the regulatory effect of mi R-124a on the expression of SRSF.Methods:1.The m RNA levels of SRSF3 in peripheral blood CD14~+monocytes of HC group,CHB group,HBV-ACLF group and A-ACLF group were detected separately by q RT-PCR.2.The m RNA levels of SRSF3 in in liver tissues of HC group and ACLF group were detected separately by q RT-PCR.3.After adding 0,100,300 and 500 ng/m L LPS for 24 hours,the m RNA levels of SRSF3 in Hep G2 cells and U937 cells were detected separately by q RT-PCR.4.After transfecting mi R-124-3p mimic,mi R-124-3p inhibitor and transfection reagent blank in Hep G2 cells and U937 cells by liposome transient transfection technique for 6 hours,and adding 500 ng/m L LPS to continue cultivate for 24 hours,the m RNA levels of SRSF3 were detected in each group.Results:1.The m RNA levels of SRSF3 in peripheral blood CD14~+monocytes were compared,which were significantly lower in HBV-ACLF group than in HC group,which were lower in CHB group than in HC group,which were lower in HBV-ACLF group and A-ACLF group than in HC group(P<0.05 or0.01),which were no significant difference between HBV-ACLF group and A-ACLF group.Correlation analysis showed that SRSF3 was negatively correlated with mi R-124a(P<0.05).2.The m RNA levels of SRSF3 in liver tissue were compared,which were lower in ACLF group than in HC group(P<0.05 or 0.01).3.With LPS stimulation concentration increased,SRSF3 m RNA levels gradually decreased in treatment groups.4.In U937 cells and Hep G2 cells,the m RNA levels of SRSF3 decreased in the transfected mi R-124-3p mimic group;the m RNA levels of SRSF3increased in the transfected mi R-124-3p inhibitor group.Summary:1.The expressions of SRSF3 m RNA in ACLF patients were down regulated,which were negatively correlated with mi R-124a levels.2.mi R-124a negatively regulated the expression of SRSF3 m RNA,which inhibited GR hn RNA splicing into GR?subtype.Conclusions:1.There was secondary glucocorticoid resistance in ACLF patients,which was which mainly caused by decreasing GR?expression and increasing GR? expression.2.The levels of mi R-124a were increased in ACLF patients,which were positively correlated with the level of inflammatory factors.3.LPS stimulated inflammatory response of U937 cells and Hep G2 cells,which induced the increase of mi R-124a levels.4. mi R-124a negatively regulated GR?expression and positively regulated GR? expression,which mediated the secondary glucocorticoid resistance.5. mi R-124a negatively regulated the expression of SRSF3 m RNA,which inhibited GR hn RNA splicing into GR? subtype. |
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