| Object: To study the function and molecular mechanism of HMGB1-LPS complex of enhancing cell autophagy level in rheumatoid arthritis,and to explore new intervention targets for clinical diagnosis and treatment in RA.Material and methods: In vivo and in vitro experiments were designed to explore and validate the molecular mechanism of HMGB1-LPS complex of enhancing cell autophagy level in RASF.This study mainly consists of two parts:Part 1: Paraffin slices of OA and RA patients were enrolled,the protein expression levels of HMGB1,Atg5,Beclin1 and LC3 were assayed by immunohistochemical staining in synovium tissue of RA and OA patients.Real-Time PCR was applied to detect the expression level of autophagy related proteins(Beclin1,Atg5,LC3).TUNEL apoptosis assay was performed to detect the cell apoptosis level in OASF and RASF of synovium tissue.We also analysed the percentage of positive cells and their prevalent degree of staining in the pathological section of RA and OA patients,the immunohistochemical reaction of synovium tissue sections form the RA and OA patients were scored and analyzed.To analyze the relationship between the HMGB1 expression level in RASF/OASF and the disease activity parameters(CRP,RF,CCP,ESR)in RA/OA patients,and the relationship between the apoptosis/autophagy level in RASF and the disease activity parameters in RA patientsPart 2: OASF and RASF were primary cultured and passaged in six well plates.When cell confluence was approximately 70-80%,it could be used in experiments.HMGB1,LPS,HMGB-LPS complex were added into the cell culture media for 48 hours,then collect the protein of each sample groups.Western Blot were performed to detect the content and grayscale change of LC3Ⅰ,LC3Ⅱ and Beclin1.The autophagy level of each group were measured by transmission electron microscope.RASF and OASF were collected,annexin V-FITC/PI apoptosis assays kit were used to measure the cell apoptosis rate by flow cytometer.At the same time,western blot was performed to detect the expression of Bax and Bcl-2.The cell surface receptors TLR-2,TLR-4 and RAGE were analysed by flow cytometry.The TLR-2,TLR-4 and RAGE si RNA were designed and used to reduce the expression of these receptors.After the down-regulation of these receptors and co-cultured with HMGB1-LPS complex,the autophagy level of RASF were measured.Path Scan Akt signaling antibody array kit were used to identify the signaling pathway.Western Blot were performed to affirm the result of Path Scan Akt signaling antibody array.Immunochemiluminometric assays were performed to detect the expression level of IL-1β,IL-2R,IL-6,IL-8,IL-10 and TNF-α of the cell culture supernatant.Result:1.Clinical characteristics of RA and OA patientsA total of 20 RA and 16 OA patients were enrolled in this study.Serum CRP and ESR levels were significantly higher in RA patients than in OA patients,the serum levels of CRP in all 20 RA patients were higher above 8mg/L,and the ESR levels in 18 out of 20(90%)RA patients were above 20 mm/h.Furthermore,the positive rate of serum CCP in RA patients was 90%(18 out of 20 patients),while its concentration range was 25-273.01 RU/ml.The positive rate of serum RF in RA patients was 95%(19 out of 20 patients),while its concentration range was 25-143.3 KIU/L.Thus,the positive rate of serum CCP and RF were significantly higher in RA patients than in OA patients.2.The expression level of autophagy related proteins and HMGB1 in synovium tissue were significantly higher in RA patients than in OA patientsTo evaluate the differences of autophagy level and HMGB1 expression level between RA and OA patients,real-time PCR and immunochemistry were applied to detect the expression level of autophagy related proteins(Beclin1,Atg5,LC3 and HMGB1).The m RNA relative expression levels of beclin1,Atg5,LC3 and HMGB1 in synovium tissue were statistically higher in RA patients than in OA patients(P<0.001).According to the results of immunochemistry,the protein expression levels of Atg5,Beclin1,LC3 and HMGB1 in synovium tissue were significantly higher in RA patients than in OA patients.The immunohistochemical reaction score of autophagy related proteins(Beclin1,Atg5 and LC3)were significantly higher in RA patients than in OA patients(P<0.001).3.The cell apoptosis level in synovium tissue were significantly higher in OA patients than in RA patientsTo evaluate the cell apoptosis level of synovium tissue in RA and OA patients,we used the In Situ Cell Death Detection kit(POD)to detect the cell apoptosis of synovium tissue in RA and OA patients.The cell apoptosis levels in synovium tissue were statistically lower in RA patients(n=20)than in OA patients(n=16)(P<0.001).The ratio of TUNEL positive cells of synovium tissue sections from the RA and OA patients were scored and analyzed.The TUNEL reaction score of cell apoptosis were significantly higher in OA patients than in RA patients(P<0.001).autophagy related protein and HMGB1 in RASF: 4.The relationship of RA disease activity parameters with the expression level ofIn this study we performed Pearson bivariate correlation analysis to analyze the relationship between the LC3 immunohistochemical reaction scores and the disease activity parameters in RA patients.The results showed that the LC3 immunohistochemical reaction scores were strongly correlated with serum ESR,CRP and HMGB1 levels(the P value were all <0.05),and the LC3 immunohistochemical reaction scores were also strongly correlated with serum CCP and RF levels.(P<0.001,P=0.002).5.The relationship of RA disease activity parameters with the cell apoptosis level of RASF:In this study,we performed Pearson bivariate correlation analysis to analyze the relationship between the TUNEL reaction score of cell apoptosis and the disease activity parameters in RA patients.The results showed that the TUNEL reaction score of cell apoptosis were negatively correlated with serum ESR and CRP levels(the P value were all <0.05),and the TUNEL reaction score of cell apoptosis were also strongly negatively correlated with serum CCP and RF levels.(the P value were all <0.05).6.HMGB1-LPS complex could regulate cell autophagy,apoptosis and the speed of propagation in RASFWestern Blot and transmission electron microscope were performed to detect the autophagy level of each group.The results showed that HMGB-LPS complex could strongly increase the autophagy level of RASF,a lot of autophagosome and autolysosome could be observed by transmission electron microscope.Each group of RASF were subjected to immunofluorescence staining,the expression level of Beclin1 and Atg5 were measured.Annexin V-FITC/PI apoptosis assays kit were used to measure the cell apoptosis rate by flow cytometer.At the same time,western blot was performed to detect the the content and grayscale change of Bax and Bcl-2.The results showed that HMGB-LPS complex could decrease the cell apoptosis level of RASF.Cell cycle analysis kit were used to measure the speed of RASF’s propagation.The results showed that HMGB-LPS complex could induce more RASFs into S/ G2 phase,the speed of RASF’s propagation were accelerated significantly.Thus,HMGB-LPS complex could strongly enhance the autophagy level of RASF,decrease its apoptosis and accelerate the speed of its propagation.7.HMGB1-LPS complex could increase the expression level of cell-surface receptor TLR-4 in RASF,and it could also increase the autophagy level of RASF by binding with TLR-4The expression level of RASF cell-surface receptors: TLR-4,TLR-2 and RAGE were detected by flow cytometry.The results showed that HMGB-LPS complex could significantly increase the expression level of cell-surface receptor TLR-4 in RASF.TLR-4 si RNA(Human),TLR-2 si RNA(Human)and RAGE si RNA(Human)were used to inhibit the expression of TLR-2,TLR-4 and RAGE.HMGB1,LPS,HMGB-LPS complex were added into the cell culture media for 48 hours,then collect the protein of each sample groups.Western Blot were performed to detect the content and grayscale change of LC3Ⅱ/GADPH.The results showed that the down-regulated TLR-2 and RAGE in RASF had no connection with the autophagy level of RASF,while the down-regulated TLR-4 in RASF could strongly decrease the autophagy level of RASF.Thus,HMGB1-LPS complex could increase the expression level of cell-surface receptor TLR-4 in RASF,and it could also increase the autophagy level of RASF by binding with TLR-4.8.HMGB1-LPS complex could activate the autophagy associated signaling pathway: Akt-m TOR,and the inflammatory signaling pathways: NF-κB,p38 MAPKPathScan Akt signaling antibody array kit were used to identify the signaling pathways.Western Blot were performed to affirm the result of Path Scan Akt signaling antibody array.The results showed that HMGB-LPS complex could significantly increase the expression level of Akt Ser473,m TOR Ser2481,p70 S6 Kinase Thr389 in RASF.Beclin1 si RNA(Human)were used to inhibit the expression of Beclin1.HMGB1,LPS,HMGB-LPS complex were added into the cell culture media for 48 hours.The results showed that blocking the expression of Beclin1 in RASF could strongly decrease the expression level of Akt Ser 473、m TOR Ser 2481、p70 S6 Kinase Thr389 in RASF.Western Blot were also performed to detect the expression level of NF-κB,p-NF-κB,p38 MAPK,p-p38 MAPK.The results showed that HMGB-LPS complex could significantly increase the expression level of p-NF-κB,p-p38 MAPK in RASF,while blocking the expression of Beclin1 in RASF strongly decrease the expression level of p-NF-κB,p-p38 MAPK in RASF.9.HMGB1-LPS complex could enhance the proinflammatory fuction of RASF,blocking autophagy could down-regulate the proinflammatory fuction of RASFBeclin1 siRNA(Human)were used to inhibit the expression of Beclin1.HMGB1,LPS,HMGB-LPS complex were added into the cell culture media for 48 hours,then collect cell culture supernatant of each sample groups.Immunochemiluminometric assays were performed to detect the expression level of IL-1β,IL-2R,IL-6,IL-8,IL-10 and TNF-α of the cell culture supernatant.The results showed that IL-1β,IL-2R and IL-10 levels of cell culture supernatant were below the detection limit in all samples tested.HMGB-LPS complex could significantly increase the expression level of IL-6,IL-8 and TNF-α in RASF,while blocking the expression of Beclin1 in RASF could strongly decrease the expression level of IL-6,IL-8 and TNF-α.Conclusion: Based on our preliminary findings,the expression levels of autophagy related proteins(Atg5,Beclin1 and LC3)in synovium tissue were significantly higher in RA patients than healthy people,and were positively correlated with the RA disease activity parameters.The apoptosis level was significantly lower in RASFs than in normal cells,and was negatively correlated with the RA disease activity parameters.There lies a balance disorder between autophagy and apoptosis in RA patients,it could promote the development and progression of RA.In vitro,HMGB-LPS complex were added into the cell culture media of RASF,it could increase the TLR4 expression level on the surface of RASF,it could also activate the autophagy associated signaling pathway: Akt-m TOR,and the inflammatory signaling pathways: NF-κB,p38 MAPK by binding with TLR-4.HMGB-LPS complex can increase the autophagy level in and decrease the apoptosis level in RASF,accelerate the speed of its propagation,and also significantly increase the production of pro-inflammatory cytokines in RASF.Blocking autophagy progress could reduce the activation rate of the autophagy associated signaling pathway,and the inflammatory signaling pathways.Thus,it could down-regulate the proinflammatory fuction of RASF,inhibit the disease-causing capability of RASF.This study is considered to be a promising approach of RA’s therapy. |
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