Role Of Plasma Exosomes In OSAHS Induced Liver Fibrosis And Its Mechanism

Objectives:1.To investigate the changes of serum liver function and liver fibrosis indicators in patients with Obstructive Sleep Apnea-hypopnea Syndrome（OSAHS）.2.To clarify the effects of OSAHS patients on human hepatic stellate cell LX-2 fibrosis,proliferation,migration and apoptosis via plasma exosomes and the possible molecular mechanisms underlying their effects.Methods.1.The patients attending the Department of Otolaryngology,Second Xiangya Hospital of Central South University from January to June 2021were collected and divided into OSAHS patients and non-OSAHS patients according to the results of polysomnography monitoring,and the liver function and liver fibrosis indexes in the sera were detected,and the relationship between liver function and liver fibrosis indexes and polysomnography monitoring parameters was analyzed statistically.2.Plasma was collected from 10 OSAHS patients and 10non-OSAHS patients,and the exosomes in the plasma were separated using ultracentrifugation,and the two groups of exosomes were treated with human hepatic stellate cells LX-2,and an equal amount of 2%de-exosomal serum medium was used to treat LX-2 cells as a blank control group.The protein and nucleic acid expression levels of fibrosis-related genes FN,COL1A1 and MMP9,apoptotic genes Caspase3,Bax and Bcl-2 were detected in the above three groups of cells by Western-blot as well as qPCR,and the differences in proliferation and migration ability of the three groups of cells were also observed by EdU staining and scratch assay.3.High-throughput sequencing was used to sequence plasma exosomes from OSAHS patients and non-OSAHS patients,respectively,and bioinformatics was used to analyze the differential expression.And the expression levels of differential miRNAs in human hepatic stellate cell LX-2 as well as plasma exosomes were verified by qPCR.4.miR-184 mimics and inhibitors were designed and synthesized.miR-184 mimics and inhibitors were transfected into human hepatic stellate cells LX-2 using liposome transfection reagents.miR-184mimics,miR-184 mimics,inhibitor control and miR-184 inhibitor groups were examined by Western-blot and qPCR,respectively,for cell fibrosis.The protein and nucleic acid expression levels of FN,COL1A1 and MMP9,apoptosis genes Caspase 3,Bax and Bcl-2 were measured by Western blot and qPCR,and the differences in the proliferation and migration ability of the above groups were observed by EdU staining and scratch assay.Results:1.There was no statistical difference in age and sex between OSAHS patients and non-OSAHS patients,and the ALT,AST,ALT/AST,and hemoglobin levels were significantly higher in the moderate and severe OSAHS groups than in the non-OSAHS and mild OSAHS groups（P<0.05）.2.The serum hyaluronidase（Hyaluronan,HA）levels were higher in the mild,moderate and severe OSAHS groups than in the non-OSAHS group.Serum collagen type IV（CIV）and chitinase-3-like protein 1（CHI3L1）levels were significantly higher in the severe OSAHS group than in the non-OSAHS group（P<0.05）.Further correlation analysis showed that HA and CHI3L1 serological levels were positively correlated with Apnea Hypoxia Index（AHI）but negatively correlated with Lowest saturation oxygen（LSaO₂）.3.Compared with the blank control and non-OSAHS patients plasma exosome-treated groups,WB and qPCR results of OSASH patients plasma exosomes treated with human hepatic stellate cells LX-2showed increased expression of fibrosis genes（FN,COL1A1,MMP9）and anti-apoptotic genes Bcl-2 protein and nucleic acid levels,while pro-apoptotic genes Bax and Caspase 3 The results of EdU staining and scoring experiments showed that the proliferation and migration ability were enhanced.4.High-throughput sequencing analysis showed that a total of 8miRNAs were up-regulated and 7 miRNAs were down-regulated in the OSAHS exosome group compared with the non-OSAHS exosome group,with miR-184 being the most significantly differentially expressed in the two groups.5.Compared with the mimic control,miR-184 mimics transfected with human hepatic stellate cells LX-2 showed an increase in protein and nucleic acid expression of fibrosis genes（FN,COL1A1,MMP9）and a decrease in protein and nucleic acid expression of the pro-apoptotic gene Bax and caspase 3 in WB and qPCR.The results showed enhanced proliferation and migration ability.Compared with the inhibitor control group,after transfection of human hepatic stellate cells LX-2 with miR-184 inhibitor,WB and qPCR results showed decreased expression of protein and nucleic acid levels of fibrosis genes（FN,COL1A1,MMP9）,while protein and nucleic acid levels of pro-apoptotic genes Bax and Caspase 3 increased,and EdU staining results showed proliferation ability was decreased.Conclusions.1.OSAHS may induce liver injury,and the severity of liver injury is positively correlated with the severity of OSAHS.2.plasma exosomes from OSAHS patients induce LX-2 activation in human hepatic stellate cells,as evidenced by increased expression levels of fibrotic genes,inhibition of apoptosis,and enhanced cell proliferation and migration.3.miR-184 can cause LX-2 activation in human hepatic stellate cells,as evidenced by increased expression levels of fibrotic genes and enhanced cell proliferation and migration ability.