FNDC5/irisin Ameliorates Liver Fibrosis Via Hepatic Stellate Cell-derived Extracellular Vesicles And Endoplasmic Reticulum Stress

Part Ⅰ FNDC5/irisin inhibits extracellular vesicles release by promoting ubiquitination and degradation of Rab27 b in hepatic stellate cellsObjective:Liver fibrosis is an abnormal repair process in the liver in response to injury stimuli,characterized by activation of hepatic stellate cells（HSCs）and deposition of extracellular matrix（ECM）.Extracellular vesicles（EVs）,as key mediators of intercellular communication,have been shown to be involved in the regulation of liver fibrosis.Irisin is a fragment secreted by fibronectin type Ⅲ domain containing protein 5（FNDC5）cleavage and is an actin mainly released from skeletal muscle into circulating blood.Recent studies have found that irisin may be involved in the regulation of liver fibrosis in mice with nonalcoholic fatty liver disease.However,how irisin alleviates liver fibrosis and its molecular mechanism,and the regulatory mechanism of irisin on HSCs activation and EVs release were still unclear.To this end,this study observed the effect of FNDC5/irisin on the gene expression profile of HSCs,and explored the effect of irisin and its molecular mechanism from the perspective of HSCs-derived EVs.Methods:（1）Primary HSCs were isolated from healthy C57BL/6 mice livers,and the size distribution of EVs was analyzed by dynamic light scattering（DLS）using a Zetasizer Nano series particle size analyzer.（2）Mouse primary HSCs were treated with three different treatment methods（solvent PBS,20 ng/m L PDGF-BB,20 ng/m L PDGF-BB+100 ng/m L irisin）for 12 h,and separated by ultra-centrifugation for EVs,nanoparticle tracking analysis（NTA）technology was used to detect the size,shape and quantity of EVs released by PDGF-stimulated and activated HSCs,and Western blotting（WB）was used to detect markers that distinguish EVs from microvesicles.The expression levels of TSG101 and CD81 were verified and the effect of irisin on the EVs release of HSCs stimulated and activated by PDGF was also observed.（3）Total RNA of HSCs in each treatment group was extracted,RNA was detected by Agilent biospectrum analyzer,RNA-SEQ（transcriptome sequencing technology）library was generated by Illumina m RNA Tru SEQ Directed Library Kit and sequenced by Illumina Hi SEQ4000.RNA-seq was used to screen the differential expression gene profiles of primary HSCs of mice treated in three different ways,and gene aggregation enrichment analysis（GSEA）was used to analyze the related signaling pathways,to observe the effect of irisin on the expression profile of PDGF-BB stimulated activated HSCs gene,and to explore the possible regulatory mechanism and target genes.WB was used to verify the expression of Pdgfra and Pdgfrb proteins in HSCs and their released EVs after three different treatments.（4）The morphology and number of multivesicular bodies（MVB）and intraluminal vesicles（ILV）in primary HSCs of mice after three different treatments were observed by transmission electron microscope（TEM）.The subcellular distribution of MVB in HSCs after three different treatments was analyzed by immunofluorescence（IF）technique using CD63 as a marker of MVB.The expression of CD63 protein in HSCs was verified by WB detection.（5）Protein expression of Rab GTPase family members in mouse primary HSCs after three different treatments was analyzed by protein mass spectrometry（PMS）,and Rab27 a and Rab27 b protein expressions in HSCs after three different treatments were detected by WB.The m RNA expression levels of Rab27 a and Rab27 b in HSCs were analyzed by real-time quantitative polymerase chain reaction（q RT-PCR）.（6）The protein synthesis inhibitor cycloheximide（50 μg/m L CHX）,the autophagy inhibitor bafilomycin A1（10 μmol/L Baf A1）and the ubiquitin protease inhibitor lactosin（10 μmol/L Lac）mouse primary HSCs were pretreated for 8 h,and then treated with 20 ng/m L PDGF-BB and 20 ng/m L PDGF-BB+100 ng/m L irisin for12 h,respectively.HSCs from each treatment group were collected,and Rab27 b protein expression was detected by WB.The Rab27 b ubiquitination level in mouse primary HSCs was further detected by co-immunoprecipitation（Co-IP）.Results:（1）Zetasizer Nano series particle size analyzer found that the average diameter of EVs released by HSCs was about 100 nm through dynamic light scattering（DLS）analysis.（2）Nanoparticle tracking analysis（NTA）technology shown that compared with the PBS group,the number of EVs in the PDGF-BB group was significantly increased（P<0.05）,Compared with the PDGF-BB group,the number of EVs in the irisin group was significantly reduced（P<0.05）,however,the size and morphology of EVs were not significantly affected by different treatment groups（P>0.05）.The results of WB detection showed that compared with the cell lysate control,the protein levels of TSG101 and CD81 in EVs were significantly increased,and the endoplasmic reticulum protein Calnexin was not detected in the EVs,compared with the control group,the concentrations of TSG101 and CD81 in the EVs of the PDGF-BB group were significantly increased（P<0.05）,the protein expressions of TSG101 and CD81 in the EVs of the PDGF-BB+irisin group were significantly lower than those of the PDGF-BB group（P<0.05）.（3）RNA-Seq shown that there were 3,611 genes with significantly different expression between PDGF-BB group and PDGF-BB+irisin group,of which 1,556 genes were down-regulated and 2,055 genes were up-regulated.KEGG pathway analysis found that differentially expressed genes were significantly enriched in pro-inflammatory and pro-fibrosis-related pathways.The results of WB verification showed that the expression levels of Pdgfra and Pdgfrb proteins in HSCs and their released EVs after PDGF-BB treatment were significantly increased,but PDGF-BB+irisin treatment only reversed HSCs and their released EVs.The increase in Pdgfra expression in vivo did not affect the increase in Pdgfrb expression.（4）Transmission electron microscopy（TEM）observation showed that the number of MVB in HSCs and the number of ILV in MVB in PDGF-BB and PDGF-BB+irisin groups were significantly increased compared with the control group（P<0.05）.The number of MVB in HSCs and the number of ILV in MVB in PDGF-BB+irisin group increased more significantly than that in PDGF-BB group,and the difference was statistically significant（P<0.05）.Immunofluorescence（IF）results showed that compared with the control group,the number of CD63 positive spots in each HSCs in PDGF-BB and PDGF-BB+irisin groups was significantly increased（P<0.05）,and the number of CD63 positive spots in PDGF-BB+irisin group was more significantly increased than that in PDGF-BB group.The difference was statistically significant（P<0.05）.WB assay also showed that the expression level of CD63 protein in HSCs of irisin treatment group was significantly increased（P<0.05）.（5）Protein mass spectrometry（PMS）and WB assay showed that the expression levels of Rab27 a and Rab27 b in the primary HSCs of mice in PDGF-BB group were significantly increased（P<0.05）,especially Rab27 b.Compared with PDGF-BB group,the expression of Rab27 b protein in PDGF-BB+irisin group was significantly decreased（P<0.05）,but the expression of Rab27 a was not significantly decreased.However,q RT-PCR results showed that the expression of Rab27 a m RNA in HSCs in PDGF-BB group was slightly increased compared with the control group,but the difference was not statistically significant（P>0.05）,while the expression of Rab27 b m RNA was significantly increased compared with the control group（P<0.05）.Compared with PDGF-BB group,m RNA expression levels of Rab27 a and Rab27 b in PDGF-BB+irisin group did not change significantly（P>0.05）.（6）WB assay results showed that the expression level of Rab27 b protein in HSCs of CHX and CHX+Baf A1 groups was significantly decreased compared with the control group（P<0.05）,regardless of whether PDGF-BB and PDGF-BB+irisin treatment was used.Compared with CHX group and CHX+Baf A1 group,Rab27 b protein expression level in HSCs was significantly increased in CHX+Lac group（P<0.05）.The results of Co-IP experiment showed that compared with PDGF-BB group,Rab27 b protein expression level of HSCs in PDGF-BB+irisin group,PDGF-BB+Lac group and PDGF-BB+irisin+Lac group was significantly higher（P<0.05）.Compared with PDGF-BB+irisin+Lac and PDGF-BB+irisin+Lac groups,Rab27 b protein expression in HSCs was significantly higher（P<0.05）.These results indicated that Lac could inhibit Rab27 b degradation,but Baf A1 could not.The ubiquitin level of Rab27 b in irisin treatment group was significantly increased regardless of Lac（P<0.05）.Conclusions:（1）Irisin can reduce the release of HSCs-derived EVs induced by PDGF-BB activation,but has no effect on the morphology and size of EVs.（2）Irisin inhibits the transmembrane process of ILV in MVB in HSCs by increasing the ubiquitination and degradation of Rab27 b,which may be one of the molecular mechanisms by which irisin affects the number of EVs released from HSCs.Part Ⅱ FNDC5/irisin inhibits hepatic stellate cell activation by inhibiting the release of extracellular vesicles from hepatic stellate cellsObjective:At present,studies have shown that EVs derived from HSCs may be involved in the process of liver fibrosis,but how EVs participate in the regulation of the development of liver fibrosis in this process and the role of activated HSCs EVs after irisin treatment remains unclear.Therefore,this part focuses on the dynamic process of EVs generated by activated HSCs being taken up by recipient HSCs,and further discusses the influence of EVs derived from activated HSCs on the migration ability of recipient HSCs,and discusses the regulation of HSCs-derived EVs after irisin treatment on the activation of recipient HSCs.Methods:（1）The primary HSCs were isolated from the liver of healthy C57BL/6 mice according to the method in the first part,and 100 μg（about 1 × 10⁹ EVs）of different groups of EVs were obtained by different treatment of the primary HSCs of the mice.After labeling them with PKH26,they were incubated with mouse primary HSCs（1 ×10⁶）for 1 h,3 h,and 6 h,respectively,and the endocytosis of recipient HSCs into EVs was observed by immunofluorescence.（2）Different groups of EVs were isolated after treating primary mouse HSCs for12 h with three different treatment methods（solvent PBS,20 ng/m L PDGF-BB,20ng/m L PDGF-BB+100 ng/m L irisin）respectively.Take 100 μg respectively（about 1× 10⁹）and incubate with 1 × 10⁶ mouse primary HSCs for 24 hours,collected HSCs from each group,and detected the following indicators:（a）The expression of receptor HSCs Pdgfra protein in EVs obtained by different treatments was detected by WB.（b）q RT-PCR was used to detect the gene expression related to extracellular matrix proteins（α-SMA and Collagen I）and inflammatory factors（TNF-α and IL-1β）of receptor HSCs in each group to verify that different treatments activate HSCs-derived EVs regulation of receptor HSCs activation.（3）Scratch test was used to observe the effect of EVs obtained by different treatments on the migration ability of recipient HSCs.Images were taken at 0 h and24 h,respectively,and Image J software was used to measure the cell boundary distance.Results:（1）Recipient HSCs were incubated with PKH26-labeled donor HSCs-derived EVs for 1 h,3 h,and 6 h,respectively.The immunofluorescence results showed that EVs were taken up by recipient HSCs at 1 h,and the uptake increased significantly after 3 h.Continue to reach the maximum at 6h,which is time-dependent.（2）When the receptor HSCs were co-incubated with EVs derived from HSCs treated with three different treatments,the protein expression level of Pdgfra in the activated（PDGF-BB EVs）group was significantly increased compared with blank（Vehicle EVs）group（P<0.05）.Compared with activated（PDGF-BB EVs）group,the protein expression level of Pdgfra in receptor HSCs in irisin-treated（PDGF-BB+irisin EVs）group was significantly decreased（P<0.05）.（3）The results of qRT-PCR analysis showed that the m RNA expressions ofα-SMA,Collagen I,TNF-α and IL-1β of receptor HSCs in activated（PDGF-BB EVs）group were significantly increased compared with control（Vehicle EVs）group（P<0.05）.Compared with activated（PDGF-BB EVs）group,the m RNA expressions of α-SMA Collagen I,TNF-α and IL-1β of receptor HSCs in irisin（PDGF-BB+irisin EVs）group were significantly decreased（P<0.05）.（4）The scratch test results showed that compared with the control（Vehicle EVs）group,the migration ability of receptor HSCs in the activated（PDGF-BB EVs）group was significantly improved（P<0.05）,Compared with the activated（PDGF-BB EVs）group,the migration ability of recipient HSCs was significantly decreased（P<0.05）.Conclusions:Primary mouse HSCs showed time-dependent endocytosis to EVs derived from activated HSCs.The EVs derived from activated HSCs treated with irisin can be taken up by the recipient HSCs and significantly inhibit the activation of the recipient HSCs through paracrine.This effect of irisin may be related to the fact that the EVs regulate the signal transduction of the recipient HSCs Pdgfra and change the gene expression of fibrosis and inflammation.Part Ⅲ Intraperitoneal injection of FNDC5/irisin ameliorated CCl₄-induced liver fibrosis in mice by regulating the distribution of immune cells and inflammatory factor genes in the liverObjective:Previous studies have shown that irisin can inhibit the release of EVs from activated HSCs by regulating the ubiquitination of Rab27 b protein in activated HSCs in vitro,at the same time,the EVs released from irisin-treated activated HSCs could inhibit the activation of receptor HSCs in a paracrine manner.The role of HSCs activation.In order to explore whether irisin can improve liver fibrosis in mice in vivo,in this part,the liver fibrosis models of wild mice and FNDC5 knockout(FNDC5^-/-)mice were constructed to observe the effect of irisin on CCl₄-induced liver fibrosis in mice.Fibrosis-modifying effects and possible mechanisms of action.Methods:18 wild mice and 18 FNDC5^-/-mice were randomly divided into three groups:olive oil control group,CCl₄ group and CCl₄⁺irisin treatment group.Mice in olive oil control group were intraperitoneally injected with the same amount of olive oil twice a week,and mice in CCl₄ group and CCl₄⁺irisin group were intraperitoneally injected with 0.01 m L/g olive oil containing 10% CCl₄ twice a week,for modeling for 8weeks.From the first week of the experiment,mice in the CCl₄⁺irisin group were intraperitoneally injected with 0.5 mg/kg/d of recombinant human irisin prepared by normal saline,and mice in the olive oil control group and the CCl₄ group were intraperitoneally injected with the same amount of normal saline.Finally,24 h after administration,all mice were anesthetized with pentobarbital（100 mg/kg）and sacrificed,and their blood and liver were collected.The serum was separated,the right lobe tissue of each mouse was about 3 mm × 3 mm,fixed with 4% neutral formaldehyde,and the rest of the liver tissue was stored in a refrigerator at-80℃.Detect the following indicators:（1）Routine pathological sections were stained with HE,Masson and Tianwolf scarlet to observe the pathological changes of liver in each group.（2）m RNA and protein expressions of Rab27 b and Pdgfra in liver of mice in each group were detected by q RT-PCR and WB,respectively.（3）The expression of extracellular matrix proteins α-SMA and Collagen I in liver of mice in each group was detected by immunofluorescence and WB.（4）The m RNA expressions of extracellular matrix proteins α-SMA and Collagen I and inflammatory cytokines TNF-α and IL-1β in liver of mice in each group were detected by q RT-PCR.（5）The levels of CD4⁺ T lymphocytes,CD8⁺ T lymphocytes,M1（CD11c⁺）macrophages and M2（CD206⁺）macrophages in liver of mice in each group were detected by flow cytometry.Results:（1）In wild-type mice,histopathological observation under HE,Masson and Tianwolf scarlet staining revealed obvious hepatic steatosis,fibrous connective tissue hyperplasia and obvious hepatic fibrosis in the liver of CCl₄ group compared with the control group,The degree of hepatocyte degeneration and fibrosis in irisin-treated mice was significantly reduced compared with that in CCl₄-induced mice.In FNDC5^-/-mice,liver steatosis and fibrosis of mice in the CCl₄ group were significantly higher than those in the corresponding control group,and the fibrosis was significantly more serious than that in the wild-type CCl₄ group.Irisin treatment significantly improved CCl₄-induced liver fibrosis in FNDC5^-/-mice,but not as much as in wild-type mice.（2）In wild-type and FNDC5^-/-mice,the expression of Rab27 b m RNA and Pdgfra m RNA in liver of CCl₄ group were significantly increased compared with olive oil group（P<0.05）.Compared with CCl₄ group of wild-type mice,the expressions of Rab27 b m RNA and Pdgfra m RNA in liver of CCl₄ group of FNDC5^-/-mice were more significantly increased（P<0.05）.In wild-type and FNDC5^-/-mice,the expression of Rab27 b m RNA in liver of CCl₄⁺irisin group was not significantly changed compared with that of CCl₄ group（P>0.05）,but the expression of Pdgfra m RNA was significantly decreased compared with that of CCl₄（P<0.05）,but it was still higher than that of control group.WB test results showed that in wild-type and FNDC5^-/-mice,compared with olive oil group,the expression of Rab27 b and Pdgfra protein in liver of CCl₄ group was significantly increased（P<0.05）,especially in FNDC5^-/-mice.The protein expression level of Rab27 b in liver of CCl₄⁺irisin group was significantly lower than that of CCl₄ group（P<0.05）,and the decrease of FNDC5^-/-mice was more obvious.In wild-type mice,the protein expression level of Pdgfra in liver of CCl₄⁺irisin group was significantly lower than that of CCl₄ group（P<0.05）.In FNDC5^-/-mice,the protein expression level of Pdgfra in liver of CCl₄⁺irisin group had no significant change compared with that of CCl₄ group（P>0.05）.（3）Compared with olive oil group,fibrotic marker proteins α-SMA（red）and Collagen I（green）were significantly expressed in wild type and FNDC5^-/-mice CCl₄ group,especially in FNDC5^-/-mice.Irisin treatment significantly reduced the fluorescence intensity of the expression of these two proteins,but the decrease of FNDC5^-/-mice was not as significant as that of the wild type.WB test results showed that in wild-type and FNDC5^-/-mice,the protein expressions of α-SMA and Collagen I in liver of CCl₄ group were significantly higher than those of olive oil group（P<0.05）.The protein expressions of α-SMA and Collagen I in liver of mice in CCl₄⁺irisin group were significantly lower than those in CCl₄ group（P<0.05）.However,irisin treatment of FNDC5^-/-mice had less relief on α-SMA and Collagen I in liver than wild-type mice.（4）q RT-PCR results showed that in wild-type and FNDC5^-/-mice,the m RNA expressions of α-SMA,Collagen I,TNF-α and IL-1β in liver of CCl₄ group were significantly higher than those of olive oil group（P<0.05）.Compared with CCl₄ group,the m RNA expressions of α-SMA,Collagen I,TNF-α and IL-1β in liver of CCl₄⁺irisin group were significantly decreased（P<0.05）.（5）Flow cytometry results showed that compared with olive oil group,the number of CD8⁺ T lymphocytes in liver of CCl₄ group was significantly increased（P<0.05）,and the number of CD4⁺ T lymphocytes was significantly decreased（P<0.05）in wild-type and FNDC5^-/-mice.Irisin treatment significantly restored a decrease in the number of CD4⁺ T lymphocytes,but did not reverse the increase in the number of CD8⁺ T lymphocytes.In both kinds of mice,compared with olive oil group,the number of inflammatory M1（CD11c⁺）type macrophages in the liver of CCl₄ group was significantly increased（P<0.05）,and the number of anti-inflammatory M2（CD206⁺）type macrophages was significantly decreased（P<0.05）,irisin treatment significantly reduced the number of M2（CD206⁺）type macrophages and increased the number of wild-type CD11c⁺ macrophages,but irisin did not reverse the increase of CD11c⁺ macrophages in FNDC5^-/-mice.Conclusions:（1）Intraperitoneal injection of irisin can improve the process of CCl₄-induced liver fibrosis in mice,and its mechanism may be related to regulating the distribution of immune cells in the liver,reducing the expression of inflammatory-related genes,and regulating the secretion of EVs.（2）Deletion of FNDC5 gene in vivo can further accelerate the development of liver fibrosis in mice,and the therapeutic effect of irisin is related to the regulatory function of FNDC5 gene in vivo.Part Ⅳ FNDC5/irisin inhibits hepatic stellate cell activation and liver fibrosis by regulating stability of HNRNP A1 and endoplasmic reticulum stressObjective:Endoplasmic reticulum stress（ERs）and ERs-activated unfolded protein response（UPR）have been shown to be drivers of liver injury,inflammation,and fibrosis,and an important response that often accompanies the development of acute and chronic liver diseases.Studies have found that ERs can participate in the development of fibrosis by inducing the degradation of heterogeneous nuclear nucleoprotein A1（HNRNP A1）.In the above studies,we have confirmed the effect of irisin on the release of EVs from activated HSCs and the process of liver fibrosis,but the regulation of the expression of ERs and HNRNPA1,an important link in the activation of HSCs,is still unclear.Therefore,the study in this part intends to investigate the mechanism of irisin improving liver fibrosis from the perspective of ERs by observing the regulation of irisin on HNRNP A1 protein stability and ERs.Methods:（1）The mouse primary HSCs were cultured in vitro,and the HSCs were treated with 100 ng/m L irisin or an equal volume of PBS for 12 days.Three replicate wells were set in each group.The HSCs were collected at the beginning of the experiment and on the 12 th day,and the protein was extracted.ERs-related proteins GRP78,PERK and their phosphorylation expression,regulation of CHOP.（2）The human hepatic stellate cell line LX-2 was cultured in vitro,the cells were pretreated with 100 ng/m L irisin or an equal volume of PBS for 2 h,and then incubated with 2 μg/m L tunicamycin（Tun）for 12 h to induce cell ERs.The cells were collected,the protein was extracted,and the protein expressions of PERK,p-PERK and HNRNP A1 were detected by WB.（3）FLAG-HNRNP A1 was transfected into LX-2 cells.After transfection,the cells were pretreated with 100 ng/m L irisin and an equal volume of PBS for 2 h,and then incubated with 2 μg/m L Tun for 12 h.After exposure to 50 μg/m L CHX,the cells were collected at the designated time（0 h,1 h,3 h and 6 h）,the protein expression level of HNRNP A1 in cells at each time point was detected by western blotting method,and the half-life curve of FLAG-HNRNP A1 was drawn.（4）Trans Fast transfection reagent Lipofectamine 2000 was used to transfect PERK overexpression plasmid into LX-2 cells.After confirming the transfection efficiency,cells were pretreated with 100 ng/m L irisin or an equal volume of PBS for2 h,and then 2 μg /m L Tun was incubated for 12 h,cells were collected,and the protein levels of p-PERK,PERK and HNRNP A1 in LX-2 cells in each group were detected by WB.（5）Trans Fast transfection reagent Lipofectamine 2000 was used to transfect short hairpin RNA（sh RNA）HNRNP A1 interfering sequence（sh RNA-HNRNP A1）and its negative control sequence sh RNA-NC into LX-2 cells.After transfection,cells were pretreated with 100 ng/m L irisin or an equal volume of PBS for 2 h,and then incubated with 2 μg/m L Tun for 12 h.The expressions of HNRNP A1 and α-SMA in LX-2 cells were detected by immunofluorescence staining,and the The m RNA levels of TGF-β,α-SMA,Collagen I and TIMP-1 in LX-2 cells were detected by q RT-PCR,and the protein expressions of Collagen I,GRP78 and CHOP in LX-2 cells were detected by WB method.（6）The third part of the three different treated wild-type mouse liver pathological sections were used to observe the expression and localization of p-PERK,HNRNP A1 and α-SMA in the liver by immunofluorescence staining.（7）Based on wild-type mice and HNRNP A1^-/-mice,10% CCl₄ dissolved in olive oil was injected intraperitoneally according to 0.01 m L/g body weight,2 times a week for a total of 8 weeks,to construct CCl₄-induced mice.In the liver fibrosis model,starting from the first day of CCl₄ injection,the CCl₄⁺irisin group was also intraperitoneally injected with 0.5 mg/kg/d of recombinant human irisin prepared with normal saline every day,and the olive oil control group and CCl₄ group were also given the same amount respectively.Intraperitoneal injection of normal saline.After the experiment,the mice were killed to collect blood and liver,routine pathological sections,Sirius red and Masson staining were used to observe the fibrosis of mice liver tissue.WB was used to detect the expression of liver fibrosis and ERs-related proteins（α-SMA,Collagen I,GRP78 and CHOP）in the liver of mice in each group.Results:（1）Compared with the freshly isolated mouse primary HSCs（resting state）,the protein expression levels of GRP78,p-PERK,PERK and CHOP,α-SMA and Collagen I in cultured 12 d HSCs（activated state）were significantly increased.Compared with the primary HSCs of mice cultured for 12 d,the protein expressions of GRP78,p-PERK,PERK,CHOP,α-SMA and Collagen I in HSCs treated with irisin were significantly down-regulated（P<0.05）.（2）WB detected the expression levels of p-PERK and PERK protein and HNRNP A1 protein in LX-2 cells.Compared with the control group,the expression levels of p-PERK and PERK protein in Tun group were significantly increased（P<0.05）,and the expression levels of HNRNP A1 protein were significantly decreased（P<0.05）.The expression levels of p-PERK and PERK protein in irisin group were significantly lower than those in Tun group（P<0.05）,and the expression levels of HNRNP A1 protein were significantly higher than those in Tun group（P<0.05）.（3）The results of analyzing the stability of HNRNP A1 protein after CHX treatment showed that in the control group,the protein expression level of FLAG-tagged HNRNP A1 began to decrease with time prolonging for 3 h,and decreased significantly at 6 h,The protein expression level of tagged HNRNP A1 began to decline at 1 h and almost disappeared at 6 h.It can be seen that Tun treatment accelerated the degradation of FLAG-tagged HNRNP A1,while the irisin pretreatment group significantly slowed down the protein expression level of FLAG-tagged HNRNP A1.（4）Under Tun exposure,p-PERK and PERK protein expression levels of LX-2cells were significantly increased,while HNRNP A1 protein expression level was significantly decreased（P<0.05）,which was consistent with the previous results.After Tun+irisin treatment,p-PERK and PERK protein expression levels of LX-2cells were significantly decreased compared with Tun group,while HNRNP A1 protein expression level was significantly increased（P<0.05）.Compared with Tun+irisin group,p-PERK and PERK protein expression levels of LX-2 cells transfected with PERK overexpressed plasmid were significantly increased,and HNRNP A1 protein expression level was significantly decreased（P<0.05）.（5）Immunofluorescence staining showed that Tun could induce the expression of α-SMA in LX-2 cells and down-regulate the expression of HNRNP A1（P<0.05）.irisin treatment could effectively inhibit the increase of α-SMA expression and decrease of HNRNP A1 expression in Tun induced LX-2 cells（P<0.05）.However,in LX-2 cells transfected with SHRNA-HNRNP A1,irisin treatment inhibited the expression of α-SMA and HNRNP A1 in Tun induced LX-2 cells,and its expression pattern was consistent with that of Tun group.q RT-PCR results showed that compared with the control group,m RNA expressions of TGF-β,α-SMA,Collagen I and TIMP-1 in LX-2 cells in Tun treatment group were significantly increased（P<0.05）.The m RNA expressions of TGF-β,α-SMA,Collagen I and TIMP-1 in LX-2cells in Tun+irisin treatment group were significantly decreased compared with Tun treatment group（P<0.05）,The m RNA expressions of TGF-β,α-SMA,Collagen I and TIMP-1 in LX-2 cells transfected with sh RNA-HNRNP A1 were significantly higher than those in Tun+irisin group（P<0.05）.WB results showed that compared with the control group,the protein expression levels of Collagen I,GRP78 and CHOP in LX-2cells in Tun treatment group were significantly increased（P<0.05）.Compared with Tun treatment group,Collagen I,GRP78 and CHOP protein expression levels in LX-2cells in Tun+irisin treatment group were significantly decreased（P<0.05）.Collagen I,GRP78 and CHOP protein expression levels of LX-2 cells in Tun+irisin+sh RNA-HNRNP-A1 treatment group were significantly increased compared with Tun+irisin treatment group（P<0.05）.（6）The results of tissue immunofluorescence staining showed that the expression levels of p-PERK and α-SMA in the liver tissue of the wild-type CCl₄ group and the co-localization intensity in HSCs were significantly higher than those of the olive oil control group,CCl₄⁺irisin Compared with the CCl₄ group,the expression levels of p-PERK and α-SMA and their co-localization in HSCs were significantly attenuated in the treatment group.The observation of the co-localization of HNRNP A1 andα-SMA in the liver of wild-type mice showed that compared with the olive oil group,the expression of HNRNP A1 in the liver tissue of the mice in the CCl₄ group was significantly decreased,while the expression of HNRNP A1 in the liver tissue of the mice in the CCl₄⁺irisin treatment group was significantly lower than that in the olive oil group.The expression of A1 was partially restored,and the strength of colocalization with α-SMA in HSCs was also significantly increased.（7）Histopathological results of mouse liver stained with Sirius red and Masson showed that CCl₄ induced significant liver fibrosis in wild-type mice,and irisin treatment could significantly improve the degree of liver fibrosis in mice（P<0.05）,In HNRNP A1^-/-mice,CCl₄ could also induce more obvious liver fibrosis than wild-type mice,and the improvement of fibrosis after irisin treatment was significantly worse than that of wild-type mice（P<0.05）.The results of WB detection showed that compared with wild-type mice,HNRNP A1^-/-further induced the expression ofα-SMA,Collagen I,GRP78 and CHOP in the liver tissue of CCl₄-induced liver fibrosis model mice,while irisin was in the liver of CCl₄-induced liver fibrosis model mice.The inhibition of these proteins in HNRNP A1^-/-mice was also significantly weaker than that in wild-type mice（P<0.05）.Conclusions:（1）Irisin can improve liver fibrosis in mice by inhibiting the degradation of HNRNP A1 protein mediated by HSCs ERs protein PERK,thereby regulating HSCs activation and extracellular matrix production.（2）HNRNP A1 is a new intermediate substrate of HSCs ERs signaling protein PERK,and it is also a key protein for irisin to improve liver fibrosis in mice.