Anthocyanins Regulate The Autophagy Flux Level Of Hepatic Stellate Cells Through Mmu＿circ＿0000623 To Improve The Mechanism Of Liver Fibrosis In Mice

Objective:Hepatic fibrosis refers to the cicatricial repair reaction that occurs when the liver is damaged,mainly manifested by excessive deposition of Extracellular matrix（ECM）.Hepatic fibrosis is closely related to inflammation and oxidative stress.Previous studies have shown that changes in the level of autophagy can regulate the activation of HSCs,thus regulating liver fibrosis.Transcription factor EB（TFEB）is a major regulator of lysosome biosynthesis and autophagy,and its high expression has been confirmed to cure liver injury.Anthocyanins are a class of water-soluble flavonoids widely found in fruits and vegetables.A large number of studies have shown that anthocyanins have extensive antioxidant and anti-inflammatory properties.The benefits of anthocyanins on human health have been confirmed by many fields of research,but the inhibitory effect of anthocyanins on liver fibrosis and the molecular mechanism of anthocyanins are still blank.In this study,anthocyanin was selected as an intervention condition to explore the role of anthocyanin in regulating autophagy flux and TFEB in hepatic fibrosis.Methods:Twenty-four 8-week-old C57BL/6J male mice were randomly divided into four groups:control group,CCl₄group,CCl₄+Antho100 group,and CCl₄+Antho200group.CCl₄group,CCl₄+Antho100 group and CCl₄+Antho200 group were intrabitoneally injected with 10%olive oil dissolved CCl₄at 2 m L/kg body weight into 6 8-week-old C57BL/6J male mice,3 times a week until 12 weeks.Control group was given control（10%olive oil,2m L/kg）.CCl₄+Antho100 group and CCl₄+Antho200 group were given 100 and 200 mg/kg body weight anthocyanins per day by gavage during CCl₄injection.The control group and the CCl₄group were given the same amount of normal saline every day.After the last injection of chronic CCl₄,all mice were anesthetized with pentobarbital（100mg/kg）and their blood and liver were kept.The serum was separated,and the right lobe tissue of each mouse liver was about 3mm×3mm,fixed with 4%neutral formaldehyde,and the remaining liver tissue was stored in the refrigerator at-80℃.Detect the following indicators:（1）Routine pathological sections were stained with HE,Masson and Sirius scarlet,and the pathological changes of liver in each group were observed.（2）Shear wave elastography（SWE）was used to evaluate the hardness of liver in different treatment groups.（3）m RNA expressions of CollagenⅠandα-SMA in liver of mice in each group were detected by q RT-PCR.（4）The expressions of CollagenⅠandα-SMA were observed by IHC and WB.（5）The expression levels of AST and ALT in the liver of mice in each group were detected by ELISA.Mouse hepatic stellate cells（m HSCs）were randomly divided into three groups:PBS group（treated with solvent PBS）,PDGF group（treated with PDGF 20ng/m L）and PDGF+anthocyanin group（treated with PDGF 20ng/m L+100μg/m L）.After treatment,the following tests were performed:（6）CCK-8 and Ed U staining were used to detect the proliferation of m HSCs cells in each group.（7）m RNA and protein expressions of CollagenⅠandα-SMA in different treatment groups were detected by q RT-PCR and WB,respectively.（8）Scratch test was used to verify the effects of different treatment groups on the migration ability of m HSCs.（9）TUNEL staining was used to detect apoptosis of m HSCs in different treatment groups（10）m RNA and protein expressions of several common autophagy markers（ATG7,ATG10,ATG5,BECN1）were detected by q RT-PCR and WB.（11）The protein expressions of several autophagy related proteins（LC3Ⅰ,LC3Ⅱ,P62）were detected by WB.（12）Autophagy fluxes in different treatment groups were detected by m RFP-GFP-LC3 method.（13）m RNA expression of lysosome markers（LAMP1 and LAMP2）in m HSCs of different treatment groups was detected by q RT-PCR.（14）m RNA expressions of lysosome markers（LAMP1 and LAMP2）in the liver of mice in the first part of animal model were detected by q RT-PCR.（15）m RNA and protein expression of TFEB in m HSCs of different treatment groups were detected by q RT-PCR and WB.（16）q RT-PCR and WB were used to detect the expression of TFEB m RNA and protein in the liver of mice in the first animal model group.Results:（1）The pathological observation of routine pathological sections,HE,Masson and sinophora scarlet staining showed that the liver of mice in CCl₄group had obvious fibrous connective tissue hyperplasia and liver fibrosis compared with the control group.The degree of liver fibrosis in the CCl₄+Antho100 group and the CCl₄+Antho200 group treated with 100 mg/kg and 200 mg/kg anthocyanins by intragastric administration was significantly reduced compared with that in the CCl₄group,and this phenomenon was more obvious in Masson staining and Sirius red staining of collagen fibers.（2）The SWE test results showed that the liver hardness of CCl₄group was significantly increased compared with the control group（P<0.05）.Compared with CCl₄group,liver hardness in CCl₄+Antho100 and CCl₄+Antho200 groups was significantly decreased（P<0.05）.（3）m RNA expressions of CollagenⅠandα-SMA,markers of liver fibrosis,were detected by q RT-PCR.The results showed that the m RNA expressions of CollagenⅠandα-SMA in liver of CCl₄group were significantly increased compared with control group（P<0.05）.Compared with CCl₄group,the m RNA expressions of CollagenⅠandα-SMA in liver of CCl₄+Antho100 and CCl₄+Antho200 groups were significantly decreased（P<0.05）.（4）The results of IHC and WB showed that compared with the control group,there were significant expressions of CollagenⅠandα-SMA in the liver of CCl₄group（P<0.05）.Compared with CCl₄group,the expression levels of collagenⅠandα-SMA in liver of CCl₄+Antho100 and CCl₄+Antho200 groups were significantly decreased（P<0.05）.（5）ELISA assay of AST and ALT in liver of mice showed that compared with the control group,the expressions of AST and ALT in CCl₄group were significantly increased（P<0.05）.Compared with CCl₄group,the expression levels of AST and ALT in CCl₄+Antho100 and CCl₄+Antho200 groups were significantly decreased（P<0.05）.（6）The proliferation of m HSCs cells was detected by CCK-8 and Ed U staining,and the results showed that compared with PBS group,the cells in PDGF group were significantly enhanced（P<0.05）.Compared with PDGF group,cell proliferation in PDGF+Antho group was significantly decreased（P<0.05）.（7）q RT-PCR results showed that m RNA expressions of CollagenⅠandα-SMA in PDGF group were significantly higher than those in PBS group（P<0.05）.Compared with PDGF group,m RNA expressions of CollagenⅠandα-SMA in PDGF+Antho group were significantly decreased（P<0.05）.WB test results showed that compared with PBS group,the expressions of CollagenⅠandα-SMA in PDGF group were significantly increased（P<0.05）.Compared with PDGF group,the expressions of CollagenⅠandα-SMA in PDGF+Antho group were significantly decreased（P<0.05）.（8）Scratch test results showed that migration ability of m HSCs in PDGF group was significantly enhanced compared with PBS group（P<0.05）.Compared with PDGF group,migration ability of m HSCs in PDGF+Antho group was significantly decreased（P<0.05）.（9）TUNEL staining showed that although the number of positive cells in PDGF+Antho group increased,there was no statistical difference between the groups.（10）q RT-PCR and WB detection results showed that compared with PBS group,m RNA and protein expressions of autophagy related proteins ATG7,ATG10,ATG5and BECN1 in PDGF group and PDGF+Antho group were significantly increased（P<0.05）.There were no significant differences in m RNA and protein expressions of autophagy related proteins ATG7,ATG10,ATG5 and BECN1 between PDGF group and PDGF+Antho group.（11）WB detection results showed that compared with PBS group,the LC3Ⅱ/Ⅰvalues in PDGF group and PDGF+Antho group were significantly increased（P<0.05）;There was no significant difference in LC3Ⅱ/Ⅰvalues between PDGF group and PDGF+Antho group.Compared with PBS group,P62 protein expression in PDGF group was significantly increased（P<0.05）.Compared with PDGF group;The expression of P62 protein in PDGF+Antho group was significantly decreased（P<0.05）.（12）m RFP-GFP-LC3 assay showed that compared with PBS group,autophagy flux in PDGF group was decreased（P<0.05）.Compared with PDGF,autophagy flux in PDGF+Antho group was significantly increased（P<0.05）.（13）The results of q RT-PCR showed that compared with PBS group,the cell lysosome markers（LAMP1 and LAMP2）in PDGF group were significantly decreased（P<0.05）;Compared with PDGF group,m RNA expressions of lysosome markers（LAMP1 and LAMP2）in PDGF+Antho group were significantly increased（P<0.05）.（14）Compared with the control group,m RNA expressions of lysosome markers（LAMP1 and LAMP2）in CCl₄and CCl₄+Antho 100 groups were significantly decreased（P<0.05）.Compared with CCl₄group,m RNA expressions of lysosome markers（LAMP1 and LAMP2）in CCl₄+Antho 200 group were significantly increased（P<0.05）.（15）Compared with PBS group,the m RNA and protein expression of TFEB in PDGF group were significantly decreased（P<0.05）.Compared with PDGF group,m RNA and protein expressions of TFEB in PDGF+Antho group were significantly increased（P<0.05）.（16）Compared with the control group,the m RNA and protein expression of TFEB in CCl₄group were significantly decreased（P<0.05）.Compared with CCl₄group,m RNA and protein expression of TFEB in CCl₄+Antho 100 group and CCl₄+Antho 200 group were significantly increased（P<0.05）.Conclusions:（1）Anthocyanins can slow the progression of liver fibrosis in mice.In vitro cell culture studies have shown that anthocyanins inhibit the activation and migration of hepatic stellate cells.（2）Anthocyanins can improve autophagy blocking of m HSCs by PDGF and lysosome damage.（3）Anthocyanins significantly increased the expression of TFEB in vivo and in vitro models,and TFEB may play an important role in anthocyanin regulation of autophagy flux.Objective:Previous studies have shown that anthocyanins may play a therapeutic role in liver fibrosis by regulating circ₀000623,but the specific regulatory mechanism of circ₀000623 remains unclear.Numerous studies have shown that circ RNA can play relevant biological roles by competing with synthetic mirnas.Existing studies have confirmed that TFEB is a major regulator of lysosome biosynthesis and autophagy.This part aims to investigate whether anthocyanins play a therapeutic role in mouse liver fibrosis through circ₀000623/ mi R-351-5p /TFEB axis.Methods:（1）The ring formation stability of circ₀000623 was verified by exonuclease tolerance assay.（2）m HSCs were used and randomly divided into three groups: PBS group,PDGF group and PDGF+Antho group（the grouping and treatment methods were the same as in Part 1）.After 48 h of treatment,the expression of circ₀000623 in different groups was detected by q RT-PCR.m HSCs were used and randomly divided into three groups: PDGF group（PDGF20ng/m L treatment）,PDGF+Antho group（PDGF 20ng/m L treatment +100μg/m L）and PDGF+circ₀000623 group（PDGF 20ng/m L treatment +25μg/m L）,Follow-up detection of m HSCs was performed 48 h after treatment:（3）CCK-8 and Ed U staining were used to detect the proliferation of m HSCs cells in each group.（4）Scratch test was used to verify the effects of different treatment groups on the migration ability of m HSCs.（5）m RNA expressions of Collagen Ⅰ and α-SMA in hepatic stellate cells of mice in each group were detected by q RT-PCR.（6）Autophagy fluxes in different treatment groups were detected by m RFP-GFP-LC3 method.（7）m RNA expression of lysosome markers（LAMP1 and LAMP2）in m HSCs of different treatment groups was detected by q RT-PCR.（8）q RT-PCR and WB were used to detect the expression of TFEB m RNA and protein in m HSCs of different treatment groups.（9）The cell nucleus and cytoplasm were separated,and the distribution of circ₀000623 in the nucleus and cytoplasm subcellular components was detected by q RT-PCR.（10）We attempted to explore the common target mi RNA of circ₀000623 and TFEB proteins using starbase database,and after overexpression of circ₀000623,q RT-PCR was used to verify the intermediate role of mi RNA.（11）m HSCs were used and randomly divided into four groups: PBS group,PDGF group,PDGF+Antho group and PDGF+circ₀000623 group（the treatment of each group was the same as the first and second parts）.After 48 h of treatment,the expression of mi R-351-5p in different groups was detected by q RT-PCR.（12）FISH assay verified the sublocalization of circ₀000623 and mi R-351-5p in m HSCs.（13）Dual luciferase detection and RIP detection were used to verify the presence of targeted binding between circ₀000623 and mi R-351-5p.（14）Dual luciferase assay and RIP assay verified the presence of targeted binding of mi R-351-5p and TFEB m RNA.m HSCs were used and randomly divided into four groups: si-NC group（transfected with invalid small interfering RNA）,si-cicr₀000623 group（transfected with si-cicr₀000623）,si-cicr₀000623+mi R-351-5p inhibitor group（transfection of si-cicr₀000623+ transfection of mi R-351-5p inhibitor）and si-cicr₀000623+TFEB（transfection of si-cicr₀000623+ transfection of GFP-TFEB plasmid）were treated with different methods.Conduct the following follow-up tests:（15）CCK-8 and Ed U staining were used to detect the proliferation of m HSCs cells in each group.（16）Scratch experiments were used to verify the effects of different treatment groups on the migration ability of m HSCs.（17）m RNA expressions of Collagen I and α-SMA in m HSC were detected by q RT-PCR.（18）Autophagy fluxes in different treatment groups were detected by m RFP-GFP-LC3 method.（19）The protein and m RNA expressions of lysosome markers（LAMP1 and LAMP2）in m HSCs of different treatment groups were detected by q RT-PCR and WB.（20）m RNA expressions of cicr₀000623,mi R-351-5p and TFEB in m HSCs of different treatment groups were detected by q RT-PCR,respectively.In the animal experiment,24 8-week-old C57BL/6J male mice were randomly divided into four groups: control group,NC group,CCl4 group and CCl4+cicr₀000623 group.In CCl4 group and CCl4+cicr₀000623 group,68-week-old C57BL/6J male mice were intrabitoneally injected with 10% olive oil dissolved CCl4 at 2 m L/kg body weight,3 times a week until 12 weeks.control group and NC group were given control agent（10% olive oil,2m L/kg）.During the injection of CCl4 group and CCl4+cicr₀000623 group,100μL control AAV8-negative control virus particles were injected into CCl4 group via caudal vein at the same time of each intraperitoneal injection.The CCl4+cicr₀000623 group was injected with100μLAAV8-cicr₀000623 virus particles via caudal vein（the above adenovirus particles were constructed by Gene Pharma with a concentration of 1.5×1012TU/m L）.The following tests shall be carried out after the molding is completed:（21）The routine pathological sections were stained with HE,Masson and sinophora scarlet,and the pathological changes of liver in each group were observed.（22）Ultrasonic SWE examination was performed to evaluate the hardness of the liver of mice in different treatment groups.（23）The protein and m RNA expressions of Collagen Ⅰ and α-SMA in liver of mice in each group were detected by q RT-PCR and WB.（24）The expressions of Collagen Ⅰ and α-SMA in extracellular matrix were observed by IHC.（25）The expression levels of AST and ALT in liver of mice in each group were detected by ELISA.（26）The m RNA expression of mi R-351-5p and TFEB and the m RNA expression of lysosome markers（LAMP1 and LAMP2）in the liver of mice in each group were detected by q RT-PCR..Results:（1）The expression level of RNA after RNase R treatment was detected by q RT-PCR.The results showed that there was no statistical significance in the expression level of circ₀000623 group after RNase R treatment.The expression level of Tnrc6 b group was significantly down-regulated by RNase R treatment.（2）The expression of circ₀000623 in different treatment groups was detected by q RT-PCR.The results showed that the expression of circ₀000623 in PDGF group was significantly decreased compared with that in PBS group（P < 0.05）.Compared with PDGF group,circ₀000623 expression in PDGF+Antho group was significantly increased（P < 0.05）.（3）The proliferation of m HSCs was detected by CCK-8 and EdU staining.The results showed that compared with PDGF group,the proliferation of m HSCS in PDGF+Antho group and PDGF+circ₀000623 group was significantly decreased（P <0.05）.（4）Scratch test results showed that compared with PDGF group,migration ability of m HSCs in PDGF+Antho group and PDGF+circ₀000623 group was significantly enhanced（P < 0.05）.（5）q RT-PCR results showed that m RNA expressions of Collagen Ⅰ and α-SMA in PDGF+Antho and PDGF+circ₀000623 groups were significantly increased compared with PDGF group（P < 0.05）.（6）Compared with PDGF group,autophagy flux level of m HSCs in PDGF+Antho group and PDGF+circ₀000623 group was significantly increased by m RFP-GFP-LC3（P < 0.05）.（7）Compared with PDGF group,m RNA expressions of lysosome markers（LAMP1 and LAMP2）in m HSCs in PDGF+Antho group and PDGF+circ₀000623 group were significantly increased（P < 0.05）.（8）Compared with PDGF group,m RNA expression of TFEB in m HSCs in PDGF+Antho group and PDGF+circ₀000623 group was significantly increased（P <0.05）.Compared with the PDGF group,the expression of TFEB protein in m HSCs in PDGF+Antho group and PDGF+circ₀000623 group was significantly increased（P <0.05）.（9）The expression of circ₀000623 in different subcellular components was detected by q RT-PCR after karyoplasmic separation.It was found that circ₀000623was mainly distributed in the cytoplasm.（10）Three mirnas were screened out by Venn diagram,namely mi R-125b-5p,mi R-351-5p and mi R-665-3p.We verified the expression of these three mirnas through overexpression of circ₀000623,and q RT-PCR showed that the downregulation of mi R-351-5p was the most significant.（11）The expression of mi R-351-5p in different treatment groups was detected by q RT-PCR.The results showed that the expression of mi R-351-5p in PDGF group was significantly increased compared with that in PBS group（P < 0.05）.Compared with PDGF group,the expression of mi R-351-5p in PDGF+Antho group was significantly decreased（P < 0.05）.Compared with PDGF group,the expression of mi R-351-5p in PDGF+circ₀000623 group was significantly decreased（P < 0.05）.（12）FISH assay results showed that circ₀000623 and mi R-351-5p shared common sublocalization in m HSCs.（13）Dual luciferase detection and RIP detection results showed that circ₀000623 had targeted binding with mi R-351-5p.（14）Dual luciferase assay and RIP assay confirmed the presence of targeted binding of mi R-351-5p to TFEB m RNA.（15）The proliferation of m HSCs cells was detected by CCK-8 and Ed U staining.The results showed that the proliferation of m HSCs cells in the si-cicr₀000623 group was significantly increased compared with that in the si-NC group（P < 0.05）.Compared with si-cicr₀000623+mi R-351-5p inhibitor group,the proliferation of m HSCs in si-cicr₀000623+TFEB group and si-cicr₀000623+ TFEB inhibitor group was significantly decreased（P < 0.05）.（16）The scratch test results showed that the migration ability of m HSCs in the si-cicr₀000623 group was significantly enhanced compared with the si-NC group（P< 0.05）.Compared with si-cicr₀000623+mi R-351-5p inhibitor group,migration ability of m HSCs in si-cicr₀000623+TFEB group and si-cicr₀000623+ TFeb group was significantly decreased（P < 0.05）.（17）Compared with the si-NC group,m RNA expressions of Collagen Ⅰ andα-SMA in m HSCs in the si-cicr₀000623 group were significantly increased（P <0.05）.Compared with the si-cicr₀000623 group,The m RNA expressions of Collagen Ⅰ and α-SMA in m HSCs were decreased significantly in si-cicr₀000623+mi R-351-5p inhibitor group and si-cicr₀000623+TFEB group（P <0.05）.（18）Compared with the si-NC group,the autophagy flux level of m HSCs in the si-cicr₀000623 group was significantly decreased（P < 0.05）.Compared with si-cicr₀000623+mi R-351-5p inhibitor group and si-cicr₀000623+TFEB group,autophagy flux levels of m HSCs were significantly increased（P < 0.05）.（19）Compared with the si-NC group,the protein and m RNA expressions of lysosome markers（LAMP1 and LAMP2）in m HSCs cells in the si-cicr₀000623group were significantly decreased（P < 0.05）.Compared with the si-cicr₀000623group,Protein and m RNA expressions of m HSCs lysosome markers（LAMP1 and LAMP2）were significantly increased in si-cicr₀000623+mi R-351-5p inhibitor group and si-cicr₀000623+TFEB group（P < 0.05）.（20）The results of q RT-PCR showed that compared with the si-NC group,The expression of cicr₀000623 was significantly decreased in si-cicr₀000623+mi R-351-5p inhibitor group and si-cicr₀000623+TFEB group（P <0.05）.Compared with the si-NC group,the expression of mi R-351-5p in the si-cicr₀000623 group and the si-cicr₀000623+TFEB group was significantly increased（P < 0.05）.Compared with the si-cicr₀000623+mi R-351-5p inhibitor group,the expression of mi R-351-5p was significantly decreased（P < 0.05）;Compared with the si-NC group,the m RNA expression of TFEB in the si-cicr₀000623 group was significantly decreased（P < 0.05）.Compared with si-cicr₀000623+mi R-351-5p inhibitor group,TFEB m RNA expression was significantly increased in si-cicr₀000623+TFEB inhibitor group and si-cicr₀000623+ TFEB inhibitor group（P < 0.05）.（21）In the routine pathological sections,HE,Masson and sinophora scarlet staining showed that the liver of mice in CCl4 group and CCl4+ transfected no-carrier group had obvious fibrous connective tissue hyperplasia and liver fibrosis compared with the control group.The degree of liver fibrosis in the CCl4+ in vivo transfected with the overexpressed vector group was significantly reduced compared with that in the CCl4 and CCl4+ in vivo transfected with no carrier group.（22）Compared with the control group or NC group,the liver hardness of CCl4 group was significantly increased（P < 0.05）.Compared with CCl4 group,the liver hardness of CCl4+cicr₀000623 group was significantly decreased（P < 0.05）.（23）m RNA and protein expressions of Collagen Ⅰ and α-SMA,markers of liver fibrosis,were detected by q RT-PCR and WB.The results showed that,compared with the control group,The m RNA and protein expressions of Collagen Ⅰ and α-SMA in liver of CCl4 group and CCl4+ group were significantly increased（P < 0.05）.Compared with CCl4 group and CCl4+ group transfected with no-carrier in vivo,the m RNA and protein expressions of Collagen Ⅰ and α-SMA in liver of CCl4+ group transfected with overexpressed vector were significantly decreased（P < 0.05）.（24）The IHC results showed that compared with the control group,Collagen Ⅰand α-SMA were significantly expressed in the liver of CCl4 group and CCl4+transfected empty carrier group（P < 0.05）.Compared with CCl4 group and CCl4+ in vivo transfected with no-carrier group,the expression levels of ollagenⅠ and α-SMA in liver of CCl4+ in vivo transfected with overexpressed vector were significantly decreased（P < 0.05）.（25）ELISA results of AST and ALT in liver injury markers of mice showed that compared with the control group,the expressions of AST and ALT in liver of mice in CCl4 group and CCl4+ transfected no-carrier group were significantly increased（P <0.05）.Compared with CCl4 group and CCl4+ group transfected with no-carrier in vivo,the expression levels of AST and ALT in liver of CCl4+ group transfected with overexpressed vector were significantly decreased（P < 0.05）.（26）Compared with the control group,the expression of mi R-351-5p in CCl4 group was significantly increased（P < 0.05）.Compared with the CCl4 group,mi R-351-5p in the CCl4+cicr₀000623 group was significantly decreased（P < 0.05）.Compared with the control group,the m RNA expression of TFEB in CCl4 group was significantly decreased（P < 0.05）.Compared with CCl4 group,TFEB m RNA in CCl4+cicr₀000623 group was significantly increased（P < 0.05）.Compared with the control group,the expressions of lysosome markers（LAMP1 and LAMP2）in CCl4 group were significantly decreased（P < 0.05）.Compared with CCl4 group,lysosome markers（LAMP1 and LAMP2）were significantly increased in CCl4+cicr₀000623group（P < 0.05）.Conclusions:（1）Anthocyanins can promote the expression of circ₀000623,improve lysosome function and promote autophagy flux to treat hepatic fibrosis.（2）Anthocyanins play a role in the treatment of mouse liver fibrosis through circ₀000623/ mi R-351-5p /TFEB axis.