| Background Alcoholic hepatitis(AH)is a liver-damaging disease caused by long-term alcohol consumption.It belongs to the early stage of alcoholic liver disease and is also the focus of clinical intervention and treatment.Despite years of research,there is still a lack of effective clinical methods to prevent or reverse AH in patients.The continuous over activation of liver macrophages(kupffer cells)plays a crucial role in inducing and promoting AH.Recent studies have shown that exosomes can directly activate liver macrophages in AH,which is an important factor in the excessive activation of macrophages.Inhibiting the production of secretions and reducing the activation of macrophages can alleviate the excessive inflammatory response of the liver in AH,which is an important research direction in the treatment of AH.Objective Balanophora polysaccharide(BPP)is a whole plant of Balanophora involucrata Hook.f.(BP)and its similar plants,which has been used as a folk medicine for a long time and has been proven to reduce the mortality of alcohol fed mice.The purpose of this study is to study the mechanism of BPP,an extract of BP,in improving AH and providing experimental evidence for the application of BPP.Methods C57BL/6 mice were fed a Lieber-De Carli diet to establish a chronic AH mouse model.During the modeling period,the mice in the dietary control group were given a control liquid feed,while the other groups were given an alcohol liquid feed with an alcohol concentration of 5%(volume/volume).The feed was prepared on the same day,and daily changes in food consumption and body weight of the mice were recorded.Specific pathogen free(SPF)grade C57BL/6 male mice(20-22 g)were randomly divided into a liquid diet control group(PF),an alcoholic feeding model group(AF),an alcoholic feeding +low dose of Balanophora polysaccharide group(AF+BPP-L),and an alcoholic feeding+high dose of Balanophora polysaccharide group(AF+BPP-H),with 12 mice in each group.In the drug action group,mice were administered 200 mg/kg and 400 mg/kg of Balanophora polysaccharide by gavage,while mice in the other groups were administered the corresponding drug solvent,0.5% sodium carboxymethyl cellulose(CMC-Na),once a day,for4 weeks.After the end of the last administration,the mice were killed by eyeball blood extraction,and the following indicators were observed: 1)Hematoxylin eosin staining(HE)and oil red O staining in liver tissue were used to detect liver damage and lipid deposition in mice;2)Biochemical enzyme method was used to detect the content of alanine transaminase(ALT)in serum,total cholesterol(TC),and triglyceride(TG)in liver tissue of mice;3)Real-time PCR was used to detect the expression of inflammatory factors,lipid synthesis related genes,and miR-155 in liver tissue;4)Western blot was used to detect the expression of autophagy related proteins,such as the mammalian target of rapamycin(m TOR)and Ras homologous enriched in brain(Rheb).Detection of autophagy marker protein,selective autophagy junction protein Sequencosome 1(p62/SQSTM1).Detect the expression of lysosomal associated membrane proteins such as Recombinant Lysosomal Associated Membrane Protein1(LAMP1)and Recombinant Lysosomal Associated Membrane Protein 2(LAMP2);Detect the expression of recombinant cluster of differentiation 40 ligand(CD40L)in liver tissue;5)Immunohistochemistry and immunofluorescence were used to detect the expression of CD40 L and CD63 in liver tissue,respectively;6)The size,concentration,and surface markers of the exosomes were characterized by transmission electron microscopy and nanoparticle tracking analysis(NTA)techniques.The concentration and size distribution of the exosomes were analyzed using the Nano Sight NS300 system(Nano Sight,Amesbury).Cell experiments used alcohol to stimulate AML-12 cells to establish a model of hepatocyte autophagy damage,and added 100 μg/m L 、 200 μg/m L Balanophora polysaccharide was used for intervention.THP-1 cells were cultured in RPMI-1640 complete medium and subjected to 5 μg/m L phorbol myristate acetate(PMA)stimulated for 12 hours to form PMA differentiated THP-1 cells(pd-TPH-1).The exosomes of normal and alcohol stimulated AML-12 cells were collected,labeled with PKH26,and co cultured with pd TPH-1cells.The exosomes were interfered with with Luxiacao polysaccharide.The following indicators were observed: 1)Real time PCR was used to detect the expression of miR-155 in AML-12 cells and the expression of pd-TPH-1 inflammatory related factors;2)Western blot was used to detect the expression of lysosomal associated proteins LAMP1,LAMP2,and secreted body associated protein CD40 L in AML-12 cells;3)PKH26 labeled exosomes were used to observe the uptake of pd-TPH-1 cell exosomes;4)Transmission electron microscopy and nanoparticles tracking analysis techniques were used to characterize the size,concentration,and surface markers of the exosomes.Nano Sight NS300 system(Nano Sight,Amesbury)was used to analyze the concentration and size distribution of the exosomes.Results BPP effectively protected the liver injury of AH mice: 1)Compared with the control diet group mice,HE staining and oil red O staining results showed that the alcohol diet model group mice had severe liver injury and lipid deposition,and after the intervention of BPP,liver injury and lipid deposition were significantly reduced;2)Compared with the control diet group,the alcohol diet model group had higher levels of serum ALT and liver TC,TG.After intervention with BPP,the levels of serum ALT and liver TC,TG were significantly decreased,with a statistically significant difference(P <0.05);3)Real-time PCR results showed that compared with the control diet group,the alcohol diet group had indicators related to liver tissue inflammation and macrophage activation(such as TNF-α,IL-1β,NLRP3,MCP-1,etc.)and lipid synthesis related genes(such as SREBP1 c,Fasn,etc.)increased significantly,and decreased significantly after intervention with BPP(P <0.05).BPP significantly improved the autophagic damage of liver cells in AH mice and inhibited the production of liver secretions: 1)The expression level of miR-155 in the liver of mice fed with alcohol increased,and after the intervention of BPP,the expression of miR-155 in mice fed with alcohol significantly decreased(P <0.05);2)Compared with the control diet group,the expression of m TOR,Rheb,and p62 in the liver tissue of mice in the alcohol diet group significantly increased,while the expression of LAMP1 and LAMP2 decreased.After the intervention of BPP,LAMP1 and LAMP2 significantly increased,and p62 significantly decreased(P <0.05).3)Immunohistochemical and Western blot results showed that compared with PF group,the expression of CD40 L in liver tissue of AF group mice was significantly increased,while the expression of CD40 L in alcohol diet mice decreased after the intervention of BPP;4)Compared with the PF group,the number of exocrine bodies in the AF group significantly increased,while the number of BPP decreased after intervention,which was statistically significant in the high-dose group(P <0.05).BPP reduced the autophagic damage and increased secretion of alcohol induced AML-12cells: 1)BPP significantly inhibited the increase in miR-155 expression level in alcohol induced AML-12 cells;2)Compared with the normal group,alcohol inhibited the expression of LAMP1 and LAMP2 in AML-12 cells,while the effect of BPP could be reversed(P <0.05);3)Compared with the normal group,the number of exocrine bodies in the alcohol group significantly increased,and after the intervention of BPP,the number of exocrine bodies decreased(P <0.05);4)Compared with the normal group,the protein expression of CD40 L in the alcohol group significantly increased,and after intervention with BPP,the expression of CD40 L in the alcohol group decreased(P <0.05);5)BPP can inhibit the expression of inflammatory factors in pd-TPH-1 cells,and its inflammatory factor is found in pd-TPH-1cells co cultured with the secretion of alcohol stimulated AML-12 cells.The expression of TNF-α,IL-1β was significantly decreased after intervention with BPP(P <0.05).Conclusion 1)BPP can effectively inhibit alcohol induced liver inflammation,reduce liver lipid deposition,and improve liver damage in chronic AH mice;2)BPP can effectively reduce the alcohol induced increase in the production of liver exosome-CD40 L,inhibit the activation of liver macrophages,and improve the excessive inflammatory response of the liver;3)BPP can inhibit the alcohol induced expression of miR-155 in hepatocytes,improve the function of autophagic lysosomes,and thereby inhibit the secretion of exosomes. |
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