基于LncRNA Meg3调控PI3K/Akt/mTOR信号通路探讨肝豆灵介导肝星状细胞自噬改善肝豆状核变性肝纤维化的作用机制OA

Objective To investigate how Gandouling activates the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway by regulating the long non-coding RNA maternal expression gene 3(LncRNA Meg3),thereby regulating autophagy in hepatic stellate cells and improving liver fibrosis in Wilson disease(WD).Methods Using a random number table,24 SD rats were divided into the blank control(n=6)and Gandouling groups(n=18).The Gandouling group rats were administered Gandouling suspension(0.96 g/kg)via oral gavage,whereas the blank control group rats received an equal volume of physiological saline via oral gavage.A copper-loaded cell model was established using copper sulfate pentahydrate(CuSO4·5H2 O)to stimulate the human hepatic stellate cell line LX-2.The Cell Counting Kit-8(CCK-8)assay was used to determine the optimal modeling concentration and intervention duration for CuSO4·5H2 O,as well as the optimal concentration and intervention duration for Gandouling-containing serum.LX-2 cells were divided into the control,model,Gandouling,pcDNA3.1-Meg3,and pcDNA3.1-NC groups for respective interventions.Cell proliferation was assessed using the 5-ethynyl-2'-deoxyuridine assay;changes in cellular autophagy flux were observed using confocal laser microscopy.Transmission electron microscopy was used to examine cellular ultrastructure;Western blotting was employed to detect key autophagy molecules,including the yeast Atg6 homolog(Beclin-1),Microtubule-associated protein 1 light clain 3(LC3)-Ⅱ/LC3-Ⅰ,ubiquitin-binding protein p62(p62),hepatic stellate cell activation markers collagen Ⅰ(Collagen Ⅰ)and alpha-smooth muscle actin(α-SMA),and phosphorylated proteins(p-PI3K,p-Akt,and p-mTOR)in the PI3K/Akt/mTOR pathway relative to total protein.Real-time quantitative PCR was used to detect mRNA expression of Collagen Ⅰ,α-SMA,LncRNA Meg3,PI3K,Akt,and mTOR.Results The optimal modeling condition for LX-2 cells was 100 μmol/L CuSO4·5H2 O treatment for 48 h,whereas the optimal intervention condition was 10%Gandouling-containing serum treatment for 48 h.Compared with the model group,the Gandouling and pcDNA3.1-Meg3 groups exhibited a reduction in green fluorescent puncta,and both groups showed a decrease in relative fluorescence intensity(P<0.05).Laser confocal microscopy revealed a reduction in the number of autophagolysosomes(P<0.05).Transmission electron microscopy showed reduced numbers of autophagosomes and autophagolysosomes.Beclin-1 and LC3-Ⅱ/LC3-Ⅰ protein expression were downregulated(P<0.05),p62 protein expression increased(P<0.05),and Collagen Ⅰ and α-SMA protein and mRNA expressions decreased(P<0.05).In contrast,PI3K/Akt/mTOR pathway mRNA expression and p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR were upregulated(P<0.05).Additionally,compared with the model group,the Gandouling group showed increased LncRNA Meg3 mRNA expression(P<0.05).Conclusion Gandouling delays the progression of hepatic fibrosis in WD by upregulating LncRNA Meg3 expression,activating the PI3K/Akt/mTOR signaling pathway,and suppressing abnormal autophagy in copper-loaded LX-2 cells.LncRNA Meg3 may represent a potential key target for Gandouling in improving hepatic fibrosis associated with WD.