Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-β/Smad signaling pathway

BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.

Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats

AIM:To observe the effect of Danshao Huaxian capsule(DHC)on the expression of Gremlin and bone morphogenetic protein-7(BMP-7)in the liver of hepatic fibrosis rats.METHODS:A total of 75 male Wistar rats were randomly divided into a normal control group(A),a CCl4-induced hepatic fibrosis model group(B),a natural recovery group(C),a low-dose DHC-treated group(D),and a high-dose DHC-treated group(E),with 15 rats in each group.Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride(CCl4)and a highlipid/low-protein diet for 8 wk,except for the rats in group A.Then,the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks,respectively.By the end of the experiment,the level of transforming growth factorβ1(TGF-β1)in the liver homogenate was determined by an enzyme-linked immunosorbent assay.The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reversetranscription polymerase chain reaction,an immunohistochemical assay,and Western blot analysis.RESULTS:Compared with group A,the level of TGF-β1and the mRNA and protein expression of Gremlin were significantly higher in group B(TGF-β1:736.30±24.40μg/g vs 284.20±18.32μg/g,P<0.01;mRNA of Gremlin:80.40±5.46 vs 49.83±4.20,P<0.01;positive protein expression rate of Gremlin:38.46%±1.70%vs 3.83%±0.88%,P<0.01;relative protein expression of Gremlin:2.81±0.24 vs 0.24±0.06,P<0.01),and the mRNA and protein expression of BMP-7was significantly lower in group B(mRNA:54.00±4.34vs 93.99±7.03,P<0.01;positive protein expression rate:28.97%±3.14%vs 58.29%±6.02,P<0.01;relative protein expression:0.48±0.31 vs 1.05±0.12,P<0.01).Compared with groups B and C,the degree of hepatic fibrosis was significantly improved,and the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHCtreated groups(TGF-β1:523.14±21.29μg/g,441.86±23.18μg/g vs 736.30±24.40μg/g,651.13±15.75μg/g,P<0.01;mRNA of Gremlin:64.86±2.83,55.82±5.39 vs 80.40±5.46,70.37±4.01,P<0.01;positive protein expression rate of Gremlin:20.78%±1.60%,17.43%±2.02%vs 38.46%±1.70%,29.50%±2.64%,P<0.01;relative protein expression of Gremlin:1.95±0.26,1.65±0.20 vs 2.81±0.24,2.22±0.63,P<0.01),and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups(mRNA:73.52±4.56,81.78±5.38 vs 54.00±4.34,62.28±4.51,P<0.01;positive protein expression rate:41.44%±4.77%,47.49%±4.59%vs28.97%±3.14%,35.85%±3.50%,P<0.01;relative protein expression:0.71±0.06,0.81±0.07 vs 0.48±CONCLUSION:The therapeutic mechanism of DHC forhepatic fibrosis in rats may be associated with inhibitionof the expression of Gremlin and up-regulation of the expression of BMP-7.

Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells

BACKGROUND Hepatic stellate cells(HSCs)are the key effector cells mediating the occurrence and development of liver fibrosis,while aerobic glycolysis is an important metabolic characteristic of HSC activation.Transforming growth factor-β1(TGF-β1)induces aerobic glycolysis and is a driving factor for metabolic reprogramming.The occurrence of glycolysis depends on a high glucose uptake level.Glucose transporter 1(GLUT1)is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism,thus affecting cell proliferation and growth.However,little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIM To investigate the mechanisms of action of GLUT1,TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue.A Seahorse extracellular flux(XF)analyzer was used to examine changes in aerobic glycolytic flux,lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation.The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothersagainst-decapentaplegic-homolog 2/3(Smad2/3)signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase(PI3K)/AKT signaling pathways.In addition,GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs.Finally,a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues.In addition,immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins,indicating that GLUT1 expression was related to the development of liver fibrosis.TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad,p38 MAPK and P13K/AKT signaling pathways.The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression.GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration.A GLUT1 inhibitor was administered in a mouse model of liver fibrosis,and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSION TGF-β1 induces GLUT1 expression in HSCs,a process related to liver fibrosis progression.In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs.In addition,in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.